Int. J. Exp. Pathol. (2016), 97, 139–149
ORIGINAL ARTICLE
Immunohistochemical study of hepatic fibropoiesis associated with canine visceral leishmaniasis Igor M. V. M. Madeira, Debora. M. O. Pereira, Aline. A. Sousa, Cesar A. Vilela, Izabela F. G. Amorim, Marcelo V. Caliari, Carolina C. Souza and Wagner L. Tafuri Departamento de Patologia Geral, Instituto de Ci^encias Biol ogicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil
INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY
SUMMARY
Hepatic fibropoiesis has been confirmed in canine visceral leishmaniasis. In fibrotic disease, hepatic stellate cells (HSC) play an important role in fibropoiesis, undergoing activation by TGF-b to acquire characteristics of myofibroblasts. These cells show extensive capacity for proliferation, motility, contractility, collagen synthesis and extracellular matrix component synthesis. The aim of this work was to identify markers of HSC activation in 10 symptomatic and 10 asymptomatic dogs naturally doi: 10.1111/iep.12179 infected with Leishmania (Leishmania) infantum. Eight uninfected dogs were used as controls. Alpha-actin (a-SMA), vimentin and cytokeratin were investigated by immunohistochemistry as HSC markers. The cytokine TGF-b in tissue was also evaluated by immunohistochemistry. All infected dogs showed higher numbers of reticular fibres than controls. Fibropoiesis found in infected dogs was always associated with the presence of parasites and chronic granulomatous hepatitis. Positive correlation Received for publication: 14 July 2015 Accepted for publication: 13 February was found among fibropoiesis, parasite tissue load and expression of a-SMA. There 2016 was no correlation between fibropoiesis, vimentin and cytokeratin markers. The expression of cytokine TGF-b was higher in infected dogs than in controls, but not Correspondence: Wagner Luiz Tafuri significantly different between symptomatic and asymptomatic dogs. These results Departamento de Patologia Geral confirm previous work describing the intense hepatic fibropoiesis in dogs naturally Instituto de Ci^encias Biol ogicas Universidade Federal de Minas Gerais infected with Leishmania infantum, but now associated them with overexpression of Av. Ant^ onio Carlos 6627 TGF-b, where a-SMA may be a superior marker for activated HSC cells in CVL. Belo Horizonte, CEP 31270-901 MG, Brasil Tel.: +55 31 3409 2889 Fax: +55 31 3409 2879 E-mail: [email protected]
Keywords alpha-actin (a-SMA), canine visceral leishmaniasis, cytokeratin (CK19), hepatic fibrosis, TGF-b, vimentin
Liver disease and various associated histopathological changes have been described in canine visceral leishmaniasis (CVL) (Tryphonas et al. 1977; Anosa & Idowu 1983; Keenan et al. 1984; Sanchez et al. 2004; Rallis et al. 2005; Melo et al. 2008, 2009) and human visceral disease (Rogers 1908; Bogliolo 1956; Andrade & Andrade 1966; Duarte & Corbett 1987; Corbett et al. 1993; Duarte et al. 2009). Pathological changes in the liver extracellular matrix (ECM) of individuals diagnosed with leishmaniasis have long been described in the literature, with the first report by Rogers (1908), studying an Indian kala azar case, and later described by Nattan-Larrier (1918) as a diffuse hepatic fibrosis which he called Rogers cirrhosis. In Brazil, Bogliolo (1956) provided a detailed description of intense and diffuse hepatic intralobular fibrosis in a 19-year-old male diagnosed with visceral
leishmaniasis. He described the formation of reticular collagen fibres on the sinusoidal walls and around the hepatocytes, where they induced atrophy of the hepatocytes. These alterations have since been shown in six isolated Brazilian cases of human visceral leishmaniasis (Da Silva & De Paola 1958; Raso & Siqueira 1964; Duarte & Corbett 1987). In CVL, studies conducted by our group showed intense collagen deposition in many organs including liver, lung, kidney, lymph nodes and spleen, regardless of the clinical form of the disease (Tafuri et al. 1996; Goncßalves et al. 2003; Melo et al. 2008, 2009; Silva et al. 2013). Injury-associated liver collagen is synthesized by portal fibroblasts or sinusoid hepatic stellate cells (HSC) differentiated in myofibroblasts (Guyot et al. 2006). In addition to HSC, liver precursor cells (LPC) play an important role in
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liver fibrosis, differentiating into hepatocytes or cholangiocytes, which are epithelial cells involved in bile duct formation. Thus, many studies have shown a strong relationship between severity of fibrosis and bile duct formation (Clouston et al. 2005; Richardson et al. 2007; Knight et al. 2007; Friedman 2000; Van Hul et al. 2009). Because most liver hepatocytes have decreased replicating capacity, liver regeneration following injury depends mainly on LPC activation. It was recently shown that inhibition of HSC activation drastically reduced the proliferation of LPCs (Lorenzini et al. 2010; Pintilie et al. 2010), suggesting a major role of these cells in the pathogenesis of liver fibrogenesis. This relationship has been shown to increase fibrogenesis indirectly through the recruitment of innate immune cells (Ruddell et al. 2009). Although the cellular basis of liver fibrogenesis has been revealed for some diseases, it has not been explored for CVL. Here, we investigated some aspects of this question by analysing the expression pattern of different known fibrosis markers in livers of dogs naturally infected with Leishmania (L.) infantum.
Materials and methods Animals Adult mixed-breed dogs of unknown age of both sexes were obtained from the Control Zoonosis Center of the Municipality of Ribeir~ao das Neves, in the metropolitan area of Belo Horizonte, Minas Gerais (MG) State, Brazil. Leishmania (Leishmania) infantum infection was diagnosed using the indirect immunofluorescence antibody test (IFAT) (titre >1:40) and enzyme-linked immunosorbent assay (ELISA) (optical density >100; 1:400 dilutions). Other eight uninfected dogs with negative serological and parasitological examinations (IFAT and ELISA) from the city of Carandaı, MG (non-endemic area), Brazil, were used as controls. The dogs were screened for parasites using impression smears of bone marrow, ear biopsies and immunohistochemistry. Dogs were quarantined for at least 40 days in kennels at the Instituto de Ci^encias Biol ogicas (ICB), Universidade Federal de Minas Gerais, with drinking water and balanced commercial food (Nero Refeicß~ao, Total Alimentos, Brazil) provided ad libitum prior to the initiation of the study. Dogs were treated for intestinal helminths (Helfine c~aesâ, Agener Uni~ ao, Apucarana, PR, Brazil) and ectoparasites (Frontline Top Spotâ, Merial, Brazil) and vaccinated against rabies (Defensorâ, Pfizer Sa ude Animal, S~ao Paulo, SP, Brazil) and other infectious diseases (Vanguard HTLP 5/CV-Lâ, Pfizer Sa ude Animal). We did not carry out serological examinations for Ehrlichia for these dogs. At the end of the quarantine period, in a group of twenty infected dogs, these animals were physically examined and classified as asymptomatic (n = 10) (no clinical signs of disease) or symptomatic (n = 10), with classical disease: (i) body condition (weight loss or cachexia and clinical anaemia); (ii) enlarged cervical and popliteal lymph nodes; and (iii) dermatological symptoms (alopecia, dry exfoliative
dermatitis or ulcers, onychogryphosis, keratoconjunctivitis) (Amorim et al. 2011).
Parasitological analysis To confirm diagnosis of Leishmania infection, all dogs were anesthetized with 2.5% (0.5 ml/kg) intravenous thiopental and bone marrow aspirates were taken. The smears were air-dried and stained with 10% Giemsa. Leishmania amastigotes were detected in all infected animals by light microscopy (1000 9 magnification). The entire slide fields of bone marrow smears were examined. Control animals were parasite-negative.
Histology Dogs were anesthetized with 0.5 ml/kg ketamine and xylazine and euthanized with thiopental i.v. (2.5%). Liver samples were collected and fixed in 10% neutral buffered formalin, prepared conventionally for histology, cut into 4-lm sections and stained with haematoxylin and eosin (H&E). Liver lesions were detected and graded. Sample fragments were stained with Gomori’s ammoniacal silver for collagen detection as described previously (Melo et al. 2008, 2009) and analysed morphometrically to characterize intralobular collagen deposition, excluding perivascular collagen.
