This is intriguing, since both TLRs and IL-1b converge on MyD88-dependent signaling pathways, which prompted our investigation into the functional impact of MyD88 during the course of S. aureus biofilm infection. Here we demonstrate that MyD88 influences the course of S. aureus catheter-associated biofilm infection. Specifically, bacterial burdens were significantly increased on indwelling catheters and surrounding tissues of MyD88 knockout compared to wild type mice during early stages of infection, which coincided with enhanced dissemination to the heart and kidney. The expression of several proinflammatory Ponatinib mediators was decreased in biofilm-infected tissues of MyD88 KO mice and immunofluorescence staining revealed an increased fibrotic response in MyD88 KO animals concomitant with enhanced recruitment of alternatively activated M2 macrophages. These studies advance our understanding of the hierarchy of MyD88-dependent pathways and, in the context of earlier work with TLR and IL-1b KO animals, suggest that MyD88 plays a dual role during biofilm infection. First, MyD88-dependent signals are responsible for early biofilm containment as typified by increased titers and infection dissemination in MyD88 KO animals. In addition, MyD88 normally induces M1 macrophage polarization, which is revealed by exaggerated M2 macrophage infiltrates and extensive fibrosis following MyD88 loss. Collectively, these findings indicate that the lack of MyD88 augments macrophage polarization towards an anti-inflammatory/pro-fibrotic M2 phenotype, which may impede bacterial clearance and contributes to biofilm persistence in vivo, in part, by enhancing the fibrotic encapsulation of biofilm infections. Biofilm infections are associated with a high morbidity, with surgical debridement or implant removal in conjunction with longterm antibiotic regimens representing the most successful therapies. Limited information is currently available regarding innate immune responses to S. aureus biofilms, which may prove critical for developing new therapeutic modalities for infection. To our knowledge, this study is the first to report the involvement of MyD88-dependent signals in orchestrating innate immune responses to S. aureus biofilm infections. This was an important issue to address in the context of recent evidence demonstrating that IL-1b, but not TLR2 or TLR9, affected S. aureus biofilm development. Because these molecules all converge on MyD88 for signal transduction, addressing the importance of MyD88 during biofilm growth may elucidate key pathways for novel biofilm therapeutics. In particular, bacterial burdens on catheters and surrounding tissues were significantly elevated in MyD88 KO mice during acute infection, which correlated with a failure in infection containment, as evidenced by increased dissemination to the kidney and heart at days 3 and 7 postinfection. Similar increases in bacterial burdens on infected pins and surrounding tissues in MyD88 KO mice were also observed using an orthopedic model of S. aureus biofilm infection. Interestingly, histological analysis revealed an exaggerated host fibrotic response surrounding infected catheters of MyD88 KO animals, which agreed with a bias towards alternatively activated M2 macrophages that are known to promote fibrosis. Together, these results indicate that signals emanating from MyD88 are involved in orchestrating several aspects of the host response to S. aureus biofilm infection, including bacterial containment, fibrosis, and M2 macrophage polarization. In the absence of MyD88, these responses become dysregulated leading to increased bacterial burdens and infection dissemination during acute infection.