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.

Necrotic cell death during Mycobacterium tuberculosis (Mtb) infection is considered host detrimental since it facilitates mycobacterial spread. Ferroptosis is a type of regulated necrosis induced by accumulation of free iron and toxic lipid peroxides. We observed that Mtb-induced macrophage necrosis is associated with reduced levels of glutathione and glutathione peroxidase-4 (Gpx4), along with increased free iron, mitochondrial superoxide, and lipid peroxidation, all of which are important hallmarks of ferroptosis. Moreover, necrotic cell death in Mtb-infected macrophage cultures was suppressed by ferrostatin-1 (Fer-1), a well-characterized ferroptosis inhibitor, as well as by iron chelation. Additional experiments in vivo revealed that pulmonary necrosis in acutely infected mice is associated with reduced Gpx4 expression as well as increased lipid peroxidation and is likewise suppressed by Fer-1 treatment. Importantly, Fer-1–treated infected animals also exhibited marked reductions in bacterial load. Together, these findings implicate ferroptosis as a major mechanism of necrosis in Mtb infection and as a target for host-directed therapy of tuberculosis.

BackgroundEffective treatment of Mycobacterium tuberculosis (Mtb) infection requires at least 6 months of daily therapy with multiple orally administered antibiotics. Although this drug regimen is administered annually to millions worldwide, the impact of such intensive antimicrobial treatment on the host microbiome has never been formally investigated. Here, we characterized the longitudinal outcome of conventional isoniazid-rifampin-pyrazinamide (HRZ) TB drug administration on the diversity and composition of the intestinal microbiota in Mtb-infected mice by means of 16S rRNA sequencing. We also investigated the effects of each of the individual antibiotics alone and in different combinations.ResultsWhile inducing only a transient decrease in microbial diversity, HRZ treatment triggered a marked, immediate and reproducible alteration in community structure that persisted for the entire course of therapy and for at least 3 months following its cessation. Members of order Clostridiales were among the taxa that decreased in relative frequencies during treatment and family Porphyromonadaceae significantly increased post treatment. Experiments comparing monotherapy and different combination therapies identified rifampin as the major driver of the observed alterations induced by the HRZ cocktail but also revealed unexpected effects of isoniazid and pyrazinamide in certain drug pairings.ConclusionsThis report provides the first detailed analysis of the longitudinal changes in the intestinal microbiota due to anti-tuberculosis therapy. Importantly, many of the affected taxa have been previously shown in other systems to be associated with modifications in immunologic function. Together, our findings reveal that the antibiotics used in conventional TB treatment induce a distinct and long lasting dysbiosis. In addition, they establish a murine model for studying the potential impact of this dysbiosis on host resistance and physiology.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0286-2) contains supplementary material, which is available to authorized users.

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.

dCutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a strong Th1 response that leads to skin lesion development. In areas where L. braziliensis transmission is endemic, up to 15% of healthy subjects have tested positive for delayed-type hypersensitivity to soluble leishmania antigen (SLA) and are considered to have subclinical (SC) infection. SC subjects produce less gamma interferon (IFN-␥) and tumor necrosis factor alpha (TNF-␣) than do CL patients, but they are able to control the infection. ؉ T cells was higher in CL than in SC cells. While the use of a granzyme B inhibitor decreased the number of apoptotic cells in the CL group, the use of z-VAD-FMK had no effect on the frequency of these cells. These results suggest that CL CD8 ؉ T cells are more cytotoxic and may be involved in pathology. L eishmaniasis is caused by infection with parasites of the genusLeishmania. Leishmaniasis is a neglected tropical disease; 214,000 new cases of cutaneous leishmaniasis (CL) are reported annually worldwide, and the estimated incidence of leishmaniasis is 690,000 to 1,200,000 cases. Approximately 67,000 cases are reported in South America, Central America, and the Caribbean (1). In mice, the majority of Leishmania-specific CD4 ϩ T cells differentiate into T-helper 1 (Th1) cells that secrete gamma interferon (IFN-␥) and contribute to the elimination of the parasite through the activation of macrophages (2, 3). Although protective immunity has predominantly been related to IFN-␥-producing CD4 ϩ T cells, infection with Leishmania also results in the activation and expansion of parasite-specific CD8 ϩ T cells (4, 5). Human CL caused by Leishmania braziliensis is characterized by a strong Th1 response with the production of high levels of IFN-␥ and tumor necrosis factor alpha (TNF-␣) (6, 7). This exaggerated Th1 response is associated with the development of lesions and the severity of the disease (6, 8-10). In patients with CL caused by L. braziliensis, there are more CD4 ϩ than CD8 ϩ T cells, but this ratio reaches an equilibrium due to the increase in CD8 ϩ T cells that occurs during the healing process (11). The enrichment of Leishmania-reactive CD8 ϩ T cells in older lesions suggests that these cells may play a role in the healing process (12). In contrast, other studies have associated CD8ϩ T cell functions with pathology. For example, the cytotoxicity mediated by CD8 ϩ T cells is greater in mucosal leishmaniasis (ML), a more severe form of L. braziliensis infection, than in CL (13,14). More recently, it was shown that the frequency with which CD8 ϩ T cells express granzyme in the lesions of CL patients is greater than that in patients in the early phase of CL (i.e., before the ulcer has developed) and that the frequency with which CD8 ϩ T cells express granzyme is directly associated with the intensity of the inflammatory reaction observed in CL ulcers (15,16). This controversy regarding the role of cytotoxicity in the pathogenesis of human leishmaniasis indicates that the functions of CD...

