Differential involvement of LUBAC‐mediated linear ubiquitination in intestinal epithelial cells and macrophages during intestinal inflammation

Abstract

Disruption of the intestinal epithelial barrier and dysregulation of macrophages are major factors contributing to the pathogenesis of inflammatory bowel diseases (IBDs). Activation of NF-κB and cell death are involved in maintaining intestinal homeostasis in a cell type-dependent manner. Although both are regulated by linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination, the physiological relevance of linear ubiquitination to intestinal inflammation remains unexplored. Here, we used two experimental mouse models of IBD (intraperitoneal LPS and oral dextran sodium sulfate [DSS] administration) to examine the role of linear ubiquitination in intestinal epithelial cells (IECs) and macrophages during intestinal inflammation. We did this by deleting the linear ubiquitination activity of LUBAC specifically from IECs or macrophages. Upon LPS administration, loss of ligase activity in IECs induced mucosal inflammation and augmented IEC death. LPS-mediated death of LUBAC-defective IECs was triggered by TNF. IEC death was rescued by an anti-TNF antibody, and TNF (but not LPS) induced apoptosis of organoids derived from LUBAC-defective IECs. However, augmented TNF-mediated IEC death did not overtly affect the severity of colitis after DSS administration. By contrast, defective LUBAC ligase activity in macrophages ameliorated DSS-induced colitis by attenuating both infiltration of macrophages and expression of inflammatory cytokines. Decreased production of macrophage chemoattractant MCP-1/CCL2, as well as pro-inflammatory IL-6 and TNF, occurred through impaired activation of NF-κB and ERK via loss of ligase activity in macrophages. Taken together, these results indicate that both intraperitoneal LPS and oral DSS administrations are beneficial for evaluating epithelial integrity under inflammatory conditions, as well as macrophage functions in the event of an epithelial barrier breach. The data clarify the cell-specific roles of linear ubiquitination as a critical regulator of TNF-mediated epithelial integrity and macrophage pro-inflammatory responses during intestinal inflammation.

Systematic HOIP interactome profiling reveals critical roles of linear ubiquitination in tissue homeostasis

“…Interestingly, our study also demonstrated that Hoip deficiency in osteoblasts caused a reduction in bone mass and that this defect was caused by reduced linear ubiquitination of SMURF1. Although some articles have identified functions of HOIP mediated linear ubiquitination in Treg cells, B cells, liver parenchymal cells, macrophages, epithelial cells and endothelial cells 9 , 11 – 16 , our report describes a undefined role of linear ubiquitination in osteoblast cells.…”

“…Most previous studies have focused on the functional elucidation of linear ubiquitination in a single organ, thereby limiting the generalization and comparison of the findings to many other tissues simultaneously 9 , 11 – 16 . Our HOIP PPI analysis provided systematic insights into key roles of linear ubiquitination in physiological and pathological processes across tissues.…”

“…Deletion of epithelial HOIP during influenza A virus (IAV) infection worsens lung injury and decreases survival 15 . Defective HOIP catalytic activity in macrophages, but not in intestinal epithelial cells, ameliorates dextran sodium sulfate-induced colitis 16 . Moreover, bacteria and viruses always hijack linear ubiquitination within host cells to cause the disturbance of tissue homeostasis 1 .…”

OTULIN protects the intestinal epithelium from apoptosis during inflammation and infection

“…Similar to the phenotype of OTULIN IEC-KO mice, mice with intestinal epithelial deficiency in A20, a crucial ubiquitin-binding anti-inflammatory and cytoprotective protein that has been associated with IBD [ 40 , 41 ], do not develop spontaneous intestinal pathology, but are also hypersensitive to TNF and DSS [ 18 ] and to infection with Salmonella [ 38 ]. In addition, a recent study showed that mice that lack the C-terminal catalytic center of HOIP, abolishing LUBACs E3 ligase activity, specifically in IECs are also normal but sensitized to TNF and DSS, indicating that linear ubiquitination in IECs protects against intestinal inflammation by suppressing TNF-induced IEC apoptosis under inflammatory conditions [ 42 ]. Together, our study identifies OTULIN as a major antiapoptotic protein in the intestinal epithelium that ensures maintenance of epithelial barrier integrity in inflammatory conditions, and suggest that OTULIN defects may increase susceptibility to IBD.…”

Attenuation of HOIL-1L ligase activity promotes systemic autoimmune disorders by augmenting linear ubiquitin signaling

“…BMDMs were prepared as described previously ( 33 ). Briefly, bone marrow cells were isolated from the humerus, femurs, and tibias of mice of the indicated genotype.…”