LUBAC Mediated Epithelial Signaling During Influenza Infection

Introduction: Influenza A virus (IAV) infection is characterized by the rapid development of acute lung injury (ALI) with poor outcomes. As the primary target for IAV replication, alveolar epithelial cells (AEC) are important in mounting the initial response to infection, leading to recruitment of immune cells to the infection site to orchestrate host defenses. Dysregulated production of cytokines or “cytokine storm” is an important contributor to host mortality. The Linear Ubiquitin Chain Assembly Complex (LUBAC) has been identified as a mediator of NF-kB activation. LUBAC is composed of SHARPIN, HOIL-1L and HOIP. The role of LUBAC in regulating the immune response to influenza in the lung epithelium is unknown. Using a mouse model of IAV infection as well as in vitro approaches, we studied the role of LUBAC in regulating the epithelial-driven inflammatory response to IAV. Methods and Results: We found a significant survival benefit in SPCCre+HOIL-1Lfl/fl mice during IAV infection with a corresponding reduction in protein and recruited cells in bronchoalveolar lavage fluid (BALF) as compared to wild-type (WT) mice. Contributing to enhanced survival, we observe an altered profile of recruited immune cells to the lungs, lower viral load, and decreased levels of pro-inflammatory cytokines and interferon in BALF as compared to WT. IAV infection increased HOIL-1L expression in AEC which is independent of direct viral infection, as both infected and uninfected populations of AEC display increased HOIL-1L expression. IFN-alpha produced by infected cells is sufficient to upregulate HOIL-1L. Involvement of the interferon receptor (IFNAR1) signaling axis was confirmed by silencing IFNAR1 or the downstream transcription factor, IRF1, which prevented increased HOIL-1L expression during IAV. Additionally, we detected increased binding of IRF1 to the HOIL-1L promoter during IAV treatment in vitro. Silencing of either HOIL-1L or HOIP in AEC decreases LUBAC activity, as measured by linear ubiquitination of NEMO (NF-kB Essential Modulator), and activation of NF-kB and IRF pathways as determined by phosphorylation of IkB-alpha and IRF3 respectively. However, unlike SPCCre+HOIL-1Lfl/fl mice, SPCCre+HOIPfl/fl mice have reduced survival during a mild IAV infection, with increases in protein levels and number of recruited cells in BALF, and higher viral titers. Conclusions: Our results show that while the absence of HOIL-1L in AEC markedly improve survival, complete loss of defensive signaling from the epithelium due to loss of HOIP hinders recovery. Additionally IAV induced interferon signaling increases HOIL-1L expression and represents a maladaptation that contributes to enhanced LUBAC formation, increased inflammation and mortality. These findings suggest that the lung epithelium plays a major role regulating the amplitude of the host immune response to IAV-infection. This work is supported by HL-48129, HL71643, HL-76139, and HL-132454