Catherine Ronet

Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis.

An RNA virus of a parasite binds to human Toll-like receptor 3 and modulates host immune responses to the parasite. Mucocutaneous leishmaniasis is caused by infections with intracellular parasites of the Leishmania Viannia subgenus, including Leishmania guyanensis. The pathology develops after parasite dissemination to nasopharyngeal tissues, where destructive metastatic lesions form with chronic inflammation. Currently, the mechanisms involved in lesion development are poorly understood. Here we show that metastasizing parasites have a high Leishmania RNA virus–1 (LRV1) burden that is recognized by the host Toll-like receptor 3 (TLR3) to induce proinflammatory cytokines and chemokines. Paradoxically, these TLR3-mediated immune responses rendered mice more susceptible to infection, and the animals developed an increased footpad swelling and parasitemia. Thus, LRV1 in the metastasizing parasites subverted the host immune response to Leishmania and promoted parasite persistence.

NKT Cells Are Critical for the Initiation of an Inflammatory Bowel Response against Toxoplasma gondii

We demonstrated in this study the critical role of NKT cells in the lethal ileitis induced in C57BL/6 mice after infection with Toxoplasma gondii. This intestinal inflammation is caused by overproduction of IFN-γ in the lamina propria. The implication of NKT cells was confirmed by the observation that NKT cell-deficient mice (Jα281−/−) are more resistant than C57BL/6 mice to the development of lethal ileitis. Jα281−/− mice failed to overexpress IFN-γ in the intestine early after infection. This detrimental effect of NKT cells is blocked by treatment with α-galactosylceramide, which prevents death in C57BL/6, but not in Jα281−/−, mice. This protective effect is characterized by a shift in cytokine production by NKT cells toward a Th2 profile and correlates with an increased number of mesenteric Foxp3 lymphocytes. Using chimeric mice in which only NKT cells are deficient in the IL-10 gene and mice treated with anti-CD25 mAb, we identified regulatory T cells as the source of the IL-10 required for manifestation of the protective effect of α-galactosylceramide treatment. Our results highlight the participation of NKT cells in the parasite clearance by shifting the cytokine profile toward a Th1 pattern and simultaneously to immunopathological manifestation when this Th1 immune response remains uncontrolled.

Leishmania major induces distinct neutrophil phenotypes in mice that are resistant or susceptible to infection

Polymorphonuclear neutrophils (PMN) are key components of the inflammatory response contributing to the development of pathogen‐specific immune responses. Following infection with Leishmania major, neutrophils are recruited within hours to the site of parasite inoculation. C57BL/6 mice are resistant to infection, and BALB/c mice are susceptible to infection, developing unhealing, inflammatory lesions. In this report, we investigated the expression of cell surface integrins, TLRs, and the secretion of immunomodulatory cytokines by PMN of both strains of mice, in response to infection with L. major. The parasite was shown to induce CD49d expression in BALB/c‐inflammatory PMN, and expression of CD49d remained at basal levels in C57BL/6 PMN. Equally high levels of CD11b were expressed on PMN from both strains. In response to L. major infection, the levels of TLR2, TLR7, and TLR9 mRNA were significantly higher in C57BL/6 than in BALB/c PMN. C57BL/6 PMN secreted biologically active IL‐12p70 and IL‐10. In contrast, L. major‐infected BALB/c PMN transcribed and secreted high levels of IL‐12p40 but did not secrete biologically active IL‐12p70. Furthermore, IL‐12p40 was shown not to associate with IL‐23 p19 but formed IL‐12p40 homodimers with inhibitory activity. No IL‐10 was secreted by BALB/c PMN. Thus, following infection with L. major, in C57BL/6 mice, PMN could constitute one of the earliest sources of IL‐12, and in BALB/c mice, secretion of IL‐12p40 could contribute to impaired, early IL‐12 signaling. These distinct PMN phenotypes may thus influence the development of L. major‐specific immune response.

