Eliane Bourreau

Differential Production of Systemic and Intralesional Gamma Interferon and Interleukin-10 in Nodular and Ulcerative Forms of Buruli Disease

ABSTRACT Buruli disease, caused by Mycobacterium ulcerans, is the third most important mycobacterial disease in humans besides tuberculosis and leprosy. We have compared systemic and intralesional cytokine production in patients presenting with a nodular form and a necrotizing, ulcerative form of the disease. Gamma interferon (IFN-γ) levels in response to whole M. ulcerans and Mycobacterium bovis BCG bacilli and in response to purified Ag85 protein from BCG were lower in peripheral blood mononuclear cells (PBMC) cultures from Buruli disease patients than in PBMC from healthy purified protein derivative-positive contacts. Interleukin-4 (IL-4) and IL-13 content was below the detection threshold in these PBMC cultures. IFN-γ production after stimulation with M. ulcerans was significantly lower (P < 0.05) in PBMC cultures from patients with ulcers than in those from patients with nodules. On the other hand, PBMC from Buruli disease patients produced significant levels of IL-10 in response to M. ulcerans (but not to M. bovis BCG) and production was highest in patients with the ulcerative form. Third, semiquantitative reverse transcription-PCR analysis demonstrated a similar difference in the local, intralesional cytokine profile for the two forms of the disease: high IFN-γ but low IL-10 mRNA levels in nodular lesions and high IL-10 but low IFN-γ mRNA levels in ulcerative lesions. Intralesional IL-4 and IL-13 mRNA levels were low and only detected in patients with the ulcerative form. Our results indicate, although they do not formally prove, that production of IL-10 rather than production of IL-4 or IL-13 by Th2-type T cells may be involved in the low M. ulcerans-specific IFN-γ response in Buruli disease patients.

High Intralesional Interleukin-10 Messenger RNA Expression in Localized Cutaneous Leishmaniasis Is Associated with Unresponsiveness to Treatment

The intralesional expression of cytokines (interleukin [IL]-4, IL-13, IL-10, and interferon-gamma) was analyzed in 65 patients with localized cutaneous leishmaniasis due to Leishmania guyanensis before specific treatment with pentamidine isethionate. The local expression of IL-10 was significantly higher in patients who responded poorly to treatment than in patients whose lesions were regressing. When an IL-10 level >10 (ratio of the concentration of IL-10 [pg/microL] to that of beta-actin [pg/microL]) was used as an indicator of treatment failure, the sensitivity of this test was 78.6, and the specificity was 72.5. Thus, high intralesional expression of IL-10 might predict a poor response to conventional treatment.

Interleukin (IL)-13 is the predominant Th2 cytokine in localized cutaneous leishmaniasis lesions and renders specific CD4+ T cells unresponsive to IL-12

Semiquantitative reverse transcription-polymerase chain reaction analysis of leishmania lesion cytokine profile showed a Th2 cytokine expression pattern, as reflected by interleukin (IL)-4 and IL-13 mRNA expression. There was a predominance of IL-13 in most lesions from patients with American localized cutaneous leishmaniasis caused by Leishmania guyanensis. IL-13 production by peripheral blood mononuclear cells in response to specific leishmania antigens was confirmed in these patients. The absence of the second chain of the IL-12 receptor (IL-12Rbeta2) mRNA expression in lesions and the presence of specific IgE and IgG4 in some serum samples demonstrated the functional role of these Th2 cytokines. IL-13, unlike IL-4, rendered specific T cells unresponsive to IL-12 by inhibiting the expression of the IL-12Rbeta2 chain. These data establish the crucial role of IL-13 in human cutaneous leishmaniasis.

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.

In Leishmaniasis due to Leishmania guyanensis infection, distinct intralesional interleukin-10 and foxp3 mRNA expression are associated with unresponsiveness to treatment

The presence of intralesional natural regulatory T cells, characterized by the expression of Foxp3 mRNA, was analyzed in patients with localized leishmaniasis due to Leishmania guyanensis infection that was unresponsive to treatment with pentamidine isethionate. Foxp3 mRNA levels were associated with unresponsiveness to treatment among patients with a lesion duration of 1 month, but this association was not observed among patients with a lesion duration of <1 month. In conclusion, high intralesional expression of Foxp3 might be an indicator of poor response to treatment, depending on the duration of lesions.