Immunohistochemistry: anti-Leishmania amastigotes, TGF-b, vimentin, a-SMA and cytokeratin (CK19) We carried out distinct immunohistochemical protocols in accordance with each antigen. However, all slides with tissue sections were previously dewaxed, hydrated, incubated in 4% hydrogen peroxide (30 v/v) in 0.01 M phosphate-buffered saline (PBS; pH 7.2) to block endogenous peroxidase activity and incubated with goat normal serum (1:100 dilution) to block non-specific immunoglobulin absorption. For the detection of Leishmania amastigotes, serum from dogs naturally infected with Leishmania (Leishmania) infantum (infected dog obtained from metropolitan area of Belo Horizonte, MG, Brazil) was diluted 1:100 with 0.01 M PBS and used as the primary antibody (Tafuri et al. 2004). Slides were incubated in humid conditions at 4°C for 18–22 h, washed in PBS, incubated with biotinylated goat anti-mouse and anti-rabbit Ig (Dako, Carpinteria, CA, 192 USA; LSAB2 kit), washed and then incubated with streptavidin–peroxidase complex (Dako; LSAB2 kit) for 20 min at room temperature. Reactions were visualized by applying the Liquid DAB + Substrate Chromogen System (Dako, K3468) for 5–10 min, with Harris’ haematoxylin counterstaining for 5 min. For vimentin, a-SMA and cytokeratin (HSC markers) immunohistochemical characterization, we employed specific primary antibodies (Table 1). Slides were dewaxed and hydrated, and antigen retrieval was conducted by a 30-min water bath (98°C) in Target Retrieval Solution 1% (Dako). The slides were treated with methanol containing 0.3% hydrogen International Journal of Experimental Pathology, 2016, 97, 139–149
Dog Leishmania Liver Fibrosis peroxide for 15 min at room temperature to inactivate endogenous peroxidase. The following steps were as described above. Leishmania amastigotes or cells positive for vimentin, aSMA, and cytokeratin stained brown, and all fields were examined from each tissue sample. The assay for TGF-b was similar, but washing was with 0.01 M PBS and Tweenâ 1%. Antigen retrieval, in this case, was performed using Pascal pressure cooker (DakoCytomation) according to the manufacturer’s instructions.
Morphometric analysis for interstitial matrix alterations (collagen deposits), a-SMA, vimentin, cytokeratin and the cytokine TGF-b Twenty random images from slides of liver tissue were used for morphometric analysis using an Axiolab light microscope (Zeiss) with 440 9 resolution. The images were relayed to an image analysis system (Kontron Elektronik image analyser, Carl Zeiss, Germany – KS300 software). Using a digital pad, the total area stained for collagen, a-SMA, vimentin, cytokeratin and TGF-b was quantified from real images and segmented to generate binary images (Caliari 1997). These results were expressed in lm2 according to Melo et al. (2008, 2009).
Liver parasite load Immunolabelled amastigotes were counted in 20 random images (Figueiredo et al. 2010). Horizontal and vertical movements were carried out using the microscope stage – XY translation to avoid overlapping fields. The images were analysed as described for TGF-b, viewed on a screen and relayed to a computer-assisted image analysis system (Kontron Elektronik).
Statistical analysis Statistical analyses were carried out with GraphPad Prism version 5.0. Two pathologists made the reading of each slide in each blade without prior knowledge of clinical status of dogs. All analyses were performed randomly, and the results grouped into symptomatic infected, asymptomatic infected or uninfected (controls). For parametric data (Pearson’s coefficient) obtained, we compared all groups using one-way ANOVA and Tukey’s post hoc tests followed by residual analyses to check error distribution and appropriateness of the normal model. Student’s t-test was used for parasite load data (immunolabelled amastigotes). For nonparametric data (Spearman’s coefficient), Kruskal–Wallis test was carried out on morphometric analysis of collagen deposits (Gomori’s ammoniacal silver staining). Differences were considered significant when P ≤ 0.05.
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Care and Use Committee, which also reviewed and approved this work (CETEA, Universidade Federal de Minas Gerais, protocol no. 285/2010 approved at 8 June 2011 and validated through at 8 June 2016).
Results Histology Macroscopically, the livers of all infected animals were generally enlarged. Thus, hepatomegaly was not necessarily found in symptomatic dogs; it varied markedly in association with dark red coloration, indicating congestion. Haematoxylin–eosin staining showed a general chronic inflammatory reaction involving the entire architecture of the liver including the capsule, portal tracts, central veins and perisinusoidal spaces. In general, infected animals showed a chronic inflammatory exudate of mononuclear cells (plasma cells, macrophages and lymphocytes) diffuse in the Glisson’s capsule, focal in the hepatic portal tract or present as an intralobular granuloma formation. Degenerative hepatocyte lesions (hydropic and steatosis), hypertrophy and hyperplasia of Kupffer cells and congestion with deposits of hemosiderin pigment in the sinusoids were also observed (Figure 1a,b). Histological screening focused on the detection and grading of the following abnormalities in livers: (i) capsular inflammation; (ii) portal inflammation; (iii) presence of intralobular granulomas; (iv) hypertrophy and hyperplasia of Kupffer cells; (v) degenerative hepatocyte lesions (hydropic and steatotic); (vi) congestion; and (vii) haemosiderin deposits. Changes were evaluated semiquantitatively as absent or present and graded according to intensity over the entire tissue slide: (N) no histological alterations; (Si) slight (20–30%); (M), moderate (31–60%); and (Sv), severe (>60%) (Table 2). Intralobular granulomas were observed in 90% of infected dogs. They were present with variable size and morphology (oval or ellipsoid nodule exudate) constituted by macrophages, some epithelioid cells, small numbers of lymphocytes, plasma cells and, rarely, neutrophils (Figure 1c–e). Intracellular amastigote forms of Leishmania were found inside macrophages of intralobular granulomas or in Kupffer cells (data not shown). No significant differences were found in the parasite load of asymptomatic and symptomatic dogs.
Table 1 Primary antibodies used for immunohistochemistry Molecules/Markers*
Source (clone)
Dilution
Ethical approval statement
a-SMA 1A4 Vimentina V9 Cytokeratin CK 19 (AE1/AE3) TGF-b
Dako Dako Dako Santa
1/100 1/100 1/100 1/2500
Housing, anaesthesia and all procedures were in compliance with the guidelines established by the Institutional Animal
*For all immunohistochemistry assays, we used goat normal serum. or PBS as a control of antibodies
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(M0851) (M0725) (M3515) Cruz (sc-146)
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(a)
(b)
(c)
(d)
Figure 1 (a–e) Liver sections of dogs naturally infected with L. (L) infantum: (a) Asymptomatic dog: (A) Portal space with a cellular infiltrated of plasma cells, lymphocytes and macrophages (cross). Note congestion of a branch of portal vein (PV) (bar = 32 lm). (b) Higher magnification showing the mononuclear cellular exudate (bar = 16 lm). (c) Symptomatic dog. Observe three granulomas’ formation into the hepatic lobule (intralobular granulomas) (bar = 32 lm). (d) Higher magnification showing intralobular granuloma comprising of mononuclear cell macrophages, lymphocytes and plasma cells). Note an intense swelling of hepatocytes with hydropic degeneration (‘balloon cells’) (crosses) (bar = 16 lm). (e) Highest magnification (oil immersion objective) showing an epithelioid cell (*) with an elongated or oval nucleus and a pink cytoplasm with indistinct cell boundaries (appearing to merge into one another) (arrow) confirming ‘an epithelioid granuloma’ formation, lymphocytes (**) and plasma cells (***) (bar = 6 lm). Haematoxylin–eosin (HE) staining.
(e)
Extracellular matrix alterations (fibropoiesis) Staining of hepatic reticular fibres by Gomori’s ammoniacal silver demonstrated increased collagen deposits in all infected dogs compared to control dogs. Fibre deposition was found in the portal space region and in sinusoids in the hepatic lobe walls. Reticular fibres were diffusely spread in several directions, forming a compact network, and in some cases, fibres encircled groups of hepatocytes or a single cell (Figure 2a,b). As seen, hepatic reticular fibres in infected dogs were thicker than those in controls. In fact, morphometric analysis showed a higher fibropoiesis in both symptomatic and asymptomatic infected dogs than in controls (P < 0.0001) (Figure 3). Otherwise, symptomatic animals showed more intense fibropoiesis than asymptomatic dogs and controls.
There was a positive correlation between hepatic collagen deposition and the tissue parasite load (Figure 4).