Leishmania RNA virus: when the host pays the toll

The presence of an RNA virus in a South American subgenus of the Leishmania parasite, L. (Viannia), was detected several decades ago but its role in leishmanial virulence and metastasis was only recently described. In Leishmania guyanensis, the nucleic acid of Leishmania RNA virus (LRV1) acts as a potent innate immunogen, eliciting a hyper-inflammatory immune response through toll-like receptor 3 (TLR3). The resultant inflammatory cascade has been shown to increase disease severity, parasite persistence, and perhaps even resistance to anti-leishmanial drugs. Curiously, LRVs were found mostly in clinical isolates prone to infectious metastasis in both their human source and experimental animal model, suggesting an association between the viral hyperpathogen and metastatic complications such as mucocutaneous leishmaniasis (MCL). MCL presents as chronic secondary lesions in the mucosa of the mouth and nose, debilitatingly inflamed and notoriously refractory to treatment. Immunologically, this outcome has many of the same hallmarks associated with the reaction to LRV: production of type 1 interferons, bias toward a chronic Th1 inflammatory state and an impaired ability of host cells to eliminate parasites through oxidative stress. More intriguing, is that the risk of developing MCL is found almost exclusively in infections of the L. (Viannia) subtype, further indication that leishmanial metastasis is caused, at least in part, by a parasitic component. LRV present in this subgenus may contribute to the destructive inflammation of metastatic disease either by acting in concert with other intrinsic “metastatic factors” or by independently preying on host TLR3 hypersensitivity. Because LRV amplifies parasite virulence, its presence may provide a unique target for diagnostic and clinical intervention of metastatic leishmaniasis. Taking examples from other members of the Totiviridae virus family, this paper reviews the benefits and costs of endosymbiosis, specifically for the maintenance of LRV infection in Leishmania parasites, which is often at the expense of its human host.

Phenotypic and Functional Differences Between NKT Cells Colonizing Splanchnic and Peripheral Lymph Nodes

NKT cells are considered unconventional T cells. First, they are restricted by a nonclassical MHC class I molecule, CD1d, which presents glycolipids; second, their TCR repertoire is very limited. After stimulation by their TCR, NKT cells rapidly release large amounts of cytokines, such as IL-4 and IFN-γ. Little is known about NKT cells present in lymph nodes. In the present report we show that NKT cells are differently distributed in various lymph nodes and are, for instance, abundant in pancreatic and mesenteric lymph nodes of C57BL/6 mice and nonobese diabetic mice. The high frequency of NKT cells in splanchnic lymph nodes is not simply a consequence of inflammatory signals, as draining lymph nodes still contain low frequencies of NKT cells after IFA or CFA injections. NKT cells from splanchnic lymph nodes harbor a Vβ repertoire similar to that of splenic and liver NKT cells, in contrast to peripheral NKT cells that are not biased toward Vβ8 segments. Analysis of cytokine production by NKT cells from splanchnic lymph nodes reveals that they produce at least as much IL-4 as IFN-γ, in contrast to NKT cells from other organs (spleen, liver, and peripheral lymph nodes), which produce much more IFN-γ than IL-4. These specific features of NKT cells from splanchnic lymph nodes might explain their protective action against the development of pathogenic Th1 cells in type 1 diabetes.

Comparison of the T Cell Patterns in Leprous and Cutaneous Sarcoid Granulomas: Presence of Vα24-Invariant Natural Killer T Cells in T-Cell-Reactive Leprosy Together with a Highly Biased T Cell Receptor Vα Repertoire

The T-cell-reactive (eg, tuberculoid and reversal) forms of leprosy represent a well-defined granulomatous reaction pattern against an invading pathogen. The immune response in cutaneous sarcoidosis is a granulomatous condition that pathologically is very similar to T-cell reactive leprosy. However, it lacks a defined causative agent. In view of the role of NKT cells in murine granulomas induced by mycobacterial cell walls, we have searched for the presence of NKT cells in the cutaneous lesions of both leprosy and sarcoidosis. These cells were present in T-cell-reactive leprosy but were undetectable in cutaneous sarcoidosis. We have also studied the TCR Valpha repertoire in the two diseases. In addition to Valpha24(+) NKT cells, all patients with T-cell-reactive leprosy showed a very restricted T-cell-reactive Valpha repertoire with a strong bias toward the use of the Valpha6 and Valpha14 segments. Valpha6 and Valpha14(+) T cells were polyclonal in terms of CDR3 length and Jalpha usage. In contrast, most sarcoidosis patients showed a diverse usage of Valpha chains associated with clonal or oligoclonal expansions reminiscent of antigen-driven activation of conventional T cells. Thus the origin and perpetuation of the two kinds of granulomatous lesions appear to depend on altogether distinct T-cell recruiting mechanisms.

