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2022
Annual Update
in Intensive Care
and Emergency
Medicine 2022

Edited by Jean-Louis Vincent

123
Annual Update in Intensive Care
and Emergency Medicine
The series Annual Update in Intensive Care and Emergency Medicine is the
continuation of the series entitled Yearbook of Intensive Care and Emergency
Medicine in Europe and Intensive Care Medicine: Annual Update in the United States.

More information about this series at http://link.springer.com/bookseries/8901

Jean-Louis Vincent
Editor

Annual Update in
Intensive Care and
Emergency Medicine 2022
Editor
Jean-Louis Vincent
Department of Intensive Care
Erasme University Hospital
Université libre de Bruxelles
Brussels, Belgium
[email protected]

ISSN 2191-5709     ISSN 2191-5717 (electronic)

Annual Update in Intensive Care and Emergency Medicine
ISBN 978-3-030-93432-3    ISBN 978-3-030-93433-0 (eBook)
https://doi.org/10.1007/978-3-030-93433-0

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature
Switzerland AG 2022
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether
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The use of general descriptive names, registered names, trademarks, service marks, etc. in this publica-
tion does not imply, even in the absence of a specific statement, that such names are exempt from the
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This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Contents

Part I Sepsis and the Immune Response

1 The Role of Mitochondria in the Immune Response
in Critical Illness����������������������������������������������������������������������������������������   3
Y. Wang and A. S. McLean
2 Immunomodulation by Tetracyclines in the Critically Ill:
An Emerging Treatment Option?������������������������������������������������������������ 13
A. Sauer, C. Putensen, and C. Bode
3 Hit Early: Blocking Interleukin-1 in the Treatment
of COVID-19 Pneumonia�������������������������������������������������������������������������� 29
E. J. Giamarellos-Bourboulis, M. Mouktaroudi, and M. G. Netea
4 Hemoadsorption Therapy During ECMO: Emerging Evidence ���������� 39
A. Supady, T. Wengenmayer, and D. Brodie

Part II Respiratory Issues

5 The Forgotten Circulation and Transpulmonary
Pressure Gradients������������������������������������������������������������������������������������ 51
E. Bowcock, S. R. Orde, and A. S. McLean
6 Oxygen: Origin, Physiology, Pathophysiology,
and Use in the Critically Ill ���������������������������������������������������������������������� 67
H. P. M. M. Gelissen, H. J. de Grooth, and A. M. E. de Man
7 Nebulized Therapeutics for COVID-19 Pneumonia
in Critical Care������������������������������������������������������������������������������������������ 81
J. Dhanani and M. C. Reade

Part III Mechanical Ventilation

8 Positive End-Expiratory Pressure in Invasive and Non-invasive
Ventilation of COVID-19 Acute Respiratory Distress Syndrome���������� 101
L. Weaver, D. G. Bates, and L. Camporota

v
vi Contents

9 Personalized Mechanical Ventilation Settings: Slower Is Better!���������� 113

P. L. Silva, P. R. M. Rocco, and P. Pelosi
10 Spontaneous Breathing in Acute Respiratory Failure���������������������������� 129
E. Chiodaroli and D. Chiumello
11 Laryngeal Injury: Impact on Patients in the Acute and Chronic
Phases���������������������������������������������������������������������������������������������������������� 137
E. Kelly, S. Wallace, and Z. Puthucheary

Part IV Fluids and Electrolytes

12 Fluid Responsiveness as a Physiologic Endpoint
to Improve Successful Weaning���������������������������������������������������������������� 151
R. Castro, P. Born, and J. Bakker
13 Tidal Volume Challenge Test: Expanding Possibilities�������������������������� 161
S. N. Myatra, N. Prabu, and J.-L. Teboul
14 Fluid Management in COVID-19 ICU Patients�������������������������������������� 173
R. Shi, X. Monnet, and J.-L. Teboul
15 Electrolytes in the ICU������������������������������������������������������������������������������ 183
A. Reintam Blaser, A. R. H. van Zanten, and A. M. E. de Man

Part V Renal Failure

16 Hemodynamic Instability During Continuous Renal
Replacement Therapy: Is It All About Fluid? ���������������������������������������� 203
S. M. T. Nasser, N. Boyer, and L. G. Forni
17 How to Prolong Filter Life During Continuous Renal
Replacement Therapy?������������������������������������������������������������������������������ 213
Y. Tsujimoto and T. Fujii
18 Prevention of Acute Kidney Injury After Cardiac Surgery ������������������ 223
M. Ostermann, K. Weerapolchai, and N. Lumlertgul

Part VI Circulatory Shock

19 Contrast-Enhanced Renal Ultrasound for Assessment
of Renal Perfusion in Critically Ill Patients�������������������������������������������� 237
J. Watchorn, K. Bramham, and S. Hutchings
20 Focused Clinical Hemodynamic Assessment in Septic Shock���������������� 249
E. Kattan, G. Hernández, and J. Bakker
21 Vasopressor Choice and Timing in Vasodilatory Shock ������������������������ 259
P. M. Wieruszewski and A. K. Khanna
Contents vii

Part VII Cardiac Arrest

22 Advanced Life Support Update���������������������������������������������������������������� 273
G. D. Perkins and J. P. Nolan
23 Brain Injury Biomarkers for Predicting Outcome
After Cardiac Arrest���������������������������������������������������������������������������������� 285
J. Humaloja, N. J. Ashton, and M. B. Skrifvars

Part VIII Neurocritical Care and Neuromonitoring

24 The Emerging Role of the Microbiota in Neurocritical Care���������������� 301
D. Battaglini, P. Pelosi, and C. Robba
25 Brain–Multiorgan Cross-Talk in Critically Ill Patients
with Acute Brain Injury���������������������������������������������������������������������������� 317
K. Kotfis, D. Siwicka-Gieroba, and W. Dąbrowski
26 The Importance of Neuromonitoring
in Non Brain Injured Patients������������������������������������������������������������������ 333
D. Battaglini, P. Pelosi, and C. Robba

Part IX Artificial Intelligence

27 Artificial Intelligence in Critical Care Medicine������������������������������������ 353
J. H. Yoon, M. R. Pinsky, and G. Clermont
28 Artificial Intelligence in Infection Management in the ICU������������������ 369
T. De Corte, S. Van Hoecke, and J. De Waele

Part X Quality of Care

29 Acute Medical Rehabilitation in Intensive Care ������������������������������������ 385
H. M. Buyruk, Y. Buyruk, and C. Ince
30 Sepsis Performance Improvement Programs:
From Evidence Toward Clinical Implementation���������������������������������� 397
M. Schinkel, P. W. B. Nanayakkara, and W. J. Wiersinga

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Abbreviations

AKI Acute kidney injury

APACHE Acute physiology and chronic health evaluation
ARDS Acute respiratory distress syndrome
COVID Coronavirus disease
CRP C-reactive protein
CRRT Continuous renal replacement therapy
CT Computed tomography
DAMP Damage-associated molecular pattern
DO2 Oxygen delivery
ECMO Extracorporeal membrane oxygenation
EHR Electronic health record
GCS Glasgow Coma Scale
ICU Intensive care unit
IFN Interferon
IL Interleukin
LV Left ventricular
MAP Mean arterial pressure
NO Nitric oxide
NOS Nitric oxide synthase
PAMP Pathogen-associated molecular pattern
PEEP Positive end-expiratory pressure
RBC Red blood cell
RCT Randomized controlled trial
RRT Renal replacement therapy
RV Right ventricular
SARS-CoV-2 Severe acute respiratory syndrome coronavirus-2
SOFA Sequential organ failure assessment
TBI Traumatic brain injury
TNF Tumor necrosis factor
VILI Ventilator-induced lung injury
VT Tidal volume

ix
 Part I
Sepsis and the Immune Response
The Role of Mitochondria in the Immune
Response in Critical Illness 1
Y. Wang and A. S. McLean

1.1 Introduction

Immune dysregulation, characterized by an imbalance between a systemic

inflammatory response syndrome and a compensatory anti-inflammatory
response syndrome, is often observed in critically ill patients [1, 2]. This imbal-
ance between the pro- and anti-inflammatory responses frequently leads to
immunoparalysis in critically ill patients, rendering them more susceptible to
further infections, and is associated with increased mortality [3]. Currently, no
effective treatments are available to restore immune homeostasis and reduce
mortality in these patients, largely due to the heterogeneity in patients’ immune
status and more importantly the lack of understanding of the underlying cause of
such immune dysfunction [2, 4]. Immune response is not a standalone process
but is interconnected with other cellular activities, a very important one of which
is cellular metabolism. Metabolic pathways and immune response are tightly
intertwined both in health and in disease [5]. The link between immune cell func-
tion and mitochondrial function is now well recognized and a field known as
“immunometabolism” is dedicated to understanding the relationship between
immune and metabolic pathways [6–8]. Mitochondria play a crucial role in regu-
lating not only the growth, but also the function, of immune cells. In addition to
providing energy to support the synthesis of the macromolecules essential for
immune cell proliferation, mitochondria also act as signaling organelles, driving

Y. Wang (*)
Department of Intensive Care Medicine, Nepean Hospital, Kingswood, NSW, Australia
Centre for Immunology and Allergy Research, Westmead Institute for Medical Research,
Westmead, NSW, Australia
e-mail: [email protected]
A. S. McLean
Department of Intensive Care Medicine, Nepean Hospital, Kingswood, NSW, Australia

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 3

J.-L. Vincent (ed.), Annual Update in Intensive Care and Emergency Medicine
2022, Annual Update in Intensive Care and Emergency Medicine,
https://doi.org/10.1007/978-3-030-93433-0_1
4 Y. Wang and A. S. McLean

activation of immune cells via metabolic intermediates, mitochondrial DNA

(mtDNA), and reactive oxygen species (ROS). In addition, mitochondrial dynam-
ics (fusion and fission), biogenesis (synthesis of new mitochondria), and mitoph-
agy (degradation of damaged mitochondria) also play important roles in
regulating immune cell functions. Knowledge in immunometabolism in critical
illness, in particularly sepsis, opens up a new paradigm in patient care. Potential
therapies targeting metabolic pathways, instead of solely immune-related path-
ways, might be the way to repair cellular function and restore immune homeosta-
sis [4]. The other aspect of immunometabolism—looking at how immune
responses influence metabolic pathways—is equally important, but beyond the
scope of this review. Interaction between metabolism and immune response at
the organ level has been reviewed elsewhere [6].

1.2  itochondrial Machinery That Mediates and Regulates

M
Immune Responses in Critical Illness

Apart from being the powerhouse of the cell, the mitochondrion has emerged as a
signaling hub that shapes and modulates how the immune system responds to
infection or trauma. Mitochondrial dysfunction is evident in leukocytes from criti-
cally ill patients, and is believed to be the underlying cause of immunoparalysis
and may account for the development of organ dysfunction [7–9]. Early recovery
of mitochondrial function correlates with improved recovery in critically ill
patients [10].

1.2.1 Metabolic Reprogramming

The immune-regulating mitochondrial machinery is a complex network involving

many pathways and mechanisms that diverge and converge at various levels.
Metabolic reprogramming is one mechanism that has been well studied in both
innate and adaptive immune cells. Immune cells at different activation states (quies-
cent vs. activated), or with different functions (pro-inflammatory vs. anti-inflamma-
tory), and different cell types (granulocytes, macrophages, dendritic cells, T- and
B-lymphocytes), make use of different metabolic pathways (e.g., glycolysis, oxida-
tive phosphorylation, fatty acid metabolism) to produce ATP [11]. The choice of
different metabolic pathways, supports the energy demand of cells at different acti-
vation state. For example, upon infection or stimulation, immune cells become acti-
vated and produce cytokines and hence tend to favor glycolysis over oxidative
phosphorylation for fast turnaround of ATP. Although the same amount of starting
material, such as glucose, is used, oxidative phosphorylation generates 18 times
more ATP than glycolysis, although is a lot slower. On the other hand, the choice of
metabolic pathway determines the fate of the immune cells, i.e., naïve or memory,
effector or regulatory, etc. However, the environment that the cells are in in the first
place, triggers the changes in the metabolic pathways. The overall trend is that
1 The Role of Mitochondria in the Immune Response in Critical Illness 5

neutrophils, inflammatory macrophages (M1 macrophages), activated effector T

cells, and dendritic cells rely more on aerobic glycolysis, whereas alternatively
polarized macrophages (M2 macrophages), regulatory T cells (Tregs), and memory
T cells prefer oxidative phosphorylation and fatty acid oxidation for energy produc-
tion [8, 11, 12]. Metabolic reprogramming serves an important role in catering for
the immune cells’ energy demand at different phases of their activation and prolif-
eration. However, imbalance across the metabolic pathways could have serious
pathological impact. One example may be the hyperlactatemia often seen in criti-
cally ill patients. Increased aerobic glycolysis in the activated immune cells during
the initial hyper-­inflammatory response is believed to contribute to the increase in
blood lactate levels in sepsis [13, 14].

1.2.2 Mitochondrial ROS and mtDNA

Metabolic reprogramming sets the scene for the immune response, which is then
subjected to many more modifications and regulations by factors that are directly
or indirectly related to mitochondrial metabolism. Two important mitochondria-
related immune regulators that have been well studied are mitochondrial ROS and
mtDNA. Mitochondrial ROS are produced in healthy mitochondria, as a by-prod-
uct of oxidative phosphorylation. At low dose, mitochondrial ROS serve important
signaling functions, especially in the innate immune response. They are known to
mediate NLRP3 inflammasome activation, leading to production of the pro-­
inflammatory cytokines, interleukin (IL)-1β and IL-18 [8, 15]. Mitochondrial ROS
also induce a type-I interferon (IFN) response via mitochondrial antiviral-signal-
ing (MAVS) and the IFN regulatory factor 3 (IRF3) pathway [16]. However, the
level of mitochondrial ROS needs to be tightly regulated by the antioxidant system.
Excessive mitochondrial ROS can cause oxidative damage to proteins/enzymes
involved in oxidative phosphorylation and create mutations in mtDNA, contribut-
ing to the immune dysregulations as seen in critical illness [17]. Like mitochon-
drial ROS, mtDNA also plays an important role in innate immunity [12]. In healthy
cells, mtDNA is located in the matrix of mitochondria, encoding 13 proteins, all of
which are components of oxidative phosphorylation. mtDNA is released to the
cytosol upon mitochondrial dysfunction which involves changes to the integrity or
permeability of the mitochondrial membrane. mtDNA, released into the cytosol,
can activate the NLRP3 inflammasome with release of IL-1β and IL-18. Due to its
bacterial origin, cytosolic mtDNA also serves as a damage-associated molecular
pattern (DAMP), which can be recognized by intracellular pattern recognition
receptors (PRRs), such as Toll-like receptor 9 (TLR9), and initiate the nuclear
factor-kappa B (NF-κB)-dependent pro-inflammatory signaling pathway. In addi-
tion, cytosolic mtDNA can also be sensed by cyclic GMP-AMP synthase (cGAS)
and activate the cGAS/stimulator of IFN genes (cGAS/STING) pathway and its
downstream IFN response [18]. mtDNA can also be released into the circulation
and cause systemic inflammation. Circulating mtDNA has been associated with
mortality in critically ill patients [19].
6 Y. Wang and A. S. McLean

1.2.3 Succinate and Itaconate

In addition to mitochondrial ROS and mtDNA, metabolites such as succinate and

itaconate have also emerged as part of immune-regulating mitochondrial machinery
[4, 20]. Both succinate and itaconate are intermediates from the tricarboxylic acid
(TCA) cycle with opposite effects on the immune response. The TCA cycle gener-
ates nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide
(FADH2), providing electrons to fuel oxidative phosphorylation. Succinate accumu-
lation occurs under conditions such as hypoxia or inflammation. It can be released
from mitochondria into the cytosol and functions as a signal transducer promoting
pro-inflammatory gene expression via hypoxia-inducible factor 1α (HIF-1α) activa-
tion. Accumulation and oxidation of succinate by succinate dehydrogenase (SDH)
in the mitochondria also leads to increased production of mitochondrial ROS via a
process called reverse electron transport. This further enhances the pro-­inflammatory
effect of succinate. Like ROS, the level of succinate needs to be carefully regulated
due to its inflammation aggravating effect. Plasma succinate has been proposed as a
predictor of mortality for critically ill patients who are severely injured [21].
Itaconate, which is derived from cis-aconitate of the TCA cycle, is a succinate-reg-
ulating factor. It is shown to counteract the pro-inflammatory effect of succinate by
inhibiting SDH. Itaconate can also be released into the cytosol and activate tran-
scription factor NF-E2 p45-related factor 2 (Nrf2), a master regulator of antioxidant
and anti-inflammatory responses [22]. Recently, itaconate has also been shown to
inhibit the inflammatory response in macrophages through activating transcription
factor 3 (ATF3).

