The tumour necrosis factor receptor-associated factor (TRAF) family is composed of cytoplasmic adaptor proteins involved in the transduction of downstream signals of various receptors, such as T cell receptors (TCRs), TLRs, NLRs, and IL-17 receptor (IL-17R) (161). To date, seven members of the TRAFs family have been identified, which, except for TRAF1, contain in the N-terminus a ring finger domain, the domain responsible for the catalytic activity of E3 ubiquitin ligase (162). Recent studies have also demonstrated that TRAF2, TRAF3, TRAF5 and TRAF6 function as E3 ubiquitin ligases (161). TRAFs, as adaptor proteins and E3 ubiquitin ligases, are involved in innate and adaptive immune signal transduction, leading to the activation of transcription factors such as NF-κB, AP-1and IRFs (163).

Preliminary evidence linking TRAFs with intestinal inflammation was suggested by a study reporting that Traf2 ^-/- mice, with BALB/C background, spontaneously developed severe colitis and died within 3 weeks after birth (77). The spontaneous colitis phenotype was largely dependent on TNF-α induced apoptosis of colonic epithelial cells. The production of TNF-α by colon lamina propria cells (LPCs) in response to symbiotic bacteria may have been a key event that exacerbated the development of colitis, due to increasing apoptosis of colonic epithelial cells and aggravation of the destruction of epithelial barrier function, leading to dramatic alterations in colonic microecology (77). Changes in colonic microbiota will also cause LPCs to further produce TNF-α, which will then lead to uncontrolled intestinal inflammation (77). Subsequently, Traf2^-/- mice were also found to have abnormal accumulation of IL-10-secreting neutrophils, a finding that was not surprising given that development of IL-10-secreting neutrophils is largely dependent on TNF-α signaling (78). IL-10-secreting neutrophils might induce immunosuppression under certain conditions and aggravate colitis by enhancing colonic bacterial invasion, suggesting a potentially important role for TRAF2-mediated TNF-α signaling in regulating IL-10-mediated colonic homeostasis. Additional studies found that myeloid cell-specific knockout of TRAF2 or TRAF3 aggravated colitis by promoting expression of pro-inflammatory cytokines stimulated by TLRs in macrophages (79). Investigation of the molecular mechanisms suggested that TRAF2 and TRAF3 acted synergically with E3 ubiquitin-ligase cAIPs to mediate ubiquitination of cRel and IRF5. Following TRAF2 and TRAF3 knock-out, cRel and IRF5 achieved stable expression levels, rendering macrophages highly sensitive to TLR ligands and IL-1β-induced cytokines (79). These results indicate an involvement of TRAF2 and TRAF3 in the negative feedback regulation mechanism of inflammation suppression. Another study from Jun, Qiao et al. observed TRAFs up-regulation in PBMC and colonic mucosa of IBD patients, further suggesting a role for this family of proteins in the development and progression of IBD (80, 81, 83). In addition, TRAF3 has been shown to be a negative regulator of inflammation in the TNBS-induced colitis mouse model by interfering with IL-17/IL-17R/Act1/TRAF6-mediated proinflammatory pathways though binding to IL-17R (82). Furthermore, increased sensitivity to DSS-induced colitis was observed in Traf5^-/- mice, and might be attributed to enhanced Th2 and IFN-γ/IL-17A co-producing CD4⁺T cell responses and CD4⁺T cell NF-κB activation under intestinal inflammation (84). These findings suggest that TRAF5 works as an anti-inflammatory regulator in experimental colitis in mice. Similarly, specific knockout of TRAF6 in IECs also leads to severe DSS-induced colitis in mice, suggesting that TRAF6 exhibits protective anti-inflammatory effect in intestinal epithelial cells; it is worth noting that this effect appears to be independent of TLR signals (85).

Overall, TRAFs showed a consistent anti-inflammatory effect during the occurrence and development of IBD, albeit via different pathways or target cells. How TRAFs play an anti-inflammatory role in IBD through the regulation of innate immunity and adaptive immunity remains an issue worthy of further exploration. Another question to be explored in future studies is how to separately evaluate the different roles of each TRAF protein in the separation of other functions.

