CD40 ligand is not essential for induction of type 1 cytokine responses or protective immunity after primary or secondary infection with histoplasma capsulatum

Abstract

The induction of type 1 immune responses (interleukin [IL]-12, interferon [IFN]-gamma) has been shown to be important in mediating protection against many intracellular infections including Histoplasma capsulatum. Costimulatory molecules such as CD40 ligand (CD40L) have been shown to be a central regulator of type 1 responses in vivo. To study the role of CD40L in mediating protection against infection with H. capsulatum, CD40L-deficient (CD40L-/-) and CD40L+/+ mice were infected with H. capsulatum and assessed for various parameters. After a lethal challenge of H. capsulatum, CD40L-/- mice were not substantially different from CD40L+/+ mice in terms of mortality, fungal burden, or production of IFN-gamma, IL-12, nitric oxide, or tumor necrosis factor alpha. Moreover, CD40L-/- mice treated with anti-IFN-gamma or anti-IL-12 at the time of infection had accelerated mortality, providing further evidence that IL-12 and IFN-gamma are produced in vivo in the absence of CD40L. In addition, CD40L-/- mice infected with a sublethal dose of H. capsulatum survived infection, whereas all mice infected with the same dose and treated with anti-IFN-gamma had accelerated mortality, demonstrating that IFN-gamma but not CD40L was essential for primary immunity to H. capsulatum infection. Interestingly, depletion of either CD4+ or CD8+ T cells resulted in accelerated mortality in CD40L-/- mice, suggesting a critical role for these cells in response to infection. Finally, CD40L-/- mice initially infected with a sublethal dose of H. capsulatum were protected from secondary infection with a lethal dose of H. capsulatum, demonstrating that CD40L is not required for the maintenance of memory immunity.

Figure 1

CD40L^−/− mice on a C57BL/6 background are more susceptible to infection with L. major but not H. capsulatum compared with CD40L^+/+ mice. (A) CD40L^−/− or CD40L^+/+ mice (6–10 mice/group) on a C57BL/6 background were injected intravenously with H. capsulatum yeast cells (6 × 10⁵) and followed for mortality. (B) In a parallel experiment, CD40L^−/− and CD40L^+/+ mice were challenged in the hind footpad with 10⁵ live L. major metacyclic promastigotes. Weekly footpad measurements represent the average footpad scores ± SEM. Similar results were noted in a second experiment.

Figure 2

CD40L^−/− mice are resistant to sublethal infection with H. capsulatum. CD40L^−/− or CD40L^+/+ mice (4–6 mice/group) on a C57BL/6 × 129 background were injected intravenously with varying doses of H. capsulatum yeast cells (6 × 10⁵, 6 × 10⁴, 6 × 10³) and followed for mortality. Results are combined from two individual experiments.

Figure 3

CD40L^−/− mice have similar mRNA expression for type 1 cytokines as CD40L^+/+ mice. mRNA was isolated from spleen cells combined from three individual spleens of mice at 7 and 20 d after primary infection with H. capsulatum. As a control, mRNA was prepared from uninfected mice at 7 d after infection. Cytokine mRNA was subsequently determined for IFN-γ, IL-12 p40, TNF-α, and NO by semiquantitative reverse transcription PCR as described in Materials and Methods. N.D., not done.

Figure 4

Neutralization of endogenous IFN-γ or IL-12 results in accelerated mortality in CD40L^−/− and CD40L^+/+ mice after infection with H. capsulatum. CD40L^−/− and CD40^+/+ (C57BL/6 × 129) mice (4–6 mice/group) were injected intravenously with H. capsulatum yeast cells (6 × 10⁵) and followed for mortality. Additional groups of mice were treated with anti–IFN-γ (1 mg) or anti–IL-12 (1 mg) at the time of reinfection. Results are combined from two independent experiments. To assess quantitative burden of H. capsulatum, mice were killed 7 d after infection, and spleen cells from individual mice (n = 3) were plated at various concentrations on agar plates as described in Materials and Methods. 1 wk later, colonies were counted as CFUs. Asterisk indicates that results are statistically different (P <0.001) from those of mice infected with H. capsulatum alone.

Figure 5

IFN-γ but not CD40L is required for protective immunity after sublethal infection to H. capsulatum. CD40L^−/− and CD40L^+/+ mice on a C57BL/6 × 129 or C57BL/6 background were infected intravenously with a sublethal dose of H. capsulatum yeast cells (6 × 10⁴) and treated with anti–IFN-γ (1 mg) at the time of infection. Mice were followed for mortality.

Figure 6

CD4 or CD8⁺ T cells are required for effective immunity to H. capsulatum infection in CD40L^−/− mice. In results combined from two independent experiments, CD40L^−/− and CD40L^+/+ mice (4–6 mice/ group) intravenously infected with H. capsulatum yeast cells (6 × 10⁵) were treated with anti-CD4 (1 mg) or anti-CD8 (1 mg) 3 d before, at the time of, and 5–7 days after infection and were followed for mortality. Quantitative burden of H. capsulatum was assessed as described in Fig. 4. Similar results were noted in a second experiment.

Figure 7

CD40L is not required for effective immunity after secondary infection with a lethal challenge of H. capsulatum. In results combined from two independent experiments, CD40L^−/− mice initially were infected intravenously with 6 × 10⁴ yeast cells and then reinfected (secondary infection) 3 wk later with H. capsulatum yeast cells (6 × 10⁵). As a control, CD40L^−/− mice were infected primarily with 6 × 10⁵ yeast cells at the same time mice were undergoing secondary infection.