Expression of TGF-b TGF-b expression was measured by morphometric analysis taking into consideration two staining patterns in the hepatic parenchyma: a diffuse staining mostly localized along the sinusoids and the localized staining expressed by the mononuclear cells of the granulomas (hepatic intralobular granulomas). In fact, we found intense expression (positive cells) in the entire liver parenchyma, mainly along the sinusoids. As intralobular granulomas were formed from Kupffer cells, we observed intense positivity in all cases of chronic hepatitis (Figure 5a,b). Thus, both asymptomatic and symptomatic dogs showed higher expression of TGF-b than did the control
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Table 2 Histopathological evaluation of liver in canine visceral leishmaniosis (Leishmania L. infantum) Symptomatic n = 10
Asymptomatic n = 10
Control n = 8
Histopathology
N
Si
M
Sv
P ≤ 0.05a D / F
N
Si
M
Sv
P ≤ 0.05a D / F
N
Sl
M
Sv
Capsular inflammation Portal inflammation Presence of intralobular granulomas Hypertrophy and hyperplasia of Kupffer cells Degeneration Congestion Hemosiderin
2 1 1 3
3 3 6 3
4 5 2 3
1 1 1 1
* * * *
* * * *
3 1 1 2
2 5 5 5
5 3 3 3
0 1 1 0
* * * *
* * * *
5 4 8 5
3 4 0 3
0 0 0 0
0 0 0 0
1 1 3
2 3 3
4 3 3
3 3 1
* **/ * */* */*
2 1 3
3 3 4
3 4 2
2 2 1
*/* */* */*
4 3 6
3 4 2
0 1 0
1 0 0
/ / / /
/ / / /
a
Qualitative analyses Frequency (F): presence or absence and semi-quantitative analyses of tissue changes, taking into account their distribution throughout the histological section and classified as follows: – Distribution (D): no histological alterations (N); slight (Si), moderate (M) and severe (Sv) histological alterations. Significant differences (P ≤ 0.05) relative to control are represented by * and relative to asymptomatic by**.
(a)
(b)
Figure 2 (a-b) Liver sections of uninfected and infected dogs naturally infected with L. (L.) infantum: (a) Control dog – note a delicate network (weak colour black staining) of intralobular hepatic reticular fibres (arrows). (b) Symptomatic dog – note conspicuous collagen thickening in the space of Disse (stronger black staining of intralobular reticular fibres) (arrows). Some reticular fibres are coiled (arrowhead). Gomori’s ammoniacal silver staining (bar = 32 lm). (S) Sinusoid blood vessel. International Journal of Experimental Pathology, 2016, 97, 139–149
Figure 3 Morphometric analyses of collagen (reticular fibres) deposition in livers of dogs naturally infected with L. infantum. *Significant difference between asymptomatic and symptomatic dogs compared to controls. ** Significant difference between asymptomatic and symptomatic dogs. One-way ANOVA and Tukey’s post hoc tests (P < 0.001).
Figure 4 Positive correlation between tissue parasite load and collagen deposition in livers. (Pearson’s r = 0.4906; P = 0.0281).
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(a)
(b)
Figure 5 (a, b) Liver sections of a symptomatic infected dog naturally infected with L. (L) infantum: (a) Intralobular hepatic granulomas showing intense expression of TGF-b (arrows). (b) Higher magnification showing positive cells of the intralobular granulomas. Also note TGF-b positivity in sinusoid blood wall. Immuno-streptavidin–peroxidase method (bar = 16 lm). (S) Sinusoid blood vessels.
Figure 6 Morphometric analyses of TGF-b in livers of naturally infected dogs with L. (L.) infantum. (*) Significant difference between asymptomatic and symptomatic dogs compared to controls. One-way ANOVA and Tukey’s post hoc tests (P < 0.001).
(a)
(b)
(c)
Figure 7 Liver sections of a symptomatic dog naturally infected with L. (L) infantum: (a) Note vimentin-positive focal cells in the sinusoids and in the portal area mainly around vessels indicating spindle-shaped stromal cells (arrows) (bar = 32 lm). (b) Now observe a-actin-positive cells diffusely distributed throughout the entire liver parenchyma (arrows). (c) Detail of letter (b) showing the positivity of a-actin in the hepatic lobule in the sinusoid vessels (arrowheads) (bar = 16 lm). Immunostreptavidin–peroxidase method.
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(a)
Figure 8 Morphometric analyses of a-actin in livers of naturally infected dogs with L. (L.) infantum. *Significant difference between asymptomatic and symptomatic dogs compared to controls. One-way ANOVA and Tukey’s post hoc tests (P < 0.001).
(b)
(c)
Figure 9 Positive correlation between positive a-SMA reactivity and collagen deposition in livers. (Pearson’s r = 0.651; P = 0.001).
group (Figure 6) (P < 0.05). However, we did not find differences between asymptomatic and symptomatic dogs.
Expression of vimentin Vimentin expression observed by immunohistochemistry showed variable intensity among and within all groups, although the tissue distribution pattern was similar. As a result, we did not observe differences among the groups. Thus, in all groups, we observed positive focal cells in the sinusoids and in the portal area, mainly around vessels, indicating spindle-shaped stromal cells (fibroblasts) (Figure 7a). There was no positive correlation between vimentin reactivity and collagen deposition (Pearson’s r = 0.051, P = 0.827).
Expression of a-SMA Positive cells were observed diffusely distributed throughout the liver parenchyma in the hepatic lobule in the sinusoid
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Figure 10 a,c: Liver sections of an asymptomatic dog naturally infected with L. (L) infantum: (a) Note cytokeratin biliary ducts’ epithelial positive cells in the portal area (arrows) and biliary tree (arrowheads). (b) Observe epithelial positivity cells throughout the biliary tree. (c) No positive cells in hepatocytes, in sinusoid cell wall and/or in intralobular granulomas formation were noticed (arrow). Immuno-streptavidin–peroxidase method. (bar = 32 lm).
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Figure 11 Morphometric analyses of cytokeratin (CK19) in livers of naturally infected dogs with L. (L.) infantum. *Significant difference between asymptomatic and symptomatic dogs compared to controls. One-way ANOVA and Tukey’s post hoc tests (P < 0.001).
vessels and in the portal area, notably around the biliary ducts. Endothelial cells were always negative (Figures 7b–c). The a-SMA expression was higher in infected groups than in controls. We did not find differences between asymptomatic and symptomatic dogs (Figure 8). There was a positive correlation between positive a-SMA reactivity and collagen deposition (Figure 9).
Expression of cytokeratin We observed a similar pattern of cytokeratin tissue expression in all cases. Immunolabelled cells in the hepatic portal space were restricted to epithelial cells of intrahepatic bile ducts (Figure 10a,b). Thus, we did not find cytokeratin in hepatocytes, in sinusoid cell walls and/or in intralobular granuloma formations (Figure 10c). However, infected animals showed higher expression of cytokeratin than did control dogs. There were no significant differences between asymptomatic and symptomatic dogs (Figure 11). There was no positive correlation between positive cytokeratin reactivity and collagen deposition (Spearman’s r = 0.2060; P = 0.3835) or positive cytokeratin reactivity and a-SMA (Pearson r = 0.1474; P = 0.5352).
