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Contribution of astrocytes and macrophage migration inhibitory factor to immune-mediated canine encephalitis caused by the distemper virus
Veterinary Immunology and Immunopathology 221 (2020) 110010
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Research paper
Contribution of astrocytes and macrophage migration inhibitory factor to immune-mediated canine encephalitis caused by the distemper virus
T
Tatianna F.S. De Nardoa, Paulo H.L. Bertoloa, Priscila A. Bernardesa, Danísio P. Munaria, Gisele F. Machadob, Luciana S. Jardimc, Pamela R.R. Moreiraa, Mayara C. Rosolema, Rosemeri O. Vasconcelosa,* a School of Agrarian and Veterinary Sciences (FCAV), São Paulo State University (UNESP), Via de Acesso Paulo Donato Castellane s/n, 14884-900, Jaboticabal, SP, Brazil b School of Veterinary Medicine of Araçatuba (FMVA), UNESP, Araçatuba, SP, Brazil c Autonomous Veterinarian, Ribeirão Preto, SP, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Demyelination Neuroinflammation Virus Immune response Dog
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that is produced by many cell types in situations of homeostasis or disease. One of its functions is to act as a proinflammatory molecule. In humans, several studies have shown that MIF levels become elevated in the serum, urine, cerebrospinal fluid and tissues of patients with chronic inflammatory diseases (systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, sepsis, atheromas, diabetes and cancer). In dogs, distemper is a viral infectious condition that may lead to demyelination and inflammation in the central nervous system (CNS). In addition to the action of the virus, the inflammatory process may give rise to lesions in the white matter. Therefore, the objectives of the present study were to evaluate the role of MIF in the encephalitis that the canine distemper virus causes and to compare this with immunodetection of major histocompatibility complex-II (MHC-II), CD3 T lymphocytes, MMP-9 and glial fibrillary acidic protein (GFAP; astrocytes) in demyelinated areas of the encephalon, in order to ascertain whether these findings might be related to the severity of the encephalic lesions. To this end, a retrospective study on archived paraffinized blocks was conducted, in which 21 encephala from dogs that had been naturally infected with the canine distemper virus (infected group) and five from dogs that had been free from systemic or CNS-affecting diseases (control group) were used. In the immunohistochemical analysis on the samples, the degree of marking by GFAP, MHC-II, MMP-9 and MIF was greater in the demyelinated areas and in the adjacent neuropil, and this was seen particularly in astrocytes. Detection of CD3 was limited to perivascular cuffs. In areas of liquefactive necrosis, Gitter cells were positive for MMP-9, MIF and MHC-II. Hence, it was concluded that activated astrocytes influenced the afflux of T lymphocytes to the encephalon (encephalitis). In the more advanced phases, activated phagocytes in the areas of liquefactive necrosis (Gitter cells) continued to produce inflammatory mediators even after the astrocytes in these localities had died, thereby worsening the encephalic lesions. Distemper virus-activated astrocytes and microglia produce MIF that results in proinflammatory stimulus on glial cells and brain-infiltrating leukocytes. Therefore, the effect of the inflammatory response is potentiated on the neuropil, resulting in neurological clinical signs.
1. Introduction Distemper is a highly contagious viral disease caused by the canine distemper virus (CDV), which is a morbillivirus in the family Paramyxoviridae. It affects domestic dogs and other species of wild animals (Appel and Summers, 1995) such as foxes, wolves, racoons, hyenas, ferrets, wild felids and aquatic mammals like dolphins and sealions, among others (Carvalho et al., 2014; Beineke et al., 2015). ⁎
Dogs are the main reservoir for CDV and act as a source of infection for wild carnivores (Moll et al., 1995). Cats that are subjected to experimental infection present viral replication with pronounced lymphopenia, but without showing clinical signs of the disease (Carvalho et al., 2014). Distemper can affect dogs of both sexes and any age or breed (Tipold et al., 1992; Thomas et al., 1993), but it is more common in puppies (Gillespie, 1962; Appel, 1969; Krakowka and Koestner, 1976)
Corresponding author. E-mail address: [email protected] (R.O. Vasconcelos).
https://doi.org/10.1016/j.vetimm.2020.110010 Received 24 March 2019; Received in revised form 10 January 2020; Accepted 14 January 2020 0165-2427/ © 2020 Published by Elsevier B.V.
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2005). In previous studies, acute demyelination of the CNS was causally correlated with circulatory disorders relating to presence of the virus in the vascular endothelium and in the ependymal cells. This results in metabolic disorders regarding oligodendrocytes (reductions of enzyme production and transcriptional activity of myelin) and astrocytes, with consequential apoptosis of these cells (Carvalho et al., 2012; Pan et al., 2013). In parallel, infection of neurons by CDV results in apoptosis of neurons and death of axons, which also evolve to demyelination (Pan et al., 2013). Diffusion of the virus in nerve tissue has been verified in vitro and has been attributed to cell-to-cell contact, through the cytoplasmic processes of astrocytes (Wyss-Fluehmann et al., 2010). CDV may stimulate the microglia and, through this, lesions to the neuropil become more frequent due to expression of MHC-II by these cells and due to production of lytic enzymes and reactive oxygen species (ROS), which are toxic to glial cells, including oligodendrocytes (Carvalho et al., 2012). Macrophage migration inhibitory factor (MIF), which is a pro-inflammatory cytokine secreted by sensitized lymphocytes and macrophages, among other cells, has been evaluated in patients with demyelinating diseases of immunomediated origin (multiple sclerosis). In these cases, this cytokine presented high levels, which showed that it might have a relationship with the pathogenesis of immunomediated and demyelinating diseases of the CNS (Cox et al., 2013). In the literature on the mechanisms for development of demyelinating lesions in encephalitis due to canine distemper, the action of the virus on cells of the CNS has been highlighted. In particular, the action of astrocytes at the initial phase of infection has been highlighted. Subsequently, the local immune response has been incriminated with regard to worsening of the lesions of nerve tissues (Lempp et al., 2014). MIF secretion is responsible for increased expression of toll-like receptors (TLRs) and adhesion molecules in macrophages (Mitchell et al., 1999; Kleemann et al., 2000). MIF-stimulated murine macrophages have been found to induce expression of matrix metallopeptidase-9 (MMP-9), through the MEK-ERK mitogen-activated protein kinase (MAPK) pathway. Activation of this pathway is necessary for MMP-9 expression and activation in response to MIF stimulation (Yu et al., 2007). Therefore, the objective of the present study was to evaluate the role of MIF in encephalitis caused by CDV, in comparison with the immunodetection of GFAP (astrocytes), MHC-II, T lymphocytes (CD3) and MMP-9 in areas of demyelination of the encephalon, in order to ascertain whether these findings might be related to the severity of encephalic lesions in dogs.
