Hyper-ISGylation promotes acute pulmonary pathology during chronic viral infection

ISG15 is one of the strongest type I interferon (IFN-I) stimulated genes (ISGs) which is conjugated to proteins in an enzymatic process termed ISGylation. Protein ISGylation is subsequently reversed by the enzyme Usp18 which acts in a de-ubiqutinase fashion to remove SIG15 from target proteins. Many proteins are ISGylated following IFN-I signaling however, immunological studies have been limited to the anti-viral properties of ISG15 conjugation while its regulation of innate and adaptive immune responses has been poorly studied. Here using Usp18−/− and enzymatically inactive (Usp18C61A/C61A Knock-in) mouse models, we show that hyper-ISGylation during chronic viral infection leads to severe immune pathology characterized by hematological disruptions, cytokine amplification, lung vascular leakage and death. The pathology associated with chronic infection required T cells but not T cell-intrinsic Usp18 expression. We reveal that lack of Usp18 in LysM+ cells reproduced the pathological manifestations observed in Usp18−/− or Usp18C61A/C61A mice. Using single cell sequencing, we observed that hyper-ISGylation during chronic infection altered neutrophil maturation and promoted an inflammatory neutrophil lung infiltrate. Importantly, neutrophil depletion reversed morbidity and mortality in Usp18C61A/C61A mice. In summary, we reveal Usp18’s enzymatic function is crucial for regulating inflammatory neutrophil differentiation and preventing immune hyperactivation during chronic viral infection. Moreover, our results suggest that hyper-ISGylation may drive the inflammatory pathology observed in Usp18-null patients and indicate that regulation of ISG15 expression or conjugation could lessen disease.