Molecular Immunology 156 (2023) 61–76

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Molecular Immunology
journal homepage: www.elsevier.com/locate/molimm

MCP-1/IL-12 ratio expressions correlated with adventitial collagen

depositions in renal vessels and IL-4/IFN-γ expression correlated with
interstitial collagen depositions in the kidneys of dogs with
canine leishmaniasis
Barbara Laurice Araújo Verçosa a, b, *, Maria Imaculada Muniz-Junqueira b,
Daniel Menezes-Souza a, Ricardo Toshio Fujiwara a, Luciano de F. Borges c, 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
Instituto de Ciências Biológicas, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil

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

Keywords: Collagen deposition is a common event in chronic inflammation, and canine Leishmaniosis (CanL) is generally
Canine leishmaniosis associated with a long and chronic evolution. Considering that the kidney shows fibrinogenic changes during
Inflammation CanL, and the balance of cytokines/chemokines regulates the profibrinogenic and antifibrinogenic immune re­
Fibrosis
sponses differently, it can be hypothesized that the balance of cytokines/chemokines can be differentially
Kidney
Dog
expressed in the renal tissue in order to determine the expression of collagen depositions in the kidneys. This
Cytokines study aimed to measure collagen deposition and to evaluate cytokine/chemokine expressions in the kidney by
Chemokines means of qRT-PCR in sixteen Leishmania-infected dogs and six uninfected controls. Kidney fragments were
stained with hematoxylin & eosin (H&E), Masson’s Trichrome, Picrosirius Red, and Gomori’s reticulin. Inter­
tubular and adventitial collagen depositions were evaluated by the morphometric approach. Cytokine RNA
expressions were measured by means of qRT-PCR to identify molecules involved in chronic collagen depositions
in kidneys with CanL. Collagen depositions were related to the presence of clinical signs, and more intense
intertubular collagen depositions occurred in infected dogs. Adventitial collagen deposition, as morphometrically
measured by the average area of the collagen, was more intense in clinically affected dogs than in subclinically
infected dogs. TNF-α/TGF-β, MCP1/IL-12, CCL5/IL-12, IL-4/IFN-γ, and IL-12/TGF-β expressions were associated
with clinical manifestations in dogs with CanL. The IL-4/IFN-α ratio was more commonly expressed and upre­
gulated in clinically affected dogs, and downregulated in subclinically infected dogs. Furthermore, MCP-1/IL-12
and CCL5/IL-12 were more commonly expressed in subclinically infected dogs. Strong positive correlations were
detected between morphometric values of interstitial collagen depositions and MCP-1/IL-12, IL-12, and IL-4
mRNA expression levels in the renal tissues. Adventitial collagen deposition was correlated with TGF-β, IL-4/
IFN-γ, and TNF-α/TGF-β. In conclusion, our results showed the association of MCP-1/IL-12 and CCL5/IL-12 ratios
with an absence of clinical signs, as well as an IL-4/IFN-α ratio with adventitial and intertubular collagen de­
positions in dogs with visceral leishmaniosis.

1. Introdution Leishmania genus. The annual report of global VL indicates that there are
50,000–90,000 new cases each year worldwide (WHO, 2023). The
Visceral leishmaniasis (VL) or kala-azar is a neglected tropical dis­ canine form of VL (CanL) usually involves the systemic and conspicuous
ease caused by an intracellular protozoan parasite that belongs to the deposition of collagen in the liver, spleen, lymph nodes, lungs, and

* Correspondence to: Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Morfologia, Av. Antônio Carlos, 6627, Campus
Pampulha, CEP: 31270-010 Belo Horizonte, Minas Gerais, Brazil.
E-mail address: brbaravet@yahoo.com.br (B.L.A. Verçosa).