and in unvaccinated dogs (Chappuis, 1995). Infected dogs develop clinical signs and respiratory, gastrointestinal, cutaneous, ophthalmological and neurological lesions, which can occur sequentially, simultaneously or separately (Decaro et al., 2004; Carvalho et al., 2014). It has been suggested in some studies that failure of the immunological system might cause evolution of the virus to the central nervous system (CNS) (Mangia et al., 2014). The duration of infections caused by CDV depends on the dog’s age, its immunocompetence and the virulence of the pathogen (Beineke et al., 2015). Transmission of CDV between susceptible animals takes place via aerosols and, 24 h afterwards, the virus will have become replicated in macrophages and B and T lymphocytes. Subsequently, it disseminates to lymphoid organs, thus resulting in immunosuppression and viremia. It then reaches parenchymatous organs such as the lungs and other epithelial tissues, via a hematogenous route. It enters the CNS either through lymphocytes or through platelets, which alter the permeability of the vascular endothelium (meninges and choroid plexus) of the fourth ventricle, via periventricular ependymal cells (Carvalho et al., 2014). Invasion of the CNS is associated with tropism of the viral strain to the nerve cells and with the animal’s immunological state. CDV can affect neurons, astrocytes, oligodendrocytes, cells of the choroid plexus and the microglia (Beineke et al., 2009). The differences between the neurological clinical signs of dogs with distemper may be related to the viral strain involved. Thus, the Snyder Hill strain generally affects neuron and the R252 strain has the capacity to affect glial cells (Summers et al., 1984; Vandevelde and Zurbriggen, 2005). Astrocytes account for 95 % of the cells infected by the virus (Lempp et al., 2014). CDV may cause lesions in both the white and the grey matter of the CNS. The damage caused to the white matter commonly consists of demyelination, while in the grey matter it relates to neuronal infection and necrosis of the nerve tissue. Astrocytic reactivity commonly appears, along with the presence of cytoplasmic and/or nuclear inclusions (Vandevelde and Zurbriggen, 2005; Beineke et al., 2015). In the acute phase, the lesions are characterized by discrete increases in the numbers of astrocytes and microglia (Ulrich et al., 2014). Experimental studies on ferrets inoculated with virulent strains of CDV have shown that in the acute phase of the infection, neuron death, activation of microglia, reactive gliosis and release of pro-inflammatory cytokines in infected areas trigger a strong local immune response. This is independent of the systemic immunosuppression that has been described in young animals. It has been suggested that cell death due to direct damage caused by the virus may be amplified through the local immune response (Rudd et al., 2010). In the chronic phase, infected dogs may present demyelinating polioencephalitis and/or leukoencephalomyelitis (Beineke et al., 2015), which may often be multifocal. This frequently affects adult dogs, which may or may not present systemic clinical signs. In this phase, the viral antigen is restricted to only a few astrocytes (Stein et al., 2006). Expression of major histocompatibility complex class II (MHC-II) occurs predominantly in the microglia. This is responsible for continuous demyelination and for dissemination of perivascular mononuclear infiltrate. The alterations begin with hyperplasia of astrocytes and proliferation of microglia in the white matter, in the subpial and subependymal regions (Stein et al., 2006). Astrocytes also express MHC, and increased expression of glial fibrillary acidic protein (GFAP) occurs in the neuropil adjacent to the demyelinated areas, mainly with involvement of the cerebellum and brainstem (Orsini et al., 2007) Demyelination in the chronic phase coincides with recovery of the immune system, at around six to seven weeks after infection. The virus initially induces the perivascular cuffs, composed of lymphocytes, plasmocytes and monocytes. Inflammation in the demyelinated areas may lead to progression of tissue destruction, since increased production of pro-inflammatory cytokines occurs in these areas. It is possible that astrocytes, which are the first target of the virus, participate in amplification of the immune response (Vandevelde and Zurbriggen,
2. Material and methods 2.1. Material A retrospective study on archived paraffinized blocks was conducted, in which 21 encephala from dogs that had been naturally infected with the CVD (infected group) and five from dogs that had been free from systemic or CNS-affecting diseases (control group) were used. All the blocks from the dogs in both groups came from the archives of the Department of Animal Clinical and Surgical Medicine and Animal Reproduction, School of Veterinary Medicine of Araçatuba (FMVA), São Paulo State University (UNESP), Araçatuba campus, state of São Paulo, Brazil, and from the Department of Veterinary Pathology, School of Agrarian and Veterinary Sciences (FCAV), UNESP, Jaboticabal campus, state of São Paulo, Brazil. The archived material was selected through histopathological analysis on all the tissue samples coming from these animals. To select samples for the infected group, only animals with lesions characteristic of distemper and without any other intercurrent encephalic or systemic disease were considered. The samples for the control group were selected based on complete absence of lesions in the encephalon and of 2
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(DakoCytomation, code S302283-2), replacing the primary antibody. Positive controls were run using tissues that had been suggested by the manufacturers of each antibody. To determine the number of immunomarked cells for all the antibodies tested, counts were performed in five microscope fields at high magnification (objective lens 40x), with an area of 0.19625 μm2. This was done using a Nikon Eclipse E200 microscope (Moreira et al., 2010). The values obtained from these fields were used to determine the average number of immunomarked cells per animal, and these averages were determined separately for the infected and control groups.
systemic or neoplastic diseases. To standardize the anatomical areas of the canine encephala, it was decided to select the cerebellum and adjacent areas, because the samples were not uniform. Likewise, it was not possible to correlate the histological findings with the clinical signs or with the ages of the dogs in the infected group because this information was not stated in all the medical files that accompanied the animals to the necropsies. The paraffin blocks were sliced into sections of thickness 5 μm, and these were stained with hematoxylin and eosin (HE) for analysis under an optical microscope. In the histopathological analysis on the encephala in the infected group, the following were considered: type of lesion (degenerative, inflammatory or demyelinating); the distribution of these lesions (focal or multifocal), the presence of viral inclusion corpuscles and the intensity of the lesions, which was determined according to their score: 0 (absent), 1 (slight), 2 (moderate) or 3 (severe).
2.3. Statistical analysis The effects of the group (infected or control) on the characteristics studied were analyzed using the least-squares method (ANOVA), with weighting for heterogenous variance. The analyses were performed using the Guided Data Analysis procedure (SAS 9.1, SAS Institute, Cary, NC, USA). The distribution of the residuals for each analysis model was non-normal and, for this reason, the data were subjected to logarithmic transformation. Differences between the groups were considered significant when P < 0.05. The data were normalized and then subjected to multivariate analyses (principal component analysis and factor analysis), which were performed to describe the combined variance of the characteristics studied. Principal component analysis can be used to analyze interrelationships among a large number of variables and to explain these variables in terms of their common underlying dimensions (Hair et al., 2010). The multivariate analyses were done using the R Core Team software (2015).
2.2. Immunohistochemical analysis The general procedure for developing this technique consisted of deparaffinization of the encephalon sections in a heated chamber at 60 °C for one hour, followed by two baths in xylol for 10 min each. The samples were then hydrated in a decreasing series of alcohol solutions until reaching a bath of distilled water. After this, antigen recovery was performed by means of heating (Table 1), in a sodium citrate buffer solution (10 mM; pH 6.0). In a microwave oven, the incubation was for 2 min at maximum power and 10 min at minimum power (750 W), opening every 5 min to replenish the buffer. Endogenous peroxidase blocking (Table 1) was performed using an 8 % solution consisting of 92 mL of methyl alcohol (Synth) and 8 mL of hydrogen peroxide (30 volumes; Merck) for 30 min at room temperature in a darkened chamber. Blocking of nonspecific reactions (Table 1) was done using a commercial product (Protein Block, DakoCytomation, code. × 0909), for 30 min at room temperature. Following this, the sections were incubated with the primary antibodies, with specific dilutions and incubation times, at 4 °C or at room temperature, according to the specifications for each antibody (Table 1). For all the antibodies, a peroxidase-binding polymer complex was used (Envision Dual Link System-HRP, DakoCytomation, code K406189-2). Between each of these steps, baths in distilled water and in buffer solution (Tris HCl; pH 7.4) were used. To view the reaction, the chromogen DAB (3,3-diaminobenzidine; DakoCytomation, code K3468-1) was used. Harris’s hematoxylin was used for counterstaining and the material was mounted on slides using Entellan (Merck). Between each of these steps, baths in distilled water and in phosphate-buffered saline solution (pH 7.4) were used. A negative control on the immunohistochemical reaction was performed in order to rule out possible nonspecific binding between the secondary antibody and canine tissue epitopes. These negative controls for the different immunomarkings were run using the antibody diluent
3. Results The analysis under an optical microscope on the encephala of the control group did not show any lesions in the nerve tissue. However, in the infected group, areas of demyelination were seen, characterized by multiple pleomorphic vacuoles in the white matter of the cerebellum, always associated with proliferation of gemistocytic astrocytes and/or areas of liquefactive necrosis. These lesions showed predominantly multifocal distribution and were located in the cerebellum, cerebellar peduncle and neuropil adjacent to the fourth ventricle (Fig. 1A) and meninges. The gemistocytic astrocytes were characterized by presenting plentiful acidophilic cytoplasm and a normochromic to hypochromic nucleus that was generally peripheral. These cells were specifically located within the areas of demyelination and often presented multiple intracytoplasmic and intranuclear inclusion corpuscles (Fig. 1B) that were acidophilic and pleomorphic. On rare occasions, intranuclear inclusions were observed in oligodendrocytes. In the adjacent neuropil, glial reactivity, reactive microgliosis and astrocyte proliferation were
Table 1 Antibodies used to detect different markers of inflammation in brains of dogs with distemper. Primary antibodies
Target cell
Species of origin
Antigen recovery
Dilution
Length of incubation
MIF
Microglia Astrocytes Leukocytes Astrocytes Endothelium Leukocytes Glia cells T lymphocytes
Polyclonal mouse (Santa Cruz, code SC20121)
Pressure pan (Dako)
1:300
18 h (4 °C)
Polyclonal cow (Dako, code Z0334) Polyclonal rabbit (Cell Signaling, code 9661)
Microwave Pressure pan (Dako)
1:700 1:200
2 h at room temperature 18 h (4 °C)
Monoclonal mouse (Dako, code M7254)
Pressure pan (Dako)
1: 350
Monoclonal mouse (Dako, code M0746)
Pressure pan (Dako)
1:200
18 h (4 °C) 18 h (4 °C)
GFAP MMP9
CD3 MHC-II
Leukocytes Glia cells
MIF – macrophage migration inhibitory factor; GFAP – glial fibrillary acidic protein; MMP9 – matrix metalloproteinase 9; CD3 – cluster of differentiation 3; MHC-II – major histocompatibility complex II. 3
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Fig. 1. Photomicrographs of the brain of dogs with distemper. A) Note the area of demyelination in the white matter adjacent to the fourth ventricle (*, bar =200 μm). B) Area similar to that described in A. Notice numerous pleomorphic acidophilic inclusions in the cytoplasm of reactive astrocytes (arrows, bar =20 μm). C) Area of liquefactive necrosis rich in Gitter cells (arrows, bar =20 μm). D) Note perivascular cuffs and vascular hyperemia in the neuropil (arrows, bar =50 μm). Hematoxylin and Eosin.
