Maria Bellio

Expression of functional TLR4 confers proinflammatory responsiveness to Trypanosoma cruzi glycoinositolphospholipids and higher resistance to infection with T. cruzi.

TLRs function as pattern recognition receptors in mammals and play an essential role in the recognition of microbial components. We found that the injection of glycoinositolphospholipids (GIPLs) from Trypanosoma cruzi into the peritoneal cavity of mice induced neutrophil recruitment in a TLR4-dependent manner: the injection of GIPL in the TLR4-deficient strain of mice (C57BL/10ScCr) caused no inflammatory response. In contrast, in TLR2 knockout mice, neutrophil chemoattraction did not differ significantly from that seen in wild-type controls. GIPL-induced neutrophil attraction and MIP-2 production were also severely affected in TLR4-mutant C3H/HeJ mice. The role of TLR4 was confirmed in vitro by testing genetically engineered mutants derived from TLR2-deficient Chinese hamster ovary (CHO)-K1 fibroblasts that were transfected with CD14 (CHO/CD14). Wild-type CHO/CD14 cells express the hamster TLR4 molecule and the mutant line, in addition, expresses a nonfunctional form of MD-2. In comparison to wild-type cells, mutant CHO/CD14 cells failed to respond to GIPLs, indicating a necessity for a functional TLR4/MD-2 complex in GIPL-induced NF-κB activation. Finally, we found that TLR4-mutant mice were hypersusceptible to T. cruzi infection, as evidenced by a higher parasitemia and earlier mortality. These results demonstrate that natural resistance to T. cruzi is TLR4 dependent, most likely due to TLR4 recognition of their GIPLs.

Inflammasome-derived IL-1 beta production induces nitric oxide-mediated resistance to Leishmania

Parasites of the Leishmania genus are the causative agents of leishmaniasis in humans, a disease that affects more than 12 million people worldwide. These parasites replicate intracellularly in macrophages, and the primary mechanisms underlying host resistance involve the production of nitric oxide (NO). In this study we show that the Nlrp3 inflammasome is activated in response to Leishmania infection and is important for the restriction of parasite replication both in macrophages and in vivo as demonstrated through the infection of inflammasome-deficient mice with Leishmania amazonensis, Leishmania braziliensis and Leishmania infantum chagasi. Inflammasome-driven interleukin-1β (IL-1β) production facilitated host resistance to infection, as signaling through IL-1 receptor (IL-1R) and MyD88 was necessary and sufficient to trigger inducible nitric oxide synthase (NOS2)-mediated production of NO. In this manuscript we identify a major signaling platform for host resistance to Leishmania spp. infection and describe the molecular mechanisms underlying Leishmania-induced NO production.

The Immune Response to Trypanosoma cruzi: Role of Toll-Like Receptors and Perspectives for Vaccine Development

In the past ten years, studies have shown the recognition of Trypanosoma cruzi-associated molecular patterns by members of the Toll-like receptor (TLR) family and demonstrated the crucial participation of different TLRs during the experimental infection with this parasite. In the present review, we will focus on the role of TLR-activated pathways in the modulation of both innate and acquired immune responses to T. cruzi infection, as well as discuss the state of the art of vaccine research and development against the causative agent of Chagas disease (or American trypanosomiasis).

Impaired Innate Immunity in Tlr4−/− Mice but Preserved CD8+ T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8+ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-γ secreting CD8+ T cells specific for H-2Kb-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2−/−, Tlr4−/−, Tlr9−/ − or Myd88−/− mice generated both specific cytotoxic responses and IFN-γ secreting CD8+ T cells at levels comparable to WT mice, although the frequency of IFN-γ+CD4+ cells was diminished in infected Myd88−/− mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-γ, TNF-α and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4−/− mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi.

Chloroquine, an Endocytosis Blocking Agent, Inhibits Zika Virus Infection in Different Cell Models

Zika virus (ZIKV) infection in utero might lead to microcephaly and other congenital defects. Since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. Chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. Here we show that chloroquine exhibits antiviral activity against ZIKV in Vero cells, human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres. We demonstrate that chloroquine reduces the number of ZIKV-infected cells in vitro, and inhibits virus production and cell death promoted by ZIKV infection without cytotoxic effects. In addition, chloroquine treatment partially reveres morphological changes induced by ZIKV infection in mouse neurospheres.

