Vanessa Carregaro

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.

CCR5 Plays a Critical Role in the Development of Myocarditis and Host Protection in Mice Infected with Trypanosoma cruzi

Abstract The pathogenesis of myocarditis during Trypanosoma cruzi infection is poorly understood. We investigated the role played by chemokine receptor 5 (CCR5) in the influx of T cells to the cardiac tissue of T. cruzi—infected mice. mRNA and protein for the CCR5 ligands CCL3, CCL4, and CCL5 were detected in the hearts of infected mice in association with CD4+ and CD8+ T cells. There was a high level of CCR5 expression on CD8+ T cells in the hearts of infected mice. Moreover, CCR5 expression on CD8+ T cells was positively modulated by T. cruzi infection. CCR5-deficient mice infected with T. cruzi experienced a dramatically inhibited migration of T cells to the heart and were also more susceptible to infection. These results suggest that CCR5 and its ligands play a central role in the control of T cell influx in T. cruzi-infected mice. Knowledge of the mechanisms that trigger and control the migration of cells to the heart in patients with Chagas disease may help in the design of drugs that prevent myocarditis and protect against the development of severe disease.

Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta. Anti-Trypanosoma cruzi activity without cytotoxicity to mammalian cells.

Amphibian skin secretions are known as a rich source of biologically active molecules, most of which are alkaloids, biogenic amines, and peptides. Dermaseptins are a class of antimicrobial peptides present in tree frogs of thePhyllomedusa genus. They are cationic molecules of 28–34 residues that permeabilize the membrane of Gram-positive and Gram-negative bacteria, yeasts, and filamentous fungi, showing little or no hemolytic activity. This work reports the isolation, molecular mass analysis, primary structure determination, biological activities, and potential therapeutic applications of an antimicrobial peptide found in the skin secretion of Phyllomedusa oreades, which is a newly described amphibian species endemic of the Brazilian savanna. DS 01 is a 29-residue-long peptide with a molecular mass of 2793.39 Da showing antibacterial properties against Gram-positive and Gram-negative bacteria in the range of 3–25 μm. Anti-protozoan activity was investigated using T. cruzi in its trypomatigote and epimastigote forms cultivated in both cell culture and blood media. Within 2 h after incubation with DS 01 at a final concentration of ∼6 μm, no protozoan cells were detected. Two synthetic dermaseptins, described previously by our group and named dermadistinctins K and L (DD K and DD L), also had their anti-Trypanosoma cruzi activity investigated and demonstrated similar properties. Toxicity of DS 01 to mouse erythrocytes and white blood cells was evaluated by means of atomic force microscopy and flow cytometry. No morphological alterations were observed at a lytic concentration of DS 01, suggesting its therapeutic value especially as an anti-T. cruzi agent to prevent infections during blood transfusion.

Deconstructing Tick Saliva: NON-PROTEIN MOLECULES WITH POTENT IMMUNOMODULATORY PROPERTIES*

Dendritic cells (DCs) are powerful initiators of innate and adaptive immune responses. Ticks are blood-sucking ectoparasite arthropods that suppress host immunity by secreting immunomodulatory molecules in their saliva. Here, compounds present in Rhipicephalus sanguineus tick saliva with immunomodulatory effects on DC differentiation, cytokine production, and costimulatory molecule expression were identified. R. sanguineus tick saliva inhibited IL-12p40 and TNF-α while potentiating IL-10 cytokine production by bone marrow-derived DCs stimulated by Toll-like receptor-2, -4, and -9 agonists. To identify the molecules responsible for these effects, we fractionated the saliva through microcon filtration and reversed-phase HPLC and tested each fraction for DC maturation. Fractions with proven effects were analyzed by micro-HPLC tandem mass spectrometry or competition ELISA. Thus, we identified for the first time in tick saliva the purine nucleoside adenosine (concentration of ∼110 pmol/μl) as a potent anti-inflammatory salivary inhibitor of DC cytokine production. We also found prostaglandin E2 (PGE2 ∼100 nm) with comparable effects in modulating cytokine production by DCs. Both Ado and PGE2 inhibited cytokine production by inducing cAMP-PKA signaling in DCs. Additionally, both Ado and PGE2 were able to inhibit expression of CD40 in mature DCs. Finally, flow cytometry analysis revealed that PGE2, but not Ado, is the differentiation inhibitor of bone marrow-derived DCs. The presence of non-protein molecules adenosine and PGE2 in tick saliva indicates an important evolutionary mechanism used by ticks to subvert host immune cells and allow them to successfully complete their blood meal and life cycle.

Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis

Significance Methotrexate (MTX) is the first-line therapy for rheumatoid arthritis (RA). However, about 40% of patients are resistant to MTX. Furthermore, MTX resistance is only apparent after a prolonged continuous MTX treatment (>3 mo), by which time the disease of the nonresponders would have aggravated. Thus, there is a considerable unmet need for a biomarker to select MTX-resistant patients and place them immediately on alternative therapy. We found here that the low density of CD39 on peripheral regulatory T cells in RA patients is a rapid, convenient, and reliable (P < 0.01) biomarker for MTX resistance. Our findings also provide previously unrecognized information on aspects of immune regulation in RA and the mechanism of action of MTX. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4+CD25+FoxP3+) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39+CD4+CD25+FoxP3+ Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients.

