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

Severe Cutaneous Leishmaniasis in a Human Immunodeficiency Virus Patient Coinfected with Leishmania braziliensis and Its Endosymbiotic Virus.

Leishmania parasites cause a broad range of disease, with cutaneous afflictions being, by far, the most prevalent. Variations in disease severity and symptomatic spectrum are mostly associated to parasite species. One risk factor for the severity and emergence of leishmaniasis is immunosuppression, usually arising by coinfection of the patient with human immunodeficiency virus (HIV). Interestingly, several species of Leishmania have been shown to bear an endogenous cytoplasmic dsRNA virus (LRV) of the Totiviridae family, and recently we correlated the presence of LRV1 within Leishmania parasites to an exacerbation murine leishmaniasis and with an elevated frequency of drug treatment failures in humans. This raises the possibility of further exacerbation of leishmaniasis in the presence of both viruses, and here we report a case of cutaneous leishmaniasis caused by Leishmania braziliensis bearing LRV1 with aggressive pathogenesis in an HIV patient. LRV1 was isolated and partially sequenced from skin and nasal lesions. Genetic identity of both sequences reinforced the assumption that nasal parasites originate from primary skin lesions. Surprisingly, combined antiretroviral therapy did not impact the devolution of Leishmania infection. The Leishmania infection was successfully treated through administration of liposomal amphotericin B

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

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

MyD88 and TLR9 are involved in IL-12 production following infection and their absence increases susceptibility to infection.

<p>At indicated time points RNA from draining popliteal lymph node cells of wild-type, TLR9^−/− and Myd88^−/− mice infected by <i>L.g.</i> LRV^high (Fig3A left panel) or <i>L.g.</i> LRV^low (Fig3A right panel) were reverse-transcribed into cDNA and relative transcript levels were determined using gene specific primers by quantitative real time PCR. Results are expressed as mean± SEM of the individual mice analyzed per group with the average value for the C57BL/6 mice given a value of 1. A.U refers to Arbitrary Units of fold change. (B) Mice (n≥4) were infected in the hind footpads and footpad swelling was measured weekly over 8 weeks using a Vernier caliper. (D) At 8 weeks post-infection mice were sacrificed and footpad parasite burden was determined using parasite specific Kmp11 primers. Results are expressed as the mean ± SEM. Significance determined at *p≤0.05, **p≤0.01, ***p≤0.005.</p

Detection of Leishmania RNA virus in Leishmania parasites.

BACKGROUND: Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence. METHODOLOGY/PRINCIPAL FINDINGS: This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice. CONCLUSIONS/SIGNIFICANCE: We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis

LRV1-dependent infectious metastasis occurs in the absence of IFN-γ.

<p>Mice deficient in IFN-γ and their WT controls (C57BL/6) were infected in the hind footpads with LRV1+ or LRV1-<i>L</i>.<i>g</i> stationary-phase promastigotes. <b>(A)</b> The change in footpad swelling was measured weekly as a proxy for disease progression. Statistical analysis is indicated for comparisons between WT LRV1+ infected mice in black and IFN-γ^-/- mice in grey. <b>(B)</b> At the peak of WT infection (week 4) <i>in vivo</i> parasite luminescence was determined as a measure of parasite burden. <b>(D)</b> At 7 weeks of infection, LRV1+<i>L</i>.<i>g</i> infected IFN-γ^-/- mice developed secondary lesions in their tails which were positive for parasite luminescence. <b>(C)</b> The number of secondary lesions was counted in all groups of mice. <b>(E)</b> At 4 weeks post infection, lymphocytes were extracted from popliteal LNs draining the primary lesion and then re-stimulated <i>ex vivo</i>. IL-17A secretion was quantified by ELISA in cell-free supernatants. <b>(F-G)</b> At 7 weeks post infection, lymphocytes were extracted from <b>(F)</b> popliteal LNs and <b>(F)</b> iliac LNs draining metastatic lesions, and restimulated. IL-17A production was quantified by ELISA as above. Graphs are representative of a minimum of 3 independent experiments, using at least 5 mice per condition and presented as mean ± SEM. Significance tested by an unpaired, parametric <i>t</i>-test, (bar graphs) or one-way Anova (disease score), and indicated as *: P<0.05, **P<0.005, ***P<0.0001.</p

IL-17A mediates LRV1-dependent metastasis.

