bThe relationship established between Leishmania infantum and the vertebrate host can lead to a self-healing infection or to the manifestation of visceral leishmaniasis, a chronic systemic infection associated with high rates of mortality. We hypothesized that regulatory cytokines, such as interleukin-27 (IL-27), play a role in susceptibility to L. infantum infection. IL-27 is a heterodimeric cytokine composed of IL-27p28 and EBi3 subunits which, when combined, bind to IL-27R, leading to STAT-1 and -3 activation, playing a role in the regulation of the immune response. We observed in this work that IL-27 regulates the Th1/Th17 profiles in a mouse model of visceral leishmaniasis (VL) caused by L. infantum. We showed here that the pathogen recognition by endosomal Toll-like receptors triggers a type I interferon (IFN) response, which acts through the type I IFN receptor and interferon regulatory factor 1 to induce IL-27 production by macrophages. Furthermore, IL-27 plays a major regulatory role in vivo, because Ebi3 ؊/؊ mice can efficiently control parasite replication despite reduced levels of IFN-␥ compared to wild-type mice. On the other hand, the absence of Ebi3 leads to exacerbated IL-17A production in the infected organs as well as in a coculture system, suggesting a direct regulatory action of IL-27 during L. infantum infection. As a consequence of exacerbated IL-17A in Ebi3 ؊/؊ mice, a greater neutrophil influx was observed in the target organs, playing a role in parasite control. Thus, this work unveiled the molecular steps of IL-27 production after L. infantum infection and demonstrated its regulatory role in the IL-17A-neutrophil axis. Visceral leishmaniasis (VL), or kala-azar, is a systemic chronic disease with high mortality rates if not treated. It is caused by the parasites Leishmania infantum and L. donovani, and it is estimated that 300,000 new cases and 20,000 deaths occur annually (http://www.who.int/mediacentre/factsheets/fs375/en/). After dermal inoculation by the sandfly vector, the parasite disseminates to the liver, spleen, bone marrow, and lymph nodes of susceptible hosts, causing symptoms such as hepatosplenomegaly, lymphadenopathy, anemia, constant fever, and immunosuppression (1). Even though it is considered one of the six most important parasitic diseases affecting humans, the immunobiology of VL is not completely understood and novel therapeutic approaches are desired.The immune response against Leishmania spp. is critically mediated by gamma interferon (IFN-␥)-producing Th1 cells, which activate macrophages to produce leishmanicidal compounds, such as nitric oxide (NO) (2). Together with IFN-␥, the inflammatory cytokine interleukin-17A (IL-17A) also mediates protection against L. infantum (3, 4). On the other hand, Th2 cytokines are involved in susceptibility to leishmaniasis (2, 5). Moreover, the regulatory cytokines IL-10, IL-21, and IL-27 are produced in the bone marrow of VL patients, suggesting their association with the disease (6).The cytokine IL-27 is a dimer composed of p28...

