A20 (TNFAIP3) is a protein that is involved in the negative feedback regulation of NF-κB signaling in response to specific proinflammatory stimuli in different cell types and has been suggested as a susceptibility gene for rheumatoid arthritis. To define the contribution of A20 to rheumatoid arthritis pathology, we generated myeloid-specific A20-deficient mice and show that specific ablation of Tnfaip3 in myeloid cells results in spontaneous development of a severe destructive polyarthritis with many features of rheumatoid arthritis. Myeloid-A20-deficient mice have high levels of inflammatory cytokines in their serum, consistent with a sustained NF-κB activation and higher TNF production by macrophages. Destructive polyarthritis in myeloid A20 knockout mice was TLR4-MyD88 and IL-6 dependent but was TNF independent. Myeloid A20 deficiency also promoted osteoclastogenesis in mice. Together, these observations indicate a critical and cell-specific function for A20 in the etiology of rheumatoid arthritis, supporting the idea of developing A20 modulatory drugs as cell-targeted therapies.

Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.

Dendritic cells (DCs) regulate both immunity and tolerance. Here we have shown that the ubiquitin editing enzyme A20 (Tnfaip3) determines the activation threshold of DCs, via control of canonical NF-κB activation. Tnfaip3(fl/fl)Cd11c-cre(+) mice lacking A20 in DCs demonstrated spontaneous proliferation of conventional and double-negative T cells, their conversion to interferon-γ (IFN-γ)-producing effector cells, and expansion of plasma cells. They developed ds-DNA antibodies, nephritis, the antiphospholipid syndrome, and lymphosplenomegaly-features of systemic lupus erythematosus-and extramedullary hematopoiesis. A20-deficient DCs were resistant to apoptosis, caused by increased sensitivity to CD40L and RANKL prosurvival signals and upregulation of antiapoptotic proteins Bcl-2 and Bcl-x. They captured injected apoptotic cells more efficiently, resisted the inhibitory effects of apoptotic cells, and induced self-reactive effector lymphocytes. Because genetic polymorphisms in TNFAIP3 are associated with human autoimmune disorders, these findings identify A20-mediated control of DC activation as a crucial checkpoint in the development of systemic autoimmunity.

A20 is a nuclear factor κB (NF-κB) target gene that encodes a ubiquitin-editing enzyme that is essential for the termination of NF-κB activation after tumor necrosis factor (TNF) or microbial product stimulation and for the prevention of TNF-induced apoptosis. Mice lacking A20 succumb to inflammation in several organs, including the intestine, and A20 mutations have been associated with Crohn’s disease. However, ablation of NF-κB activity, specifically in intestinal epithelial cells (IECs), promotes intestinal inflammation. As A20 deficiency sensitizes cells to TNF-induced apoptosis yet also promotes NF-κB activity, it is not clear if A20 deficiency in IECs would exacerbate or ameliorate intestinal inflammation. We generated mice lacking A20 specifically in IECs. These mice did not show spontaneous intestinal inflammation but exhibited increased susceptibility to experimental colitis, and their IECs were hypersensitive to TNF-induced apoptosis. The resulting TNF-driven breakdown of the intestinal barrier permitted commensal bacterial infiltration and led to systemic inflammation. These studies define A20 as a major antiapoptotic protein in the intestinal epithelium and further indicate that defects in A20 might contribute to inflammatory bowel disease in humans.