Discussion We demonstrated hepatic disorders in dogs with L. infantum resulting from chronic inflammation associated with fibrotic disease characterized as perihepatocytic deposits of extracellular matrix components, mainly collagen fibrils. These data are similar to the findings of our previous works (Tafuri et al. 1996; Melo et al. 2008, 2009; Souza et al. 2014) and others (Rallis et al. 2005). Otherwise, Souza et al. (2014) working with thirty naturally infected dogs with L. infantum showed deposition of fibrous connective
tissue in all organs of infected dogs, correlated with grade of inflammation and iron (haemosiderin) deposition in the tissues. They worked with dogs with negative serologicy for Ehrlichiosis. In fact, we have not found any previous evidence in the literature which correlates fibrosis and Ehrlichosis. For example, Mylonakis et al. (2010) described absence of myelofibrosis in dogs with myelosuppression induced by Ehrlichia canis infection. Thus taking the previous literature, and our own previous work (Souza et al. 2014) into account, we did not do Ehrlichosis serology as part of the present study. The livers of asymptomatic and symptomatic infected dogs generally displayed a chronic inflammatory reaction characterized by mononuclear cell infiltration in the capsule, hepatic portal space and entire hepatic parenchyma. Intralobular granuloma formations were observed in the majority of the cases (90%). A similar finding has been described in experimental murine visceral leishmaniasis models (Murray 2001), in dogs experimentally infected with Leishmania donovani and L. infantum (Oliveira et al. 1993; Tafuri et al. 1996) and in dogs naturally infected with L. infantum (Tafuri et al. 1996; Sanchez et al. 2004; Lima et al. 2007). In conjunction with this chronic inflammation, Gomori’s ammoniacal silver staining revealed higher numbers of collagen fibres in livers of infected dogs than in controls. In naturally infected dogs, there was a positive correlation between hepatic collagen deposits and parasite load. Infected dogs showed more hepatic collagenogenesis (intralobular fibrosis), probably stimulated by the tissue parasite load, as suggested by Bogliolo (1956) and Corbett et al. (1993) in human visceral leishmaniasis. To investigate the possible origin of the fibropoiesis and better understand the role of HSCs and myofibroblasts, immunohistochemical assays characterizing TGF-b, vimentin, a-SMA and CK19 were performed. Injury-induced liver collagen is synthesized by portal fibroblasts or sinusoidlocated HSCs differentiated into myofibroblasts (Guyot et al. 2006). In addition to HSCs, LPCs play an important role in liver fibrosis. In spite of the current debate regarding the relationship between LPCs and HSCs, it is widely accepted that TGF-b plays a central role in the development of liver fibrosis after HSC and LPC activation (Valkova 2002; Schuppan et al. 2003; Gui et al. 2006). TGF-b initiates signalling through its interaction with receptors on the surface of HSCs, inducing differentiation of HSC into myofibroblasts and enhancing ECM synthesis (Niu and Zhao, 2007). TGF-b also inhibits the expression of collagenase and tissue inhibitors and increases the expression of matrixdegrading enzymes such as plasminogen activator and metalloproteinases. In addition, acting as a stimulant to change the phenotype of quiescent cells to myofibroblasts, TGF-b participates in the maintenance of this phenotype. Thus, TGF-b data in parallel with other myofibroblast marker results might play a role in hepatic fibrosis in chronic CVL. We observed expression of TGF-b in cells of intralobular granulomas. These data suggest that in contrast to other hepatic fibrosis models, granulomas probably International Journal of Experimental Pathology, 2016, 97, 139–149
Dog Leishmania Liver Fibrosis persist in the progression of CVL disease and actively express TGF-b (Gressner et al. 2002; Matsuzaki 2009). The HSCs in normal liver constitute the largest reserves of retinoids (vitamin A) in the body. Under stress and injury, they become activated and undergo phenotypic changes, including loss of vitamin A, production of fibrogenic cytokines and expression of new proteins such as alpha-actin (aSMA) and acquire myofibroblastic characteristics (Lepreux et al. 2001; Guyot et al. 2006; Zhao & Burt 2007). Our results showing increased staining of a-SMA indicate that HSC-derived myofibroblasts might also play a role in CVL liver fibrogenesis. Increased expression of a-SMA in perisinusoidal HSCs in areas of fibrosis around portal spaces and fibrous septa has been reported in chronic hepatitis in humans. In the course of the development of hepatic fibrosis in humans and rats, the number of HSCs increases, and they differentiate into myofibroblast-like cells with significant expression of a-SMA (Burt 1999; Boisclair et al. 2001; Lepreux et al. 2001). Activated HSCs in humans also synthesize tissue inhibitors of metalloproteinases 1 and 2 that inhibit interstitial collagenase. Thus, these higher metalloproteinase inhibitors’ tissue-level activity contributes to the accumulation of ECM proteins (Bedossa & Paradis 2003; Guyot et al. 2006; Wipff et al. 2007). The etiopathogenesis of chronic liver disease in dogs has not been sufficiently clarified, particularly the role of ECM proteins in the progression of hepatic fibrosis (Ijzer et al. 2006; Mekonnen et al. 2007). Van Hul et al. (2009) reported a close association between positive aSMA immunostaining and the differentiation of LPC cells. Possibly, the higher labelling of the cytokeratin (CK AE1/ AE3) observed in our study could indicate that fibrogenesis in liver also involves LPC differentiation into cholangiocytes, detected by the appearance of bile ducts. The cytoskeleton protein vimentin is considered a classic marker of cells of mesenchymal origin cells such as myofibroblasts and portal fibroblasts. Studies have shown the involvement of these cells in collagen deposition in fibrotic liver through the analysis of vimentin expression (Lepreux et al. 2001; Zhao & Burt 2007). However, we did not observe increased expression of vimentin in the livers of CVL-infected dogs. Moreover, expression of vimentin by portal fibroblasts did not vary between control and infected dogs. We did not find correlation between vimentin expression and collagen deposition. Thus, vimentin does not appear to be an adequate marker of myofibroblasts. Immunopositivity of perisinusoidal HSCs to a-SMA was seen in both the infected and control dogs; however, intensity and density in the infected group were higher than those in the controls. a-SMA is a known marker for myofibroblasts. The role of myofibroblasts is notable in hepatic fibrotic disease in dogs. Research indicates that inflammation in canine liver, as well as in livers of humans and rats, is connected to the activation of periductal myofibroblasts and probably to their proliferation of bile ducts (Burt 1999; Ijzer et al. 2006; Libbrecht et al. 2002; Mekonnen et al. 2007; Wipff et al. 2007). Myofibroblasts in normal livers of dogs, humans and rats are located around blood vessels and bile International Journal of Experimental Pathology, 2016, 97, 139–149
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ducts, and we observed their increased immunopositivity with intense fibropoiesis. Periductal myofibroblasts appear to be important in hepatic fibrosis, and when activated by various cytokines, the synthesis and secretion of proteins of the extracellular matrix are increased (Bedossa & Paradis 2003; Guyot 2006). In this work, we have confirmed the hepatic fibropoiesis in CVL. In fact, we still have found a positive correlation between hepatic collagen deposition and tissue parasite load. The origin of the fibropoiesis (injury-induced liver collagen) comes from portal fibroblasts or sinusoid-located HSCs differentiated into myofibroblasts. As mentioned, LPCs also play an important role in liver fibrosis and it is widely accepted in the literature that TGF-b plays a central role in the development of liver fibrosis after HSC and LPC activation. Thus, our TGF-b data showing an overexpression has suggested that this cytokine plays a central role in the development of liver fibrosis of dogs naturally infected with L. infantum where a-SMA may be a superior marker for activated HSC cells in CVL.
Acknowledgments The authors thank FAPEMIG for grant APQ-01378-12, CNPq for grant 474665/2012-7 and grant 303022/2013–2 and Lucidus Consulting for the English language revision.
Conflict of interest The authors declare that we do not have any conflict of interest in this work.
