Toll-like receptor 4 signaling leads to severe fungal infection associated with enhanced proinflammatory immunity and impaired expansion of regulatory T cells

Abstract

Toll-like receptors (TLRs) present in innate immune cells recognize pathogen molecular patterns and influence immunity to control the host-parasite interaction. The objective of this study was to characterize the involvement of TLR4 in the innate and adaptive immunity to Paracoccidioides brasiliensis, the most important primary fungal pathogen of Latin America. We compared the responses of C3H/HeJ mice, which are naturally defective in TLR4 signaling, with those of C3H/HePas mice, which express functional receptors, after in vitro and in vivo infection with P. brasiliensis. Unexpectedly, we verified that TLR4-defective macrophages infected in vitro with P. brasiliensis presented decreased fungal loads associated with impaired synthesis of nitric oxide, interleukin-12 (IL-12), and macrophage chemotactic protein 1 (MCP-1). After intratracheal infection with 1 million yeasts, TLR4-defective mice developed reduced fungal burdens and decreased levels of pulmonary nitric oxide, proinflammatory cytokines, and antibodies. TLR4-competent mice produced elevated levels of IL-12 and tumor necrosis factor alpha (TNF-alpha), besides cytokines of the Th17 pattern, indicating a proinflammatory role for TLR4 signaling. The more severe infection of TLR4-normal mice resulted in increased influx of activated macrophages and T cells to the lungs and progressive control of fungal burdens but impaired expansion of regulatory T cells (Treg cells). In contrast, TLR4-defective mice were not able to clear their diminished fungal burdens totally, a defect associated with deficient activation of T-cell immunity and enhanced development of Treg cells. These divergent patterns of immunity, however, resulted in equivalent mortality rates, indicating that control of elevated fungal growth mediated by vigorous inflammatory reactions is as deleterious to the hosts as low fungal loads inefficiently controlled by limited inflammatory reactions.

FIG. 1.

TLR4 deficiency leads to less-severe fungal infection of macrophages (Mφ) associated with decreased synthesis of NO. The phagocytic and fungicidal abilities of macrophages from mice with defective TLR4 signaling (C3H/HeJ) or defective TLR4 expression (C57BL/6 TLR4^−/−) were compared with those of their TLR4-normal controls (C3H/HePas and C57BL/6 TLR4^+/+, respectively). (A and D) For phagocytic assays, IFN-γ-primed (20 ng/ml, overnight) and unprimed macrophage cultures were infected with P. brasiliensis yeasts at a macrophage/yeast ratio of 25:1. The cells were cocultivated for 4 h at 37°C under 5% CO₂ to allow adhesion and ingestion of fungi. Cells were washed, fixed, and stained with Giemsa stain; an average of 1,000 macrophages were analyzed, and the number of macrophages with adhered or ingested yeasts was determined. (B and E) For fungicidal assays, IFN-γ-primed and unprimed macrophages were infected with yeast cells as described for panel A. After 48 h at 37°C under 5% CO₂, plates were centrifuged, and supernatants were used to determine levels of nitrite and cytokines. The monolayers were washed with distilled water to lyse macrophages, and 100 μl of cell homogenates was assayed for the presence of viable yeasts by a CFU assay. (C and F) Supernatants from fungicidal assays were used to determine the levels of nitrites using the Griess reagent. Data are means ± standard errors of the means for quintuplicate samples from one experiment representative of three independent determinations. *, P < 0.05.

FIG. 2.

Macrophages (Mφ) from TLR4-defective mice secrete diminished levels of IL-12 and MCP-1. IFN-γ-treated (20.00 ng/ml) or untreated macrophages of TLR4-competent (C3H/HePas) and TLR4-defective (C3H/HeJ) mice were challenged with viable P. brasiliensis yeasts (fungus/macrophage ratio, 1:25) for 4 h, washed, and further cultivated for 48 h at 37°C under 5% CO₂. Plates were then centrifuged, and supernatants were used for cytokine measurements by ELISA. Data are means ± standard errors of the means for triplicate samples from one experiment representative of three independent determinations. *, P < 0.05.

FIG. 3.

