Vanessa Carregaro

Interleukin-1 (IL-1) is a pleiotropic cytokine important in inflammatory and infectious diseases, but its roles in visceral leishmaniasis (VL) remain poorly understood. Here, we evaluated the role of IL-1 cytokines during experimental visceral leishmaniasis. We showed that IL-1 receptor 1 (IL-1R1) deficiency enhanced the gamma interferon (IFNγ)-mediated response and restricted parasites in an IL-1α-dependent manner, whereas IL-1β deficiency had no effect. The absence of IL-1R1 and IL-1α led to intestinal microbiota dysbiosis. The infection induced IL-1α expression in the gut. The absence of IL-1α led to reduced expression of tight-junction molecules, increased intestinal permeability, and bacterial translocation. These alterations amplified non- Leishmania -specific IFN-γ production, which was reversed by broad-spectrum antibiotic treatment. Bone marrow chimeric transplantation showed that hematopoietic cells are the source of IL-1α during VL. Together, our findings reveal that IL-1α acts as a key regulator of gut barrier integrity in VL and indirectly restrains systemic IFNγ-driven inflammation. This work highlights IL-1α-dependent intestinal protection as an overlooked component of VL pathogenesis. IMPORTANCE: Visceral leishmaniasis (VL) is a systemic and chronic disease caused by Leishmania parasites. Most infected individuals remain asymptomatic due to an appropriate IFNγ-mediated response. By contrast, the disease progression is characterized by excessive inflammation, with high levels of cytokines, among them IFNγ. IL-1 cytokines play a role in modulating immune responses and epithelial barriers. We report that IL-1α protects the intestinal environment and indirectly regulates the IFNγ-mediated response during experimental VL. The infection induces IL-1α expression in the intestine, and its absence leads to dysbiosis, gut permeability, and bacterial translocation. Overall, this study reveals the role of IL-1α in intestinal integrity during VL and in regulating the IFNγ-mediated response. Expanding the knowledge of IL-1α in intestinal protection may support therapeutic approaches to improve the outcomes of infection.

A key point of immunity against protozoanLeishmaniaparasites is the development of an optimal T cell response, which includes a low apoptotic rate, high proliferative activity and polyfunctionality. During acute infection, antigen-specific T cells recognize the pathogen resulting in pathogen control but not elimination, promoting the development and the maintenance of a population of circulating effector cells that mount rapid response quickly after re-exposure to the parasite. However, in the case of visceral disease, the functionality of specific T cells is lost during chronic infection, resulting in inferior effector functions, poor response to specific restimulation, and suboptimal homeostatic proliferation, a term referred to as T cell exhaustion. Multiple factors, including parasite load, infection duration and host immunity, affect T lymphocyte exhaustion. These factors contribute to antigen persistence by promoting inhibitory receptor expression and sustained production of soluble mediators, influencing suppressive cell function and the release of endogenous molecules into chronically inflamed tissue. Together, these signals encourage several changes, reprogramming cells into a quiescent state, which reflects disease progression to more severe forms, and development of acquired resistance to conventional drugs to treat the disease. These points are discussed in this review.

Leishmania major (L. major) causes cutaneous leishmaniasis in the Old World. The infection mostly induces a localized lesion restricted to the sand fly bite. The costs and the side effects of current treatments render imperative the development of new therapies that are affordable and easy to administrate. Topical treatment would be the ideal option for the treatment of cutaneous leishmaniasis. MF29 is a 3-haloacetamidobenzoate that was shown in vitro to inhibit tubulin assembly in Leishmania. Here, we tested a topical cream formulated with MF29. BALB/c mice were infected in the ear dermis with L. major metacyclic promastigotes and once the lesion appeared, mice were treated with different concentrations of MF29 and compared to the control group treated with the cream used as the vehicle. We observed that topical application of MF29 reduced the progression of the infection while control groups developed an unhealing lesion that became necrotic. The treatment decreased the type 2 immune response. Comparison with SinaAmphoLeish, another topical treatment, revealed that MF29 treatment once a day was sufficient to control lesion development, while application SinaAmphoLeish needed applications twice daily. Collectively, our data suggest that MF-29 topical application could be a promising topical treatment for cutaneous leishmaniasis.

