Cellular Immunology 408 (2025) 104909

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Cellular Immunology
journal homepage: www.elsevier.com/locate/ycimm

Enhanced apoptosis and inflammation allied with autophagic and apoptotic

Leishmania amastigotes in the seemingly undamaged ear skin of clinically
affected dogs with canine visceral Leishmaniasis
Barbara Laurice Araújo Verçosa a,b,c,* , Maria Imaculada Muniz-Junqueira b,
Ana Lys Bezerra Barradas Mineiro d , Maria Norma Melo a , Anilton Cesar Vasconcelos a
a
Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
b
Laboratório de Imunologia Celular, Faculdade de Medicina, Universidade de Brasília, Brasília, Brazil
c
Faculdade de Ciências da Saúde Pitágoras, Campus Codó, Codó, Maranhão, Brazil
d
Departamento de Clínica e Cirurgia Veterinária, Centro de Ciências Agrárias, Universidade Federal do Piauí, Teresina, Piauí, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Programmed cell death plays a relevant role in the pathogenesis of visceral Leishmaniasis. Apoptosis selects
Leishmania suitable parasites, regulating parasite density, whereas autophagy eliminates pathogens. This study aimed to
Apoptosis assess the inflammation and apoptosis in inflammatory cells and presents a unique description of the presence of
Autophagy
autophagic and apoptotic Leishmania amastigotes in naturally Leishmania-infected dogs. Fragments from seem­
Pyroptosis
NETosis
ingly undamaged ear skin of sixteen Leishmania-infected dogs and seven uninfected dogs were evaluated through
histomorphometry, ultrastructural, immunohistochemical and transmission electron microscopy (TEM) analyses.
Leishmania amastigotes were present on seemingly undamaged ear skin only in clinically affected dogs. Parasite
load, morphometrical parameters of inflammation and apoptotic index of inflammatory cells were higher in
clinically affected animals and were related to clinical manifestations. Apoptotic index and morphometric pa­
rameters of the inflammatory infiltrate in undamaged ear skin were positively correlated with parasite load.
Apoptotic and non-apoptotic Leishmania amastigotes were observed within neutrophils and macrophages.
Leishmania amastigotes were positive for Bax, a marker for apoptosis, by immunohistochemistry. Morphological
characteristics of apoptosis and autophagy in Leishmania amastigotes were observed only in phagocytes of
clinically affected dogs. Positive correlations were found between histomorphometry and clinical manifestations.
Our results showed that apoptosis and autophagy in Leishmania amastigotes may be related to both the increase
in parasite load and apoptotic index in inflammatory cells, and with the intensity of the inflammatory response in
clinically affected dogs. Thus, our study suggests that apoptotic and autophagy Leishmania within phagocytes
may have facilitate the survival of the parasite and it appears to play an important role in the process of
Leishmania infection.

1. Introduction its clinical characteristics, its degree of transmissibility, and its zoonotic
potential. The number of new regions endemic to CanL has grown
Leishmaniosis is still a global health problem, mainly affecting low- significantly in recent years [2]. The Canidae are considered the most
income populations worldwide, causing approximately one million in­ important reservoirs for zoonotic VL due to the presence of parasites in
fections and 20,000–30,000 deaths per year. Visceral leishmaniosis (VL) the skin, especially in the ears, which can infect the vector [3].
is a potentially fatal zoonosis, with an estimated incidence of Cell death is morphologically classified in six types, depending on
50,000–90,000 new cases per year in humans worldwide [1]. The World their morphophysiological and immunological characteristics: type 1)
Organization for Animal Health (OIE) recognizes the canine form of apoptosis (Programmed cell death- PCD); type 2) autophagy (PCD); type
visceral leishmaniosis (CanL) as an extremely important disease due to 3) necrosis (Accidental cell death - ACD); type 4) pyroptosis (PCD); type

* Corresponding author at: Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Patologia Geral, Av. Antônio Carlos, 6627,
Campus Pampulha, CEP: 31270-010, Belo Horizonte, Minas Gerais, Brazil.
E-mail addresses: [email protected], [email protected] (B.L.A. Verçosa).

https://doi.org/10.1016/j.cellimm.2024.104909
Received 16 September 2024; Received in revised form 30 November 2024; Accepted 6 December 2024
Available online 15 December 2024
0008-8749/© 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
B.L.A. Verçosa et al. Cellular Immunology 408 (2025) 104909