(P = 0.0052). Only in relation to detection of the enzyme MMP-9 was there no statistically significant difference between the groups (P = 0.8071). In the factor analysis (Table 2), it was seen regarding the first factor (F1) that increased magnitude of GFAP was related to increased levels of MIF and CD3 (positive values greater than 0.6), with a significant difference between the groups (P = 0.0001). Regarding the second factor (F2), a relationship between the variables MHC and MMP-9 was noted. However, the groups did not differ in relation to this factor (P = 0.3012). In the principal component analysis (Fig. 4), the distribution of the groups between components 1 and 2 was ascertained. It was found that principal component 1 (PC1) explained 41.86 % of the variability of the data, such that the control individuals were grouped in a different region from the other animals (infected group), as a function of the combined variation of the characteristics studied. Thus, these characteristics made it possible to group the diseased animals differently from the control group. All the immunomarkings evaluated varied in the same manner (Fig. 5). Greater associations were observed between GFAP and CD3 and between MIF and MHC-II. In other words, the magnitudes of these characteristics increased in step with increasing degrees of disease.
seen. The areas of liquefactive necrosis contained large numbers of Gitter cells (Fig. 1C). Presence of inflammation was characterized by perivascular cuffs surrounding the vessels of the neuropil (Fig. 1D), with predominant involvement of the white matter, the meninges and occasionally the choroid plexus. The infiltrate that was observed was predominantly mononuclear (lymphocytes and plasmocytes). Two animals in the infected group showed lesions suggestive of acute evolution in the white matter of the cerebellum, characterized by intense astrocytic reactivity together with large numbers of intracytoplasmic viral inclusions in these cells. In these cases, no areas or demyelination had yet appeared, nor was there any presence of perivascular cuffs. The immunohistochemical analysis on the immunomarkings of the different antibodies showed that there was a high level of marking for GFAP (Fig. 2A), which occurred in gemistocytic astrocytes that were present in the areas of demyelination (Fig. 2A1) and in the adjacent neuropil (Fig. 2A2). However, in areas of liquefactive necrosis, debris from filamentous structures that were positive for GFAP and associated with Gitter cells were observed. CD3 T lymphocytes were mostly detected in the perivascular cuffs (Fig. 2B). Occasionally, positive cells appeared dispersed in the neuropil. The enzyme MMP-9 was detected in gemistocytic astrocytes or in activated astrocytes on the periphery of the demyelinated lesions (Fig. 2C), and in microglia, in Gitter cells in demyelinated areas (Fig. 2C) and in leukocytes in the perivascular cuffs. The cytokine MIF was detected in astrocytes (Fig. 2D) in the areas of lesions and in adjacent regions, in microglia and in leukocytes in the perivascular cuffs (Fig. 2D). The marking of MHC-II was intense and diffuse in the areas of demyelination and in tissues adjacent to these areas (Fig. 3A and B). In regions more distant from the lesions, this marking was focal and only slight or absent. Positivity for this antibody was observed in reactive astrocytes, in microglia and in the vascular wall. Gemistocytic astrocytes presented weak marking. They showed positive marking for MIF, GFAP, MMP-9 (Fig. 1) and MHC-II (Fig. 2). Comparison between the groups showed significant differences between them (i.e. P < 0.05) in relation to the following parameters: GFAP (P = 0.0006), MIF (P = 0.0120), CD3 (P = 0.0113) and MHC-II
4. Discussion The presence of viral inclusions associated with areas of demyelination in areas adjacent to the fourth ventricle indicates that the virus can penetrate into the encephalon via the cerebrospinal fluid. This corroborates reports in the literature (Vandevelde and Zurbriggen, 2005, Carvalho et al., 2012; Pan et al., 2013). This finding is backed up by reports of detection of viral particles in cells of the choroid plexus and in ependymal cells, relating to the ventricular system (Carvalho et al., 2012). Some authors have highlighted that demyelination resulted from infection of oligodendrocytes, which are myelin-producing cells (Vandevelde and Zurbriggen, 2005). However, in other studies, it was observed that the majority of the infected cells were astrocytes (Pan et al., 2013; Lemp et al., 2014). Through electron microscopy, it was 4
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Fig. 2. Photomicrographs of the brain of dogs with distemper. A) Note astrocytic reactivity (GFAP) in the areas of demyelination (A1 = detail of gemistocytic astrocytes) and adjacent to the lesion. (A2 = detail of reactive astrocytes, bar =100 μm). B) T-lymphocyte (CD3) labeling in the meningeal inflammatory infiltrate (detail), adjacent to demyelination (*, bar =50 μm). C) Detection of MMP-9 in gemistocytic astrocytes from the demyelination area and in Gitter cells from the areas of liquefactive necrosis (detail, arrow, bar =50 μm). D) Marking of MIF in the gemistocytic astrocytes of the demyelinated areas (*) and in the adjacent neuropil, as well as in the perivascular cuffs (detail, bar =50 μm). Peroxidase-linked polymer complex.
seen that infection of oligodendrocytes was rare in distemper cases and it was proposed that the virus caused infection in these cells but that, even so, this could influence demyelination (Lempp et al., 2014). Another line of investigation emphasized the hypothesis that demyelination was related to metabolic alterations that impeded oligodendrocytes from performing their functions. Such alterations would be secondary to vascular damage or damage to the ventricular system, due to the effect of the virus. In the present study, high levels of astrocyte activation were observed in the areas of demyelination: this corroborates the findings of other authors, who reported that astrocytes contributed towards the demyelination process through being in close contact with these localities (Pan et al., 2013). On the other hand, involvement of oligodendrocytes was rare among the dogs of the present study. Oligodendrocytes with intranuclear inclusions were only rarely observed, which was contrary to what had been reported by other authors (Vandelvelde and Zurbrigen, 1995). According to the literature, the neurological lesions of distemper in young animals are caused through direct viral injury, with involvement of the grey and white matter, including the presence of encephalic necrosis (polioencephalomalacia) and absence of encephalic inflammatory infiltrate, possibly due to immaturity of the immunological system and systemic immunosuppression (Rudd et al., 2010). In the present study, only two animals presented lesions suggestive of acute-
Table 2 Comparison of variation of characteristics according to factor (F1 and F2). Characteristic
F1
F2
GFAP MIF CD3 MHC MMP9 Total proportion
0.781035 0.608419 0.614181 0.552647 −0.036735 0.332835
−0.080777 0.440450 0.007401 0.612321 0.951087 0.296016
phase infection. In these cases, only a large number of viral inclusions in reactive astrocytes was noted, along with proliferation and reactivity of these cells, which was concordant with the report from Rudd et al. (2010). Most of the dogs in the infected group presented areas of liquefactive necrosis that were always associated with areas of demyelination, with large numbers of gemistocytic astrocytes and perivascular cuffs. According to the literature, presence of this type of astrocyte in the lesions indicates that the process has become chronic (Pan et al., 2013). In the present study, in the areas of demyelination, gemistocytic astrocytes were a constant and notable finding. These cells expressed MIF, MMP-9, GFAP and MHC-II. Astrocytes are possibly the first cells to Fig. 3. Photomicrographs of the brain of dogs with distemper. A) Note intense positive labeling for MHC-II in the area of demyelination and in the areas adjacent to the lesion. In detail, gemistocytic positive astrocytes are found in a demyelinated area (bar =100 μm). B) Same as A, in an area adjacent to the fourth ventricle (bar =200 μm). Peroxidase-linked polymer complex.
5
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Fig. 4. Variation of principal component 1 (PC1) in relation to principal component 2 (PC2) for the characteristics studied. Labels indicate the classification according to group; Group 1 = infected group (GI); Group 2 = control group (GC).