A striking feature of human visceral leishmaniasis (VL) is chronic inflammation in the spleen and liver, and VL patients present increased production levels of multiple inflammatory mediators, which contribute to tissue damage and disease severity. Here, we combined an experimental model with the transcriptional profile of human VL to demonstrate that the TLR4-IFN-β pathway regulates the chronic inflammatory process and is associated with the asymptomatic form of the disease. Tlr4-deficient mice harbored fewer parasites in their spleen and liver than wild-type mice. TLR4 deficiency enhanced the Th1 immune response against the parasite, which was correlated with an increased activation of dendritic cells (DCs). Gene expression analyses demonstrated that IRF1 and IFN-β were expressed downstream of TLR4 after infection. Accordingly, IRF1- and IFNAR-deficient mice harbored fewer parasites in the target organs than wild-type mice due to having an increased Th1 immune response. However, the absence of TLR4 or IFNAR increased the serum transaminase levels in infected mice, indicating the presence of liver damage in these animals. In addition, IFN-β limits IFN-γ production by acting directly on Th1 cells. Using RNA sequencing analysis of human samples, we demonstrated that the transcriptional signature for the TLR4 and type I IFN (IFN-I) pathways was positively modulated in asymptomatic subjects compared with VL patients and thus provide direct evidence demonstrating that the TLR4-IFN-I pathway is related to the nondevelopment of the disease. In conclusion, our results demonstrate that the TLR4-IRF1 pathway culminates in IFN-β production as a mechanism for dampening the chronic inflammatory process and preventing immunopathology development. Visceral leishmaniasis (VL) is one of the most lethal neglected tropical diseases and is caused by Leishmania parasites. Most subjects infected with Leishmania present subclinical VL symptoms, and their immune response is mediated by Th1 cells and immunoregulatory mechanisms. However, when infection progresses to disease, VL patients present increased levels of inflammatory mediators in the serum which are related to the severity of disease. During infection, Toll-like receptors (TLRs) interact with Leishmania parasites and contribute to the outcome of the disease. Herein, we report that TLR4 signaling hampers the chronic immune response during VL to prevent immunopathology. TLR4 triggers the activation of IRF1 and thus induces the transcription of IFN-β, which in turn acts directly on Th1 cells to limit the production of IFN-γ. In addition, a transcription analysis of human VL samples provides direct evidence demonstrating that the TLR4-IFN-I pathway is related to the asymptomatic form of the disease. Collectively, our findings reveal that TLR4 hampers the Th1 immune response through IRF1 and IFN-β to prevent immunopathology during VL.

Zika virus (ZIKV) Infection has several outcomes from asymptomatic exposure to rash, conjunctivitis, Guillain-Barré syndrome or congenital Zika syndrome. Analysis of ZIKV immunity is confounded by the fact that several related Flaviviruses infect humans, including Dengue virus 1–4, West Nile virus and Yellow Fever virus. HLA class II restricted T cell cross-reactivity between ZIKV and other Flaviviruses infection(s) or vaccination may contribute to protection or to enhanced immunopathology. We mapped immunodominant, HLA class II restricted, CD4 epitopes from ZIKV Envelope (Env), and Non-structural (NS) NS1, NS3 and NS5 antigens in HLA class II transgenic mice. In several cases, ZIKV primed CD4 cells responded to homologous sequences from other viruses, including DENV1–4, WNV or YFV. However, cross-reactive responses could confer immune deviation - the response to the Env DENV4 p1 epitope in HLA-DR1 resulted in IL-17A immunity, often associated with exacerbated immunopathogenesis. This conservation of recognition across Flaviviruses, may encompass protective and/or pathogenic components and poses challenges to characterization of ZIKV protective immunity.

Adenosine is an endogenously released purine nucleoside that signals through four widely expressed G protein-coupled receptors: A1, A2_A, A2_B, and A3. Of these, A_2AR is recognized as mediating major adenosine anti-inflammatory activity. During cutaneous leishmaniasis, adenosine induces immunosuppression, which promotes the establishment of infection. Herein, we demonstrated that A_2AR signaling is exploited by Leishmania infantum parasites, the etiologic agent that causes Visceral Leishmaniasis, to successfully colonize the vertebrate host. A_2AR gene-deleted mice exhibited a well-developed cellular reaction with a strong Th1 immune response in the parasitized organs. An intense infiltration of activated neutrophils into the disease-target organs was observed in A_2AR^−/− mice. These cells were characterized by high expression of CXCR2 and CD69 on their cell surfaces and increased cxcl1 expression. Interestingly, this phenotype was mediated by IFN-γ on the basis that a neutralizing antibody specific to this cytokine prevented neutrophilic influx into parasitized organs. In evaluating the immunosuppressive effects, we identified a decreased number of CD4⁺ FOXP3⁺ T cells and reduced il10 expression in A_2AR^−/− infected mice. During ex vivo cell culture, A_2AR^−/− splenocytes produced smaller amounts of IL-10. In conclusion, we demonstrated that the A_2AR signaling pathway is detrimental to development of Th1-type adaptive immunity and that this pathway could be associated with the regulatory process. In particular, it promotes parasite surveillance.

Visceral leishmaniasis (VL) is a chronic and fatal disease caused by Leishmania infantum in Brazil. Leukocyte recruitment to infected tissue is a crucial event for the control of infections such as VL. Among inflammatory cells, neutrophils are recruited to the site of Leishmania infection, and these cells may control parasite replication through oxidative or non-oxidative mechanisms. The recruitment, activation and functions of the neutrophils are coordinated by pro-inflammatory cytokines and chemokines during recognition of the parasite by pattern recognition receptors (PRRs). Here, we demonstrated that the Toll-like receptor 2 (TLR2) signaling pathway contributes to the development of the innate immune response during L. infantum infection. The protective mechanism is related to the appropriate recruitment of neutrophils to the inflammatory site. Neutrophil migration is coordinated by DCs that produce CXCL1 and provide a prototypal Th1 and Th17 environment when activated via TLR2. Furthermore, infected TLR2-/- mice failed to induce nitric oxide synthase (iNOS) expression in neutrophils but not in macrophages. In vitro, infected TLR2-/- neutrophils presented deficient iNOS expression, nitric oxide (NO) and TNF-α production, decreased expression of CD11b and reduced L. infantum uptake capacity. The non-responsive state of neutrophils is associated with increased amounts of IL-10. Taken together, these data clarify new mechanisms by which TLR2 functions in promoting the development of the adaptive immune response and effector mechanisms of neutrophils during L. infantum infection.