Most member of the tripartite motif protein superfamily (TRIMs) exhibit E3 ubiquitin ligase activity due to the presence of ring finger domain, which is involved in the regulation of a variety of cell biological processes, including cell homeostasis, cell cycle, apoptosis, senescence (164). Increasing evidence suggests that some TRIM family members promote or inhibit the development of IBD through different mechanisms. Firstly, knockout of TRIM14 alleviated acute colitis and CAC by weakening the nonclassical NF-κB pathway mediated inflammatory response; this is due to the ability of TRIM14 to recruit USP14 to deubiquitination p100/p52, thereby preventing it from p62 mediated autophagy degradation (86). Secondly, TRIM21 was found to inhibit Th1/Th17 differentiation in the intestinal mucosa by ubiquitinating IRF3 in CD4⁺T cells, thus playing an anti-inflammatory role in the pathogenesis of IBD (87). Similarly, Zhou et al. reported that TRIM21 expression in colitis-associated colorectal cancer (CAC) decreased and was negatively associated with colon cancer occurrence, further suggesting an anti-cancer effect of TRIM21 (88). Subsequently, TRIM22 mutants have been reported to be impaired in their ability to bind NOD2 and mediate NOD2 K63-linked polyubiquitination, leading to NOD2-dependent activation of IFN-β and NF-κB signals, which were involved in the occurrence of with VEOIBD (165). In addition, TRIM26-mediated K11-linked TAB1 polyubiquitination has been found to enhance TAK1 activation and subsequent activation of NF-κB and MAPK signaling pathways in macrophages, pointing to a pro-inflammatory role of TRIM26 in DSS-induced colitis (89). Furthermore, TRIM27 expression was elevated in the colon tissues of Crohn’s patients and in CD4⁺T cells in the mesenteric lymph nodes of DSS-induced colitis mice (90, 91). In a study by Zaman et al., Trim27^-/- mice were not sensitive to DSS-induced colitis, again implicating TRIM27 in the occurrence and development of IBD (92). Further research reported that the knockout of TRIM27 reduced DSS induced intestinal inflammation and inhibited tumorigenesis of CAC induced by AOM/DSS, mainly due to repression of signal transducer and activator of transcription 3 (STAT3) activation in hematopoietic cells (93). However, hyperactivation of STAT3 has been recognised as an important mechanism in the transition from colonic inflammation to colonic neoplasia (166). These results suggest that TRIM27 is not only a pro-inflammatory factor, but also a potential oncoprotein. Moreover, TRIM31 was found to suppress the activation of NLRP3 inflammasome by promoting NLRP3 polyubiquitination and proteasome degradation, thus alleviating DSS-induced colitis (94). Additionally, research by Petit et al. showed down-regulation of TRIM33 expression in PBMC of CD patients, and that specific knockout of TRIM33 in myeloid cells caused impaired monocyte recruitment and macrophage differentiation, leading to a continuous state of colonic inflammation (95). These results indicated that expression of TRIM33 in myeloid cells was important in the maintenance of intestinal immune homeostasis. More recently, the expression level of TRIM34 was also found to be significantly decreased in the colonic mucosa of patients with UC (96). TRIM34 knock-out contributed to decreased secretion of Muc2 by goblet cells, leading to defects in the internal mucus layer. This phenotype makes mice more susceptible to DSS-induced colitis and inflammation-associated colorectal cancer, suggesting that TRIM34 in IECs plays an important role in maintaining the integrity of intestinal barrier, and in preventing severe colitis and tumorigenesis (96). Furthermore, mRNA and protein levels of TRIM58 were significantly reduced in colon tissues of mild or active UC patients (97). Additional mechanistic studies revealed that TRIM58 regulates TLR2 in myeloid cells by ubiquitination, and inhibits intestinal inflammation though terminating the overactivation of NF-κB/AP-1 signaling pathway induced by TLR2 (97). Finally, Trim62^-/- mice exhibited reduced cytokine production dependent on caspase recruitment domain-containing protein 9 (CARD9) signaling pathway, and increased susceptibility to fungal infection and DSS-induced colitis (98). CARD9 is a susceptibility gene for IBD and a critical component of anti-fungal innate immune signaling (167). Further research found that TRIM62 mediated K27-linked ubiquitination of CARD9 at K125 site was critical for CARD9 activation in DCs, suggesting an involvement of TRIM62 in mucosal anti-fungal immune response and intestinal inflammation (98).

In conclusion, the TRIM protein family play important roles in the development of IBD by regulating innate and adaptive immune systems. More research is needed to elucidate the exact mechanisms by which members of the TRIM family influence the occurrence or progression of inflammation at a molecular level. It will be interesting to see whether these processes occur in an E3 ubiquitin ligase activity dependent manner.