Human cutaneous leishmaniasis: interferon-dependent expression of double-stranded RNA-dependent protein kinase (PKR) via TLR2

We investigated the type I interferon (IFN‐1)/PKR axis in the outcome of the Leishmania (Leishmania) amazonensis infection, along with the underlying mechanisms that trigger and sustain this signaling pathway. Reporter assays of cell extracts from RAW‐264.7 macrophages infected with L. (L.) amazonensis or HEK‐293T cells cotransfected with TLR2 and PKR promoter constructions were employed. Primary macrophages of TLR2‐knockout (KO) or IFNR‐KO mice were infected, and the levels of PKR, IFN‐1, and superoxide dismutase 1 (SOD1) transcript levels were investigated and compared. Immunohistochemical analysis of human biopsy lesions was evaluated for IFN‐1 and PKR‐positive cells. Leishmania infection increased the expression of PKR and IFN‐β on induction of PKR‐promoter activity. The observed effects required the engagement of TLR2. TLR2‐KO macrophages expressed low IFN‐β and PKR levels postinfection with a reduced parasite load. We also revealed the requirement of PKR signaling for Leishmania‐induced IFN‐1 expression, responsible for sustaining PKR expression and enhancing infection. Moreover, during infection, SOD1 transcripts increased and were also enhanced when IFN‐1 was added to the cultures. Remarkably, SOD1 expression was abrogated in infected, dominant‐negative PKR‐expressing cells. Finally, lesions of patients with anergic diffuse cutaneous leishmaniasis exhibited higher levels of PKR/IFN‐1‐expressing cells compared to those with single cutaneous leishmaniasis. In summary, we demonstrated the mechanisms and relevance of the IFN‐1/PKR axis in the Leishmania infection.—De Carvalho Vivarini, A., Pereira, R. M. S., Dias Teixeira, K. L., Calegari‐Silva, T. C., Bellio, M., Laurenti, M. D., Corbett, C. E. P., de Castro Gomes, C. M., Soares, R. P., Mendes Silva, A., Silveira, F. T., Lopes, U. G. Human cutaneous leishmaniasis: interferon‐dependent expression of double‐stranded RNA‐kinase (PKR) via TLR2. FASEB J. 25, 4162–4173 (2011). www.fasebj.org

NF-κB-mediated repression of iNOS expression in Leishmania amazonensis macrophage infection

Host invasion by pathogens is frequently associated with the activation of nuclear factor kappaB (NF-kappaB), which modulates the expression of genes involved in the immunological response of the host. However, pathogens may also subvert these mechanisms to secure their survival. We describe the effect of Leishmania amazonensis infection on NF-kappaB transcriptional factor activation in macrophages and the subsequent reduction in inducible nitric oxide synthase (iNOS) expression. L. amazonensis promastigote infection activates the p50/p50 NF-kappaB complex, a classic transcriptional repressor. Interestingly, L. amazonensis promotes the change of the classical p65/p50 NF-kappaB dimer induced by LPS, leading to the p50/p50 NF-kappaB complex activation in macrophages stimulated with LPS. Moreover, this parasite promotes the reduction of p65 total levels in infected macrophages. All these effects contribute to the observation that this parasite is able to restrain the NF-kappaB-dependent transcriptional activity induced by LPS. Strikingly, L. amazonensis reduces the mRNA levels of the iNOS in addition to protein expression and the production of nitric oxide in LPS-stimulated macrophages. Accordingly, as revealed by reporter-gene assays, L. amazonensis-induced iNOS repression requires NF-kappaB sites in the iNOS promoter region. In summary, our results suggest that L. amazonensis has developed an adaptive strategy to escape from host defense by activating the NF-kappaB repressor complex p50/p50. The activation of this specific host transcriptional response negatively regulates the expression of iNOS, favoring the establishment and success of L. amazonensis infection.