significantly in situations of injury. This led to activation of the microglia and to an afflux of leukocytes to the nerve tissue. In addition, this cytokine has been implicated in immunomediated anti-myelin response mechanisms in multiple sclerosis and in murine experimental models for allergic encephalitis (Cox et al., 2013). This has also been observed in animals that were experimentally infected with, for example, the Ross river fever and chikungunya viruses, which also have an immunomediated component. Mice presenting greater expression of MIF were found to present clinical signs and tissue inflammatory processes of greater severity (Herrero et al., 2011). Therefore, from the findings of the present study, it can be suggested that MIF would be a good marker for severe neurological lesions, given that its immunodetection was greater when lesions of greater extent were present in the neuropil of dogs with distemper. In the areas of liquefactive necrosis, remains of astrocytes that were positive for GFAP were noted, interspersed in the lesion. Gemistocytic astrocytes possibly died through the effects of activation of macrophages and microglia in these localities, given that Gitter cells also expressed MMP-9, MIF and MHC-II. The deleterious effects of the inflammatory mediators (reactive oxygen species, nitric oxide and cytokines) may have been responsible for destruction of the demyelinated nerve tissue and for the death of astrocytes in these localities. Activated astrocytes may have contributed towards demyelination in the initial phase of the lesion, due to the direct action of the virus on these glial cells and on axons, thus resulting in secondary metabolic dysfunction of oligodendrocytes (Ulrich et al., 2014). Subsequently, the tissue injury would become amplified through local release of inflammatory mediators. Lastly, activated macrophages (Gitter cells) would act in the areas of liquefactive necrosis. Pan et al. (2013) also noted that GFAP was absent or only detected at low levels in the areas of liquefactive necrosis that resulted from demyelinating encephalopathy in distemper cases. Other authors have highlighted that activation of microglia, induced by CDV, may have some relevance regarding the pathogenesis of acute demyelination within distemper, through diffusely increased regulation of MHC in the white matter and through release of factors that are toxic to myelin (Vandevelde and Zurbriggen, 2005; Stein et al., 2006; Qesla et al., 2014). Those reports are in line with the results from the present study, since reactive microgliosis and increased expression of MHC II were observed in the demyelinated areas and in adjacent areas, thus confirming the participation of microglia in this process of expansion of the nerve lesion. The enzyme MMP-9 was possibly responsible for breaking the
Fig. 5. Variation of principal component 1 (PC1) in relation to principal component 2 (PC2) for the characteristics studied. Vectors indicate the relationships between the characteristics evaluated (GFAP, CD3, MIF, MHC-II and MMP-9).
be activated after the virus has arrived in the encephalon. These activated cells subsequently produce MIF, which would contribute towards activation of the vascular endothelium, microglia and lymphocyte influx after MMP-9 vascular basement membrane degradation, as described in the literature (Mitchell et al., 1999; Kleemann et al., 2000; Yu et al., 2007). A study on encephalitis in old dogs showed through immunohistochemical analysis that the virus was present in neurons, astrocytes, leukocytes in perivascular cuffs, vascular endothelium and areas of demyelination. Presence of cells positive for CD3 (T lymphocytes), CD79 (B lymphocytes) and MAC387 (macrophages) was observed in the inflammatory infiltrate of the neuropil, in response to chemiotactic stimuli that were released from the areas of the lesions. Moreover, presence of marking for vimentin in lymphocytes in the cuffs suggested that vimentin promoted migration of leukocytes to the site of injury (Headley et al., 2009). In parallel, marking for MIF in astrocytes in the areas of demyelination and adjacent areas was detected. This pro-inflammatory cytokine was detected at baseline levels in the CNS, and its levels increased 6
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demyelinating lesions in the white matter of the encephalon and spinal cord. Inflammation and involvement of the grey matter have also been reported. These alterations are generally preceded by viral or bacterial infection, or they may occur after vaccination with attenuated viruses (measles, rabies, influenza, rubella, etc.). Although the pathogenesis has not yet been fully elucidated, it has been suggested that the local immune response may be responsible for lesions to the myelin sheath of the axons, which would result in neurological lesions (Lee, 2011). Dogs with distemper may provide a model for understanding the pathogenesis of human demyelinating diseases.
blood-brain barrier and degrading the vascular basal membrane, thereby enabling an afflux of leukocytes to injured areas. However, it could not be confirmed whether the activated form of the enzyme predominated in the lesions, because the material studied had been embedded in paraffin, which cannot be subjected to the zymography technique. In the literature, it has been highlighted that MMP-9 expression levels are higher in inflammatory diseases of the CNS, thus suggesting that this inducible gelatinase is a systemic marker of inflammation in animals (Opdenakker et al., 2001). A study evaluating the presence of MMP-2 and MMP-9 in the cerebrospinal fluid of dogs with subacute distemper showed that the levels of pro-MMP-2 forms (cerebrospinal fluid and cerebellum) and pro-MMP-9 forms (cerebellum) were high in all the animals. The levels of active forms of these gelatinases were elevated in the cerebrospinal fluid. These findings confirm that these enzymes participate in breaking the blood-brain barrier. Thus, they contribute towards development of neuroinflammation and demyelination (Machado et al., 2013). In some studies on humans, the presence of metalloproteinases has been positively correlated with worsening of the lesion, in processes affecting the CNS (Yong et al., 2001). Marking for CD3+ T lymphocytes was observed in the encephala of the animals in the infected group, and this showed a significant association with marking for GFAP (Table 2), i.e. when the parameters studied were grouped and comparisons were made between the groups. This suggests that activation of astrocytes contributed towards migration of T lymphocytes to the encephalon. Expression of MHC-II in astrocytes and microglia has already been described, which demonstrates their potential for activation of T lymphocytes, through antigenic presentation via MHC (Hamo et al., 2007). MIF stimulates inflammatory response in brain by microglia and astrocytes activation. In astrocytes this activation is by the CD74 receptor. These authors believe that MIF would be a new target for therapeutic interference in neuroinflammation (Su et al., 2017). Thus, in the present study, it can be inferred that one of the CDV access routes in the nervous system was through the ventricular system. This hypothesis is reinforced by the presence of lesions and viral inclusions in the neuropil adjacent to the ependymal cells. A hematogenic route could be another possibility: astrocytes form part of the glia limitans and are also present in the blood-brain barrier, which thus confirms that they come into close contact with antigens that arrive via this route. Astrocytes thus become activated, which is shown in the areas with lesions through thickening of the cytoplasmic prolongations, marked with GFAP. From this point, the process of the immune response of the encephalon begins with production of MIF and MMP-9 and expression of MHC-II. This enables the breaking of the blood-brain barrier and migration of leukocytes into the nerve tissue. The leukocytes of the perivascular cuffs amplify the production of cytokines and inflammatory mediators. This further activates the microglia. The tissue damage caused by the mediators and by the harmful components released by the microglia may give rise to areas of liquefactive necrosis in the neuropil, thereby resulting in death of the astrocytes and oligodendrocytes at this locality. The Gitter cells at these localities continue to produce cytokines and inflammatory mediators, thus causing even greater deleterious effects on the nerve tissue. In multivariate analysis, it was evident that the immunoinflammatory parameters of the CNS vary at the same time and are directly related to the state of the disease. The cytokine MIF was possibly a link between glial cells (astrocytes and microglia), MMP-9 expression and lymphocyte influx into the CNS. Recent studies have highlighted that MIF regulates chemokine release (CCL5) and modulation of toll-like receptor 4 in astrocytes, which in turn favors recruitment of inflammatory cells to the injured neuropil (Zhou et al., 2018). It would be of interest to makes parallels regarding immunomediated evolution between the encephalic lesions of canine distemper and cases of acute disseminated encephalomyelitis (ADEM) in children. In ADEM, children develop neurological signs relating to