5) necroptosis (PCD); and type 6) NETosis (PCD) [4,5]. Additionally, response to limited resources in the sandfly gut [31–33]. The term
these types of cell death processes can interact each other through “selfish altruism” was introduced to qualify regulated cellular death in
interconnected or even overlapping signaling pathways, and the final protozoan parasites: an altruistic phenomenon implemented for the
product is the result of the interaction of different types of cell death, entire population that is clonal [34]. Therefore, PCD appears to be an
which is closely related to the development of diseases [6], inclusive in “altruistic” mechanism for selecting suitable parasites for disease
Leishmaniosis [7]. transmission and it appears also to regulate parasite density within the
Apoptosis is morphologically characterized by generating specific host, to avoid hyperparasitism that would kill the host and thus prevent
morphological changes, such as, alteration in mitochondrial function, parasite transmission [35]. Additionally, PCD appears to maintain the
rounding of the cell, reduction in cell volume (pyknosis), condensation clonality of the parasite population, removing unsuitable cells and thus
of chromatin, and nuclear fragmentation. Membrane blebbing culmi­ ensuring the propagation of the fittest and most virulent cells [36].
nates in the formation of apoptotic bodies that are phagocytosed by Autophagy is a fundamental biological process that eliminates un­
nearby cells [8]. In contrast, autophagy is restricted, parts of the cyto­ wanted cellular components and pathogens to maintain cellular ho­
plasm are sequestered within double-membrane vacuoles and digested meostasis, having a relationship functionally complex with apoptosis
by lysosomal hydrolases [9,10]. However, some authors point out the and inflammatory response [37–39]. There are three forms of auto­
morphological characters as insufficient to classify autophagy as death phagy: (1) autophagy mediated by chaperone, which requires interac­
pathway, since its greatest characteristic is to promote cell survival tion between thermal shock protein (HSP70, heat shock protein) and
through autophagic flux [11]. altered macromolecules in the cytoplasm, to trigger the autophagic
Apoptosis or programmed cell death (PCD) is a regulated mechanism process; (2) microautophagy, in which parts of the cytoplasm are
of silent cell elimination. This phenomenon is naturally controlled, directly encompassed by lysosomes and (3) macroautophagy or auto­
initiated, or inhibited by various physiological or pathological stimuli phagy, described above as a catabolic process in which a cell degrades
[12]. The apoptotic process is an intricate web of intracellular signaling and recycles its own contents and which consists of the most common
pathway that encompasses three phases: activation, execution and and best studied type [40,41].
cellular demolition that may be triggered by extrinsic or intrinsic The autophagy is a conserved process by which substrates in the
pathways [12]. This process represents a type of cellular self-destruction cytosol are transported to the lysosome via a double membrane-bound
dependent on energy, and synthesis, and degradation of proteins. As an intermediate vesicle called the autophagosome [10]. The selection of
active process, apoptosis requires ATP reserves, at least in the early autophagosomal content can occur in a relatively nonselective manner
stages, whereas necrosis occurs when ATP is depleted [13]. (known as “bulk autophagy”) or involve the tightly regulated elimina­
PCD may be triggered by the extrinsic or an intrinsic apoptotic tion of individual cellular components (known as “selective auto­
pathway. In the extrinsic pathway, which is triggered by death receptors phagy”), depending on the inducing factor [42].
stimulation, there are activation of caspase 8 that triggers the caspase Autophagy participates in the immune response to pathogen inva­
cascade and molecules of the Bcl-2 (B-cell lymphoma-2) family, result­ sion. Studies show that strategies that hinder the autophagic reaction
ing in cell apoptosis [14]; Extrinsic pathway is activated through Fas predispose cells to infectious processes caused by bacteria, protozoa,
receptor (CD95/Apo-1), and by inflammatory cytokines or immune cy­ viruses and fungi [43–45]. It is caused probably by the multiplicity of
tokines, such as TNF and IFN-γ [15]. In the mitochondrial intrinsic actions exerted by the autophagic machinery within specialized (i.e.,
pathway, the activation of caspases occurs through the release of cyto­ adaptive and innate immune cells) and parenchymal cells [43,44]. This
chrome c and other pro-apoptotic proteins, primarily components of the fact can be explained by three reasons: (1) autophagy mediates quin­
Bcl-2 family, and permeabilization of mitochondrial outer membrane tessential (and cell type-defining) functions in virtually all subtypes of
occurs, resulting in the release of molecules from mitochondria, such as immune cells, both at sites of hematopoiesis and in peripheral tissues
cytochrome c and activation of caspases [16]. The intrinsic pathway is [46,47]. Consequently, autophagy deficiency affects the generation,
activated by intracellular stress, as oxidative stress, and high calcium survival, maturation and effector properties of core cellular components
(Ca+) concentration in the cytoplasm. Caspase (Casp) 8 and 9 are among of innate and adaptive immunity [46–48]; (2) impaired autophagy re­
initiator caspases and play a role in initial signaling events of apoptosis, sponses impair the ability of infected cells to eliminate invading path­
whereas Casp 3 is an effector or executioner caspase and causes pro­ ogens (or components thereof) within the lysosome [43–45,48]. Finally,
teolytic cleavages [17,18]. The process is controlled by anti-apoptotic (3) cases of derailed autophagy exacerbate the organism’s response to
molecules (Bcl-2), and apoptotic ones (Bax) [19,20]. infection by altering the extinction of the inflammatory cascade, thus
Cellular apoptosis functions as a defense mechanism against viruses exacerbating the local and systemic harmful effects linked to infection
and other infectious agents, such as intracellular bacteria and some by invading pathogens [48].
protozoa [21]. Parasites interact with multiple regulatory mechanisms Autophagy is implicated in multiple aspects of processes that are
to make their host cells refractory to apoptosis during critical phases of relevant to the pathogenesis of infectious and inflammatory diseases,
infection, including cell invasion, stabilization, and pathogen growth such as, lymphocyte development, innate immune signaling and antigen
[22]. Studies revealed that Leishmania uses different survival strategies presentation [48]. Furthermore, autophagy processes are involved in
to inhibit macrophage apoptosis [23,24]. Leishmania, by preventing strategies to eliminate invasive pathogens through the production of
homooligomerization or upregulation of the Bcl-2 antiapoptotic mole­ reactive oxygen species, adjustment of vital nutrients and cations,
cule can impair host cell apoptosis proteins, such as Bax, which is destruction by proteolytic enzymes [49] and playing a crucial role in
required for mitochondrial permeabilization during apoptosis [25]. host-pathogen interaction, especially in intracellular pathogenesis
Evidence has demonstrated the occurrence of apoptosis in parasites [43,50].
within cells, which may represent a potential use as a tool for population The autophagy pathway is well described in Leishmania major [51],
control, which can also be generated by nutritional constraints [26,27]. Toxoplasma gondii [52], Dictyostelium discoideum [53,54], Plasmodium
Apoptosis of amastigotes was observed in hamsters during experimental falciparum [55–57], and Trypanosoma brucei [58]. Pathogen recognition-
infection with L. chagasi. Host cell apoptosis was only observed in the associated surface receptors (PRRs) such as toll-like receptors (TLRs) are
final stages of the infection, while the apoptosis process in parasites capable to associate, for example, with the beclin-1 protein and to
occurred in the early stages [28]. Some factors promote the apoptosis of induce the autophagic process. Different pro-inflammatory cytokines,
intracellular amastigotes, such as the high production of NO [29] and such as IFN-γ and TNF-α, more expressed during infection, also
hydrogen peroxide [30], both resulting from the activation of the host contribute to the activation of the autophagic pathway [59]. In macro­
cell. phages infected with L. amazonensis, autophagy induces parasite pro­
In Leishmania, PCD appears to regulate the parasite population in liferation. In vivo, the infection mechanism appears to be dependent on

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B.L.A. Verçosa et al. Cellular Immunology 408 (2025) 104909

