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Leishmania infantum amastigotes trigger a subpopulation of human B cells with an immunoregulatory phenotype.

Andreani G, Ouellet M, Menasria R, Gomez AM, Barat C, Tremblay MJ - PLoS Negl Trop Dis (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Axe des Maladies Infectieuses et Immunitaires, Centre de recherche du Centre Hospitalier Universitaire (CHU) de Québec-pavillon CHUL, Québec, Canada.

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.

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Related in: MedlinePlus

Incubation of human B cells with L. infantum amastigotes induces IL-10 production.A) Purified human B cells were either left alone (used as a control) or incubated overnight with L. infantum amastigotes at the listed parasite:host cell ratios. Next, IL-10 mRNA expression was measured by quantitative real-time PCR. Results are expressed as fold increase relative to cells left alone. P values were calculated by two-tailed Student’s t-test (n = 5). B) Cells were treated as in a panel A and IL-10 secretion was measured in cell-free supernatants by ELISA. Results are expressed as means +/- standard errors of the means (SEM) and P values were calculated by two-tailed Student’s t-test (n = 7). C) Purified human B cells were incubated overnight either with live L. infantum amastigotes, heat-killed (HK) L. infantum amastigotes, live L. infantum promastigotes (PRO), or heat-killed L. infantum promastigotes at a final parasite:host cell ratio of 3:1 before assessing IL-10 mRNA by quantitative real-time PCR. Results are expressed as fold increase relative to cells left alone. P values were calculated by two-tailed Student’s t-test (n = 5). D) Purified human B cells were incubated overnight either with live L. infantum amastigotes or live L. infantum promastigotes at a final parasite:host cell ratio of 3:1 before testing IL-10 production by ELISA. Results are expressed as means +/- SEM and P values were calculated by two-tailed Student’s t-test (n = 7). E) A representative dot plot of IL-10-expressing CD19+ B cells either left alone or incubated with L. infantum amastigotes (AMA) is shown on the left portion while percentages of IL-10-positive cells are displayed on the right portion of the panel. Results are expressed as means +/- SEM and P value is calculated by two-tailed Student’s t-test (n = 7).
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pntd.0003543.g002: Incubation of human B cells with L. infantum amastigotes induces IL-10 production.A) Purified human B cells were either left alone (used as a control) or incubated overnight with L. infantum amastigotes at the listed parasite:host cell ratios. Next, IL-10 mRNA expression was measured by quantitative real-time PCR. Results are expressed as fold increase relative to cells left alone. P values were calculated by two-tailed Student’s t-test (n = 5). B) Cells were treated as in a panel A and IL-10 secretion was measured in cell-free supernatants by ELISA. Results are expressed as means +/- standard errors of the means (SEM) and P values were calculated by two-tailed Student’s t-test (n = 7). C) Purified human B cells were incubated overnight either with live L. infantum amastigotes, heat-killed (HK) L. infantum amastigotes, live L. infantum promastigotes (PRO), or heat-killed L. infantum promastigotes at a final parasite:host cell ratio of 3:1 before assessing IL-10 mRNA by quantitative real-time PCR. Results are expressed as fold increase relative to cells left alone. P values were calculated by two-tailed Student’s t-test (n = 5). D) Purified human B cells were incubated overnight either with live L. infantum amastigotes or live L. infantum promastigotes at a final parasite:host cell ratio of 3:1 before testing IL-10 production by ELISA. Results are expressed as means +/- SEM and P values were calculated by two-tailed Student’s t-test (n = 7). E) A representative dot plot of IL-10-expressing CD19+ B cells either left alone or incubated with L. infantum amastigotes (AMA) is shown on the left portion while percentages of IL-10-positive cells are displayed on the right portion of the panel. Results are expressed as means +/- SEM and P value is calculated by two-tailed Student’s t-test (n = 7).

