Heme sequestration by hemophilin from Haemophilus haemolyticus reduces respiratory tract colonization and infection with non-typeable Haemophilus influenzae.

Iron acquisition is a key feature dictating the success of pathogen colonization and infection. Pathogens scavenging iron from the host must contend with other members of the microbiome similarly competing for the limited pool of bioavailable iron, often in the form of heme. In this study, we identify a beneficial role for the heme-binding protein hemophilin (Hpl) produced by the non-pathogenic bacterium Haemophilus haemolyticus against its close relative, the opportunistic respiratory tract pathogen non-typeable Haemophilus influenzae (NTHi). Using a mouse model, we found that pre-exposure to H. haemolyticus significantly reduced NTHi colonization of the upper airway and impaired NTHi infection of the lungs in an Hpl-dependent manner. Further, treatment with recombinant Hpl was sufficient to decrease airway burdens of NTHi without exacerbating lung immunopathology or systemic inflammation. Instead, mucosal production of the neutrophil chemokine CXCL2, lung myeloperoxidase, and serum pro-inflammatory cytokines IL-6 and TNFα were lower in Hpl-treated mice. Mechanistically, H. haemolyticus suppressed NTHi growth and adherence to human respiratory tract epithelial cells through the expression of Hpl, and recombinant Hpl could recapitulate these effects. Together, these findings indicate that heme sequestration by non-pathogenic, Hpl-producing H. haemolyticus is protective against NTHi colonization and infection. IMPORTANCE: The microbiome provides a critical layer of protection against infection with bacterial pathogens. This protection is accomplished through a variety of mechanisms, including interference with pathogen growth and adherence to host cells. In terms of immune defense, another way to prevent pathogens from establishing infections is by limiting the availability of nutrients, referred to as nutritional immunity. Restricting pathogen access to iron is a central component of this approach. Here, we uncovered an example where these two strategies intersect to impede infection with the respiratory tract bacterial pathogen Haemophilus influenzae. Specifically, we find that a non-pathogenic (commensal) bacterium closely related to H. influenzae called Haemophilus haemolyticus improves protection against H. influenzae by limiting the ability of this pathogen to access iron. These findings suggest that beneficial members of the microbiome improve protection against pathogen infection by effectively contributing to host nutritional immunity.

Neutrophil responsiveness to IL-10 impairs clearance of Streptococcus pneumoniae from the lungs.

The early immune response to bacterial pneumonia requires a careful balance between pathogen clearance and tissue damage. The anti-inflammatory cytokine interleukin (IL)-10 is critical for restraining otherwise lethal pulmonary inflammation. However, pathogen-induced IL-10 is associated with bacterial persistence in the lungs. In this study, we used mice with myeloid cell specific deletion of IL-10R to investigate the cellular targets of IL-10 immune suppression during infection with Streptococcus pneumoniae, the most common bacterial cause of pneumonia. Our findings suggest that IL-10 restricts the neutrophil response to S. pneumoniae, as neutrophil recruitment to the lungs was elevated in myeloid IL-10 receptor (IL-10R)-deficient mice and neutrophils in the lungs of these mice were more effective at killing S. pneumoniae. Improved killing of S. pneumoniae was associated with increased production of reactive oxygen species and serine protease activity in IL-10R-deficient neutrophils. Similarly, IL-10 suppressed the ability of human neutrophils to kill S. pneumoniae. Burdens of S. pneumoniae were lower in myeloid IL-10R-deficient mice compared with wild-type mice, and adoptive transfer of IL-10R-deficient neutrophils into wild-type mice significantly improved pathogen clearance. Despite the potential for neutrophils to contribute to tissue damage, lung pathology scores were similar between genotypes. This contrasts with total IL-10 deficiency, which is associated with increased immunopathology during S. pneumoniae infection. Together, these findings identify neutrophils as a critical target of S. pneumoniae-induced immune suppression and highlight myeloid IL-10R abrogation as a mechanism to selectively reduce pathogen burdens without exacerbating pulmonary damage.

Airway Prevotella promote TLR2-dependent neutrophil activation and rapid clearance of Streptococcus pneumoniae from the lung.

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.

Corynebacterium Species Inhibit Streptococcus pneumoniae Colonization and Infection of the Mouse Airway.

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.