the autophagic process, seeing an increase in LC3 expression in the autophagic pathway has also been described in metacyclogenesis, being
lesion area [60,61]. Apoptotic-like Leishmania may also uses host cell associated with the virulence of the pathogen [91,92]. However, the
autophagy machinery inhibiting T cell proliferation and favoring the apoptotic or autophagic parasite still was not shown in natural infection
overall parasite population survival [62]. in vivo. This study aimed to evaluate the apoptosis of the inflammatory
The crucial role of the autophagic pathway has been demonstrated in cells and of the Leishmania amastigotes and the inflammatory response,
apicomplexa parasites (e.g. Plasmodium sp., Toxoplasma gondii) and in and to show images of live and apoptotic and autophagic Leishmania
trypanosomatids, such as, protozoa of the genus Leishmania, T. brucei amastigotes in macrophages and neutrophils by transmission electron
and T. cruzi [63]. In pathogenic protozoa, the importance of autophagy microscopy, and to correlate these results with the parasite load and
in different processes, such as differentiation, pathogenicity and infec­ clinical features of stray dogs naturally infected by Leishmania.
tion, has been demonstrated. Trypanosoma cruzi, the protozoan that
causes Chagas disease, requires autophagy-mediated recruitment of ly­ 2. Material and methods
sosomes to the pathogen-containing vacuole to activate the necessary
virulence factors to establish infection [64]. The autophagic pathway is 2.1. Ethical issues, samples, and experimental design
a key component in the lysosomal dependent entry of Trypanosoma cruzi
into the host cell. Adult male and female stray dogs, of unknown ages, were randomly
In pyroptosis there are classical and non-classical pathways [65]. The examined for Leishmaniosis in an epidemiological study conducted by
most proteins are induced to be expressed by activation of the NF-κB the Zoonotic Disease Control Center of the city of Timon in the state of
pathway and they will form part of the inflammatory bodies in the Maranhão, Brazil. Efforts were made to avoid all unnecessary distress to
classical pathway [66]. Inflammatory bodies have a cytoplasmic pattern the dogs. All procedures concurred with the guidelines established by
recognition receptor (PRR), an adapter protein and pro-caspase-1 [67]. the local Institutional Animal Care and Use Committee, which reviewed
In the second moment, caspase-1 will cleave the N-terminal fragments of and approved this work (CETEA, Universidade Federal de Minas Gerais,
Gasdermin D (GSDMD) located in the plasma membrane, forming pores, logged under protocol number 198/2007, approved on March 27, 2008)
and consequently, leading to pyroptosis. Temporarily, the hydrolysis (Bill number 3964/97).
and activation of pro-IL-1β and pro-IL-18 will generate their pro- Sixteen positive (16/23, 69.56 %) and seven negative-tested dogs
inflammatory forms - IL-1β and IL-18, thus triggering an inflammatory were evaluated (7/23, 30.43 %). After clinical analysis, dogs were
process and immune responses [68–70]. In addition, pyroptosis can also divided into three groups: a) Eight clinically affected CanL-positive dogs
be carried out by the non-classical LPS pathway, in which Caspases- (8/23, 34.78 %), which presented clinical signs of CanL such as lym­
4,5,11 are activated to cleave GSDMD to induce pyroptosis [71–73]. phadenomegaly, conjunctivitis, onychogryphosis, hepatosplenomegaly,
The caspase family of proteins has long believed to be closely related fever, skin lesions in various body regions (nose, pinna, extremities, and
only to apoptosis. However, surprisingly, caspase-8 protein can directly nail), alopecia, ulcerations, dry flaky skin, seborrhea, and discolored
cleave GSDMD to induce pyroptosis [74]. Activation of caspase-9 nose; b) Eight subclinically infected CanL positive dogs (8/23, 34.78 %),
through cleavage and activation of caspase-3 is also involved in which showed no clinical manifestation of the disease but the laboratory
pyroptosis [75]. Furthermore, the caspase-6 mediates the cleavage of tests were positive for CanL; and c) Seven CanL-negative dogs (7/23,
GSDMC [76]. 30.43 %) that showed negative clinical and laboratory test results. All of
The DNA damage and chromatin condensation are some of the the clinically affected dogs in this study presented three or more clinical
similarities between pyroptosis and apoptosis [77]. Furthermore, in manifestations of the CanL. The dogs were grouped according to the
pyroptotic cells many bleb-like protrusions appear on the surface of the Verçosa et al. [3] classification. Clinical evaluation and sample collec­
cell membrane before its rupture [78]. Membrane blebbing also occurs tion for anatomopathological and sorological/parasitological analyses
during apoptosis, and caspase-3 is required for this process [78–81]. were performed concomitantly. Clinical, histopathological, and anato­
However, apoptosis is considered a physiological form of death, but mopathological analyses were performed in this study.
pyroptosis can cause inflammation, activated by extracellular or intra­
cellular stimulation [82], such as during infection by Leishmania [7]. 2.2. Serological and parasitological diagnosis for CanL
Unlike the explosive rupture associated with cell motility due to ne­
crosis, in pyroptosis, flattening of the cytoplasm is observed due to To confirm L. infantum infection, blood samples were taken to detect
leakage of the plasma membrane [83]. Caspase inhibition does not anti-Leishmania antibodies by means of an indirect fluorescent antibody
prevent cell death caused by death receptors, and this cell death has test (IFI to Leishmaniose Visceral Canina, Bio-Manguinhos, Fiocruz, Rio
certain necrotic characteristics [84]. de Janeiro, Brasil) and enzyme-linked immunosorbent assay (ELISA,
Necroptosis has similar morphological characteristics with necrosis Bio-Manguinhos, Fiocruz, Rio de Janeiro, Brasil). Needle aspiration and
but are regulated independently of the caspases [85]. Necrosome for­ culture for promastigotes in Novy-MacNeal-Nicolle (NNN) medium and
mation in necroptosis will occur under stimulation of TNF-α, which is Schneider’s liquid medium were also performed [93]. Polimerase Chain
transferred from the cytoplasm to the cell membrane or organelle Reaction (PCR) (NucleoSpin®Tissue, Macherey-Nagel, Durën, Ger­
membrane to form permeable pores, destroying the integrity of the many) was performed to detect parasites in the undamaged skin, using a
membrane and thus resulting in cell death. [86–88]. target sequence of the L. donovani complex.
NETosis is characterized by the release of neutrophil extracellular
traps (NETs), which consist of decondensed chromatin associated with 2.3. Processing post-mortem samples
modified histone with bactericidal proteins from granules. During
NETosis, as in apoptosis, coordinated changes occur in the nucleus and The dogs were tranquilized with 1 % acepromazine (0.25 mg/kg IM,
cytoplasm, although the nature of the changes is different [89] and does Acepran, Univet) and anesthetized with 2.5 % sodium thiopental (25
not require caspase activation [89]. Furthermore, in NETosis there is no mg/kg IV, Thiopentax). Skin fragments from the ear region were
DNA fragmentation, as observed by the TUNEL method, and there is no collected for histological analysis. After this procedure, the animals were
exposure of phosphatidylserine before the death of the neutrophil. The euthanized with an overdose of 7.5 % sodium thiopental (75 mg/kg) for
nuclear membrane fragments into vesicles during NETosis, allowing the further clinical examination. Macroscopic abnormalities were assessed,
nuclear content to mix with the content of the granules present in the and these changes were included as parameters used in the classification
cell’s cytoplasm. But the nuclear membrane remains intact, unlike ne­ of animals and the subsequent distribution into study groups.
crosis [90]. After sedation with sodium thiopental, bone marrow aspirates were
In several species of the genus Leishmania, the participation of the collected for myelograms, stained with Giemsa, cultured in an NNN

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B.L.A. Verçosa et al. Cellular Immunology 408 (2025) 104909

