Dog hepatocytes are key effector cells in the liver innate immune response to Leishmania infantum.

Hepatocytes constitute the majority of hepatic cells, and play a key role in controlling systemic innate immunity, via pattern-recognition receptors (PRRs) and by synthesizing complement and acute phase proteins. Leishmania infantum, a protozoan parasite that causes human and canine leishmaniasis, infects liver by establishing inside the Kupffer cells. The current study proposes the elucidation of the immune response generated by dog hepatocytes when exposed to L. infantum. Additionally, the impact of adding leishmanicidal compound, meglumine antimoniate (MgA), to parasite-exposed hepatocytes was also addressed. L. infantum presents a high tropism to hepatocytes, establishing strong membrane interactions. The possibility of L. infantum internalization by hepatocytes was raised, but not confirmed. Hepatocytes were able to recognize parasite presence, inducing PRRs [nucleotide oligomerization domain (NOD)1, NOD2 and Toll-like receptor (TLR)2] gene expression and generating a mix pro- and anti-inflammatory cytokine response. Reduction of cytochrome P 450s enzyme activity was also observed concomitant with the inflammatory response. Addition of MgA increased NOD2, TLR4 and interleukin 10 gene expression, indicating an immunomodulatory role for MgA. Hepatocytes seem to have a major role in coordinating liver's innate immune response against L. infantum infection, activating inflammatory mechanisms, but always balancing the inflammatory response in order to avoid cell damage.

Leishmania infantum exerts immunomodulation in canine Kupffer cells reverted by meglumine antimoniate.

Kupffer cells (KC) are the liver macrophage population that resides in the hepatic sinusoids and efficiently phagocyte pathogens by establishing an intimate contact with circulating blood. KC constitute the liver host cells in Leishmania infection, nevertheless little is described about their role, apart from their notable contribution in granulomatous inflammation. The present study aims to investigate how canine KC sense and react to the presence of Leishmania infantum promastigotes and amastigotes by evaluating the gene expression of specific innate immune cell receptors and cytokines, as well as the induction of nitric oxide and urea production. Complementarily, the impact of a leishmanicidal drug - meglumine antimoniate (MgA) - in infected KC was also explored. KC revealed to be susceptible to both parasite forms and no major differences were found in the immune response generated. L. infantum parasites seem to interact with KC innate immune receptors and induce an anergic state, promoting immune tolerance and parasite survival. The addition of MgA to infected KC breaks the parasite imposed silence and increased gene expression of Toll-like receptors (TLR) 2 and TLR4, possibly activating downstream pathways. Understanding how KC sense and react to parasite presence could bring new insights into the control or even elimination of canine leishmaniasis.

Leishmania infantum antigens modulate memory cell subsets of liver resident T lymphocyte.

In the recent years, the liver has been recognized as an important immune organ with major regulatory functions and immune memory, adding to the well-described vital metabolic functions. There are evidences from experimental infections performed with visceral Leishmania species that immune responses to parasite infection can be organ-specific. The liver is the compartment of acute resolving infection, with minimal tissue damage and resistance to reinfection, whereas the spleen is the compartment of parasite persistence. Control of hepatic infection in mice requires a coordinated immune response that involves the development of inflammatory granulomas. It is also described that the liver harbors populations of resident lymphocytes, which may exhibit memory characteristics. Therefore, the present study aims to address the role of the liver as an immune memory organ in the context of Leishmania infantum infection, by characterizing phenotypically resident liver T lymphocytes. The dynamics of memory T cells in L. infantum infected BALB/c mice and the effect of anti-leishmanial treatment in the differentiation of memory cell subsets were analyzed. The potential of recognition, differentiation and selection of memory lymphocytes by three L. infantum recombinant proteins were also explored. L. infantum infection generates effector and central memory T cells, but the cells did not expand when recalled, demonstrating a possible parasite silencing effect. The treatment with a leishmanicidal drug (antimoniate meglumine) increases the levels of memory and effector T cells, eliciting a more robust hepatic immune response. L. infantum parasites with a decreased sensitivity to the leishmanicidal drug favor the expansion of memory CD8+ T cell subset, but inhibit the proliferation of CD8+ T effector cells, possibly assuring their own survival. The recombinant proteins LirCyp1 and LirSOD are strongly recognized by memory cells of treated mice, indicating that these proteins might be used in a prophylactic or therapeutic vaccine formulation. Thus, L. infantum released antigens induce the development of immune memory subsets in the liver resident T cell population that specifically recognized parasite antigens, including recombinant proteins.

Immunization with the Leishmania infantum recombinant cyclophilin protein 1 confers partial protection to subsequent parasite infection and generates specific memory T cells.

Control of zoonotic visceral leishmaniosis can be achieved using several available drugs. These drugs present high toxicity and require longer treatment regimens which complicate compliance to the treatment. Other control measures directed to the vector or the reservoirs are useful tools to restrain the spreading of this disease but the effects are transitory. A safe, affordable and efficient vaccine conferring long lasting immunity should be the most cost effective way of controlling zoonotic visceral leishmaniosis. The present study aims at characterizing a cyclophilin protein 1 of Leishmania infantum (LiCyP1) and investigating whether recombinant LiCyP1 (LirCyP1) is able to confer protection against infection by evaluating viable parasite load and the generation of specific CD4(+) and CD8(+) effector and central memory T cells in rodent model. LiCyP1 is present in the cytoplasm of L. infantum amastigotes and promastigotes. Immunization of BALB/c mice with LirCyP1 confers high protection to L. infantum infection, causing a marked reduction in parasite replication in the liver and spleen. Furthermore, helper and cytotoxic memory T cell subsets able to specifically recognize parasite antigens expanded in immunized and in challenged mice. CD4(+) T cell subpopulation of intermediate phenotype (CD62L(high)CD127(low)) of challenging mice also presented an accentuated expansion after the recall. This study demonstrated that LirCyP1 confers partial protection to L. infantum infection, promoting the generation of a desired long lasting immunity. LirCyP1 can be considered a potential candidate for the design of a vaccine against zoonotic visceral leishmaniosis.