5. Conclusions Under the conditions of this study, it was possible to conclude that: - Astrocytes are key cells in activating the immune response against CDV, since they were detected in association with demyelinating lesions and in nerve tissue adjacent to these lesions. - Activated astrocytes expressed MIF, MMP-9 and MHC-II and possibly initiated neuroinflammation, which was subsequently maintained by microglia and inflammatory cells. - Most of the animals presented chronic lesions characterized by constant presence of gemistocytic astrocytes, inflammation and multifocal areas of demyelination and/or liquefactive necrosis. - The cytokine MIF was possibly the main modulator of neuroinflammation in dogs with distemper. - MIF could be used as a marker for inflammation in the CNS, since its expression in cases of severe demyelination and inflammation of the neuropil was greater. Ethical standards The design for this study was approved by the Ethics and Animal Welfare Committee (CEUA no. 00703/17), of FCAV - UNESP, Jaboticabal campus, state of São Paulo, Brazil. Declaration of Competing Interest The authors declare that they did not have any conflict of interest. Acknowledgements The authors would like to thank Dr. G.F. Machado, who kindly provided the paraffin blocks of canine brains of the infected group that were used in this study. T.F.S. De Nardo was supported by a grant from the National Council for Scientific and Technological Development (Conselho Nacional do Desenvolvimento Científico e Tecnológico, CNPq). P.H.L. Bertolo was a CAPES grantee. D.P. Munari held a productivity research fellowship from CNPq. The authors also wish to acknowledge Mr. Antonio Roveri Neto for his histotechnical assistance. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetimm.2020.110010. References Appel, M.J.G., 1969. Pathogenesis of canine distemper. Am. J. Vet. Res. 30, 1167–1182. Appel, M.J., Summers, B.A., 1995. Pathogenicity of morbiliviruses for terrestrial carnivores. Vet. Microbiol. 44, 187–191. Beineke, A., Puff, C., Seehusen, F., Baumgärtner, W., 2009. Pathogenesis and immunopathology of systemic and nervous canine distemper virus. Vet. Immunol. Immunopathol. 127, 1–18. https://doi.org/10.1016/j.vetimm.2008.09.023. Beineke, A., Baumgärtner, W., Wohlsein, P., 2015. Cross-species transmission of canine distemper virus – an update. One Health 1, 49–59. https://doi.org/10.1016/j.onehlt. 2015.09.002. Carvalho, O.V., Botelho, C.V., Ferreira, C.G.T., Scherer, P.O., Soares-Martins, J.A.P.,
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Research paper
Contribution of astrocytes and macrophage migration inhibitory factor to immune-mediated canine encephalitis caused by the distemper virus
T
Tatianna F.S. De Nardoa, Paulo H.L. Bertoloa, Priscila A. Bernardesa, Danísio P. Munaria, Gisele F. Machadob, Luciana S. Jardimc, Pamela R.R. Moreiraa, Mayara C. Rosolema, Rosemeri O. Vasconcelosa,* a School of Agrarian and Veterinary Sciences (FCAV), São Paulo State University (UNESP), Via de Acesso Paulo Donato Castellane s/n, 14884-900, Jaboticabal, SP, Brazil b School of Veterinary Medicine of Araçatuba (FMVA), UNESP, Araçatuba, SP, Brazil c Autonomous Veterinarian, Ribeirão Preto, SP, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Demyelination Neuroinflammation Virus Immune response Dog
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that is produced by many cell types in situations of homeostasis or disease. One of its functions is to act as a proinflammatory molecule. In humans, several studies have shown that MIF levels become elevated in the serum, urine, cerebrospinal fluid and tissues of patients with chronic inflammatory diseases (systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, sepsis, atheromas, diabetes and cancer). In dogs, distemper is a viral infectious condition that may lead to demyelination and inflammation in the central nervous system (CNS). In addition to the action of the virus, the inflammatory process may give rise to lesions in the white matter. Therefore, the objectives of the present study were to evaluate the role of MIF in the encephalitis that the canine distemper virus causes and to compare this with immunodetection of major histocompatibility complex-II (MHC-II), CD3 T lymphocytes, MMP-9 and glial fibrillary acidic protein (GFAP; astrocytes) in demyelinated areas of the encephalon, in order to ascertain whether these findings might be related to the severity of the encephalic lesions. To this end, a retrospective study on archived paraffinized blocks was conducted, in which 21 encephala from dogs that had been naturally infected with the canine distemper virus (infected group) and five from dogs that had been free from systemic or CNS-affecting diseases (control group) were used. In the immunohistochemical analysis on the samples, the degree of marking by GFAP, MHC-II, MMP-9 and MIF was greater in the demyelinated areas and in the adjacent neuropil, and this was seen particularly in astrocytes. Detection of CD3 was limited to perivascular cuffs. In areas of liquefactive necrosis, Gitter cells were positive for MMP-9, MIF and MHC-II. Hence, it was concluded that activated astrocytes influenced the afflux of T lymphocytes to the encephalon (encephalitis). In the more advanced phases, activated phagocytes in the areas of liquefactive necrosis (Gitter cells) continued to produce inflammatory mediators even after the astrocytes in these localities had died, thereby worsening the encephalic lesions. Distemper virus-activated astrocytes and microglia produce MIF that results in proinflammatory stimulus on glial cells and brain-infiltrating leukocytes. Therefore, the effect of the inflammatory response is potentiated on the neuropil, resulting in neurological clinical signs.
1. Introduction Distemper is a highly contagious viral disease caused by the canine distemper virus (CDV), which is a morbillivirus in the family Paramyxoviridae. It affects domestic dogs and other species of wild animals (Appel and Summers, 1995) such as foxes, wolves, racoons, hyenas, ferrets, wild felids and aquatic mammals like dolphins and sealions, among others (Carvalho et al., 2014; Beineke et al., 2015). ⁎
Dogs are the main reservoir for CDV and act as a source of infection for wild carnivores (Moll et al., 1995). Cats that are subjected to experimental infection present viral replication with pronounced lymphopenia, but without showing clinical signs of the disease (Carvalho et al., 2014). Distemper can affect dogs of both sexes and any age or breed (Tipold et al., 1992; Thomas et al., 1993), but it is more common in puppies (Gillespie, 1962; Appel, 1969; Krakowka and Koestner, 1976)
Corresponding author. E-mail address: [email protected] (R.O. Vasconcelos).
https://doi.org/10.1016/j.vetimm.2020.110010 Received 24 March 2019; Received in revised form 10 January 2020; Accepted 14 January 2020 0165-2427/ © 2020 Published by Elsevier B.V.
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2005). In previous studies, acute demyelination of the CNS was causally correlated with circulatory disorders relating to presence of the virus in the vascular endothelium and in the ependymal cells. This results in metabolic disorders regarding oligodendrocytes (reductions of enzyme production and transcriptional activity of myelin) and astrocytes, with consequential apoptosis of these cells (Carvalho et al., 2012; Pan et al., 2013). In parallel, infection of neurons by CDV results in apoptosis of neurons and death of axons, which also evolve to demyelination (Pan et al., 2013). Diffusion of the virus in nerve tissue has been verified in vitro and has been attributed to cell-to-cell contact, through the cytoplasmic processes of astrocytes (Wyss-Fluehmann et al., 2010). CDV may stimulate the microglia and, through this, lesions to the neuropil become more frequent due to expression of MHC-II by these cells and due to production of lytic enzymes and reactive oxygen species (ROS), which are toxic to glial cells, including oligodendrocytes (Carvalho et al., 2012). Macrophage migration inhibitory factor (MIF), which is a pro-inflammatory cytokine secreted by sensitized lymphocytes and macrophages, among other cells, has been evaluated in patients with demyelinating diseases of immunomediated origin (multiple sclerosis). In these cases, this cytokine presented high levels, which showed that it might have a relationship with the pathogenesis of immunomediated and demyelinating diseases of the CNS (Cox et al., 2013). In the literature on the mechanisms for development of demyelinating lesions in encephalitis due to canine distemper, the action of the virus on cells of the CNS has been highlighted. In particular, the action of astrocytes at the initial phase of infection has been highlighted. Subsequently, the local immune response has been incriminated with regard to worsening of the lesions of nerve tissues (Lempp et al., 2014). MIF secretion is responsible for increased expression of toll-like receptors (TLRs) and adhesion molecules in macrophages (Mitchell et al., 1999; Kleemann et al., 2000). MIF-stimulated murine macrophages have been found to induce expression of matrix metallopeptidase-9 (MMP-9), through the MEK-ERK mitogen-activated protein kinase (MAPK) pathway. Activation of this pathway is necessary for MMP-9 expression and activation in response to MIF stimulation (Yu et al., 2007). Therefore, the objective of the present study was to evaluate the role of MIF in encephalitis caused by CDV, in comparison with the immunodetection of GFAP (astrocytes), MHC-II, T lymphocytes (CD3) and MMP-9 in areas of demyelination of the encephalon, in order to ascertain whether these findings might be related to the severity of encephalic lesions in dogs.