Mentions: As circulating IL-10 levels are elevated in VL patient, we assessed whether the parasite can drive IL-10 expression and secretion in human tonsillar B cells. A dose-dependent increase in IL-10 mRNA expression was seen in response to L. infantum amastigotes (Fig. 2A). Similar results were obtained when measuring IL-10 secretion in cell-free supernatants using a commercial ELISA kit (Fig. 2B). Interestingly, the parasite-dependent induction of IL-10 mRNA was not seen when using heat-killed parasites (Fig. 2C), thus suggesting that induction of IL-10 in B cells is an active mechanism mediated by live parasites. Although promastigotes can promote IL-10 mRNA synthesis at lower levels than amastigotes (Fig. 2C), they are unable to induce IL-10 at the protein level (Fig. 2D). Experiments performed with cell culture inserts to separate amastigotes and B cells indicate that a physical contact between human B cells and L. infantum amastigotes is necessary to achieve IL-10 secretion (S2 Fig.). The production of IL-10 was also determined by intracellular staining. The percentage of IL-10-expressing cells was higher in B cells exposed to the parasite compared to untreated controls (Fig. 2E), and were identified as CD19-expressing cells as previously described [46,48]. Importantly, human peripheral blood (circulating) B cells were also capable of secreting IL-10 upon incubation with the parasite and activation markers were also enhanced upon an exposure to L. infantum amastigotes (S3 Fig.). It has been previously described in mice that TGF-β is secreted by B cells with modulatory functions and is able to modulate CD4+ T-cell functions [53]. However, we were not able to detect TGF-β secretion or activation of latent TGF-β in B-cell culture media following exposure to parasites or in the supernatant of CD4+ T cells exposed to culture media from human B cells treated with amastigotes. Taken together these results demonstrate that a contact with L. infantum amastigotes activates human B cells and leads to secretion of T-cell suppressive factors such as IL-10. However, we have no evidence for a contribution of TGF-β in the suppressive phenotype.


Leishmania infantum amastigotes trigger a subpopulation of human B cells with an immunoregulatory phenotype.

Andreani G, Ouellet M, Menasria R, Gomez AM, Barat C, Tremblay MJ - PLoS Negl Trop Dis (2015)

Incubation of human B cells with L. infantum amastigotes induces IL-10 production.A) Purified human B cells were either left alone (used as a control) or incubated overnight with L. infantum amastigotes at the listed parasite:host cell ratios. Next, IL-10 mRNA expression was measured by quantitative real-time PCR. Results are expressed as fold increase relative to cells left alone. P values were calculated by two-tailed Student’s t-test (n = 5). B) Cells were treated as in a panel A and IL-10 secretion was measured in cell-free supernatants by ELISA. Results are expressed as means +/- standard errors of the means (SEM) and P values were calculated by two-tailed Student’s t-test (n = 7). C) Purified human B cells were incubated overnight either with live L. infantum amastigotes, heat-killed (HK) L. infantum amastigotes, live L. infantum promastigotes (PRO), or heat-killed L. infantum promastigotes at a final parasite:host cell ratio of 3:1 before assessing IL-10 mRNA by quantitative real-time PCR. Results are expressed as fold increase relative to cells left alone. P values were calculated by two-tailed Student’s t-test (n = 5). D) Purified human B cells were incubated overnight either with live L. infantum amastigotes or live L. infantum promastigotes at a final parasite:host cell ratio of 3:1 before testing IL-10 production by ELISA. Results are expressed as means +/- SEM and P values were calculated by two-tailed Student’s t-test (n = 7). E) A representative dot plot of IL-10-expressing CD19+ B cells either left alone or incubated with L. infantum amastigotes (AMA) is shown on the left portion while percentages of IL-10-positive cells are displayed on the right portion of the panel. Results are expressed as means +/- SEM and P value is calculated by two-tailed Student’s t-test (n = 7).
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Related In: Results  -  Collection