Toll‐like receptor 4 (TLR4)‐dependent proinflammatory and immunomodulatory properties of the glycoinositolphospholipid (GIPL) from Trypanosoma cruzi

We have demonstrated recently that the glycoinositolphospholipid (GIPL) molecule from the protozoan Trypanosoma cruzi is a TLR4 agonist with proinflammatory effects. Here, we show that GIPL‐induced neutrophil recruitment into the peritoneal cavity is mediated by at least two pathways: one, where IL‐1β acts downstream of TNF‐α, and a second, which is IL‐1β‐ and TNFRI‐independent. Moreover, NKT cells participate in this proinflammatory cascade, as in GIPL‐treated CD1d−/− mice, TNF‐α and MIP‐2 levels are reduced significantly. As a consequence of this inflammatory response, spleen and lymph nodes of GIPL‐treated mice have an increase in the percentage of T and B cells expressing the CD69 activation marker. Cell‐transfer experiments demonstrate that T and B cell activation by GIPL is an indirect effect, which relies on the expression of TLR4 by other cell types. Moreover, although signaling through TNFRI contributes to the activation of B and γδ+ T cells, it is not required for increasing CD69 expression on αβ+ T lymphocytes. It is interesting that T cells are also functionally affected by GIPL treatment, as spleen cells from GIPL‐injected mice show enhanced production of IL‐4 following in vitro stimulation by anti‐CD3. Together, these results contribute to the understanding of the inflammatory properties of the GIPL molecule, pointing to its potential role as a parasite‐derived modulator of the immune response during T. cruzi infection.

Miltefosine induces programmed cell death in Leishmania amazonensis promastigotes

In the current study, we evaluated the mechanism of action of miltefosine, which is the first effective and safe oral treatment for visceral leishmaniasis, in Leishmania amazonensis promastigotes. Miltefosine induced a process of programmed cell death, which was determined by the externalization of phosphatidylserine, the incorporation of propidium iodide, cell-cycle arrest at the sub-G0/G1 phase and DNA fragmentation into oligonucleosome-sized fragments. Despite the intrinsic variation that is detected in Leishmania spp, our results indicate that miltefosine causes apoptosis-like death in L. amazonensis promastigote cells using a similar process that is observed in Leishmania donovani.

TLR4 Promotes B Cell Maturation: Independence and Cooperation with B Lymphocyte-Activating Factor

We have previously shown that TLR4 triggering promotes the generation of CD23+CD93+ transitional T2-like cells in vitro from mouse B cell precursors, suggesting a possible role for this receptor in B cell maturation. In this study, we perform an extensive study of cell surface markers and functional properties of B cells matured in vitro with LPS, comparatively with the well-known B cell maturation factor B lymphocyte-activating factor (BAFF). LPS increased generation of CD23+ transitional B cells in a TLR4-dependent way, upregulating IgD and CD21 and downregulating CD93, without inducing cell proliferation, in a manner essentially equivalent to BAFF. For both BAFF and LPS, functional maturation of the IgM+CD23+CD93+ cells was confirmed by their higher proliferative response to anti-CD40 plus IL-4 compared with IgM+CD23negCD93+ cells. BAFF-R-Fc–mediated neutralization experiments showed that TLR4-induced B cell maturation was independent of BAFF. Distinct from BAFF, maturation by LPS relied on the activation of canonical NF-κB pathway, and the two factors together had complementary effects, leading to higher numbers of IgM+CD23+CD93+ cells with their simultaneous addition. Importantly, BCR cross-linking abrogated the generation of CD23+ B cells by LPS or BAFF, indicating that signals mimicking central tolerance act on both systems. Addition of cyclosporin A reverted BCR-mediated inhibition, both for BAFF and LPS, suggesting similar regulation of signaling pathways by calcineurin. Finally, LPS-injected mice showed a rapid increase of mature B cells in the bone marrow, suggesting that TLR4 signaling may effectively stimulate B cell maturation in vivo, acting as an accessory stimulus in B cell development, complementary to the BAFF physiological pathway.