culture medium enriched with Schneider’s medium, and examined different antibodies diluted in PBS in a humid atmosphere for at least 12
under an optical microscope (Olympus CX 23). Growth of the promas­ h. Immune expression of Caspase (Casp) 3 (Lyophilized mouse mono­
tigote forms of Leishmania was observed on days 5, 8, 10, and 15. All clonal antibody CPP32, Novocastra) and Bax (Polyclonal Rabbit anti-
infected dogs (clinically affected and subclinical infected) showed pos­ human Bax, DAKO) antigens were assessed. Monoclonal antibodies
itive results in PCR tests and in at least one of the two parasitological were used in 1:100 dilution and a LSAB® System - HRP (Biotinylated
tests (smear stained with Giemsa and culture) in at least one of the Link Streptavidin – HRP, DAKO) was revealed with a 3.3′- dia­
different organs (bone marrow, spleen, liver, skin, and lymph nodes). minobenzidine (DAB) solution (0.024 %) in PBS. The slides for negative
The animals were then euthanized with an overdose of sodium thio­ controls were incubated only with PBS. Counterstaining was performed
pental. After, to confirm the existence or not of infection, all Leishmania- using Harry’s hematoxylin (Sigma Chemical, USA).
infected dogs were euthanized by the Zoonotic Disease Control Center in
Teresina, PI, Brazil, according to Brazilian law (Decree n◦ 51.838 of the 2.6. Polymerase Chain Reaction (PCR)
Ministry of Health) and submitted to necropsy for sample collection. The
uninfected control animals were stray dogs from the same area, which Fragments from undamaged ear skin samples, which tested negative
were captured and euthanized for rabies control. for bacterial and fungal infection, were submitted to DNA extraction.
Prior to fixation with methyl alcohol, samples of seemingly undam­ The “Genomic DNA from tissue” kit (NucleoSpin®Tissue, Macherey-
aged ear skin were collected, and impression smears of the cut surface Nagel, Durën, Germany) was used, following the manufacturer’s in­
were taken on clean slides to directly view the parasite [64]. After this structions. The PCR was performed with a GoTaq® Green Master Mix kit
procedure, undamaged ear skin impression smears were stained with (Promega Corporation, cat. M7122. Madison, WI. USA). It was used 1 μL
Giemsa, for Leishmania amastigotes. Skin samples were collected and of the DNA sample and 1 μL of each prime from the specific Donovani
fixed in 10 % formaldehyde buffered with 0.01 M phosphate, pH 7.4, for complex DNA sequence described by Piarroux et al. [96]. The DNA
analysis under a conventional light microscope and immunohisto­ samples from dog skin naturally infected with Leishmania with an
chemistry. Skin fragments were processed for histological and molecular accentuated cutaneous parasitism, as well as a L. chagasi DNA, MHOM/
analyses. Tissue (seemingly undamaged ear skin) sections (5-μm thick) BR/BH46 strain, which had been previously tested and contained 1 ng/
were stained with Hematoxylin and eosin (HE), Trichomes of Shorr (for mL, were used as PCR-generated positive controls. The DNA from un­
conjunctive tissue), Giemsa Lemnert (for Leishmania amastigotes and infected dog skin was used as a negative control, along with a control
mast cells), and immunoperoxidase (for apoptotic protein - Bax and with no DNA, in which all the reaction components were present, except
Caspase 3 - and Leishmania amastigotes), and were analyzed under light the DNA. The DNA amplification was performed according to protocols
microscopy (Olympus CX23). Immunohistochemical staining was per­ described by Laskay et al. [97], with some alterations. The PCR-
formed by the Goodpasture method (Gram staining) and by the Grocott amplified products were analyzed through electrophoresis in non-
method (fungi staining) to discard the possibility of contamination with denaturing 5 % polyacrylamide gel in TBE buffer (89 mM Tris-Borato,
bacterial or fungal infection. 2 mM EDTA pH 8.0). Gels were transferred to a fixed solution and
Another fragment was fixed with 5 % glutaraldehyde in phosphate digitally photographed for further analysis.
buffer 0.05 M, pH 7.3, for 24 h. The fixative was then renewed after 12 h
of fixation and kept at 4 ◦ C. At the time of inclusion, 1–2 mm thick skin 2.7. Description of morphological patterns of histopathological lesions in
fragments were cut from the surface, dehydrated in an increasing series seemingly undamaged ear skin
of alcohols, and included in a plastic resin composed primarily of glycol
methacrylate (Historesine, Leica) [94]. Histological sections of 3 and 5 The composition, intensity, and distribution of the inflammatory
μm of thickness were stained with HE. response in seemingly undamaged ear skin were evaluated from sections
stained with HE. The inflammatory response was the presence of three
2.4. Immunohistochemistry to detect Leishmania amastigotes in or more inflammatory cells localized in the interstitial space. The
undamaged ear skin tissues seemingly undamaged ear skin slides were analyzed under an optical
microscope and the histopathological lesions were classified according
The Immunohistochemical technique was first used to define amas­ to the morphological pattern into: (a) discrete and focal: with a small,
tigote forms of Leishmania in material embedded in paraffin [95]. The isolated foci of inflammatory cells, (b) moderate and multifocal: with
slices were incubated with an anti-Leishmania chagasi polyclonal dog coalescent foci and (c) severe and diffuse: with large diffuse areas, as
antibody in a 1:100 dilution. The reaction was optimized with a LSAB® described by Solano-Gallego et al. [98].
System - HRP (Biotinylated Link Streptavidin – HRP, cat. K0675, DAKO
corporation, Carpinteria, USA), revealed with a 3.3′- diaminobenzidine 2.8. Morphometric analysis
(DAB) solution in 0.024 % PBS (Sigma Chemical, USA), and stained with
Harris hematoxylin (Sigma Chemical, USA). Fragments of dog skin The morphometric analysis was done in eight clinically affected,
infected with L. chagasi were used as positive and negative controls, and eight subclinical infected, and seven uninfected dogs in a blind assay.
were incubated with primary antibodies and only PBS, respectively. Images were digitalized in an image analyzer Kontron KS300 2.0
(extreme values of area, perimeter, and diameter of the inflammatory
2.5. Detection of Caspase 3 and Bax in undamaged ear skin fragments by lesion) and Media Cybernetics Image-Pro Plus 4.5 (cellularity, apoptotic
immunohistochemistry index in the inflammatory infiltrate, and parasite load). Cellularity of
the inflammatory infiltrate referring to the number of inflammatory
Histological slides of skin samples embedded in paraffin were cells per field. Twenty fields per animal were analyzed by morphometry,
deparaffinized in xylene and rehydrated in alcohol. After this procedure, showing a representative value according to Moro et al. [99], with
the slides were incubated with a solution containing 0.03 % hydrogen modifications. The criterion used in the selection of the samples was the
peroxide in methanol for 30 min in the dark in order to block the activity presence of inflammatory infiltrate, which, through morphometric
of endogenous peroxidase. Antigenic recovery was performed using 1.2 analysis, presented a group of three or more inflammatory cells. The cell
mg/mL Tris-HCl (pH 1.0) in a microwave oven (Sanyo, Brazil), in count of the inflammatory region was done in an area of 356,207 μm2
consecutive cycles of 10 and 5 min. After washing with 0.01 M phos­ per field, analyzed with an objective lens 10×, averaging out a total skin
phate buffered saline, pH 7.2 (PBS), the sections were treated with Lock area of 7,124,140 μm2.
Kit (Vector Laboratories, Inc., Burlingame, USA) and a protein block The quantification of the parasite load was performed on slides
(Dako Corporation). The slides were then incubated at 4 ◦ C with stained by immunoperoxidase under a light microscope. The 20 fields

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B.L.A. Verçosa et al. Cellular Immunology 408 (2025) 104909

were randomly selected, adding a total skin area of 468,752 μm2, from 3. Results
which 23,437.6 μm2 was by field.
The apoptotic index was assessed by Trichomes of Shorr, which 3.1. Leishmaniosis diagnostic and clinical analysis
shows the best characteristics of apoptotic cells. A single observer
(BLAV) performed the apoptotic cell quantification. The simultaneous In this study, the most frequent clinical manifestations of Leishmania-
presence of at least three peculiar morphological features of the process infected dogs were weight loss (7/8, 87.5 %), skin ulcerations in body
was considered: shrunken anoikic cells (cell retraction and loss of regions, except ear (7/8, 87.5 %), conjunctivitis (3/8, 37.5 %), ony­
adherence between cells and basement membrane); cytoplasmatic and chogryphosis (4/8, 50.0 %), and apathy (4/8, 50.0 %). Summary of the
nuclear condensation (compression of nuclear chromatin in homoge­ diagnostic test by clinical groups is shown in Supplementary Table 1.
nous dense masses, aligned on the internal side of the nuclear mem­
brane, sometimes forming decreasing images); nuclear fragmentation 3.2. Morphological patterns and histomorphometrical analysis of
(convolution and fragmentation of the nuclear membrane, without inflammatory response
karyorrhexis or rupture); cell fragmentation (with the formation of
apoptotic bodies). The apoptotic index (AI) was determined by the Histopathological sections of undamaged ear skin of all groups
following formula: showed inflammatory infiltrates. Inflammation was observed in all of
the evaluated dogs and was directly related to clinical signs in Leish­
AI = (Ʃ#of apoptotic cells/Ʃ total#of cells) × 100
mania-infected dogs (p < 0.05, Fisher test, Supplementary Table 2). In
the clinically affected group, multifocal inflammatory infiltrate was
2.9. Detection of the apoptosis by terminal deoxynucleotidyl transferase found around the hair follicles and blood vessels, while it was focal in
(TdT)-mediated dUTP nick end labeling (TUNEL method) the subepidermal region, it was slightly diffuse in the dermis, consisting
of macrophages, lymphocytes, plasmocytes, and polymorphonuclear
Apoptotic cells were detected using a specific in situ cell death neutrophils. In subclinically infected dogs, the inflammatory infiltrate
detection kit (“Apoptag® Peroxidase in situ, Apoptosis Detection Kit”, was multifocal, perifollicular, periglandular, perivascular, and, in rare
Chemicon, Temecula, CA, USA), and the assay was performed following case, subepidermal, consisting of macrophages, lymphocytes, and plas­
the protocols provided by the manufacturer. The development was then mocytes. The uninfected dogs presented focal, perifollicular, peri­
performed with DAB+ (Substrate Chromogen System, DAKO Corpora­ vascular, and subepidermal inflammatory infiltrate, consisting of
tion, Carpinteria, USA), and stained with Methyl Green, for 3 min, at macrophages, lymphocytes, and rare plasmocytes (Fig. 1A–F).
room temperature. A negative control was performed, omitting TdT in The number of inflammatory foci, mean area, perimeter, extreme
the reaction [100]. Positive control contained in the kit was used. diameters, and cellularity of the inflammatory infiltrate in undamaged
ear skin of Leishmania-infected and uninfected dogs are shown in
2.10. Transmission Electronic Microscopy Fig. 2A–F.