and in unvaccinated dogs (Chappuis, 1995). Infected dogs develop clinical signs and respiratory, gastrointestinal, cutaneous, ophthalmological and neurological lesions, which can occur sequentially, simultaneously or separately (Decaro et al., 2004; Carvalho et al., 2014). It has been suggested in some studies that failure of the immunological system might cause evolution of the virus to the central nervous system (CNS) (Mangia et al., 2014). The duration of infections caused by CDV depends on the dog’s age, its immunocompetence and the virulence of the pathogen (Beineke et al., 2015). Transmission of CDV between susceptible animals takes place via aerosols and, 24 h afterwards, the virus will have become replicated in macrophages and B and T lymphocytes. Subsequently, it disseminates to lymphoid organs, thus resulting in immunosuppression and viremia. It then reaches parenchymatous organs such as the lungs and other epithelial tissues, via a hematogenous route. It enters the CNS either through lymphocytes or through platelets, which alter the permeability of the vascular endothelium (meninges and choroid plexus) of the fourth ventricle, via periventricular ependymal cells (Carvalho et al., 2014). Invasion of the CNS is associated with tropism of the viral strain to the nerve cells and with the animal’s immunological state. CDV can affect neurons, astrocytes, oligodendrocytes, cells of the choroid plexus and the microglia (Beineke et al., 2009). The differences between the neurological clinical signs of dogs with distemper may be related to the viral strain involved. Thus, the Snyder Hill strain generally affects neuron and the R252 strain has the capacity to affect glial cells (Summers et al., 1984; Vandevelde and Zurbriggen, 2005). Astrocytes account for 95 % of the cells infected by the virus (Lempp et al., 2014). CDV may cause lesions in both the white and the grey matter of the CNS. The damage caused to the white matter commonly consists of demyelination, while in the grey matter it relates to neuronal infection and necrosis of the nerve tissue. Astrocytic reactivity commonly appears, along with the presence of cytoplasmic and/or nuclear inclusions (Vandevelde and Zurbriggen, 2005; Beineke et al., 2015). In the acute phase, the lesions are characterized by discrete increases in the numbers of astrocytes and microglia (Ulrich et al., 2014). Experimental studies on ferrets inoculated with virulent strains of CDV have shown that in the acute phase of the infection, neuron death, activation of microglia, reactive gliosis and release of pro-inflammatory cytokines in infected areas trigger a strong local immune response. This is independent of the systemic immunosuppression that has been described in young animals. It has been suggested that cell death due to direct damage caused by the virus may be amplified through the local immune response (Rudd et al., 2010). In the chronic phase, infected dogs may present demyelinating polioencephalitis and/or leukoencephalomyelitis (Beineke et al., 2015), which may often be multifocal. This frequently affects adult dogs, which may or may not present systemic clinical signs. In this phase, the viral antigen is restricted to only a few astrocytes (Stein et al., 2006). Expression of major histocompatibility complex class II (MHC-II) occurs predominantly in the microglia. This is responsible for continuous demyelination and for dissemination of perivascular mononuclear infiltrate. The alterations begin with hyperplasia of astrocytes and proliferation of microglia in the white matter, in the subpial and subependymal regions (Stein et al., 2006). Astrocytes also express MHC, and increased expression of glial fibrillary acidic protein (GFAP) occurs in the neuropil adjacent to the demyelinated areas, mainly with involvement of the cerebellum and brainstem (Orsini et al., 2007) Demyelination in the chronic phase coincides with recovery of the immune system, at around six to seven weeks after infection. The virus initially induces the perivascular cuffs, composed of lymphocytes, plasmocytes and monocytes. Inflammation in the demyelinated areas may lead to progression of tissue destruction, since increased production of pro-inflammatory cytokines occurs in these areas. It is possible that astrocytes, which are the first target of the virus, participate in amplification of the immune response (Vandevelde and Zurbriggen,
2. Material and methods 2.1. Material A retrospective study on archived paraffinized blocks was conducted, in which 21 encephala from dogs that had been naturally infected with the CVD (infected group) and five from dogs that had been free from systemic or CNS-affecting diseases (control group) were used. All the blocks from the dogs in both groups came from the archives of the Department of Animal Clinical and Surgical Medicine and Animal Reproduction, School of Veterinary Medicine of Araçatuba (FMVA), São Paulo State University (UNESP), Araçatuba campus, state of São Paulo, Brazil, and from the Department of Veterinary Pathology, School of Agrarian and Veterinary Sciences (FCAV), UNESP, Jaboticabal campus, state of São Paulo, Brazil. The archived material was selected through histopathological analysis on all the tissue samples coming from these animals. To select samples for the infected group, only animals with lesions characteristic of distemper and without any other intercurrent encephalic or systemic disease were considered. The samples for the control group were selected based on complete absence of lesions in the encephalon and of 2
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(DakoCytomation, code S302283-2), replacing the primary antibody. Positive controls were run using tissues that had been suggested by the manufacturers of each antibody. To determine the number of immunomarked cells for all the antibodies tested, counts were performed in five microscope fields at high magnification (objective lens 40x), with an area of 0.19625 μm2. This was done using a Nikon Eclipse E200 microscope (Moreira et al., 2010). The values obtained from these fields were used to determine the average number of immunomarked cells per animal, and these averages were determined separately for the infected and control groups.
systemic or neoplastic diseases. To standardize the anatomical areas of the canine encephala, it was decided to select the cerebellum and adjacent areas, because the samples were not uniform. Likewise, it was not possible to correlate the histological findings with the clinical signs or with the ages of the dogs in the infected group because this information was not stated in all the medical files that accompanied the animals to the necropsies. The paraffin blocks were sliced into sections of thickness 5 μm, and these were stained with hematoxylin and eosin (HE) for analysis under an optical microscope. In the histopathological analysis on the encephala in the infected group, the following were considered: type of lesion (degenerative, inflammatory or demyelinating); the distribution of these lesions (focal or multifocal), the presence of viral inclusion corpuscles and the intensity of the lesions, which was determined according to their score: 0 (absent), 1 (slight), 2 (moderate) or 3 (severe).
2.3. Statistical analysis The effects of the group (infected or control) on the characteristics studied were analyzed using the least-squares method (ANOVA), with weighting for heterogenous variance. The analyses were performed using the Guided Data Analysis procedure (SAS 9.1, SAS Institute, Cary, NC, USA). The distribution of the residuals for each analysis model was non-normal and, for this reason, the data were subjected to logarithmic transformation. Differences between the groups were considered significant when P < 0.05. The data were normalized and then subjected to multivariate analyses (principal component analysis and factor analysis), which were performed to describe the combined variance of the characteristics studied. Principal component analysis can be used to analyze interrelationships among a large number of variables and to explain these variables in terms of their common underlying dimensions (Hair et al., 2010). The multivariate analyses were done using the R Core Team software (2015).
2.2. Immunohistochemical analysis The general procedure for developing this technique consisted of deparaffinization of the encephalon sections in a heated chamber at 60 °C for one hour, followed by two baths in xylol for 10 min each. The samples were then hydrated in a decreasing series of alcohol solutions until reaching a bath of distilled water. After this, antigen recovery was performed by means of heating (Table 1), in a sodium citrate buffer solution (10 mM; pH 6.0). In a microwave oven, the incubation was for 2 min at maximum power and 10 min at minimum power (750 W), opening every 5 min to replenish the buffer. Endogenous peroxidase blocking (Table 1) was performed using an 8 % solution consisting of 92 mL of methyl alcohol (Synth) and 8 mL of hydrogen peroxide (30 volumes; Merck) for 30 min at room temperature in a darkened chamber. Blocking of nonspecific reactions (Table 1) was done using a commercial product (Protein Block, DakoCytomation, code. × 0909), for 30 min at room temperature. Following this, the sections were incubated with the primary antibodies, with specific dilutions and incubation times, at 4 °C or at room temperature, according to the specifications for each antibody (Table 1). For all the antibodies, a peroxidase-binding polymer complex was used (Envision Dual Link System-HRP, DakoCytomation, code K406189-2). Between each of these steps, baths in distilled water and in buffer solution (Tris HCl; pH 7.4) were used. To view the reaction, the chromogen DAB (3,3-diaminobenzidine; DakoCytomation, code K3468-1) was used. Harris’s hematoxylin was used for counterstaining and the material was mounted on slides using Entellan (Merck). Between each of these steps, baths in distilled water and in phosphate-buffered saline solution (pH 7.4) were used. A negative control on the immunohistochemical reaction was performed in order to rule out possible nonspecific binding between the secondary antibody and canine tissue epitopes. These negative controls for the different immunomarkings were run using the antibody diluent
3. Results The analysis under an optical microscope on the encephala of the control group did not show any lesions in the nerve tissue. However, in the infected group, areas of demyelination were seen, characterized by multiple pleomorphic vacuoles in the white matter of the cerebellum, always associated with proliferation of gemistocytic astrocytes and/or areas of liquefactive necrosis. These lesions showed predominantly multifocal distribution and were located in the cerebellum, cerebellar peduncle and neuropil adjacent to the fourth ventricle (Fig. 1A) and meninges. The gemistocytic astrocytes were characterized by presenting plentiful acidophilic cytoplasm and a normochromic to hypochromic nucleus that was generally peripheral. These cells were specifically located within the areas of demyelination and often presented multiple intracytoplasmic and intranuclear inclusion corpuscles (Fig. 1B) that were acidophilic and pleomorphic. On rare occasions, intranuclear inclusions were observed in oligodendrocytes. In the adjacent neuropil, glial reactivity, reactive microgliosis and astrocyte proliferation were
Table 1 Antibodies used to detect different markers of inflammation in brains of dogs with distemper. Primary antibodies
Target cell
Species of origin
Antigen recovery
Dilution
Length of incubation
MIF
Microglia Astrocytes Leukocytes Astrocytes Endothelium Leukocytes Glia cells T lymphocytes
Polyclonal mouse (Santa Cruz, code SC20121)
Pressure pan (Dako)
1:300
18 h (4 °C)
Polyclonal cow (Dako, code Z0334) Polyclonal rabbit (Cell Signaling, code 9661)
Microwave Pressure pan (Dako)
1:700 1:200
2 h at room temperature 18 h (4 °C)
Monoclonal mouse (Dako, code M7254)
Pressure pan (Dako)
1: 350
Monoclonal mouse (Dako, code M0746)
Pressure pan (Dako)
1:200
18 h (4 °C) 18 h (4 °C)
GFAP MMP9
CD3 MHC-II
Leukocytes Glia cells
MIF – macrophage migration inhibitory factor; GFAP – glial fibrillary acidic protein; MMP9 – matrix metalloproteinase 9; CD3 – cluster of differentiation 3; MHC-II – major histocompatibility complex II. 3
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Fig. 1. Photomicrographs of the brain of dogs with distemper. A) Note the area of demyelination in the white matter adjacent to the fourth ventricle (*, bar =200 μm). B) Area similar to that described in A. Notice numerous pleomorphic acidophilic inclusions in the cytoplasm of reactive astrocytes (arrows, bar =20 μm). C) Area of liquefactive necrosis rich in Gitter cells (arrows, bar =20 μm). D) Note perivascular cuffs and vascular hyperemia in the neuropil (arrows, bar =50 μm). Hematoxylin and Eosin.
