Upon host infection with IAV, alveolar macrophages are an important source of cytokines and chemokines, attracting innate immune cells to the lung during the primary phase of infection. To test whether A20 directly controls IAV-induced gene expression in alveolar macrophages, we isolated these cells from lungs of A20myel-KO and control littermates and infected them in vitro with IAV X-47. Expression and secretion of the proinflammatory cytokines IL-6 and TNF, the type-I IFN IFNβ and IFNα4 and the chemokines MCP-1 (ccl2) and KC (cxcl1) was significantly higher in IAV infected cells lacking A20 compared to infected wild type cells (figure 2C and figure S1). Taken together these results demonstrate that A20 negatively regulates IAV-induced proinflammatory and antiviral gene expression in alveolar macrophages, consistent with the inhibitory effect of A20 seen on RIG-I-induced NF-κB and IRF3 activation.