Pro-Cellular Exhaustion Markers are Associated with Splenic Microarchitecture Disorganization and Parasite Load in Dogs with Visceral Leishmaniasis

Tainã Luís De Souza,Renato Porrozzi,Elisa Cupolillo,Daniella Areas Mendes-Da-Cruz,Fabiano Borges Figueiredo,Artur Augusto Velho Mendes Junior,Rodrigo Caldas Menezes,Fernanda Nazaré Morgado,Aurea Virginia Andrade Da Silva,Mariana Côrtes Boité,Luiza De Oliveira Ramos Pereira

doi:10.1038/s41598-019-49344-1

Tainã Luís De Souza, Renato Porrozzi + Show 9 more

Open Access

https://doi.org/10.1038/s41598-019-49344-1

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Abstract

In canine visceral leishmaniasis (CVL), splenic white pulp (SWP) disorganization has been associated with disease progression, reduced cytokine and chemokine expression and failure to control the parasite load. This profile is compatible with the cellular exhaustion previously shown in human visceral leishmaniasis. The present study aimed to evaluate the in situ expression of cellular exhaustion markers and their relation to clinical signs, SWP disorganization and parasite load. Forty dogs naturally infected by Leishmania infantum were grouped according to levels of SWP organization and parasite load. SWP disorganization was associated with reductions in the periarteriolar lymphatic sheath and lymphoid follicles/mm2 and worsening of the disease. Apoptotic cells expressing CTLA-4+ increased in dogs with disorganized SWP and a high parasite load. In the same group, PD-L1 and LAG-3 gene expression were reduced. A higher number of CD21+TIM-3+ B cells was detected in disorganized spleens than in organized spleens. Apoptosis is involved in periarteriolar lymphatic sheath reduction and lymphoid follicle atrophy and is associated with CTLA-4+ cell reductions in the splenic tissue of dogs with visceral leishmaniasis (VL). Failure to control the parasite load was observed, suggesting that cell exhaustion followed by T and B cell apoptosis plays a role in the immunosuppression observed in CVL.

Highlights

Zoonotic visceral leishmaniasis (ZVL) is a tropical and subtropical disease caused by Leishmania infantum, an intracellular protozoan that is transmitted to its vertebrate hosts by the blood feeding of female sand flies of the genera Phlebotomus and Lutzomyia
The expression of programmed death 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) by peripheral and splenic CD8+ cells has been demonstrated in human visceral leishmaniasis (VL)[16] and in mice experimentally infected with Leishmania donovani, another Leishmania species that causes VL15–17
We hypothesized that failures in mounting an effective immune response and in controlling the parasite load are associated with the disorganization of the splenic white pulp (SWP) and cellular exhaustion in dogs presenting with VL

Summary

Introduction

Zoonotic visceral leishmaniasis (ZVL) is a tropical and subtropical disease caused by Leishmania infantum, an intracellular protozoan that is transmitted to its vertebrate hosts by the blood feeding of female sand flies of the genera Phlebotomus and Lutzomyia. Recent studies have correlated the progression of disease with the disorganization of the splenic microarchitecture[3,10], leading to increased parasite load and reduced expression of cytokines, chemokines and chemokine receptors[11], which is compatible with the cellular exhaustion profile. Administration of anti-CTLA-4 blocking antibodies in vivo led to increased frequencies of IFN-γ- and IL-4-producing cells in the liver and spleen of the experimentally infected mice and accelerated the development of the hepatic granulomatous response associated with a reduction in the parasite load[17]. We evaluated the expression of the exhaustion markers PD-1, PD-L1, CTLA-4, TIM-3 and LAG-3 in the spleen of dogs naturally infected with L. infantum and correlated the results with the clinical signs, organization of the SWP and parasite load observed in the animals

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