Identification and characterization of GLDC and SPINK6 as host factors important for the pathogenesis of human influenza

Abstract

Influenza viruses (IAVs), including human IAVs and occasionally avian IAVs are the major cause of human respiratory infections. Human IAVs usually lead to upper respiratory infection with mild to moderate symptoms, and occasionally causing pneumonia with fatal outcome; whilst avian IAV infections are associated with more severe disease and higher fatality. We aim to understand the pathogenesis of influenza and identify the host susceptibility genes to severe influenza. We have previously performed two genome-wide association studies (GWASs) in the H7N9 cohort and the pandemic H1N1 cohort. We integrated GWAS discovery and expression quantitative trait loci (eQTL) datasets to prioritize two candidate genes, glycine decarboxylase (GLDC) and serine protease inhibitor Kazal-type 6 (SPINK6) that may impact the host susceptibility to severe influenza. Subsequently, I performed a series of in vitro and in vivo characterization to investigate the role of GLDC and SPINK6 in the pathogenesis of influenza. The integrative analysis suggested higher GLDC expression might confer a higher risk to H7N9 and severe H1N1 infections. I demonstrated that GLDC inhibitor AOAA and siRNA depletion boosted IFNβ and IFN stimulated genes (ISGs) in combination with Poly I:C stimulation. GLDC inhibition and depletion significantly amplified antiviral response of type I IFNs and ISGs upon viral infection and suppressed H1N1 and H7N9 replication. Consistently, GLDC overexpression significantly promoted viral replication due to the attenuated antiviral responses. Moreover, GLDC inhibition in H1N1-infected BALB/c mice led to amplified antiviral response and suppressed viral growth. AOAA provided potent protection to the infected mice from lethal infection, comparable to a standard antiviral against influenza viruses. Collectively, we conclude that GLDC regulates cellular antiviral response and orchestrates viral growth. SPINK6 was previously identified in human skin as a cellular inhibitor of serine proteases of KLK family. Serine proteases cleave hemagglutinin (HA) of influenza A viruses to initiate infection. The integrative analysis of GWAS and eQTL indicates that a genetic variant correlated to higher SPINK6 expression confers protection from human H7N9 infection. These prompted me to hypothesize that SPINK6 may inhibit airway serine proteases and restrict IAV activation. My results indicate that SPINK6 restricts HA cleavage and viral replication mediated by trypsin, a prototype chymotrypsin-like serine protease commonly used for cell culture-based propagation of most IAVs as a proxy of HA cleavage serine proteases. I demonstrate SPINK6 specifically suppresses the proteolytic activity of HAT and KLK5, HAT- and KLK5-mediated HA cleavage, virion maturation, and IAV replication. SPINK6 constrains activation of progeny virions and impairs viral growth in physiological-relevant human airway organoids where SPINK6 is intrinsically expressed; and vice versa, blockage of endogenous SPINK6 enhances HA cleavage and viral growth. In IAV-infected BALB/c mice, SPINK6 significantly suppresses viral growth and improves mouse survival. Therefore, we conclude that SPINK6 is a novel host inhibitor of airway serine proteases that restricts activation and replication of IAVs. Taken together, the integration of GWAS results and eQTLs datasets provide a robust platform to identify host susceptibility genes to severe influenza in a hypothesis-free manner and to elucidate the pathogenesis to severe influenza.