Ring finger protein 5 (RNF5) expression was found to be decreased in the colonic inflammatory mucosa of IBD patients, and was negatively associated with S100 calcium binding protein A8 (S100A8) expression (99). S100A8 can induce neutrophils chemotaxis as well as promoting the expression of pro-inflammatory cytokines in immune cells (168). The inverse correlation of RNF5/S100A8 was consistent with the clinical severity of IBD patients, suggesting that the RNF5/S100A8 axis may play a role in the development of IBD (99). Studies using animal models found that RNF5 deletion aggravated DSS-induced colitis, increased the production of Th1-type inflammatory cytokines, and impaired intestinal epithelial regeneration in the inflammatory recovery stage (99). Further mechanistic studies revealed that lack of RNF5 in IECs weakened mediated ubiquitination and proteasomal degradation of S100A8, which in turn promoted S100A8 secretion and induced activation of CD4⁺T cells (99). Decreased expression of RNF8 in colonic tissue was associated with impaired autophagy and elevated levels of phosphorylated Akt/mTOR in the TNBS-induced colitis mouse model (100). Overexpression of RNF8 reversed these phenotypes and reduced intestinal inflammation, possibly due to RNF8 ubiquitin degradation of AKT1 and inhibition of Akt/mTOR signal pathway to enhance autophagy (100). In addition, the expression of RNF20 and RNF40 was down-regulated in the colonic epithelium and in the stroma of UC and colitis-associated colorectal cancer (CAC) patients (101). The susceptibility of RNF 20 heterozygote (Rnf20^+/- ) mice to colitis and inflammatory-associated tumour was increased, likely because RNF20 deletion in non-cancerous epithelial cells, intestinal organoids, and innate immune cells promoted p65 binding to the κB site, leading to the transcriptional activation of NF-κB target genes (101). Intestinal epithelial cells-specific knockout of RNF40 resulted in local and systemic protective effects on DSS induced colonic inflammation, and these effects were different from the anti-inflammatory and tumour suppressive effects of RNF20 (102). RNF40 deficiency in IECs not only reduced the burden of colonic inflammation by reducing NF-κB transcription activity via delaying the nuclear translocation of RelA, but also alleviated bone fragility induced by inflammation (102). RNF128 was also found to be involved in the pathogenesis of IBD. On one hand, RNF128 expression was found to be up-regulated in the CD4⁺T cells of the intestinal layer propria in CD patients, while down-regulated in CD4⁺T cells of the peripheral blood (103). On the other hand, the expression of RNF128 in CD4⁺T cells in UC patients in remission stage was significantly higher than patients with active UC and healthy subjects, indicating that RNF128 may be involved in maintaining remission in UC patients (104). Furthermore, the expression of RNF183 was up-regulated in IECs of IBD patients and TNBS-induced colitis mouse model, and promoted NF-κB signaling mediated intestinal inflammation by increasing ubiquitination -proteasome degradation of IκB (105). Similarly, RNF183 expression in colonic epithelial cells was up-regulated in IBD patients and DSS-induced colitis mouse model, leading to caspase-8 mediated apoptosis by promoting K63-linked ubiquitination-mediated lysosomal degradation of death receptor 5 (DR5) (106). Consistent with this, DR5 was downregulated in IECs of IBD patients (169). Previous research has shown that translocation of DR5 to lysosomes leads to the release of lysosomal proteases into the cytoplasm, thereby promoting apoptosis (170). These findings imply that RNF183 may play a pro-inflammatory role in the context of IBD.

Notably, GWAS identified RNF186 as a UC susceptibility gene (171). Further studies have shown that R179X, a truncated mutant of RNF186, had a protective effect on UC, possibly due to R179X mislocalization and impaired RNF186 function or altered associations with interacting proteins and subsequent substrate protein ubiquitination (172). Besides, the UC-associated variation (A64T, substitution of alanine with threonine at the 64th position) of RNF186, which was identified in North American and European UC patients (173), impaired the E3 ubiquitin ligase activity of RNF186 and was associated with increased sensitivity to DSS-induced intestinal inflammation in mice (107). Meanwhile, Rnf186^-/- mice showed increased colon permeability to organic solutes and high sensitivity to DSS-induced colitis (107). The reason for increased colon permeability might be due to the role of RNF186 in controlling occludin homeostasis through the K48-linked polyubiquitination, hence RNF186 absence will result in increased amounts of occludin and abnormal distribution (concentrated in the cytoplasm) in IECs (107). With respect to high sensitivity to DSS-induced colitis, RNF186 deficiency led to disturbed proteostasis and thus increased endoplasmic reticulum (ER) stress in IECs (107). Recently, it has been reported that RNF186 knockout mice were found to have increased bacterial loads in their mesenteric lymph nodes and spleen during DSS-induced colitis, and RNF186-deficient macrophages were impaired in bacterial phagocytosis and intracellular bacterial clearance (108). Further mechanistic studies revealed that the ER localization of RNF186 in macrophages and its mediated ubiquitination of activating transcription factor 6 (ATF6) were crucial steps in NOD2-induced antimicrobial effect (108). In addition, increased sensitivity to DSS colitis in Rnf186^-/- mice was linked to reduced autophagy in the colonic epithelia attributed to RNF186 mediating K27-linked ubiquitination of EphB receptor B2 (EPHB2) at K892 site and further recruiting MAP1LC3B for autophagy (174). This RNF186-dependent, EPHB2-induced autophagy helped to promote the clearance of bacteria from the colonic epithelium (174). These results suggest a clear role for RNF186 in regulating intestinal homeostasis, albeit through different regulatory mechanisms.