Interleukin 17A Acts Synergistically With Interferon γ to Promote Protection Against Leishmania infantum Infection

Interleukin 17 (IL-17) is an inflammatory cytokine that plays a protective role against intracellular parasites. The role of IL-17 during Leishmania infection remains controversial and poorly defined. We evaluated whether IL-17 participates in the host immune response to Leishmania infantum. IL-17A is present in sera from patients with visceral leishmaniasis and decreases after successful treatment. In C57BL/6 infected mice, higher production of IL-17A coincided with the peak of parasitism. Il17ra(-/-) mice were more susceptible to infection and also exhibited reduced inflammatory infiltration and interferon γ (IFN-γ)-expressing CD4+ T-cell frequencies than wild-type mice. The frequencies of FoxP3+ CD4+ T cells and interleukin 10 (IL-10)-expressing CD4+ T cells were increased in Il17ra(-/-) mice. We also demonstrated that IL-17A acts synergistically with IFN-γ to potentiate NO production and leishmanicidal activity in infected macrophages. Therefore, our results indicate that L. infantum induces IL-17A production, which promotes the control of parasite replication by strengthening T-helper type 1 responses and NO production and prevents regulatory T-cell and IL-10-expressing T-cell expansion.

Targeting endothelin ETA and ETB receptors inhibits antigen-induced neutrophil migration and mechanical hypernociception in mice

Endothelin may contribute to the development of inflammatory events such as leukocyte recruitment and nociception. Herein, we investigated whether endothelin-mediated mechanical hypernociception (decreased nociceptive threshold, evaluated by electronic pressure-meter) and neutrophil migration (myeloperoxidase activity) are inter-dependent in antigen challenge-induced Th1-driven hind-paw inflammation. In antigen challenge-induced inflammation, endothelin (ET) ETA and ETB receptor antagonism inhibited both hypernociception and neutrophil migration. Interestingly, ET-1 peptide-induced hypernociception was not altered by inhibiting neutrophil migration or endothelin ETB receptor antagonism, but rather by endothelin ETA receptor antagonism. Furthermore, endothelin ETA, but not ETB, receptor antagonism inhibited antigen-induced PGE2 production, whereas either selective or combined blockade of endothelin ETA and/or ETB receptors reduced hypernociception and neutrophil recruitment caused by antigen challenge. Concluding, this study advances knowledge into the role for endothelin in inflammatory mechanisms and further supports the potential of endothelin receptor antagonists in controlling inflammation.

Neutrophils recruited by CXCR1/2 signalling mediate post-incisional pain

Neutrophil recruitment mediated by the CXCL1/KC chemokine and its receptors CXCR1/CXCR2 plays a critical role in inflammatory diseases. Recently, neutrophil migration and activation triggered by CXCL1‐CXCR1/2 signalling was implicated in inflammatory nociception; however, their role in post‐surgical pain has not been elucidated. In this study, we addressed the function of neutrophils in the genesis of post‐incisional pain in an experimental model of post‐surgical pain.

Phlebotomine salivas inhibit immune inflammation-induced neutrophil migration via an autocrine DC-derived PGE2/IL-10 sequential pathway

In the present study, we investigated whether saliva from Phlebotomus papatasi and Phlebotomus duboscqi inhibited antigen‐induced neutrophil migration and the mechanisms involved in these effects. The pretreatment of immunized mice with salivary gland extracts (SGE) of both phlebotomines inhibited OVA challenge‐induced neutrophil migration and release of the neutrophil chemotactic mediators, MIP‐1α, TNF‐α, and leukotriene B4 (LTB4). Furthermore, SGE treatment enhanced the production of anti‐inflammatory mediators, IL‐10 and PGE2. SGE treatments failed to inhibit neutrophil migration and MIP‐1α and LTB4 production in IL‐10−/− mice, also failing in mice treated with nonselective (indomethacin) or selective (rofecoxibe) cyclooxygenase (COX) inhibitors. COX inhibition resulted in diminished SGE‐induced IL‐10 production, and PGE2 release triggered by SGE remained increased in IL‐10−/− mice, suggesting that prostanoids are acting through an IL‐10‐dependent mechanism. SGE treatments in vivo reduced the OVA‐induced lymphoproliferation of spleen‐derived cells. Further, the in vitro incubation of bone marrow‐derived dendritic cells (DC) with SGE inhibited the proliferation of CD4+T cells from OVA‐immunized mice, which was reversed by indomethacin and anti‐IL‐10 antibody treatments. Supporting these results, SGE induced the production of PGE2 and IL‐10 by DC, which were blocked by COX inhibition. These effects were associated with the reduction of DC‐membrane expression of MHC‐II and CD86 by SGE treatment. Altogether, the results showed that Phlebotomine saliva inhibits immune inflammation‐induced neutrophil migration by an autocrine DC sequential production of PGE2/IL‐10, suggesting that the saliva constituents might be promising therapeutic molecules to target immune inflammatory diseases.

Antileishmanial activity of ruthenium(II)tetraammine nitrosyl complexes.

The complexes trans-[Ru(NO)(NH(3))(4)L](X)(3) (X = BF(4)(-), PF(6)(-) or Cl(-) and L = N-heterocyclic ligands, P(OEt)(3), SO(3)(-2)), and [Ru(NO)Hedta)] were shown to exhibit IC(50pro) in the range of 36 (L = imN) to 5000 microM (L = imC). The inhibitory effects of trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3) and of the Angelis salt on the growth of the intramacrophage amastigote form studied were found to be similar while the trans-[Ru(NH(3))(4)imN(H(2)O)](2+) complex was found not to exhibit any substantial antiamastigote effect. The trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3) compound, administered (500 nmol kg(-1) day(-1)) in BALB/c mice infected with Leishmania major, was found to exhibit a 98% inhibition on the parasite growth. Furthermore, this complex proved to be at least 66 times more efficient than glucantime in in vivo experiments.