Leishmania infantum is a protozoan parasite that causes visceral leishmaniasis (VL). This infection triggers dendritic cell (DC)activation through the recognition of microbial products by Toll-like receptors (TLRs). Among the TLRs, TLR9 is required for DC activation by different Leishmania species. We demonstrated that TLR9 is upregulated in vitro and in vivo during infection. We show that C57BL/6 mice deficient in TLR9 expression (TLR9 ؊/؊ mice) are more susceptible to infection and display higher parasite numbers in the spleen and liver. The increased susceptibility of TLR9 ؊/؊ mice was due to the impaired recruitment of neutrophils to the infection foci associated with reduced levels of neutrophil chemoattractants released by DCs in the target organs. Moreover, both Th1 and Th17 cells were also committed in TLR9 ؊/؊ mice. TLR9-dependent neutrophil recruitment is mediated via the MyD88 signaling pathway but is TIR domain-containing adapter-inducing interferon beta (TRIF) independent. Furthermore, L. infantum failed to activate both plasmacytoid and myeloid DCs from TLR9 ؊/؊ mice, which presented reduced surface costimulatory molecule expression and chemokine release. Interestingly, neutrophil chemotaxis was affected both in vitro and in vivo when DCs were derived from TLR9 ؊/؊ mice. Our results suggest that TLR9 plays a critical role in neutrophil recruitment during the protective response against L. infantum infection that could be associated with DC activation. L eishmania intracellular protozoan parasites cause a heterogeneous disease that can range from cutaneous lesions to visceral disease. Visceral disease can be caused by both Leishmania donovani and Leishmania infantum and is the most severe manifestation of this type of parasite infection. Therefore, Leishmania infections remain an important cause of human mortality and morbidity around the world (www.paho.org/english/ad/dpc/cd /res-dch-leish-priorities.pdf).Host defenses against Leishmania spp. depend on the activation of an inflammatory response initiated by the innate immune system (1), followed by specific immune responses mediated by gamma interferon (IFN-␥)-or interleukin-17 (IL-17)-producing CD4 ϩ T lymphocytes (2, 3). The innate immune system has specific mechanisms to rapidly recognize the parasite. A major component of pathogen recognition comprises a family of Toll-like receptors (TLRs) that detect common pathogen-associated molecular patterns (PAMPs) of various groups of microorganisms (4-7), including Leishmania spp. (8, 9).The role of TLRs in Leishmania infection control has been supported by the observations that mice lacking MyD88, an adaptor molecule required for TLR signaling, display enhanced susceptibility to infection (10, 11). It has been described that resistance to Leishmania species infection is dependent on parasite lipophosphoglycan (LPG) recognition by TLR2 (8), the induction of IL-12, and the development of a Th1 immune response (12) together with NO production (13). Previous studies demonstrated a role for TLR2 and TLR3 i...

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.

A novel T cell-secreted cytokine, termed secreted osteoclastogenic factor of activated T cells (SOFAT) that induces osteoclastic bone resorption in a RANKL-independent manner, has been described. Our group have previously reported that SOFAT is highly expressed in gingival tissues of patients with chronic periodontitis suggesting a putative role in the bone loss associated with periodontal disease. The aim of the present study was to identify other potential cellular sources of SOFAT in the bone resorptive lesions of patients with periodontal disease. Gingival tissues were biopsied from systemically healthy subjects without periodontal disease (n=5) and patients with chronic periodontitis (n=5), and the presence of SOFAT was analyzed by immunohistochemistry and immunofluorescence staining. The present data demonstrated marked SOFAT staining in diseased periodontal tissues that was predominantly associated with the lymphocytic infiltration of gingival tissues. Notably, in addition to CD3+ T cells, B-lineage cells including plasma cells also exhibited strong staining for SOFAT. As SOFAT has not previously been reported in B-lineage cells, splenic T cells and B cells were further purified from BALB/c mice and activated using CD3/CD28 and lipopolysaccharide, respectively. SOFAT was quantified by reverse transcription-quantitative polymerase chain reaction and was shown to be significantly expressed (P<0.05) in both activated T cells and B cells compared with unstimulated cells. These data support a putative role of SOFAT in the bone loss associated with chronic periodontal disease. In addition, to the best of our knowledge, this study demonstrates for the first time that in addition to T cells, B-lineage cells may also be a significant source of SOFAT in inflammatory states.

