CCR5 deficiency mitigates the deleterious effects of tumor necrosis factor α antagonism in murine histoplasmosis

Abstract

In murine histoplasmosis, tumor necrosis factor α (TNF-α) antagonism increases the number of regulatory T cells (Tregs) in lungs, and these cells profoundly hinder protective immunity. Because CCR5 mediates Treg homing and proliferation, we determined the outcome of antagonizing TNF-α in CCR5(-/)(-) mice infected with Histoplasma capsulatum. The absence of CCR5 attenuated the severity of infection associated with TNF-α neutralization. Infected controls given anti-TNF-α had a 10-fold increase in the number of Tregs in lungs compared with a <2-fold increase in CCR5(-/)(-) lungs. This difference was partially attributable to impaired homing in the absence of CCR5. Neutralization of TNF-α-enhanced CCR5 ligands in wild-type lungs thus promotes a gradient between lungs and the thymus. This study elucidates the interplay between TNF-α and CCR5 in histoplasmosis. The data suggest that targeting CCR5 may improve host immunity in individuals receiving TNF-α antagonists during infection.

Figure 1.

CCR5^–/– mice display prolonged survival in the absence of tumor necrosis factor α (TNF-α). Wild-type (WT) and CCR5^–/– mice were given a monoclonal antibody to TNF-α, interferon γ (IFN-γ), or rat immunoglobulin G (IgG) and intranasally infected with 2 × 10⁶ yeasts to determine fungal burden and survival. Lungs were harvested at day 7 postinfection to determine colony-forming units (CFU) (A and C). For survival studies, animals were given anti–TNF-α, anti–IFN-γ, or rat IgG once a week and monitored for 40 days (B and D). Data represent the mean ± SEM (n = 6–12) from 2–3 experiments. *P < .05; **P < .01; ***P < .001.

Figure 2.

Tumor necrosis factor α (TNF-α) antagonism increases the number of regulatory T cells (Tregs) in the lungs. Wild-type (WT) and CCR5^–/– mice were given a monoclonal antibody to TNF-α or rat immunoglobulin G (IgG) and intranasally infected with 2 × 10⁶ yeasts. At day 7 postinfection, the proportion (A) and absolute number (B) of Tregs were characterized in the lungs by flow cytometry. Tregs were defined as CD4⁺ Foxp3⁺ T cells. Data represent the mean ± SEM (n = 8–12) from 2–3 experiments. **P < .01; ***P < .001.

Figure 3.

Neutralization of tumor necrosis factor α (TNF-α) enhances regulatory T-cell (Treg) proliferation in wild-type (WT) and CCR5^–/– lungs. WT and CCR5^–/– mice were given a monoclonal antibody to TNF-α or rat immunoglobulin G (IgG) and intranasally infected with 2 × 10⁶ yeasts. Mice were intraperitoneally given bromodeoxyuridine (BrdU) for 2 consecutive days before being killed. At day 7 postinfection, the percentage of Tregs (CD4⁺ Foxp3⁺ T cells) that incorporated BrdU was measured by flow cytometry (A). RNA was extracted from lung leukocytes at day 7 postinfection and interleukin 2 (IL-2) expression was measured by quantitative real-time polymerase chain reaction. Hypoxanthine-guanine phosphoribosyltransferase was used as an endogenous control, and values represent log increase compared with respective IgG controls (B). Data represent the mean ± SEM (n = 6–12) from 2–3 experiments. **P < .01; ***P < .001.

Figure 4.

Tumor necrosis factor α (TNF-α) antagonism alters thymic cellularity in wild-type (WT) and, to a lesser extent, CCR5^–/– mice during infection. Wild-type and CCR5^–/– mice were given a monoclonal antibody to TNF-α or rat immunoglobulin G (IgG) and intranasally infected with 2 × 10⁶ yeasts. At day 7 postinfection, thymi were isolated from mice to determine the total number of thymocytes (A) and number of CD4⁺ Foxp3⁺ cells (B). RNA was extracted from lung leukocytes at day 7 postinfection and CCR5, CCL3, CCL4, and CCL5 expression was measured by quantitative real-time polymerase chain reaction. Hypoxanthine-guanine phosphoribosyltransferase was used as an endogenous control, and values represent log increase compared with IgG control lungs (C). Data represent the mean ± SEM (n = 6–12) from 2–3 experiments. **P < .01; ***P < .001.

Figure 5.

Tumor necrosis factor α (TNF-α) signals via TNF receptor (TNFR) 1 to regulate regulatory T cell (Treg) emergence in infected lungs. Wild-type (WT), TNFR1^–/–, and TNFR2^–/– mice were intranasally infected with 2 × 10⁶ yeasts. Mice were intraperitoneally given bromodeoxyuridine (BrdU) for 2 consecutive days before being killed. At day 7 postinfection, the percentage of CD4⁺ cells that were Foxp3⁺ (A), the absolute number of CD4⁺ Foxp3⁺ cells (B), and the percentage of CD4⁺ Foxp3⁺ that incorporated BrdU (C) in the lungs were measured by flow cytometry. Data represent the mean ± SEM (n = 8–12) from 2–3 experiments. *P < .05; **P < .01; ***P < .001.