Barbara A. Malynn

A20, encoded by the TNFAIP3 gene, is a protein linked to Crohn’s disease and celiac disease in humans. We now find that mice expressing point mutations in A20’s M1-ubiquitin–binding zinc finger 7 (ZF7) motif spontaneously develop proximal enteritis that requires both luminal microbes and T cells. Cellular and transcriptomic profiling reveals expansion of Th17 cells and exuberant expression of IL-17A and IL-22 in intestinal lamina propria of A20 ^ZF7 mice. While deletion of IL-17A from A20 ^ZF7/ZF7 mice exacerbates enteritis, deletion of IL-22 abrogates intestinal epithelial cell hyperproliferation, barrier dysfunction, and alarmin expression. Colonization of adult germ-free mice with microbiota from adult WT specific pathogen–free mice drives duodenal IL-22 expression and duodenitis. A20 ^ZF7/ZF7 Th17 cells autonomously express more RORγt and IL-22 after differentiation in vitro. ATAC sequencing identified an enhancer region upstream of the Il22 gene, and this enhancer demonstrated increased activating histone acetylation coupled with exaggerated Il22 transcription in A20 ^ZF7/ZF7 T cells. Acute inhibition of RORγt normalized histone acetylation at this enhancer. Finally, CRISPR/Cas9–mediated ablation of A20 ^ZF7 in human T cells increases RORγt expression and IL22 transcription. These studies link A20’s M1-ubiquitin binding function with RORγt expression, expansion of Th17 cells, and epigenetic activation of IL-22–driven enteritis.

Polymorphisms in the TNFAIP3 locus encoding the A20 protein are strongly associated with psoriatic skin and joint disease. Reduced A20 expression, driven by both genetic and epigenetic factors, underscores its critical role as a negative regulator of psoriatic disease (PsD). Our recent study using a germline knockin mouse model harboring a mutation in A20’s seventh zinc finger, which impairs A20 binding to linear (M1) ubiquitin, revealed a spontaneous phenotype resembling psoriatic arthritis. These mice demonstrated sustained nuclear factor–κB signaling in response to transient tumor necrosis factor stimulation, leading to inappropriate transcription of mid- and late-response inflammatory genes. These findings highlight the role of dysregulated innate immune-signaling kinetics as a potential driver of PsD pathogenesis. These findings, together with distinct inflammatory mouse models resulting from temporally extended inflammatory gene activation, highlight the role of dysregulated innate immune-signaling kinetics as a potential driver of tissue inflammation with relevance to psoriatic skin and joint disease. This newly developed mouse model of PsD was presented at the Group for Research and Assessment of Psoriasis and Psoriatic Arthritis (GRAPPA) 2024 annual meeting.

A20, encoded by the TNFAIP3 gene, is a protein linked to Crohn’s disease and celiac disease in humans. We now find that mice expressing point mutations in A20’s M1 ubiquitin binding motif (ZF7) spontaneously develop proximate enteritis that requires both luminal microbes and T cells. Cellular and transcriptomic profiling reveal expansion of TH17/22 cells and aberrant expression of IL-17A and IL-22 in intestinal lamina propria of A20 ^ZF7 mice. While deletion of IL-17A from A20 ^ZF7/ZF7 mice exacerbates enteritis, deletion of IL-22 abrogates intestinal epithelial cell hyperproliferation, barrier dysfunction, and alarmin expression. A20 ^ZF7/ZF7 TH17/22 cells autonomously express more RORψt and IL-22 after differentiation in vitro . ATAC sequencing identified an enhancer region upstream of the Il22 gene in A20 ^ZF7/ZF7 T cells, and this enhancer demonstrated increased activating histone acetylation coupled with exaggerated Il22 transcription. Finally, CRISPR/Cas9-mediated ablation of A20 ^ZF7 in human T cells increases RORψt expression and IL22 transcription. These studies link A20’s M1 ubiquitin binding function with RORψt expression, epigenetic activation of TH17/22 cells, and IL-22 driven enteritis.

Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of 2 IBD susceptibility genes, A20 (Tnfaip3) and Abin-1 (Tnip1), in intestinal epithelial cells (IECs) sensitized mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion was rescued by germ-free derivation or deletion of MyD88, while deletion of Trif provided only partial protection. Combined deletion of Ripk3 and Casp8, which inhibits both apoptotic and necroptotic death, completely protected against death after acute deletion of A20 and Abin-1 in IECs. A20- and Abin-1–deficient IECs were sensitized to TNF-independent, TNFR1-mediated death in response to lymphotoxin α (LTα) homotrimers. Blockade of LTα in vivo reduced weight loss and improved survival when combined with partial deletion of MyD88. Biopsies of inflamed colon mucosa from patients with IBD exhibited increased LTA and IL1B expression, including a subset of patients with active colitis on anti-TNF therapy. These data show that microbial signals, MyD88, and LTα all contribute to TNF-independent intestinal injury.

Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of two IBD susceptibility genes, A20 ( Tnfaip3 ) and Abin-1 ( Tnip1 ), in intestinal epithelial cells (IECs) sensitizes mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion is rescued by germ-free derivation or deletion of MyD88 , while deletion of Trif provides only partial protection. Combined deletion of Ripk3 and Casp8 , which inhibits both apoptotic and necroptotic death, completely protects against death after acute deletion of A20 and Abin-1 in IECs. A20 and Abin-1 -deficient IECs are sensitized to TNF-independent, TNFR-1-mediated death in response to lymphotoxin alpha (LTα) homotrimers. Blockade of LTα in vivo reduces weight loss and improves survival when combined with partial deletion of MyD88 . These data show that microbial signals, MyD88 , and LTα all contribute to TNF-independent intestinal injury. SUMMARY: Here we show that germ-free derivation, MyD88 deletion, combined Ripk3 and Casp8 deletion, or anti-LTα, all reduce TNF-independent intestinal injury after A20 and Abin-1 deletion.

A20 is an anti-inflammatory protein that is strongly linked to human disease. Here, we find that mice expressing three distinct targeted mutations of A20’s zinc finger 7 (ZF7) ubiquitin-binding motif uniformly developed digit arthritis with features common to psoriatic arthritis, while mice expressing point mutations in A20’s OTU or ZF4 motifs did not exhibit this phenotype. Arthritis in A20^ZF7 mice required T cells and MyD88, was exquisitely sensitive to tumor necrosis factor and interleukin-17A, and persisted in germ-free conditions. A20^ZF7 cells exhibited prolonged IκB kinase activity that drove exaggerated transcription of late-phase nuclear factor-κB response genes in vitro and in prediseased mouse paws in vivo. In addition, mice expressing double-mutant A20 proteins in A20’s ZF4 and ZF7 motifs died perinatally with multi-organ inflammation. Therefore, A20’s ZF4 and ZF7 motifs synergistically prevent inflammatory disease in a non-catalytic manner.

OTUB1 is a deubiquitinating enzyme that cleaves K48-linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, non-catalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin conjugating enzymes and inhibits their activity by trapping the E2~ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to members of the UBE2D (UBCH5) and UBE2E families, as well as to UBC13 (UBE2N). Cellular roles have been identified for the interaction of OTUB1 with UBC13 and members of the UBE2D family, but not for UBE2E E2 enzymes. We report here a novel role for OTUB1-E2 interactions in modulating E2 protein ubiquitination. We find that depletion of OTUB1 dramatically destabilizes the E2 conjugating enzyme UBE2E1 (UBE2E1) in cells and that this effect is independent of the catalytic activity of OTUB1 but depends on the ability of OTUB1 to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we have found a new role for OTUB1 in rescuing specific E2s from degradation in vivo .

A20 (TNFAIP3) and ABIN-1 (TNIP1) are candidate susceptibility genes for inflammatory bowel disease and other autoimmune or inflammatory diseases, but it is unclear how these proteins interact in vivo to prevent disease. Here we show that intestinal epithelial cell (IEC)-specific deletion of either A20 or ABIN-1 alone leads to negligible IEC loss, whereas simultaneous deletion of both A20 and ABIN-1 leads to rapid IEC death and mouse lethality. Deletion of both A20 and ABIN-1 from enteroids causes spontaneous cell death in the absence of microbes or hematopoietic cells. Studies with enteroids reveal that A20 and ABIN-1 synergistically restrict death by inhibiting TNF-induced caspase 8 activation and RIPK1 kinase activity. Inhibition of RIPK1 kinase activity alone, or caspase inhibition combined with RIPK3 deletion, abrogates IEC death by blocking both apoptosis and necroptosis in A20 and ABIN-1 double-deficient cells. These data show that the disease susceptibility proteins A20 and ABIN-1 synergistically prevent intestinal inflammation by restricting IEC death and preserving tissue integrity.