Date Published: February 24, 2015
Publisher: Public Library of Science
Author(s): Guadalupe Andreani, Michel Ouellet, Rym Menasria, Alejandro Martin Gomez, Corinne Barat, Michel J. Tremblay, Peter C. Melby. http://doi.org/10.1371/journal.pntd.0003543
Abstract: Visceral leishmaniasis is caused by the protozoan parasites Leishmania infantum and Leishmania donovani. This infection is characterized by an uncontrolled parasitization of internal organs which, when left untreated, leads to death. Disease progression is linked with the type of immune response generated and a strong correlation was found between disease progression and serum levels of the immunosuppressive cytokine IL-10. Other studies have suggested a role for B cells in the pathology of this parasitic infection and the recent identification of a B-cell population in humans with regulatory functions, which secretes large amounts of IL-10 following activation, have sparked our interest in the context of visceral leishmaniasis. We report here that incubation of human B cells with Leishmania infantum amastigotes resulted in upregulation of multiple cell surface activation markers and a dose-dependent secretion of IL-10. Conditioned media from B cells incubated with Leishmania infantum amastigotes were shown to strongly inhibit CD4+ T-cell activation, proliferation and function (i.e. as monitored by TNF and IFNγ secretion). Blockade of IL-10 activity using a soluble IL-10 receptor restored only partially TNF and IFNγ production to control levels. The parasite-mediated IL-10 secretion was shown to rely on the activity of Syk, phosphatidylinositol-3 kinase and p38, as well as to require intracellular calcium mobilization. Cell sorting experiments allowed us to identify the IL-10-secreting B-cell subset (i.e. CD19+CD24+CD27-). In summary, exposure of human B cells to Leishmania infantum amastigotes triggers B cells with regulatory activities mediated in part by IL-10, which could favor parasite dissemination in the organism.
Partial Text: Leishmaniasis is an infection caused by protozoan parasites of the genus Leishmania and is one of the most significant neglected tropical diseases, with 350 million people in 98 countries worldwide at risk of developing one of the forms of the disease . Visceral leishmaniasis (VL) is the most severe form of the disease and it represents nearly 40,000 deaths per year . VL is characterized by an uncontrolled parasitization of organs, such as spleen, liver and bone marrow, and is caused by the species Leishmania infantum (L. infantum) (known as L. chagasi in South America) and L. donovani. All parasites of the genus Leishmania are obligate, intracellular protozoa that infect cells of the macrophage-dendritic cell lineage of their vertebrate hosts (primarily macrophages) [2,3]. The parasite exists under two distinct morphologic forms, i.e. either as motile promastigotes within the alimentary canal of their phlebotomine sandfly vector or as nonmotile amastigotes that reside within phagolysosomes of mammalian mononuclear phagocytes. Infection of the mammalian host is initiated when the female sandfly regurgitates infectious promastigotes during its blood meal. Promastigotes are quickly internalized by tissue phagocytes recruited to the site of infection. Following phagocytosis, promastigotes are engulfed in phagolysosomes, where they transform into the non-motile intracellular amastigotes. Thereafter, amastigotes replicate within acidic phagolysosomes, eventually lysing the cell and freeing themselves to interact with adjacent cells. According to a recent report, amastigotes could also be transferred directly to other target cells via LAMP-rich parasitophorous extrusions , exploiting an alternative mechanism of transmission that would minimize exposure to the immune system.
The biological contribution of IL-10 for the visceralization of L. infantum in mice has been well established [29,68–70]. Moreover, IL-10 levels correlate with disease progression in humans [6,71] and dogs . Finally, the importance of B cells as a source of IL-10 in the susceptible Balb/c murine model of L. major infection has been already described  and their potential to regulate T-cell responses was described in mice infected with L. donovani . However, while Deak and colleagues have recently demonstrated the role of B cells in the visceralisation of Leishmania in a murine model of VL, they have shown that this effect is not relying on IL-10 . In the spleen of VL patients, IL-10 mRNA was mostly associated with T lymphocytes and a reduced B/T ratio was observed compared to exposed controls. This would suggest a minor role for B cells in the elevated plasma IL-10 levels. However, our results in Fig. 2 (panels C and D) with B cells exposed to L. infantum promastigotes suggest that IL-10 mRNA levels are not necessarily correlated to IL-10 cytokine production. The direct involvement of human B cells to the elevated levels of plasma IL-10 following L. infantum infection and their role in the visceralization of L. infantum in humans still remains unclear. In the current study, we first demonstrate that in vitro incubation of L. infantum amastigotes with purified human B cells increases expression of numerous activation markers (Fig. 1) and the parasite-directed effect on B-cell activation requires an intimate contact between the two distinct entities (S1 Fig.). This is in agreement with the demonstrated B-cell activation in patients with localized cutaneous leishmaniasis .