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correlation with necro-inflammatory activity, fibrotic stage, and expression of alpha-smooth muscle actin, cytokeratin 7, and CD3 + cells. Vet. Pathol. 44, 803–813. Melo F., Amaral M., Oliveira P. et al. (2008) Diffuse intralobular liver fibrosis in dogs naturally infected with Leishmania (Leishmania) chagasi. Am. J. Trop. Med. Hyg. 79, 198–204. Melo F.A., Moura E.P., Ribeiro R.R. et al. (2009) Hepatic extracellular matrix alterations in dogs naturally infected with Leishmania (Leishmania) chagasi. Int. J. Exp. Pathol. 90, 538–548. Murray H.W. (2001) Tissue granuloma structure-function in experimental visceral leishmaniasis. Int. J. Exp. Pathol. 82, 249–267. Mylonakis M.E., Day M.J., Siarkou V., Vernau W., Koutinas A.F. (2010) Absence of myelofibrosis in dogs with myelosuppression induced by Ehrlichia canis infection. J. Comp. Pathol. 142, 328– 331. Nattan-Larrier L. (1918) Les cirrhoses hepatiques due au Kalazar. Bull. Acad. Med. 89, 402–408. Niu Z.W. & Zhao H.W. (2007) [The effects of transforming growth factor-beta1 on the differentiation of BALB/c 3T3 fibroblasts into myofibroblasts and the change of Egr-1 expression]. Zhonghua Jie He He Hu Xi Za Zhi 30, 269–272. Oliveira G.G., Santoro F. & Sadigursky M. (1993) The subclinical form of experimental visceral leishmaniasis in dogs. Mem. Inst. Oswaldo Cruz 88, 243–248. Pintilie D.G., Shupe T.D., Oh S.H., Salganik S.V., Darwiche H., Petersen B.E. et al. (2010) Hepatic stellate cells’ involvement in progenitor-mediated liver regeneration. Lab. Invest. 90, 1199– 1208. Rallis T., Day M.J., Saridomichelakis M.N. et al. (2005) Chronic hepatitis associated with canine leishmaniosis (Leishmania infantum): a clinicopathological study of 26 cases. J. Comp. Pathol. 132, 145–152. Raso P. & Siqueira J.T. (1964) Subsıdio ao conhecimento da anatomia patol ogica da leishmaniose visceral, com especial refer^encia as les~ oes pulmonares e cardıacas. O Hospital 65, 1291–1309. Richardson M.M., Jonsson J.R., Powell E.E. et al. (2007) Progressive fibrosis in nonalcoholic steatohepatitis: association with altered regeneration and a ductular reaction. Gastroenterology 133, 80–90. Rogers L. (1908) A peculiar intralobular cirrhosis of the liver produced by the protozoal parasite of Kala-azar. Ann. Trop. Med. Parasitol. 2, 147–152. Ruddell R.G., Knight B., Tirnitz-Parker J.E. et al. (2009) Lymphotoxin-beta receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury. Hepatology 49, 227–239. Sanchez M.A., Diaz N.L., Zerpa O., Negron E., Convit J., Tapia F.J. (2004) Organ-specific immunity in canine visceral leishmaniasis: analysis of symptomatic and asymptomatic dogs naturally infected with Leishmania chagasi. Am. J. Trop. Med. Hyg. 70, 618–624. Schuppan D., Krebs A., Bauer M. & Hahn E.G. (2003) Hepatitis C and liver fibrosis. Cell Death Differ. 10, S59–S67. Silva L.C., Castro R.S., Figueiredo M.M., Michalick M.S. & Tafuri W.L. (2013) Canine visceral leishmaniasis as a systemic fibrotic disease. Int. J. Exp. Pathol. 94, 133–143. Souza C.C., Barreto T.O., da Silva S.M. et al. (2014) A potential link among antioxidant enzymes, histopathology and trace elements in canine visceral leishmaniasis. Int. J. Exp. Pathol. 95, 260–270. Tafuri W.L., Barbosa A.J., Michalick M.S. et al. (1996) Histopathology and immunocytochemical study of type 3 and
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ORIGINAL ARTICLE
Immunohistochemical study of hepatic fibropoiesis associated with canine visceral leishmaniasis Igor M. V. M. Madeira, Debora. M. O. Pereira, Aline. A. Sousa, Cesar A. Vilela, Izabela F. G. Amorim, Marcelo V. Caliari, Carolina C. Souza and Wagner L. Tafuri Departamento de Patologia Geral, Instituto de Ci^encias Biol ogicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil
INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY
SUMMARY
Hepatic fibropoiesis has been confirmed in canine visceral leishmaniasis. In fibrotic disease, hepatic stellate cells (HSC) play an important role in fibropoiesis, undergoing activation by TGF-b to acquire characteristics of myofibroblasts. These cells show extensive capacity for proliferation, motility, contractility, collagen synthesis and extracellular matrix component synthesis. The aim of this work was to identify markers of HSC activation in 10 symptomatic and 10 asymptomatic dogs naturally doi: 10.1111/iep.12179 infected with Leishmania (Leishmania) infantum. Eight uninfected dogs were used as controls. Alpha-actin (a-SMA), vimentin and cytokeratin were investigated by immunohistochemistry as HSC markers. The cytokine TGF-b in tissue was also evaluated by immunohistochemistry. All infected dogs showed higher numbers of reticular fibres than controls. Fibropoiesis found in infected dogs was always associated with the presence of parasites and chronic granulomatous hepatitis. Positive correlation Received for publication: 14 July 2015 Accepted for publication: 13 February was found among fibropoiesis, parasite tissue load and expression of a-SMA. There 2016 was no correlation between fibropoiesis, vimentin and cytokeratin markers. The expression of cytokine TGF-b was higher in infected dogs than in controls, but not Correspondence: Wagner Luiz Tafuri significantly different between symptomatic and asymptomatic dogs. These results Departamento de Patologia Geral confirm previous work describing the intense hepatic fibropoiesis in dogs naturally Instituto de Ci^encias Biol ogicas Universidade Federal de Minas Gerais infected with Leishmania infantum, but now associated them with overexpression of Av. Ant^ onio Carlos 6627 TGF-b, where a-SMA may be a superior marker for activated HSC cells in CVL. Belo Horizonte, CEP 31270-901 MG, Brasil Tel.: +55 31 3409 2889 Fax: +55 31 3409 2879 E-mail: [email protected]
Keywords alpha-actin (a-SMA), canine visceral leishmaniasis, cytokeratin (CK19), hepatic fibrosis, TGF-b, vimentin
Liver disease and various associated histopathological changes have been described in canine visceral leishmaniasis (CVL) (Tryphonas et al. 1977; Anosa & Idowu 1983; Keenan et al. 1984; Sanchez et al. 2004; Rallis et al. 2005; Melo et al. 2008, 2009) and human visceral disease (Rogers 1908; Bogliolo 1956; Andrade & Andrade 1966; Duarte & Corbett 1987; Corbett et al. 1993; Duarte et al. 2009). Pathological changes in the liver extracellular matrix (ECM) of individuals diagnosed with leishmaniasis have long been described in the literature, with the first report by Rogers (1908), studying an Indian kala azar case, and later described by Nattan-Larrier (1918) as a diffuse hepatic fibrosis which he called Rogers cirrhosis. In Brazil, Bogliolo (1956) provided a detailed description of intense and diffuse hepatic intralobular fibrosis in a 19-year-old male diagnosed with visceral
leishmaniasis. He described the formation of reticular collagen fibres on the sinusoidal walls and around the hepatocytes, where they induced atrophy of the hepatocytes. These alterations have since been shown in six isolated Brazilian cases of human visceral leishmaniasis (Da Silva & De Paola 1958; Raso & Siqueira 1964; Duarte & Corbett 1987). In CVL, studies conducted by our group showed intense collagen deposition in many organs including liver, lung, kidney, lymph nodes and spleen, regardless of the clinical form of the disease (Tafuri et al. 1996; Goncßalves et al. 2003; Melo et al. 2008, 2009; Silva et al. 2013). Injury-associated liver collagen is synthesized by portal fibroblasts or sinusoid hepatic stellate cells (HSC) differentiated in myofibroblasts (Guyot et al. 2006). In addition to HSC, liver precursor cells (LPC) play an important role in
© 2016 The Authors. International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology
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liver fibrosis, differentiating into hepatocytes or cholangiocytes, which are epithelial cells involved in bile duct formation. Thus, many studies have shown a strong relationship between severity of fibrosis and bile duct formation (Clouston et al. 2005; Richardson et al. 2007; Knight et al. 2007; Friedman 2000; Van Hul et al. 2009). Because most liver hepatocytes have decreased replicating capacity, liver regeneration following injury depends mainly on LPC activation. It was recently shown that inhibition of HSC activation drastically reduced the proliferation of LPCs (Lorenzini et al. 2010; Pintilie et al. 2010), suggesting a major role of these cells in the pathogenesis of liver fibrogenesis. This relationship has been shown to increase fibrogenesis indirectly through the recruitment of innate immune cells (Ruddell et al. 2009). Although the cellular basis of liver fibrogenesis has been revealed for some diseases, it has not been explored for CVL. Here, we investigated some aspects of this question by analysing the expression pattern of different known fibrosis markers in livers of dogs naturally infected with Leishmania (L.) infantum.
Materials and methods Animals Adult mixed-breed dogs of unknown age of both sexes were obtained from the Control Zoonosis Center of the Municipality of Ribeir~ao das Neves, in the metropolitan area of Belo Horizonte, Minas Gerais (MG) State, Brazil. Leishmania (Leishmania) infantum infection was diagnosed using the indirect immunofluorescence antibody test (IFAT) (titre >1:40) and enzyme-linked immunosorbent assay (ELISA) (optical density >100; 1:400 dilutions). Other eight uninfected dogs with negative serological and parasitological examinations (IFAT and ELISA) from the city of Carandaı, MG (non-endemic area), Brazil, were used as controls. The dogs were screened for parasites using impression smears of bone marrow, ear biopsies and immunohistochemistry. Dogs were quarantined for at least 40 days in kennels at the Instituto de Ci^encias Biol ogicas (ICB), Universidade Federal de Minas Gerais, with drinking water and balanced commercial food (Nero Refeicß~ao, Total Alimentos, Brazil) provided ad libitum prior to the initiation of the study. Dogs were treated for intestinal helminths (Helfine c~aesâ, Agener Uni~ ao, Apucarana, PR, Brazil) and ectoparasites (Frontline Top Spotâ, Merial, Brazil) and vaccinated against rabies (Defensorâ, Pfizer Sa ude Animal, S~ao Paulo, SP, Brazil) and other infectious diseases (Vanguard HTLP 5/CV-Lâ, Pfizer Sa ude Animal). We did not carry out serological examinations for Ehrlichia for these dogs. At the end of the quarantine period, in a group of twenty infected dogs, these animals were physically examined and classified as asymptomatic (n = 10) (no clinical signs of disease) or symptomatic (n = 10), with classical disease: (i) body condition (weight loss or cachexia and clinical anaemia); (ii) enlarged cervical and popliteal lymph nodes; and (iii) dermatological symptoms (alopecia, dry exfoliative
dermatitis or ulcers, onychogryphosis, keratoconjunctivitis) (Amorim et al. 2011).