(P = 0.0052). Only in relation to detection of the enzyme MMP-9 was there no statistically significant difference between the groups (P = 0.8071). In the factor analysis (Table 2), it was seen regarding the first factor (F1) that increased magnitude of GFAP was related to increased levels of MIF and CD3 (positive values greater than 0.6), with a significant difference between the groups (P = 0.0001). Regarding the second factor (F2), a relationship between the variables MHC and MMP-9 was noted. However, the groups did not differ in relation to this factor (P = 0.3012). In the principal component analysis (Fig. 4), the distribution of the groups between components 1 and 2 was ascertained. It was found that principal component 1 (PC1) explained 41.86 % of the variability of the data, such that the control individuals were grouped in a different region from the other animals (infected group), as a function of the combined variation of the characteristics studied. Thus, these characteristics made it possible to group the diseased animals differently from the control group. All the immunomarkings evaluated varied in the same manner (Fig. 5). Greater associations were observed between GFAP and CD3 and between MIF and MHC-II. In other words, the magnitudes of these characteristics increased in step with increasing degrees of disease.
seen. The areas of liquefactive necrosis contained large numbers of Gitter cells (Fig. 1C). Presence of inflammation was characterized by perivascular cuffs surrounding the vessels of the neuropil (Fig. 1D), with predominant involvement of the white matter, the meninges and occasionally the choroid plexus. The infiltrate that was observed was predominantly mononuclear (lymphocytes and plasmocytes). Two animals in the infected group showed lesions suggestive of acute evolution in the white matter of the cerebellum, characterized by intense astrocytic reactivity together with large numbers of intracytoplasmic viral inclusions in these cells. In these cases, no areas or demyelination had yet appeared, nor was there any presence of perivascular cuffs. The immunohistochemical analysis on the immunomarkings of the different antibodies showed that there was a high level of marking for GFAP (Fig. 2A), which occurred in gemistocytic astrocytes that were present in the areas of demyelination (Fig. 2A1) and in the adjacent neuropil (Fig. 2A2). However, in areas of liquefactive necrosis, debris from filamentous structures that were positive for GFAP and associated with Gitter cells were observed. CD3 T lymphocytes were mostly detected in the perivascular cuffs (Fig. 2B). Occasionally, positive cells appeared dispersed in the neuropil. The enzyme MMP-9 was detected in gemistocytic astrocytes or in activated astrocytes on the periphery of the demyelinated lesions (Fig. 2C), and in microglia, in Gitter cells in demyelinated areas (Fig. 2C) and in leukocytes in the perivascular cuffs. The cytokine MIF was detected in astrocytes (Fig. 2D) in the areas of lesions and in adjacent regions, in microglia and in leukocytes in the perivascular cuffs (Fig. 2D). The marking of MHC-II was intense and diffuse in the areas of demyelination and in tissues adjacent to these areas (Fig. 3A and B). In regions more distant from the lesions, this marking was focal and only slight or absent. Positivity for this antibody was observed in reactive astrocytes, in microglia and in the vascular wall. Gemistocytic astrocytes presented weak marking. They showed positive marking for MIF, GFAP, MMP-9 (Fig. 1) and MHC-II (Fig. 2). Comparison between the groups showed significant differences between them (i.e. P < 0.05) in relation to the following parameters: GFAP (P = 0.0006), MIF (P = 0.0120), CD3 (P = 0.0113) and MHC-II
4. Discussion The presence of viral inclusions associated with areas of demyelination in areas adjacent to the fourth ventricle indicates that the virus can penetrate into the encephalon via the cerebrospinal fluid. This corroborates reports in the literature (Vandevelde and Zurbriggen, 2005, Carvalho et al., 2012; Pan et al., 2013). This finding is backed up by reports of detection of viral particles in cells of the choroid plexus and in ependymal cells, relating to the ventricular system (Carvalho et al., 2012). Some authors have highlighted that demyelination resulted from infection of oligodendrocytes, which are myelin-producing cells (Vandevelde and Zurbriggen, 2005). However, in other studies, it was observed that the majority of the infected cells were astrocytes (Pan et al., 2013; Lemp et al., 2014). Through electron microscopy, it was 4
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Fig. 2. Photomicrographs of the brain of dogs with distemper. A) Note astrocytic reactivity (GFAP) in the areas of demyelination (A1 = detail of gemistocytic astrocytes) and adjacent to the lesion. (A2 = detail of reactive astrocytes, bar =100 μm). B) T-lymphocyte (CD3) labeling in the meningeal inflammatory infiltrate (detail), adjacent to demyelination (*, bar =50 μm). C) Detection of MMP-9 in gemistocytic astrocytes from the demyelination area and in Gitter cells from the areas of liquefactive necrosis (detail, arrow, bar =50 μm). D) Marking of MIF in the gemistocytic astrocytes of the demyelinated areas (*) and in the adjacent neuropil, as well as in the perivascular cuffs (detail, bar =50 μm). Peroxidase-linked polymer complex.
seen that infection of oligodendrocytes was rare in distemper cases and it was proposed that the virus caused infection in these cells but that, even so, this could influence demyelination (Lempp et al., 2014). Another line of investigation emphasized the hypothesis that demyelination was related to metabolic alterations that impeded oligodendrocytes from performing their functions. Such alterations would be secondary to vascular damage or damage to the ventricular system, due to the effect of the virus. In the present study, high levels of astrocyte activation were observed in the areas of demyelination: this corroborates the findings of other authors, who reported that astrocytes contributed towards the demyelination process through being in close contact with these localities (Pan et al., 2013). On the other hand, involvement of oligodendrocytes was rare among the dogs of the present study. Oligodendrocytes with intranuclear inclusions were only rarely observed, which was contrary to what had been reported by other authors (Vandelvelde and Zurbrigen, 1995). According to the literature, the neurological lesions of distemper in young animals are caused through direct viral injury, with involvement of the grey and white matter, including the presence of encephalic necrosis (polioencephalomalacia) and absence of encephalic inflammatory infiltrate, possibly due to immaturity of the immunological system and systemic immunosuppression (Rudd et al., 2010). In the present study, only two animals presented lesions suggestive of acute-
Table 2 Comparison of variation of characteristics according to factor (F1 and F2). Characteristic
F1
F2
GFAP MIF CD3 MHC MMP9 Total proportion
0.781035 0.608419 0.614181 0.552647 −0.036735 0.332835
−0.080777 0.440450 0.007401 0.612321 0.951087 0.296016
phase infection. In these cases, only a large number of viral inclusions in reactive astrocytes was noted, along with proliferation and reactivity of these cells, which was concordant with the report from Rudd et al. (2010). Most of the dogs in the infected group presented areas of liquefactive necrosis that were always associated with areas of demyelination, with large numbers of gemistocytic astrocytes and perivascular cuffs. According to the literature, presence of this type of astrocyte in the lesions indicates that the process has become chronic (Pan et al., 2013). In the present study, in the areas of demyelination, gemistocytic astrocytes were a constant and notable finding. These cells expressed MIF, MMP-9, GFAP and MHC-II. Astrocytes are possibly the first cells to Fig. 3. Photomicrographs of the brain of dogs with distemper. A) Note intense positive labeling for MHC-II in the area of demyelination and in the areas adjacent to the lesion. In detail, gemistocytic positive astrocytes are found in a demyelinated area (bar =100 μm). B) Same as A, in an area adjacent to the fourth ventricle (bar =200 μm). Peroxidase-linked polymer complex.
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Fig. 4. Variation of principal component 1 (PC1) in relation to principal component 2 (PC2) for the characteristics studied. Labels indicate the classification according to group; Group 1 = infected group (GI); Group 2 = control group (GC).
significantly in situations of injury. This led to activation of the microglia and to an afflux of leukocytes to the nerve tissue. In addition, this cytokine has been implicated in immunomediated anti-myelin response mechanisms in multiple sclerosis and in murine experimental models for allergic encephalitis (Cox et al., 2013). This has also been observed in animals that were experimentally infected with, for example, the Ross river fever and chikungunya viruses, which also have an immunomediated component. Mice presenting greater expression of MIF were found to present clinical signs and tissue inflammatory processes of greater severity (Herrero et al., 2011). Therefore, from the findings of the present study, it can be suggested that MIF would be a good marker for severe neurological lesions, given that its immunodetection was greater when lesions of greater extent were present in the neuropil of dogs with distemper. In the areas of liquefactive necrosis, remains of astrocytes that were positive for GFAP were noted, interspersed in the lesion. Gemistocytic astrocytes possibly died through the effects of activation of macrophages and microglia in these localities, given that Gitter cells also expressed MMP-9, MIF and MHC-II. The deleterious effects of the inflammatory mediators (reactive oxygen species, nitric oxide and cytokines) may have been responsible for destruction of the demyelinated nerve tissue and for the death of astrocytes in these localities. Activated astrocytes may have contributed towards demyelination in the initial phase of the lesion, due to the direct action of the virus on these glial cells and on axons, thus resulting in secondary metabolic dysfunction of oligodendrocytes (Ulrich et al., 2014). Subsequently, the tissue injury would become amplified through local release of inflammatory mediators. Lastly, activated macrophages (Gitter cells) would act in the areas of liquefactive necrosis. Pan et al. (2013) also noted that GFAP was absent or only detected at low levels in the areas of liquefactive necrosis that resulted from demyelinating encephalopathy in distemper cases. Other authors have highlighted that activation of microglia, induced by CDV, may have some relevance regarding the pathogenesis of acute demyelination within distemper, through diffusely increased regulation of MHC in the white matter and through release of factors that are toxic to myelin (Vandevelde and Zurbriggen, 2005; Stein et al., 2006; Qesla et al., 2014). Those reports are in line with the results from the present study, since reactive microgliosis and increased expression of MHC II were observed in the demyelinated areas and in adjacent areas, thus confirming the participation of microglia in this process of expansion of the nerve lesion. The enzyme MMP-9 was possibly responsible for breaking the
Fig. 5. Variation of principal component 1 (PC1) in relation to principal component 2 (PC2) for the characteristics studied. Vectors indicate the relationships between the characteristics evaluated (GFAP, CD3, MIF, MHC-II and MMP-9).