https://doi.org/10.1016/j.molimm.2023.02.010
Received 2 November 2022; Received in revised form 4 February 2023; Accepted 13 February 2023
0161-5890/© 2023 Elsevier Ltd. All rights reserved.
B.L.A. Verçosa et al.
kidneys (Silva et al., 2013; Melo et al., 2008), which may induce local
fibrosis.
Fibrosis is induced by the formation and accumulation of the extra­
cellular matrix (ECM) in tissues, and is essential for wound healing as a
secondary response to any type of tissue injury, providing a scaffold for
tissue regeneration after lesions. Fibrosis is stimulated by several in­
juries, including infectious lesions and inflammatory response, in the
local tissue, which may induce molecular pathways, initiating and
driving the fibrosis process (Djudjaj and Boor, 2019). Following the
initial injury, various tissues and organs, such as the kidney, undergo a
dynamic process of wound healing, resulting in either a controlled tissue
repair and regeneration, or in uncontrolled tissue or organ damage
(Schaaf, 2000). Fibrosis is triggered and orchestrated by the crosstalk of
several cell types. In the kidney, there are epithelial, endothelial, and
inflammatory cells that trigger and sustain fibrosis and mesenchymal
cells, such as fibroblasts, pericytes, and myofibroblasts, which execute
fibrosis (Djudjaj and Boor, 2019). When the tissue repair programs are
activated, fibroblastic cells produce a highly cross-linked extracellular
matrix, which is stiff and shows molecular features that stimulate
myofibroblast activation. Myofibroblasts are contractile cells that stiffen
the extracellular matrix, secrete and activate pro-fibrotic growth factors,
and produce an extracellular matrix that stimulates further myofibro­
blast activation (Walraven and Hinz, 2018). Fibrosis may affect all
kidney compartments, causing glomerulosclerosis, tubulointerstitial
fibrosis, and arteriosclerosis. Furthermore, in each compartment, the
basement membrane can suffer fibrotic thickening (Djudjaj and Boor,
2019).
Glomerulosclerosis and tubulointerstitial fibrosis involve inflamma­
tion, proliferation, apoptosis, and collagen deposition (Zeisberg et al.,
2000a; El Nahas, 2003). Glomerular and tubulointerstitial fibrosis start
due to the infiltration of monocytes and macrophages and progress with
an excessive extracellular matrix accumulation (Benigni and Remuzzi,
2001; Eddy, 2000). Tubulointerstitial cells are then replaced by myofi­
broblasts, producing scarring and fibrosis, and causing impairment of
the renal function. Pericytes may reside subjacent to the endothelium of
large vessels (Andreeva et al., 1998) and may participate as a precursor
for myofibroblasts. Vascular injury triggers pericyte migration and dif­
ferentiation into myofibroblasts. Attracted inflammatory cells in the
renal interstitium initiate interactions with interstitial fibroblasts.
Activation and proliferation of fibroblasts and myofibroblasts culminate
in interstitial fibrosis, which compresses tubules, generating apoptosis
and tubular atrophy. In addition, glomeruli become distorted and
tubular-like (Zeisberg et al., 2000b).
Renal fibroblasts produce a variety of cytokines and chemokines
under basal and pathological conditions (Qi et al., 2006) that may
regulate tissue fibrosis. The production of TGF-β, IL-4, IL-6 and IL-13 is
augmented at the site of fibrosis. IL-6 induces fibroblast proliferation
and collagen production (Duncan and Berman, 1991), as well as the
synthesis of tissue inhibitor of metalloproteinase-1 (Lotz and Guerne,
1991). In addition, IL-4 suppress TNF-α and IL-6 production and can also
positively induces tissue fibrosis (Gordon and Martinez, 2010; Biswas
and Mantovani, 2010; Murray and Wynn, 2011). Both IL-13 and TGF-β
can bind to its receptors in fibroblasts and stimulate collagen deposition.
On the other hand, IL-12 upregulates IFN-γ expression, inhibiting
collagen production by fibroblasts to suppress fibrosis (She et al., 2021).
IL-10, an anti-inflammatory and anti-fibrotic cytokine, inhibits the
downregulation of IFN-γ and upregulation of TGF-β (Steen et al., 2020).
Many cytokines (IL-1, IL-12, and TNF-α) and chemokines (MCP-1
and CCL-5) have been reported to be involved in the process of tissue
regeneration (Tedgui and Mallat, 2006). In addition to chemotaxis,
chemokines also perform a variety of functions. Studies have shown that
chemokines promote the resolution of the inflammatory response
(Anders et al., 2003; Wintges et al., 2013, 2010). CCL-2 promotes the
engulfment of apoptotic cells by macrophages (Amano et al., 2004),
while CCL-5 plays an important anti-inflammatory role (Anders et al.,
2003; Wintges et al., 2013; Kim et al., 2015). Chemokines and their
62
Molecular Immunology 156 (2023) 61–76
receptors play an important role in the recruitment of T cells, macro­
phages, and dendritic cells during the development of chronic kidney
disease (CKD) (Chung and Lan, 2011; Holdsworth, 2007). Alterations in
the cytokine profile, especially chemokines, may contribute to the
development of severe glomerular lesions (Araya et al., 2006; Vaidya
et al., 2011). In cases of chronic infections caused by Leishmania spp., the
infection induces the expression of several chemokine genes (Bre­
nier-Pinchart et al., 2001; Ritter and Korner, 2002). An enhanced
expression of CCL-2 (MCP-1), CCL-4 (macrophage inflammatory protein
(MIP)− 1β), and CCL-5 (RANTES) in human and mice infected with
Leishmania major was identified (Ji et al., 2003; Matte and Oliver, 2002).
To the best of our knowledge, the relationship between the levels of
cytokines/chemokines and collagen deposition promoting fibrosis in
renal tissues in CanL has not yet been studied. Considering that the
kidney shows fibrinogenic changes during CanL and that the balance of
cytokines/chemokines regulates the profibrinogenic and anti­
fibrinogenic immune responses differently, it could be hypothesized that
the balance of cytokines/chemokines can be differentially expressed in
the renal tissues, thus determining the expression of collagen deposition
in the kidneys, which may depend on the clinical form of canine leish­
maniasis. In this context, this study aimed to measure collagen de­
positions and to evaluate the cytokine expression involved in collagen
depositions by means of RT-qPCR in renal tissue fragments of Leishma­
nia-infected dogs.
2. Materials and methods
2.1. Animals
All procedures agreed with the guidelines established by our Insti­
tutional Committee for Animal Care and Use so as to avoid any unnec­
essary suffering for the dogs. The Ethics Committee on Animal
Experimentation (CETEA, in Portuguese) also reviewed and approved
this work (national guidelines, Law 11.794, implemented on August 10,
2008, by the Federal University of Minas Gerais (UFMG); logged under
protocol number 224/2009).
Sixteen naturally Leishmania-infected dogs, with or without clinical
manifestations, along with six uninfected dogs, used as controls, were
obtained from the Zoonotic Disease Control Center (CCZ) in Teresina,
Piaui, Brazil. All dogs were adults and mongrel, but varied in breed,
weight, age, and sex.
Blood samples were collected to detect anti-Leishmania antibodies by
IFAT (titers ≥ 1:40) and ELISA. Samples of needle aspirates from the
popliteal lymph node and bone marrow were obtained from each dog for
culture in NNN and in Schneider liquid medium. Once the infection was
confirmed, the dogs were euthanized, and the necropsy was performed.
Before fixing the samples in formaldehyde, impressions were made of
the cut surface on clean slides. Myelograms and impressions on slides
were stained with Giemsa, for parasitological visualization. PCR was
performed to detect parasites in the kidneys, using a target sequence of
the L. donovani complex.
All infected animals (with or without clinical manifestation) showed
positive results in PCR and in at least one of the parasitological tests
(myelogram, impression smears, and culture) in different organs
(spleen, liver, skin, and lymph nodes). All uninfected controls evaluated
in this study presented negative results in all diagnostic analyses,
including PCR. Anti-Leishmania antibodies were detected by IFAT in the
sera from all of the infected animals, with titers ranging from
1:40–1:640.
According to their clinical signs, the Leishmania-infected dogs were
divided into two groups: (a) Clinically affected dogs – with clinical signs
of VL (n = 8), (b) Subclinically infected dogs – when they were not
presenting any clinical manifestation of the disease (n = 8), according to
Verçosa et al. (2008). Six animals were used as uninfected controls.
Leishmania-infected and uninfected dogs were tranquilized with 1%
acepromazine and later anesthetized with a 2.5% sodium thiopental
B.L.A. Verçosa et al.
solution. After this procedure, the animals were euthanized with an
overdose of 7.5% sodium thiopental (75 mg/kg) for subsequent post-
mortem examination and sample collection. Renal fragments were
collected, processed, and stained for histological analysis. Serial sec­
tions, 5 µm thick, were stained with hematoxylin and eosin (H&E), PAS,
Masson’s trichrome, Picrosirius Red, and Gomori’s reticulin. PAS
staining was used to highlight glomerular and tubular basement mem­
branes. Masson Trichrome was used to stain collagen in blue. Picrosirius
Red was used to visualize collagen fibers. Gomori’s reticulin was used
for the histological demonstration of reticular fibers.
2.2. Creatinine, urea, total protein, albumin, and globulin quantification
in serum
Blood samples were collected without anticoagulant from the jugular
vein. Sera were stored at 20ºC until tested. Creatinine, urea, total pro­
tein, albumin, and globulin were quantified by the LABTEST kit (Labtest
Diagnostic SA, Lagoa Santa, MG, Brazil), following manufacturer in­
structions. Biochemical tests were carried out in the semi-automatic
biochemical analyzer (TP analyzer, Thermo Plate). The serum globulin
concentration was calculated by subtracting the albumin from the total
proteins. The normal reference values of Bistner et al. (2002) were used
for biochemical analyses.
2.3. PCR
Kidney samples from all dogs were subjected to DNA extraction to
confirm the presence of the parasite. The genomic DNA was extracted
using the "NucleoSpin®Tissue kit" (Macherey-Nagel, Durën, Germany).
PCR was performed with a GoTaq® Green Master Mix Kit (Promega
Corporation. Madison, WI) using primers from the specific DNA
sequence of L. donovani, as previously described (Verçosa et al., 2022).
2.4. Descriptive and semiquantitative analyses of the histopathological
alterations
Characteristics, such as the intensity, composition, and distribution
of the renal inflammatory response, were evaluated on slides stained
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Molecular Immunology 156 (2023) 61–76
Fig. 1. Analysis of interstitial and adventitial
collagen depositions in the renal vessels stained
by Picrossirius Red. Initially, the total area of
the vessel (adventitia area + vascular lumen)
was measured. The value obtained was sub­
tracted from the luminal area to determine the
filled area occupied by the adventitial layer of
the vessel, which was expressed as: Area of
adventitial collagen deposition = Σ total vessel
area - luminal area. The vascular lumen was
defined as the set comprised of the vascular
lumen, endothelium, and inner and muscular
layers of the vessels. Letters A - C show repre­
sentative images of adventititial collagen
deposition quantification. To quantify the
collagen fibrils in the interstitial space, digita­
lized images (10x magnification) of Picrossirius
Red stained slides of the cortical region of the
renal tissue were analyzed by densitometric
methods. Interstitial collagen deposition was
calculated by the formula: Insterstitial collagen
area = Σ areas with the same density of pixels/
total area. Quantification was performed using
the Kontron KS300 software, evaluating 20
microscopic fields.
with H&E. The inflammatory foci were characterized as: (1) discrete and
focal: when the foci were small and isolated; (2) moderate and multi­
focal: with more evident and coalescent foci; and (3) intense and diffuse:
with large and extensive areas, as described by Verçosa et al. (2011).
The intensity of the histopathological findings was classified as fol­
lows: absent, + - discrete, + + - moderate, and + ++ - severe, according
to Verçosa et al. (2011).
2.5. Semiquantitative analysis of Glomerulosclerosis
Glomerulosclerosis on slides stained with PAS was assessed using a
semiquantitative technique, as described previously by Wu et al. (1997).
The degree of sclerosis in each glomerulus was subjectively graded on a
scale from 0 to 4, where: grade 0 corresponds to normal; 1 represents a
minimal degree, as the sclerotic area occupies up to 25% of the
glomerulus; 2: moderate intensity, corresponding to 25–50% of the
sclerotic glomerular area; 3: moderate to severe, with the sclerotic area
representing 50–75% of the total glomerular area; and 4: severe in­
tensity, with the sclerotic area ranging from 75% to 100%. Glomerulo­
sclerosis was considered when the glomerular basement membrane
thickening, mesangial hypertrophy, and capillary occlusion appeared.
2.6. Descritive and histomorphometric analysis of the interstitial and
adventitial collagen fibrils of blood vessels in renal tissues
Slides stained with Gomori thrichome were analyzed under an op­
tical microscope, and the general collagen deposition was classified
according to the morphological pattern into: (1) mild - focal distribu­
tion, affecting a small area of the parenchyma; (2) moderate - multifocal
distribution, compromising a large area of the parenchyma; and (3)
intense - more diffuse distribution, compromising a large area of the
parenchyma (Verçosa et al., 2008).
Morphometry was conducted in a blind assay by a single observer
(BLAV), using digitalized pictures in an image analyzer (Kontron KS300
version 2.0, Karl Zeiss). Microscopic images stained with Picrossirius
Red were obtained with a digital camera (Sony DSC-W7/7.2 Mega
Pixels) connected to a light microscope (Olympus BX41) using 10x and
40x plan achromatic objectives. Images were transferred to a computer
B.L.A. Verçosa et al. Molecular Immunology 156 (2023) 61–76

Fig. 2. Histopathological findings in

clinically affected Leishmania-infected
dogs. In A: Diffuse and moderate
mononuclear inflammatory infiltrate (*)
(PAS) is located in the perivascular,
periglomerular, and peritubular areas.
Intertubular and glomerular atrophy
(white arrow) and tubular dilatation
associated with hyaline cylinders (black
arrow). In B: Intertubular collagen
deposition (blue color) and hyaline
cylinders in red color (white arrow) are
shown on a slide stained by Masson
trichome. In C: Hyaline cylinders asso­
ciated with tubular atrophy/dilation
(white arrow) are shown (PAS). In D:
Hydropic degeneration in renal tubules
(white arrow) and glomerular collagen
deposition (black arrow) are shown on a
slide stained by Masson trichome. In E:
Representative images of the thickness
of the Bowman capsule (white arrow) is
shown (PAS). In F: A representative
image of tubular atrophy associated
with the thickness of the tubular mem­
brane (white arrow) is shown in a clin­
ically affected Leishmania-infected dog
(PAS). Bars = 38 µm in A and B; 50 µm
in C and D; and 100 µm in E and F.

