Immune modulation via exposure to a primary stimulus can modify innate immune cell responses to a subsequent unrelated infection. Previously, we found that conserved mammalian proteins which display a pattern of repeating protein subunits (RPSP) enhance clearance of a secondary respiratory bacterial infection in a TLR2/1-dependent manner. RPSPs like filamentous actin or ferritin are expressed intracellularly in a steady-state, thus their detection by extracellular pattern recognition receptors should elicit danger-like signals. Unlike classical DAMPs, which prompt an inflammatory response, TLR2/1-based recognition of RPSPs did not result in proinflammatory cytokine production or neutrophil lung infiltration. Yet, upon challenge with Staphylococcus aureus, RPSP inoculated mice exhibited a more robust inflammatory response and better bacterial clearance than control mice. This TLR2/1-based RPSP recognition was not limited to mammalian proteins. Enhanced S. aureus clearance was achieved with a synthetic peptide (Q11; QQKFQFQFEQQ) that organizes into RPSP fibers and is non-immunogenic. Further, we found that RPSP enhanced S. aureus clearance was dependent on non-classical Type I IFN signaling whereby IFNβ production was required, but absence of the IFNAR1 subunit of the Type I IFN receptor did not abrogate S. aureus clearance. In vivo depletion and bacterial clearance assays showed that macrophages were essential for this subsequent improved response, which persisted after resting cells for days post RPSP stimulation. Our results indicate that RPSP stimulation of macrophages enhances these cell’s abilities to respond to a secondary infection in an IFNβ dependent manner and has features suggestive of trained immunity.

Previously we found that conserved viral architecture, namely the repeating protein subunit pattern (RPSP) of virus capsids is recognized by the cell-surface receptor TLR2/6. This RPSP is common to most, if not all virus capsids (vc) potentially serving as a conserved mechanism of extracellular recognition for viral pathogen-associated molecular patterns (PAMPs). TLR2/6-dependent recognition of vcRPSP induced an antiviral state that resulted in recruitment of immune cells and induction of type I IFN which we found to be required for reduced susceptibility to subsequent bacterial infections (BSI) in murine model of respiratory S. aureus infections. The improved BSI clearance after RPSP recognition was also seen in vcRPSP-treated PBMC-derived human macrophages, indicating the RPSP response is likely similar in humans. TLR2 is unique in that it recognizes an array of PAMPs, signals from the cell surface or endosome (proinflammatory or type I IFN response, respectively), and interacts with co-receptors, including CD14. Indeed, using bone marrow macrophages (BMMs) deficient in CD14, we found that CD14 was required for improved BSI clearance post-vcRPSP recognition. Interestingly, while blocking endosome maturation and acidification did not reduce RPSP-dependent improved clearance of BSI, using knockout BMMs we also found that MyD88, a canonical cell surface TLR signaling molecule, but also TRAM that is associated with endosomal signaling, were both required for improved BSI clearance post-vcRPSP recognition. This indicates that CD14, and the TLR signaling adaptors, MyD88 and TRAM play a role in the recognition and response to RPSP for a TLR2/6-vcRPSP mediated antiviral response but that endosome is not involved in the response.