1.2.4 Mitochondrial Dynamics

The above mentioned immune-regulating mitochondrial factors are centered around

the biochemical aspect of mitochondrial biology. Another important aspect of
immune-regulating mitochondrial machinery is mitochondrial dynamics, which is
to maintain and provide infrastructural support for the immune response. The size
and shape of mitochondria undergo constant change through fusion and fission,
which is important for maintaining the health and function of mitochondria. First,
fusion incorporates newly synthesized mitochondria (from mitochondrial biogene-
sis) into the current mitochondrial network. Second, fusion also allows for mixing
of proteins and/or mtDNA between the existing mitochondria, which on one hand
enhances the metabolic capacity of the mitochondria, and on the other enables the
damaged proteins and/or mutated mtDNA to be segregated from the healthy ones.
Finally, segregation is achieved via fission and the damaged mitochondria can be
destroyed through a process known as mitophagy. The proportion of mitochondria
with damaged proteins or mutated mtDNA is kept below a critical threshold level
through this process to maintain mitochondrial function [23, 24]. In addition to
quality control, mitochondrial fusion and fission also participate in immune regula-
tion. In activated T cells, there is an increase in fission, which creates round and
1 The Role of Mitochondria in the Immune Response in Critical Illness 7

fragmented mitochondria with loose cristae, favoring aerobic glycolysis. And in

memory T cells, increased fusion generates elongated mitochondria which favors
oxidative phosphorylation and fatty acid oxidation [8, 25].

1.3 I mmunometabolism: The Perfect World Scenario vs.

the Critical Illness Scenario

So far, we have presented a list of mitochondrial components that are thought to

play important roles in regulating the immune response. Our list is far from com-
plete, but does highlight a few mechanisms that could relate to the development of
immune dysregulation in critical illness. Figure 1.1 illustrates what we think would

Perfect world scenario

Infection Pro-inflammatory Anti-inflammatory

cytokines cytokines

Glycolysis OXPHOS, FAO

Fission Fusion
Naive cells Effector Regulatory
cells cells

Infection resolved and immune homeostasis restored

Critical illness scenario

+ +
Pro-inflammatory Anti-inflammatory
Infection cytokines
cytokines

Glycolysis + OXPHOS, FAO

Succinate

mtDNA
+
Fission + Delayed Fusion
Naive cells Effector Regulatory
cells cells

ROS

Nutrient
Exhaustion

Cell death Cell hibernation

Fig. 1.1 Immunometabolism in the ‘perfect world scenario’ vs. the ‘critical illness scenario’.
OXPHOS oxidative phosphorylation, FAO fatty acid oxidation
8 Y. Wang and A. S. McLean

happen to the immune response when metabolism was in perfect control (the perfect
world scenario) and when it became inconsistent and changeable (the critical illness
scenario). In the perfect world scenario, the presence of an insult (e.g., infection or
a trauma-related stress signal), would trigger metabolic reprogramming, switching
from oxidative phosphorylation to glycolysis. This would enable activation of
immune cells and production of pro-inflammatory cytokines and other mediators.
At the same time, mitochondrial fission would increase to keep up with the meta-
bolic reprogramming. The slightly elevated mitochondrial ROS and succinate in
response to initial insult or cytokines would promote the pro-inflammatory response.
Once the insult was eliminated, mitochondrial fusion would increase to create fused
elongated mitochondria that favor oxidative phosphorylation and fatty acid oxida-
tion. This would allow activation of regulatory immune cells and production of
anti-inflammatory cytokines and other mediators. And itaconate would counteract
the effect of succinate, activate the Nrf2-mediated antioxidant pathway to dampen
down mitochondrial ROS, and activate ATF3 to inhibit the inflammatory response
in macrophages. Immune homeostasis would be achieved as a result.
In the critical illness scenario, initial metabolic reprogramming from oxidative
phosphorylation to glycolysis would go on for longer than necessary, generating
excessive lactate (hyperlactatemia) and pro-inflammatory cytokines and mediators. A
disrupted mitochondrial fusion/fission cycle could be to blame, one which could not
support the timely switch to oxidative phosphorylation and fatty acid oxidation. The
anti-inflammatory response would eventually kick in but by then damage would
already have occurred to mitochondria and mtDNA because of excessive production
of ROS in response to stress or cytokines. Excessive ROS and released mtDNA would
aggravate the pro-inflammatory response, which in turn would trigger a more aggres-
sive anti-inflammatory response to try and salvage the situation. The competition
between pro- and anti-inflammatory responses would exhaust the nutrients and lead to
shutdown of the whole metabolic system. Cells would either die or go into hibernation
to preserve energy [26]. This scenario is an over-simplified version of what might hap-
pen in the actual disease setting, without considering the crosstalk between cells and
organs and many other factors that are not included here. It is designed to shed light
on the interaction between the immune response and metabolism.

1.4 Potential of Mitochondria-Targeting Therapy

in Critical Care

Our understanding thus far leads us to think that targeting mitochondria could
perhaps correct the underlying cause of immune dysfunction in critical illness and
lead to better recovery of the patients. The central role of mitochondrial dynamics
in supporting and initiating metabolic reprogramming would make it the perfect
therapeutic target. To get the fusion/fission cycle going, the mitochondrial network
needs to be replenished by newly synthesized mitochondria via biogenesis.
Therapies that could potentially boost mitochondrial biogenesis are mitochondrial
1 The Role of Mitochondria in the Immune Response in Critical Illness 9

transplantation, metformin, nitric oxide (NO), and carbon monoxide. Mitochondrial

transplantation has been used successfully in pediatric patients with myocardial
ischemia-­reperfusion injury [27]. Metformin can activate peroxisome proliferator-
activated receptor (PPAR)-gamma coactivator-1α (PGC-1α), and Nrf2, the master
regulator of mitochondrial biogenesis and antioxidant systems [28]. Premorbid use
of metformin is associated with lower mortality in sepsis [29]. NO and carbon
monoxide can also enhance mitochondrial biogenesis [30–32]. Dietary nitrite has
been trialed in patients with coronary artery disease (ClinicalTrials.gov Identifier:
NCT00069654). Other therapies, such as mitochondria-targeted antioxidant
(MitoQ) [33], could also be beneficial in protecting mtDNA and oxidative
phosphorylation from oxidative damage. MitoQ has been trialed in people with
Parkinson’s disease (ClinicalTrials.gov Identifier: NCT00329056).

1.5  hallenges of Applying Mitochondria-Targeting

C
Therapy in Critical Care

There are challenges to overcome before mitochondria-targeting therapy would be

possible. First, how do we assess mitochondrial dysfunction in the clinic and iden-
tify patients who would benefit from such therapy? A few possible ways could be
considered. Non-invasive assessment of mitochondrial oxygen metabolism using a
novel device called the COMET monitor was tested on 40 patients during the acute
phase of sepsis. This device is based on the protoporphyrin IX-triplet state lifetime
technique (PpIX-TSLT) and has been shown to be feasible [33]. This technology is
still in its early phase of clinical application but does offer some hope. Another pos-
sible biomarker that could potentially be used for assessing mitochondrial dysfunc-
tion is plasma mtDNA, but its sensitivity and specificity need further investigation
[19, 34, 35]. Furthermore, we could consider using immune response markers as a
surrogate markers, one such example could be IFNα inducible protein 27 (IFI27)
[36]. If we could overcome the first challenge, the second would be how to deliver
mitochondria-targeting therapies to the right organ at the right time.

1.6 Conclusion

In this chapter, we have demonstrated the important role of mitochondria in

regulating the immune response and proposed a scenario that explains immune–
metabolism crosstalk in the context of critical illness. We have highlighted the role
of mitochondrial dynamics in overseeing and supporting metabolic reprogramming
during immune cell activation. Mitochondrial ROS can be friend or foe when it
comes to immune regulation. Two TCA intermediates—succinate and itaconate—
with opposite effects have emerged as important players of the immune-regulating
mitochondrial machinery. Our understanding in immunometabolism could take us
to the next era of critical care: mitochondria-targeting therapy.
10 Y. Wang and A. S. McLean

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Immunomodulation by Tetracyclines
in the Critically Ill: An Emerging 2
Treatment Option?

A. Sauer, C. Putensen, and C. Bode

2.1 Introduction

Sepsis and acute respiratory distress syndrome (ARDS) are still the most common
causes of death in critically ill patients. Although our knowledge of the underlying
immunopathogenesis has grown tremendously and we have made substantial
advances in supportive care, the overall mortality for sepsis and ARDS remains high
[1, 2]. In 2016, sepsis was redefined as life-threatening organ dysfunction caused by
a dysregulated host response to infection. Hyperinflammation occurring concur-
rently with immunosuppression puts patients at risk for developing fatal secondary
infections and chronic critical illness syndrome [1]. An extension of sepsis in terms
of pathogenesis has made ARDS similarly resistant to therapy and the prognosis for
patients with this syndrome remains equally dismal [2]. Despite over 30 years of
preclinical and clinical trials, no effective pharmacotherapies exist to improve out-
comes in patents with sepsis or ARDS [1, 2]. New therapeutic agents are desper-
ately needed, even more so in the light of the ongoing coronavirus disease 2019
(COVID-19) pandemic.
Tetracyclines are a family of bacteriostatic antibiotics that inhibit protein synthe-
sis by reversibly binding to the bacterial ribosome. Upon binding they allosterically
prevent the binding of the amino acyl-tRNA to the mRNA ribosome complex. They
exhibit broad-spectrum antibacterial activity against a wide-range of Gram-positive
and Gram-negative bacteria as well as atypical pathogens, such as chlamydiae, spi-
rochaetes, and rickettsiae [3]. Additionally, tetracyclines exert pleiotropic immuno-
modulatory effects that may be able to rebalance immune homeostasis in critically
ill patients. Their beneficial anti-inflammatory effects have been reported for chronic

A. Sauer · C. Putensen · C. Bode (*)

Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn,
Bonn, Germany
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 13

J.-L. Vincent (ed.), Annual Update in Intensive Care and Emergency Medicine
2022, Annual Update in Intensive Care and Emergency Medicine,
https://doi.org/10.1007/978-3-030-93433-0_2
14 A. Sauer et al.

pulmonary diseases, chronic inflammatory skin diseases, autoimmune disorders, as

well as neurodegenerative diseases, and they have become a standard of care in the
treatment of periodontitis, acne, and rosacea [3, 4]. Recently, evidence has emerged
that tetracyclines could potentially be beneficial in ARDS and sepsis [5–7].
In this chapter, we provide an overview of the current preclinical and clinical
studies on the immunomodulatory effects of tetracyclines in the critical care setting
(Tables 2.1 and 2.2). We elucidate the underlying mechanisms of the immunomodu-
latory properties of tetracyclines that may have therapeutic effects in sepsis and
ARDS. Finally, we discuss future research perspectives including the role of non-­
antibiotic tetracyclines.

2.2 Immunopathogenesis of Sepsis

Sepsis is a heterogenous syndrome characterized by an unbalanced hyperinflamma-

tory state and profound immunosuppression. Pathogen-associated molecular pat-
terns (PAMPs) released by sepsis-inducing microorganisms activate pattern
recognition receptors (PRRs) expressed by various immune cells and trigger a
strong innate immune response. The best known PAMPs include lipopolysaccharide
(LPS), lipoteichoic acid (LPA), and microbial DNA. PRRs can also sense cell
injury-associated endogenous molecules referred to as damage-associated molecu-
lar patterns (DAMPs), such as ATP, mitochondrial DNA, hyaluronan, heat shock
proteins, and fibrinogen [1, 8]. Upon ligand binding, activation of downstream sig-
nalling pathways (e.g., nuclear factor kappa-B [NF-κB] and mitogen-activated pro-
tein kinase [MAPK]) leads to the transcription of genes encoding pro-inflammatory
cytokines and chemokines such as tumor necrosis factor (TNF)-α, interleukin
(IL)-1ß, IL-8, IL-18 and interferons (IFN). The expression of IL-1ß and IL-18 is
tightly regulated by inflammasomes, which execute a unique form of programmed
cell-death called pyroptosis. In most cases, these processes aid in neutralizing
invading pathogens and apoptotic cells. However, in sepsis they can lead to an
unbalanced host response that can potentially trigger a life-threatening “cytokine
storm” [1, 9]. Another hallmark of the innate immune response in sepsis is the acti-
vation of the complement system, which results in the recruitment of leukocytes,
endothelial cells, and platelets and ultimately in sepsis-induced endothelial barrier
dysfunction. Loss of vascular integrity leads to tissue edema and reduced microvas-
cular perfusion. Coagulation activation is tightly interconnected with complement
activation and predisposes patients to disseminated intravascular coagulation (DIC),
microvascular immunothrombosis, and hemorrhage.
The initial hyperinflammatory state is counterbalanced by immunosuppression
which involves both innate and adaptive immunity. One key phenomenon is the
apoptosis of B and CD4+ and CD8+ T cells and dendritic cells causing an acquired
immune deficiency syndrome linked to an unfavorable prognosis. Depletion of T
cells is further augmented by increased expression of programmed cell death 1
(PD1) and upregulation of its ligand (PDL1) on various immune and epithelial cells.
The reprogramming of antigen-presenting cells results in reduced human leukocyte
2 Immunomodulation by Tetracyclines in the Critically Ill: An Emerging Treatment… 15

Table 2.1 Immunomodulatory effects of tetracyclines in preclinical and clinical models of acute
respiratory distress syndrome (ARDS)
Author Year Tetracycline Model Stimulants or pathogens Immune response
Peukert 2021 Tetracycline Mouse, LPS, H1N1 influenza IL-1ß, IL-18, caspase-1
et al. [5] human virus (mouse); viral, activation, neutrophil
(ex vivo) bacterial, and non-­ influx ↓, survival ↑
pulmonary ARDS
(human)
Zhang 2019 Doxycycline Mouse Paraquat MMP-9, MPO,
et al. [20] neutrophil influx ↓
Wang 2014 Doxycycline Rat Cardiopulmonary bypass TNF-α, IL-1ß,
et al. [24] MMP-9 ↓
Zhang 2014 Doxycycline Dog Cardiopulmonary bypass MMP-9, MPO,
et al. [17] neutrophil influx ↓
Roy et al. 2012 CMT-3 Pig Ischemia by clamping of TNF-α, IL-1ß, Il-6,
[16] SMA, placement of fecal IL-8, IL-10 ↓, MMP-2,
clot in peritoneum -9, NE, survival ⇔
Ng et al. 2012 Doxycycline Mouse H3N2 influenza virus MMP-2, MMP-9,
[27] T1-α, thrombomodulin,
neutrophil influx ↓
Moon 2012 Doxycycline Mouse LPS Syndecan-1 (MMP-7
et al. [22] substrate), neutrophil
influx ↓
Zhou et al. 2010 CMT-3 Sheep 3rd degree burn, smoke MMP-2 ↓, MMP-9 ⇔,
[25] inhalation, barotrauma survival ↑
Sochor 2009 Doxycycline Rat Acute pancreatitis MMP-9, neutrophil
et al. [21] (intraductal influx ↓
glycodeoxycholic acid,
cerulein)
Fujita 2007 Doxycycline Mouse LPS or Streptococcus MMP-2, -9, neutrophil
et al. [26] pneumoniae influx ↓, survival ↑
Kim et al. 2006 CMT-3 Rat Ventilation MMP-9, MPO,
[14] neutrophil influx ↓
Fujita 2006 Doxycycline Mouse Bleomycin MMP-2, -9, neutrophil
et al. [28] influx ↓
Steinberg 2005 CMT-3 Pig Ischemia by clamping of IL-6, IL-8, IL-10, NE
et al. [19] SMA, placement of fecal ↓, IL-1, MMP-2, -9,
clot in peritoneum neutrophil influx ⇔,
survival ↑
Steinberg 2003 CMT-3 Rat Cecal ligation and MMP-2, MMP-9 ↓,
et al. [15] puncture survival ↑
Carney 2001 CMT-3 Pig LPS MMP-2, MMP-9,
et al. [23] neutrophil influx ↓
McCann 1999 CMT-3 Pig Cardiopulmonary bypass, Neutrophil influx ↓
et al. [29] LPS
Carney 1999 CMT-3 Pig Cardiopulmonary bypass, MMP-2, MMP-9, NE,
et al. [13] LPS neutrophil influx ↓,
survival ↑
↑ significant increase, ↓ significant decrease, ⇔ no significant difference, CMT-3 chemically modi-
fied tetracycline 3, IL interleukin, LPS lipopolysaccharide, MMP metalloproteinase, MPO myelo-
peroxidase, NE neutrophil elastase, SMA superior mesenteric artery, TNF-α tumor necrosis factor
alpha, T1-α membrane protein of alveolar type I epithelium
16 A. Sauer et al.