cIAP1, cIAP2 and XIAP, members of the inhibitor of apoptosis (IAP) family, were initially identified as anti-apoptotic proteins. However, accumulating studies have shown that cIAP1, cIAP2 and XIAP are key and universal regulatory factors in inflammatory and innate immune signaling pathways, which is attributed to their E3 ubiquitin ligase activity (43). For example, XIAP is indispensable for NOD-mediated NF-κB activation. Briefly, the research conducted by Bauler et al. showed that XIAP facilitated maximal production of pro-inflammatory cytokines during bacterial infection in vivo and in vitro, or during combined treatment with NOD2 and TLR2 ligands, taking the lead in suggesting a role for XIAP in NOD signaling (175). Subsequently, Krieg et al. revealed that XIAP mediated NOD signaling by interacting with RIPK2 in vitro ( 176). Furthermore, Damgaard et al. demonstrated that XIAP was an essential ubiquitin ligase in the NOD2 signaling pathway in vivo ( 115). Mechanistically, Once the NOD receptors activated by specific PAMPs, XIAP is recruited to the NOD receptor signaling complex (NOD-SC) containing RIPK2 (115). On the one hand, XIAP promotes the ubiquitination of RIPK2, which is a critical step in NOD-mediated NF-κB activation (115). On the other hand, XIAP recruits LUBAC to the NOD-SC to further activate the NOD downstream signaling cascade (115). Therefore, dysregulation of XIAP results in impaired NOD signal transduction, reduced response to bacteria and increased intestinal inflammation. Indeed, numerous studies have shown that XIAP mutations are associated with IBD (177). XIAP mutations may cause a primary immunodeficiency disease, X-linked lymphoproliferative disease type 2 (XLP-2), which is often characterized by hemophagocytic lymphohistiocytosis (HLH), EBV infection and recurrent splenomegaly (178). However, patients with XIAP deficiency may also have Crohn’s disease (4% ~20%) (179, 180). In some patients, Crohn’s disease-like enteritis is found to be the first or only clinical manifestation, with the characteristics of early age of onset, severe illness and unresponsiveness to standard treatment, including biological treatment (180–182). Interestingly, missense mutations of XIAP are mainly concentrated in two domains, BIR2 and RING, which are the key to XIAP-mediated NOD signaling pathway (183). Therefore, XIAP deficiency is considered to be the Mendelian cause of IBD. Unlike XIAP, the requirements of cIAP1/2 for the NOD signaling pathway are controversial. An experimental study performed by Bertrand et al. showed macrophages derived from ciap1^{-/ –} or ciap2^-/- mice, or human colonocytes depleted cIAP1 or cIAP2 through RNAi were defective in mediating NOD signaling pathways characterized by a sharp decrease in the production of NOD-dependent pro-inflammatory cytokines and chemokine. And this blunted inflammatory response was also observed in vivo when ciap1^-/– or ciap2^-/- mice were stimulated by NOD agonists (109). Nevertheless, an in vitro study by Damgaard et al. found that chemical depletion of cIAP1/2 in cells via Smac mimetic compounds (leading to rapid degradation of cIAP1/2 without affecting the stability of XIAP) did not block NOD-mediated NF-κB activation (184). Furthermore, Stafford et al. showed that cIAP1 and cIAP2 were dispensable for NOD signaling pathway using cIAP1- or cIAP2-deficient mice in vivo ( 110). Recently, Goncharov et al. showed that XIAP-selective antagonists (which do not affect the stability of cIAP1) can block NOD2 signalling by interfering with the binding of XIAP to RIP2, further suggesting that XIAP may be the most critical IAP required for NOD signalling (185). This discrepancy may, on the one hand, be due to the fact that these authors used different strains of cIAP-deficient mice and, on the other hand, may be due to the fact that the protein’s physiological role may differ from its role in the controlled environment of biochemical experiments. Of note, recent research has shown that cIAP1 plays a more important role in the regulation of TNF-α induced IECs death (111). In this study, compared with ciap2^{-/ –}, Xiap^{-/ -}mice and wild-type mice, ciap1^-/- mice showed more intestinal epithelial cell apoptosis when injected with TNF-α (111). In vitro studies also found that intestinal epithelial cells derived from ciap1^-/- mice were more sensitive to TNF-induced apoptosis (111). However, whether the specific mechanism of cIAP1 regulating the sensitivity of IECs to TNF-a depends on the its E3 ubiquitin ligase activity remains to be further studied. Moreover, cIAP2 were found to be up-regulated in colonic epithelial cells of patients with UC (112, 113). However, research on the role of cIAP2 in colitis and CAC reported inconsistent results. The ciap2^-/- mice showed increased susceptibility to DSS induced acute and chronic colitis, but were resistant to AOM/DSS induced CAC (114). The effects of both inhibition of gut inflammation and promotion of tumorigenesis appear contradictory, but one possible explanation is that cIAP2 protects the IECs from inflammatory damage and promotes cell proliferation during the recovery stage of inflammation, but its expression in the CAC microenvironment promotes tumorigenesis by maintaining cancer cells survival (114).

