Live nontypeable Haemophilus influenzae exacerbates respiratory syncytial virus-associated lower airway inflammation

Abstract Background Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract disease in infants and immunocompromised adults, and secondary bacterial infections are recognized as important contributors to disease severity. Recent microbiome studies have shown that Haemophilus influenzae is frequently co-detected in severe RSV cases and ranks among the bacteria most closely correlated with disease severity; however, its causal role and underlying mechanisms remain unclear. Here, we used a murine model to examine the manner in which H. influenzae influences RSV-associated airway inflammation. Methods We established a murine respiratory infection model by infecting mice with RSV and subsequently intratracheally inoculating them with live, ultraviolet (UV)-killed, or heat-killed nontypeable H. influenzae (NTHi). Body weight, viral and bacterial loads, bronchoalveolar lavage fluid (BALF) cytokine levels, and immune cell populations were analyzed. Results Live NTHi inoculation following RSV infection induced severe lower airway inflammation, characterized by pronounced body weight loss and lung injury along with increased CD8 + T-cell accumulation and enhanced interleukin (IL)-6 inflammatory mediator production in BALF. Additionally, CD8 + T-cell depletion modestly attenuated body weight loss, altered the recruited innate immune cell composition, and reduced BALF IL-6 levels. However, RSV-infected mice inoculated with UV- or heat-killed NTHi exhibited CD8 + T-cell accumulation without developing body weight loss, which suggested that CD8 + T-cell accumulation alone may be insufficient to drive full disease manifestation. In contrast to that observed for non-viable NTHi, live NTHi induced robust neutrophil recruitment along with strong production of inflammatory mediators. Additionally, BALF IL-6 levels showed a strong negative correlation with body weight; hence, IL-6 was identified as a potential marker associated with disease severity. Conclusions This mouse model shows that pulmonary inoculation of live NTHi after RSV infection induces severe lower airway inflammation partially involving CD8 + T cells and that IL-6 is a potential biomarker associated with disease severity. Overall, this study suggests a novel pathological role for NTHi in the exacerbation mechanism of secondary bacterial infection in RSV infection.