Table 2.2 Immunomodulatory effects of tetracyclines in preclinical and clinical models of sepsis
Stimulants or
Author Year Tetracycline Model pathogens Immune response
Colaço et al. 2021 Doxycyline Mouse E. coli, H1N1 Liver, lung, kidney
[6] influenza virus, injury ↓, mitochondrial
C. albicans, protein synthesis ↓;
Plasmodium FAO, steroid
berghei sensitivity, survival ↑
Patel et al. 2020 Doxycycline Mouse Cecal ligation TNF-α, IL-1ß, IL-6,
[7] and puncture MPO ↓, survival ↑
Sun et al. 2020 Minocycline Human THP-1 LPS TNF-α, IL-8, MIP-1α,
[35] monocytes MIP-1ß ↓, modulated
NF-κB-, p38-,
ERK1/2-pathways
Sun et al. 2015 Minocycline, Human THP-1 LPS Autophagy ↑ by
[34] tigecycline, monocytes inhibiting mTOR;
doxycycline TNF-α, IL-8 ↓
Nukarinen 2015 Doxycycline RCT Severe sepsis or MMP-8, -9, TIMP-1
et al. [48] septic shock ⇔
Fredeking 2015 Doxycycline RCT Dengue virus IL-6, TNF-α, mortality
et al. [47] ↓
Bode et al. 2014 Doxycycline Human THP-1 LPS, E. coli Phagocytosis, IL-1ß,
[45] monocytes, IL-6 ↓, TLR-1, TLR-4,
PBMCs (ex TLR-6 ↓
vivo)
Tai et al. 2013 Minocycline Human THP-1 LPS TNF-α, IL-6, IFN-γ,
[41] monocytes IL-8, IP-10, MCP-1,
MIP-1α, MIP-1ß,
RANTES, eotaxin ↓,
IKKα/β
phosphorylation
inhibited
Pang et al. 2012 Minocycline Human LPS TNF-α, IL-1ß, IL-6,
[33] monocytes (ex COX-2, PGE2 ↓,
vivo) LOX-1, NF-κB,
LITAF, Nur77, PI3K/
Akt-, p38-MAPK
pathway ↓
Castro et al. 2011 Tetracycline, RCT Dengue virus IL-6, Il-1ß, TNF-α ↓,
[46] doxycycline IL-1ra ↑, TNF-R1 ⇔
Maitra et al. 2005 CMT-3 Rat Cecal ligation Liver injury, MMP-9,
[40] and puncture MMP-2, TGF-ß1,
caspase-3 ↓, survival ↑
Maitra et al. 2004 CMT-3 Rat Cecal ligation TNF-α ↓, p38-,
[38] and puncture p42/44–MAPK
activation inhibited,
survival ↑
Maitra et al. 2003 CMT-3 Rat Cecal ligation Liver injury, NO,
[39] and puncture MMP-9 ↓, survival ↑
2 Immunomodulation by Tetracyclines in the Critically Ill: An Emerging Treatment… 17

Table 2.2 (continued)

Stimulants or
Author Year Tetracycline Model pathogens Immune response
D’Agostino 2001 CMTs, Murine J774 LPS TNF-α, IL-10 ⇔,
et al. [44] macrophages iNOS, nitrite, NO,
Il-12 ↓, cytotoxity ↑
Patel et al. 1999 CMTs, Murine RAW LPS PGE2, nitrite ↓
[42] minocycline 264.7 cells, (CMT-3)
human A 549
cells
D’Agostino 1998 Doxycycline Mouse, murine LPS NO ↓, survival ↑
et al. [36] macrophages
Amin et al. 1997 CMTs, Murine LPS iNOS mRNA
[43] doxycycline macrophages accumulation and
protein expression ↓
Milano 1997 Tetracycline Mouse, murine LPS TNF-α, IL-1α, nitrate,
et al. [37] macrophages iNOS activity ↓,
macrophages: NO ↓,
TNF-α, IL-1α ⇔,
survival ↑
↑ significant increase, ↓ significant decrease, ⇔ no significant difference, C. albicans Candida
albicans, CMT-3 chemically modified tetracycline 3, COX-2 cyclooxygenase 2, E. coli Escherichia
coli, ERK extracellular-signal regulated kinases, FAO fatty acid oxidation, IFN interferon, IKK
inhibitor of nuclear factor kappa B kinase, IL interleukin, IL-1ra interleukin-1 receptor antagonist,
iNOS inducible nitric oxide synthase, IP-10 interferon gamma induced protein 10, LITAF lipopoly-
saccharide induced TNF factor, LPS lipopolysaccharide, LOX-1 lectin-like oxidized low density
lipoprotein receptor-1, MMP metalloproteinase, MAPK mitogen-activated protein kinase, MCP
monocyte chemoattractant protein, MIP macrophage inflammatory protein, MPO myeloperoxi-
dase, mTOR mammalian target of rapamycin, NF-κB nuclear factor kappa-light-chain-enhancer of
activated B-cells, NO nitric oxide, PBMCs peripheral blood mononuclear cells, PGE2 prostaglan-
din E2, PI3k phosphatidylinositol-3-kinase, RANTES regulated upon activation, normal T cell
expressed and presumably secreted, RCT randomized controlled trial, TGF-ß1 transforming
growth factor beta 1, TIMP-1 tissue inhibitor of metalloproteinase-1, TLR toll-like receptor, TNF-α
tumor necrosis factor alpha, TNF-R1 tumor necrosis factor receptor 1

antigen-antigen D related (HLA-DR) expression on monocytes and impaired pro-

duction of pro-inflammatory mediators, including TNF-α, IL-6, Il-1ß, and IFN-γ
referred to as “immunoparalysis”. Although these compensatory mechanisms
attempt to restore immune homeostasis, a subtype of patients develops persistent
inflammation, immunosuppression, and catabolism syndrome (PICS), which is pre-
dictive of a poor outcome. It is most likely caused by persistent inflammation
through a constant release of DAMPs driving organ injury [1].

2.3 Immunopathogenesis of ARDS

Sepsis and ARDS have similar underlying mechanisms: ARDS, defined as a life-­
threatening form of respiratory failure, is driven by an uncontrolled inflammatory
host response induced by direct (pulmonary) or indirect (extrapulmonary) insults.
18 A. Sauer et al.

The most common causes include sepsis, viral and bacterial pneumonia, aspiration
of gastric contents, and major trauma [2].
Inflammasome activation plays a central role in the development of ARDS [5]. In
general, inflammasomes are multiprotein complexes that consist of a sensor NOD-,
LRR- and pyrin domain-containing protein 3 (NLRP3), an adaptor apoptosis-­
associated speck-like protein containing a CARD (ASC), and an effector (caspase-
­1) [10]. Inflammasome activation generates IL-1ß and IL-18 which both drive the
inflammatory cascade forward and are linked to an unfavorable prognosis [5]. This
process involves two signals. Inflammasomes assemble downstream of PRRs in
response to PAMPs and DAMPs. For example, LPS binding to Toll-like receptor 4
(TLR4) leads to the translocation of NF-κB into the nucleus and the transcription of
pro-inflammatory mediators and inflammasome components including pro-­
caspase-­1, pro–IL-1β, and pro-IL-18 (signal 1). Various stimuli such as ATP, viral
RNA, and pore-forming toxins activate the sensor NLRP3, resulting in inflamma-
some assembly via ASC oligomerization (signal 2). Active capase-1 converts pro-­
IL-­1β and pro-IL-18 into their mature forms causing pyroptotic cell death [5, 10,
11]. Inflammation and pyroptosis mediate substantial epithelial and endothelial
injury with a subsequent loss of the alveolar-capillary barrier integrity, leading to
influx of protein-rich edema fluid and immune cells into the alveoli [2]. This exuda-
tive edema causes dysfunctional surfactant and atelectasis, which in turn can predis-
pose patients to biophysical injury of the lungs [2].
The influx of immune cells (especially neutrophils) triggered by the activation of
TLRs on alveolar type II cells and resident macrophages is a salient feature of
ARDS [12]. As neutrophils begin their transepithelial migration into the lungs, they
become primed to phagocytose invading microbes and release toxic mediators
including reactive oxygen species (ROS), neutrophil elastase, proteases, and nitric
oxide (NO). Proteases such as metalloproteinases (MMPs) contribute to the disrup-
tion of the barrier integrity and lung parenchyma by degrading collagen [12–15].
Both neutrophil elastase and MMPs are known to promote lung injury in patients
with ARDS [15]. Lastly, persistent inflammation and unbalanced immune homeo-
stasis can further intensify existing lung damage and cause lasting injury and
fibrosis [2].

2.4 Mechanisms of Action of Tetracyclines in ARDS

In vitro and in vivo studies have highlighted the wide-range of immunomodulatory

effects of tetracyclines in models of ARDS, pneumonia, and sepsis [7, 16, 17]. They
improve survival and organ injury by modulating a plethora of inflammatory pathways
that become dysregulated in critically ill patients [1, 5, 6, 18]. In ARDS, tetracyclines
decrease a variety of inflammatory mediators, including inflammasome-dependent
IL-1ß and IL-18 secretion, which drives ARDS development [5, 16, 19]. Furthermore,
they impair the breakdown of extracellular matrix components and inhibit neutrophil
infiltration [14, 17, 20–23] (Fig. 2.1).
2 Immunomodulation by Tetracyclines in the Critically Ill: An Emerging Treatment… 19

Alveolar
macrophage

Neutrophil
Sloughing of
Monocyte bronchial epithelium
Tetracyclines
Lymphocyte

Erythrocytes

Endothelial
Apoptosis of AEC
cell
Tetracyclines
1
DAMPs Inflammasome
activation
PAMPs

BEC
Edema fluid Tetracyclines
2 2
Chemokine Proinflammatory
AEC I release cytokine release

AEC II Increased
5 permeability
4 Extracellular matrix
Neutrophil
breakdown
degranulation

Damage to
Tetracyclines basement membrane

3
Neutrophil influx

Disrupted endothelium

Fig. 2.1 The immunomodulatory effects of tetracyclines in ARDS. ① By sensing pathogen-­

associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), Toll-­
like receptors (TLRs) become activated, thereby triggering the activation of the NLRP3
inflammasome. Tetracyclines inhibit the activation of nuclear factor-kappa B (NF-κB) and the
NLRP3 inflammasome and subsequent ② release of proinflammatory cytokines and chemokines
causes impaired ③ chemotaxis of immune cells including neutrophils. Tetracyclines further block
④ neutrophil degranulation and ⑤ extracellular matrix breakdown. ⊥ inhibition, AEC I type I alve-
olar epithelial cell, AEC II type II alveolar epithelial cell, BEC bronchial epithelial cell

2.4.1 In Vivo Models

2.4.1.1 E ffects on Inflammatory Cytokines and NLRP3

Inflammasome Caspase-1 Signaling
Tetracyclines significantly reduce the secretion of pro-inflammatory cytokines,
including TNF-α, IL-1ß, IL-6, and IL-8, thereby improving survival and lung injury
in indirect models of ARDS [16, 19, 24].
Current research suggests that a key mechanism underlying the immunomodula-
tory effects of tetracyclines is the inhibition of the secretion of the pro-inflammatory
20 A. Sauer et al.

cytokines IL-1ß and Il-18 via the NLRP3 inflammasome pathway. In a recent study,
tetracycline significantly reduced both LPS- and influenza-induced lung injury in
mice by inhibiting inflammasome-caspase-1 dependent IL-1ß and IL-18 produc-
tion. This effect was mediated by direct inhibition of caspase-1 activation by tetra-
cycline [5].

2.4.1.2 Effects on MMPs

The best described property of tetracyclines is the inhibition of MMPs in
ARDS. MMPs are a family of zinc-dependent endopeptidases that degrade the base-
ment membrane as well as extracellular matrix components and are involved in
numerous pathological conditions including inflammation, tissue remodeling, and
tumorigenesis. They are produced by a variety of cells including stromal, epithelial,
and inflammatory cells. Tetracyclines directly inhibit MMP activity by chelating
Zn2+ ions from their active site and by inhibiting their transcription [3].
The potential role of tetracyclines as MMP inhibitors in the pathogenesis of
ARDS has been investigated in several animal studies. Carney et al. [23] showed
that pigs pretreated with chemically modified tetracycline 3 (CMT-3) 12 h prior to
intravenous LPS developed less lung injury, less edema and hypoxia by inhibiting
MMP-9 and MMP-2. Additionally, plateau airway pressure was decreased [23].
Similar results were achieved by the same group through the inhibition of gelatin-
ases and neutrophil elastase by CMT-3 in a porcine cardiopulmonary bypass and
LPS-induced lung injury model. The survival rate was increased from 60 to 100%
by CMT-3 treatment [13]. Steinberg et al. [15] demonstrated that blockage of
MMP-2 and MMP-9 by CMT-3 was associated with less edema and histological
lung injury as well as increased survival in an indirect model of ARDS in rats sub-
jected to cecal ligation and puncture. Of note, CMT-3 prevented all the histopatho-
logical changes seen in ARDS [15]. Although not statistically significant, the
authors observed a 64% reduction in MMP-2 activity and a 34% reduction in
MMP-9 activity in bronchoalveolar lavage (BAL) fluid in a porcine model of ARDS
[19]. Furthermore, administration of CMT-3 improved hemodynamics, gas
exchange, lung histology, and survival through the inhibition of MMP-2 in an ovine
ARDS model induced by third-degree burns, smoke inhalation and barotrauma inju-
ries [25]. MMP-9 levels were not affected by CMT-3 but levels were only measured
in plasma and not in BAL fluid as opposed to in the studies described earlier [25].
Levels of MMP-2, MMP-9, and neutrophil elastase measured in plasma were also
not affected by CMT-3 in a cecal ligation and puncture-induced ARDS model [16].
The authors concluded that this might be due to the use of ketamine, which has been
shown to weaken the effects of cecal ligation and puncture in rats via the inhibition
of NF-κB.
Doxycycline has also been described as another potent MMP inhibitor in various
animal models of primary and secondary ARDS [17, 20, 21, 24, 26, 27]. In a
pancreatitis-­induced ARDS model, doxycycline reduced MMP-9 levels which cor-
related with decreased pulmonary edema and hemorrhage [21]. Similar results were
reproduced in cardiopulmonary bypass-induced ARDS models [20, 24]. The posi-
tive influence of doxycycline on endothelial barrier integrity was also demonstrated
2 Immunomodulation by Tetracyclines in the Critically Ill: An Emerging Treatment… 21

by decreased levels of endothelial protein. Not only were MMP-2 and MMP-9 lev-
els in BAL fluid reduced in a H3N2 influenza-induced ARDS model, so were con-
centrations of endothelial protein thrombomodulin and T1-α, a membrane protein
of alveolar type I epithelium, indicating less alveolar capillary membrane damage
[27]. Furthermore, doxycycline might attenuate the development of pulmonary
fibrosis in ARDS through the inhibition of gelatinases [28].

2.4.1.3 Effects on Neutrophil Transmigration

One of the hallmarks of ARDS is the influx of neutrophils into the lungs. Tetracyclines
attenuate neutrophil infiltration and thereby prevent ARDS, an effect possibly linked
to the concomitant decrease of MMP levels [13, 14, 23, 26]. In a ventilation-­induced
lung injury (VILI) model, pretreatment with CMT-3 decreased neutrophil infiltra-
tion and myeloperoxidase levels, which correlated significantly with MMP-9 activ-
ity. The role of MMP-9 during neutrophil migration is, however, not well defined.
As a proteinase, MMP-9 could potentially degrade the basement membrane and
thereby facilitate migration [14]. In an in vitro experiment, neutrophil transmigra-
tion across Matrigel and MMP-9 levels in the Matrigel invasion chamber were
reduced by doxycycline [21].
Pretreatment with CMT-3 inhibited neutrophil influx in models of bacterial- and
cardiopulmonary bypass-induced ARDS [13, 23, 29]. Additionally, doxycycline
prevented neutrophil infiltration in models of viral-, bacterial-, cardiopulmonary
bypass- and pancreatitis-induced ARDS [17, 21, 22, 27, 30].

