Cardiac dysfunction is a common complication of severe influenza virus infection, but whether this occurs due to direct infection of cardiac tissue or indirectly through systemic lung inflammation remains unclear. To test the etiology of this aspect of influenza disease, we generated a novel recombinant heart-attenuated influenza virus via genome incorporation of target sequences for miRNAs expressed in cardiomyocytes. Compared with control virus, mice infected with miR-targeted virus had significantly reduced heart viral titers, confirming cardiac attenuation of viral replication. However, this virus was fully replicative in the lungs and induced similar systemic inflammation and weight loss compared to control virus. The miR-targeted virus induced fewer cardiac conduction irregularities and significantly less fibrosis in mice lacking interferon-induced transmembrane protein 3 (IFITM3), which serve as a model for influenza-associated cardiac pathology. We conclude that robust virus replication in the heart is required for pathology, even when lung inflammation is severe.

Influenza A virus activates cellular inflammasome pathways, though it remains unknown whether many of the effector proteins downstream of inflammasome activation promote virus clearance or instead promote pathological inflammation. We investigated the role of gasdermin D (GSDMD), a pore-forming inflammasome effector protein that allows cellular release of inflammatory molecules and eventual pyroptotic lysis of cells. GSDMD knockout (KO) mice infected with influenza virus exhibited reduced weight loss and mortality compared to infected wild type (WT) mice. Lung viral titers were similar between genotypes, indicating that GSDMD does not directly affect virus replication. Instead, we observed that GSDMD KO mice had less severe lung inflammation, histopathology, and immune cell infiltration, suggesting that GSDMD promotes tissue-damaging immune responses following infection. Global transcriptomic analysis revealed significant decreases in specific inflammatory gene programs in GSDMD KO lungs, including decreased neutrophil chemotaxis and activation gene signatures, which were confirmed by decreased neutrophil elastase measurements and decreased neutrophil numbers in the lung. Indeed, exogenous depletion of neutrophils starting at day 3 post infection in WT mice recapitulated the protective phenotype observed in GSDMD KO mice, implicating neutrophils as central players in the GSDMD-dependent pathological response to influenza virus. Overall, these findings reveal an important role for GSDMD during influenza virus-induced lung inflammation, pathogenesis, and neutrophil accumulation. Therapeutic interventions targeting the GSDMD/neutrophil axis may provide an effective means to treat severe influenza virus infection.

Cardiac dysfunction is a common extrapulmonary complication of severe influenza virus infection. Prevailing models propose that influenza-associated heart dysfunction is indirectly triggered by cytokine mediated cardiotoxicity downstream of the inflamed lung, rather than by direct infection of cardiac tissue. To test the etiology of cardiac dysfunction resulting from influenza virus infection, we generated a novel recombinant H1N1 influenza A virus that was attenuated in cardiomyocytes by incorporation of target sequences for miRNAs expressed specifically in that cell type (miR133b and miR206). Compared with control virus, mice infected with the miR-targeted virus had significantly reduced heart viral titers, confirming cardiac attenuation of viral replication. The miR-targeted virus, however, was fully replicative and inflammatory in lungs when compared to control virus, and induced similar systemic weight loss. The miR-targeted virus induced considerably lower levels of cardiac arrhythmia, fibrosis, and inflammation, compared with control virus, in mice lacking interferon induced transmembrane protein 3 (IFITM3), which serve as the only available model for severe influenza-associated cardiac pathology. We conclude that robust replication of virus in the heart is required for pathology even when lung inflammation is severe. Indeed, we show that human stem cell-derived cardiomyocytes are susceptible to influenza virus infection. This work establishes a fundamental new paradigm in which influenza virus damages the heart through direct infection of cardiomyocytes.