for morphometry. Twenty histological fields per animal were digitalized
and assessed according to Moro et al. (2004). These were quantified in
fields with 356,207 µm2, obtained with an 10x objective, evaluating a
final total kidney area of 7,124,140 µm2.
Areas of fibrosis were quantified in the renal tubules and around the
glomeruli (interstitial fibrosis), as well as in the adventitia of blood
vessels (Fig. 1), as described by Farris et al. (2011). Digitalized images
(10x) of Picrossirius Red stains of the cortical region of the renal tissue
were analyzed by densitometric methods for the quantification of
collagen fibrils in the interstitial space. The same criteria were used both
for control and Leishmania-infected specimens. Areas occupied by large
blood vessels and glomeruli were not considered. Interstitial collagen
deposition was calculated by the formula: Insterstitial collagen area = Σ
areas with the same density of pixels/total area.
Initially, the total area of the vessel (adventitia area + vascular
lumen) was measured. The value obtained was subtracted from the
luminal area to determine the filled area occupied by the adventitial
layer of the vessel, expressed as: Area of adventitial collagen deposition
= Σ total vessel area - luminal area. Values of adventitial collagen in
blood vessels were obtained from the ratio between the adventitial area
and the luminal area, expressed as: Adventitial collagen = Σ Adventitial
collagen deposition total area of vessel/artery cross-sectional area. The
artery’s cross-sectional area was defined as the set comprised of the
vascular lumen, endothelium, internal elastic lamina, and intima and
media layers of the vessel (Fig. 1).
2.7. Molecular analysis of cytokines and chemokines related to collagen
deposition
2.7.1. Tissue collection, extraction of total RNA, and synthesis of first
strand cDNAs
Kidney samples were collected and immediately placed in liquid
nitrogen. Subsequently, the samples were stored at − 80 ◦ C until use for
RNA extraction. Renal fragments (20 mg) from each studied dog were
used for RNA extraction. The samples were homogenized and treated
with the NucleoSpin® RNA II Kit (Macherey-Nagel, Germany). The
cDNAs were synthesized from 1.0 µg of total RNA, using the High-
Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA)
with Real Time Random primers, and stored at − 20 ◦ C until use.

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B.L.A. Verçosa et al. Molecular Immunology 156 (2023) 61–76

Table 1 concentration in relation to the antagonic cytokine/chemokine, so that,

Serum biochemical quantification of naturally Leishmania-infected (clinically it is important also to analise its ratios, as it was shown.
affected and subclinical infected) and uninfected dogs. All specific primers for each studied gene were designed using se­
Biochemical Control Subclinical Clinically Reference quences obtained from GeneBank, according to Alves et al. (2009). For
parameters (n = 7) infected affected Values* PCR controls, primers were designed with the aid of Gene Runner,
(n = 8) (n = 8) version 3.05 (Hasting Software Inc. 2004), using GeneBank specific
Mean Mean ± SD Mean ANOVA canine sequences. The primers were synthesized by Eurofins MWG
± SD (N, %) ± SD (N, p operon (Huntsville, Al, USA) and reconstituted in water free of nuclease.
(N, %) %)

Urea (mg/ 38.7 43.8 139.6 21.4 – Clinically 2.7.3. Quantitative real-time PCR
dL) ± 10.9 ± 11.6 (1/ ± 88.3 59.9 affected vs Quantitative RT-qPCR was performed with 100 mM of each primer
(0) 8, 12.5) (6/8, 75) Uninfected
p ¼ 0.002
(Supplementary Table 1) and 1:10 diluted cDNA in an ABI Prism 7500
Clinically DNA Sequence Detection System, using the Master Mix SYBR Green PCR
affected vs (PE Applied Biosystems, Foster City, CA, USA). The samples were
Subclinical incubated at 95 ◦ C for 10 min and subjected to 40 cycles of 95 ◦ C for 15 s
infected
and 60 ◦ C for 1 min, during which time fluorescence data were
p ¼ 0.002
Creatinine 1.0 1.1 ± 0.4 1.4 0.50 – ns collected. Three replicated analyses were performed, and the amount of
(mg/dL) ± 0.2 (0) ± 0.69 1.50 target RNA was normalized in relation to the GAPDH, HPRT, RPS18,
(0) (2/8, 25) RPL1, and β-Actin genes of the endogenous control genes (reference
Total 6.8 10.0 ± 2.3 11.8 5.40 – Clinically gene), as defined by Vandesompele et al. (2002). Five internal controls
Proteins ± 0.4 (7/8, 87.5) ± 3.94 7.10 affected vs
(g/dL) (0) (7/8, Uninfected
were used in each run of RT-qPCR to evaluate the possible genomic
87.5) p < 0.001 contamination of DNA (without adding reverse transcriptase) and the
Albumin (g/ 2.7 2.5 ± 0.7 2.4 2.60 – ns purity of the reagents (without adding cDNA). Single peak dissociation
dL) ± 0.1 (4/8, 50) ± 0.68 3.30 curves confirmed the specificity of PCR products. The results of RT-qPCR
(0) (4/8, 50)
were analyzed as median in relation to the expression of each messenger
Globulin 4.0 8.3 ± 2.0 9.3 2.70 – Clinically
(g/dL) ± 0.4 (8/8, 100) ± 4.22 4.40 affected vs RNA of the cytokines according to the clinically affected and subclini­
(0) (7/8, Uninfected cally infected groups as compared to the values of the uninfected group
87.5) p < 0.001 (Menezes-Souza et al., 2011). The data were expressed according to the
*Normal reference values described by Bistner et al. (2002). Control: group of 2-ΔΔCT method (Delta delta CT method), using the mean ΔCt value of the
uninfected dogs; Subclinical infected: infected dogs with any clinical manifes­ control group as a calibrator (Bustin et al., 2009). Relative quantifica­
tation; Clinically affected: infected dogs with clinical manifestations of CanL. tion relates the PCR signal of the target transcript in a treatment group to
n = total number of samples; N = Number of animals outside the reference that of another sample, such as an untreated control. The 2-ΔΔCT method
values. Bold: ANOVA and Tukey test. ns: p > 0.05 is a convenient way to analyze relative changes in gene expression from
quantitative real-time PCR experiments. This method assumes 100%
efficiency of qPCR assays, as described by Livak and Schmittgen (2001).
Each gene was normalized with a caliper using the formula ΔCT
(target gene Ct - reference gene Ct). As an exemple, first average the ΔCT
for IL-4 in the experimental group against the 5 constitutive genes,
which were called ΔCT1: ΔCT1 = [(IL-4 Ct - GAPDH Ct) + (IL-4 Ct -
HPRT Ct) + (IL-4 Ct - RPS18 Ct) (IL-4 Ct - RPL1 Ct) (IL-4 Ct - β-Actin
Ct)]/[5]. Then average the ΔCT for IL-4 in the control group (calibrator)
against the 5 constitutive genes, which were called ΔCT2: ΔCT2 = [(IL-
4 Ct - GAPDH Ct) + (IL-4 Ct - HPRT Ct) + (IL-4 Ct - RPS18 Ct) (IL-4 Ct -
RPL1 Ct) (IL-4 Ct - β-Actin Ct)]/[5]. Then it was applied the value in the
formula 2-ΔΔCT, which is 2-(ΔCt1-ΔCT2). The values were expressed in
graphs using Log in base 10 to facilitate data visualization, as used by
Menezes-Souza et al. (2012).
After normalization, the expression levels of each gene in the infec­
ted groups, as compared to the expression levels in the control group,
were regulated up or down (DelPuerto et al., 2010; Verçosa et al., 2021a,
Verçosa et al., 2021b). The qPCR data were evaluated as mean
fold-differences relative to each messenger RNA expression of the cy­
tokines according to the clinical groups (clinically affected versus sub­
Fig. 3. Positive correlation between the number of clinical signs and high levels clinically infected dogs) in relation to the values of the uninfected group
of urea (mg/dL) in Leishmania-infected dogs (clinically affected and subclini­
(Menezes-Souza et al., 2011). It must be emphasized that the presenta­
cally infected), n = 16, p < 0.05 (Pearson test).
tion as up and down is in comparison with values of normal uninfected
control, so that, the normal value is not shown. Data that were consid­
2.7.2. Primers for gene evaluation [IFN-γ, IL-4, IL-6, IL-12, IL-13, TGF-β, ered significant outliers were excluded from the statistical analysis.
TNF-α, CCL2 (MCP1), CCL4 (MIP-1 beta), and CCL5 (MCP2)]
The genes were chosen considering their pro and anti-fibrotic ac­
2.8. Statistical analysis
tivity. The ratios evaluated followed the criterion of antagonistic action,
using pro-fibrotic gene expression in the numerator (IL-13, TGF-β, IL-4,
All data collection and statistical tests were performed using blind
MCP1, CCL4, and CCL5) and its antagonist in the denominator (IFN-γ,
analyses. All results were subjected to the Lilliefors test for Gaussian
TNF-α and IL-12). It should be considered that cytokines/chemokines
distribution and the Cochran-Bartlett test to assess homoscedasticity.
are effective by its direct action on its receptor, however, the final
The Grubbs test - ESD method (Extreme Student Deviation) was per­
consequence on the cellular function depends on its relative
formed to determine significant outliers. The morphometric data were