2.4.2 Human Data

Recently, Peukert et al. described the effect of tetracycline on the NLRP3 inflamma-
some pathway in patients with direct ARDS (Fig. 2.1). Human alveolar leukocytes
were isolated within 24 h of onset of direct ARDS. Cultured leukocytes continued
to produce IL-1ß and IL-18 suggesting that the NLRP3 inflammasome pathway
remained intact. Tetracycline inhibited the production of IL-1ß and IL-18 by alveo-
lar leukocytes in a dose-dependent manner. This study indicates that the inhibition
of caspase-1-dependent IL-1ß and IL-18 by tetracyclines might be a new therapeu-
tic approach in patients with direct ARDS [5].
A randomized clinical trial is currently investigating whether doxycycline can
limit the NF-κB dependent release of pro-inflammatory cytokines and thereby
prevent evolution towards ARDS in patients with COVID-19 (ClinicalTrials.gov
Identifier: NCT04371952). In 89 high-risk COVID-19 patients living in long-
term care facilities, it was recently shown that the administration of doxycycline
within 12 h after symptom onset was associated with early clinical recovery,
decreased hospitalization and reduced mortality [31]. These findings contradict
the results of a randomized controlled trial which suggested doxycycline was not
effective for suspected COVID-19 [32]. In this study, 798 participants received
doxycycline compared to 994 participants randomized to standard care. However,
the trial had several limitations: first, the trial included participants recruited
22 A. Sauer et al.

within 14 days after symptom onset. Second, almost half of the participants were
accrued without PCR-confirmed severe acute respiratory syndrome coronavirus
2 (SARS-CoV-2) infection [32]. Because the inflammasome-caspase-1 pathway
is activated early in ARDS [5, 8], this might explain why doxycycline was not
beneficial.

2.5 Mechanisms of Action of Tetracyclines in Sepsis

Tetracyclines exert their pleiotropic immunomodulatory effects via several inflam-

matory pathways such as NF-κB and MAPKs downstream of PRRs whereby they
inhibit the secretion of inflammatory mediators including cytokines, chemokines,
MMPs, prostaglandin E2 (PGE2), and NO [7, 33–38]. They also ameliorate sepsis-­
induced liver injury by inhibiting apoptotic pathways [39, 40]. Mild perturbation of
mitochondrial function by tetracyclines can install disease tolerance mechanisms
like tissue repair and metabolic reprogramming [6].

2.5.1 In Vitro Models

2.5.1.1 Effects on Cytokine and Chemokine Production

An uncontrolled host response to infection can trigger a so-called cytokine storm
which is one of the main characteristics of sepsis [1]. Mounting evidence has identi-
fied autophagy as an important regulator of excessive inflammation. Sun et al. [35]
have shown that minocycline, which induces autophagy by inhibiting the mamma-
lian target of rapamycin (mTOR) signaling pathway, suppresses cytokine produc-
tion and cell proliferation, and protects human THP-1 cells from LPS-toxicity.
Additionally, the study suggests that the IKK/NF-κB signal pathway was linked to
minocycline-induced autophagy [35]. A previous study also demonstrated that
minocycline decreased cytokine and chemokine production by inhibiting IKKα/β
phosphorylation in LPS-stimulated THP-1 cells [41]. The influence of tetracyclines
on certain signaling pathways was further characterized by Sun et al. [34]. The
modulated phosphorylation of the NF-κB-, p38- and ERK1/2-pathways by doxycy-
cline, minocycline, and tigecycline significantly inhibited the expression of TNF-α,
IL-8, macrophage inflammatory protein 1α (MIP-1α) and MIP-1ß by LPS-stimulated
THP-1 cells [34].

2.5.1.2 Effects on Arachidonic Acid Metabolites and NO Production

Metabolites of arachidonic acid, such as PGE2 and NO, are inhibited by tetracy-
clines and play a role in inflammatory processes [42]. CMT-3 inhibited both nitrite
and the cyclooxygenase 2 (COX-2) mediated PGE2 accumulation in murine macro-
phages stimulated with LPS [42]. Moreover, tetracyclines regulate inducible NO
synthase (iNOS) at the post-transcriptional level, thereby decreasing NO levels in
LPS-stimulated murine macrophages [36, 37, 43, 44].
2 Immunomodulation by Tetracyclines in the Critically Ill: An Emerging Treatment… 23

2.5.2 In Vivo Models

2.5.2.1 E ffects on MAPK Signaling Pathways

and Inflammatory Mediators
Doxycycline ameliorated systemic and pulmonary inflammation in a murine sepsis
model induced by cecal ligation and puncture [7]. By decreasing levels of IL-1ß,
IL-6, TNF-α, myeloperoxidase (MPO), and the antioxidant glutathione in plasma
and lung homogenates, doxycycline improved survival. The anti-inflammatory
effect of CMT-3 is possibly mediated through the inhibition of MAPKs. In rats
subjected to cecal ligation and puncture, pretreatment with CMT-3 inhibited TNF-α
secretion and activation of p38 and p42/44–MAPK pathways, thereby preventing
the progression to septic shock [38].
Tetracyclines also act as inhibitors of NO synthesis. In mice injected intraperito-
neally with LPS, doxycycline prevented septic shock by inhibiting nitrate produc-
tion by an IL-10 independent mechanism [36]. Furthermore, tetracyclines caused a
decrease in iNOS activity in a similar sepsis model [37].

2.5.2.2 Effects on Organ Dysfunction

Maitra et al. [39] showed that CMT-3 improved survival and was hepatoprotective
in rats subjected to sepsis by cecal ligation and puncture. They demonstrated that
the underlying mechanisms by which CMT-3 improved survival and hepatic injury
were the CMT-3 induced reduction of MMP-9 and NO. The hepatoprotective effect
of CMT-3 was further characterized by the same group. Administration of CMT-3 in
septic rats caused decreased levels of MMP-9 and increased the expression of tissue
inhibitor of metalloproteinase-1 (TIMP-1), which is an in vivo inhibitor of MMP-9.
Furthermore, transforming growth factor beta-1 (TGF-ß1) and caspase-3 were
reduced, thereby preventing liver injury and increasing survival in septic rats [40].
Recently, Colaço et al. [6] demonstrated how doxycycline can protect against sepsis
by inducing disease tolerance without diminishing bacterial load in a mouse model
of bacterial sepsis. In this study, tissue damage of the liver, lungs, and kidneys on a
molecular and histopathological level was reduced by treatment with doxycycline.
Furthermore, the authors demonstrated similar protective effects in influenza-­
induced sepsis in contrast to fungal- or cerebral malaria-induced infection models.
Bulk RNA sequencing showed that doxycycline altered the expression of genes
involved in epithelial cell differentiation suggesting more effective lung repair with-
out the development of lung fibrosis. Functional analysis found a cluster of down-
regulated genes related to decreased liver collagen production indicating that
doxycycline potentially plays a role in limiting liver fibrosis. During infection, liv-
ers of septic mice accumulated acylcarnitines and steroids. Administration of doxy-
cycline partially decreased this accumulation suggesting that it might reverse the
block in mitochondrial import of fatty acids during sepsis. It also increased the
activation of glucocorticoid receptors through serine phosphorylation. Furthermore,
it was shown that mild perturbation of mitochondrial function, like the electron
transport chain, by doxycycline can activate disease tolerance mechanisms, such as
24 A. Sauer et al.

tissue repair and metabolic reprogramming in sepsis. One of the underlying mecha-
nisms could be the doxycycline-induced sensitization to adrenergic agonists that
reduces lipid accumulation in the liver. This elegant study demonstrates how doxy-
cycline may rebalance immune homeostasis in sepsis [6].

2.5.3 Human Data

Minocycline significantly ameliorated the LPS-induced inflammatory response in

human monocytes obtained from healthy volunteers by decreasing the production
of TNF-α, IL-1ß, IL-6, COX-2, and PGE2, as well as reducing activation of lectin-­
like oxidized low density lipoprotein receptor-1 (LOX-1), TNF-α factor (LITAF),
and Nur77 nuclear receptor. The immunomodulatory effects of minocycline were
mediated through the blocked activation of NF-κB, p38 MAPK, and phos-
phoinositide 3-kinase (PI3K)/Akt pathways [33]. Consistent with this, another
study demonstrated that doxycycline reduced the LPS-induced gene expression of
IL-1ß and IL-6 as well as phagocytosis of heat-inactivated Escherichia coli by
monocytes [45].
The Fredeking group investigated the impact of tetracyclines on the inflamma-
tory response in patients with dengue hemorrhagic fever in two randomized con-
trolled trials [46, 47]. In the first trial, hospitalized patients received usual care or
usual care combined with doxycycline or tetracycline. Serum cytokine and cytokine
receptor levels were determined on days 1, 3, and 7 of treatment. Doxycycline was
significantly more effective in modulating levels of IL-6, IL-1ß, TNF-α, IL-1 recep-
tor antagonist (IL-1ra) and TNF-R1 than tetracycline. In the second trial with 231
participants, doxycycline treatment was associated with lower mortality, which
positively correlated with reduced levels of pro-inflammatory cytokines. Patients in
the doxycycline arm had a 46% lower mortality than those in the usual care arm [47].
In a prospective randomized placebo-controlled pilot trial, intravenous doxycy-
cline did not have an impact on MMP-8, MMP-9, or TIMP-1 in patients with severe
sepsis or septic shock [48]. This finding might be explained by the small sample size
and large variance of disease severity that made it nearly impossible to detect a
statistical significance. Only 23 patients were included in the analysis of this pilot
trial. The studied population was also very heterogenous in terms of disease severity
and disease onset, which was reflected in the detected variation in baseline concen-
trations and activities of MMP-8, MMP-9, and TIMP-1 [48].

2.6 Future Research Perspectives

ARDS and sepsis are both heterogenous syndromes characterized by significant

variability in the degree of immune dysregulation, disease severity, and prognosis.
As described in this chapter, tetracyclines modulate immunity at multiple levels
involving the inhibition of concurrent pro- and anti-inflammatory pathways.
Although our knowledge of the underlying mechanisms has increased, we need
2 Immunomodulation by Tetracyclines in the Critically Ill: An Emerging Treatment… 25

more clinical trials before we can adopt tetracyclines as routine treatment in sepsis
and ARDS.
To prevent the development of antibiotic resistance, we need to further investi-
gate the use of non-antibacterial tetracycline derivatives such as CMT-3; their effec-
tiveness has already been observed in preclinical trials [5, 16, 25]. Furthermore,
research should focus on developing tetracyclines with minimal adverse effects and
on augmenting certain immunomodulatory effects.
Most clinical trials of novel sepsis and ARDS therapies have focused on large
patient cohorts without considering the heterogenous nature of both syndromes and
the varying immune responses of each patient. This has likely contributed to the fact
that immunomodulatory drugs have not been demonstrated to have a clinical benefit
in the past. Each patient needs to be longitudinally evaluated by using biomarkers
and establishing baseline immune cell responsiveness [1]. In other words, immune
profiling is necessary to stratify critical care patients in future trials and to identify
biological subphenotypes in ARDS and sepsis. Calfee et al. [49] have described dif-
ferential responses to treatment according to phenotype in ARDS. Predictive enrich-
ment strategies are recommended to promote the efficiency of clinical trials and to
enable precision medicine.

2.7 Conclusion

In summary, tetracyclines have been shown to be potent drugs with pleiotropic

immunomodulatory effects inhibiting multiple inflammatory pathways that trigger
an uncontrolled immune response in ARDS and sepsis. In preclinical studies, tetra-
cyclines have been described as promising immunomodulatory agents that seem to
have the potential to correct the unbalanced immune homeostasis present in criti-
cally ill individuals. Future trials need to further investigate tetracyclines in terms of
dosing and side effects as well as focus on preventing antibiotic resistance. Finally,
the extent of inflammation and immunosuppression varies between each patient
explaining why so many trials have failed to show a survival benefit for immuno-
modulatory drugs in the past. We need to identify subphenotypes of ARDS and
sepsis that will respond to adjunctive tetracycline treatment.

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Hit Early: Blocking Interleukin-1
in the Treatment of COVID-19 3
Pneumonia

E. J. Giamarellos-Bourboulis, M. Mouktaroudi,
and M. G. Netea

3.1 Introduction

The rise of the pandemic by the novel severe acute respiratory syndrome coronavi-
rus-2 (SARS-CoV-2) generated concern in the medical community for patients who
are admitted to the intensive care unit (ICU) because of severe respiratory failure.
The majority of these patients have unfavorable outcomes and this led to the consid-
eration of how progression into severe respiratory failure and ICU admission may
be prevented. The current approach for bacterial sepsis advocates that early initia-
tion of treatment is a life-saving strategy [1]. This opinion was based on the obser-
vation that starting antimicrobials in the first hour from hypotension was associated
with 79.1% survival and that every hour of delay impacted by 7.6% the relative
increase in the odds for death [2]. What are the similarities between coronavirus
disease 2019 (COVID-19) and sepsis that could indicate that the principles of early
treatment may apply, and how feasible is this in COVID-19? One recent meta-anal-
ysis described that almost 80% of hospitalized patients with COVID-19 meet the
Sepsis-3 definition using the Sequential Organ Failure Assessment (SOFA) score
[3]. Despite the similarity in definition, the major obstacle for early treatment in
COVID-19 would be the lack of extremely potent antiviral drugs, even though a
recent retrospective analysis of 475 hospitalized patients showed that start of

E. J. Giamarellos-Bourboulis (*) · M. Mouktaroudi

4th Department of Internal Medicine, National and Kapodistrian University of Athens
Medical School, Athens, Greece
e-mail: [email protected]
M. G. Netea
Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud
University, Nijmegen, The Netherlands
Department of Immunology and Metabolism, Life and Medical Sciences Institute, University
of Bonn, Bonn, Germany

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 29

J.-L. Vincent (ed.), Annual Update in Intensive Care and Emergency Medicine
2022, Annual Update in Intensive Care and Emergency Medicine,
https://doi.org/10.1007/978-3-030-93433-0_3
30 E. J. Giamarellos-Bourboulis et al.

remdesivir in the first 3 days from positive testing for SARS-­CoV-­2 was associated
with lower odds for death [4].
Early treatment of COVID-19 thus needs to rely on the early recognition of the
activation of the pro-inflammatory cascade and of the immediate start of anti-­
inflammatory treatment aiming to prevent progression into severe respiratory failure
and unfavorable outcome. The current chapter provides existing evidence about the
kinetics of cytokines for disease progression, to suggest novel biomarkers for prog-
nosis and to present clinical evidence on the strategy of early anti-inflammatory
treatment for COVID-19, with a focus on interleukin (IL)-1-dependent pathways.

3.2 Cytokines and Disease Progression in COVID-19

Early since the beginning of the SARS-CoV-2 pandemic, it was realized that the more
severe patients were suffering from hyperinflammation, which was associated with
unfavorable outcome [5]. Since then, several prospective studies have been conducted
to associate the concentrations of circulating cytokines with disease severity and dis-
ease prognosis. A PubMed search entering the keywords “plasma cytokines” and
“COVID-19” on 22 October 2021 retrieved 697 results. Pre-prints, reviews, and case
reports were excluded leaving 587 peer-reviewed articles. Following the reading of the
abstracts, only studies associating plasma cytokines with disease severity and progno-
sis were retained. Some of these studies, summarized in Table 3.1, compare the circu-
lating levels of cytokines with disease severity. It needs to be highlighted that not all
existing cytokines were measured in all studies; however, one common denominator of
the studies analyzed in Table 3.1 is that concentrations of IL-6 and IL-1 receptor antag-
onist (IL-1ra) increase with increasing disease severity [6–11].
Another set of studies tried to identify which of the circulating cytokines may be
a biomarker of prognosis in COVID-19. All these studies used 30-day mortality as
an endpoint and their results are summarized in Table 3.2. Most of the enrolled
patients were severe on enrolment and analyses indicated that circulating concentra-
tions of IL-6 and, to a lesser extent other cytokines, were increased from the day of

Table 3.1 Synopsis of studies measuring circulating cytokines in the plasma of patients with
coronavirus disease 2019 (COVID-19) on the day of hospital admission
Number of Cytokines increased in
Ref. Design Study period Country patients severe/critical ICU patients
6 Single-center, NR USA 36 M-CSF, IL-1ra, IP-10,
biobank MCP-1, IL-2, TNFα, IL-6,
7 Single-center, March–May Turkey 100 IL-1ra, IL-18
prospective 2020
8 Prospective NR USA 15 IL-1ra, IL-6, IL-19
9 Prospective NR USA 40 IL-1β, IL-6, IL-10, sTNFR1
10 Prospective February– China 1472 IL-6
March 2020
11 Prospective February–May Italy 175 IL-2, IL-6, IL-8, IL-16,
2020 sTNFR1
IL interleukin, TNF tumor necrosis factor, ICU intensive care unit, NR not reported, M-CSF mac-
rophage colony-stimulating factor, IP interferon-inducible protein, MCP monocyte chemoattrac-
tant protein, IL-1ra IL-1 receptor antagonist
3 Hit Early: Blocking Interleukin-1 in the Treatment of COVID-19 Pneumonia 31

Table 3.2 Synopsis of studies associating cytokine admission levels with unfavorable outcome in
coronavirus disease 2019 (COVID-19)
Suggested
Cytokine prognostic
Number greater in cut-off for
Study of Disease non-­ unfavorable
Ref. Design period Country patients severity survivors outcome
12 Single-­ April– Italy 46 Stage I to IL-6 NR
center, May 2020 III
prospective
13 Single-­ January– China 31 Severe IL-6, IL-8, NR
center, June 2020 IL-10
prospective
14 Three February Japan 102 Moderate IL-6 49 pg/ml for
centers, 2020–July and severe IL-6
prospective 2021
15 Three March– France 150 Severe IL-6, CRP 212 pg/ml for
centers, April IL-6
prospective 2020
16 Single-­ March– USA 1484 Mild to IL-6, IL-8, 70 pg/ml for
center, April severe TNF-α IL-6, 50 pg/ml
prospective 2020 for IL-8; 35 pg/
ml for TNF-α
17 Multicenter, March– Greece 178 Severe IL-6 30 pg/ml for
prospective May 2020 IL-6
IL interleukin, TNF tumor necrosis factor, CRP C-reactive protein, NR not reported

admission among non-survivors [12–17]. However, the results of these studies need
to be interpreted under the notion that the research question was “which measured
cytokines predicted an unfavorable outcome among patients who were already
either severe or critical?”. None of these studies searched for a biomarker that could
identify which patients with moderate illness will progress over time into severe or
critical illness.

