Roles of Toll-Like Receptor 2 (TLR2), TLR4, and MyD88 during Pulmonary Coxiella burnetii Infection

Abstract

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular, primarily pulmonary, bacterial pathogen. Although much is known about adaptive immune responses against this bacterium, our understanding of innate immune responses against C. burnetii is not well defined, particularly within the target tissue for infection, the lung. Previous studies examined the roles of the innate immune system receptors Toll-like receptor 2 (TLR2) and TLR4 in peripheral infection models and described minimal phenotypes in specific gene deletion animals compared to those of their wild-type controls (S. Meghari et al., Ann N Y Acad Sci 1063:161-166, 2005,http://dx.doi.org/10.1196/annals.1355.025; A. Honstettre et al., J Immunol 172:3695-3703, 2004,http://dx.doi.org/10.4049/jimmunol.172.6.3695) . Here, we assessed the roles for TLR2, TLR4, and MyD88 in pulmonary C. burnetii infection and compared responses to those that occurred in TLR2- and TLR4-deficient animals following peripheral infection. As observed previously, neither TLR2 nor TLR4 was needed for limiting bacterial growth after peripheral infection. In contrast, TLR2 and, to a lesser extent, TLR4 limited growth (or dissemination) of the bacterium in the lung and spleen after pulmonary infection. TLR2, TLR4, and MyD88 were not required for the general inflammatory response in the lungs after pulmonary infection. However, MyD88 signaling was important for infection-induced morbidity. Finally, TLR2 expression on hematopoietic cells was most important for limiting bacterial growth in the lung. These results expand on our knowledge of the roles for TLR2 and TLR4 in C. burnetii infection and suggest various roles for these receptors that are dictated by the site of infection.

FIG 1

Weight loss and bacterial counts after peritoneal C. burnetii infection. TLR2 and TLR4 do not promote C. burnetii clearance after peritoneal infection. C57BL/6, TLR2^−/−, and TLR4^−/− (n = 5 per group) mice were injected i.p. with 10⁴ GE of phase I C. burnetii. (A) Mice were weighed daily, and results were recorded over 9 days. The graph represents average weights at each day postinfection, and error bars indicate standard errors of the means (SEM). Significance was determined by two-way ANOVA with Bonferroni's posttest. (B) C. burnetii burden in the heart, liver, and spleen was determined by immunohistochemistry. The graph represents pooled data, and error bars indicate SEM. Twenty images per slide were counted, and if total values exceeded 250, we were unable to distinguish differences with confidence. Therefore, when sample values were >250, we recorded them as such. Significance was determined by one-way ANOVA with Bonferroni's posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

FIG 2

Weight loss and tissue weights after pulmonary C. burnetii infection. C57BL/6, TLR2^−/−, TLR4^−/−, and MyD88^−/− (n = 5 per group) mice were left uninfected or were infected i.t. with 10³ or 10⁵ GE of phase I C. burnetii. (A) Mice were weighed daily, and results were recorded over 9 days. (B) On day 9, mice were euthanized and spleens and lungs were removed and weighed. The graph represents pooled data normalized to body weight, and error bars indicate SEM. Significance was determined by two-way ANOVA with Bonferroni's posttest. P values were <0.05 (*) and <0.001 (***) compared to PBS for each mouse strain. P values were <0.01 (##) and <0.001 (###) compared to C57BL/6 mice for each infectious dose.

FIG 3

Liver damage after pulmonary C. burnetii infection. Representative histology pictures of C57BL/6, TLR2^−/−, TLR4^−/−, and MyD88^−/− livers after i.t. infection with 10⁵ GE of phase I C. burnetii. Arrows indicate sites of necrosis, which are absent from MyD88^−/− livers. Images were taken at ×4 magnification.

FIG 4

Immune cell infiltration after pulmonary C. burnetii infection. Representative histology pictures of C57BL/6, TLR2^−/−, TLR4^−/−, and MyD88^−/− lungs after i.t. infection with 10⁵ GE of phase I C. burnetii. Arrows indicate recruited leukocytes observed around airways and blood vessels in all infected strains. Wild-type, TLR2^−/−, and TLR4^−/− infected tissues contain greater mononuclear cell infiltrate and loss of lacy architecture than MyD88^−/− infected tissue. Images were taken at ×4 magnification.

FIG 5

C. burnetii burden after 9 days of pulmonary infection. C57BL/6, TLR2^−/−, TLR4^−/−, and MyD88^−/− (n = 5 per group) mice were left uninfected or were infected i.t. with 10³ or 10⁵ GE of phase I C. burnetii. (A) Spleens and lungs were collected on day 9 postinfection, and C. burnetii-containing vacuoles were identified by immunohistochemistry and counted. Twenty images per slide were counted, and if total values exceeded 250, we were unable to distinguish differences with confidence. Therefore, when sample values were >250, we recorded them as such. (B) Spleens and lungs were collected on day 9 postinfection, and C. burnetii bacterial load was determined by PCR. (C) Image analysis of C. burnetii-stained area in immunohistochemistry images from mice infected i.t. with 10³ GE. Three representative images were used from three separate experiments. Error bars indicate standard deviations (SD). Significance between groups was determined by two-way Student's t test compared to the corresponding wild type (WT). *, P < <0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

C. burnetii burden in TLR2^−/− mice after 16 days of pulmonary infection. C57BL/6 and TLR2^−/− (n = 5 per group) mice were infected i.t. with 10³ or 10⁵ GE of phase I C. burnetii. Spleens and lungs were collected on day 16 postinfection, and the C. burnetii bacterial load was determined by PCR. Error bars indicate SD, and significance between groups was determined by two-way Student's t test compared to the corresponding wild type. *, P < 0.05; ***, P < 0.001.

FIG 7

C. burnetii burden in TLR4^−/− mice after 16 days of pulmonary infection. C57BL/6 and TLR4^−/− (n = 5 per group) mice were infected i.t. with 10³ or 10⁵ GE of phase I C. burnetii. Spleens and lungs were collected on day 16 postinfection, and the C. burnetii bacterial load was determined by PCR. Error bars indicate SD, and significance between groups was determined by two-way Student's t test compared to the corresponding wild type. *, P < 0.05.

FIG 8

C. burnetii burden in TLR2^−/− chimera mice. Chimera mice were infected i.t. with 10³ GE of phase I C. burnetii. Spleens and lungs were collected on day 9 postinfection, and the C. burnetii bacterial load was determined by PCR. Error bars indicate SD, and significance between groups was determined by two-way Student's t test compared to the corresponding wild type. *, P < 0.05.