Similarly, the role of F-box/WD repeat containing protein 7 (FBW7) in the pathogenesis of IBD also appears contradictory. FBW7 is a substrate recognition component of the SCF ubiquitin ligase, and can ubiquitinate and degrade a variety of proteins that play a role in differentiation and proliferation, including c-Jun, c-Myc, Notch and Cyclin E1, among others (186, 187). In the DSS-induced colitis mouse model, specific knockout FBW7 in IECs led to NF-κB pathway activation and exacerbated intestinal inflammation, suggesting that FBW7 may play a protective role in IBD (116). In contrast, one study by Meng et al. showed increased FBW7 expression in IECs of IBD patients and TNBS-induced colitis mice (117). In this study up-regulation of FBW7 in IECs was linked to the severity of colonic mucosal inflammation in IBD patients (117). Further molecular mechanistic studies revealed that up-regulated FBW7 activated the NF-κB signaling pathway by mediating ubiquitination degradation of IκBα, suggesting a pro-inflammatory effect for FBW7 (117). Although different colitis mouse models were used in the two studies and might account for this discrepancy, a more plausible explanation is that FBW7 homeostasis in IECs is critical in regulating intestinal inflammation, and too much or little might aggravate intestinal inflammation. Notably, another study also found that increased expression of FBW7 in IBD patients and in the DSS-induced colitis mouse model, and this expression level was significantly correlated with the severity of IBD, further suggesting that FBW7 up-regulation may be an intermediate or pathogenic factor in the pathogenesis of IBD (118). In the same study, the authors engineered mice with myeloid cell-specific FBW7 knockout and found that FBW7 deficiency in macrophages attenuated experimental colitis induced by DSS and TNBS (118). This effect was mainly attributed to FBW7 promoting K48-linked polyubiquitination and proteasome degradation of enhancer of zeste homolog 2 (EZH2) in CX3RC1^hi macrophages, inhibiting H3K27me3 modification of EZH2, enhancing the expression of CCL2 and CCL7, and therefore promoting the recruitment of CXCR1^int proinflammatory macrophages to inflamed colon tissues (118). These results suggest that, unlike the role of FBW7 in intestinal epithelial cells, FBW7 in myeloid cells has a pro-inflammatory effect. In addition, Pellino3, an important intermediate signal protein in the innate immune response pathway, was found to be down-regulated in the colon of patients with CD. Mechanistic studies revealed that Pellino3 in macrophages mediated RIPK2 ubiquitination promotes NOD2 signal transduction and plays a protective role in colitis (119). Recently, Hrd1, an E3 ubiquitin-ligase responsible for the degradation of unfolded proteins in the endoplasmic reticulum, has been found to be reduced in the intestinal tissues of IBD patients, DSS and TNBS-induced mice, as well as in the lipopolysaccharide (LPS)-induced intestinal epithelial inflammation model (120, 121). The specific molecular mechanisms underlying these phenotypes remain unclear and need further investigation.

CYLD encodes one deubiquitinase of the USP family. The N-terminal of this enzyme contains three Cap-Gly domains (the first two are responsible for binding to microtubules, and the third is responsible for binding to IκB kinase IKK adaptor protein NEMO) and two proline-rich motifs. The C-terminal contains a catalytic USP domain that preferentially recognizes the polyubiquitination linked by K63 and M1 ( Figure 4 ) (199). Of note, only the two DUBs, CYLD and OTU deubiquitinase with linear linkage specificity (OTULIN) can remove LUBAC mediated -M1-linked polyubiquitin chains on proteins. CYLD was initially considered to be a typical recessive tumor suppressor gene, because the mutations of this gene were linked to skin adnexal tumors in humans (200). However, CYLD has also been found to be an important negative regulator of the NF-κB signaling pathway (201), and NF-κB activation has a strong pro-inflammatory effect in IBD. Therefore, the role of CYLD in inflammatory bowel disease has also received attention from researchers.

As early as in 2005, Costello et al. found that CYLD was significantly down-regulated in the intestinal mucosal tissues of IBD patients based on genome-wide cDNA microarray data (202). Subsequently, Zhang et al. observed that CYLD-deficient mice were more susceptible to DSS-induced colonic inflammation, due to impaired negative regulation of the NF-KB signaling pathway in the B cells, T cells, and myeloid cells, and also displayed significantly increased incidence of colonic tumors compared with the control group (134). In addition, Patrick et al. found that CYLD’s deubiquitase catalytic activity was necessary for the necrosis of colonic epithelial cells and the occurrence of colitis in FADD^IEC-KO mice (135). FADD is an adaptor protein required for death receptor-induced apoptosis. Colonic epithelial cell necrosis and colitis were observed to occur spontaneously following IEC specific knockout of FADD in mice (135). Moreover, Cleynen et al. conducted a large multicenter GWAS in CD and UC patients, and found that SNPs of the CYLD gene were significantly associated with CD, among which the rs12324931 was the strongest SNP (203). Importantly, Demetrios et al. investigated the role of the deubiquitination enzyme activity of CYLD in colitis -associated colorectal cancer using a conditioned CYLD inactivation mouse model (IEC-CYLD^Δ9 mice, which harbors a mutation that eliminated the CYLD deubiquitination domain in IECs) (136). Their study found that IEC-CYLD ^Δ9 mice did not exhibit spontaneous intestinal lesions before the age of 1 year, but showed an incidence of colon tumors was significantly higher than in WT mice under the pressure of AOM/DSS (136). These results suggested that the deubiquitinase activity of CYLD in IECs plays an important inhibitory role in the process of colitis-associated carcinogenesis. Furthermore, the research results from Yilang et al. revealed a new physiological role of the CYLD spliceosome sCYLD in regulating intestinal inflammation via TGF-β signaling (137). Their data showed that expression of spliceosome of CYLD (sCYLD) and Smad7 in the colonic mucosa lamina propria T cells of CD patients was increased and correlated with disease severity. Mice overexpressing scyld and Smad7 (scyld/smad7 mice) developed severe spontaneous colitis caused by overactivation of effector T cells due to impaired Treg inhibition resulting from alteration of TGF-β signaling (137). In this model, sCYLD in CD4⁺T cells could not mediate deubiquitination of Smad7 linked by K63, and enhance the nuclear translocation of Smad7 and forming complex with Smad7 in the nucleus, which impaired the DNA-binding ability of Smad3 and thus negatively regulated Smad-dependent TGF-β signaling (137). Research from by Sandip et al. further clarified the anti-inflammatory mechanism of CYLD (138). Their research discovered that CYLD expression in the colonic mucosal of UC patients was decreased and negatively correlated with IL-18 abundance (138). In line with the studies described above (134), CYLD knockout caused increased destruction of intestinal epithelia and severe colonic inflammation when challenged by pathological factors. Mechanistically, CYLD-mediated K63-linked deubiquitination of NLRP6 negatively regulated NLRP6-ASC-dependent inflammasome activation and IL-18 production in IECs, thereby inhibiting intestinal inflammation (138).