3.3  ontribution of Monocytes and Macrophages in Early

C
Cytokine Response

The hyperinflammation of COVID-19 has features of cytokine storm syndrome

or macrophage activation syndrome. Macrophage activation syndrome is often
described as an auto-inflammatory entity due to sudden stimulation of tissue
macrophages with over-production of IL-1β and the generation of a vicious
cycle where IL-1β propagates further production of cytokines by macrophages
through its receptor [18]. Inflammasomes play a central role in the production
of IL-1β since they activate caspase-1, which cleaves inactive pro-IL-1β to
active IL-1β.
There is scarce evidence regarding the participation of the NACHT, LRR, and
PYD domains-containing protein (NLRP) inflammasome in the pathogenesis of
COVID-19. In one post-mortem study of only four patients, lung sections were
stained for components of the NLRP3 inflammasome mainly apoptosis-associated
32 E. J. Giamarellos-Bourboulis et al.

speck-like protein containing a caspase recruitment domain (ASC) and caspase-1;

lung deposition was significantly higher than in control subjects [19]. It has been
suggested that several particles of other coronaviruses may stimulate components of
the NLRP3 inflammasome. More precisely, ORF3a binds to ASC and ORF8b binds
to leucine-rich repeats and this stimulates the assembly of NLRP3 [20]. Although
activation of NLRP3 leads to activation of the proteolytic activity of caspase-1, the
excess production of IL-β is a dual process, because for caspase-1 to act, priming of
the cell cytoplasm with pro-IL-1β is needed. It has been suggested that alarmins or
damage-associated molecular patterns (DAMPs) that are released from the lung epi-
thelial cells during rapid viral replication act on Toll-like receptor-4 (TLR4) and
lead to intracellular accumulation of pro-IL-1β [21]. Indeed, increased levels of the
DAMP S100A9 (also known as calprotectin) circulate in the plasma of patients with
COVID-19 [11]. In parallel to the production of pro-IL-1β under the stimulation of
DAMPs, one more cytokine of the IL-1 family is produced, namely IL-1α. This is
already active and does not necessitate any further processing. Abundant release of
IL-1α is described in lung infections, including COVID-19 [22].
Both IL-1α and IL-1β act on the IL-1 receptor in tissue macrophages and per-
petuate the inflammatory process. In an attempt of the host to attenuate this phe-
nomenon, IL-1ra is produced to limit the binding of IL-1α and IL-1β to their receptor
and to minimize biological function [23]. There are three main observations that
suggest that early production of IL-1α and IL-1β drives progression into severe
respiratory failure:

• IL-1ra is increased in many severe cases of COVID-19 (Table 3.1).

• Treatment with anakinra, a recombinant human IL-1ra, has been associated with
survival benefit in severe COVID-19 [24], suggesting that the IL-1 pathway is
already activated in severe disease and that IL-1 activation may start before signs
of severity appear.
• When severe disease develops, IL-1β is not detectable at high concentrations in
the circulation by contrast to the other cytokines [16]. Since IL-1β and IL-6 are
secreted in parallel from tissue macrophages, it may be hypothesized that the
peak of IL-1β precedes the peak of the other cytokines.

3.4 How Can Early Activation of IL-1 Be Identified?

Not all patients with moderate-to-severe COVID-19 are at risk for progression into
severe respiratory failure. This observation introduces the need for a biomarker that
can identify activation of the IL-1 pathway early and that can prognosticate early
which patients will deteriorate to develop severe respiratory failure. During March
2020, blood was drawn from 57 patients from three study sites in Greece on hospital
admission for pneumonia. The concentrations of the biomarker suPAR (soluble uro-
kinase plasminogen activator receptor) were measured. Despite the lack of signs of
severe respiratory failure or respiratory distress at the time of admission, patients
with concentrations of suPAR ≥6 ng/ml had 85.7% risk of progression into severe
respiratory failure or death in the first 14 days of follow-up. This value was only
3 Hit Early: Blocking Interleukin-1 in the Treatment of COVID-19 Pneumonia 33

8.3% among patients with suPAR <6 ng/ml [25]. This finding was further validated
in two later studies. The first study enrolled 352 patients and was multicenter in the
United States, Denmark, Germany and Greece. Patients with suPAR ≥6.8 ng/ml on
admission had 44.9% risk of progression into severe respiratory failure [26]. Finally,
in the phase 2 study, SAVE (suPAR-guided anakinra treatment for validation of the
risk and early management of severe respiratory failure by COVID-19), 59.2% of
patients with suPAR ≥6 ng/ml receiving usual care progressed into severe respira-
tory failure or died the first 14 days contrary to only 3.7% of patients with suPAR
<6 ng/ml [27]. Unpublished data from our group show that suPAR ≥6 ng/ml indi-
cates increased circulating levels of the DAMP S100A8/A9 [28]. The above analy-
sis on the role of DAMPs in priming tissue macrophages for pro-IL-1β led us to
consider suPAR as an early biomarker of activation of the IL-1 pathway in patients
which generates the risk for progression into severe respiratory failure.

3.5  AVE Strategy: Early Blockade of IL-1 Guided

S
by Biomarkers

The knowledge that suPAR may be an early biomarker for the risk of progression
into severe respiratory failure led us to study its use to guide treatment with anakinra
in one phase 2 and one phase 3 study. The therapeutic strategy targets hospitalized
patients with COVID-19 pneumonia with a PaO2:FiO2 ratio ≥150 who do not need
ICU treatment. If these patients have a plasma suPAR ≥6 ng/ml, early treatment
with anakinra is started with one subcutaneous daily dose of 100 mg for 10 days.
The phase 2 study was given the acronym SAVE and was a single-arm,
open-­label trial (EudraCT number: 2020-001466-11; ClinicalTrials.gov Identifier:
NCT04357366). Parallel comparators treated using standard-of-care in other
departments of academic hospitals were recruited using the inclusion and exclusion
criteria of the SAVE trial and were propensity-score matched for age, comorbidi-
ties, admission severity scores (Acute physiology and chronic health evaluation
[APACHE] II, SOFA, pneumonia severity index, World Health Organization [WHO]
scale) and for treatment with azithromycin, hydroxychloroquine, and dexametha-
sone. The incidence of severe respiratory failure or death after 14 days was 22.3%
among anakinra-treated patients and 59.2% among comparators [27].
Following advice from the Emergency Task Force for COVID-19 of the European
Medicine Agency, the pivotal, multicenter confirmatory phase III randomized con-
trolled trial with the acronym SAVE-MORE was designed (EudraCT number:
2020-005828-11; ClinicalTrials.gov Identifier: NCT04680949). The study enrolled
594 patients who were subject to 1:2 stratified randomization into standard-of-care and
placebo or standard-of-care and anakinra [29]. Stratified randomization was done tak-
ing into consideration, apart from allocation into placebo or anakinra, severity as
defined by the need of oxygen therapy, intake of dexamethasone, body mass index, and
geographic origin. On the day the study medication was started, 91.6% of participants
were in need of supplemental oxygen. The primary study endpoint was comparison of
the distribution of the scores from the 11-point WHO Clinical Progression ordinal
Scale (CPS) on Day 28 as defined by ordinal multiple regression analysis. The adjusted
34 E. J. Giamarellos-Bourboulis et al.

proportional odds of having a worse score with anakinra than with placebo was 0.36
(95% confidence interval [CI] 0.26–0.50; p < 0.0001). Three supporting analyses of the
primary endpoint were done. These were: (a) the distribution of the 11-point WHO-
CPS on Day 14. The adjusted odds ratio was 0.58 (95% CI 0.42–0.79; p = 0.001); (b)
proportion of patients with persistent disease at day 28 and with severe disease or death
on day 28. Multivariate logistic regression analysis indicated that anakinra significantly
reduced the risk of persistence (odds ratio 0.36; 95% CI 0.25–0.53; p < 0.0001) and of
severe disease or death (odds ratio 0.46; 95% CI 0.26–0.83; p = 0.010); and (c) inci-
dence of severe respiratory failure or death by day 14. This was 31.2% in the placebo
arm and 20.7% in the anakinra arm (adjusted hazard ratio 0.67; p = 0.017). Secondary
endpoints also favored anakinra treatment. More precisely: (1) the median absolute
decrease in WHO-CPS by day 28 from baseline was 3 and 4 in the placebo and anakinra
groups, respectively (odds ratio 0.40; p < 0.0001); (2) median absolute decrease in
WHO-CPS by day 14 from baseline was 2 and 3 in the placebo and anakinra groups,
respectively (odds ratio 0.64; p = 0.007); (3) median absolute decrease in SOFA score
by day 7 from baseline was 0 and 1 in the placebo and anakinra groups, respectively
(odds ratio 0.63; p = 0.004); (4) median time to hospital discharge was 12 and 11 days
in the placebo and anakinra groups respectively (odds ratio 1.22; p = 0.033); and (5)
median time for discharge from the ICU in case of ICU admission was 14 and 10 days
in the placebo and anakinra groups, respectively (odds ratio 2.33; p = 0.026) [28]. It has
to be noted that 11.1% of participants in the SAVE-MORE trial were not in need of
supplemental oxygen at the time of screening for eligibility but had progressed into
need for supplemental oxygen by mask or nasal prongs before the start of the study
drug. As such, sudden need for oxygen may be considered a potential indication for
anakinra treatment. The SAVE-MORE investigators also developed a prognostic score
to help identify patients who may receive most benefit from anakinra treatment [29].
This score comprises aspartate aminotransferase >44 U/l; neutrophil to lymphocyte
ratio > 5.5; C-reactive protein (CRP) >50 mg/l; and ferritin >700 ng/ml. Patients scor-
ing positive for at least two of these elements had the greatest chance for improvement
with anakinra treatment [29].
The CORIMUNO-ANA-1 study, which preceded the SAVE-MORE trial, did not
give such favorable results. The patient population was similar to patients enrolled
in the SAVE and SAVE-MORE trials, i.e., they were hospitalized and receiving
nasal oxygen or oxygen by mask; 59 patients were randomized to treatment with
anakinra and usual care and 55 patients to treatment with usual care alone [30]. The
study was negative for the primary endpoint albeit showing a trend for anakinra
benefit; 36% and 38% of patients respectively had a WHO-CPS score > 5 on day 4;
and 47% and 51% respectively were receiving non-invasive ventilation or mechani-
cal ventilation or had died by day 14 [30]. There are several possible explanations
why the CORIMUNO-ANA-1 study was negative: (a) no biomarker was used for
the selection of patients, so it is possible that some of the enrolled patients were not
at a stage of early IL-1 activation; (b) no placebo was used; and (c) the duration of
anakinra treatment was just 5 days.
In the CAN-COVID trial, 454 patients with hypoxic COVID-19 not requiring
mechanical ventilation and with signs of hyperinflammation were randomized to a
single injection of placebo or the anti-IL-1β monoclonal antibody, canakinumab
3 Hit Early: Blocking Interleukin-1 in the Treatment of COVID-19 Pneumonia 35

[31]. The primary endpoint was survival without need for non-invasive ventilation
or mechanical ventilation by day 29. This endpoint was met in 88.8% of patients in
the canakinumab arm and 85.7% of patients in the placebo arm. The superior effi-
cacy of anakinra over canakinumab in a similar patient population may be explained
by the activity of anakinra against IL-1α, which is not inhibited by canakinumab. An
alternative explanation for this discrepancy is that suPAR was not used as a criterion
for enrichment of the patient population.

3.6 Conclusion

The above analysis suggests there is great heterogeneity of patients at the time of
hospital admission and that we need to understand early which patients are at risk
for progression into severe respiratory failure so as to initiate early treatment aimed
at preventing deterioration. This requires a personalized approach with the use of
appropriate biomarkers (Fig. 3.1). In the case of anakinra, we suggest that the drug
may be of benefit in patients admitted with COVID-19 pneumonia who do not

Stage II Stage III

Stage I
(Pulmonary Phase) (Hyperinflammation Phase)
(Early Viral Phase)
Suggested personalized
strategy for “Early Hit”

Trace the patient AT RISK

for progression

• suPAR ≥ 6ng/ml
• Sudden need for oxygen
• Increase of AST, NLR, CRP, ferritin

ANAKINRA
Severity

Viral response phase

Hyper-inflammation

Clinical Constitutional symptoms PaO2/Fio2<150-300mmHg ARDS, Shock, Cardiac Failure

Symptoms Fever, Cough
Laboratory
Lymphopenia Abnormal chest imaging ↑↑↑ CRP, LDH, IL-6, D-dimers, ferritin
Signs Troponin, NT-proBNP
Potential
Therapies Dexamethasone Dexamethasone
ANAKINRA IL-6 receptor antagonists

Fig. 3.1 Suggestion for early treatment with anakinra during the disease course of COVID-19
pneumonia. The results of the SAVE and SAVE-MORE trials [27, 29] suggest that anakinra could
potentially be beneficial in patients who are at the pulmonary stage of the disease and at risk of
progression to the hyperinflammatory stage. The patient at risk could be identified by at least one
of following: soluble urokinase plasminogen activator receptor (suPAR) ≥6 ng/ml; sudden need
for oxygen; increased circulating concentrations of at least two of aspartate aminotransferase
(AST), neutrophil to lymphocyte ratio (NLR), C-reactive protein (CRP) and ferritin. ARDS acute
respiratory distress syndrome, BNP brain natriuretic peptide, FiO2 fraction of inspired oxygen, IL
interleukin, LDH lactate dehydrogenase, PaO2 partial oxygen pressure, ↑↑↑ extremely elevated
36 E. J. Giamarellos-Bourboulis et al.

require ICU treatment. Existing evidence does not support the use of anakinra for
patients already in severe respiratory failure.

Acknowledgements M. G. Netea is supported by an ERC Advanced Grant (#833247) and a

Spinoza grant from the Netherlands Organization for Scientific Research.

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Hemoadsorption Therapy During ECMO:
Emerging Evidence 4
A. Supady, T. Wengenmayer, and D. Brodie

4.1 Introduction

Over the past decade, venovenous extracorporeal membrane oxygenation (ECMO) has
evolved to become a cornerstone for the treatment of patients with severe respiratory
failure, and venoarterial ECMO has increasingly been used for cardiocirculatory sup-
port in patients with cardiogenic shock or cardiac arrest [1–3]. During the coronavirus
disease 2019 (COVID-19) pandemic, there appears to be a continued role for ECMO in
the treatment of patients with severe respiratory failure, although mortality remains high
and appears to be worsening over the course of the pandemic [4, 5]. Therefore, it will be
crucial to further refine the treatments and adjunctive therapies available to complement
ECMO, with the potential to improve both survival and quality of life [6–8].