Ear skin fragments were collected and processed for Transmission 3.3. Morphometric analysis
Electronic Microscopy (TEM). Initially, thick sections (1 μm) were taken
to determine the area to be sectioned for TEM analysis and examined Leishmania parasites were observed in all CanL-positive dogs by
under optical microscope. Semi-thin sections have better resolution by parasitological tests (smear stained with Giemsa and culture) in different
optical microscopy, because it favours better visualization of the organs (bone marrow, spleen, liver, skin, and lymph nodes) and the
morphology of Leishmania and its host cell [94]. The sections were made parasite load in dermis of undamaged ear skin was related to clinical
in an ultramicrotome using glass knives, placed on slides under water, manifestations (p < 0.05, Fisher test). Associations among the parasite
dried on a hot plate, and stained in HE. load, inflammation, and positive PCR results in undamaged ear skin
TEM analyses of seeming undamaged ear skin fragments of dogs fragments in all groups are shown in Supplementary Table 2. None of the
clinically affected with a high parasite load were fixed in 5 % glutaral­ animals in the subclinical and uninfected groups presented amastigote
dehyde in 0.1 M cacodylate buffer, pH 7.2, and processed following forms of Leishmania in the undamaged skin. Nonetheless, only in clini­
standard protocols, including post-fixation in osmium tetroxide, fol­ cally affected CanL-positive group, Leishmania was observed in dermis of
lowed by block counterstaining with uranyl acetate and embedding in undamaged ear skin. In the clinically affected dogs, the parasite load was
Epon resin [101]. Ultrathin sections were counterstaining with lead higher than that observed in the subclinical infected and uninfected
citrate and analyzed in the FEI Tecnai G2–12 Spirit BioTwin (120 kV) animals (*p < 0,05, ANOVA and Tukey). In clinically affected dogs,
Transmission Electron Microscope. TEM was used to evaluate pro­ amastigote forms inside the macrophages (Fig. 3 A) and neutrophils
grammed cells death in inflammatory infiltrate, and better characterize (Fig. 3 B) were detected by an anti-Leishmania antibody, in dermis of
apoptosis and autophagy of Leishmania amastigotes. undamaged ear skin by immunohistochemistry. Neutrophil presents
segmented nuclei, a morphological characteristic that goes before
2.11. Statistical analysis NETosis. In Fig. 4, it is shown a preparation of Leishmania amastigotes in
thick sections (1 μm) stained with HE. Inflammatory cells infected with
The sample size was calculated according to the method used by Dell Leishmania amastigotes presents condensed chromatin and ruptured
et al. [102]. The power was 90 % (0.9) and α = 0.05; the standard de­ cytoplasmic membranes, both characteristics suggestive of pyroptosis.
viation of the variables was estimated as 20 % (0.2), and the magnitude The apoptotic index was performed in cells of the inflammatory
of the difference (d) was estimated as 40 % (0.4). The minimum number infiltrate in all of the analyzed groups. The apoptotic index of inflam­
of animals per group was found to be seven. matory cells was higher in the clinically affected animals than that in
Data were submitted to the Lilliefors test for a Gaussian distribution subclinical infected and uninfected dogs (Fig. 5 A). Inflammatory cells in
and to a Cochran-Bartlett test to evaluate the homoscedasticity. Data dermis of undamaged ear skin showed morphological features of
with Gaussian distribution were analyzed by ANOVA and Tukey multi­ apoptosis and suggestive of pyroptosis, such as cytoplasmic and nuclear
ple comparison. Non-parametric data were analyzed by Kruskal- Wallis condensation, cell retraction, and anoikis in Leishmania-infected dogs
and Dunn tests. The Fisher test was used to test the association of vari­ (Fig. 5 B).
ables. To verify correlation between variables, the Pearson test was Apoptosis was confirmed by in situ fragmentation of genomic DNA
used. All statistical analyses were performed using the GraphPad Prism 5 by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick
program and were considered significant up to p < 0.05. end labeling, TUNEL reaction, within inflammatory foci in dermis of

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B.L.A. Verçosa et al. Cellular Immunology 408 (2025) 104909

Fig. 1. Morphological patterns of inflammatory response in the undamaged ear skin of a Leishmania-infected dog. In A, diffuse inflammatory infiltrate in the
subepidermal region and around the hair follicles and blood vessels (*) in a clinically affected dog. In B, multifocal medium perifollicular, periglandular and per­
ivascular inflammatory infiltrate (*) in a subclinical infected dog. In C, focal and moderate inflammatory infiltrate consisting of macrophages, lymphocytes, plas­
mocytes, and polymorphonuclear neutrophils (*) in a clinically affected dog. Shorr Trichome. In D, discrete inflammatory focus around the hair follicles and blood
vessels, composed by mononuclear cells (macrophages and lymphocytes) (*) in a subclinical infected dog. Shorr Trichome. In E, multifocal and moderate inflam­
matory infiltrate consisting of macrophages, lymphocytes, plasmocytes, and polymorphonuclear neutrophils (*) in a clinically affected dog. HE. In F, Focal, peri­
follicular and moderate inflammatory infiltrate mainly consisting of polymorphonuclear neutrophils (yellow arrow) and macrophages (blue arrow) in a clinically
affected dog with high parasite load. Bar = 64 μm (A and B), 32 μm (C, E and F) and 16 μm (D). f = hair follicles; b = blood vessels; ep = epidermis; g = glands. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

undamaged ear skin. Positive cells showed brownish intranuclear uninfected dogs, as shown in Supplementary Table 3.
staining. Apoptosis was confirmed by the observation of inflammatory
cells with similar morphological features, presenting brownish intra­
nuclear clusters, assessed by the TUNEL method in inflammatory infil­ 3.4. Transmission Electronic Microscopy (TEM)
trate foci (Fig. 5 C).
Inflammatory cells in dermis of undamaged ear skin showed positive Ultrastructurally, in clinically affected dogs, apoptotic inflammatory
immunostaining by Casp 3 and Bax in Leishmania-infected dogs. In­ cells in dermis of undamaged ear skin were shrunk, with condensed
flammatory cells (macrophage and neutrophils) showed intense cyto­ nuclear chromatin and cytoplasm. Condensed nuclei were frequently
plasmic expression of Caspase 3 and Bax (Fig. 5 D and E, respectively). fragmented. Leishmania amastigotes were observed within inflammatory
Peculiar morphological features of apoptosis (anoikis, chromatin cells (neutrophils and macrophages). Representative images of cells in
condensation, nuclear fragmentation, and nuclear retraction) were dermis presenting shrunken anoikic cells, cytoplasmic and nuclear
associated with a strong expression of Bax in mononuclear cells and condensation, and nuclear convolution and fragmentation in the un­
neutrophils (Fig. 5 F). In addition to the inflammatory cells, it was damaged ear skin slides of Leishmania-infected dogs by TEM are shown
observed that, in most cases, the apoptotic Leishmania were also labeled in Fig. 7. Fig. 7 shows ultrastructural morphological pattern suggestive
for Bax (Fig. 6). of pyroptosis (chromatin condensation, segmented nuclei, ruptured
Apoptotic index of inflammatory cells and morphometric parameters cytoplasmic membrane, and nuclear retraction) in inflammatory cells
of the inflammatory infiltrate in dermis of undamaged ear skin were (neutrophil). In addition, Leishmania amastigotes also show ultrastruc­
correlated with parasite load in the ear skin of Leishmania-infected and tural morphological pattern of apoptosis. In clinically affected dogs with
a high parasite load, both apoptotic and non-apoptotic Leishmania