In addition to research on CYLD, studies of other USP family members in the context of IBD also show good progress. Firstly, studies have reported that SNPs in USP3, USP25, and USP40 were associated with IBD (203, 204). In addition, based research using whole genome sequencing data from Chinese patients, the mutation frequency of USP48 gene was significantly different between ulcerative colitis-associated colorectal cancer and scattered colorectal cancer, suggesting that a potential role for this mutation in the transition from inflammation to cancer (139). Secondly, T cell-specific USP8-deficient mice spontaneously developed colitis, likely due to imbalance of T cell homeostasis, increase of intestinal CD8⁺T cells, and impaired immunosuppression of Tregs (205). These results indicate that USP8 may have a function in maintaining intestinal homeostasis by regulating T cell homeostasis. Besides, USP9X negatively regulated c-Myc by directly stabilizing FBW7 to restore damaged intestinal epithelium and inhibit the development of colitis-associated colon cancer in animal models (140). Besides, USP16 was observed to be up-regulated in colonic macrophages of IBD patients and the deletion of USP16 in macrophages alleviated DSS-induced colitis and inflammation-associated colon carcinogenesis (141). Mechanistically, USP16 selectively removed the K33-linked polyubiquitin chains from IKKβ thereby facilitating the interaction of IKKβ with p105 and phosphorylating p105, thus activating the NF-κB signaling pathway (141). Similarly, a recent study showed that USP22 exerted an antitumor effect in colorectal cancer by reducing mTOR activity (142). Subsequently, specific knockout UPS22 in IECs increased the severity of inflammation in mice with DSS colitis and promoted colitis-related colorectal cancer, further confirming the role of USP22 in repressing intestinal inflammation and tumors (143). Finally, a recent study found an increased susceptibility to DSS-induced colitis in USP38 KO mice, accompanied by higher levels of IL-6 and IL-23A in colon tissue and peripheral blood (144). Mechanistic studies uncovered a role for USP38 in stabilizing the lysine demethylase 5B (KDM5B) by removing the K48-linked polyubiquitin chains, thus promoting KDM5B-mediated histone demethylation to inhibit the expression of IL-6 and IL-23A in bone marrow-derived cells, and ultimately inhibiting the occurrence and progression of intestinal inflammation (144).

Among all the deubiquitinating enzymes, A20 is by far the most intensively studied in the pathogenesis of IBD. A20, also known as TNFAIP3 (TNF-α-induced protein 3), was originally thought to be a protein that protected cells from TNF-α-induced cytotoxicity (209). However, accumulating evidence showed that A20 was not only an inhibitor of TNF-α -dependent NF-κB activation, but also a negative regulator of IL-1, PRRs, and T- and B-cell antigen-induced NF-κB activation (210–212). Therefore, A20 is widely believed to exhibit anti-inflammatory properties, which are generally attributed to its role as a ubiquitin-regulating enzyme with E3 ubiquitin ligase and deubiquitinase activity (213). Structurally, A20 contains one N-terminal OTU domain and seven C-terminal ZnF domains. The OTU domain is mainly responsible for the A20 deubiquitination enzyme activity and can specifically catalyze the hydrolysis of K11-, K48- and K63- linked ubiquitination chains (214). The ZnF domain mainly mediates the E3 ubiquitin ligase activity of A20; more specifically, the ZnF4 domain of A20 has a high affinity for K63 ubiquitin, and the ZnF7 domain is able to bind to the M1 chain in an efficient manner ( Figure 4 ) (214).