A. Supady (*)
Department of Medicine III (Interdisciplinary Medical Intensive Care), Medical Center –
University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Department of Cardiology and Angiology, Heart Center, University of Freiburg,
Freiburg, Germany
Heidelberg Institute of Global Health, University of Heidelberg, Heidelberg, Germany
e-mail: [email protected]
T. Wengenmayer
Department of Medicine III (Interdisciplinary Medical Intensive Care), Medical Center –
University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Department of Cardiology and Angiology, Heart Center, University of Freiburg,
Freiburg, Germany
D. Brodie
Division of Pulmonary, Allergy and Critical Care Medicine, Columbia University College of
Physicians and Surgeons, New York, NY, USA
Center for Acute Respiratory Failure, New York-Presbyterian Hospital, New York, NY, USA

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 39

J.-L. Vincent (ed.), Annual Update in Intensive Care and Emergency Medicine
2022, Annual Update in Intensive Care and Emergency Medicine,
https://doi.org/10.1007/978-3-030-93433-0_4
 Another Random Document on
Scribd Without Any Related Topics
 I burn nor am consumed, I live and die,
Far from myself am I and yet so near,
I sink to hell, I rise to Heaven on high,
One thing alone I hope, and yet I fear.
Gentle, yet fierce—for what I loathe I sigh,
To love thee racks my soul with torment drear,
Thus step by step already am I come,
Drawn in these different ways to my last doom.
ERASTRO.
Elicio, mark! how gladly would I pour
At Galatea's feet all that she hath left
To me in life, if but she would restore
The heart and soul whereof I am bereft.
My herd I would bestow, and furthermore
My Spot and Hawk, if she would but the theft
Forego: but ah! the goddess on her throne
More than aught else would have my soul alone.
ELICIO.
Erastro, mark! if once the heart on high
Be placed by fate, or chance, or what you will,
To pluck it down 'twere foolishness to try
By force, or art, or any human skill.
Rejoice that she is blessed; though thou canst die
In truth without her, 'tis my thought that still
No life on earth can be more full of bliss
Than death for such a noble cause as this.

Erastro was already setting himself to follow on in his song when

they perceived, by a thickly wooded hillock which was at their back,
no slight clamour and sound; and, both rising to their feet to see
what it was, they saw a shepherd descending from the mountain,
running at the greatest speed in the world, with a naked knife in his
hand, and the hue of his countenance changed, and, coming after
him, another shepherd swift of foot, who in a few strides overtook
the first, and seizing him by the collar of his skin-coat, raised his arm
in the air as high as he could, and a sharp dagger which he carried
unsheathed, and buried it twice in his body, saying:
'Receive, oh ill-starred Leonida, the life of this traitor, which I offer
up in vengeance of your death.'
This happened with such rapidity that Elicio and Erastro had not the
opportunity to stop him; for they came up at the time when the
stricken shepherd was already giving out his last breath, struggling
to utter these few ill-formed words:
'Would that you had allowed me, Lisandro, to satisfy Heaven with a
longer repentance for the wrong I did you, and had then taken from
me the life which, for the reason I have said, now departs from this
flesh ill-content.'
And without being able to say more he closed his eyes in everlasting
night. By these words Elicio and Erastro fancied that for no small
cause had the other shepherd inflicted on him so cruel and violent a
death. And the better to inform themselves of the whole occurrence,
they would fain have inquired of the murderous shepherd; but he,
with retreating step, leaving the shepherd dead and the two
wondering, turned to go back into the hillock beyond. And when
Elicio desired to follow him, and to learn from him what he wished,
they saw him come again out of the wood, and, being a good space
distant from them, in a loud voice he said to them:
'Pardon me, gentle shepherds, if I have not been gentle in having
wrought in your presence that which you have seen, for the just and
mortal rage which I had conceived against that traitor did not permit
a more moderate course on my part. What I counsel you is, that, if
you would not anger the Deity that dwells in high Heaven, you
should not offer the last rites and accustomed prayers for the
traitorous soul of that body which you have before you, nor give it
burial, if here in your country it is not the custom to give it to
traitors.'
And, saying this, he turned with all speed to go into the forest, with
so much haste as to take away from Elicio the hope of overtaking
him, even though he followed him. And so the twain with tender
hearts turned to perform the pious office, and to give burial, as best
they could, to the wretched body, which had so suddenly ended the
course of its short days. Erastro went to his hut which was not far
away, and, bringing sufficient implements, made a grave at the very
spot where the body was; and, bidding it the last farewell, they
placed it therein. Not without compassion for his hapless lot they
returned to their herds, and, collecting them again with some haste
(for the sun was already entering with all speed by the gates of the
west), betook themselves to their accustomed shelters, where
neither the comfort they felt therein, nor the little that his cares
allowed him, could keep Elicio from wondering what causes had
moved the two shepherds to come to so desperate a pass; and
already he regretted that he had not followed the murderous
shepherd, and learnt from him, if possible, what he wished. With this
thought, and with the many that his love caused in him, after
leaving his herd in a place of safety, he went out from his hut, as
was his wont at other times, and by the light of the beauteous
Diana, who showed herself resplendent in the sky, he entered the
denseness of a dense wood beyond, seeking some solitary spot
where, in the silence of the night, with greater peace he might give
rein to his amorous fancies: for it is an assured fact that, to sad,
fanciful hearts, there is no greater joy than solitude, the awakener of
sad or happy memories. And thus going little by little, enjoying a
gentle breeze which blew against his face, full of most delicate
scents, which from the scented flowers wherewith the green earth
was heaped it gently stole, as it passed through them wrapped in
the delicate air, he heard a voice as of one who grievously
complained, and checking for a while his breath within him, so that
the sound might not hinder him from hearing what it was, he
perceived that from some thickset bramble bushes, a little way off,
the mournful voice proceeded, and though interrupted by endless
sighs, he understood that it uttered these sad words:
'Cowardly and craven arm, mortal enemy of that which you owe to
yourself, look, naught now remains on which to take vengeance,
save yourself! What does it profit you to prolong the life I hold in so
great abhorrence? If you think that our ill is of those that time is
wont to heal, you live deceived, for there is nothing more remote
from cure than our misfortune: seeing that she who might have
made mine pleasant, had a life so short that, in the green years of
her joyous youth, she offered it to the blood-thirsty knife, that it
might take it from her, through the treason of the wicked Carino. He
to-day, by losing his own, will have in part appeased that blessed
soul of Leonida, if, in the heavenly region where she dwells, she can
cherish desire for any vengeance. Ah, Carino, Carino! I beseech the
high Heavens, if by them just prayers are heard, not to heed the
plea, if any you offer, for the treachery you have done me, and to
suffer that your body may lack burial, even as your soul lacked
mercy. And you, fair and hapless Leonida, receive, in token of the
love I bore you in life, the tears I shed at your death; and put it not
down to lack of feeling that I do not end my life, with all I feel at
your death: for a grief that should end so soon would be a scant
return for what I ought and wish to feel. You will see, if you take
account of things here, how this wretched body will one day be
consumed by grief, little by little, for its greater grief and suffering:
even as powder, moist and kindled, which, without making a noise,
or raising a flame on high, is consumed in itself, without leaving of
itself aught save the traces of consumed ashes. It grieves me as
much as it can grieve me, oh soul of my soul, seeing that I could not
enjoy you in life, that in death I cannot perform for you the last rites
and honours which befitted your goodness and virtue; but I promise
to you, and swear, for the short time—and it will be very short—that
this impassioned soul of mine shall rule the heavy burden of this
wretched body, and my weary voice have breath to form it, not to
treat aught else in my sad and bitter songs save your praises and
deserts.'
At this point the voice ceased, from the sound of which Elicio clearly
perceived that it was the murderous shepherd; whereat he was
much rejoiced, because it seemed to him that he was in a position to
learn from him what he desired. And, wishing to approach more
closely, he needs must stop again, for it seemed to him that the
shepherd was tuning a rebeck, and he wished first to hear if he
should say anything to its sound. And he did not wait long before he
heard him, with gentle and tuneful voice, singing after this wise:

LISANDRO.
Blest soul, that from the veil
Of human life below
Free to the realms above didst, deathless, wing,
Leaving as in a jail
Of misery and woe
This life of mine which yet to thee did cling!
The bright light of the spring,
When thou art gone is dead,
And beaten to the ground
The hope I thought to found
On that firm seat where joy its radiance shed.
Alas! when thou wert gone,
My life died too: naught lived save grief alone.

Death claimed thee for his prey,

He revelled in his prize,
Thy loveliness beyond compare he marred;
He came to take away
The light of these mine eyes
Which gazed on thee and did their riches hoard.
Swiftly beneath his sword,
Like wax in summer's sun
Or cloud before the wind,
The fancies of my mind
Which sprang from glorious Love have been undone.
The stone above thy tomb
Shuts in my fortune and declares my doom.

How could thy brother speed

His cruel, ruthless hand
In hot revengeful purpose 'gainst thy heart?
How came the wicked deed
To tear thee from the land
And set thee from thy mortal veil apart?
Why sought he with his dart
Two lovers thus to sever?
Our love had had no end,
Our pathway would we wend
In holy wedlock hand in hand for ever.
Command why didst thou give,
Cruel, scornful hand! that dying I should live?

My hapless soul shall spend

 y ap ess sou s a spe d
The days, the months, the years,
In sad laments that ne'er shall reach their close.
'Midst joys that have no end
Thy soul shall know no fears
Of stubborn time—forgot for aye thy woes;
Secure in thy repose,
The bliss thou shalt behold
That thy good life hath won
Which ne'er shall be undone:
Him that so loved thee in remembrance hold,
If unto thee be given
To keep remembrance of the earth in Heaven.

Blest, lovely soul above!

How foolish have I been
To ask that thou shouldst mind thee of thy swain;
Who gave thee all his love.
Eternally, I ween,
Shall I, if thou art kind, thus feel my pain.
'Twere better for my gain
That I should be forgot,
That woe should waste away
The life that yet doth stay,
That I should perish 'neath my cruel lot,
Since in my bitter grief
Death's ill I count not ill, but sweet relief.

Amidst the holy choir,

Amongst the sainted dead,
Dear soul! enjoy the wealth of Heaven's delight,
That fears nor time nor fire;
The mercies that are shed
On all who flee not from the path of right.
I hope to reach that height,
To dwell with thee in bliss,
Amidst eternal spring,
If to thy steps I cling
And know no dread nor yet the pathway miss.
Oh lead me to this goal!
For such a deed as this befits thy soul.

And then, blest souls that dwell in Heaven, behold

The good that I desire,
Enlarge the wings of this my good desire
 Enlarge the wings of this my good desire.

Here ceased the voice, but not the sighs of the hapless swain who
had sung, and both served to increase in Elicio the desire to know
who he was. And bursting through the thorny brambles so as to
reach more quickly the spot whence the voice proceeded, he came
to a little meadow which, in the fashion of a theatre, was girt all
round with very dense and tangled shrubs; and there he saw a
shepherd who was standing in an attitude of great vigour, with his
right foot advanced and his left behind, his right arm raised in the
manner of one hoping to make a mighty throw. And such was the
truth, for at the noise which Elicio had made in bursting through the
bushes, he, thinking it was some wild beast (against which the
woodland shepherds were forced to defend themselves), had placed
himself in a position to hurl at him a weighty stone he was holding in
his hand. Elicio, perceiving his intent by his posture, before he could
accomplish it, said to him: 'Calm your bosom, hapless shepherd, for
he who comes hither, brings a bosom ready for all you might ask of
it; desire to learn your fortune has made him break in upon your
tears, and disturb the solace which might attend upon you in
solitude.'
With these gentle and courteous words of Elicio the shepherd was
calmed, and with no less gentleness replied to him, saying: 'I
gratefully acknowledge your kind offer, whoever you be, courteous
shepherd; but, as for fortune, if you desire to learn mine who never
had any, you will scarce be able to have your wish.' 'You speak true,'
answered Elicio, 'since from the words and plaints I this night have
heard from you, you clearly show the little or none that you have.
But you will no less satisfy my desire by telling me your troubles
than by making known to me your joys. May fortune give you these
in what you desire, so that you do not deny me what I beg of you, if
indeed your not knowing me do not prevent it; although I would
have you know, so as to reassure and move you, that I have not a
soul so happy as not to feel as much as it should the miseries you
would recount to me. This I tell you, for I know that nothing is more
wasted, nay thrown away, than for an unhappy man to recount his
woes to one whose heart is brimful with joys.' 'Your kindly words,'
answered the shepherd, 'compel me to satisfy you in what you ask
me, not only that you may not fancy that from a mean and craven
soul spring the complaints and lamentations you say you have heard
from me, but also that you may realise that the feeling I show is but
small as compared with the cause I have for showing it.'
Elicio thanked him heartily, and after some more courteous words
had passed between the two, Elicio giving proof that he was a true
friend of the woodland shepherd, the latter, recognising that they
were not feigned promises, granted in the end what Elicio asked.
The twain sate them down on the green grass, covered with the
splendour of the fair Diana, who could that night rival her brother in
brightness, and the woodland shepherd, with tokens of a tender
grief, began to speak in this wise:
'On the banks of the Betis, a stream exceeding rich in waters, which
enriches great Vandalia, was born Lisandro (for that is my luckless
name), and of parents so noble that I would to Almighty God I had
been begotten in a lowlier station; for ofttimes nobility of lineage
lends wings and strength to the soul to raise the eyes to where a
humble lot would never dare to raise them, and from such boldness
calamities are often wont to spring such as you shall hear from me,
if with attention you will listen to me. In my village was also born a
shepherdess, whose name was Leonida, the sum of all the beauty
which, as I fancy, could be found in a great part of the world,—born
of parents no less noble and wealthy than her beauty and virtue
deserved. Whence it came to pass that, the parents of both being
among the chief people of the place, and the rule and government
of the village being vested in them, envy, the deadly enemy of a
peaceful life, brought about strife and mortal discord between them
over some differences concerning the administration of the village, in
such a manner that the village was divided into two factions; the
one followed that of my parents, the other that of Leonida's, with so
deep-rooted a hatred and malice that no human effort has been able
to bring about peace between them. Fate then decreed, as though
to shut out every prospect of friendship, that I should fall in love
with the fair Leonida, daughter of Parmindro, the head of the
opposite faction; and my love was, indeed, so great that, though I
strove in countless ways to put it from my heart, they all ended in
my remaining yet more vanquished and enslaved. Before me rose a
mountain of difficulties, which hindered me from gaining the end of
my desire, such as Leonida's great worth, the inveterate enmity of
our parents, the few or no occasions which presented themselves to
me for disclosing my thoughts to her: and yet, whenever I turned
the eyes of fancy towards the rare beauty of Leonida, every difficulty
was made smooth, so that it seemed to me a little thing to break
through sharp points of adamant, that I might reach the goal of my
loving and honourable thoughts. Having then for many days battled
with myself, to see if I could turn my soul from a design so arduous,
and seeing that it was impossible, I set all my skill on considering
how I might give Leonida to understand the secret love in my
breast. And even as, in any matter, the beginnings are always
difficult, so in those that relate to love they are for the most
exceedingly difficult, until Love himself, when he wishes to show
himself favourable, opens the gates of the remedy, where they seem
most closely barred. Thus it appeared in my case, for my thought
being guided by his, I came to fancy that no better means presented
themselves to my desire than to make friends with the parents of
Silvia, a shepherdess who was a bosom friend of Leonida, and often
they visited each other at their houses, in company with their
parents. Silvia had a kinsman called Carino, a very close companion
of Crisalvo, fair Leonida's brother, whose boldness and harshness of
manner had gained him the nickname of cruel, and so, by all those
who knew him, he was generally called cruel Crisalvo; and in the
same way they called Carino, Silvia's kinsman and Crisalvo's
companion, the cunning Carino, from his being officious and sharp-
witted. With him and with Silvia (for it seemed to serve my purpose)
by means of many presents and gifts I forged a friendship, to
outward seeming: at least on Silvia's side it was stronger than I
desired, for the presents and favours, which with pure heart she
bestowed on me, constrained by my unceasing services, were by my
fortune taken as instruments to place me in the misery where now I
see myself. Silvia was passing fair, and adorned with graces so many
that the hardness of Crisalvo's savage heart was moved to love her
(but this I did not learn save to my hurt); and many days later, after
that from long experience I was sure of Silvia's good-will, an
opportunity offering itself one day, in the tenderest words I could, I
disclosed to her the wound in my stricken breast, telling her that,
though it was so deep and dangerous, I did not feel it so much, only
because I thought that in her solicitude lay its cure. I informed her,
too, of the honourable goal to which my thoughts were tending,
which was to unite myself in lawful wedlock with the beauteous
Leonida; and that, since it was a cause so just and good, she must
not disdain to take it under her care. Finally, not to weary you, love
furnished me with such words to say to her, that she, being
overcome by them and more by the pain which she, like a clever
woman, recognised from the signs of my face as dwelling in my soul,
determined to take charge of my cure, and to tell Leonida what I felt
for her, promising to do for me all that her power and skill might
achieve, even though such an undertaking was fraught with
difficulties for her, by reason of the great enmity she knew to exist
between our parents; though, on the other hand she thought that it
might put an end to their differences, if Leonida were to marry me.
Moved then by this good intention, and softened by the tears I shed,
as I have said before, she dared to intercede on behalf of my
happiness, and, discussing with herself how she would approach
Leonida, she made me write her a letter, which she offered to give
her at the moment she thought fitting. Her counsel seemed to be for
my good, and that same day I sent her a letter, which I have always
known by heart, as having been the beginning of the happiness I felt
at the reply to it, though it would be better not to remember happy
things at a time so sad as that in which I now find myself. Silvia
received the letter, and awaited the opportunity for placing it in
Leonida's hands.'
'Nay,' said Elicio, interrupting Lisandro's discourse, 'it is not right that
you should fail to repeat to me the letter you sent to Leonida, for,
seeing that it was the first, and that you were so deeply in love at
that time, it must undoubtedly be eloquent. And since you have told
me that you know it by heart, and of the pleasure you obtained from
it, do not now withhold it from me by not repeating it.'
'You say well, my friend,' replied Lisandro, 'for I was then as deeply
in love and timid as now I am unhappy and despairing; and, on that
account, it seems to me that I did not succeed in uttering any
eloquent words, though it was sufficient success that Leonida should
believe those which were in the letter. Since you wish so much to
hear them, it ran as follows:

LISANDRO TO LEONIDA.

