CD40 restrains in vivo growth of Toxoplasma gondii independently of gamma interferon

Abstract

CD40-CD154 interaction is pivotal for resistance against numerous pathogens. However, it is not known if this pathway can also enhance in vivo resistance in gamma interferon (IFN-gamma)-deficient hosts. This is an important question because patients and mice with defects in type 1 cytokine response can control a variety of pathogens. While blockade of endogenous CD154 resulted in a remarkable increase in parasite load in IFN-gamma-/- mice infected with Toxoplasma gondii, in vivo administration of a stimulatory anti-CD40 monoclonal antibody markedly reduced parasite load. This latter effect took place even in T-cell-depleted mice and was accompanied by induction of macrophage toxoplasmacidal activity. CD40 stimulation restricted T. gondii replication independently of STAT1, p47 GTPases, and nitric oxide. In vivo CD40 ligation enhanced tumor necrosis factor alpha (TNF-alpha) production by T. gondii-infected macrophages. In addition, CD40 stimulation required the presence of TNF receptor 2 to reduce parasite load in vivo. These results suggest that CD40-CD154 interaction regulates IFN-gamma-independent mechanisms of host protection through induction of macrophage antimicrobial activity and modulation of TNF-alpha signaling.

FIG. 1.

Blockade of endogenous CD154 increases load of T. gondii in IFN-γ^−/− mice. Animals were infected with 1 × 10³ tachyzoites of the ts4 strain of T. gondii and were treated with either control or anti-CD154 MAb. Peritoneal cells were obtained 8 days postinfection to determine the percentage of infected cells, number of tachyzoites per 100 peritoneal cells, and total number of tachyzoites per peritoneal cavity. Experiments were repeated twice with similar results.

FIG. 2.

Induction of CD154 in response to T. gondii. Purified CD4⁺ T cells from IFN-γ^−/− mice were isolated 1 week after infection with 1 × 10³ tachyzoites of the ts4 strain of T. gondii. CD4⁺ T cells from uninfected mice were used as controls. CD4⁺ T cells were incubated with either uninfected or T. gondii-infected autologous bone marrow-derived macrophages. Expression of CD154 on CD4⁺ T cells was assessed by flow cytometry after 18 h of in vitro stimulation. Each dot represents an individual mouse.

FIG. 3.

CD40 stimulation reduces T. gondii load in vivo independently of T cells. IFN-γ^−/− mice infected with 4 × 10⁴ tachyzoites of the ts4 strain of T. gondii were treated with either a stimulatory anti-CD40 or control MAb (25 μg i.p.) daily beginning 1 day prior to infection. T cells were depleted in some mice by administration of anti-CD4 and anti-CD8 MAbs. Peritoneal cells were obtained 5 days postinfection to determine the percentage of infected cells, number of tachyzoites per 100 peritoneal cells, and total number of tachyzoites per peritoneal cavity. Experiments were repeated twice with similar results.

FIG. 4.

In vivo CD40 stimulation induces anti-T. gondii activity in macrophages. IFN-γ^−/− mice received 1 dose of either a stimulatory anti-CD40 or control MAb (25 μg i.p.). Peritoneal cells were collected after 24 h and plated on eight-chamber slides. After removal of nonadherent cells, macrophages were challenged with tachyzoites of the RH strain of T. gondii. The percentage of infected macrophages and number of tachyzoites per 100 macrophages were determined 1 and 18 h postinfection. Experiments were repeated twice with similar results.

FIG. 5.

CD40 stimulation reduces T. gondii load in vivo independently of STAT1. STAT1^−/− mice infected with 4 × 10⁴ tachyzoites of the ts4 strain of T. gondii were treated either with a stimulatory anti-CD40 (@CD40) or control MAb (25 μg i.p.) daily beginning 1 day prior to infection. Peritoneal cells were obtained 5 days postinfection to determine the total number of tachyzoites per peritoneal cavity. Experiments were repeated twice with similar results.

FIG. 6.

In vivo CD40 ligation increases TNF-α production in macrophages. IFN-γ^−/− mice received 1 dose of either a stimulatory anti-CD40 (@CD40) or control MAb (25 μg i.p.). Peritoneal cells were collected after 24 h and cultured in Teflon vessels with or without T. gondii-DsRed tachyzoites in the presence of brefeldin A. After 6 h, cells were stained with either FITC-conjugated F4/80 or isotype control MAb, followed by permeabilization and incubation with either anti-TNF-α-allophycocyanin or isotype control MAbs. Levels of expression of intracellular TNF-α in either uninfected or T. gondii-infected F4/80⁺ macrophages are expressed as corrected mean fluorescence intensity (cMFI). Peritoneal cells incubated with IFN-γ/LPS used as positive controls revealed an average cMFI for TNF-α of 709 in F4/80⁺ macrophages. Experiments were repeated twice with similar results.

FIG. 7.

TNFR2 is required for CD40-induced reduction of T. gondii parasite load. B6 or TNFR2^−/− mice were treated with a neutralizing anti-IFN-γ MAb. Mice were infected with 4 × 10⁴ tachyzoites of the ts4 strain of T. gondii and were treated with either a stimulatory anti-CD40 (@CD40) or control MAb. Peritoneal cells were obtained 5 days postinfection to determine the total number of tachyzoites per peritoneal cavity. Experiments were repeated twice with similar results.