Dysregulation or dysfunction of A20 expression is associated with several autoimmune diseases and cancers. SNPs at the A20 locus increase susceptibility to a number of human autoimmune diseases, including IBD, type I diabetes, rheumatoid arthritis, and systemic lupus erythematosus (215–218). In the context of IBD, an earlier genome-wide scan of the IBD family showed that the occurrence of this disease was associated with mutations in the chromosome 6q region, which contained the A20 locus (219). In addition, GWAS further suggested that A20 is a susceptibility gene for CD (145, 220). More recently, a case report found novel three heterozygous A20 mutations associated with VIOIBD (221). Importantly, a number of A20 SNPs were located in the upstream, downstream or intron regions of the A20 coding region, implying that they may affect the regulatory elements or conformation of A20 expression, and may therefore affect A20 expression or function. For example, it has been found that A125V mutation may lead to conformational changes in A20 that impaired its ability to deubiquitinate and degrade the target protein TRAF2 (222). Furthermore, recent studies have shown that a A20 gene polymorphism was correlated with the efficacy of anti-TNF therapy in IBD patients (223, 224), indicating suggesting a link between abnormal A20 expression and immune activity. However, most of disease-related SNPs within the A20 locus are located in the non-coding region, and it remains unclear how they affect A20 expression and IBD pathogenesis. Therefore, A20 expression in IBD patients has the target of several research studies. Zheng et al. found that A20 expression level, at both mRNA and protein level, in the intestinal tissues of children with IBD was down-regulated either in active stage or in remission stage compared with intestinal mucosa in children without IBD (146). In a related study, Deenaz et al. found that A20 mRNA expression was increased but protein expression was lower in children with CD compared with UC and non-IBD patients (147). This difference may be due to the different experimental detection methods used, since immunohistochemistry used in the study by Zheng et al. and enzyme-linked immunosorbent assay (ELISA) in the study by Deenaz et al. These differences between mRNA and protein expression were also seen in adult IBD patients. Indeed, studies have found that while A20 mRNA expression in UC patients is significantly up-regulated and negatively associated with disease activity, its protein expression level is down-regulated (148). In addition, Garcia et al. reported that A20 expression level in the inflamed intestinal tissues of IBD patients was increased and was related to increased apoptosis of IECs (149). Regardless, it was indisputable that A20 expression is dysregulated in IBD patients, suggesting the involvement of this protein in the pathogenesis of IBD.

Many studies in recent years investigated how A20 affected the pathogenesis of IBD in different cell types. Firstly, A20 knockout mice provided important knowledge regarding biological function of A20. A20 knockout mice die prematurely due to spontaneous multiorgan inflammation and cachexia (150). A20 knockout mouse was are unable to terminate TNF-α -induced NF-κB activation, suggesting serious deficiencies in the management of inflammatory responses. Subsequently, experiments based on A20^{-/ -} mice and A20 Tg mice (overexpression A20) showed that A20 maintained the intestinal barrier function and supported the tight junctions of IECs by deubiquitinating non-k48 linked polyubiquitinated of occludin (151). Mice where A20 has been specifically knockout in IECs did not develop spontaneous intestinal inflammation, but showed increased sensitivity to DSS-induced colitis and hindered recovery from acute DSS-induced intestinal inflammation, suggesting a role for A20 in alleviating intestinal inflammation and in the recovery following intestinal epithelial injury (152). In addition, Kattah et al. found that mice with IECs depleted of A20 or A20-binding inhibitor of NF-κB-1 (ABIN-1) alone appeared normal, while deletion of A20 and ABIN-1 together resulted in rapid death of IECs (153). These observations suggested that A20 and ABIN-1 have a synergistic effect in maintaining the survival of IECs. Findings derived from mechanistic studies revealed that, on the one hand, A20 and ABIN-1 jointly prevented IEC death by restraining caspase8 activation and apoptosis signal transduction (153). On the other hand, A20 and ABIN-1 may also regulate different ubiquitination events of RIPK1, which affect RIPK1 phosphorylation and RIPK1 kinase activity, repressing RIPK1-mediated necrosis (153). The synergistic effect between A20 and its chaperone factor ABIN-1 may be a manifestation of genetic epistasis, that is, expression of ABIN-1 increased in the absence of A20 to compensate for A20 deficiency and the resulting decreased cell survival. Similarly, ABIN-3 has also been reported to negatively regulate intestinal inflammation caused by necrosis; this effect appears to be via recruitments of A20 into the TNF-RSC and cooperation with deubiquitination enzyme A20 to limit RIPK3’s ubiquitination in IBD (154).

Overexpression of A20 has also been found to sensitize IECs and intestinal organoids to TNF-α -induced apoptosis (149). Under the stimulation of TNF-α, A20 overexpression in IECs resulted in the formation of more A20 dimers, which enhanced Ripoptosome complex assembly and RIPK1-dependent apoptosis through the ZnF7 binding to linear ubiquitin (149). Therefore, a balance of A20 expression in IECs is important to protect these cells against the challenge of pathological factors. Spontaneous intestinal inflammation, characterized by loss of Paneth and goblet cells, IEC proliferation, and crypt apoptosis, have been reported in the double-knockout A20/ATG16L1 mice (155). In this study, A20 and ATG16L1 were found to reduce the expression level of each other through their OTU domain and WD40 domain, respectively (155). This post-transcriptional cross-regulation may be a novel and important control mechanism in intestinal homeostasis.