In vivo, TLR4 dysfunction leads to less-severe fungal infection. (A) Recovery of CFU from the lungs of TLR4-defective and TLR4-normal control mice infected i.t. with 1 × 10⁶ yeasts. (B) Lung homogenates were used to determine the levels of nitrites using the Griess reagent. Data are means ± standard errors of the means for groups of six to eight mice at 96 h, 2 weeks, and 11 weeks after infection. The results are representative of three independent experiments. *, P < 0.05.

FIG. 4.

Increased numbers of activated macrophages, CD4⁺ T lymphocytes, and CD8⁺ T lymphocytes were detected in the lungs of TLR4-competent mice at weeks 2 and 11 of infection. Leukocyte subsets in the lung-infiltrating leukocytes (LIL) from TLR4-defective and TLR4-normal mice inoculated i.t. with 1 million P. brasiliensis yeast cells were characterized by flow cytometry. Lungs of C3H/HePas and C3H/HeJ mice (six to eight mice per group) were excised, washed in PBS, minced, and digested enzymatically. At weeks 2 and 11 after infection, lung cell suspensions were obtained and stained as described in Materials and Methods. The acquisition and analysis gates were restricted to lymphocytes or macrophages. The data are mean results from six to eight mice per group ± standard errors of the means and are representative of two independent experiments. *, P < 0.05.

FIG. 5.

TLR4-defective mice presented increased numbers of Treg cells in the lungs. FoxP3 expression by lung lymphocytes from TLR4-defective (C3H/HeJ) and normal (C3H/HePas) mice inoculated i.t. with 1 million P. brasiliensis yeast cells was determined by flow cytometric analysis. Lungs of six to eight mice per group were excised, washed in PBS, minced, and digested enzymatically; at 2 and 11 weeks after infection, cell suspensions were obtained and stained as described in Materials and Methods. The expression of leukocyte markers on the cell surface, as well as intracellular FoxP3 expression in lung-infiltrating leukocytes, was analyzed by flow cytometry. Surface staining of CD25⁺ cells and intracellular FoxP3 expression were backgated on the CD4⁺ T-cell population. The data are numbers of CD4⁺ CD25⁺ FoxP3⁺ cells for individual mice (five or six per group) and are representative of two independent experiments.

FIG. 6.

Lung homogenates of TLR4-competent mice presented increased levels of proinflammatory cytokines. At 96 h, 2 weeks, and 11 weeks after i.t. infection with 10⁶ P. brasiliensis yeast cells, lungs from TLR4-defective and TLR4-competent mice were collected and disrupted in 5.0 ml of PBS, and supernatants were analyzed for cytokine contents by capture ELISAs. Data are mean cytokine levels ± standard errors of the means (six to eight animals per group). The results are representative of three independent experiments. *, P < 0.05.

FIG. 7.

TLR4 deficiency determines impaired humoral immunity. Levels of P. brasiliensis-specific antibodies in the sera of TLR4-defective (C3H/HeJ) and normal (C3H/HePas) mice at week 11 after i.t. infection with 1 × 10⁶ yeast cells are shown. Sera were assayed for total IgG, IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 by using an isotype-specific ELISA as detailed in Materials and Methods. Data are mean serum titers (log₂) ± standard errors (six to eight mice per group). *, P < 0.05 for comparison with controls.

FIG. 8.

Compared with TLR4-normal mice, TLR4-defective mice present decreased fungal loads and lung pathology but equivalent survival times. (A) Survival times of TLR4-defective and control mice after i.t. infection with 1 × 10⁶ P. brasiliensis yeast cells were determined for a period of 250 days. No significant differences were seen between the median survival times of the two mouse strains. The results are representative of two independent experiments. (B) By 250 days after infection, survivor mice (three to six per group) were sacrificed, and CFU counts in tissues were determined. No viable yeasts were recovered from livers and spleens. (C to F) Photomicrographs of pulmonary lesions of TLR4-competent C3H/HePas mice (C and D) and TLR4-defective C3H/HeJ mice (E and F) at week 11 of infection with 1 million P. brasiliensis yeasts. At this period, the morphology of lesions was equivalent in the two mouse strains; fungal cells were surrounded by confluent or isolated granulomas scattered through the lung tissue. Lesions were stained with H&E (C and E) or with Grocott stain (D and F); magnification, ×100. (G) Total areas of lesions in the lungs of mice (n = 10) at week 11 after infection. *, P < 0.05.