MyD88 deficiency results in tissue-specific changes in cytokine induction and inflammation in interleukin-18-independent mice infected with Borrelia burgdorferi

Abstract

Toll-like receptors (TLRs) play an important role in the control of infection with Borrelia burgdorferi. Deficiencies in TLR-2 or the shared TLR adapter molecule MyD88 have been shown to result in greatly increased bacterial burdens in mice. However, although in vitro studies have shown that the activation of TLR pathways by B. burgdorferi results in the release of inflammatory cytokines, studies in deficient mice have shown either no change or increased rather than decreased inflammation in infected animals. In this study, we looked at mechanisms to explain the increase in inflammation in the absence of MyD88. We found that MyD88-deficient mice infected with B. burgdorferi did not show increased inflammation at sites typically associated with Lyme disease (joints and heart). However, there was markedly increased inflammation in the muscles, kidneys, pancreas, and lungs of deficient animals. Muscle inflammation was typically seen perivascularly and perineuronally similar to that seen in infected humans. Chemotactic chemokines and cytokines were greatly increased in the muscle and kidneys of MyD88-deficient animals but not in the joints or heart tissue, suggesting that MyD88-independent pathways for recognizing B. burgdorferi and inducing these chemokines are present in the muscle and kidneys. Interleukin-18 signaling through MyD88 does not appear to play a role in either control of infection or inflammation.

FIG. 1.

MyD88 deficiency does not cause increased arthritis or carditis in B. burgdorferi-infected mice. MyD88^−/− and MyD88^+/+ mice were infected with B. burgdorferi as described in Materials and Methods and were sacrificed 2 weeks after infection. Representative HE-stained sections of joints (A to D) and heart (E and F) of both wild-type (A, B, and E) and MyD88^−/− (knockout) (C, D, and F) mice are shown. B and D are the magnified pictures of the boxed areas of A and B, respectively. The insets are the magnified views of the areas in the boxes.

FIG. 2.

MyD88 deficiency results in the B. burgdorferi infection-induced development of myositis, glomerulonephritis, alveolitis, and pancreatitis. MyD88^−/− and MyD88^+/+ mice were infected with B. burgdorferi as described in Materials and Methods and were sacrificed 3 weeks after infection. Representative HE-stained sections of muscle (A and B), kidney (C and D), lungs (E and F), and pancreas (G and H) of both MyD88^−/− mice (A, C, E, and G) and wild-type littermate MyD88 control mice (B, D, F, and H) are shown. The insets are the magnified views of the areas in the boxes.

FIG. 3.

B. burgdorferi-infected MyD88^−/− mice have higher bacterial loads in all inflamed organs. MyD88^−/− and MyD88^+/+ mice were infected with B. burgdorferi as described in Materials and Methods and sacrificed 3 weeks after infection. DNA was isolated from joint, muscle, and kidney tissues and processed to quantitate B. burgdorferi DNA levels using real-time RT-PCR for a single-copy B. burgdorferi gene, recA. Numbers represent copies of B. burgdorferi recA normalized to the weight of tissue used to isolate DNA. Ten mice each were used for studies of wild-type (WT) and MyD88^−/− (knockout [KO]) joints and muscle tissue; five mice each were used for studies of kidney tissue. ***P < 0.001; **P < 0.01.

FIG. 4.

MyD88 deficiency alters B. burgdorferi-induced proinflammatory cytokine expression patterns. MyD88^−/− (knockout [KO]) and MyD88^+/+ (wild-type [WT]) mice were injected with B. burgdorferi (Bb) or sham as described in Materials and Methods and sacrificed 2 weeks after infection. Total RNA was extracted from the muscle (A), kidney (B), and joint (cartilage) (C), and expression of IFN-γ, IL-10, TNF-α, IL-12p40, IL-12p35, and GM-CSF was examined by real-time RT-PCR. P values at the top of individual graphs are comparisons between infected wild-type and knockout mice. The scales on the y axis are different for each individual graph. ICAM-1, intercellular adhesion molecule 1.

FIG. 5.

MyD88 deficiency alters B. burgdorferi-induced proinflammatory chemokine expression patterns. MyD88^−/− (knockout [KO]) and MyD88^+/+ (wild-type [WT]) mice were injected with B. burgdorferi (Bb) or sham as described in Materials and Methods and sacrificed 3 weeks after infection. Total RNA was extracted from the muscle (A), kidney (B), and joint (cartilage) (C), and expression of CXCL-1, CXCL-2, MIP-1α, MIP-1β, MCP-1, and RANTES was examined by real-time RT-PCR. P values at the top of individual graphs represent comparisons between infected wild-type and knockout mice. The scales on the y axis are different for each individual graph.

FIG. 6.

B. burgdorferi-infected mice develop arthritis and carditis in the absence of IL-18. IL-18^−/− mice and their wild-type littermates (IL-18^+/+) were infected with B. burgdorferi as described in Materials and Methods and sacrificed 3 weeks after infection, and their joints and hearts were processed for either histology or DNA isolation. Representative HE-stained sections of joints (A to D) and heart (E and F) of both wild-type (A, B, and E) and IL-18^−/− (knockout) (C, D, and F) mice are shown. B and D are the magnified pictures of the boxed areas of A and B, respectively. The insets are the magnified views of the areas in the boxes. (G) DNA was isolated from joints of IL-18^−/− (n = 6) and wild-type (n = 5) mice and processed to quantitate B. burgdorferi DNA levels using real-time RT-PCR for a single-copy B. burgdorferi gene, recA. Numbers represent copies of B. burgdorferi recA normalized to the weight of tissue used to isolate DNA.