Biofilms are complex communities of bacteria encased in a matrix composed primarily of polysaccharides, extracellular DNA, and protein. Staphylococcus aureus (S. aureus) can form biofilm infections, which are often debilitating due to their chronicity and recalcitrance to antibiotic therapy. Currently, the immune mechanisms elicited during biofilm growth and their impact on bacterial clearance remains to be defined. We utilized a mouse model of catheter-associated biofilm infection to assess the functional importance of Toll-like receptors 2 and 9 in the host immune response during biofilm formation, since ligands for both receptors are present within the biofilm. Interestingly, neither receptor impacted bacterial density or inflammatory mediator secretion during biofilm growth in vivo, suggesting that S. aureus biofilms circumvent these traditional bacterial recognition pathways. Several potential mechanisms were identified to account for biofilm evasion of innate immunity, including significant reductions in IL-1β, TNF-α, CXCL2, and CCL2 expression during biofilm infection compared to the wound healing response elicited by sterile catheters, limited macrophage invasion into biofilms in vivo, and a skewing of the immune response away from a microbicidal phenotype as evidenced by decreases in iNOS expression concomitant with robust arginase-1 induction. Co-culture studies of macrophages with S. aureus biofilms in vitro revealed that macrophages successful at biofilm invasion displayed limited phagocytosis and gene expression patterns reminiscent of alternatively activated M2 macrophages. Collectively, these findings demonstrate that S. aureus biofilms are capable of attenuating traditional host proinflammatory responses, which may explain why biofilm infections persist in an immunocompetent host.

The discovery of mammalian Toll-like receptors (TLRs), first identified in 1997 based on their homology with Drosophila Toll, greatly altered our understanding of how the innate immune system recognizes and responds to diverse microbial pathogens. TLRs are evolutionarily conserved type I transmembrane proteins expressed in both immune and non-immune cells and are typified by N-terminal leucine-rich repeats and a highly conserved C-terminal domain termed the Toll/interleukin (IL)-1 receptor (TIR) domain. Upon stimulation with their cognate ligands, TLR signaling elicits the production of cytokines, enzymes, and other inflammatory mediators that can impact several aspects of central nervous system (CNS) homeostasis and pathology. For example, TLR signaling plays a crucial role in initiating host defense responses during CNS microbial infection. Furthermore, TLRs are targets for many adjuvants which help shape pathogen-specific adaptive immune responses in addition to triggering innate immunity. Our knowledge of TLR expression and function in the CNS has greatly expanded over the last decade, with new data revealing that TLRs also impact non-infectious CNS diseases/injury. In particular, TLRs recognize a number of endogenous molecules liberated from damaged tissues and, as such, influence inflammatory responses during tissue injury and autoimmunity. Also, recent studies have implicated TLR involvement during neurogenesis and learning and memory in the absence of any underlying infectious etiology. Due to their presence and immune regulatory role within the brain, TLRs represent an attractive therapeutic target for numerous CNS disorders and infectious diseases. However, it is clear that TLRs can exert either beneficial or detrimental effects in the CNS, which likely depend on the context of tissue homeostasis or pathology. Therefore, any potential therapeutic manipulation of TLRs will require an understanding of the signals governing specific CNS disorders to achieve tailored therapy.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature monocytes and granulocytes that are potent inhibitors of T cell activation. A role for MDSCs in bacterial infections has only recently emerged and nothing is known about MDSC function in the context of Staphylococcus aureus (S. aureus) infection. Since S. aureus biofilms are capable of subverting immune-mediated clearance, we examined whether MDSCs could play a role in this process. CD11b+Gr-1+ MDSCs represented the main cellular infiltrate during S. aureus orthopedic biofilm infection, accounting for over 75% of the CD45+ population. Biofilm-associated MDSCs inhibited T cell proliferation and cytokine production, which correlated with a paucity of T cell infiltrates at the infection site. Analysis of FACS-purified MDSCs recovered from S. aureus biofilms revealed increased Arg-1, iNOS, and IL-10 expression, key mediators of MDSC suppressive activity. Targeted depletion of MDSCs and neutrophils using the mAb 1A8 (anti-Ly6G) improved bacterial clearance by enhancing the intrinsic pro-inflammatory attributes of infiltrating monocytes and macrophages. Furthermore, the ability of monocytes/macrophages to promote biofilm clearance in the absence of MDSC action was revealed with RB6-C85 (anti-Gr-1 or anti-Ly6G/Ly6C) administration, which resulted in significantly increased S. aureus burdens both locally and in the periphery, since effector Ly-6C monocytes and by extension, mature macrophages, were also depleted. Collectively, these results are the first to demonstrate that MDSCs are key contributors to the chronicity of S. aureus biofilm infection, as their immunosuppressive function prevents monocyte/macrophage proinflammatory activity, which facilitates biofilm persistence.

Toll-like receptors (TLRs) are a family of pattern-recognition receptors expressed on cells of the innate immune system that allow for the recognition of conserved structural motifs on a wide array of pathogens, referred to as pathogen-associated molecular patterns, as well as some endogenous molecules. The recent emergence of studies examining TLRs in the central nervous system (CNS) indicates that these receptors not only play a role in innate immunity in response to infectious diseases but may also participate in CNS autoimmunity, neurodegeneration, and tissue injury. This review summarizes the experimental evidence demonstrating a role for TLRs in the context of CNS inflammation in both infectious and noninfectious conditions.

Astrocytes play an important role in initiating and regulating CNS immune responses through the release of proinflammatory cytokines and chemokines. Here we demonstrate that primary astrocytes are capable of recognizing the Grampositive bacterium Staphylococcus aureus and its cell wall product peptidoglycan (PGN) and respond by producing numerous proinflammatory mediators including interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), macrophage inflammatory protein-1b (MIP-1b), MIP-2, and monocyte chemoattractant protein (MCP-1). Astrocytes have recently been shown to express Toll-like receptor 2 (TLR2), a pattern recognition receptor important for recognizing structural components of various Gram-positive bacteria, fungi, and protozoa. However, the functional significance of TLR2 in mediating astrocyte activation remains unknown. Primary astrocytes from TLR2 knockout mice were used to evaluate the role of TLR2 in astrocyte responses to S. aureus and PGN. The results demonstrate that TLR2 is essential for maximal proinflammatory cytokine and chemokine production, but not phagocytosis, in primary astrocytes following S. aureus and PGN exposure. In addition, both stimuli led to a significant increase in TLR2 mRNA expression in wild-type astrocytes as assessed by real-time quantitative RT-PCR. These findings suggest that astrocytes may play a key role in the initial antibacterial immune response in the CNS through engagement of TLR2.