IFN‐γ‐producing CD45RA+ CD8+ and IL‐10‐producing CD45RA– CD4+ T cells generated in response to LACK in naive subjects never exposed to Leishmania

Leishmania guyanensis (L. g.)‐specific CD8+ T cells can be isolated from PBMC of subjects who have never been previously exposed to Leishmania. Cells that produce IFN‐γ in response to live L. g. are generated from naive CD45RA+ CD8+ T cells. The generation of L. g.‐specific CD8+ T cells requires the presence of whole L. g. or UV‐irradiated parasite but not the soluble antigens from L. g. promastigotes. The IFN‐γ‐producing T cells recognize a specific antigen, the Leishmania homologue of receptors of activated C kinases (LACK) and this antigen but not live L. g. can produce a strong IL‐10 response in CD45RA– CD4+ memory T cells from naive subjects. A single epitope (amino acid 156 – 173) is found to induce the IL‐10 synthesis. While the IFN‐γ‐producing cells are present among CD45RA+ CD8+ T cells that are CD62L– CDR7– and CLA–, the LACK‐reactive IL‐10‐producing cells are CD4+ T cells that are CD62L+ CCR7– and CLA–.

Th2 responses predominate during the early phases of infection in patients with localized cutaneous leishmaniasis and precede the development of Th1 responses

ABSTRACT Intralesional Th2 responses preceded the development of Th1 responses in localized cutaneous leishmaniasis due to Leishmania guyanensis. Although the number of parasites increased in Th2 lesions, no correlation was found between the levels of cytokine expression and the number of parasites. In contrast, the decreased number of parasites in Th1 lesions is negatively correlated to gamma interferon expression.

LACK-Specific CD4+ T Cells That Induce Gamma Interferon Production in Patients with Localized Cutaneous Leishmaniasis during an Early Stage of Infection

ABSTRACT The profile of cytokines induced by soluble leishmania antigen (SLA) and the Leishmania homologue of the mammalian receptor for activated C kinase (LACK), a candidate vaccine against leishmaniasis, and the cellular source of the cytokines produced in response to these antigens were analyzed in patients infected with Leishmania guyanensis. Gamma interferon (IFN-γ) and interleukin-10 (IL-10) were produced in response to LACK. Although LACK-specific CD4+ cells producing IFN-γ were isolated only during the early phase of infection (less than 30 days following the onset of infection), cells producing IL-10 in response to LACK were detected in all patients. CD4+ T cells producing IFN-γ and IL-13 were produced in response to SLA in all patients. SLA- and LACK-specific T cells are effector memory cells, as they are CD45RA− CCR7− CD4+ T cells. CD4+ T cells producing IFN-γ are CD62L−, and CD4+ T cells producing IL-10 are CD62L+, indicating that these cells have different tissue-homing capacities. These findings show that SLA and LACK induce both type 1 (IFN-γ) and type 2 (IL-10 or IL-13) cell responses.

Transforming growth factor beta 1 production by CD4+ CD25+ regulatory T cells in peripheral blood mononuclear cells from healthy subjects stimulated with Leishmania guyanensis.

ABSTRACT Transforming growth factor β (TGF-β) has been shown to be a central immunomodulator used by leishmaniae to escape effective mechanisms of protection in human and murine infections with these parasites. However, all the information is derived from studies of established infection, while little is known about TGF-β production in response to Leishmania stimulation in healthy subjects. In this study, TGF-β1 production was demonstrated in peripheral blood mononuclear cells from healthy subjects never exposed to leishmaniae in response to live Leishmania guyanensis, and the TGF-β1-producing cells were described as a distinct subpopulation of CD4+ CD25+ regulatory T cells. The suppressive properties of CD4+ CD25+ T cells were demonstrated in vitro by their inhibition of production of interleukin 2 (IL-2) and IL-10 by CD4+ CD25− T cells in the presence of either anti-CD3 or L. guyanensis. Although neutralization of TGF-β1 did not reverse the suppressive activity of CD4+ CD25+ T cells activated by anti-CD3, it reversed the suppressive activity of CD4+ CD25+ T cells activated by L. guyanensis. Altogether our data demonstrated that TGF-β1 is involved in the suppressive activity of L. guyanensis-stimulated CD4+ CD25+ T cells from healthy controls.

Prevalence and Distribution of Leishmania RNA Virus 1 in Leishmania Parasites from French Guiana

In South America, the presence of the Leishmania RNA virus type 1 (LRV1) was described in Leishmania guyanensis and Leishmania braziliensis strains. The aim of this study was to determine the prevalence distribution of LRV1 in Leishmania isolates in French Guiana given that, in this French overseas department, most Leishmania infections are due to these parasite species. The presence of the virus was observed in 74% of Leishmania spp. isolates, with a highest presence in the internal areas of the country.