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B.L.A. Verçosa et al. Cellular Immunology 408 (2025) 104909

Fig. 2. The morphometric parameters of inflammatory infiltrate in undamaged ear skin fragments of CanL-positive dogs. Samples were obtained from eight clinically
affected, eight subclinical infected, and seven healthy uninfected dogs (controls). In A, the number of inflammatory foci in clinically affected dogs was higher than in
the other groups and subclinical infected dogs was higher than in uninfected groups. The average area (B), perimeter (C) and major diameter (D) of the inflammatory
infiltrate in clinically affected dogs was higher than that of the uninfected group. The minor diameter (E) and cellularity (F) of the inflammatory infiltrate in clinically
affected dogs were higher than in the other groups. *ANOVA and Tukey test p < 0.05.

amastigotes were observed within the same non-apoptotic macrophage peripheral chromatin in apoptotic Leishmania amastigote. Small blebs
and neutrophil. Apoptosis and autophagy in Leishmania amastigotes were detected attached to the cell membrane. In detail, see the cyto­
were observed only in clinically affected dogs with a high parasite load. plasmic membrane intact. All these alterations are morphological
Representative images of ultrastructural morphological pattern of characteristic of apoptosis.
autophagy and apoptosis of Leishmania amastigotes in undamaged ear
skin of a clinically affected dog with a high parasite load are shown in 4. Discussion
Fig. 8 A and B, respectively. Fig. 8 A shows ultrastructural morpholog­
ical pattern of autophagic Leishmania amastigote presenting appearance To the best of our knowledge, this is the first report of the occurrence
of electron-lucent areas in the cytoplasm. Intense cytoplasm vacuoliza­ of autophagic and apoptotic amastigotes in natural infection in vivo in
tion and the presence of structures resembling autophagosomes con­ Leishmania-infected dogs. This study assessed cellular death (PCD),
taining membrane profiles in Leishmania amastigotes, which are main autophagy and inflammation in undamaged ear skin of clinically
morphological characteristic of autophagy processes. The cytoplasm affected and subclinical naturally Leishmania-infected dogs and showed
seeming to be degraded, presenting areas of cytoplasm extraction is apoptotic amastigote forms of Leishmania infantum inside macrophages
shown in Fig. 8 A. In detail, see the presence of nuclear vacuolization. In and neutrophils with and without morphological characteristics of
Fig. 8 B, observe the cytoplasmatic and nuclear condensation and apoptosis. Our results demonstrate that: (a) Inflammation was observed

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B.L.A. Verçosa et al. Cellular Immunology 408 (2025) 104909

Fig. 3. Parasite in undamaged ear skin fragments in CanL. In A and B, focal and intense inflammatory infiltrate consisting of macrophages and polymorphonuclear
neutrophils. Leishmania amastigotes are shown, stained in brown, inside the macrophage (A) and inside the neutrophil (B) by imunoperoxidase in a clinically affected
dog with high parasite load. In detail, please, note that most of Leishmania amastigotes are in cytoplasm of macrophages and neutrophil (black arrows). In addition,
observe that in this neutrophil the nuclei appears segmented, a phenomenon that mainly occur in PMN before NETosis. (red arrow). Imunoperoxidase. Bars = 125
μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

in all the evaluated dogs and was directly related to clinical signs in
Leishmania-infected dogs. (b) The parasite load and apoptotic index of
inflammatory cells was higher in clinically affected dogs than the other
groups and was related to clinical manifestations. (c) Macrophage and
neutrophils showed intense cytoplasmic expression of caspase 3 and
Bax. (d) Apoptotic Leishmania were labeled for Bax. (e) Apoptotic index
of inflammatory cells and morphometric parameters of the inflamma­
tory infiltrate in dermis of undamaged ear skin were positively corre­
lated with parasite load. (f) Inflammatory cells (neutrophil) showed
ultrastructural morphological pattern of apoptosis (chromatin conden­
sation, segmented nuclei, ruptured cytoplasmic membrane, chromatin
at the periphery of the nucleus, and nuclear retraction) by TEM; in
addition, morphological characteristics of pyroptosis and NETosis were
also observed in inflammatory cells, and (g) Leishmania amastigotes
showed ultrastructural morphological pattern of apoptosis and auto­
phagy and were observed only in clinically affected dogs.
Our data showed ultrastructural morphological patterns of apoptosis
and autophagy in Leishmania amastigotes observed in infected dogs with
a high parasite load and enhanced apoptotic index in inflammatory cells
Fig. 4. Thick sections (1 μm) of undamaged ear skin fragments of clinically in the undamaged ear skin of clinically affected dogs. Only one in vitro
affected dog with CanL. Perifollicular and perivascular inflammatory response study conducted using TEM showed the presence of promastigote forms
(*) consisting of mononuclear cells (M) associated with intense parasite load. of Leishmania major with intact structures, which was viable inside PMN
Leishmania amastigotes are shown inside the macrophage in apoptosis (white cells and, the phagocytosis by macrophages of apoptotic neutrophils
arrow). However, only by the morphological aspect of the cell death, it is not [103]. Furthermore, it has been demonstrated that phagocytosis of
possible to exclude that the mechanism of the death was by pyroptosis, so that it apoptotic Leishmania induces the secretion, by macrophages, of anti-
is better to consider the possibility of apoptosis/pyroptosis. Semi-thin sections
inflammatory molecules, such as IL-10 and TGF-β, or lipids, such as
have better resolution when it is assessed by optical microscopy. HE. Bar =
lipoxin A4. In addition, it induces inhibition of the secretion of pro-
30 μm.

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B.L.A. Verçosa et al. Cellular Immunology 408 (2025) 104909

Fig. 5. The graph in A shows that the apoptotic index in undamaged ear skin fragments was higher in clinically affected dogs than in subclinical infected and
uninfected groups. *ANOVA and Tukey test p < 0,05. Samples were obtained from eight clinically affected, eight subclinical infected, and seven healthy uninfected
dogs (controls). Apoptotic inflammatory cells were quantified in HE stained slides, considering the simultaneous presence of at least three cells with peculiar
morphological features of apoptosis. It should be noted that for cells evaluated only by its morphological aspect, it cannot be excluded that some of these cells might
have presented the mechanisms of pyroptosis for cell death. In B, inflammatory cells, macrophage, show morphological features of apoptosis, such as cytoplasmic and
nuclear condensation, cell retraction, and anoikis (In detail, green arrow) in a Leishmania-infected dog. Shorr trichome. Please note in C that cells in apoptosis in
inflammatory infiltrate foci characterized by morphological features (cytoplasmic and nuclear condensation, cell retraction, and anoikis) present also brownish
intranuclear clusters (In detail, black arrow), the apoptosis marker by staining with the TUNEL method for in situ fragmentation of genomic DNA. In D, inflammatory
cells show positive immunostaining for Caspase 3 in a Leishmania-infected dog. Macrophage (red arrow) and neutrophil (black arrow) show intense cytoplasmic
expression of caspase 3. Imunoperoxidase. In E, the apoptotic macrophage shows a positive immunostaining for Bax (In detail, black arrow) in a Leishmania-infected
dog. Imunoperoxidase. In F, apoptotic inflammatory cells (macrophages and neutrophils) show intense cytoplasmic expression of Bax. Morphological features of
apoptosis (anoikis, chromatin condensation, nucleolar fragmentation, and nuclear retraction) are associated with a strong expression of Bax in mononuclear cells (In
detail, black arrow) and neutrophils (In detail, blue arrow). Imunoperoxidase. Bars = 10 μm (D); 12 μm (C); 16 μm (B, F) 32 μm; and (C and E). f = hair follicles; b =
blood vessel. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

