Gut commensal bacteria direct a protective immune response against Toxoplasma gondii

Abstract

Toxoplasma gondii is a universally distributed pathogen that infects over one billion people worldwide. Host resistance to this protozoan parasite depends on a Th1 immune response with potent production of the cytokines interleukin-12 and interferon gamma. Although Toll-like receptor 11 (TLR11) plays a major role in controlling Th1 immunity to this pathogen in mice, this innate immune receptor is nonfunctional in humans, and the mechanisms of TLR11-independent sensing of T. gondii remain elusive. Here, we show that oral infection by T. gondii triggers a TLR11-independent but MyD88-dependent Th1 response that is impaired in TLR2xTLR4 double knockout and TLR9 single knockout mice. These mucosal innate and adaptive immune responses to T. gondii rely on the indirect stimulation of dendritic cells by normal gut microflora. Thus, our results reveal that gut commensal bacteria can serve as molecular adjuvants during parasitic infection, providing indirect immunostimulation that protects against T. gondii in the absence of TLR11.

Figure 1

TLR11 is essential for in vivo DC-IL-12 responses to T. gondii during systemic infection, but is dispensable for mucosal immune responses to the parasite. (A) WT, TLR11-/-, and MyD88–/– mice (five animals per group) were infected orally or intraperitoneally (IP) with an average of 20 T. gondii ME49 strain cysts per mouse. Serum IL-12p40 responses were measured 5 days later by ELISA. The data shown are the mean ± SD; the results are representative of four independent experiments. (B) Visualization of IL-12p40-producing cells during oral infection with T. gondii. WT IL-12p40-YFP reporter animals (Yet40) or TLR11-/-×Yet40 mice were infected orally with T. gondii, as described above. Small intestines were removed for microscopic analysis on day 5 post-infection to visualize IL-12p40-producing cells. Images were acquired with a Leica SPE with a 63× objective. The experiment shown is representative of the twelve performed. (C) DCs are the major IL-12-producing cells in WT and TLR11-/- mice. The different mucosal cell populations in the lamina propria (LP) of the small intestine and in the mLNs were examined for their ability to produce IL-12 in response to T. gondii. WT Yet40 and TLR11-/-×Yet40 mice were infected orally with T. gondii, as described above. On day 5 post-infection, small intestine and mLN single cell suspensions were prepared, stained with antibodies to I-A^b, CD11c, CD11b, and CD8α cell surface markers, and analyzed using flow cytometry. The data shown are the mean ± SD; the results are representative of four independent experiments, each involving four or five animals per group. * P< 0.05; ** P< 0.01.

Figure 2

Natural peroral route infection by T. gondii results in TLR11-independent Th1 responses to the parasite. (A) Recall response to STAg in WT, TLR11-/-, and MyD88-/- mice during oral infection with T. gondii. mLNs were harvested from WT, TLR11-/-, and MyD88-/- mice on day 7 post infection and restimulated with STAg (10 ug/ml) alone or in the presence of blocking anti-CD4 mAb or anti-CD8 mAb (20 ug/ml) for 48 hours. IFN-γ levels were quantified by ELISA. (B) Single-cell analysis of IFN-γ secretion by CD4+ T lymphocytes revealed that TLR11 regulates the Th1 response to T. gondii during systemic, but not oral, infection. WT, TLR11-/-, and MyD88–/– mice were infected orally or intraperitoneally (IP) with an average of 20 T. gondii ME49 strain cysts per mouse, and IFN-γ production by CD4+ T cells was analyzed on day 7 post-infection. (C) TLR2, -4, and -9 play important roles in regulating IFN-γ responses during T. gondii peroral infection. The indicated animals were infected as described above and the percentage of IFN-γ+ cells was analyzed and quantified using flow cytometry. The data shown are the mean ± SD; the results are representative of four independent experiments performed, each involving three animals per group. * P< 0.05; ** P< 0.01.

Figure 3

Commensal bacteria activate IL-12 production by DCs following oral T. gondii infection of TLR11-deficient mice. (A) Sensing of T. gondii in vitro by lamina propria DCs depends upon TLR11. WT and TLR11-/- LP DCs were prepared from small intestines and incubated with media alone, T. gondii profilin (TgPRF, 1 ug/ml), or live parasites (ME49 strain, 1:1 ratio). After 20 hr, cell culture supernatants were harvested and IL-12 production was analyzed by ELISA. (B) WT DCs were stimulated with conditional T. gondii null mutants for profilin (ΔTgPRFe/TgPRFi +ATc) for 20 hr and IL-12 responses were analyzed, as described above. (C) The DC IL-12 response to T. gondii in vitro does not require TLR2, TLR4, or TLR9. LP DCs were prepared from TLR2, TLR4, TLR2×4, TLR9, and TLR11–deficient animals and stimulated with live T. gondii (ME49) for 20 hr. Analysis of cell culture supernatants revealed that only TLR11 is required for DC IL-12 responses to T. gondii in vitro. (D) Commensal bacteria are responsible for TLR11-independent IL-12 production by DCs in vivo during T. gondii infection. Gut commensal microflora in WT and TLR11-/- mice were depleted by antibiotic treatment prior to T. gondii infection. DC IL-12 responses from control and antibiotic-treated mice were analyzed by intracellular staining on day 3 post-T. gondii infection. (E) IL-12 secretion from WT and TLR11-/- mLN cultures on day 3 after oral infection with the parasite demonstrated that antibiotic treatment reduced the production of this cytokine in WT animals and abolished IL-12 secretion in gut commensal microflora-depleted TLR11-/- mice. The data shown are the mean ± SD. The results are representative of three experiments performed, each involving at least four animals per group. * P< 0.05; ** P< 0.01.

Figure 4

Lack of TLR11 prevents the development of T. gondii-initiated acute ileitis and liver damage. (A) WT, TLR11-/-, MyD88-/-, TLR2×4-/-, and TLR9-/- mice (five animals per group) were infected orally with an average of 20 T. gondii ME49 strain cysts per mouse, and the immunopathology of the small intestines and liver tissues was analyzed on day 7 post-infection. The results are representative of five experiments performed, each involving at least four animals per group