This study investigates how specific members of the lung microbiome influence the early immune response to infection. Prevotella species are a major component of the endogenous airway microbiota. Increased abundance of Prevotella melaninogenica correlates with reduced infection with the bacterial pathogen Streptococcus pneumoniae, indicating a potentially beneficial role. Here, we show that P. melaninogenica enhances protection against S. pneumoniae, resulting in rapid pathogen clearance from the lung and improved survival in a mouse lung co-infection model. This response requires recognition of P. melaninogenica lipoproteins by toll-like receptor (TLR)2, the induction of TNFα, and neutrophils, as the loss of any of these factors abrogates Prevotella-induced protection. Improved clearance of S. pneumoniae is associated with increased serine protease-mediated killing by lung neutrophils and restraint of P. melaninogenica-induced inflammation by IL-10 in co-infected mice. Together, these findings highlight innate immune priming by airway Prevotella as an important protective feature in the respiratory tract.

The stability and composition of the airway microbiome is an important determinant of respiratory health. Some airway bacteria are considered to be beneficial due to their potential to impede the acquisition and persistence of opportunistic bacterial pathogens such as Streptococcus pneumoniae. Among such organisms, the presence of Corynebacterium species correlates with reduced S. pneumoniae in both adults and children, in whom Corynebacterium abundance is predictive of S. pneumoniae infection risk. Previously, Corynebacterium accolens was shown to express a lipase which cleaves host lipids, resulting in the production of fatty acids that inhibit growth of S. pneumoniae in vitro. However, it was unclear whether this mechanism contributes to Corynebacterium-S. pneumoniae interactions in vivo. To address this question, we developed a mouse model for Corynebacterium colonization in which colonization with either C. accolens or another species, Corynebacterium amycolatum, significantly reduced S. pneumoniae acquisition in the upper airway and infection in the lung. Moreover, the lungs of co-infected mice had reduced pro-inflammatory cytokines and inflammatory myeloid cells, indicating resolution of infection-associated inflammation. The inhibitory effect of C. accolens on S. pneumoniae in vivo was mediated by lipase-dependent and independent effects, indicating that both this and other bacterial factors contribute to Corynebacterium-mediated protection in the airway. We also identified a previously uncharacterized bacterial lipase in C. amycolatum that is required for inhibition of S. pneumoniae growth in vitro. Together, these findings demonstrate the protective potential of airway Corynebacterium species and establish a new model for investigating the impact of commensal microbiota, such as Corynebacterium, on maintaining respiratory health.

Repair program(s) in "alternatively activated" macrophages can help with short-term wound responses after sterile damage events. Clot resolution by fibrinolysis and phagocytosis by tissue macrophages can activate anti-inflammatory and pro-fibrotic signals like Transforming Growth Factor- β. It has been hypothesized by the late Andrew Tager and Rachel Chambers that some forms of human pulmonary fibrosis may involve chronic alveolar damage, leakage of blood components, clots, and subsequent pro-fibrotic events that then fail to remodel back to normal alveolar structure. We found evidence of chronic bleeds and hemosiderin-positive macrophages in the lungs of Platelet Endothelial Cell Adhesion Molecule-1 deficient mice that developed a progressive and fatal pulmonary fibrosis. We are testing this hypothesis in vitro using murine broncho-alveolar macrophages exposed to aged clots. Interestingly, fresh blood and clots were not efficiently phagocytosed by the murine myeloid leukemia cell line RAW264 nor by broncho-alveolar macrophages. Clots needed to be aged for two days first. Ingestion also causes the macrophages to become fluorescent, which is a useful way to assess phagocytosis. We are now developing mRNA sequencing protocols to assess the gene programs of macrophages over time.

Commensal (non-pathogenic) bacteria play an important role in protecting against disease. In both humans and animal models, the depletion of commensal bacteria by antibiotics increases susceptibility to pathogen infection of the lung. However, the specific commensal bacteria which contribute to this protective effect remain undefined. In the respiratory tract, Prevotella species are frequently associated with health rather than disease, indicating a potentially beneficial role. Specifically, some Prevotella species negatively correlate with infection with the bacterial pathogen Streptococcus pneumoniae, the most common cause of bacterial pneumonia. We recapitulated this relationship in mice, where we find that exposure to an airway commensal Prevotella species improves rapid clearance of S. pneumoniae from the lung. The use of heat-killed Prevotella was sufficient for inducing protection against S. pneumoniae, which was lost in mice depleted of the pro-inflammatory cytokine TNF-α. These results suggest that Prevotella exposure primes an immune response that increases protection against S. pneumoniae infection in the lung. We further find that mouse neutrophils both secrete TNF-α and are improved in their ability to kill S. pneumoniae following Prevotella exposure. Together, these findings indicate that airway Prevotella species can enhance protection against S. pneumoniae lung infection by activating an innate immune response that increases bacterial clearance.