Leishmania guyanensis suppressed inducible nitric oxide synthase provoked by its viral endosymbiont

Inducible nitric oxide synthase (iNOS) is essential to the production of nitric oxide (NO), an efficient effector molecule against intracellular human pathogens such a

Leishmaniavirus-dependent metastatic leishmaniasis is prevented by blocking IL-17A

Cutaneous leishmaniasis has various outcomes, ranging from self-healing reddened papules to extensive open ulcerations that metastasise to secondary sites and are often resistant to standard therapies. In the case of L. guyanensis (L.g), about 5-10% of all infections result in metastatic complications. We recently showed that a cytoplasmic virus within L.g parasites (LRV1) is able to act as a potent innate immunogen, worsening disease outcome in a murine model. In this study, we investigated the immunophenotype of human patients infected by L.g and found a significant association between the inflammatory cytokine IL-17A, the presence of LRV1 and disease chronicity. Further, IL-17A was inversely correlated to the protective cytokine IFN-γ. These findings were experimentally corroborated in our murine model, where IL-17A produced in LRV1+ L.g infection contributed to parasite virulence and dissemination in the absence of IFN-γ. Additionally, IL-17A inhibition in mice using digoxin or SR1001, showed therapeutic promise in limiting parasite virulence. Thus, this murine model of LRV1-dependent infectious metastasis validated markers of disease chronicity in humans and elucidated the immunologic mechanism for the dissemination of Leishmania parasites to secondary sites. Moreover, it confirms the prognostic value of LRV1 and IL-17A detection to prevent metastatic leishmaniasis in human patients

Exacerbated leishmaniasis caused by a viral endosymbiont can be prevented by immunization with Its viral capsid

Recent studies have shown that a cytoplasmic virus called Leishmaniavirus (LRV) is present in some Leishmania species and acts as a potent innate immunogen, aggravating lesional inflammation and development in mice. In humans, the presence of LRV in Leishmania guyanensis and in L. braziliensis was significantly correlated with poor treatment response and symptomatic relapse. So far, no clinical effort has used LRV for prophylactic purposes. In this context, we designed an original vaccine strategy that targeted LRV nested in Leishmania parasites to prevent virus-related complications. To this end, C57BL/6 mice were immunized with a recombinant LRV1 Leishmania guyanensis viral capsid polypeptide formulated with a T helper 1-polarizing adjuvant. LRV1-vaccinated mice had significant reduction in lesion size and parasite load when subsequently challenged with LRV1+ Leishmania guyanensis parasites. The protection conferred by this immunization could be reproduced in naïve mice via T-cell transfer from vaccinated mice but not by serum transfer. The induction of LRV1 specific T cells secreting IFN-γ was confirmed in vaccinated mice and provided strong evidence that LRV1-specific protection arose via a cell mediated immune response against the LRV1 capsid. Our studies suggest that immunization with LRV1 capsid could be of a preventive benefit in mitigating the elevated pathology associated with LRV1 bearing Leishmania infections and possibly avoiding symptomatic relapses after an initial treatment. This novel anti-endosymbiotic vaccine strategy could be exploited to control other infectious diseases, as similar viral infections are largely prevalent across pathogenic pathogens and could consequently open new vaccine opportunities

Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation

The biological function of Z-DNA and Z-RNA, nucleic acid structures with a left-handed double helix, is poorly understood(1-3). Z-DNA-binding protein 1 (ZBP1; also known as DAI or DLM-1) is a nucleic acid sensor that contains two Z alpha domains that bind Z-DNA(4,5) and Z-RNA(6-8). ZBP1 mediates host defence against some viruses(6,7,9-14) by sensing viral nucleic acids(6,7,10). RIPK1 deficiency, or mutation of its RIP homotypic interaction motif (RHIM), triggers ZBP1-dependent necroptosis and inflammation in mice(15,16). However, the mechanisms that induce ZBP1 activation in the absence of viral infection remain unknown. Here we show that Z alpha-dependent sensing of endogenous ligands induces ZBP1-mediated perinatal lethality in mice expressing RIPK1 with mutated RHIM (Ripk1(mR/mR)), skin inflammation in mice with epidermis-specific RIPK1 deficiency (RIPK1(E-KO)) and colitis in mice with intestinal epithelial-specific FADD deficiency (FADD(IEC-KO)). Consistently, functional Z alpha domains were required for ZBP1-induced necroptosis in fibroblasts that were treated with caspase inhibitors or express RIPK1 with mutated RHIM. Inhibition of nuclear export triggered the Z alpha-dependent activation of RIPK3 in the nucleus resulting in cell death, which suggests that ZBP1 may recognize nuclear Z-form nucleic acids. We found that ZBP1 constitutively bound cellular double-stranded RNA in a Z alpha-dependent manner. Complementary reads derived from endogenous retroelements were detected in epidermal RNA, which suggests that double-stranded RNA derived from these retroelements may act as a Z alpha-domain ligand that triggers the activation of ZBP1. Collectively, our results provide evidence that the sensing of endogenous Z-form nucleic acids by ZBP1 triggers RIPK3-dependent necroptosis and inflammation, which could underlie the development of chronic inflammatory conditions-particularly in individuals with mutations in RIPK1 and CASP8(17-20)

A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis through its ZnF7 ubiquitin-binding domain

Deficiency in the deubiquitinating enzyme A20 causes severe inflammation in mice, and impaired A20 function is associated with human inflammatory diseases. A20 has been implicated in negatively regulating NF-.B signalling, cell death and inflammasome activation; however, the mechanisms by which A20 inhibits inflammation in vivo remain poorly understood. Genetic studies in mice revealed that its deubiquitinase activity is not essential for A20 anti-inflammatory function. Here we show that A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis and that this function depends on its zinc finger 7 (ZnF7). We provide genetic evidence that RIPK1 kinase-dependent, RIPK3-MLKL-mediated necroptosis drives inflammasome activation in A20-deficient macrophages and causes inflammatory arthritis in mice. Single-cell imaging revealed that RIPK3-dependent death caused inflammasome-dependent IL-1 beta release from lipopolysaccharide-stimulated A20-deficient macrophages. Importantly, mutation of the A20 ZnF7 ubiquitin binding domain caused arthritis in mice, arguing that ZnF7-dependent inhibition of necroptosis is critical for A20 anti-inflammatory function in vivo

Type I interferons induced by endogenous or exogenous viral infections promote metastasis and relapse of leishmaniasis

The presence of the endogenous Leishmania RNA virus 1 (LRV1) replicating stably within some parasite species has been associated with the development of more severe forms of leishmaniasis and relapses after drug treatment in humans. Here, we show that the disease-exacerbatory role of LRV1 relies on type I IFN (type I IFNs) production by macrophages and signaling in vivo. Moreover, infecting mice with the LRV1-cured Leishmania guyanensis (LgyLRV1(-) ) strain of parasites followed by type I IFN treatment increased lesion size and parasite burden, quantitatively reproducing the LRV1-bearing (LgyLRV1(+) ) infection phenotype. This finding suggested the possibility that exogenous viral infections could likewise increase pathogenicity, which was tested by coinfecting mice with L. guyanensis and lymphocytic choriomeningitis virus (LCMV), or the sand fly-transmitted arbovirus Toscana virus (TOSV). The type I IFN antiviral response increased the pathology of L. guyanensis infection, accompanied by down-regulation of the IFN-γ receptor normally required for antileishmanial control. Further, LCMV coinfection of IFN-γ-deficient mice promoted parasite dissemination to secondary sites, reproducing the LgyLRV1(+) metastatic phenotype. Remarkably, LCMV coinfection of mice that had healed from L. guyanensis infection induced reactivation of disease pathology, overriding the protective adaptive immune response. Our findings establish that type I IFN-dependent responses, arising from endogenous viral elements (dsRNA/LRV1), or exogenous coinfection with IFN-inducing viruses, are able to synergize with New World Leishmania parasites in both primary and relapse infections. Thus, viral infections likely represent a significant risk factor along with parasite and host factors, thereby contributing to the pathological spectrum of human leishmaniasis

Innate effector cells and proinflammatory cytokines are elevated in colitic pIV−/− K14 CIITA Tg mice.

