Research paperToll-like receptors and cytokines in the brain and in spleen of dogs with visceral leishmaniosis
Introduction
Visceral leishmaniosis (VL) is an multisystemic disease caused by the Leishmania infantum (L. chagasi) protozoan (Mauricio et al., 2000). Domestic dogs can present with several systemic manifestations (Garcia-Alonso et al., 1996; Ciaramella et al., 1997; Blavier et al., 2001; Reis et al., 2009), including some rare reports of neurological clinical signs (Font et al., 2004; Ikeda et al., 2007; José-lópez et al., 2012; Melo et al., 2012; Gianuzzi et al., 2017), or the infection may remain asymptomatic.
Neurological clinical signs include walking in circles, seizures, paresis, tetraplegia, head tilt, motor incoordination, intention tremor, nystagmus, strabismus and myoclonia (Font et al., 2004; Ikeda et al., 2007; José-lópez et al., 2012; Márquez et al., 2013; Gianuzzi et al., 2017). In addition to neurological clinical signs, brain inflammation has also been reported in dogs with VL, where meningitis and choroiditis are the usual histopathological findings (Nieto et al., 1996; Viñuelas et al., 2001; Melo et al., 2009, 2013, 2015a; Grano et al., 2016; Gianuzzi et al., 2017). Considering these findings, the focus of our research group in recent years has been to investigate the question about why infected dogs present neurological clinical signs and brain inflammation. We have previously suggested two hypotheses. One is that parasite migration from macrophage-rich infected tissues to the brain stimulates inflammation. However, the presence of the Leishmania parasite in the brain is still unclear, although we have detected its DNA in this compartment (Grano et al., 2014; Melo et al., 2015a). The second hypothesis is that peripheral stimuli, such as inflammatory mediators, can reach the central nervous system (CNS), developing local inflammation (Melo et al., 2015a).
Pattern recognition receptors (PRRs) are an important group of innate immune receptors that recognize pathogen-associated molecular patterns (PAMPs), which are conserved structures present in fungi, viruses, bacteria and protozoans (reviewed by Ospelt and Gay, 2010) and recognize damage-associated molecular patterns (DAMPs), which are host-derived proteins (Miyake, 2007; Piccinini and Midwood, 2010) in chronic inflammation, triggering a cascade of immune reactions (Fukata et al., 2009).
TLRs are PRRs that act on the recognition of microbial structures and induce innate and adaptive immune responses (Tuon et al., 2008). Different TLRs detect distinct molecules from viruses, bacteria, fungi and parasites (Kawai and Akira, 2011). Signaling activation through TLRs causes the production of chemokines, inflammatory cytokines, adhesion molecules and co-stimulatory molecules (Ospelt and Gay, 2010). Gene expression of cytokines and chemokines has already been characterized during canine VL in parasite-infected tissues (Panaro et al., 2009; Barbosa et al., 2011; Melo et al., 2015a), as well as in the CNS (Melo et al., 2013; 2015a), where there was an increase in the transcripts of proinflammatory cytokines such as TNF-α, IFN-γ and IL-1β in the brain (Melo et al., 2013).
Concerning TLRs, there are only a few study reports in canine VL. Most studies have focused on TLRs 2, 4 and 9, especially in peripheral blood mononuclear cells (PBMCs) (Melo et al., 2014a), jejunum and colon (Figueiredo et al., 2013), skin (Esteve et al., 2015; Hosein et al., 2015), liver (Hosein et al., 2015), spleen, lymph nodes (Melo et al., 2014b; Hosein et al., 2015) and brain (Melo et al., 2014b).
Studies evaluating the immune response in the CNS of dogs with leishmaniosis are scarce. Despite the growing number of studies about PRRs in recent years, the Toll-like receptor profile in the brain, or even in the spleen, remains to be characterized in order to elucidate which receptors might be related to the recognition of the Leishmania parasite or its antigens in the brain during canine VL. Therefore, the aim of this study was to determine the gene expression of TLR 1–10 and the production of proinflammatory cytokines (TNF-α, IFN-γ, IL-1β and IL-6) in two target compartments of our previous studies: 1) CNS, which is represented here by the brain, the main focus of our studies, and 2) spleen, which is considered an organ bearing the bulk of parasite burden, representing the peripheral system, in which we investigated previously and detected an up-regulation of TLR-2 (Melo et al., 2014b).
Section snippets
Animals
Twenty-one dogs, 13 males and 8 females, ranging in age from 1 to 4 years old, were selected from the Zoonosis Control Center in the municipality of Araçatuba, São Paulo State, Brazil and included in this study. Seventeen naturally infected dogs by the Leishmania parasite were euthanized with the owner’s permission, in compliance with state law (Brasil, 2008), as soon as the VL diagnosis was confirmed. All infected dogs were symptomatic, but they did not present a history of neurological signs.
Clinical staging
We found in the macroscopic examination that 70.58% (12/17) of the infected dogs presented cachexia or weight loss, followed by 64.70% (11/17) with skin disease, including alopecia, nasal hyperkeratosis, ulcers and seborrhea, 35.29% (6/17) with splenomegaly, 35.29% (6/17) with conjunctivitis and onychogryphosis, and 29.41% (5/17) with lymphadenopathy.
Concerning the laboratory findings, we observed that 88.23% (15/17) of the infected dogs presented anemia, 52.94% (9/17) presented lymphocytopenia
Discussion
We detected brain inflammation, as well as TLR gene expression and Leishmania DNA in the brain and spleens of dogs with VL. The population of dogs evaluated here included animals belonging to the four clinical stages of the classification proposed by Solano-Gallego et al. (2011). However, there was no correlation between the clinical stage or parasite load and TLR expression. An absence of correlation was also observed between the clinical classification and the intensity of brain inflammation.
Conclusions
Our data provide support to explain the involvement of innate immune sensors in the immune response against L. infantum. The data presented herein provide important evidence that TLR-5 and TLR-9 play a role in splenic innate immunity during canine VL and that TLR-4 might be related to the pathogenesis of brain lesions, although the number of brain samples that presented up-regulation of TLR-4 was low, and future investigations should be performed. Concerning cytokines, IFN-γ may be related to
Conflict of interest statement
The authors of this work do not have any financial, personal or other relationship with organizations or people that could inadequately influence the content of this paper.
Acknowledgments
F.G. Grano was supported by a Ph.D.’s degree scholarship from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant number 2013/25498-1). The research project was supported by FAPESP, grant number 2016/02384-9 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant number 420970/2016-9.
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