"So long as I have been able (though with very great grief to
myself) to resist with my own strength the amorous flame which for
you, fair Leonida, consumes me, fearful of the exalted worth which I
recognise in you, I have never had the boldness to discover to you
the love I bear you; but now that the virtue, which up till now has
made me strong, is consumed, it has become necessary for me to
disclose the wound in my breast, and thus, by writing to you, to
make trial of the first and last remedy in your power. What the first
may be, you know, and to be the last is in your hand, from which I
hope for the pity that your beauty promises, and my honourable
desires merit. What they are, and the goal to which they tend, you
shall learn from Silvia, who will give you this: and since she has been
so bold, being who she is, as to bring it to you, know that they are
as honourable as is due to your merit".'
The words of this letter did not seem bad to Elicio, and Lisandro,
continuing the story of his love, said:
'Many days did not pass before this letter came into the fair hands of
Leonida, by means of the kindly hands of Silvia, my true friend. In
giving it, she told her such things that she largely assuaged the rage
and emotion which Leonida had felt at my letter, such as telling her
how good it would be if through our marriage the enmity of our
parents were to cease, and that an object so well meant should lead
her not to reject my desires; all the more as it should not be
compatible with her beauty to allow one who loved her as much as I
to die, without more consideration; adding to these other
reasonings, which Leonida recognised as just. But, so as not to show
herself vanquished in the first encounter, and won in the first
advance, she did not give to Silvia as pleasant a reply as she wished.
But still, at the intercession of Silvia, who forced her to it, she replied
with this letter which I shall now repeat to you:

LEONIDA TO LISANDRO.

"If I had thought, Lisandro, that your great daring had sprung from
my lack of modesty, I would have carried out on myself the
punishment that your fault deserves; but as what I know of myself
makes me sure on this point, I have come to the conclusion that
your great boldness has proceeded more from idle thoughts, than
from thoughts of love; and though they may be as you say, think not
that you can move me to cure them, as you did Silvia to believe
them. I complain more of her for having made me answer you, than
of you who dared to write to me, for silence had been fit answer to
your folly. If you draw back from your purpose, you will act wisely,
for I would have you know that I deem my honour of more account
than your empty thoughts."
This was Leonida's reply, which, together with the hopes that Silvia
gave me, though it seemed somewhat harsh, made me count myself
the happiest man on earth. Whilst these matters were passing
between us, Crisalvo did not neglect to woo Silvia with countless
messages, gifts and services; but so hard and severe was Crisalvo's
disposition that he could never move Silvia to grant him the smallest
favour. Whereat he was as desperate and impatient as a bull when
speared and vanquished. For the sake of his love he had formed a
friendship with the cunning Carino, Silvia's kinsman, though these
two had first been mortal enemies, for in a wrestling-bout, which on
a great feast-day the deftest swains of the place held before all the
village, Carino was vanquished by Crisalvo, and mauled: so that he
conceived in his heart undying hatred for Crisalvo, and no less was
the hatred he felt against another person, a brother of mine, for
having thwarted him in a love-affair, in which my brother carried off
the fruit Carino hoped for. This rancour and ill-will Carino kept secret
till time disclosed to him the opportunity when he might avenge
himself on both at once, in the cruellest way imaginable. I kept
friends with him, so that admission to Silvia's house might not be
denied me; Crisalvo adored him, so that he might further his designs
with Silvia; and his friendship was such that whenever Leonida came
to Silvia's house, Carino accompanied her: wherefore it seemed
good to Silvia to tell him, since he was my friend, of my love-affair
with Leonida, which was by this time prospering with such ardour
and good fortune, through Silvia's good offices, that we now awaited
but the time and place to cull the honourable fruit of our pure
desires. On hearing of this, Carino used me as an instrument to
commit the greatest treason in the world. For one day (feigning to
be true to Crisalvo, and giving him to understand that he rated his
friendship higher than his kinswoman's honour), he told him that the
chief reason why Silvia did not love or favour him, was that she was
in love with me; he knew it unmistakably, and our love-affair was
going on so openly that if he had not been blinded by his amorous
passion he would by now have perceived it from a thousand signs;
and the more to assure himself of the truth he was telling him, he
bade him look to it henceforward, for he would see clearly how Silvia
without any restraint granted me exceptional favours. At this news
Crisalvo must have been quite beside himself, as appeared from
what followed therefrom. Henceforward he employed spies to watch
my dealings with Silvia; and as on many occasions I sought to be
alone with her, in order to speak not of the love he thought, but of
things concerning mine, these were reported to Crisalvo, together
with other favours prompted by pure friendship, which Silvia showed
me at every step. Whereat Crisalvo came to so desperate a pass,
that many times he sought to kill me, though I did not think it was
for such a cause, but on account of the long-standing enmity of our
parents. But as he was Leonida's brother, I was more concerned to
guard myself than to harm him, thinking it certain that if I married
his sister our enmities would have an end. Of this he was quite
ignorant, thinking rather that, because I was his enemy, I had
sought to make love to Silvia, and not because I was really fond of
her; and this increased his anger and resentment to such a degree
that it robbed him of reason, though he had so little that little was
needed to destroy it. And this evil thought wrought so strongly in
him, that he came to loath Silvia as much as he had loved her,
merely because she favoured me, not with the good-will he thought,
but as Carino told him. And so, in whatever circle or assembly he
was, he spoke ill of Silvia, giving her dishonourable names and
epithets. But as all knew his ugly character and Silvia's goodness,
they lent little or no belief to his words. Meanwhile Silvia had
arranged with Leonida that we two should be married, and, in order
that it might be done with more safety to ourselves, that it would be
well for Leonida, one day when she came with Carino to her house,
not to return that night to that of her parents, but to go thence in
Carino's company to a village half a league distant from ours, where
some rich kinsmen of mine lived, in whose house we could with
greater peace effect our designs. For if Leonida's parents were not
pleased at the issue, it would at least be easier, when she was away
from them, to come to terms. This resolve having been taken,
Carino was informed of it, and, displaying the greatest spirit, offered
to Silvia to escort Leonida to the other village as she desired. The
services I did to Carino for the good-will he showed, the promises I
uttered to him, the embraces I gave him, would methinks have
sufficed to extinguish in a heart of steel any evil purpose it might
cherish against me. But that traitor of a Carino, casting behind him
my words, deeds and promises, without regarding what he owed
himself, planned the treason which now you shall hear. Having
informed himself of Leonida's wish, and seeing that it agreed with
what Silvia had told him, he planned that on the first night which
from the appearance of the day promised to be dark, Leonida's
departure should be effected, offering once more to maintain all
possible secrecy and loyalty. After making this agreement which you
have heard, he went off to Crisalvo, as I have since learnt, and told
him that his kinswoman Silvia had gone so far in her love-affair with
me, that I had determined on a certain night to steal her from her
parents' house, and take her to another village where my kinsmen
dwelt. There an opportunity offered itself to avenge his feelings on
both, on Silvia for the small account she had made of his services,
on me for our long-standing enmity, and for the injury I had done
him in robbing him of Silvia, since she was leaving him on my
account alone. Carino knew how to exaggerate to him, and to say
what he wanted, in such a way as, even with less effort, would have
moved to any evil purpose a heart not so cruel as his. The day being
now arrived which I thought was to be the day of my greatest bliss,
after having told Carino not what he actually did do, but what he
was to do, I went off to the other village to give orders how to
receive Leonida. And to leave her entrusted to Carino was like
leaving the innocent lamb in the power of the hungry wolves, or the
gentle dove in the claws of the fierce hawk, who tears it to pieces.
Ah, friend! when I come to this point with my imagination, I know
not how I have strength to sustain life, nor thought to think of it,
much more tongue to tell it! Ah, ill-advised Lisandro! How did you
not know Carino's duplicity? Yet, who would not have trusted his
words, since he risked so little in proving them true by deeds! Ah, ill-
starred Leonida! how little did I know how to enjoy the favour you
did me, in choosing me for your own! Finally, to end with the
tragedy of my misfortune, you must know, discreet shepherd, that
on the night Carino was to take Leonida with him to the village
where I was expecting her, he summoned another shepherd, called
Libeo, who ought to have considered him an enemy, though Carino
concealed it beneath his wonted false dissimulation, and asked him
to accompany him that night, for he was resolved to carry off a
shepherdess, his sweetheart, to the village I have told you, where
he purposed to marry her. Libeo, a man of spirit and a lover himself,
readily offered him his company. Leonida bade farewell to Silvia with
close embraces and loving tears, an omen, as it were, that it was to
be the last farewell. The hapless maid must needs have thought
then of the treason she was committing against her parents; not of
that Carino was planning against her,—and how bad a return she
was making for the good opinion that was held about her in the
village. But, passing over all these thoughts, constrained by the
loving thought that vanquished her, she entrusted herself to the care
of Carino, who was to conduct her to where I awaited her. How
often do I call to mind when I reach this point, what I dreamed the
day I would have counted fortunate, had the number of my days
ended thereon! I remember that, leaving the village a little while
before the sun withdrew his rays from our horizon, I sate me down
at the foot of a tall ash tree on the very road by which Leonida was
to come, waiting till night should close in a little more to further my
purpose and to receive her, and without knowing how or wishing it, I
fell asleep. Scarce had I yielded my eyes to slumber when,
methought, the tree against which I leaned, bending before the fury
of a fierce wind that was blowing, tearing its deep roots out of the
earth, fell upon my body, and attempting to get away from the
heavy weight, I rolled from side to side. While in this plight
methought I saw a white hind beside me, which I earnestly implored
to lift, as well as it could, the heavy burden from my shoulders, and
when moved with compassion, it was about to do it, at the same
moment a fierce lion sprang from the thicket, and seizing it in his
sharp claws, marched off with it through the forest. After I had
escaped with great toil from the heavy burden, I went to look for it
in the mountain, and found it torn and wounded in a thousand
places. Whereat I felt so much grief that my soul was wrung from
me merely by reason of the pity it had shown at my plight: and thus
I began to weep in my dreams, so that the tears themselves awoke
me, and finding my cheeks bathed with sorrow I was beside myself,
pondering on what I had dreamed; but in the joy I hoped to have in
seeing my Leonida, I failed to see then that fortune was showing me
in dreams what was to happen in a short time to me awake. At the
moment when I awoke night had just closed in with such darkness,
with such terrible thunder and lightning as furthered the
perpetration of the cruel deed which that night was perpetrated. As
Carino left Silvia's house with Leonida, he entrusted her to Libeo,
telling him to go with her by the road to the village I have
mentioned, and though Leonida was perturbed at seeing Libeo,
Carino assured her that Libeo was no less a friend of mine than he
was, and that in security she could go with him slowly whilst he
went forward to give me tidings of her approach. The guileless maid,
being after all in love, believed the words of the treacherous Carino,
and with less mistrust than was fitting, guided by the courteous
Libeo, advanced her timid steps, which were to be the last of her
life, thinking they led her to the height of her bliss. Carino went on
before the two, as I have already told you, and gave information of
what was happening to Crisalvo, who with four of his kinsmen was in
ambush on the very road by which they were to pass, this being
wholly shut in by forest on either side. He told them how Silvia was
coming and I was the only one with her, and that they should rejoice
at the good opportunity fate put in their hands to avenge the wrong
we two had done him, and that he should be the first to prove the
edge of his knife on Silvia, though she was a kinswoman of his.
Immediately the five cruel butchers prepared to stain themselves in
the innocent blood of the pair who came along the road all
unsuspicious of such treason; when they reached the place where
the ambush was, at once the traitorous murderers were on them,
and surrounded them. Crisalvo came up to Leonida, thinking she
was Silvia, and with insulting and excited words, in the hellish rage
which mastered him, left her stretched on the ground with six mortal
wounds, whilst Libeo weltered on the earth with countless stabs
dealt by the other four, who thought they were inflicting them on
me. When Carino saw how well his traitorous intent had turned out,
without awaiting words, he went away, and the five traitors, fully
satisfied as if they had done some notable exploit, returned to their
village. Crisalvo went to Silvia's house himself to give her parents the
news of what he had done, so as to increase their grief and pain,
telling them to go and bury their daughter Silvia, whose life he had
taken because she had set more store on the cold esteem of
Lisandro his enemy, than on the unremitting attentions shown by
him. Silvia, who heard what Crisalvo was saying,—her soul telling
her what had happened, told him that she was alive, and free too
from all that he had accused her of; and that he should be sure he
had not killed one whose death would grieve him more than the loss
of his own life. And with this she told him that his sister Leonida had
that night left her house in unwonted apparel. Crisalvo was amazed
to see Silvia alive, thinking for sure that he had left her dead, and
being suddenly seized with great fear, immediately hastened to his
house, and not finding his sister there, returned alone in the
greatest consternation and frenzy to see who it was he had killed,
since Silvia was alive. Whilst all this was going on, I was awaiting
Carino and Leonida with strange anxiety; and as it seemed to me
that by this time they were later than they should be, I wished to go
and meet them, or learn if by any accident they had been detained
that night. I had not gone far along the road when I heard a piteous
voice saying: "Oh sovereign Maker of Heaven, withhold the hand of
thy justice and open that of thy mercy in order to show mercy to this
soul, which soon shall give account to thee of the offences it has
committed against thee! Ah Lisandro, Lisandro! surely Carino's
friendship will yet cost you your life, since it cannot be that grief for
my having lost mine for your sake will put an end to it! Ah, cruel
brother, can it be that without hearing my excuses you desired to
inflict on me so soon the punishment of my error?" When I heard
these words, I at once recognised from the voice and from them
that it was Leonida who uttered them, and—an augury of my
misfortune—with feelings in a turmoil, I set to groping where
Leonida was weltering in her own blood; and, having at once
recognised her, I let myself fall on her wounded body, and with the
greatest grief possible, said to her: "What woe is this, my joy, my
soul? what cruel hand was it that did not respect so much beauty?"
At these words I was recognised by Leonida; and raising her weary
arms with much effort, she threw them round my neck, and,
pressing with all her strength, she joined her mouth to mine, and,
with weak and broken utterance, spoke but these words to me: "My
brother has killed me, Carino ... betrayed, Libeo is without life, and
may God give you yours, Lisandro mine, for long and happy years,
and may he grant that I enjoy in another life the peace denied me
here;" and, joining her mouth closer to mine, she pressed her lips
together to give me her first and last kiss; and, as she opened them,
her soul went from her, and she lay dead in my arms. When I
perceived it, I abandoned myself to grief over her body, and
remained senseless; and if, instead of being alive, I had been dead,
whoever saw us in that plight had called to mind the hapless plight
of Pyramus and Thisbe. But on coming to myself, I had opened my
mouth to fill the air with cries and sobs, when I perceived someone
coming with hurried steps to where I was; and, when he was near,
though the night was dark, the eyes of my soul gave me assurance
that he who came there was Crisalvo, as was the truth. He was
coming back to convince himself whether perchance it was his sister
Leonida he had killed. When I recognised him, before he could
guard himself against me, I came upon him like a raging lion; and,
giving him two blows, I brought him to the ground. Before he
ceased to breathe, I dragged him to where Leonida was, and,
placing in her dead hand the dagger her brother wore—the same
with which she had been killed—I guided it and plunged it thrice
through his heart. And mine being somewhat consoled by Crisalvo's
death, without further delay I took upon my shoulders Leonida's
body, and bore it to the village where my kinsmen lived. Telling them
what had happened, I asked them to give it honourable burial, and
immediately determined to take on Carino the same vengeance as
on Crisalvo; but, since he has kept away from our village, it has
been delayed until to-day, when I found him on the skirts of this
wood, after going about in search of him for six months. Now he has
come to the end his treason deserved; and none now is left on
whom to wreak vengeance, unless it be the life I endure so much
against my will. This, shepherd, is the cause whence proceed the
laments you have heard from me. If it seems to you sufficient to
cause yet a deeper grief, I leave to your good judgment to
determine!'
Therewith he ended his discourse, and set to weeping so copiously
that Elicio could not refrain from keeping him company therein; but
after they had for a long while eased with gentle sighs, the one the
pain he suffered, the other the compassion he felt thereat, Elicio
began to console Lisandro with the best arguments he knew, though
his misfortune was as far beyond consolation as he had seen from
its issue. Amongst other things he said to him, the one which gave
Lisandro most solace was to tell him that in misfortunes beyond
remedy, the best remedy was to hope for none; and, since one
might believe from Leonida's purity and noble disposition, according
to his account, that she was enjoying a life of bliss, he should rather
rejoice at the happiness she had gained, than grieve for that which
she had lost. Whereto Lisandro replied:
'I know full well, my friend, that your arguments have power to
make me believe they are true; but not that they have—nor will all
the arguments in the world have—power to give me any consolation.
With Leonida's death began my evil fortune, which will end when I
behold her again; and since this cannot be without I die, the man
who should help me to attain death will I count the greatest friend
of my life!'
Elicio did not wish to give him more sorrow with his words of solace,
since he did not regard them as such; only he asked him to come
with him to his hut, where he might stay as long as it pleased him,
offering him his friendship in all wherein he might be able to serve
him. Lisandro thanked him as heartily as possible; and though he
was unwilling to consent to go with Elicio, yet he had to do so,
constrained by his repeated asking. And so the two arose, and came
to Elicio's cabin, where they rested for the little that remained of the
night. Now when the white dawn was leaving the couch of her
jealous husband, and beginning to give signs of the coming day,
Erastro arose and began to put in order Elicio's herd and his own to
lead them to the accustomed pasture. Elicio invited Lisandro to come
with him; and so, when the three shepherds came with their gentle
flock of sheep through a ravine below, on ascending an incline, they
heard the sound of a gentle pipe, which was straightway recognized
by the two enamoured swains, Elicio and Erastro, for it was Galatea
who was playing it. And it was not long before some sheep began to
show themselves over the crest of the hill, and immediately behind
them Galatea, whose beauty was such that it were better to leave it
to speak for itself, since words fail to enhance it. She came dressed
like a girl of the mountains, with her long hair free to the wind,
whereof the sun himself appeared to be envious, for, smiting it with
his rays, he sought to rob it of lustre if he could; but that which
came from the glimmer of it seemed another new sun. Erastro was
beside himself looking at her, and Elicio could not keep his eyes from
gazing at her. When Galatea saw the flock of Elicio and Erastro join
hers, she showed that she did not wish that day to keep them
company, and called to the pet lamb of her flock, which the rest
followed, and directed it to another spot, different from that for
which the shepherds were making. Elicio, seeing what Galatea was
doing, and being unable to endure such open contempt, came to
where the shepherdess was and said to her:
'Permit your flock, fair Galatea, to come with ours, and, if you do not
like our company, choose that which will please you better, for your
sheep will not, through your absence, lack good pasturage, since I,
who was born to serve you, will take more care of them than of my
own. Do not seek to disdain me so openly, for the pure affection I
cherish towards you does not deserve it. According to the way you
were taking, you were making for the spring of slates, but, now you
have seen me, you wish to change your road; and, if this is as I
think, tell me where you wish, to-day and always, to graze your
herd, for I swear to you never to take mine there.'
'I assure you, Elicio,' replied Galatea, 'that it was not to shun your
company or that of Erastro that I have changed the way you think I
was taking, for my intention is to spend the noon-tide of to-day by
the stream of palms, in the company of my friend Florisa, who is
awaiting me there, for as early as yesterday we two agreed to graze
our flocks there to-day. As I came along, heedlessly playing my pipe,
the pet lamb took the road of slates, as more accustomed for it. For
the affection you bear me and the offers you make me I thank you,
and count it no small thing that I have justified myself against your
suspicion.'
'Ah, Galatea!' replied Elicio, 'how well you invent what seems good to
you, though you have so little need to use stratagem with me, for
after all I do not seek to wish more than you wish! Now, whether
you go to the stream of palms, to the wood of council, or to the
spring of slates, be assured that you cannot go alone, for my soul
accompanies you always; and, if you do not see it, it is because you
do not wish to see it, so that you may not be obliged to heal it.'
'Until now,' said Galatea, 'I have yet to see my first soul, and so I am
not to blame if I have healed none.'
'I do not know how you can say that, fair Galatea,' replied Elicio,
'since you see them to wound them, and not to heal them.'
'You accuse me falsely,' replied Galatea, 'in saying that I have
wounded anyone without arms, seeing that these are not granted to
women.'
'Ah, discreet Galatea,' said Elicio, 'how you jest at what you perceive
of my soul, which you have invisibly wounded, and with no other
arms than those of your beauty! I do not so much complain of the
wrong you have done me, as that you hold it in little account.'
'I would hold myself in less account, if I held it in more,' replied
Galatea.
At this moment Erastro came up, and, seeing that Galatea was going
off and leaving them, said to her:
'Where are you going, whom do you flee, fair Galatea? If you part
from us who adore you, who shall hope for your company? Ah fair
foe! how heedlessly you go your way, triumphing over our
affections! May Heaven destroy the warm affection I bear you, if I
do not long to see you in love with some one who may value your
plaints in the same degree as you value mine! Do you laugh at what
I say, Galatea? Then I weep at what you do.'
Galatea could not answer Erastro, for she was going away, guiding
her flock towards the stream of palms; and bowing her head from
afar in token of farewell, she left them. When she saw herself alone,
whilst she was making for the spot where her friend Florisa thought
she would be, with the exquisite voice Heaven had pleased to give
her, she went along singing this sonnet:

