The balance between IL-12/IL4 in renal tissue switches the inflammatory response arm and shows relationship with the clinical signs in Leishmania-infected dogs
Graphical abstract
Introduction
Leishmaniasis is among the most neglected diseases, affecting the poorest populations predominantly in developing countries. Visceral, cutaneous, and mucocutaneous leishmaniasis cause high morbidity and mortality in man, and approximately 2 million new cases occur annually worldwide (World Health Organization (WHO, 2010). Visceral leishmaniasis systemically affects humans and dogs, with the latter being the prevalent domestic reservoirs of the parasite (Burza et al., 2018). Recently, the term leishmaniosis was applied to the canine form of the disease, whereas leishmaniasis was used to refer to human disease (Miró and López-Vélez, 2018). So that, this terminology was used in this work.
Leishmania may affect several organs, including the kidneys in humans and dogs. They may affect renal glomeruli and tubules and may also show interstitial nephritis in cases of visceral leishmaniasis (VL) (Clementi et al., 2011). Patients with VL have demonstrated mild to moderate proteinuria, hematuria, and increased urine leukocytes in over 50 % of cases (Salgado Filho et al., 2003). It was observed that more than 11 % of patients with chronic leishmaniasis had decreased renal filtration rates upon hospital admission, although these symptoms disappeared after anti-parasitic therapy (Silva Jr et al., 2014).
In symptomatic CanL, the kidney may also be compromised as demonstrated in membranoproliferative glomerulonephritis, mesangial proliferative glomerulonephritis, and interstitial nephritis (Soares et al., 2005). Furthermore, amastigotes of Leishmania spp can be found in the inflammatory infiltrate in the renal parenchyma (Soares et al., 2005). In CanL, interstitial nephritis and glomerular alterations were predominantly localized in the cortex region, similar to that observed in infected humans (Costa et al., 2003).
The presence of parasites or their antigens in renal tissues may trigger an immune response. Successful immunity to Leishmania spp depends on the recruitment of the appropriate immune effector cells to the site of infection, a process in which chemokines play a crucial role. The pathogenesis of VL is complex and often associated with altered cytokine levels and chemokine expression profiles and defective immune cell migration (Racoosin and Beverley, 1997; Ritter and Korner, 2002; Antoniazi et al., 2004; Stanley and Engwerda, 2007).
The expression of IL-2, IL-12, IFN-γ, and TNF-α mRNAs is higher in asymptomatic infected dogs associated with lower parasite load in the pre-scapular lymph nodes, peripheral blood, and skin (Chamizo et al., 2005; Sanches-Robert et al. (2008), Alves et al., 2009; Menezes-Souza et al., 2011). Barbosa et al. (2011) evaluated gene expression in PBMCs, bone marrow, and lymph nodes of symptomatic and asymptomatic dogs and found that the expression of IFN-γ, IL-4, IL-12, and IL-2 were different in each tissue type. There was an increase in IL-4 expression in PBMCs, and the expression levels of other cytokines were similar to those found in the uninfected control. In the bone marrow, the expression levels of all evaluated cytokines were similar to those in the controls. IFN-γ and IL-2 were the predominantly expressed cytokines in the lymph nodes of symptomatic dogs, whereas IL-12 and IL-4 were more highly expressed in the infected groups without clinical manifestations. These data suggest that the immune response is compartmentalized (Reis et al., 2009), possibly depending on the presence of the parasite or its antigens in a specific organ. To our knowledge, the cytokine levels in renal tissue in CanL were not evaluated yet. Taking into consideration that the kidney shows alterations in CanL, and the balance of cytokine differently regulate pro- and anti-inflammatory immune responses, we hypothesized that cytokine balance might be differentially expressed in renal tissue determining the clinical expression of the renal disease.
High anti-Leishmania antibody titers, which are not immunoprotective, were detected in symptomatic dogs (Abranches et al., 1991; Martínez-Moreno et al., 1995). Many diseases that affect the kidneys, including CanL, are caused by the "in situ" deposition of circulating immune complexes (McCluskey and Bhan, 1982; Macianti et al., 1989; Lopes et al., 1996; Soares et al., 2009). Furthermore, studies on pathogenesis of canine glomerulonephritis caused by L. infantum have revealed the involvement of T cells (Costa et al., 2000), cytokines, adhesion molecules, and the process of apoptosis (Costa et al., 2010). Interstitial nephritis is a frequent lesion in VL (Olsen et al., 1986), although the pathogenesis of tubule-interstitial injury remains unclear. However, the immune mechanism mediated by inflammatory cells has been considered in humans (Boucher et al., 1986) and experimental models (Neilson et al., 1984).
Although some studies have shown renal alterations in leishmaniosis, the relationship between cytokines and the inflammatory response is unclear. Therefore, the aim of this study was to evaluate the inflammation and expression of cytokines using histomorphometry and quantitative polymerase chain reaction (qPCR), respectively, and to correlate these findings with the clinical manifestations in dogs naturally infected with L. infantum.
Section snippets
Ethical issues and study groups
This study involved only stray dogs captured as a part of a program to control CanL and urban rabies. Efforts were made to avoid unnecessary distress to the dogs. Housing, anesthesia, and all procedures conformed to the guidelines established by our local Institutional Dog Care and Use Committee, which reviewed and approved this study (“Ethical Committee in Dog Experimentation” – CETEA, national guidelines, Law number 11.794, 8/10/2008 from Federal University of Minas Gerais- UFMG; protocol n°
Clinical findings
Sixteen dogs (out of 23) were found infected (69.56 %) and 7 were uninfected (30.44 %). The majority of dogs were male (20/23, 86.86 %). The uninfected control and symptomatic and symptomatic infected groups weighed 10.43 ± 1.32 kg, 10.29 ± 2.09 kg, and 7.98 ± 1.22 kg, respectively. All dogs were adult mongrels. Half of the infected dogs were classified as asymptomatic, whereas the remaining half presented clinical signs for CanL (symptomatic). Seven of eight symptomatic dogs (87.50 %)
Discussion
In this novel study, inflammation and the expression of certain cytokines in the kidneys of naturally infected dogs with CanL were evaluated using histomorphometry and qPCR, respectively.
There was a higher expression of IL-4 in kidney tissues of symptomatic infected dogs associated with a high inflammatory response and more severe renal histopathological lesions than those of the dogs without clinical signs. IL-4 induces cell differentiation to the Th2 profile, and this cytokine may cause
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
The study was supported by the Fundação de Amparo a Pesquisa do Estado de Minas Gerais - FAPEMIG, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES, and Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq. Bárbara L. A. Verçosa was supported by CAPES (Programa de Pós-gradução em Biologia Celular – ICB/UFMG) and was an investigator supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológica (CNPq), Brazil (process number 168270/2017-0). Maria
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