Antigen-presenting dendritic cells rescue CD4-depleted CCR2-/- mice from lethal Histoplasma capsulatum infection

Abstract

Excessive production of interleukin-4 impairs clearance of the fungal pathogen Histoplasma capsulatum in mice lacking the chemokine receptor CCR2. An increase in the interleukin-4 level is associated with decreased recruitment of dendritic cells to lungs; therefore, we investigated the possibility that these cells influence interleukin-4 production. Adoptive transfer of wild-type or CCR2(-/-) bone marrow-derived dendritic cells loaded with heat-killed yeast cells to infected CCR2(-/-) mice suppressed interleukin-4 transcription. Surprisingly, transfer of cells did not reduce the fungal burden despite the fact that it limited interleukin-4 transcription. Yeast cell-loaded bone marrow-derived dendritic cell-mediated regulation of interleukin-4 transcription was dependent on major histocompatibility complex II antigen presentation to CD4(+) T cells. We previously showed that CD4(+) T cells were a source of interleukin-4 in infected CCR2(-/-) mice, but their contribution to the TH2 phenotype was unclear. Here we demonstrated that these cells were functionally important since elimination of them prior to infection, but not elimination of them at the time of infection, reduced the interleukin-4 level in infected CCR2(-/-) mice. However, the fungal burden was reduced only in CD4-depleted CCR2(-/-) mice that received yeast cell-loaded bone marrow-derived dendritic cells. Taken together, the data indicate that generation of excess interleukin-4 in lungs of H. capsulatum-infected CCR2(-/-) mice is at least partially a consequence of decreased recruitment of dendritic cells capable of antigen presentation. Furthermore, CD4(+) T cells had a deleterious impact on immunity in infected CCR2(-/-) mice.

FIG. 1.

Expression of costimulatory molecules on the surface of BMDC. WT or CCR2^−/− BMDC were not treated or exposed to LPS, anti-CD40, and heat-killed H. capsulatum for 4 h. Subsequently, a portion of the BMDC were infected with viable H. capsulatum yeast cells at a ratio of yeast cells to BMDC of 2:1. mat, matured (treated with LPS and anti-CD40); Ag, heat-killed H. capsulatum; KO, knockout. The data are data from one of three similar experiments.

FIG. 2.

Adoptive transfer of Ag-BMDC to CCR2^−/− mice suppresses IL-4 production. (A) Schematic diagram of adoptive transfer experiments. (B) Log₁₀ relative quantification (RQ) of IL-4 transcription from whole lungs 7 days postinfection compared to uninfected lung expression. Prior to infection BMDC were not treated (Ag-free) or were matured (mat) and exposed to heat-killed H. capsulatum (Ag) or ovalbumin (ova) (n = 6 to 9). (C) Levels of IL-4 protein in whole-lung homogenates as determined by ELISA (n = 6). (D) IL-12 transcription by WT BMDC prior to transfer compared to IL-12 transcription by untreated WT BMDC. **, P = 0.002 compared to CCR2^−/−; *, P < 0.05 compared to CCR2^−/−; #, P < 0.05 compared to Ag-BMDC that were not matured. The data are the means and SEM of two or three experiments.

FIG. 3.

IL-4 regulation is associated with an increase in the number of lung DC. (A) Percentage of lung CD11c⁺ CD11b⁺ I-Ab^hi DC 7 days postinfection (n = 8 or 9). mat, matured (treated with LPS and anti-CD40). (B) Absolute number of lung DC at day 7 (n = 8 or 9). **, P < 0.001 compared to CCR2^−/−. The data represent the means and SEM of two or three experiments. (C) Representative FACS plots of isolated lung leukocytes stained with CD11c and CD45.1 to identify transferred donor Ag-BMDC and Ag-free BMDC prior to infection (D0) and at days 1 (D1), 3 (D3), and 7 (D7) postinfection. CD45.1 transfer experiments were performed two times with three or four mice per group.

FIG. 4.

BMDC kill H. capsulatum in vitro. BMDC (10⁵ cells) were infected at a ratio of yeast cells to BMDC of 1:10 or 1:1. After 24 and 48 h, samples were plated on medium to enumerate the viable organisms. The data are the means and SEM of three experiments. *, P < 0.05. Hc, H. capsulatum.

FIG. 5.

Transfer of Ag-free BMDC impairs yeast clearance. The fungal burdens in lungs and spleens were determined 7 days postinfection (n = 6). **, P < 0.001; *, P < 0.005. The data are the means and SEM of two experiments. mat, matured (treated with LPS and anti-CD40).

FIG. 6.

Transfer of Ag-free BMDC to CCR2^−/− mice results in altered distribution of yeast cells within phagocyte populations. Flow cytometry was performed to determine the relative percentage of lung leukocytes infected with GFP-labeled H. capsulatum 7 days postinfection (A), the percentage of CD11c⁺ CD11b⁺ I-A^bhigh lung DC infected with GFP-labeled H. capsulatum (B), and the percentage of GFP-labeled H. capsulatum distributed in DC 7 days postinfection (C) (n = 8 or 9). **, P < 0.001 compared to WT mice; *, P < 0.05 compared to WT mice; ##, P < 0.001 compared to CCR2^−/− mice; #, P < 0.05 compared to CCR2^−/− mice. The data are the means and SEM of three experiments. mat, matured (treated with LPS and anti-CD40).

FIG. 7.

Transfer of Ag-BMDC suppresses IFN-γ and TNF-α production. The data indicate the log₁₀ RQ cytokine transcription 7 days postinfection in lungs of CCR2^−/− mice immunized with BMDC (A), WT mice immunized with BMDC (B), and CCR2^−/− mice immunized with Ag (C) (n = 6). *, P < 0.01. The data are the means and SEM of two experiments. mat, matured (treated with LPS and anti-CD40).

FIG. 8.

Depletion of CD4⁺ T cells, but not depletion of CD8⁺ T cells, prior to infection in CCR2^−/− mice that received Ag-BMDC suppressed IL-4 production and decreased the fungal burden. (A) Log₁₀ IL-4 transcription in lungs compared to transcription in uninfected lungs 7 days postinfection (n = 6). (B and C) Fungal burden in lungs at 7 days (n = 6) and 14 days (n = 8) postinfection. (D) Survival after infection with 2 × 10⁶ yeast cells (n = 12, except for CCR2^−/− [n = 6]). (E and F) Transcription of IFN-γ and TNF-α in mice (n = 8 to 10). (G and H) Percentages and absolute numbers of CD8⁺ T cells 7 days postinfection (n = 6). *, P < 0.01 compared to CCR2^−/− mice. The data are the means and SEM of two experiments.