65
B.L.A. Verçosa et al. Molecular Immunology 156 (2023) 61–76

Fig. 4. In A: Glomerulosclerosis in
clinically affected dogs. Semi-
quantitative analyses of glomerulo­
sclerosis in the kidney of Leishmania-
infected dogs and non-infected controls.
Clinically affected dogs (n = 8) and
subclinically infected dogs (n = 8) pre­
sented more glomerulosclerosis than
uninfected controls (n = 7) (Kruskal-
Wallis and Dunn, *p < 0.05) (median,
quartiles and extremes). In B: Leucocyte
infiltration in glomerulus (*). Glomer­
ulus showing morphological character­
istics of glomerulosclerosis as the
thickening of the glomerular basement
membrane (black arrow), mesangial
hypertrophy, and capillary occlusion
(white arrow) on a slide stained by PAS.
In C: Glomerular and intertubular
collagen deposition (blue color) on a
slide stained by Masson trichome. In D:
Morphological characteristics of
apoptosis (cell shrinkage, chromatin
condensation at the periphery of the
nucleus, nuclear and/or cytoplasm
fragmentation, nuclear pyknosis, and
nucleolar disintegration) are shown in a
mesangial cell (black arrow) stained by
Masson trichome. Bars = 50 µm (C) and
100 µm (B and D).

submitted to ANOVA and to the Tukey multiple comparison procedure.
When data distribution did not meet the requirements of normality, the
non-parametric Kruskal-Wallis method was applied, followed by Dunn’s
method for multiple comparisons. The qRT-PCR data were analyzed by
the Mann Whitney test. The chi-squared test was also used to check the
association of variables. To verify an eventual correlation between
variables, the Pearson and Spearman tests were used in morphometry
and molecular data, respectively. All statistical analyses were performed
using the SAEG program, version 9.1. Results were given as mean ± SD
or median, quartiles, and extreme values, and p < 0.05 was accepted as
statistically significant.
3. Results
3.1. Clinical and histopathological findings
The main clinical signs were lymphadenopathy, conjunctivitis,
onychogryphosis, hepatosplenomegaly, and fever. Renal inflammation
was observed in all groups (clinically affected, subclinically infected,
and uninfected control). A diffuse and severe inflammatory infiltrate (2/
8, 25%), with intertubular fibrosis, hyaline cylinders, and tubular at­
rophy/dilation appeared in clinically affected dogs. Multifocal or diffuse
inflammatory infiltrates, ranging from minimal to severe, located in the
perivascular, periglomerular, and peritubular regions, were observed in
clinically affected dogs. In this group, the dogs showed a discrete (1/8,
12.5%) and moderate (5/8, 62.5%) inflammatory infiltrate. Only one
uninfected dog presented discrete renal inflamation (1/6, 16.66%).
Macrophages, along with lymphocytes, were the predominant cells.
Subclinically infected dogs had only discrete (8/8, 100%) and focal
inflammation, located in the perivascular, periglomerular, and peri­
tubular regions, but rarely in the subcapsular region, comprised pre­
dominantly of macrophages and lymphocytes.
Fig. 2 shows histopathological findings in clinically affected dogs,
showing diffuse and moderate inflammatory infiltrate, located in the
perivascular, periglomerular, and peritubular regions, and comprised
predominantly of macrophages and lymphocytes (Fig. 2A). Intertubular
and glomerular collagen depositions were observed in Fig. 2 B. Hyaline
cylinders associated with tubular atrophy/dilation are shown in Fig. 2C.
Hydropic degeneration in renal tubules, associated with glomerular
collagen deposition, is shown in Fig. 2 D. The thickness of the Bowman
capsule and the tubular atrophy associated with the thickness of the
tubular membrane are shown in Fig. 2 E and F, respectively.
3.2. Serum levels of creatinine, urea, total protein, albumin, and globulin
Only clinically affected and subclinically infected dogs presented
biochemical alterations in serum. High levels of urea, total protein, and
globulin associated with a decrease in albumin levels were observed in
Leishmania-infected dogs. Clinically affected dogs showed increased
levels of urea when compared with subclinically infected and uninfected
groups (p = 0.002, ANOVA and Tukey test). The total serum protein and
globulin levels were higher in clinically affected animals than in unin­
fected dogs (p < 0.001, ANOVA and Tukey test). Data are shown in
Table 1. High levels of urea were positively correlated with the number
of clinical signs (r = 0.82, p < 0.001, Pearson test), as observed in Fig. 3.
3.3. Semiquantitative analysis of glomerulosclerosis
Glomerulosclerosis was observed in all Leishmania-infected animals
(clinically affected and subclinically infected). Only one of the seven
uninfected dogs had minimal intensity of sclerotic glomeruli. Semi-
quantitative analyses of glomerulosclerosis in the kidney of Leish­
mania-infected dogs and non-infected controls showed that both clini­
cally affected and subclinically infected dogs presented higher levels of
glomerulosclerosis than uninfected controls (Kruskal Wallis and Dunn,
p < 0.05) (Fig. 4A). Glomerulus presented a morphological character­
istic of glomerulosclerosis, such as the thickening of the glomerular
basement membrane, mesangial hypertrophy, and capillary occlusion
(representative image in Fig. 4 B). Glomerular and intertubular collagen
depositions are presented in Fig. 4C. Morphological characteristics of
apoptosis (cell shrinkage, chromatin condensation at the periphery of
the nucleus, nuclear and/or cytoplasm fragmentation, nuclear pyknosis,

66
B.L.A. Verçosa et al.
and nucleolar disintegration) are shown in mesangial cells by means of
Masson trichome stain (Fig. 4 D).
3.4. Descritive and histomorphometric analysis of collagen fibers in the
interstitium and adventitia of blood vessels
Adventitial and intertubular collagen depositions were interspersed
with inflammatory foci in both cortical and medullar regions in infected
dogs (clinically affected and subclinically infected), as shown in Figs. 5
and 6. Collagen deposition in the adventitia of blood vessels was less
frequent and less intense in uninfected controls, more frequent but not as
intense in subclinically infected dogs, and more intense and more
frequent in clinically affected dogs. Collagen deposition in perivascular
areas interspersed with elongated fibroblasts, myofibroblasts, and in­
flammatory cells were observed in the cortical region of the kidneys in
Leishmania-infected dogs. Interstitial collagen deposition observed in
67
Molecular Immunology 156 (2023) 61–76
Fig. 5. Representative images of inter­
stitial collagen depositions in the kidney
of clinically affected and subclinical-
Leishmania-infected dogs. In A: more
intense interstitial collagen deposition,
as morphometrically measured by the
average area of the collagen, is more
commonly observed in clinically
affected and subclinically infected dogs
than in the controls (p < 0.05, Tukey
test). In B - E: representative images of
renal interstitial collagen deposition in
clinically affected (B - C) and subclini­
cal-Leishmania-infected dogs (D - E) are
shown, stained by Picrosirius Red
(collagen fibers are stained in red) and
Gomori reticulin (reticulin fibers are
stained in black), respectively. In F:
intense renal interstitial collagen depo­
sition, stained in blue, is associated with
severe hyaline cylinders, stained in red,
by Masson trichome in clinically
affected dogs. In G - H: Renal interstitial
collagen deposition in clinically affected
dogs is shown on a slide stained by
Picrosirius Red and analyzed in micro­
scopy without (G) or with polarized
light (H), respectively. Collagen type I is
stained in yellow or orange, as observed
in F. Bars = 25 µm (C), 50 µm (B and D),
and 100 µm (E and F). V= vessel;
TB= renal tubule.
subclinically infected dogs was comparable with that observed in those
clinically affected with CanL.
The clinically affected dogs showed a discrete (1/8, 12.5%), mod­
erate (4/8, 50%), and severe score of interstitial collagen deposition (3/
8, 37.5%) (Supplementary Table 2). Furthermore, in the subclinically
infected group, only discrete interstitial collagen deposition (6/8, 75%)
was observed. Only one uninfected dog presented discrete renal inter­
stitial collagen deposition (1/6, 16.66%). The summary of the main
clinical signs, renal interstitial collagen deposition, and inflammatory
infiltrate scores in kidney fragments of Leishmania-infected dogs are
shown in Supplementary Table 2.
Interstitial and adventitial collagen depositions in renal vessels were
associated with the presence of clinical signs in Leishmania-infected dogs
(p < 0.05, Chi-squared test). Representative images of the interstitial
collagen deposition in clinically affected and subclinically infected dogs
are shown in Fig. 5. Interstitial collagen depositions, as
B.L.A. Verçosa et al. Molecular Immunology 156 (2023) 61–76

Fig. 6. Measured representative images of morphological parameters of adventitial collagen deposition as area (A), perimenter (B), major diameter (C), and minor
diameter (D). In A - B: adventitial collagen deposition morphometrically measured by average area and perimeter of collagen, which are more intense in clinically
affected than in subclinically infected dogs, and shows a higher collagen deposition than in the controls (p < 0.05, Tukey test). In C - D: major and minor diameter of
adventitial collagen deposition observed in Leishmania-infected dogs (clinically affected and subclinically infected) than in the controls (p < 0.05, Tukey test). In E -
H: intense adventitial collagen deposition in the blood vessels of the kidney on a slide stained by Picrosirius Red (E, G) and by Reticulin Gomori (F, H) in clinically
affected (E, F) and subclinically infected (G, H) dogs, respectively. Bar = 100 µm.