inflammatory cytokines, such as TNF-α, and lipids, such as leukotriene parasite, resulting in the progression of infection and disease evolution.
B4 [104,105]. In case of to eliminate apoptotic parasites from a virulent PCD was detected in inflammatory foci in all studied groups. How­
population, Leishmania do not survive within phagocytic cells in vitro ever, the apoptotic index associated with the expression of the pro-
and lose their ability to induce disease in vivo [104,105]. Phagocytosis apoptotic molecules in inflammatory cells, Bax and Casp 3, was
of apoptotic neutrophils with Leishmania also appears to decrease the enhanced only in clinically affected dogs, suggesting that the increase of
presentation of parasitic antigens by macrophages [106]. In addition, molecules that induce apoptosis in epithelial tissue may play a role in the
apoptotic cells activate the autophagic machinery of host cells, in a severity of the disease through the increase in the parasite load. HSP65
process known as LC3-associated phagocytosis, promoting an anti- expression in L. major-infected macrophages causes a dysfunction in the
inflammatory response with the production of IL-10 and TGF-β, hin­ control of the apoptosis mechanism [107]. Furthermore, the survival of
dering the production of pro-inflammatory cytokines, such as TNF-α, IL- Leishmania inside the cell’s host depends on the decrease of caspase-3
1β and IL-6, reducing T cell proliferation and thus favoring parasite expression, supposedly because the parasite interferes with the trans­
survival [62]. Thus, apoptotic Leishmania altruistically allow the intra­ formation of the pro-caspase 3 enzyme into its active form [104]. Aga
cellular survival of the fittest parasites. So that, it is possible that et al. [108] showed that the infection of neutrophils with L. major pro­
apoptotic Leishmania within phagocytes facilitates the survival of the mastigotes decreased caspase-3 activity and thus inhibited the

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B.L.A. Verçosa et al. Cellular Immunology 408 (2025) 104909

Fig. 6. Apoptotic Leishmania show intense cytoplasmic expression of Bax by immunohistochemistry. Please note in detail that a strong cytoplasmatic expression of
Bax was observed in a Leishmania amastigote (black arrow) that is not inside any cell. Imunoperoxidase. Bar = 125 μm.

favor Leishmania penetration [109]. Late apoptosis of cells of the defense

system could be beneficial to the host, by eliminating excessive number
of cells and thus avoiding the harmful effects of the exacerbated in­
flammatory response in the tissue they would cause, such as the dele­
terious effect of reactive oxygen species or the enhanced production of
pro-inflammatory cytokines triggered by the Leishmania. On the other
hand, the PCD of inflammatory cells could also be beneficial to the
parasite by limiting the host’s potential to mount a protective immune
response.
Leishmania displays different strategies to survive, including
apoptosis inhibition, to down-regulate host cell defense mechanisms,
which results in perpetuation of the infection. Vázquez-López et al.
[110] described the mechanisms of inhibition of apoptosis of amasti­
gotes of Leishmania mexicana in human monocyte-derived dendritic
cells. In this model, L. mexicana inhibited the mitogen-activated protein
kinase (MAPK), the Jun N-terminal kinase (JNK), and the p38 phos­
phorylation, which are pro-apoptotic mechanisms, while they activated
the phosphatidylinositol 3-kinase (PI3K)/Akt, which are antiapoptotic
mechanisms. It was also showed the participation of the MAPK in the
inhibition of apoptosis of the cell host by Leishmania in bone marrow
macrophages stimulated with interferon-gamma (IFN-y) and infected
Fig. 7. Ultrastructural morphological pattern of apoptosis/pyroptosis in in­
with Leishmania donovani promastigotes, in which, the inhibition of p38,
flammatory cells. On the left, please note in the neutrophil the ultrastructural
morphological pattern suggesting pyroptosis: degradation of the nucleus, JNK, and ERK ensured the survival of the parasite [111]. Additionally, it
condensation (*), ruptured cytoplasmic membranes and cytoplasmic pyroptotic was also observed that p38 inhibition was associated with an increase in
body (white arrows). The neutrophil is characterized by multilobed nucleus (N) the number of infected macrophages and the survival of Leishmania
and presence of azurophilic granules and specific granules (g) [Note that [112]. In addition to Leishmania’s ability for negatively regulate pro-
azurophilic granules (ga) are larger and more electron-dense when compared to apoptotic signaling pathways, such as MAPK pathway, it has been
specific granules (ge)], granular endoplasmic reticulum (rer), free ribosomes (r) demonstrated that the parasite can activate signaling pathways involved
and mitochondria (mi) in the cytoplasm. On the right, presence of a non- in cell survival. Sarkar et al. [113] demonstrated a delay in the apoptosis
apoptotic macrophage characterized by loose chromatin and presence of of neutrophils infected with L. major caused by the activation of the
electron-dense clumps (black arrows) in the nucleus (M). Ultrastructural
MAPK ERK 1/2, the activation of the anti-apoptotic proteins Bcl-2 and
morphological aspect of Leishmania amastigotes (L), which present normal ul­
Bfl-1, the inhibition of the mitochondrial release of cytochrome c, the
trastructure of organelles, nucleus (n), kinetoplast (k) and flagellum (f). Slide
from the undamaged ear skin of a clinically affected dog with a high parasite
inhibition of caspase-6 and by reducing the expression of Fas.
load. Bar = 1 μm. mf = myofilaments. l = Leishmania spp. Transmission Elec­ Our results shows that the apoptotic inflammatory cells and Leish­
tronic Microscopy. mania amastigotes were labeled by Bax, which is a pro-apoptotic protein
[114] and may induce apoptosis by four ways: (a) Bax (pro-apoptotic)
spontaneous apoptosis of these cells. It was also observed decreased and Bcl-2 (anti-apoptotic proteins) play a role in the formation of het­
expression of Casp-3 in infected inflammatory cells (macrophages and eromers that are complexes formed by different proteins. When the
neutrophils), which may have facilitated the increase in the parasite expression of Bax is higher than that of Bcl-2, the heteromers can pro­
load [108]. mote apoptosis; (b) Overexpression of Bax can lead to DNA damage and
The main modulators in the balance of apoptosis mechanisms in thus cause apoptosis [115]; (c) Overexpression of Bax can affect the
infected cells are: (1) the time of apoptosis (early or late occurrence); (2) change of the voltage-dependent anion channel (VDAC) environment,
the type of cell and (3) the type of parasitism (intracellular or not). Early which leads to the change of mitochondrial membrane permeability
apoptosis of host cells could contribute to the fight against infection by resulting in cell apoptosis [116]; (d) Overexpression of Bax can promote
intracellular parasites, as Leishmania. But early apoptosis could also the release of cytochrome C [114], which is conducive to the formation
of an apoptosis activation complex between cytochrome C and caspase-

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B.L.A. Verçosa et al. Cellular Immunology 408 (2025) 104909

Fig. 8. Ultrastructural morphological pattern of autophagy and apoptosis in Leishmania amastigotes. Ultrastructural morphological pattern of autophagy (A) and
apoptosis (B) in Leishmania amastigotes in undamaged ear skin of a clinically affected dog with a high parasite load. Please, note in A the characteristic aspect of
autophagy, which is a conserved process by which substrates in the cytosol are transported to the lysosome via a double membrane-bound intermediate vesicle called
the autophagosome. In A, observe the cytoplasm vacuolization (*) and the presence of structures resembling autophagosomes (p) and autolissosomes. The cytoplasm
seems to be degraded, presenting areas of cytoplasm extraction (+). Leishmania amastigotes present appearance of electron-lucent areas in the cytoplasm (*). In
detail, see the cytoplasmic and nuclear condensation and the presence of nuclear vacuolization (n) in the Leishmania. In B, observe the Leishmania with characteristics
of apoptosis: the cytoplasmic and nuclear condensation and peripheral chromatin in Leishmania nuclei (n). Small bleb is observed attached to the cell membrane of
Leishmania (in detail, blue arrow). Please, note that the cytoplasmic membrane of the Leishmania is intact (in detail, black arrow). Bars = 1 μm (A) and 2 μm (B). L =
Leishmania; N = Cell nuclei; n = Leishmania nuclei; K = kinetoplast and f = flagella. Transmission Electronic Microscopy. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