Parasitological analysis To confirm diagnosis of Leishmania infection, all dogs were anesthetized with 2.5% (0.5 ml/kg) intravenous thiopental and bone marrow aspirates were taken. The smears were air-dried and stained with 10% Giemsa. Leishmania amastigotes were detected in all infected animals by light microscopy (1000 9 magnification). The entire slide fields of bone marrow smears were examined. Control animals were parasite-negative.
Histology Dogs were anesthetized with 0.5 ml/kg ketamine and xylazine and euthanized with thiopental i.v. (2.5%). Liver samples were collected and fixed in 10% neutral buffered formalin, prepared conventionally for histology, cut into 4-lm sections and stained with haematoxylin and eosin (H&E). Liver lesions were detected and graded. Sample fragments were stained with Gomori’s ammoniacal silver for collagen detection as described previously (Melo et al. 2008, 2009) and analysed morphometrically to characterize intralobular collagen deposition, excluding perivascular collagen.
Immunohistochemistry: anti-Leishmania amastigotes, TGF-b, vimentin, a-SMA and cytokeratin (CK19) We carried out distinct immunohistochemical protocols in accordance with each antigen. However, all slides with tissue sections were previously dewaxed, hydrated, incubated in 4% hydrogen peroxide (30 v/v) in 0.01 M phosphate-buffered saline (PBS; pH 7.2) to block endogenous peroxidase activity and incubated with goat normal serum (1:100 dilution) to block non-specific immunoglobulin absorption. For the detection of Leishmania amastigotes, serum from dogs naturally infected with Leishmania (Leishmania) infantum (infected dog obtained from metropolitan area of Belo Horizonte, MG, Brazil) was diluted 1:100 with 0.01 M PBS and used as the primary antibody (Tafuri et al. 2004). Slides were incubated in humid conditions at 4°C for 18–22 h, washed in PBS, incubated with biotinylated goat anti-mouse and anti-rabbit Ig (Dako, Carpinteria, CA, 192 USA; LSAB2 kit), washed and then incubated with streptavidin–peroxidase complex (Dako; LSAB2 kit) for 20 min at room temperature. Reactions were visualized by applying the Liquid DAB + Substrate Chromogen System (Dako, K3468) for 5–10 min, with Harris’ haematoxylin counterstaining for 5 min. For vimentin, a-SMA and cytokeratin (HSC markers) immunohistochemical characterization, we employed specific primary antibodies (Table 1). Slides were dewaxed and hydrated, and antigen retrieval was conducted by a 30-min water bath (98°C) in Target Retrieval Solution 1% (Dako). The slides were treated with methanol containing 0.3% hydrogen International Journal of Experimental Pathology, 2016, 97, 139–149
Dog Leishmania Liver Fibrosis peroxide for 15 min at room temperature to inactivate endogenous peroxidase. The following steps were as described above. Leishmania amastigotes or cells positive for vimentin, aSMA, and cytokeratin stained brown, and all fields were examined from each tissue sample. The assay for TGF-b was similar, but washing was with 0.01 M PBS and Tweenâ 1%. Antigen retrieval, in this case, was performed using Pascal pressure cooker (DakoCytomation) according to the manufacturer’s instructions.
Morphometric analysis for interstitial matrix alterations (collagen deposits), a-SMA, vimentin, cytokeratin and the cytokine TGF-b Twenty random images from slides of liver tissue were used for morphometric analysis using an Axiolab light microscope (Zeiss) with 440 9 resolution. The images were relayed to an image analysis system (Kontron Elektronik image analyser, Carl Zeiss, Germany – KS300 software). Using a digital pad, the total area stained for collagen, a-SMA, vimentin, cytokeratin and TGF-b was quantified from real images and segmented to generate binary images (Caliari 1997). These results were expressed in lm2 according to Melo et al. (2008, 2009).
Liver parasite load Immunolabelled amastigotes were counted in 20 random images (Figueiredo et al. 2010). Horizontal and vertical movements were carried out using the microscope stage – XY translation to avoid overlapping fields. The images were analysed as described for TGF-b, viewed on a screen and relayed to a computer-assisted image analysis system (Kontron Elektronik).
Statistical analysis Statistical analyses were carried out with GraphPad Prism version 5.0. Two pathologists made the reading of each slide in each blade without prior knowledge of clinical status of dogs. All analyses were performed randomly, and the results grouped into symptomatic infected, asymptomatic infected or uninfected (controls). For parametric data (Pearson’s coefficient) obtained, we compared all groups using one-way ANOVA and Tukey’s post hoc tests followed by residual analyses to check error distribution and appropriateness of the normal model. Student’s t-test was used for parasite load data (immunolabelled amastigotes). For nonparametric data (Spearman’s coefficient), Kruskal–Wallis test was carried out on morphometric analysis of collagen deposits (Gomori’s ammoniacal silver staining). Differences were considered significant when P ≤ 0.05.
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Care and Use Committee, which also reviewed and approved this work (CETEA, Universidade Federal de Minas Gerais, protocol no. 285/2010 approved at 8 June 2011 and validated through at 8 June 2016).
Results Histology Macroscopically, the livers of all infected animals were generally enlarged. Thus, hepatomegaly was not necessarily found in symptomatic dogs; it varied markedly in association with dark red coloration, indicating congestion. Haematoxylin–eosin staining showed a general chronic inflammatory reaction involving the entire architecture of the liver including the capsule, portal tracts, central veins and perisinusoidal spaces. In general, infected animals showed a chronic inflammatory exudate of mononuclear cells (plasma cells, macrophages and lymphocytes) diffuse in the Glisson’s capsule, focal in the hepatic portal tract or present as an intralobular granuloma formation. Degenerative hepatocyte lesions (hydropic and steatosis), hypertrophy and hyperplasia of Kupffer cells and congestion with deposits of hemosiderin pigment in the sinusoids were also observed (Figure 1a,b). Histological screening focused on the detection and grading of the following abnormalities in livers: (i) capsular inflammation; (ii) portal inflammation; (iii) presence of intralobular granulomas; (iv) hypertrophy and hyperplasia of Kupffer cells; (v) degenerative hepatocyte lesions (hydropic and steatotic); (vi) congestion; and (vii) haemosiderin deposits. Changes were evaluated semiquantitatively as absent or present and graded according to intensity over the entire tissue slide: (N) no histological alterations; (Si) slight (20–30%); (M), moderate (31–60%); and (Sv), severe (>60%) (Table 2). Intralobular granulomas were observed in 90% of infected dogs. They were present with variable size and morphology (oval or ellipsoid nodule exudate) constituted by macrophages, some epithelioid cells, small numbers of lymphocytes, plasma cells and, rarely, neutrophils (Figure 1c–e). Intracellular amastigote forms of Leishmania were found inside macrophages of intralobular granulomas or in Kupffer cells (data not shown). No significant differences were found in the parasite load of asymptomatic and symptomatic dogs.