GALATEA.
 Away with noose and frost, with dart and fire,
Whereby to strangle, freeze, or wound or burn,
Love doth essay! 'Tis vain: my soul doth yearn
For no such knot, nor doth such flame desire.
Let each bind, freeze, kill, press, consume in ire,
'Gainst any other will its anger turn,
But mine shall snow or net or arrow spurn,
To hold me in its heat let none aspire.
My chaste intent will chill the burning flame,
The knot I shall break through by force or art,
My glowing zeal will melt away the snows,
The arrow shall fall blunted by my shame,
And thus nor noose nor fire, nor frost nor dart,
Shall make me fear, safe in secure repose.

With juster cause might beasts stand still, trees move and stones
unite on hearing Galatea's gentle song and sweet harmony than
when to Orpheus' lute, Apollo's lyre, or Amphion's music the walls of
Troy and Thebes of their own accord set themselves in the ground
without any craftsman laying hand thereon, and the sisters, dark
dwellers in deepest chaos, grew gentle at the exquisite voice of the
unheeding lover. Galatea finished her song, and at the moment
came to where Florisa was, by whom she was received with joyous
mien, as being her true friend, and she to whom Galatea was wont
to tell her thoughts. After the two had allowed their flocks to go at
their will to graze on the green grass, they determined, invited by
the clearness of the water of a stream flowing by, to wash their
beauteous faces; for, to enhance their beauty, they had no need of
the vain and irksome arts whereby those ladies in great cities who
think themselves most beautiful, torture theirs. They remained as
beautiful after washing as before, save that, through having rubbed
their faces with their hands, their cheeks remained aflame and
blushing-red, so that an indescribable beauty made them yet more
fair, and especially Galatea. In her were seen united the three
Graces whom the Greeks of old depicted naked to show (amongst
other purposes) that they were mistresses of beauty. Straightway
they began to gather divers flowers from the green meadow with
intent to make each a garland wherewith to bind up the disordered
tresses that flowed freely over their shoulders. In this task the two
beauteous shepherdesses were engaged when of a sudden they
saw, by the stream below, a shepherdess coming of gentle grace
and bearing, whereat they wondered not a little, for it seemed to
them that she was not a shepherdess of their village nor of the
others near by: wherefore they looked at her with more attention
and saw that she was coming gradually to where they were; and
though they were quite near, she came so absorbed and lost in
thought that she never saw them until they chose to show
themselves. From time to time she stopped, and raising her eyes to
Heaven, uttered sighs so piteous that they seemed to be torn from
her innermost soul; at the same time she wrung her white hands,
and tears like liquid pearls she let fall down her cheeks. From the
extremes of grief the shepherdess displayed Galatea and Florisa
perceived that her soul was filled with some inward grief, and to see
on what her feelings were set, both hid themselves amongst some
close-grown myrtles, and thence watched with curious gaze what
the shepherdess was doing. She came to the brink of the stream,
and with steadfast gaze stopped to watch the water running by; and
letting herself fall on its bank, as one wearied, she hollowed one of
her fair hands, and therein took up of the clear water, wherewith she
bathed her moist eyes, saying with voice low and enfeebled:
'Ah water clear and cool, how little avails your coldness to temper
the fire I feel in my soul! Vain will it be to hope from you—or indeed
from all the waters the mighty ocean holds—the remedy I need; for
if all were applied to the glowing passion that consumes me, you
would produce the same effect as do a few drops on the glowing
forge which but increase the flame the more. Ah, sad eyes, cause of
my ruin! to how lofty a height did I raise you for so great a fall! Ah
fortune, enemy of my repose! with what haste didst thou hurl me
from the pinnacle of my joy to the abyss of misery wherein I am! Ah
cruel sister! how came it that Artidoro's meek and loving presence
did not appease the anger of your breast devoid of love? What
words could he say to you that you should give him so harsh and
cruel a reply? It seems clear, sister, that you did not esteem him as
much as I; for, if it were so, you would in truth have shown as much
meekness as he obedience to you.'
All that the shepherdess said she mingled with such tears, that no
heart could listen to her and not be moved to compassion; and after
she had calmed her sorrowing breast for a while, to the sound of the
water gently flowing by, she sang with sweet and dainty voice this
gloss, adapting to her purpose an ancient verse:
Hope hath fled and will not stay
One thought only brings delight:
Time that passes swift of flight
Soon my life will take away.
Two things, all the world among,
Help the lover to attain
All that doth to Love belong:
E'en desire the good to gain,
Hope that makes the coward strong.
Both within my bosom lay.
No, 'twas in my stricken soul
That they lurked to take away
My desire to reach the goal.
Hope hath fled and will not stay.
Though desire should cease to be,
What time hope is on the wane,
Yet 'tis not the same in me.
My desire doth wax amain,
Though my hope away doth flee.
'Gainst the wounds my soul that blight
I can take nor care nor thought,
Martyr to my hapless plight,
In the school where Love hath taught,
One thought only brings delight.
Scarce the blessing from on high
Had unto my fancy come,
When, as gently they passed by,
Heaven, fate, and bitter doom,
With it from my soul did fly.
Whoso for my grievous plight
Fain would mourn, let him strike sail,
Into the haven of delight
Glide more gently 'fore the gale
Than Time that passes swift of flight.
Who that hath such woe as mine
Would not faint beneath his fate?
From such woes we may divine
Joy to be a featherweight,
Sorrow lead from deepest mine.
Though my fortune be not gay,
Though I falter to my knees,
 Yet this blessing is my stay:
He who robbed me of my peace
Soon my life will take away.
Soon the shepherdess ended her song, but not the tears which
made it more sad. Moved to compassion thereby, Galatea and Florisa
came out from where they lay concealed, and with loving and
courteous words greeted the sad shepherdess, saying to her among
other things:
'So may Heaven, fair shepherdess, show itself favourable to what
you would ask of it, and so may you obtain from it what you desire,
if you tell us (allowing that it be not displeasing to you), what
fortune or what destiny has brought you to this region, for, according
to the experience we have of it, we have never seen you on these
banks. Now that we have heard what you have just sung, gathering
from it that your heart has not the calm it needs, and by reason of
the tears you have shed, of which your lovely eyes gave witness, in
the name of fair courtesy we are bound to give you all the solace in
our power; and if your evil be of those that do not permit of
consolation you will at least perceive in us a good will to serve you.'
'I know not, fair maidens,' replied the strange shepherdess, 'how I
shall be able to repay you save by silence for the courteous offers
you make me, unless by saying no more about it, and being grateful
for it, and valuing them as much as they deserve it, and by not
withholding from you what you wish to learn from me, although it
would be better for me to pass by in silence the circumstances of my
misfortunes, than to tell them and give you cause to count me
immodest.'
'Your countenance and the gentle bearing that Heaven has given
you,' replied Galatea, 'do not betoken an intellect so coarse as to
make you do a thing in telling which afterwards you must needs lose
reputation; and since your appearance and words have in so short a
time made this impression on us, that we already count you discreet,
prove to us, by telling us your life, whether your misfortune comes
up to your discretion.'
'As far as I believe,' replied the shepherdess, 'both are on a level,
unless, indeed, fate has given me more judgment, the more to feel
the griefs that present themselves; but I am quite sure that my woes
exceed my discretion, in the same degree as all my craft is overcome
by them, since I have none wherewith to cure them. And that
experience may set you right, if you wish to hear me, fair maidens, I
will tell you, in as few words as possible, how, from the great
understanding you judge I possess, has sprung the woe which
surpasses it.'
'With nothing will you better satisfy our desires, discreet maiden,'
replied Florisa, 'than with telling us what we have asked you.'
'Let us retire, then,' said the shepherdess, 'from this spot, and seek
another, where, without being seen or disturbed, I may be able to
tell you what it grieves me to have promised you, for I foresee that
it will not cost more to lose the good opinion I have gained with you,
than to reveal my thoughts to you, however late, if perhaps yours
have not been touched by the affliction I am suffering.'
Desirous that the shepherdess should fulfil her promise, straightway
the three arose, and betook themselves to a secret and retired
place, known already to Galatea and Florisa, where, beneath the
pleasant shade of some leafy myrtles, without being seen by
anybody, all three could be seated. Forthwith, with exquisite grace
and charm, the strange shepherdess began to speak in this wise:
'On the banks of the famous Henares, which ever yields fresh and
pleasant tribute to your golden Tagus, most beauteous
shepherdesses, was I born and nurtured in a station not so lowly,
that I might count myself the meanest of the village. My parents are
labourers and accustomed to field-labour, in which occupation I
followed them, leading a flock of simple sheep over the common
pastures of our village. So well did I adapt my thoughts to the
condition in which my lot had placed me, that nothing gave me more
joy than to see my flock multiply and increase, and I had no other
thought save how to gain for them the richest and most fertile
pastures, the clearest and freshest waters I could find. I had not,
nor could I have, cares beyond those that might arise from the rustic
duties on which I was engaged. The woods were my companions, in
whose solitude, ofttimes invited by the sweet birds' gentle harmony,
I sent forth my voice in a thousand simple songs, without mingling
therein sighs or words that might give any token of a love-sick
breast. Ah! how often, merely to please myself and to allow the time
to pass away, did I wander from bank to bank, from vale to vale,
culling, here the white lily, there the purple iris, here the red rose,
there the fragrant pink, making from every kind of sweet-smelling
flowers a woven garland, wherewith I adorned and bound up my
hair; and then, viewing myself in the clear and peaceful waters of
some spring, I remained so joyous at having seen myself, that I
would not have changed my happiness for any other! And how often
did I make sport of some maidens, who, thinking to find in my
breast some manner of pity for the misery theirs felt, disclosed to
me, with abundance of tears and sighs, the love-secrets of their
soul! I remember now, fair shepherdesses, that one day there came
to me a girl friend of mine: throwing her arms round my neck, and
joining her face to mine, she said to me with streaming eyes: "Ah,
sister Teolinda!" (for this is the name of the hapless being before
you). "I truly believe the end of my days has come, since love has
not dealt with me as my desires deserved." Whereupon I, wondering
at her display of grief, thinking that some great misfortune had
befallen her, in the loss of her flock, or the death of her father or
brother, wiped her eyes with the sleeve of my smock, and asked her
to tell me what misfortune it was that caused her to lament so
much. She, continuing her tears, nor giving truce to her sighs, said
to me: "What greater misfortune, oh Teolinda, would you have
happen to me, than that the son of the chief man in our village,
whom I love more than the very eyes in my head, should have gone
away without saying a word to me; and that I have this morning
seen in possession of Leocadia, daughter of the head shepherd
Lisalco, a crimson belt which I had given to that false Eugenio,
whereby was confirmed the suspicion I had of the love-affair the
traitor was carrying on with her?" When I ceased hearing her
complaints, I swear to you, friends and ladies mine, that I could not
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