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. Leucotriens are lipid mediators synthesized by 5-lipoxygenase (5-LO) and they display a protective role against protozoan parasites by inducing several functions in leucocytes. We determined the role of 5-LO activity in parasite control, focusing on the inflammatory immune response against Leishmania infantum infection. LTB4 is released during in vitro infection. The genetic ablation of 5-LO promoted susceptibility in highly resistant mice strains, harboring more parasites into target organs. The susceptibility was related to the failure of neutrophil migration to the infectious foci. Investigating the neutrophil failure, there was a reduction of proinflammatory cytokines involved in the related Th17 axis released into the organs. Genetic ablation of 5-LO reduced the CD4+T cells producing IL-17, without interfering in Th1 subset. L. infantum failed to activate DC from 5-LO−/−, showing reduced surface costimulatory molecule expression and proinflammatory cytokines involved in Th17 differentiation. BLT1 blockage with selective antagonist interferes with DC maturation and proinflammatory cytokines release. Thus, 5-LO activation coordinates the inflammatory immune response involved in the control of VL.

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

BackgroundSand fly saliva plays a crucial role in establishing Leishmania infection. We identified adenosine (ADO) and adenosine monophosphate (AMP) as active pharmacologic compounds present in Phlebotomus papatasi saliva that inhibit dendritic cell (DC) functions through a PGE2/IL 10-dependent mechanism.Methodology/Principal FindingsHerein, we prepared a mixture of ADO and AMP in equimolar amounts similar to those present in the salivary-gland extract (SGE) form one pair of salivary glands of P. papatasi and co-injected it with Leishmania amazonensis or L. major into mouse ears. ADO+AMP mimicked exacerbative effects of P. papatasi saliva in leishmaniasis, increasing parasite burden and cutaneous lesions. Enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect associated with IL-10 enhancement. Immunosuppressive factors COX2 and IL-10 were upregulated and failed to enhance ear lesion and parasite burden in IL 10-/- infected mice. Furthermore, nucleosides increased regulatory T cell (Treg) marker expression on CD4+CD25- cells, suggesting induction of Tregs on effector T cells (T eff). Treg induction (iTreg) was associated with nucleoside-induced tolerogenic dendritic cells (tDCs) expressing higher levels of COX2 and IL-10. In vitro generation of Tregs was more efficient in DCs treated with nucleosides. Suppressive effects of nucleosides during cutaneous leishmaniasis were mediated through an A2AR-dependent mechanism. Using BALB/c mice deficient in A2A ADO receptor (A2AR–/–), we showed that co-inoculated mice controlled infection, displaying lower parasite numbers at infection sites and reduced iTreg generation.Conclusion/SignificanceWe have demonstrated that ADO and AMP in P. papatasi saliva mediate exacerbative effects of Leishmania infection by acting preferentially on DCs promoting a tolerogenic profile in DCs and by generating iTregs in inflammatory foci through an A2AR mechanism.

The prostaglandin, 15-deoxy Δ12,14-prostaglandin J2 (15d-PGJ2), is a lipid mediator that plays an important role in the control of chronic inflammatory disease. However, the role of prostanoid in rheumatoid arthritis (RA) is not well determined. We demonstrated the therapeutic effect of 15d-PGJ2 in an experimental model of arthritis. Daily administration of 15d-PGJ2 attenuated the severity of CIA, reducing the clinical score, pain, and edema. 15d-PGJ2 treatment was associated with a marked reduction in joint levels of proinflammatory cytokines. Although the mRNA expression of ROR-γt was profoundly reduced, FOXP3 was enhanced in draining lymph node cells from 15d-PGJ2-treated arthritic mice. The specific and polyclonal CD4+ Th17 cell responses were limited during the addition of prostaglandin to cell culture. Moreover, in vitro 15d-PGJ2 increased the expression of FOXP3, GITR, and CTLA-4 in the CD4+CD25− population, suggesting the induction of Tregs on conventional T cells. Prostanoid addition to CD4+CD25− cells selectively suppressed Th17 differentiation and promoted the enhancement of FOXP3 under polarization conditions. Thus, 15d-PGJ2 ameliorated symptoms of collagen-induced arthritis by regulating Th17 differentiation, concomitant with the induction of Tregs, and, consequently, protected mice from diseases aggravation. Altogether, these results indicate that 15d-PGJ2 may represent a potential therapeutic strategy in RA.