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.methotrexate | rheumatoid arthritis | adenosine | biomarker | ectonucleotidases

Background Mycobacterium tuberculosis infection is thought to induce oxidative stress. N-acetyl-cysteine (NAC) is widely used in patients with chronic pulmonary diseases including tuberculosis due to its mucolytic and anti-oxidant activities. Here, we tested whether NAC exerts a direct antibiotic activity against mycobacteria.MethodsOxidative stress status in plasma was compared between pulmonary TB (PTB) patients and those with latent M. tuberculosis infection (LTBI) or healthy uninfected individuals. Lipid peroxidation, DNA oxidation and cell death, as well as accumulation of reactive oxygen species (ROS) were measured in cultures of primary human monocyte-derived macrophages infected with M. tuberculosis and treated or not with NAC. M. tuberculosis, M. avium and M. bovis BCG cultures were also exposed to different doses of NAC with or without medium pH adjustment to control for acidity. The anti-mycobacterial effect of NAC was assessed in M. tuberculosis infected human THP-1 cells and bone marrow-derived macrophages from mice lacking a fully functional NADPH oxidase system. The capacity of NAC to control M. tuberculosis infection was further tested in vivo in a mouse (C57BL/6) model.ResultsPTB patients exhibited elevated levels of oxidation products and a reduction of anti-oxidants compared with LTBI cases or uninfected controls. NAC treatment in M. tuberculosis-infected human macrophages resulted in a decrease of oxidative stress and cell death evoked by mycobacteria. Importantly, we observed a dose-dependent reduction in metabolic activity and in vitro growth of NAC treated M. tuberculosis, M. avium and M. bovis BCG. Furthermore, anti-mycobacterial activity in infected macrophages was shown to be independent of the effects of NAC on the host NADPH oxidase system in vitro. Short-term NAC treatment of M. tuberculosis infected mice in vivo resulted in a significant reduction of mycobacterial loads in the lungs.ConclusionsNAC exhibits potent anti-mycobacterial effects and may limit M. tuberculosis infection and disease both through suppression of the host oxidative response and through direct antimicrobial activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0872-7) contains supplementary material, which is available to authorized users.

Heme oxygenase-1 (HO-1) is a stress response antioxidant enzyme which catalyzes the degradation of heme released during inflammation. HO-1 expression is upregulated in both experimental and human Mycobacterium tuberculosis infection, and in patients it is a biomarker of active disease. Whether the enzyme plays a protective versus pathogenic role in tuberculosis has been the subject of debate. To address this controversy, we administered tin protoporphyrin IX (SnPPIX), a well-characterized HO-1 enzymatic inhibitor, to mice during acute M. tuberculosis infection. These SnPPIX-treated animals displayed a substantial reduction in pulmonary bacterial loads comparable to that achieved following conventional antibiotic therapy. Moreover, when administered adjunctively with antimycobacterial drugs, the HO-1 inhibitor markedly enhanced and accelerated pathogen clearance. Interestingly, both the pulmonary induction of HO-1 expression and the efficacy of SnPPIX treatment in reducing bacterial burden were dependent on the presence of host T lymphocytes. Although M. tuberculosis expresses its own heme-degrading enzyme, SnPPIX failed to inhibit its enzymatic activity or significantly restrict bacterial growth in liquid culture. Together, the above findings reveal mammalian HO-1 as a potential target for host-directed monotherapy and adjunctive therapy of tuberculosis and identify the immune response as a critical regulator of this function.