In addition to the role of A20 in IECs, the role of A20 in immune cells and in the pathogenesis of IBD is also a hot topic of research. Mice with dendritic cells-specific A20 deficiency spontaneously exhibited DC activation and amplification of activated T cells (145). In addition, the A20^fl/fl-Myd88^fl/fl double knockout mice showed the ability of A20 to inhibit MyD88 signaling and the production of pro-inflammatory cytokines in dendritic cells to maintain the homeostasis of myeloid cells and T cells (145). Furthermore, A20^fl/fl CD11c-Cre mice developed inflammatory bowel disease at 5 months of age and exhibited increased colon diameter, expanded immune cells in lamina propria, and reduced goblet cells, suggesting that DC cells require A20 to maintain intestinal immune homeostasis and limit colitis induced by epithelial damage (145). Moreover, the absence of colitis in A20^fl/fl Cd11c-Cre Rag1 mice indicate that the absence of A20 in dendritic cells may led to the overactivation of intestinal T cells in A20^fl/fl-CD11c-Cre mice, which may lead to IBD. Notably, A20^fl/fl CD11c-Cre mice developed arthritis spontaneously, with a pathology similar to that of IBD-related arthritis in humans. Similarly, research by Vereecke et al. did not find spontaneous intestinal inflammation in A20^IEC-KO and A20^Myeol-KO mice. However, A20 ^IEC-KO mice were more susceptible to experimental colitis and had increased IEC apoptosis, while A20^Myeol-KO mice developed rheumatoid arthritis-like symptoms due to excessive activation of myeloid cells to produce high levels of pro-inflammatory cytokines, including TNF-α/IFN-γ (223). Therefore, A20^IEC/Myeol-KO mice not only produced high levels of pro-inflammatory cytokines, but also developed spontaneous enteritis characterized by loss of Paneth and goblet cells, increased apoptosis and proliferation of IECs, and intestinal microbiota imbalance (223). Interestingly, the abundance of intestinal flora of A20^IEC-KO was not altered, but the abundance of gut microbiota of A20^Myeol-KO and A20^IEC/Myeol-KO were significantly decreased, suggesting that A20 may act in myeloid cells to regulate gut microbiota homeostasis. Subsequent studies have shown that, before the onset of spontaneous intestinal inflammation in mice with A20 deficiency in dendritic cells, microbial homeostasis was found to be altered in a lymphocyte-independent manner, resulting in a decrease in α diversity of intestinal flora (156). Dendritic cells-specific A20 knockout expressed higher levels of antimicrobial molecules (e.g., Reg3β, Reg3γ, Pla2g2) in the ileum or proximal colon tissues, suggesting a role for A20 in these cells in limiting antimicrobial peptide expression in vivo and maintaining symbiotic homeostasis. Recently, A20 in macrophages has been shown to have a negative regulatory effect on DSS-induced colitis (157). A20 deficiency in myeloid cells did not affect macrophage development in the bone marrow, but A20 deficiency in macrophages contributed to increased expression of pro-inflammatory cytokines and overactivation of the NF-κB signaling pathway, thereby causing severe DSS-induced colitis (157). In general, the function of A20 in IECs seem to help maintain intestinal barrier stability by preventing cytokine induced apoptosis, while the function of A20 in immune cells seems to prevent excessive cytokines production in myeloid-derived cells.

In addition to the above-mentioned cell type-specifically knockouts of A20, Lu et al. constructed mice with A20^OTU and A20^ZnF4 mutations from the OUT domain and zinc finger domain of A20 (158). Their study found that both A20 mutated mice exhibited DSS-induced colitis, suggesting that the OTU and ZnF4 domains of A20 had a role in inflammation inhibition. Mechanistic studies further indicated that the OTU domain of A20 restricted the deubiquitination of RIPK1 at K48 and K63, while the ZnF4 domain was essential for the recruitment of A20 to ubiquitination of RIPK1, and that only the presence of both could regulate RIPK1 ubiquitination and NF-κB signal transduction. Moreover, Arne et al. recently developed A20^{ZnF4ZnF7/ZnF4ZnF 7}mice with both the K63 polyubiquitin-binding ZnF4 and M1 polyubiquitin-binding ZnF7 domains inactivated (159). Surprisingly, A20^{ZnF4ZnF7/ZnF4ZnF7} mice are phenotypically similar to A20 knockout mice: multi-organ inflammation and premature death. Then, they constructed tissue-specific ZnF4 and ZnF7 domain double inactivated mice, Tnfaip3 ^{ZnF4ZnF7/ZnF4ZnF7} LysM-Cre (Myeloid-specific), Tnfaip3 ^{ZnF4ZnF7/ZnF4ZnF7} Vil1-Cre (IEC-specific). The former phenotypically similar to A20^Myeol-KO mice: progressive polyarthritis, higher concentrations of TNF and IL-6 in serum and that BMDM is hypersensitive to LPS stimulation. The latter phenotypically similar to A20^IEC-KO mice: susceptible to DSS colitis and all died in response to sublethal doses of TNF attack (159). These findings suggested that the ubiquitin-binding properties of the ZnF4 and ZnF7 domains were required for A20 to inhibit pro-inflammatory signaling.

In contrast to A20 studies, there were only sporadic reports about the role of OTU family members in IBD. Multiple mutations in OTUD1 have been linked to autoimmune diseases, suggesting a possible involvement in the pathogenesis of IBD (225). In addition, GWAS of patients with UC in Korea revealed genetic susceptibility sites that were significantly associated with UC in OTUD3 (226). Recently, OTUD5 was found to be up-regulated in the intestinal inflammatory tissues of IBD patients and TNBS-induced colitis mice, and IFN-γ was found to up-regulate OTUD5’s expression through a p38/MAPK-dependent mechanism. Up-regulation of OTUD5 further increased TNF-α production in LPMCs of IBD patients (160). These findings suggested that OTUD5 may be a member of the positive feedback loop that amplifies the abnormal inflammatory response in IBD.