morphometrically measured by the average area of the collagen, were
more intense in clinically affected Leishmania-infected dogs than in the
controls. However, this occurred both in clinically affected and sub­
clinically infected dogs (p > 0.05, Tukey test) (Fig. 5A). Representative
images of intense interstitial collagen deposition in a clinically affected
dog is shown in Fig. 5 B and C, and in a subclinically infected dog in
Fig. 5 D and E, stained by Picrosirius Red and by Gomori, respectively.
Intense interstitial collagen deposition is associated with many hyaline
cylinders in the kidneys of clinically affected dogs, as observed in the
representative image stained by Masson trichome (Fig. 5F). Renal
interstitial collagen deposition in clinically affected dogs is shown in a
preparation stained by Picrosirius Red evaluated without (Fig. 5G) or
with polarized light (Fig. 5 H). The dense collagen fibers in the tissues
stained in yellow correspond to type I collagen. The fibrillar collagen
type I is more frequent than fibrillar collagen type III in the interstitial
space (Fig. 5 H).
Fig. 6 shows representative images of all of the measured morpho­
logical parameters of adventitial collagen depositions in the vessels,
such as area (A), perimenter (B), major diamenter (C), and minor dia­
menter (D). Adventitial collagen depositions in the vessels, morpho­
metrically measured by an average area and perimeter of the collagen,
were more intense in clinically affected than in subclinically infected
dogs, which showed a higher collagen deposition than did the controls
(p > 0.05, Tukey test) (Fig. 6A - B). The major and minor diameters of
adventitial collagen depositions were higher in Leishmania-infected dogs
(clinically affected and subclinically infected) than in the controls
(p > 0.05, Tukey test) (Fig. 6C - D). Intense renal adventitial collagen
depositions, on a slide stained by Picrosirius Red (Fig. 6 E - G) and by
reticulin Gomori (Fig. 6F -H) in clinically affected (Fig. 6 E - F) and
subclinically infected (Fig. 6 G-H) dogs, respectively, are shown in
Fig. 6.
3.5. Molecular analysis of cytokines and chemokines in kidney tissues
Bax, and Fas) expression levels in clinically affected and subclinical
infected dogs with canine leishmaniosis was showed in Supplementary
Table 3. The IL-4/IFN-γ ratio was higher in clinically affected dogs. A
more intense expression of the MCP1/IL-12 ratio and CCL5/IL-12 ratio
was observed in subclinically infected dogs (p > 0.05, Mann Whitney
test), as shown in Table 2. The TGF-β/TNF-α, MCP1/IL-12, CCL5/IL-12,
IL-4/IFN-γ, and TGF-β/IL-12 ratios were associated with clinical mani­
festations in dogs with CanL (p < 0.05, Chi-squared test). The IL-6, IL-
13, IL-4/IFN-γ, IL-13/IL-6, IL-13/IFN-γ, IL-4/TNF-α and TGF-β/IL-6 ex­
pressions were upregulated in clinically affected dogs and down­
regulated in subclinically infected dogs. CCL5/IL-12 ratios were
upregulated in subclinically infected dogs and downregulated in clini­
cally affected dogs. TGF-β/TNF-α, MCP1/IL-6, MCP1/IFN-γ, MCP-1/
TNF-α, TGF-β/IFN-γ, MCP1/IL-12, IL-13/IL-12, IL-13/TNF-α, CCL5/
TNF-α, CCL5/IFN-γ, and CCL5/IL-6 ratios were upregulated in clinically
affected and subclinically infected dogs. Furthermore, TGF-β, IL-4/IL-6,
and TGF-β/IL-12 ratio expression was downregulated in both infected
groups (Figs. 7–9).
Additionally, a positive correlation was detected between IL-6 and
CCL5 (r = 0.73, n = 10, p = 0.01), CCL4 (r = 0.87, n = 09, p = 0.002),
MCP-2 (r = 0.83, n = 10, p = 0.003), IFN-γ (r = 0.96, n = 09,
p < 0.0001), IL-12 (r = 0.87, n = 10, p = 0.0012), and mRNA expres­
sion levels in the renal tissues. TGF-β correlated with MCP-1 (r = 0.73,
n = 12, p = 0.007), Casp 3 (r = 0.76, n = 12, p = 0.003), Casp 8
(r = 0.85, n = 11, p = 0.001), Bax (r = 0.72, n = 11, p = 0.001), Fas
(r = 0.82, n = 11, p = 0.002), and IFN-γ (r = 0.76, n = 11, p = 0.006).
IL-13 was also correlated with IL-12 (r = 0.76, n = 11, p = 0.006) and
TNF-α (r = 0.65, n = 10, p = 0.04). In addition, positive correlations
were detected between IL-4 and IL-10 (r = 0.54, n = 13, p = 0.007), IL-
12 (r = 0.70, n = 13, p = 0.007) and TNF-α (r = 0.67, n = 12, p = 0.01)
mRNA expression levels in the renal tissues. IFN-γ correlated with IL-12
(r = 0.66, n = 11, p = 0.02). Furthermore, IL-12 expression was posi­
tively correlated with IL-10 (r = 0.79, n = 13, p = 0.0012).

Gene expression (Individual and ratios) of cytokines (IL-4, IL-6, IL-
12, IL-13, TGF-β, IFN-α, and TNF-α) and chemokines (MCP1, MCP2,
CCL4, and CCL5) are shown in Supplementary figures 1, 2, 3 and 4.
Renal cytokines (IL-4, IL-10, IL-12, IFN-γ, and TNF-α), chemokine (CCL-
4, CCL-5, MCP-1, and MCP-2) and apoptotic protein (Casp 3, Casp 8,
3.6. Cytokines/chemokines related to collagen depositions
Strong positive correlations were observed between morphometric
values of interstitial collagen deposition and MCP-1/IL-12 (r = 0.80,
n = 12, p = 0.0016), and IL-4 (r = 0.61, n = 12, p = 0.03) mRNA

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B.L.A. Verçosa et al. Molecular Immunology 156 (2023) 61–76

