Role for the CD28 molecule in the control of Coxiella burnetii infection

Abstract

Q fever is an infectious disease caused by Coxiella burnetii, an obligate intracellular bacterium that replicates in macrophages. As cell-mediated immune response to microbial pathogens requires signals mediated by T-cell receptors and costimulatory molecules such as CD28, we wondered if CD28 is involved in protection against C. burnetii infection. CD28-deficient (CD28-/-) mice were inoculated with C. burnetii by intraperitoneal and intravenous routes. With both wild-type and CD28-/- mice, C. burnetii organisms were detected exclusively in spleen and liver. The antibody response against C. burnetii was impaired in CD28-/- animals, but, surprisingly, the lack of CD28 decreased C. burnetii burden in the infected tissues, whatever the manner of inoculation of bacteria. The CD28 deficiency had no effect on either granuloma formation, which reflects cell-mediated immunity against C. burnetii, or the production of gamma interferon and tumor necrosis factor, two cytokines known to be involved in granuloma formation. On the other hand, the production of interleukin-10 (IL-10) by peritoneal macrophages was highly impaired in CD28-/- mice. The results suggest that CD28 initiates a signal that favors C. burnetii replication through the modulation of the IL-10 pathway.

FIG. 1.

C. burnetii and granulomas in tissues. Wild-type and CD28^−/− mice were infected i.p. with 5 × 10⁵ C. burnetii organisms and killed at day 7. (A) Bacteria were revealed by immunostaining of the spleen and the liver. (B) Granulomas were revealed by histochemical analysis of the spleen and the liver. Representative micrographs are shown.

FIG. 2.

C. burnetii loads for CD28^−/− and wt mice. (A to D) Wild-type and CD28^−/− mice were infected i.p. (A and B) or i.v. (C and D) with 5 × 10⁵ C. burnetii organisms and killed at different times. Bacteria were revealed by immunostaining and were numerated by image analysis. The results are expressed as the number of bacteria per square millimeter of spleen (A and C) and liver (B and D) and are the means ± SD from five mice/time point. *, P < 0.05; represents the difference between CD28^−/− mice and wt mice. (E and F) Wild-type and CD28^−/− mice were infected i.p. with increasing doses of C. burnetii organisms and killed at day 7. The bacterial DNA copy numbers in the spleen (E) and the liver (F) were determined by qPCR.

FIG.3.

Ab response in CD28^−/− and wt mice. (A to F) Wild-type and CD28^−/− mice were infected i.p. with 5 × 10⁵ C. burnetii organisms (primary infection) and reinfected after 30 days (secondary infection). Serum was collected at different times of infection or reinfection. The presence of IgM, IgG, and IgG subclasses directed against C. burnetii was assessed by immunofluorescence using inactivated C. burnetii. The results as titers are expressed as means ± SD from five mice/time point. (G) Wild-type and CD28^−/− mice were infected i.p. with increasing doses of C. burnetii organisms and killed at day 14. The presence of specific IgG was assessed by immunofluorescence. The results as titers are expressed as means ± SD from five mice.

FIG. 4.

Granuloma expression in CD28^−/− and wt mice. Wild-type (open bars) and CD28^−/− (filled bars) mice were infected i.p. (A and B) or i.v. (C and D) with 5 × 10⁵ C. burnetii organisms and killed at different times. Granulomas were revealed by histochemical analysis. Their numbers in the spleen (A and C) and the liver (B and D) were determined microscopically. The results are expressed as the number of granulomas per square millimeter of tissue and are the means ± SD from five mice/time point.

FIG. 5.

Cytokine production in CD28^−/− and wt mice. Splenocytes (A) and peritoneal macrophages (B and C) from wt and CD28^−/− mice were isolated from uninfected (day 0) and C. burnetii-infected mice. Cells were incubated with inactivated C. burnetii (10:1 bacterium-to-cell ratio) for 24 h. The amounts of IFN-γ (A), TNF (B), and IL-10 (C) contained in cell supernatants were determined by immunoassays. Results are expressed as pg/ml and represent the means ± SD from three experiments.