<p><b>(A-C)</b> Mice deficient in IFN-γ^-/- or IFN-γ^-/- IL-17A^-/- (DKO) were infected in footpads with either <b>(A)</b> LRV1+ or <b>(B)</b> LRV1- <i>L</i>.<i>g</i> promastigotes. Footpad swelling was measured weekly. <b>(C)</b> The number of secondary lesions was counted in all groups at 7 weeks post infection. <b>(D-H)</b> IFN-γ^-/- mice were infected in their footpads with LRV1+/- <i>L</i>.<i>g</i> parasites. At the onset of visible lesions (2.5 weeks post infection) IFN-γ^-/- mice were treated i.p with either one of the IL-17A inhibitors every 2 days, namely Digoxin (Dig) or SR1001 (SR). <b>(D)</b> The change in footpad swelling was measured weekly. <b>(E)</b> At 7 weeks post infection, the number of secondary lesions was counted in all groups of treated mice. <b>(F)</b> At week 4 post infection, popliteal lymph node cells from mice presented in <b>(D)</b>, were restimulated for 72h <i>in vitro</i> with UV inactivated <i>L</i>.<i>g</i> parasites. IL-17A production in culture supernatants was measured by ELISA. After 7 weeks, lymphocytes from <b>(G)</b> popliteal and <b>(H)</b> iliac lymph nodes were also restimulated <i>in vitro</i> in order to quantify IL-17A production by ELISA. Graphs are representative of a minimum of 2 independent experiments, using at least 5 mice per condition and represented as mean ± SEM. Significance is tested by one-way Anova (disease score) or an unpaired, parametric <i>t</i>-test (bar graphs) and indicated as *: P<0.05, **P<0.005, ***P<0.0001.</p

IL-17A production is associated with LRV1 presence and disease chronicity in human cutaneous leishmaniasis.

<p>78 cutaneous leishmaniasis patients infected with <i>L</i>.<i>g</i> parasites were separated into two groups based on their LRV1 status (n = 30 LRV1+, n = 48 LRV1-). Two further clinical groups could be distinguished based on the duration of the patients’ symptomatic infection: 64 acute patients (lesion history of 0–5 months) and 14 chronic patients (lesion history of 5–12 months). <b>(A)</b> Distribution of symptomatic cohorts among LRV1+ and LRV1- infection. <b>(B)</b> IL-17A transcripts were measured in all biopsies by qRT-PCR. Values were calculated using the ∆∆CT method and normalized to the average of LRV1-<i>L</i>.<i>g</i> infection in the acute cohort. <b>(C)</b> IFN-γ transcripts were measured in biopsies of acute patients by qRT-PCR and normalized as before. <b>(D)</b> Previous qRT-PCR results were expressed for each individual as an IL-17A/IFN-γ ratio. <b>(E)</b> Lymphocytes were extracted from blood samples of acute patients (LRV+ n = 5, LRV- n = 4) and separated into CD4+ and CD4- groups by MACS. Cells were re-stimulated <i>ex vivo</i> with <i>L</i>.<i>g</i> parasites prior to IL-17A quantification by ELISA in supernatants. Data are mean +/- SEM using at least 2 technical replicates per condition. Significance tested by an unpaired, parametric <i>t</i>-test and indicated as *: P<0.05, **P<0.005, ***P<0.0001.</p

LRV1 induces IL-17A secretion in murine leishmaniasis and contributes to LRV1-mediated disease severity.

<p>Mice deficient in IL-17A or TLR3 and their WT controls (C57BL/6) were infected in the hind footpads with 3x10⁶ of either LRV1+ or LRV1-<i>L</i>.<i>g</i> stationary-phase promastigotes. At the peak of infection (4 weeks post-inoculation), cells from the footpad lesions and popliteal LNs were re-stimulated <i>ex vivo</i>. IL-17A secretion in WT mice was quantified in the supernatant by ELISA in <b>(A)</b> intra-lesional biopsies and <b>(B)</b> popliteal LNs. <b>(C)</b> Change in footpad swelling in TLR3^-/- and WT mice was measured weekly as a proxy for disease score. <b>(D)</b> IL-17A secretion from popliteal LN cells was also compared between WT and TLR3^-/- mice. <b>(E)</b> Change in footpad swelling in IL-17A^-/- and WT mice was measured weekly as a proxy for disease score. Statistical analysis is indicated in black for comparison between WT and IL-17A^-/- LRV1+ infected mice, and in grey for comparison between IL-17A^-/- LRV+ and LRV- infected mice. <b>(F)</b> At the peak of infection (week 4), the parasite burden of the mice depicted in <b>(E)</b> was quantified by <i>in vivo</i> parasite luminescence, after injecting mice intra-peritoneally with luciferin. As previously for IL-17A, IFN-γ secretion in WT mice was quantified in the supernatant of restimulated cells by ELISA in <b>(G)</b> intra-lesional biopsies and <b>(H)</b> popliteal LNs. Graphs are representative of a minimum of 3 independent experiments, using at least 5 mice per condition and presented as mean ± SEM. Significance tested by an unpaired, parametric <i>t</i>-test (bar graphs) or one-way Anova (disease score), and indicated as *: P<0.05, **P<0.005, ***P<0.0001.</p