IL-27 and IL-21 are associated with T cell IL-10 responses in human visceral leishmaniasis

Abstract

IL-10 is believed to underlie many of the immunologic defects in human visceral leishmaniasis (VL). We have identified CD4(+)CD25(-)Foxp3(-) T cells as the major source of IL-10 in the VL spleen. IL-27, a member of the IL-6/IL-12 cytokine family, has been shown to promote development of IL-10-producing T cells, in part by upregulating their production of autocrine IL-21. We investigated whether IL-27 and IL-21 are associated with human VL. IL-27 was elevated in VL plasma, and at pretreatment, spleen cells showed significantly elevated mRNA levels of both IL-27 subunits, IL-27p28 and EBI-3, as well as IL-21, compared with posttreatment biopsies. CD14(+) spleen cells were the main source of IL-27 mRNA, whereas CD3(+) T cells were the main source of IL-21. IL-27 mRNA could be strongly upregulated in normal donor macrophages with IFN-γ and IL-1β, conditions consistent with those in the VL spleen. Last, a whole-blood assay revealed that most VL patients could produce Ag-specific IFN-γ and IL-10 and that the IL-10 could be augmented with recombinant human IL-21. Thus, proinflammatory cytokines acting on macrophages in the VL spleen have the potential to upregulate IL-27, which in turn can induce IL-21 to expand IL-10-producing T cells as a mechanism of feedback control.

FIGURE 1

Cytokines in plasma from VL patients. IFN-γ, IL-10 and IL-17 were measured by multiplex analysis of plasma obtained from paired VL patients at pre- and post -treatment (n = 16) and from ECs (n = 10). Dual antibody sandwich ELISA was used to measure plasma IL-27 levels in paired VL patients (n = 21) and ECs (n = 10). Significant differences are indicated with *, p < 0.05; **, p < 0.01, ***, p < 0.001.

FIGURE 2

Cytokine mRNA expression in VL spleen. (A–C) Ex-vivo analysis of relative mRNA levels for indicated genes in splenic aspirates from VL patients before (n = 28) or 21–30 d after treatment (n = 18) and in HOD spleen samples (n = 10). Paired pre- and post- treatment samples(n = 18) are also shown separately. Significant differences are indicated with *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 3

Ex-vivo analysis of cytokine mRNA levels in splenic lymphocytes subsets. (A) Relative expression of IL-10, IL-21, IL-27p28 and EBI-3 mRNA levels in sequential, positively selected CD19+, CD3+(CD25-) and in the remaining depleted (Innate cells: NK, Macrophages, DCs) splenic cell population before treatment (n = 8). (B) Relative expression of cell specific markers (CD19/B cell, CD14/ Monocytes/ Macrophages and CD1c/DCs on sequential, positively selected CD19⁺, CD3⁺, CD14⁺, CD1c⁺, CD56⁺ and in the remaining ‘depleted’ splenic cell population. (C) Relative expression of mRNA levels for IL-10, IL-21, IL-27p28, and EBI-3 in positively selected splenic cell fractions from VL patients prior to treatment (n = 8). Significant differences are indicated with *, p < 0.05; **, p < 0.01; ***, p < 0.001, ns, not significant.

FIGURE 4

IFN-γ and IL-1β induces IL-27 mRNA in human monocyte derived macrophages. (A) Monocyte derived macrophages from normal donors (n = 7) were stimulated with rhIFN-γ or rhTNF-α or their combination or medium alone for 8 h prior to infection with L. donovani amastigotes. Macrophages were cultured for another 16 h before cells were lysed for mRNA expression analysis. (B) Monocyte derived macrophages from normal donors (n = 11) were stimulated with rhIFN-γ or rhIL-1β or their combination or medium alone for 24 h before cells were lysed for mRNA expression analysis. Significant differences are indicated with *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5

IL-21 upregulates antigen specific IL-10 production in whole blood cell cultures from active VL cases. (A) IFN-γ and IL-10 production by peripheral blood cells from pre-treatment VL patients (n = 27) and EC (n = 13 or 19) in response to SLA. (B) IL-10 production by peripheral blood cells from pre-treatment VL patients (n = 18) and ECs (n = 7) in response to SLA, rhIL-27 or rhIL-21 alone or in combination. (C) IL-21 production by peripheral blood cells from pre-treatment VL patients (n = 13). Whole blood cells were cultured in the absence or presence of 10µg/ml L. donovani soluble antigen and/or exogenous rhL-27(100ng/ml) and/or rhIL-21 (25ng/ml). Twenty four hours later supernatant was collected for cytokine analysis. Cytokine concentrations shown are the values in the stimulated cultures minus the medium control. Significant differences are indicated with p values.