UFMylation inhibits the proinflammatory capacity of interferon-γ–activated macrophages

Abstract

Macrophages activated with interferon-γ (IFN-γ) in combination with other proinflammatory stimuli, such as lipopolysaccharide or tumor necrosis factor-α (TNF-α), respond with transcriptional and cellular changes that enhance clearance of intracellular pathogens at the risk of damaging tissues. IFN-γ effects must therefore be carefully balanced with inhibitory mechanisms to prevent immunopathology. We performed a genome-wide CRISPR knockout screen in a macrophage cell line to identify negative regulators of IFN-γ responses. We discovered an unexpected role of the ubiquitin-fold modifier (Ufm1) conjugation system (herein UFMylation) in inhibiting responses to IFN-γ and lipopolysaccharide. Enhanced IFN-γ activation in UFMylation-deficient cells resulted in increased transcriptional responses to IFN-γ in a manner dependent on endoplasmic reticulum stress responses involving Ern1 and Xbp1. Furthermore, UFMylation in myeloid cells is required for resistance to influenza infection in mice, indicating that this pathway modulates in vivo responses to infection. These findings provide a genetic roadmap for the regulation of responses to a key mediator of cellular immunity and identify a molecular link between the UFMylation pathway and immune responses.

Autophagy gene-dependent intracellular immunity triggered by interferon-γ

“…The ATG5-12-16L1 complex is crucial for IFNγ-induced control of MNoV CW3 in bone marrow-derived murine macrophages (11). Using Atg5 knockout BV-2 cells ( Atg5 -/- ) (26, 27) we confirmed that Atg5 was required for IFNγ-induced inhibition of MNoV CW3 replication (Fig. 1C).…”

“…Certain upstream components of the autophagy pathway are not required for IFNγ-induced inhibition of norovirus or T. gondii replication (10, 11, 15, 16). To confirm these observations in BV-2 cells, we determined whether IFNγ efficiently inhibits MNoV CW3 replication in clonal Atg14 -/- BV-2 cells (26, 27) and clonal Becn1 -/- cell lines (27). Neither Atg14 nor Becn1 were required for IFNγ-induced inhibition of MNoV CW3 replication (Fig.…”

“…After experimental selection (Fig. S2A), we quantified guides by DNA sequencing and determined the significance of differences in guide frequencies in different comparisons using STARS and negative binomial analysis (21, 26–28, 33) with a threshold of FDR < 0.1 to identify genes for further consideration (27).…”

“…Deficiency of IQGAP1 in human monocytic cells results in hyperactive type I IFN responses to cytosolic nucleotides (48). Ufc1 and Uba5 encode enzymes in the ubiquitin-like system that covalently conjugates UFM1 to target proteins (UFMylation herein) (26, 49–51). UFMylation is required to maintain ER homeostasis, so that in the absence of UFMylation consequent ER stress can lead to overactivation of IFNγ responses (26).…”

“…Ufc1 and Uba5 encode enzymes in the ubiquitin-like system that covalently conjugates UFM1 to target proteins (UFMylation herein) (26, 49–51). UFMylation is required to maintain ER homeostasis, so that in the absence of UFMylation consequent ER stress can lead to overactivation of IFNγ responses (26). We therefore quantified the effects of IFNγ on norovirus replication in cells lacking Ufc1 or Uba5 ( Ufc1 -/- , Uba5 -/- ) (26).…”