Table 2 adventitial and interstitial collagen deposition in subclinical infected

Renal cytokines and chemokine expression levels in clinically affected and and clinically affected Leishmania-infected dog are shown in Table 4 and
subclinical infected dogs with canine leishmaniosis. Fig. 10. Only CCL5 versus IL-6 presented significant correlation
Cytokines Groups Mann- (r = 0.77; p = 0,0093) in the clinically affected dogs.
/Chemokines Whitney test
Clinically affected Subclinical infected
*p 3.7. Correlation of cytokine/chemokine versus renal related serum
Median (m - M) (n) Median (m - M) (n)
biochemical markers: creatinine, urea, total protein, albumin, and globulin
IL-4* 1.03 (0.02–3.41) 0.08 (0.01–0.23 0.03
(6) (7)
Strong positive correlations were observed between serum levels of
IL-6 1.33 (0.91–2.77) 0.25 (0.03–0.64)
(5) (5) urea and IL-4 (r = 0.64, n = 14, p = 0.014), IL-10 (r = 0.72, n = 14,
IL-13 26.04 (0.02–36.36) 0.08 (0.01–0.29) p = 0.004), IL-12 (r = 0.69, n = 14, p = 0.007), IL-6 (r = 0.76, n = 14,
(7) (5) p = 0.002), and IL-13 (r = 0.72, n = 14, p = 0.003), mRNA expression
TGF-β 0.18 (0.09–1.31) 0.36 (0.02–1.02) levels in the renal tissues. All other serum biochemical markers analysed
(5) (7)
(creatinine, total protein, albumin, and globulin) did not present sig­
CCL-4 6.23 (0.01–17.04) 0.14 (0.01–0.37) 0.04
(7) (6) nificant correlations with individual cytokine/chemokine mRNA
IL-13/TNF-α 2.90 (0.24–233.5) 8.11 (0.9–12.88) expression levels in the renal tissues (p > 0.05).
(6) (7)
IL-13/IL-12 0.28 (0.06–3.24) 0.35 (0.06–6.89)
4. Discussion
(6) (7)
IL-13/IFN-γ 6.73 (0.14–1.98) 0.755 (0.17–1.48)
(6) (6) This novel study examined enhanced Interstitial and adventitial
IL-13/IL-6 1.65 (0.03–19.97) 0.265 (0.1–0.46) collagen depositions in the vessels of kidney tissues. Their relationship
(6) (6) with the expression of cytokines and chemokines in clinically affected
IL-4/TNF-α 3.26 (0.07–19.88) 0.59 (0.23–3.26)
(7) (6)
and subclinically infected dogs with CanL were shown using histo­
IL-4/IL-12 0.085 (0.03–1.05) 0.09 (0.01–0.95) morphometry and RT-qPCR, respectively. In addition, interstitial and
(6) (7) adventitial collagen depositions proved to be correlated with different
IL-4/IFN-γ* 0.56 (0.13–30.5) 0.16 (0.04–0.38) 0.02 cytokine/chemokine mRNA expressions in the renal tissues of Leish­
(6) (6)
mania-infected dogs, which depended on the severity of the clinical
IL-4/IL-6 0.12 (0.03–1.06) 0.07 (0.02–0.18)
(6) (7) forms of the disease.
TGF-β/IL-12 0.14 (0.02–0.68) 0.66 (0.06–2.91) Our data showed that the MCP-1/IL-12 and CCL5/IL-12 mRNA ratios
(6) (6) were more commonly expressed in subclinically infected dogs, while the
TGF-β/TNF-α 4.36 (0.62–201.9) 10.98 (0.07–36.27) IL-4/IFN-α ratio was more commonly expressed in clinically affected
(7) (6)
dogs. Higher chemokine/cytokine ratio expressions were associated
TGF-β/IFN-γ 0.955 1.435 (0.29–1.78)
(0.15 − 19.68) (6) (6) with clinical manifestations in dogs with CanL, suggesting that the
TGF-β/IL-6 0.375 (0.03–7.1) 0.65 (0.01–1.46) enhanced chemokine expression may protect from severity in
(6) (6) leishmaniosis.
MCP-1/IL-12* 3.985 (0.56–8.91) 15.48 0.015
The IL-4/IFN-γ ratio was more commonly expressed in clinically
(6) (3.38–26.19) (6)
MCP-1/TNF-α 118.5 (0.01–723.7) 370.2
affected dogs, and was associated with an intense renal inflammation
(6) (22.96–866.9) (7) and adventitial collagen deposition, as supported by morphology and
MCP-1/IFN-γ 55.99 (9.05–260–8) 34.24 (7.54–96.86) morphometry in this study. This suggests that, in addition to the switch
(6) (6) of the immune response to the Th2 branch being associated with the
MCP-1/IL-6 6.29 (0.02–93.24) 10.35 (2.95–31.02)
severity of the disease, this switch is also associated with the adventitial
(6) (6)
CCL-5/TNF-α 7.385 (0.01–35.29) 37.22 (1.12–66.09) collagen deposition in the vessels of the kidneys. In renal tissues, the IL-4
(6) (6) predominate in relation to the IFN-γ, in such a way that it is possible that
CCL-5/IL-12* 0.215 (0.04–1.36) 1.246 (0.29–5.66) 0.04 the decrease in IFN-γ could induce a Th2-mediated healing, increasing
(6) (6)
the adventitial collagen deposition in the renal tissues. The profibrotic
CCL-5/IFN-γ 2.46 (0.51–68.8) 2.435 (0.89–5.11)
(6) (6)
activity stands out through the intensification of the production of pro-
CCL-5/IL-6 0.31 (0.01–14.27) 0.925 (0.18–2.34) inflammatory and profibrotic mediators, such as TNF-α and TGF-β
(7) (6) (Nathan et al., 1984; Piguet et al., 1989). In fact, TGF-β and the
The results are expressed as scattering of individual values and median of that TNF-α/TGF-β ratio were also related to adventitial collagen deposition
group of the relative copy number of mRNA for cytokines and chemokine in the renal tissues. T cells produce these cytokines and control the
expression levels and ratios. The mean ΔCt value of the control group as a processes of differentiation, migration, and activation of macrophages,
calibrator. Data considered significant outliers by Grubbs test were excluded as well as the expression of MCP-1, which subsequently induces
from the statistical analysis Bold: Clinically affected versus Subclinical infected inflammation and fibrosis (Wynn, 2004; Han et al., 2012). Furthermore,
*Mann Whitney test (p < 0.05). (m - M) = Extreme values (minimum - whereas the TGF-β, IL/4/IFN-γ, and TNFα/TGF-β ratios related to
maximum). n = amostral number. adventitial fibrosis of the renal vessels, the IL-4, IL-12, and MCP-1/IL-12
ratio was related to interstitial fibrosis, showing that the pathophysio­
expression levels in the renal tissues. Furthermore, a positive correlation logical mechanism of fibrosis in the same organ may be different.
was detected between the area of adventitial collagen deposition and The intensity of collagen deposition depended on both the affected
TGF-β (r = 0.67, n = 13, p = 0.01), IL-12 (r = 0.67, n = 11, p = 0.02), kidney site and the severity of the clinical form, with collagen deposition
IL-4/IFN-γ (r = 0.87, n = 09, p = 0.002), and TNF-α/TGF-β (r = 0.78, being more intense in clinically affected Leishmania-infected dogs. The
n = 10, p = 0.007). All other cytokine/chemokine mRNA expression activation of macrophages by IFN-γ increases the expression of MHC
levels (individual and ratio) presented not significant correlations with class II molecules, the production of cytokines, and microbicidal activ­
interstitial and adventitial collagen deposition in the renal tissues ity. Once activated, macrophages act as effective antigen-presenting
(p > 0.05), as observed in Table 3. cells and become more effective phagocytes in killing intracellular
Correlations between agonistic (TGF-β, IL-13, IL-4, MCP-1 and CCL5) pathogens, such as Leishmania spp. (Gulati et al., 2016). IFN-γ also
and antagonist (TNF-α, IFN-γ, IL-12 and IL-6) genes related with stimulates mononuclear phagocytic functions, such as the processes of

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B.L.A. Verçosa et al. Molecular Immunology 156 (2023) 61–76

Fig. 7. : Cytokine mRNA expression levels (Individual and ratio) in kidneys of Leishmania-infected dogs. The genes were chosen considering their pro and anti-
fibrotic activity. Animals were categorized according with or without clinical signs in clinically affected and subclinically infected dogs. The data were assessed
according to the 2-ΔΔCT method, using the mean ΔCt value of the control group as a calibrator. The RT-qPCR data were evaluated as mean fold-differences relative to
each messenger RNA expression of the cytokines according to the clinical groups (clinically affected versus subclinically infected dogs) in relation to the values of the
uninfected group. All data were analysed as fold-change but graphically represented as log10 fold change to better representation. The results are expressed as mean
± SEM of log10 of the relative copy number of mRNA for IL-6, IL-13, TGF-β, TGF-β/TNF-α, TGF-β/IFN-γ, and TGF-β/IL-12. Data considered significant outliers by
Grubbs test were excluded from the statistical analysis. The presentation as up and down is in comparison with values of normal uninfected control. The expression of
TGF-β and IL-13 was upregulated in clinically affected dogs and downregulated in subclinical infected dogs. TGF-β/TNF-α was upregulated in subclinical infected
dogs and downregulated in clinically affected dogs. IL-6, IFN-γ/TGF-β, and IL-12/TGF-β ratios were upregulated in clinically affected and subclinical infected dogs.
There was no statistical diference between clinically affected versus subclinical infected groups (Mann-Whitney test, p > 0.05).

adherence, phagocytosis, secretion, respiratory explosion, and produc­
tion of nitric oxide. In addition, IFN-γ also inhibits the differentiation of
fibrocytes into myofibroblasts promoted by IL-4 and IL-13 (Shao et al.,
2008).
The MCP1/IL-12 ratio was more commonly expressed in subclini­
cally infected dogs. Renal cells, due to various pro-inflammatory stimuli,
produce MCP-1 (Rovin et al., 1992). MCP-1 expression has been iden­
tified in kidney diseases that involve a significant inflammatory process
(Rovin et al., 1994). MCP-1 plays an important role in the pathogenesis
of various diseases, the main feature of which is the infiltration of
mononuclear cells, such as rheumatoid arthritis and bronchial asthma
(Koch et al., 1992; Campbell et al., 1999). MCP-1 deficient mice exhibit
a change in Th2-like responses to Th1, and a greater resistance to
infection by Leishmania major (Lu et al., 1998; Gu et al., 2000). MCP-1 is
also a weak inducer of cytokine expression in monocytes and largely
causes a respiratory burst, which generates reactive oxygen species
(Jiang et al., 1992; Rollins et al., 1988; Thomas, 2017) and, conse­
quently, the death of Leishmania parasites (Engwerda et al., 1998;
Bacellar et al., 2000).
Chemokines play an important role in immune responses to Leish­
mania (Oghumu et al., 2010), which were increased mainly in the sub­
clinical forms of the disease (Mehrotra et al., 2011). Activated
monocytes can differentiate into DCs that migrate to lymphoid tissues,
as demonstrated in vivo (Randolph et al., 1999), while MCP-1 sup­
presses IL-12 production by activated human monocytes, but not by DCs
(Braun et al., 2000). MCP-1, by inducing the production of IL-10,
TGF-β1, or PGE2 during the differentiation of monocytes by cytokines
in DCs, can inhibit the production of IL-12 (Trinchieri, 1997). In addi­
tion, resistance to Leishmania infection depends mainly on T cells with a
Th1 response profile inducing immunity, which is initiated by IL-12 plus
DCs (Ghalib et al., 1995; Bacellar et al., 1996). Additionally, MCP-1
binding to CCR2 induces a novel transcription factor (MCP-induced
protein [MCPIP]), which promotes an M2 phenotype in macrophages by
inhibiting NF-κB activation, sequential ROS induction (Kapoor et al.,
2015). Thus, MCP-1 acts as a potent and essential factor in polarizing
Th0 cells to a Th2 phenotype (Gu et al., 2000) that control the inflam­
matory response and collagen deposition.
During the host’s physiological defense, in response to tissue injury
or infection, MCP-1 expression is induced by inflammatory stimuli,
promoting the leakage of effector cells into the bloodstream through the
endothelium (Ajuebor et al., 1998; Deshmane et al., 2009). However,
when a major increase in MCP-1 expression occurs, this chemokine is
responsible for sustaining and exacerbating cell recruitment and the
resulting inflammation. MCP-1 can play an important role in defending
the pathogen by acting as an accessory molecule in the cell response to
subsequent stimuli, increasing the effects of other molecules through
combinatorial signaling (Gschwandtner et al., 2019). MCP-1 has been
reported as an important factor for the resistance to Leishmania infection
(Ritter and Moll, 2000; Zaph and Scott, 2003).
CCL5/IL-12 was more expressed in subclinically infected dogs than
in clinically affected dogs. Chemokines can directly promote the
development of Th1 cells through the action of IFN-γ or by indirectly
increasing the production of IL-12. In both cases, chemokines act
through CCL4 (MIP-1b) and CCL5. However, MCP-1 can inhibit the
production of IL-12 by means of APCs, thus increasing the production of
IL-4 from activated T cells, consequently inducing the predominance of a
Th2 phenotype (Luther and Cyster, 2001).
IL-12 induces the expression of IFN-γ (Trinchieri, 2003). In addition,
IL-12 also favors the exchange of chemokine receptors, which facilitates
the entry of activated T cells in injured or infected sites (Kim et al.,
2003). The expression of CCR5 (CCL5 receptor) is positively regulated
by the expression of IL-12 (Yang et al., 2001), through which differen­
tiated effector T cells (Luther and Cyster, 2001) are recruited into the
tissues by means of CCL5 and MIP-1β (Uekusa et al., 2002; Marino et al.,