be activated after the virus has arrived in the encephalon. These activated cells subsequently produce MIF, which would contribute towards activation of the vascular endothelium, microglia and lymphocyte influx after MMP-9 vascular basement membrane degradation, as described in the literature (Mitchell et al., 1999; Kleemann et al., 2000; Yu et al., 2007). A study on encephalitis in old dogs showed through immunohistochemical analysis that the virus was present in neurons, astrocytes, leukocytes in perivascular cuffs, vascular endothelium and areas of demyelination. Presence of cells positive for CD3 (T lymphocytes), CD79 (B lymphocytes) and MAC387 (macrophages) was observed in the inflammatory infiltrate of the neuropil, in response to chemiotactic stimuli that were released from the areas of the lesions. Moreover, presence of marking for vimentin in lymphocytes in the cuffs suggested that vimentin promoted migration of leukocytes to the site of injury (Headley et al., 2009). In parallel, marking for MIF in astrocytes in the areas of demyelination and adjacent areas was detected. This pro-inflammatory cytokine was detected at baseline levels in the CNS, and its levels increased 6
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demyelinating lesions in the white matter of the encephalon and spinal cord. Inflammation and involvement of the grey matter have also been reported. These alterations are generally preceded by viral or bacterial infection, or they may occur after vaccination with attenuated viruses (measles, rabies, influenza, rubella, etc.). Although the pathogenesis has not yet been fully elucidated, it has been suggested that the local immune response may be responsible for lesions to the myelin sheath of the axons, which would result in neurological lesions (Lee, 2011). Dogs with distemper may provide a model for understanding the pathogenesis of human demyelinating diseases.
blood-brain barrier and degrading the vascular basal membrane, thereby enabling an afflux of leukocytes to injured areas. However, it could not be confirmed whether the activated form of the enzyme predominated in the lesions, because the material studied had been embedded in paraffin, which cannot be subjected to the zymography technique. In the literature, it has been highlighted that MMP-9 expression levels are higher in inflammatory diseases of the CNS, thus suggesting that this inducible gelatinase is a systemic marker of inflammation in animals (Opdenakker et al., 2001). A study evaluating the presence of MMP-2 and MMP-9 in the cerebrospinal fluid of dogs with subacute distemper showed that the levels of pro-MMP-2 forms (cerebrospinal fluid and cerebellum) and pro-MMP-9 forms (cerebellum) were high in all the animals. The levels of active forms of these gelatinases were elevated in the cerebrospinal fluid. These findings confirm that these enzymes participate in breaking the blood-brain barrier. Thus, they contribute towards development of neuroinflammation and demyelination (Machado et al., 2013). In some studies on humans, the presence of metalloproteinases has been positively correlated with worsening of the lesion, in processes affecting the CNS (Yong et al., 2001). Marking for CD3+ T lymphocytes was observed in the encephala of the animals in the infected group, and this showed a significant association with marking for GFAP (Table 2), i.e. when the parameters studied were grouped and comparisons were made between the groups. This suggests that activation of astrocytes contributed towards migration of T lymphocytes to the encephalon. Expression of MHC-II in astrocytes and microglia has already been described, which demonstrates their potential for activation of T lymphocytes, through antigenic presentation via MHC (Hamo et al., 2007). MIF stimulates inflammatory response in brain by microglia and astrocytes activation. In astrocytes this activation is by the CD74 receptor. These authors believe that MIF would be a new target for therapeutic interference in neuroinflammation (Su et al., 2017). Thus, in the present study, it can be inferred that one of the CDV access routes in the nervous system was through the ventricular system. This hypothesis is reinforced by the presence of lesions and viral inclusions in the neuropil adjacent to the ependymal cells. A hematogenic route could be another possibility: astrocytes form part of the glia limitans and are also present in the blood-brain barrier, which thus confirms that they come into close contact with antigens that arrive via this route. Astrocytes thus become activated, which is shown in the areas with lesions through thickening of the cytoplasmic prolongations, marked with GFAP. From this point, the process of the immune response of the encephalon begins with production of MIF and MMP-9 and expression of MHC-II. This enables the breaking of the blood-brain barrier and migration of leukocytes into the nerve tissue. The leukocytes of the perivascular cuffs amplify the production of cytokines and inflammatory mediators. This further activates the microglia. The tissue damage caused by the mediators and by the harmful components released by the microglia may give rise to areas of liquefactive necrosis in the neuropil, thereby resulting in death of the astrocytes and oligodendrocytes at this locality. The Gitter cells at these localities continue to produce cytokines and inflammatory mediators, thus causing even greater deleterious effects on the nerve tissue. In multivariate analysis, it was evident that the immunoinflammatory parameters of the CNS vary at the same time and are directly related to the state of the disease. The cytokine MIF was possibly a link between glial cells (astrocytes and microglia), MMP-9 expression and lymphocyte influx into the CNS. Recent studies have highlighted that MIF regulates chemokine release (CCL5) and modulation of toll-like receptor 4 in astrocytes, which in turn favors recruitment of inflammatory cells to the injured neuropil (Zhou et al., 2018). It would be of interest to makes parallels regarding immunomediated evolution between the encephalic lesions of canine distemper and cases of acute disseminated encephalomyelitis (ADEM) in children. In ADEM, children develop neurological signs relating to
5. Conclusions Under the conditions of this study, it was possible to conclude that: - Astrocytes are key cells in activating the immune response against CDV, since they were detected in association with demyelinating lesions and in nerve tissue adjacent to these lesions. - Activated astrocytes expressed MIF, MMP-9 and MHC-II and possibly initiated neuroinflammation, which was subsequently maintained by microglia and inflammatory cells. - Most of the animals presented chronic lesions characterized by constant presence of gemistocytic astrocytes, inflammation and multifocal areas of demyelination and/or liquefactive necrosis. - The cytokine MIF was possibly the main modulator of neuroinflammation in dogs with distemper. - MIF could be used as a marker for inflammation in the CNS, since its expression in cases of severe demyelination and inflammation of the neuropil was greater. Ethical standards The design for this study was approved by the Ethics and Animal Welfare Committee (CEUA no. 00703/17), of FCAV - UNESP, Jaboticabal campus, state of São Paulo, Brazil. Declaration of Competing Interest The authors declare that they did not have any conflict of interest. Acknowledgements The authors would like to thank Dr. G.F. Machado, who kindly provided the paraffin blocks of canine brains of the infected group that were used in this study. T.F.S. De Nardo was supported by a grant from the National Council for Scientific and Technological Development (Conselho Nacional do Desenvolvimento Científico e Tecnológico, CNPq). P.H.L. Bertolo was a CAPES grantee. D.P. Munari held a productivity research fellowship from CNPq. The authors also wish to acknowledge Mr. Antonio Roveri Neto for his histotechnical assistance. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetimm.2020.110010. References Appel, M.J.G., 1969. Pathogenesis of canine distemper. Am. J. Vet. Res. 30, 1167–1182. Appel, M.J., Summers, B.A., 1995. Pathogenicity of morbiliviruses for terrestrial carnivores. Vet. Microbiol. 44, 187–191. Beineke, A., Puff, C., Seehusen, F., Baumgärtner, W., 2009. Pathogenesis and immunopathology of systemic and nervous canine distemper virus. Vet. Immunol. Immunopathol. 127, 1–18. https://doi.org/10.1016/j.vetimm.2008.09.023. Beineke, A., Baumgärtner, W., Wohlsein, P., 2015. Cross-species transmission of canine distemper virus – an update. One Health 1, 49–59. https://doi.org/10.1016/j.onehlt. 2015.09.002. Carvalho, O.V., Botelho, C.V., Ferreira, C.G.T., Scherer, P.O., Soares-Martins, J.A.P.,
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