Immunotherapy with nebulized pattern recognition receptor agonists restores severe immune paralysis and improves outcomes in mice with influenza-associated pulmonary aspergillosis

Influenza-associated pulmonary aspergillosis (IAPA) is a potentially deadly superinfection in patients with influenza pneumonia, especially those with severe disease, underlying immunosuppression, corticosteroid therapy, or requiring intensive care support. Given the high mortality of IAPA, adjunct immunomodulatory strategies remain a critical unmet need. Previously, the desensitization of pattern recognition pathways has been described as a hallmark of IAPA pathogenesis and a predictor of mortality in IAPA patients. Therefore, we studied the impact of nebulized Toll-like receptor 2/6/9 agonists Pam2 CSK4 (Pam2) and CpG oligodeoxynucleotides (ODNs) on infection outcomes and pulmonary immunopathology in a corticosteroid-immunosuppressed murine IAPA model. Mice with IAPA receiving mock therapy showed rapidly progressing disease and a paralyzed immune response to secondary <i>Aspergillus fumigatus</i> infection. Nebulized Pam2ODN was well tolerated and significantly prolonged event-free survival. Specifically, dual-dose Pam2ODN therapy before and after <i>A. fumigatus</i> infection led to 81% survival and full recovery of all survivors. Additionally, transcriptional analysis of lung tissue homogenates revealed induction of pattern recognition receptor signaling and several key effector cytokine pathways after Pam2ODN therapy. Moreover, transcriptional and flow cytometric analyses suggested increased frequencies of macrophages, natural killer cells, and T cells in the lungs of Pam2ODN-treated mice. Collectively, immunomodulatory treatment with nebulized Pam2ODN strongly improved morbidity and mortality outcomes and alleviated paralyzed antifungal immunity in an otherwise lethal IAPA model. These findings suggest that Pam2ODN might be a promising candidate for locally delivered immunomodulatory therapy to improve outcomes of virus-associated mold infections such as IAPA.IMPORTANCEThe COVID-19 pandemic has highlighted the significant healthcare burden, morbidity, and mortality caused by secondary fungal pneumonias. Given the heightened prevalence of severe viral pneumonias, such as influenza, and poor outcomes of secondary mold pneumonias, adjunct immunotherapies are needed to prevent and treat secondary infections. We herein demonstrate severely paralyzed immunity to secondary <i>Aspergillus fumigatus</i> infection in a corticosteroid-immunosuppressed mouse model of influenza-associated pulmonary aspergillosis (IAPA), partially due to dysregulated pathogen-sensing pathways. To overcome immune paralysis and IAPA progression, we used a dyad of nebulized immunomodulators (Toll-like receptor agonists). Nebulized immunotherapy significantly improved morbidity and mortality compared to mock therapy, increased frequencies of mature mononuclear phagocytes and natural killer cells in the lung, and stimulated antimicrobial signaling. Collectively, this proof-of-concept study demonstrates the feasibility and efficacy of locally delivered immunomodulatory therapy to alleviate virus-induced immune dysregulation in the lung and improve outcomes of post-viral mold pneumonias such as IAPA.