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.

Role of the Complementarity-Determining Region 3 (CDR3) of the TCR-β Chains Associated with the Vα14 Semi-Invariant TCR α-Chain in the Selection of CD4+ NK T Cells

The NK1.1+TCRαβint CD4+, or double negative T cells (NK T cells) consist of a mixture of CD1d-restricted and CD1d-unrestricted cells. The relationships between CD4+NK1.1+ T cells and conventional T cells are not understood. To compare their respective TCR repertoires, NK1.1+TCRαβint, CD4+ T cells have been sorted out of the thymus, liver, spleen, and bone marrow of C57BL/6 mice. Molecular analysis showed that thymus and liver used predominantly the Vα14-Jα281 and Vβ 2, 7, and 8 segments. These cells are CD1d restricted and obey the original definition of NK T cells. The complementarity-determining region 3 (CDR3) sequences of the TCR Vβ8.2-Jβ2.5 chain of liver and thymus CD4+ NK T cells were determined and compared with those of the same rearrangements of conventional CD4+ T cells. No amino acid sequence or usage characteristic of NK T cells could be evidenced: the Vβ8.2-Jβ2.5 diversity regions being primarily the same in NK T and in T cells. No clonal expansion of the β-chains was observed in thymus and liver CD1d-restricted CD4+NK T cells, suggesting the absence of acute or chronic Ag-driven stimulation. Molecular analysis of the TCR used by Vα14-Jα281 transgenic mice on a Cα−/− background showed that the α-chain can associate with β-chains using any Vβ segment, except in NK T cells in which it paired predominately with Vβ 2, 7, and 8+ β-chains. The structure of the TCR of NK T cells thus reflects the affinity for the CD1d molecule rather than a structural constraint leading to the association of the invariant α-chain with a distinctive subset of Vβ segment.

The immunological, environmental, and phylogenetic perpetrators of metastatic leishmaniasis

Cutaneous leishmaniases have persisted for centuries as chronically disfiguring parasitic infections affecting millions of people across the subtropics. Symptoms range from the more prevalent single, self-healing cutaneous lesion to a persistent, metastatic disease, where ulcerations and granulomatous nodules can affect multiple secondary sites of the skin and delicate facial mucosa, even sometimes diffusing throughout the cutaneous system as a papular rash. The basis for such diverse pathologies is multifactorial, ranging from parasite phylogeny to host immunocompetence and various environmental factors. Although complex, these pathologies often prey on weaknesses in the innate immune system and its pattern recognition receptors. This review explores the observed and potential associations among the multifactorial perpetrators of infectious metastasis and components of the innate immune system.

Presence of Leishmania RNA Virus 1 in Leishmania guyanensis Increases the Risk of First-Line Treatment Failure and Symptomatic Relapse

Treatment failure and symptomatic relapse are major concerns in American tegumentary leishmaniasis (TL). Such complications are seen frequently in Leishmania guyanensis infections, in which patients respond variously to first-line antileishmanials and are more prone to develop chronic cutaneous leishmaniasis. The factors underlying this pathology, however, are unknown. Recently, we reported that a double-stranded RNA virus, Leishmania RNA virus 1 (LRV1), nested within L. guyanensis parasites is able to exacerbate experimental murine leishmaniasis by inducing a hyperinflammatory response. This report investigates the prevalence of LRV1 in human L. guyanensis infection and its effect on treatment efficacy, as well as its correlation to symptomatic relapses after the completion of first-line treatment. In our cohort of 75 patients with a diagnosis of primary localized American TL, the prevalence of LRV1-positive L. guyanensis infection was elevated to 58%. All patients infected with LRV1-negative L. guyanensis were cured after 1 dose (22 of 31 [71%]) or 2 doses (31 of 31 [100%]) of pentamidine. In contrast, 12 of 44 LRV1-positive patients (27%) presented with persistent infection and symptomatic relapse that required extended therapy and the use of second-line drugs. Finally, LRV1 presence was associated with a significant increase in levels of intra-lesional inflammatory markers. In conclusion, LRV1 status in L. guyanensis infection is significantly predictive (P = .0009) of first-line treatment failure and symptomatic relapse and has the potential to guide therapeutic choices in American TL.