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Show All Figures
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pntd.0003543.g002: Incubation of human B cells with L. infantum amastigotes induces IL-10 production.A) Purified human B cells were either left alone (used as a control) or incubated overnight with L. infantum amastigotes at the listed parasite:host cell ratios. Next, IL-10 mRNA expression was measured by quantitative real-time PCR. Results are expressed as fold increase relative to cells left alone. P values were calculated by two-tailed Student’s t-test (n = 5). B) Cells were treated as in a panel A and IL-10 secretion was measured in cell-free supernatants by ELISA. Results are expressed as means +/- standard errors of the means (SEM) and P values were calculated by two-tailed Student’s t-test (n = 7). C) Purified human B cells were incubated overnight either with live L. infantum amastigotes, heat-killed (HK) L. infantum amastigotes, live L. infantum promastigotes (PRO), or heat-killed L. infantum promastigotes at a final parasite:host cell ratio of 3:1 before assessing IL-10 mRNA by quantitative real-time PCR. Results are expressed as fold increase relative to cells left alone. P values were calculated by two-tailed Student’s t-test (n = 5). D) Purified human B cells were incubated overnight either with live L. infantum amastigotes or live L. infantum promastigotes at a final parasite:host cell ratio of 3:1 before testing IL-10 production by ELISA. Results are expressed as means +/- SEM and P values were calculated by two-tailed Student’s t-test (n = 7). E) A representative dot plot of IL-10-expressing CD19+ B cells either left alone or incubated with L. infantum amastigotes (AMA) is shown on the left portion while percentages of IL-10-positive cells are displayed on the right portion of the panel. Results are expressed as means +/- SEM and P value is calculated by two-tailed Student’s t-test (n = 7).
Mentions: As circulating IL-10 levels are elevated in VL patient, we assessed whether the parasite can drive IL-10 expression and secretion in human tonsillar B cells. A dose-dependent increase in IL-10 mRNA expression was seen in response to L. infantum amastigotes (Fig. 2A). Similar results were obtained when measuring IL-10 secretion in cell-free supernatants using a commercial ELISA kit (Fig. 2B). Interestingly, the parasite-dependent induction of IL-10 mRNA was not seen when using heat-killed parasites (Fig. 2C), thus suggesting that induction of IL-10 in B cells is an active mechanism mediated by live parasites. Although promastigotes can promote IL-10 mRNA synthesis at lower levels than amastigotes (Fig. 2C), they are unable to induce IL-10 at the protein level (Fig. 2D). Experiments performed with cell culture inserts to separate amastigotes and B cells indicate that a physical contact between human B cells and L. infantum amastigotes is necessary to achieve IL-10 secretion (S2 Fig.). The production of IL-10 was also determined by intracellular staining. The percentage of IL-10-expressing cells was higher in B cells exposed to the parasite compared to untreated controls (Fig. 2E), and were identified as CD19-expressing cells as previously described [46,48]. Importantly, human peripheral blood (circulating) B cells were also capable of secreting IL-10 upon incubation with the parasite and activation markers were also enhanced upon an exposure to L. infantum amastigotes (S3 Fig.). It has been previously described in mice that TGF-β is secreted by B cells with modulatory functions and is able to modulate CD4+ T-cell functions [53]. However, we were not able to detect TGF-β secretion or activation of latent TGF-β in B-cell culture media following exposure to parasites or in the supernatant of CD4+ T cells exposed to culture media from human B cells treated with amastigotes. Taken together these results demonstrate that a contact with L. infantum amastigotes activates human B cells and leads to secretion of T-cell suppressive factors such as IL-10. However, we have no evidence for a contribution of TGF-β in the suppressive phenotype.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Axe des Maladies Infectieuses et Immunitaires, Centre de recherche du Centre Hospitalier Universitaire (CHU) de Québec-pavillon CHUL, Québec, Canada.

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.

Show MeSH
Related in: MedlinePlus