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B.L.A. Verçosa et al. Molecular Immunology 156 (2023) 61–76

Fig. 8. Cytokine/chemokine mRNA expression levels (Ratio) in the kidneys of Leishmania-infected dogs. The genes were chosen considering their pro and anti-
fibrotic activity. Animals were categorized according with or without clinical signs in clinically affected and subclinically infected dogs. Data were assessed ac­
cording to the 2-ΔΔCT method, using the mean ΔCt value of the control group as a calibrator. The RT-qPCR data were evaluated as mean fold-differences relative to
each messenger RNA expression of the cytokines according to the clinical groups (clinically affected versus subclinically infected dogs) in relation to the values of the
uninfected group. All data were analysed as fold-change but graphically represented as log10 fold change to better representation. The results are expressed as
scattering of individual values and mean ± SEM of that group (bars) of log10 of the relative copy number of mRNA for IL-13/ IFN-γ, IL-13/IL-6, IL-4/IFN-γ, TGF-β/IL-
6, CCL5/IL-12, IL-13/TNF-α, IL-13/IL-12, MCP1/IL-12, and IL-4/IL-6 ratios. Data considered significant outliers by Grubbs test were excluded from the statistical
analysis. The presentation as up and down is in comparison with values of normal uninfected control. The expression of IL-13/ IFN-γ, IL-13/IL-6, IL-4/IFN-γ, and TGF-
β/IL-6 was upregulated in clinically affected dogs and downregulated in subclinical infected dogs. The CCL5/IL-12 ratio was upregulated in subclinical infected dogs
and downregulated in clinically affected dogs. IL-13/TNF-α, IL-13/IL-12, and MCP1/IL-12 ratios were upregulated in clinically affected and subclinical infected dogs.
The IL-4/IL-6 ratio expression was also downregulated in both infected groups. MCP1/IL-12 and CCL5/IL-12 ratios were higher in subclinically infected dogs and IL-
4/IFN-γ ratio was higher in clinically affected dogs. Statistical diferences between clinically affected versus subclinical infected groups were observed for the ratios
IL4/INFγ, CCL5/IL-12 and MCP1/IL-12 (*), (Mann-Whitney test, p < 0.05).

2004; Taubman and Kawai, 2001).
More intense renal vessel adventitial fibrosis occurred in clinically
affected dogs. The vascular endothelium is considered an anatomical
defense barrier. Circulating leukocytes need to transpose the endothe­
lium to reach the underlying tissue. In infection by microorganisms,
including Leishmanaia sp., inflammation is an event whereby, after the
activation of the endothelial cell, leukocytes overflow and induce an
inflammatory response in the microenvironment by releasing pro-
inflammatory stimuli. Vascular leakage and vasculopathies are impli­
cated in fibrosis (Tager et al., 2008; Varga and Abraham, 2007). Addi­
tionally, the function of the vascular barrier responds directly to
mechanical forces (Birukov et al., 2003; Dudek and Garcia, 2001) and to
changes in matrix stiffness (Birukova et al., 2013; Bordeleau et al., 2017;
Huynh et al., 2011).
In the present study, Leishmania-infected dogs (clinically affected
and subclinically infected) presented more glomerulosclerosis than un­
infected controls (Fig. 4 A). The glomeruli presented a morphological
characteristic of glomerulosclerosis, including the thickening of the
glomerular basement membrane, mesangial hypertrophy, and capillary
occlusion (Fig. 4B). As with glomerulosclerosis, tubulointerstitial
fibrosis involves inflammation, proliferation, apoptosis, and fibrosis
(Zeisberg et al., 2000a; El Nahas, 2003).
TGF-β was upregulated in clinically affected dogs that presented a
higher collagen deposition. This reinforced the fact that TGF-β appears
in its active form in clinically affected dogs. However, subclinically
infected dogs showed a significant expression of CCL5/IL-12 and MCP-
1/IL-12 associated with a lower collagen deposition, suggesting that,
in this group, collagen fiber deposition does not occur via the canonical
pathway of TGF-β, but rather in a secondary manner, through the
expression of CCL5 and MCP-1.

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B.L.A. Verçosa et al. Molecular Immunology 156 (2023) 61–76

Fig. 9. Cytokine/chemokine mRNA expression levels (Ratio) in the kidneys of Leishmania-infected dogs. The genes were chosen considering their pro and anti-
fibrotic activity. Animals were categorized according with or without clinical signs in clinically affected and subclinically infected dogs. The data were assessed
according to the 2-ΔΔCT method, using the mean ΔCt value of the control group as a calibrator. The RT-qPCR data were evaluated as mean fold-differences relative to
each messenger RNA expression of the cytokines according to the clinical groups (clinically affected versus subclinically infected dogs) in relation to the values of the
uninfected group. All data were analysed as fold-change but graphically represented as log10 fold change to better representation. The results are expressed as
scattering of individual values and median of that group (bars) of log10 of the relative copy number of mRNA for MCP1/IFN-γ, MCP1/IL-6, MCP-1/TNF-α, CCL5/IFN-
γ, CCL5/IL-6, and CCL5/TNF-α ratios. Data considered significant outliers by Grubbs test were excluded from the statistical analysis. The presentation as up and down
is in comparison with values of normal uninfected control. The expression of the MCP1/IFN-γ, MCP1/IL-6, MCP-1/TNF-α, CCL5/IFN-γ, CCL5/IL-6, and CCL5/TNF-α
ratios was upregulated in clinically affected and subclinical infected dogs. There was no statistical diference between clinically affected versus subclinical infected
groups (Mann-Whitney test, p > 0.05).

TGF-β correlated positively with apoptotic proteins (Casp 3, Casp 8,
Bax, and Fas) and with an area of adventitial collagen deposition
(r = 0.67, n = 13, p = 0.01). Apoptosis may contribute to the patho­
genesis of renal fibrosis (Guh et al., 2003; Tian et al., 2002; Huang et al.,
2005; Zhang et al., 2004). Macrophages with an anti-inflammatory
profile, myofibroblasts, and damaged tubular cells produce cytokines
and growth factors, such as TGF-β, inducing cell apoptosis and the
accumulation of extracellular matrix (Chevalier, 2006; Misser et al.,
2005). Thus, fibrosis occurs in a progressive and overlapping sequence.
TGF-β is overexpressed in the glomeruli and tubulointerstitial cells in
glomerulonephritis (Niemer et al., 1995; Yamamoto et al., 1996). Under
the influence of TGF-β, some glomerular and tubular cells become
transformed into an embryonic phenotype, thus acquiring mesenchymal
properties similar to those of fibroblasts and myofibroblasts (Wardle,
1996; Border and Noble, 1997; Peters et al., 1997; Schnaper et al.,
2003). Our findings in canine Leishmania-induced kidney fibrosis sug­
gest that Th2 cytokines (TGF-β, IL-4, and IL-12) and chemokines (MCP1
and CCL5) induce cell apoptosis and an accumulation of extracellular
matrix. In addition, there may be subtle differences in the mechanisms
that control fibrosis in different tissues and in different parts of the same
organ. Our study focused on kidney fibrosis, so it may well be possible
that distinct pathways are operating in other organs.
Strong positive correlations were detected between morphometric
values of collagen deposition and cytokine/chemokine (MCP-1/IL-12,
IL-12, IL-4, TGF-β, IL-4/IFN-γ, and TGF-β/TNF) mRNA expression levels
in the renal tissues. The balance of cytokines/chemokines regulates the
profibrinogenic and antifibrinogenic immune responses differently. In­
flammatory cytokines (Th1 and Th2) play a pivotal role in collagen
depositions, but they exert opposite functions. Th2 cytokines (IL-4, TGF-
β, and IL-13) are strongly profibrotic, while Th1 cytokines (IFN-γ, IL-6,
TNF-α, and IL-12) suppress the inflammation resolution and, therefore,
the deposition of tissue collagen (Caldas et al., 2008). Nevertheless, the
relative importance of the relationship among cytokines in a natural
model of fibrosis has not been previously investigated, particularly in
such diseases as CanL, where a Th2 response persists, since they may
have a major impact on both the design and long-term benefit of future
clinical interventions based on disrupting the Th2 signaling pathway.
Additionally, our results demonstrated that antagonistic cytokines (such
as IL-12 and IL-10) were positively correlated, possibly suggesting that a
Th1:Th2 type response is related to different immune responses in
different areas of the kidney. Alternatively, these antagonistic cytokines
may be concomitantly increasing as a regulatory mechanism of the
immune system.
Messenger RNA expression levels of cytokines (IL-4, IL-10, IL-12, IL-
6, and IL-13) showed a positive correlation with serum levels of urea in
the renal tissues (r ≥ 0.64). Th1 cytokines (IL-6, and IL-12) induce
inflammation. Th2 cytokines (IL-4, IL-10 and IL-13) stimulate an accu­
mulation of collagen. Exacerbated inflammatory response in the kidney
causes tissue damage (Andrade-Oliveira et al., 2019; Black et al., 2019)
that compromises physiological renal function. Collagen deposition
change the morphological structure of the kidney and therefore its renal
function is deteriorating (Bülow et al., 2019), which is being expressed
by altered biochemical serum indicators, as urea. The kidney plays a role
in the clearance of pro-inflammatory cytokines (Andres-Hernando et al.,
2012). IL-4 and IL-10 are frequently up-regulated cytokines in patients
with end-stage renal disease (Akchurin and Kaskel, 2015; Girndt et al.,
2003) and are represented as counter-regulatory mechanisms to atten­
uate uremia-induced immune activation of pro-effectors Th1 inflam­
matory agents (Krensky et al., 2007).
Our data showed that type I fibrillar collagen (dense) was more
frequent than type III fibrillar collagen (elastic) in the interstitial space.
Once the myofibroblasts, which are metabolically active cells with high