Table 1 Primary antibodies used for immunohistochemistry Molecules/Markers*
Source (clone)
Dilution
Ethical approval statement
a-SMA 1A4 Vimentina V9 Cytokeratin CK 19 (AE1/AE3) TGF-b
Dako Dako Dako Santa
1/100 1/100 1/100 1/2500
Housing, anaesthesia and all procedures were in compliance with the guidelines established by the Institutional Animal
*For all immunohistochemistry assays, we used goat normal serum. or PBS as a control of antibodies
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(M0851) (M0725) (M3515) Cruz (sc-146)
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(a)
(b)
(c)
(d)
Figure 1 (a–e) Liver sections of dogs naturally infected with L. (L) infantum: (a) Asymptomatic dog: (A) Portal space with a cellular infiltrated of plasma cells, lymphocytes and macrophages (cross). Note congestion of a branch of portal vein (PV) (bar = 32 lm). (b) Higher magnification showing the mononuclear cellular exudate (bar = 16 lm). (c) Symptomatic dog. Observe three granulomas’ formation into the hepatic lobule (intralobular granulomas) (bar = 32 lm). (d) Higher magnification showing intralobular granuloma comprising of mononuclear cell macrophages, lymphocytes and plasma cells). Note an intense swelling of hepatocytes with hydropic degeneration (‘balloon cells’) (crosses) (bar = 16 lm). (e) Highest magnification (oil immersion objective) showing an epithelioid cell (*) with an elongated or oval nucleus and a pink cytoplasm with indistinct cell boundaries (appearing to merge into one another) (arrow) confirming ‘an epithelioid granuloma’ formation, lymphocytes (**) and plasma cells (***) (bar = 6 lm). Haematoxylin–eosin (HE) staining.
(e)
Extracellular matrix alterations (fibropoiesis) Staining of hepatic reticular fibres by Gomori’s ammoniacal silver demonstrated increased collagen deposits in all infected dogs compared to control dogs. Fibre deposition was found in the portal space region and in sinusoids in the hepatic lobe walls. Reticular fibres were diffusely spread in several directions, forming a compact network, and in some cases, fibres encircled groups of hepatocytes or a single cell (Figure 2a,b). As seen, hepatic reticular fibres in infected dogs were thicker than those in controls. In fact, morphometric analysis showed a higher fibropoiesis in both symptomatic and asymptomatic infected dogs than in controls (P < 0.0001) (Figure 3). Otherwise, symptomatic animals showed more intense fibropoiesis than asymptomatic dogs and controls.
There was a positive correlation between hepatic collagen deposition and the tissue parasite load (Figure 4).
Expression of TGF-b TGF-b expression was measured by morphometric analysis taking into consideration two staining patterns in the hepatic parenchyma: a diffuse staining mostly localized along the sinusoids and the localized staining expressed by the mononuclear cells of the granulomas (hepatic intralobular granulomas). In fact, we found intense expression (positive cells) in the entire liver parenchyma, mainly along the sinusoids. As intralobular granulomas were formed from Kupffer cells, we observed intense positivity in all cases of chronic hepatitis (Figure 5a,b). Thus, both asymptomatic and symptomatic dogs showed higher expression of TGF-b than did the control
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Table 2 Histopathological evaluation of liver in canine visceral leishmaniosis (Leishmania L. infantum) Symptomatic n = 10
Asymptomatic n = 10
Control n = 8
Histopathology
N
Si
M
Sv
P ≤ 0.05a D / F
N
Si
M
Sv
P ≤ 0.05a D / F
N
Sl
M
Sv
Capsular inflammation Portal inflammation Presence of intralobular granulomas Hypertrophy and hyperplasia of Kupffer cells Degeneration Congestion Hemosiderin
2 1 1 3
3 3 6 3
4 5 2 3
1 1 1 1
* * * *
* * * *
3 1 1 2
2 5 5 5
5 3 3 3
0 1 1 0
* * * *
* * * *
5 4 8 5
3 4 0 3
0 0 0 0
0 0 0 0
1 1 3
2 3 3
4 3 3
3 3 1
* **/ * */* */*
2 1 3
3 3 4
3 4 2
2 2 1
*/* */* */*
4 3 6
3 4 2
0 1 0
1 0 0
/ / / /
/ / / /
a
Qualitative analyses Frequency (F): presence or absence and semi-quantitative analyses of tissue changes, taking into account their distribution throughout the histological section and classified as follows: – Distribution (D): no histological alterations (N); slight (Si), moderate (M) and severe (Sv) histological alterations. Significant differences (P ≤ 0.05) relative to control are represented by * and relative to asymptomatic by**.
(a)
(b)
Figure 2 (a-b) Liver sections of uninfected and infected dogs naturally infected with L. (L.) infantum: (a) Control dog – note a delicate network (weak colour black staining) of intralobular hepatic reticular fibres (arrows). (b) Symptomatic dog – note conspicuous collagen thickening in the space of Disse (stronger black staining of intralobular reticular fibres) (arrows). Some reticular fibres are coiled (arrowhead). Gomori’s ammoniacal silver staining (bar = 32 lm). (S) Sinusoid blood vessel. International Journal of Experimental Pathology, 2016, 97, 139–149
Figure 3 Morphometric analyses of collagen (reticular fibres) deposition in livers of dogs naturally infected with L. infantum. *Significant difference between asymptomatic and symptomatic dogs compared to controls. ** Significant difference between asymptomatic and symptomatic dogs. One-way ANOVA and Tukey’s post hoc tests (P < 0.001).
Figure 4 Positive correlation between tissue parasite load and collagen deposition in livers. (Pearson’s r = 0.4906; P = 0.0281).
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(a)
(b)
Figure 5 (a, b) Liver sections of a symptomatic infected dog naturally infected with L. (L) infantum: (a) Intralobular hepatic granulomas showing intense expression of TGF-b (arrows). (b) Higher magnification showing positive cells of the intralobular granulomas. Also note TGF-b positivity in sinusoid blood wall. Immuno-streptavidin–peroxidase method (bar = 16 lm). (S) Sinusoid blood vessels.
Figure 6 Morphometric analyses of TGF-b in livers of naturally infected dogs with L. (L.) infantum. (*) Significant difference between asymptomatic and symptomatic dogs compared to controls. One-way ANOVA and Tukey’s post hoc tests (P < 0.001).
(a)
(b)
(c)
Figure 7 Liver sections of a symptomatic dog naturally infected with L. (L) infantum: (a) Note vimentin-positive focal cells in the sinusoids and in the portal area mainly around vessels indicating spindle-shaped stromal cells (arrows) (bar = 32 lm). (b) Now observe a-actin-positive cells diffusely distributed throughout the entire liver parenchyma (arrows). (c) Detail of letter (b) showing the positivity of a-actin in the hepatic lobule in the sinusoid vessels (arrowheads) (bar = 16 lm). Immunostreptavidin–peroxidase method.
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(a)
Figure 8 Morphometric analyses of a-actin in livers of naturally infected dogs with L. (L.) infantum. *Significant difference between asymptomatic and symptomatic dogs compared to controls. One-way ANOVA and Tukey’s post hoc tests (P < 0.001).
(b)
(c)
Figure 9 Positive correlation between positive a-SMA reactivity and collagen deposition in livers. (Pearson’s r = 0.651; P = 0.001).
group (Figure 6) (P < 0.05). However, we did not find differences between asymptomatic and symptomatic dogs.
Expression of vimentin Vimentin expression observed by immunohistochemistry showed variable intensity among and within all groups, although the tissue distribution pattern was similar. As a result, we did not observe differences among the groups. Thus, in all groups, we observed positive focal cells in the sinusoids and in the portal area, mainly around vessels, indicating spindle-shaped stromal cells (fibroblasts) (Figure 7a). There was no positive correlation between vimentin reactivity and collagen deposition (Pearson’s r = 0.051, P = 0.827).
Expression of a-SMA Positive cells were observed diffusely distributed throughout the liver parenchyma in the hepatic lobule in the sinusoid
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Figure 10 a,c: Liver sections of an asymptomatic dog naturally infected with L. (L) infantum: (a) Note cytokeratin biliary ducts’ epithelial positive cells in the portal area (arrows) and biliary tree (arrowheads). (b) Observe epithelial positivity cells throughout the biliary tree. (c) No positive cells in hepatocytes, in sinusoid cell wall and/or in intralobular granulomas formation were noticed (arrow). Immuno-streptavidin–peroxidase method. (bar = 32 lm).
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Figure 11 Morphometric analyses of cytokeratin (CK19) in livers of naturally infected dogs with L. (L.) infantum. *Significant difference between asymptomatic and symptomatic dogs compared to controls. One-way ANOVA and Tukey’s post hoc tests (P < 0.001).
vessels and in the portal area, notably around the biliary ducts. Endothelial cells were always negative (Figures 7b–c). The a-SMA expression was higher in infected groups than in controls. We did not find differences between asymptomatic and symptomatic dogs (Figure 8). There was a positive correlation between positive a-SMA reactivity and collagen deposition (Figure 9).