<p>(A–B) Frequency of Ly6G⁺ neutrophil granulocytes (A) and CD11b⁺ Ly6C⁺ inflammatory monocytes (B) isolated from the colonic intestinal epithelium (cIE, left panel) and the colonic lamina propria (cLP, right panel) of <i>H. hepaticus</i>-infected pIV−/− K14 CIITA Tg mice or pIV+/− K14 CIITA Tg controls. (C) <i>ccl3</i>, <i>ccl4</i>, <i>ccl5</i>, <i>il1b</i> and <i>il6</i> mRNA expression levels in colon explants. (D) IL-1β, TNF-α, IL-12p40, CXCL9 and VEGF secretion upon <i>ex vivo</i> organ culture of colon explants. All data represent three pooled experiments (<i>n = </i>9–11 per group). αIL10R, anti-interleukin-10 receptor monoclonal antibodies; IL, interleukin; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor;</p

Colitic pIV−/− K14 CIITA Tg mice display elevated Th1 cells, IFN-γ, and CD4⁺ T cell: FoxP3⁺ Treg cell ratios.

<p>(A–B) Frequency of CD3⁺ CD4⁺ and CD8⁺ T cells isolated from anti-IL-10R mAb or isotype treated, <i>H. hepaticus</i>-infected pIV−/− K14 CIITA Tg mice or pIV+/− K14 CIITA Tg controls. Representative histograms from the colonic intestinal epithelium (cIE) (A) and summarized data (B) from cIE (left) and the colonic lamina propria (cLP) (right) as mean. (C) <i>ifng</i> and <i>tbet</i> mRNA expression levels in colon explants. (D) IFN-γ secretion upon <i>ex vivo</i> organ culture of colon explants as means and s.d. (A–D) Data represent three pooled experiments (<i>n = </i>9–11 per group). (E) Ratio of absolute numbers of CD4⁺ T cells:absolute numbers of CD25⁺ FoxP3⁺ Treg cells from cIE (left) and cLP (right) as mean from two pooled experiments (<i>n = </i>7–10 per group). αIL10R, anti-interleukin-10 receptor monoclonal antibodies; FoxP3, forkhead box P3; IFN, interferon;</p

Colitic pIV−/− K14 CIITA Tg mice lack inducible MHCII expression by colonic IECs.

<p>(A–B) Frequency of CD45.2⁻ EpCAM⁺ MHCII⁺ IECs isolated from anti-IL-10R mAb or isotype treated, <i>H. hepaticus</i>-infected pIV−/− K14 CIITA Tg mice or pIV+/− K14 CIITA Tg controls. Representative histograms (A) and summarized data as mean (B) from three pooled experiments (<i>n = </i>8–11 per group). αIL10R, anti-interleukin-10 receptor monoclonal antibodies; IEC, intestinal epithelial cell;</p

IFN-γ induces MHCII expression on IECs.

<p>(A) Frequency of CD45.2⁻ EpCAM⁺ MHCII⁺ intestinal epithelial cells (IEC) isolated from Rag1−/− or Rag1−/− IFN-γ−/− mice that were adoptively transferred with CD4⁺ CD45RB^hi T cells from WT or IFN-γ−/− mice shown as means and SEM in representative histograms (<i>n</i> = 3 mice per group). (B–C) Frequency of CD45.2⁻ EpCAM⁺ MHCII⁺ IECs (B) and CD4⁺ T cells from the colonic intestinal epithelium (C) isolated from <i>H. hepaticus</i>-infected, anti-IL-10R mAb-administered pIV−/− K14 CIITA Tg, pIV+/− K14 CIITA Tg or pIV+/− K14 CIITA Tg that were treated with neutralizing anti-IFN-γ mAb. Shown are representative FACS plots, means and SEM from two pooled experiments (<i>n</i> = 4–7 mice per group). αIFN-y, anti-interferon-γ monoclonal antibodies; FSC, forward scatter; IFN, interferon; Rag, recombination activating gene; WT, wild type;</p