9, thereby causing cell apoptosis [117]. Evaluation by PCR and Western depends on the level of GSDME expression. When GSDME expression is
blot confirmed proper integration and expression of mLLO-Bax-Smac high, it results in pyroptosis, when expression is low, it converts to
sequence into the 18srRNA locus of L. infantum. By flow cytometry it apoptosis [125]. Thus, GSDME could be a possible biomarker and
was shown accelerated apoptosis of transgenic Leishmania-infected therapeutic target to regulate pro- and anti-inflammatory responses
macrophages compared to the wild-type parasite. In addition, transgenic related to Leishmaniasis, since its expression regulates the change be­
parasites were less virulent, and a fewer parasitic burden was found in tween pyroptosis (pro-inflammatory) and apoptosis (anti-
the spleen and liver of transgenic-infected mice compared to the control inflammatory).
group [118]. Additionally, the Bec-1 protein, classified as a BH3-only Our ultrastructural results showed cytoplasm vacuolization and the
protein, interacts with Bcl-2. This interaction inhibits Bec1-dependent presence of structures resembling autophagosomes containing mem­
autophagy but does not inactivate the antiapoptotic effect of Bcl-2. brane profiles in Leishmania amastigotes only in clinically affected dogs
Under normal conditions, Bec-1 is inhibited and bound by Bcl-2, how­ (Fig. 8A). In Leishmania, autophagy is critical for maintaining cellular
ever, for autophagy to occur, dissociation between Bec-1 and Bcl-2 must homeostasis, recycling damaged organelles, glycosome renewal through
occur [119]. Finally, in Bec1-deficient cells, the expression of Bec-1 the Atg5 protein [126] and protein turnover [127]. Although the exact
mutants that do not bind to Bcl-2, in addition to inducing more auto­ autophagic mechanism has not been deciphered yet, numerous studies
phagy, also promote cell death, unlike the expression of wild-type Bec-1. point to a crucial role of autophagy and the protein it produces in the
Thus, Bcl-2, in addition to acting as an antiapoptotic protein, also differentiation and infectivity of parasites [91,128]. Due to its signifi­
functions as an antiautophagic protein, where it has an active role in cant influence on cell fate, autophagy still dominates many cellular
maintaining autophagy compatible with cell survival [120]. processes and signaling networks [128]. Due to the complexity of the
It could be observed in Fig. 7 that the nucleus of the neutrophil ap­ autophagic and immunological signaling mechanism in parasitic dis­
pears segmented and the cytoplasmic membrane was ruptured, that are eases associated with the growing number of studies related to the topic,
morphological characteristics of pyroptosis. Different types of cell death it is suggested that the Leishmania autophagy process can be a successful
can interact each other, since signaling pathways are interconnected or therapeutic approach for the treatment and, subsequently, cure for
even overlapping, and the result of this relationship is closely related to Leishmaniasis [91,127,129].
the development of diseases, including Leishmaniasis. [5,6,121]. Gas­ In this work, the clinically affected dogs presented higher morpho­
dermin E (GSDME) is an important and critical point of intersection metric parameters of the inflammatory infiltrate associated with PCD
between the processes of apoptosis and pyroptosis, acting in the con­ rates and parasite loads. This could result in high levels of MIP-1β
version between these two types of cell death [122]. Jiang et al. [123] secreted by infected apoptotic PMNs, the cytokine responsible for
showed that there is a type of pyroptosis caused by the connection or recruiting macrophages to the area of infection, lead to contact and
overlapping expression of GSDME and caspase-3. Caspase-3 specifically further multiplication of the parasite in the host cell [103]. Furthermore,
cleaves GSDME at the linker domain to generate N-terminal fragments of high levels of TGF-β and PGE2 may influence apoptosis, the inflamma­
GSDME, forming pores and thus perforating plasma membranes to tory response, and the parasite load, as observed in L. amazonensis cul­
induce pyroptosis, rather than apoptosis [124]. However, this fact tures. Just after apoptotic PMNs have been added in cultures, an

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B.L.A. Verçosa et al. Cellular Immunology 408 (2025) 104909

increase in the levels of TGE-β and PGE2 occurred, which further ex­ Declaration of competing interest
acerbates the parasite replication process [130].
Leishmania amastigotes were observed, in this study, in neutrophils The authors declare the following financial interests/personal re­
and macrophages, with and without apoptosis morphology, and only in lationships which may be considered as potential competing interests:
clinically affected dogs, which showed high parasite loads, associated Anilton Cesar Vasconcelos reports financial support and equipment,
with a high apoptotic index. Afonso et al. [130] observed an increase in drugs, or supplies were provided by Minas Gerais State Foundation of
infected cells and parasite load after apoptotic PMNs were added to a Support to the Research (FAPEMIG). All authors read and approved the
human culture of macrophages infected with L. amazonensis. In addition, manuscript.
parasite load increased after the clearance of apoptotic PMN by mac­
rophages, as Ribeiro-Gomes et al. [131] observed in mice infected with Data availability
L. major. Furthermore, many studies have confirmed the possibility of
PMNs actively to participate in the process of infection by Leishmania No data was used for the research described in the article.
[132].
Leishmania spp. induces an inflammatory response in the skin, of Acknowledgments
variable distribution and intensity [3]. The skin of clinically affected
dogs presented amastigote forms and inflammatory infiltrate containing The study was supported by the Fundação de Amparo a Pesquisa do
macrophages, lymphocytes, plasmacytes, and neutrophils, while the Estado de Minas Gerais - FAPEMIG, Coordenação de Aperfeiçoamento
subclinically infected animals presented a profile similar to the unin­ de Pessoal de Nível Superior-CAPES (Scholarship to BLAV), and Con­
fected controls, as observed in the present study. Accordingly, as related selho Nacional de Desenvolvimento Científico e Tecnológico - CNPq
by Xavier et al. [133] and Giuguetti [134], no granulomatous lesions (Fellowships to MNM, AVC and MIM-J). We would like to acknowledge
were found in any animal, as it was also described by Solano-Galleno the Laboratory of Histopathology at the Department of General Pa­
et al. [98] and Dos-Santos et al. [135]. thology and Laboratory of Apoptosis of Universidade Federal de Minas
For the parasites to survive in the early stages of infection, the host Gerais for their histopathologic preparations. We would also like to
cells should not be properly activated. Through phagocytosis of thank the employees of the Centro de Controle de Zoonoses (CCZ),
apoptotic inflammatory cells containing parasites, the amastigotes do Timon, Maranhao, Brazil, for supporting us with the collection of
not contact the phagocyte receptors; thus, phagocytic cells are not samples.
effectively activated, facilitating the infection and suppressing the
effective immune response to Leishmania. Our results indicate that Appendix A. Supplementary data
apoptosis is related to the increase in parasite load and to the intensity of
the inflammatory response. Association between these histo­ Supplementary data to this article can be found online at https://doi.
morphometry evaluations (inflammation, parasite load, and apoptosis) org/10.1016/j.cellimm.2024.104909.
and clinical manifestations was also observed. Our results show that the
apoptotic inflammatory cells and Leishmania amastigotes were labeled References
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