Expression of cytokeratin We observed a similar pattern of cytokeratin tissue expression in all cases. Immunolabelled cells in the hepatic portal space were restricted to epithelial cells of intrahepatic bile ducts (Figure 10a,b). Thus, we did not find cytokeratin in hepatocytes, in sinusoid cell walls and/or in intralobular granuloma formations (Figure 10c). However, infected animals showed higher expression of cytokeratin than did control dogs. There were no significant differences between asymptomatic and symptomatic dogs (Figure 11). There was no positive correlation between positive cytokeratin reactivity and collagen deposition (Spearman’s r = 0.2060; P = 0.3835) or positive cytokeratin reactivity and a-SMA (Pearson r = 0.1474; P = 0.5352).
Discussion We demonstrated hepatic disorders in dogs with L. infantum resulting from chronic inflammation associated with fibrotic disease characterized as perihepatocytic deposits of extracellular matrix components, mainly collagen fibrils. These data are similar to the findings of our previous works (Tafuri et al. 1996; Melo et al. 2008, 2009; Souza et al. 2014) and others (Rallis et al. 2005). Otherwise, Souza et al. (2014) working with thirty naturally infected dogs with L. infantum showed deposition of fibrous connective
tissue in all organs of infected dogs, correlated with grade of inflammation and iron (haemosiderin) deposition in the tissues. They worked with dogs with negative serologicy for Ehrlichiosis. In fact, we have not found any previous evidence in the literature which correlates fibrosis and Ehrlichosis. For example, Mylonakis et al. (2010) described absence of myelofibrosis in dogs with myelosuppression induced by Ehrlichia canis infection. Thus taking the previous literature, and our own previous work (Souza et al. 2014) into account, we did not do Ehrlichosis serology as part of the present study. The livers of asymptomatic and symptomatic infected dogs generally displayed a chronic inflammatory reaction characterized by mononuclear cell infiltration in the capsule, hepatic portal space and entire hepatic parenchyma. Intralobular granuloma formations were observed in the majority of the cases (90%). A similar finding has been described in experimental murine visceral leishmaniasis models (Murray 2001), in dogs experimentally infected with Leishmania donovani and L. infantum (Oliveira et al. 1993; Tafuri et al. 1996) and in dogs naturally infected with L. infantum (Tafuri et al. 1996; Sanchez et al. 2004; Lima et al. 2007). In conjunction with this chronic inflammation, Gomori’s ammoniacal silver staining revealed higher numbers of collagen fibres in livers of infected dogs than in controls. In naturally infected dogs, there was a positive correlation between hepatic collagen deposits and parasite load. Infected dogs showed more hepatic collagenogenesis (intralobular fibrosis), probably stimulated by the tissue parasite load, as suggested by Bogliolo (1956) and Corbett et al. (1993) in human visceral leishmaniasis. To investigate the possible origin of the fibropoiesis and better understand the role of HSCs and myofibroblasts, immunohistochemical assays characterizing TGF-b, vimentin, a-SMA and CK19 were performed. Injury-induced liver collagen is synthesized by portal fibroblasts or sinusoidlocated HSCs differentiated into myofibroblasts (Guyot et al. 2006). In addition to HSCs, LPCs play an important role in liver fibrosis. In spite of the current debate regarding the relationship between LPCs and HSCs, it is widely accepted that TGF-b plays a central role in the development of liver fibrosis after HSC and LPC activation (Valkova 2002; Schuppan et al. 2003; Gui et al. 2006). TGF-b initiates signalling through its interaction with receptors on the surface of HSCs, inducing differentiation of HSC into myofibroblasts and enhancing ECM synthesis (Niu and Zhao, 2007). TGF-b also inhibits the expression of collagenase and tissue inhibitors and increases the expression of matrixdegrading enzymes such as plasminogen activator and metalloproteinases. In addition, acting as a stimulant to change the phenotype of quiescent cells to myofibroblasts, TGF-b participates in the maintenance of this phenotype. Thus, TGF-b data in parallel with other myofibroblast marker results might play a role in hepatic fibrosis in chronic CVL. We observed expression of TGF-b in cells of intralobular granulomas. These data suggest that in contrast to other hepatic fibrosis models, granulomas probably International Journal of Experimental Pathology, 2016, 97, 139–149
Dog Leishmania Liver Fibrosis persist in the progression of CVL disease and actively express TGF-b (Gressner et al. 2002; Matsuzaki 2009). The HSCs in normal liver constitute the largest reserves of retinoids (vitamin A) in the body. Under stress and injury, they become activated and undergo phenotypic changes, including loss of vitamin A, production of fibrogenic cytokines and expression of new proteins such as alpha-actin (aSMA) and acquire myofibroblastic characteristics (Lepreux et al. 2001; Guyot et al. 2006; Zhao & Burt 2007). Our results showing increased staining of a-SMA indicate that HSC-derived myofibroblasts might also play a role in CVL liver fibrogenesis. Increased expression of a-SMA in perisinusoidal HSCs in areas of fibrosis around portal spaces and fibrous septa has been reported in chronic hepatitis in humans. In the course of the development of hepatic fibrosis in humans and rats, the number of HSCs increases, and they differentiate into myofibroblast-like cells with significant expression of a-SMA (Burt 1999; Boisclair et al. 2001; Lepreux et al. 2001). Activated HSCs in humans also synthesize tissue inhibitors of metalloproteinases 1 and 2 that inhibit interstitial collagenase. Thus, these higher metalloproteinase inhibitors’ tissue-level activity contributes to the accumulation of ECM proteins (Bedossa & Paradis 2003; Guyot et al. 2006; Wipff et al. 2007). The etiopathogenesis of chronic liver disease in dogs has not been sufficiently clarified, particularly the role of ECM proteins in the progression of hepatic fibrosis (Ijzer et al. 2006; Mekonnen et al. 2007). Van Hul et al. (2009) reported a close association between positive aSMA immunostaining and the differentiation of LPC cells. Possibly, the higher labelling of the cytokeratin (CK AE1/ AE3) observed in our study could indicate that fibrogenesis in liver also involves LPC differentiation into cholangiocytes, detected by the appearance of bile ducts. The cytoskeleton protein vimentin is considered a classic marker of cells of mesenchymal origin cells such as myofibroblasts and portal fibroblasts. Studies have shown the involvement of these cells in collagen deposition in fibrotic liver through the analysis of vimentin expression (Lepreux et al. 2001; Zhao & Burt 2007). However, we did not observe increased expression of vimentin in the livers of CVL-infected dogs. Moreover, expression of vimentin by portal fibroblasts did not vary between control and infected dogs. We did not find correlation between vimentin expression and collagen deposition. Thus, vimentin does not appear to be an adequate marker of myofibroblasts. Immunopositivity of perisinusoidal HSCs to a-SMA was seen in both the infected and control dogs; however, intensity and density in the infected group were higher than those in the controls. a-SMA is a known marker for myofibroblasts. The role of myofibroblasts is notable in hepatic fibrotic disease in dogs. Research indicates that inflammation in canine liver, as well as in livers of humans and rats, is connected to the activation of periductal myofibroblasts and probably to their proliferation of bile ducts (Burt 1999; Ijzer et al. 2006; Libbrecht et al. 2002; Mekonnen et al. 2007; Wipff et al. 2007). Myofibroblasts in normal livers of dogs, humans and rats are located around blood vessels and bile International Journal of Experimental Pathology, 2016, 97, 139–149
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ducts, and we observed their increased immunopositivity with intense fibropoiesis. Periductal myofibroblasts appear to be important in hepatic fibrosis, and when activated by various cytokines, the synthesis and secretion of proteins of the extracellular matrix are increased (Bedossa & Paradis 2003; Guyot 2006). In this work, we have confirmed the hepatic fibropoiesis in CVL. In fact, we still have found a positive correlation between hepatic collagen deposition and tissue parasite load. The origin of the fibropoiesis (injury-induced liver collagen) comes from portal fibroblasts or sinusoid-located HSCs differentiated into myofibroblasts. As mentioned, LPCs also play an important role in liver fibrosis and it is widely accepted in the literature that TGF-b plays a central role in the development of liver fibrosis after HSC and LPC activation. Thus, our TGF-b data showing an overexpression has suggested that this cytokine plays a central role in the development of liver fibrosis of dogs naturally infected with L. infantum where a-SMA may be a superior marker for activated HSC cells in CVL.
Acknowledgments The authors thank FAPEMIG for grant APQ-01378-12, CNPq for grant 474665/2012-7 and grant 303022/2013–2 and Lucidus Consulting for the English language revision.
Conflict of interest The authors declare that we do not have any conflict of interest in this work.
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