Influenza D viruses (IDV) are known to co-circulate with viral and bacterial pathogens in cattle and other ruminants. Currently, there is limited knowledge regarding host responses to IDV infection and whether IDV infection affects host susceptibility to secondary bacterial infections. To begin to address this gap in knowledge, the current study utilized a combination of in vivo and in vitro approaches to evaluate host cellular responses against primary IDV infection and secondary bacterial infection with Staphylococcus aureus (S. aureus). Primary IDV infection in mice did not result in clinical signs of disease and it did not enhance the susceptibility to secondary S. aureus infection. Rather, IDV infection appeared to protect mice from the usual clinical features of secondary bacterial infection, as demonstrated by improved weight loss, survival, and recovery when compared to S. aureus infection alone. We found a notable increase in IFN-β expression following IDV infection while utilizing human alveolar epithelial A549 cells to analyze early anti-viral responses to IDV infection. These results demonstrate for the first time that IDV infection does not increase the susceptibility to secondary bacterial infection with S. aureus, with evidence that anti-viral immune responses during IDV infection might protect the host against these potentially deadly outcomes.

Early innate viral recognition by the host is critical for the rapid response and subsequent clearance of an infection. Innate immune cells patrol sites of infection to detect and respond to invading microorganisms including viruses. Surface Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs) that can be activated by viruses even before the host cell becomes infected. However, the early activation of surface TLRs by viruses can lead to viral clearance by the host or promote pathogenesis. Thus, a plethora of research has attempted to identify specific viral ligands that bind to surface TLRs and mediate progression of viral infection. Herein, we will discuss the past two decades of research that have identified specific viral proteins recognized by cell surface-associated TLRs, how these viral proteins and host surface TLR interactions affect the host inflammatory response and outcome of infection, and address why controversy remains regarding host surface TLR recognition of viral proteins.

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.

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Respiratory virus infections can either reduce or increase host susceptibility to subsequent secondary bacterial infections (SBI). Recently, we discovered that the anti-viral immune response initiated by the recognition of exogenous pattern of virus architectures, namely the repeating protein subunit pattern (RPSP), results in improved clearance of S. aureus during SBI in mice. The RPSP is likely conserved in all viruses, suggesting a generalized mode of viral pattern recognition not specific to any single virus. Thus, we identified RPSP as a new PAMP and found it to be recognized by the TLR2/6 heterodimer on the surface of macrophages. We found that this RPSP recognition occurs prior to particle internalization and independent of virus infection. TLRs are known to signal from either the cell surface, through MyD88/Mal, or from the endosome, through TRAM/TRIF, for the induction of inflammatory responses or type I IFNs, respectively. We found that when compared to WT macrophages, tram−/− macrophages exhibited diminished RPSP internalization by confocal microscopy and flow cytometry. However, blocking endosomal acidification by Bafilomycin A1 did not reduce S. aureus killing by RPSP-treated macrophages. This suggests that a non-canonical endosomal signaling pathway may be induced upon RPSP exposure. Indeed, using knockout mice we found that type I IFN, but also MyD88 signaling are critical for improved S. aureus clearance post RPSP. Our results indicate that the recognition of RPSP by TLR2/6 activates a unique intracellular signaling pathway, specifically a combination of the two known pathways, that results in type I IFN-dependent improvement in S. aureus killing by macrophages.

Clinical evidence suggest that the newly emerging Influenza D viruses (IDV) co-circulate in humans with influenza A viruses (IAV). However, how IDV contributes to human disease is currently not known. Because IAV is known to create a suppressive environment permissive to development of life threatening secondary bacterial infection (SBI), it is important to understand whether IDV affects the host immune response to IAV and to SBIs. Using a combination of in vivo and ex vivo approaches we evaluated host immune responses to both primary pulmonary IDV infection and subsequent SBI with Staphylococcus aureus in mice. Consistent with lack of disease symptoms in humans, we found that IDV infection did not induce clinical signs of disease in WT mice, evidenced by no weight loss or morbidity/mortality. Contrary to IAV that is known to increase susceptibility to SBI at least partially by inhibiting type I interferon (IFN) signaling, we found that IDV infection appeared to protect mice from the typical clinical features of SBI, as demonstrated by improved weight loss, survival (100% vs 60%), and recovery when compared to S. aureus-infection alone. Consistent with our previous report of IFN-beta reducing SBI susceptibility, we found that IDV induced production of IFN-b. We also found that IDV infection resulted in increased weight loss and morbidity in mice deficient in type I IFN receptor subunit 2 (Ifnar2−/−), but not in Ifnar1−/− mice suggesting a role for IFN-b signaling through Ifnar2 in IDV anti-viral immunity. Thus, our results demonstrate for the first time that IDV infection does not increase the susceptibility to SBI with S. aureus in mice, with evidence that anti-IDV immune responses might protect the host from these potentially deadly SBIs.

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.