Interdependency of interleukin-10 and interleukin-12 in regulation of T-cell differentiation and effector function of monocytes in response to stimulation with Cryptococcus neoformans

Abstract

We previously demonstrated that the principal component of capsular material of Cryptococcus neoformans, glucuronoxylomannan (GXM), induces interleukin-10 (IL-10) secretion from human monocytes. Here we report that encapsulation of the yeast with GXM is able to down-regulate interleukin-12 (IL-12) production by monocytes that would normally occur in the absence of encapsulation. This phenomenon appeared to be the result of inhibition of the phagocytic process by encapsulation with GXM as well as of negative signals such as IL-10 secretion produced by interaction of GXM with leukocytes. Decreased secretion of IL-12 correlated with decreased release of gamma interferon (IFN-gamma) from T cells, suggesting a role for encapsulation with GXM in hindering a T helper type 1 (Th1) response. This is supported by the ability of encapsulation with GXM to limit increased expression of B7-1 costimulatory molecules that otherwise might limit IL-10 secretion. Endogenous IL-10 played a critical role in modulatory activity associated with encapsulation with GXM. Blocking IL-10 with monoclonal antibody to IL-10 resulted in increased (i) IL-12 secretion, (ii) IFN-gamma release from T cells, and (iii) killing of C. neoformans by monocytes. These results suggest that encapsulation with GXM limits development of a protective Th1-type response, an inhibitory process in which IL-10 plays a critical role. Scavengers of GXM and/or IL-10 could be useful in a protective Th1-type response in patients with cryptococcosis.

FIG. 1

IL-12 levels in supernatant fluids of monocytes (day +2) or PBMC (day +7) stimulated with encapsulated (6995) or acapsular (7698) C. neoformans, or with GXM (250 μg/ml), or with 7698 plus GXM in the presence or absence of MAb to IL-10 (5 μg/ml) or human recombinant IL-10 (25 ng/ml). The determination was performed with supernatant fluids harvested from cells after 2 or 7 days of incubation. Irrelevant MAb (2.5 μg/ml) did not affect IL-12 release in our experimental system. The results reported are the means + SD (error bars) of four experiments with samples from four different donors (each donor sample was done in duplicate). NS, not stimulated; ∗, P < 0.025 (MAb to IL-10-treated versus untreated cells); ∗∗, P < 0.025 (IL-10-treated versus untreated cells).

FIG. 2

IL-12 levels in supernatant fluids of monocytes treated for 18 h with IFN-γ (100 U/ml) and then washed and stimulated for 48 h with encapsulated (6995) or acapsular (7698) C. neoformans, or with GXM (250 μg/ml), or with 7698 plus GXM in the presence or absence of MAb to IL-10 (5 μg/ml) or human recombinant IL-10 (25 ng/ml). The determination was performed with supernatant fluids harvested from cells after 2 days of incubation using an assay specific for IL-12 p70. Irrelevant MAb (2.5 μg/ml) did not affect IL-12 release in our experimental system. The results reported are the means + SD (error bars) of four experiments with samples from four different donors (each donor sample was done in duplicate). NS, not stimulated; ∗, P < 0.025 (MAb to IL-10-treated versus untreated cells); ∗∗, P < 0.025 (IL-10-treated versus untreated cells).

FIG. 3

IL-12 levels in supernatant fluids from monocytes stimulated with acapsular (7698) C. neoformans or with 7698 plus GXM (250 μg/ml) in the presence or absence of cytochalasin B (Cytoch. B) (5 μg/ml). The determination was performed with supernatant fluids harvested from cells after 2 days of incubation. The results reported are the means + SD (error bars) of three experiments with samples from three different donors (each donor sample was done in duplicate). NS, not stimulated; ∗, P < 0.025 (7698-plus-cytochalasin B-treated versus 7698-treated cells).

FIG. 4

B7-1 expression on monocytes treated with acapsular (7698) C. neoformans, or with GXM (250 μg/ml), or with 7698 plus GXM. B7-1 expression was assessed after 24 h of incubation. (a) Dot plots are shown. (b) Percentage of positive cells. The results are reported as mean fluorescence intensity and represent the means + SD (error bars) of four experiments with samples from four different donors (each donor sample was done in duplicate). NS, not stimulated; ∗, P < 0.025 (7698 + GXM-treated versus 7698-treated cells).

FIG. 5

IL-10 levels in supernatant fluids from Cryptococcus-laden monocytes cocultured with autologous T cells in the presence or absence of MAb to B7-1 (2 μg/ml) or MAb to B7-2 (2 μg/ml). The results are the means + SD (error bars) of three experiments with samples from three different donors (each donor sample was done in duplicate). Irrelevant MAb (IgM[2 μg/ml] or [IgG1 2 μg/ml]) did not produce changes in proliferative response. NS, not stimulated. ∗, P < 0.025 (C. neoformans + MAb to B7-1-treated versus respective C. neoformans-treated cells alone).

FIG. 6

IFN-γ levels in supernatant fluids of PBMC stimulated with encapsulated (6995) or acapsular (7698) C. neoformans, or with GXM (250 μg/ml), or with 7698 plus GXM in the presence or absence of MAb to IL-10 (5 μg/ml) or human recombinant IL-10 (25 ng/ml). The determination was performed with supernatant fluids harvested from cells after 7 days of incubation. Irrelevant MAb (5 μg/ml) did not affect IFN-γ release in our experimental system. The results reported are the means + SD (error bars) of four experiments with samples from four different donors (each donor sample was done in duplicate). NS, not stimulated; ∗, P < 0.025 (MAb to IL-10-treated versus untreated cells); ∗∗, P < 0.025 (IL-10-treated versus untreated cells).