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B.L.A. Verçosa et al. Molecular Immunology 156 (2023) 61–76

Table 3
Correlation between mRNA expression of cytokines/chemokines and morpho­
metric values in kidney lesions of L. chagasi naturally infected dogs (clinically
affected and subclinical infected).
Cytokines/chemokines Interstitial collagen (area) Adventitial collagen (area)
(r, n, p)* (r, n, p)*

IL-4 0.61, 12, 0.03 ns

IL-6 ns ns
IL-12 ns 0.67, 11, 0.02
IL-13 ns ns
TGF-β ns 0.67, 13, 0.01
IFN-γ ns ns
TNF-α ns ns
MCP-1 ns ns
MCP-2 ns ns
CCL4 ns ns
CCL5 ns ns
MCP-1/IL-12 0.80, 12, 0.0016 ns
IL-4/IFN-γ ns 0.87, 09, 0.002 Fig. 10. Correlation between agonistic (CCL5) and antagonist (IL-6) genes
TGF-β/TNF-α ns 0.78, 10, 0.007
related with adventitial and interstitial collagen deposition in clinically affected
IL-13/TNF-α ns ns
Leishmania-infected dog, r = 0.77, p = 0.0093, Spearman test.
IL-13/IL-12 ns ns
IL-13/IFN-γ ns ns
IL-13/IL-6 ns ns in Leishmania sp. infection, in which there is a total replacement of
IL-4/TNF-α ns ns
elastic collagen type III for dense collagen type I.
IL-4/IL-12 ns ns
IL-4/IL-6 ns ns Inflammation and collagen deposition in kidney of controls were
TGF-β/IL-12 ns ns attributed to the fact that it was assessed homeless dogs, captured as part
TGF-β/TNF-α ns ns of a program for the control of VL and urban rabies. Therefore, it was not
TGF-β/IFN-γ ns ns possible to know previous health and nutritional conditions of the
TGF-β/IL-6 ns ns
control dogs in this study, which was a limitation of the study. However,
MCP1/TNF-α ns ns
MCP1/IFN-γ ns ns the clinical examination and autopsy showed no evidence of other dis­
MCP1/IL-6 ns ns eases, suggesting that the sparse inflammatory foci and minimum
CCL5/TNF-α ns ns collagen deposition found in only one kidney of controls could be caused
CCL5/IFN-γ ns ns
by a previous unspecified condition. Other possible limitation of this
CCL5/IL-6 ns ns
study is that to classify the dogs in the three study groups it was
* Spearman test, data are considered significant p < 0.05. ns: data without considered only the clinical condition by physical examination. It was
statistical significance. not considered the concentrations of acute phase proteins, as described
by Ceron et al. (2018) and Lamothe (2010). In addition, it would have
been interesting to evaluate proteinuria in this study.
Table 4
In conclusion, our results suggested that the renal collagen deposi­
Correlations between agonistic (TGF-β, IL-13, IL-4, MCP-1 and CCL5) and
tion is a consequence of immunopathologic mechanisms, involving
antagonist (TNF-α, IFN-γ, IL-12 and IL-6) genes related with adventitial and
interstitial collagen deposition in subclinical infected and clinically affected much more the injury of the vasculature structure and the stroma, rather
Leishmania-infected dog. than the injury of the epithelium. These results support the role played
by CCL5/IL12 and MCP1/IL-12 in the cascade of events that culminate
Cytokines/chemokines Groups
in the host’s defense against Leishmania’s action. Moreover, the IL-4/
Subclinical infected Clinically affected IFN-γ ratio was more commonly expressed in clinically affected dogs,
agonistic x antagonist p-value* r-squared p-value* r-squared clearly associated with an intense renal inflammation, coupled with
TGF-β x TNF-α 0,4622 0,1125 0,5374 0,1019 adventitial and interstitial collagen depositions. These data broaden our
TGF-β x IFN-γ 0,9568 0,0006475 0,3061 0,2557
knowledge on physiopathogenic mechanisms involved in determining
TGF-β x IL-12 0,3655 0,1652 0,0725 0,5949
IL-13 x IFN-γ 0,6344 0,04871 0676 0,03783 fibrosis in Leishmania-infected dogs and may suggest a novel possibility
IL-13 x IL-6 0,1741 0,3341 0,9457 0,001023 of treatment for chronic kidney disease caused by fibrosis.
IL-13 x TNF-α 0,0588 0,5433 0,2207 0,2813
IL-4 x IFN-γ 0,9464 0,000997 0,6536 0,0435 Funding
IL-4 x IL-6 0,1745 0,3335 0,0826 0484
CCL5 x IL − 12 0,0619 0623 0,0903 0,4674
IL-13 x IL-12 0,7963 0,01462 0,8716 0,005752 This study was supported by the Fundação de Amparo à Pesquisa do
MCP-1 x IL-12 0,4009 0,1441 0,3445 0,1789 Estado de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento
TGF-β x IL-6 0799 0,01422 0,4793 0,1046 de Pessoal de Nível Superior (CAPES), and Conselho Nacional de
MCP-1 x TNF-α 0,3185 0197 0,6716 0,03892
Desenvolvimento Científico e Tecnológico (CNPq). Bárbara L. A. Ver­
MCP-1 x IL-6 0,9319 0,001611 0,6573 0,04252
CCL5 x TNF-α 0,9865 0,00008155 0,7366 0,02468 çosa was supported by CAPES (Programa de Pós-graduação em Biologia
CCL5 x IL-6 0833 0,0125 0,0093 0771 Celular – ICB/UFMG) and was an investigator supported by the Con­
selho Nacional de Desenvolvimento Científico e Tecnológica (CNPq),
* Spearman test, Bold: data are considered significant p < 0.05.
Brazil (process number 168270/2017-0). Maria Imaculada Muniz-
Junqueira was an investigator supported by the Conselho Nacional de
proinflammatory and fibrogenic capacity, are related to the deposition
Desenvolvimento Científico e Tecnológico (CNPq), Brazil (process
of type III collagen (Wynn, 2008), our data suggest that the factors
number 308344/2016-2).
involved in interstitial collagen depositions in Leishmania-infected dogs
were not linked to myofibroblasts, but possibly to other factors, such as
Th2 cytokines and chemokines. Moreover, these factors may be related
to the process of development and evolution of the renal fibrotic process

73
B.L.A. Verçosa et al.
CRediT authorship contribution statement
Barbara Laurice Araújo Verçosa: Conceptualization, Data cura­
tion, Formal analysis Investigation, Methodology, Validation, Visuali­
zation, Writing – original draft, Writing – review & editing. Maria
Imaculada Muniz-Junqueira: Writing – review & editing. Daniel
Meneses-Sousa: Data curation, Formal analysis. Ricardo Toshio Fuji­
wara: Data curation, Formal analysis. Luciano de F. Borges: Data
curation, Formal analysis; Writing – review & editing. Maria Norma
Melo: Resources, Supervision, Validation, Visualization. Anilton Cesar
Vasconcelos: Funding acquisition, Project administration, Resources;
Supervision, Validation, Visualization.
Conflict of interest
The authors claim no conflict of interests.
Data Availability
No data was used for the research described in the article.
Acknowledgments
The study was supported by the FAPEMIG, CAPES (Scholarship to
BLAV), and CNPq (fellowships to MNM and AVC). Bárbara L. A. Verçosa
was supported by CAPES (ICB/UFMG) and was an investigator sup­
ported by CNPq, Brazil (process number 168270/2017–0). Maria Ima­
culada Muniz-Junqueira was an investigator supported by the CNPq,
Brazil (process number 308344/2016–2). We acknowledge the Labo­
ratory of Histopathology at the Department of General Pathology of the
Universidade Federal de Minas Gerais for their histopathological prep­
arations. We would also like to thank Antônio Francisco de Sousa,
Francisco Assis Lima Costa (in memorian), and Ivete Lopes Mendonça
(Departamento de Clínica e Cirurgia Veterinária, Centro de Ciências
Agrárias, Universidade Federal do Piauí, Teresina, Piauí, Brasil) for
supporting us with the collection of samples.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.molimm.2023.02.010.
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