A20 is an anti-inflammatory protein linked to multiple human diseases; however, the mechanisms by which A20 prevents inflammatory disease are incompletely defined. We found that A20-deficient T cells and fibroblasts were susceptible to caspase-independent and kinase RIPK3-dependent necroptosis. Global deficiency in RIPK3 significantly restored the survival of A20-deficient mice. A20-deficient cells exhibited exaggerated formation of RIPK1-RIPK3 complexes. RIPK3 underwent physiological ubiquitination at Lys5 (K5), and this ubiquitination event supported the formation of RIPK1-RIPK3 complexes. Both the ubiquitination of RIPK3 and formation of the RIPK1-RIPK3 complex required the catalytic cysteine of A20's deubiquitinating motif. Our studies link A20 and the ubiquitination of RIPK3 to necroptotic cell death and suggest additional mechanisms by which A20 might prevent inflammatory disease.

Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.

Natural killer (NK) cells are innate immune effectors that mediate rapid responses to viral antigens. Interleukin (IL)-15 and its high affinity IL-15 receptor, IL-15Ralpha, support NK cell homeostasis in resting animals via a novel trans presentation mechanism. To better understand how IL-15 and IL-15Ralpha support NK cell activation during immune responses, we have used sensitive assays for detecting native IL-15 and IL-15Ralpha proteins and developed an assay for detecting complexes of these proteins. We find that IL-15 and IL-15Ralpha are preassembled in complexes within the endoplasmic reticulum/Golgi of stimulated dendritic cells (DCs) before being released from cells. IL-15Ralpha is required for IL-15 production by DCs, and IL-15 that emerges onto the cell surface of matured DCs does not bind to neighboring cells expressing IL-15Ralpha. We also find that soluble IL-15-IL-15Ralpha complexes are induced during inflammation, but membrane-bound IL-15-IL-15Ralpha complexes, rather than soluble complexes, support NK cell activation in vitro and in vivo. Finally, we provide in vivo evidence that expression of IL-15Ralpha specifically on DCs is critical for trans presenting IL-15 and activating NK cells. These studies define an unprecedented cytokine-receptor biosynthetic pathway in which IL-15Ralpha serves as a chaperone for IL-15, after which membrane-bound IL-15Ralpha-IL-15 complexes activate NK cells via direct cell-cell contact.

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48-linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic 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 E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1-E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1-/- knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability 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 provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

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 A20ZF7 mice required T cells and MyD88, was exquisitely sensitive to tumor necrosis factor and interleukin-17A, and persisted in germ-free conditions. A20ZF7 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.

Microbial dysbiosis commonly occurs in patients with inflammatory bowel diseases (IBD). Exogenous causes of dysbiosis such as antibiotics and diet are well described, but host derived causes are understudied. A20 is a potent regulator of signals triggered by microbial pattern molecules, and A20 regulates susceptibility to intestinal inflammation in mice and in humans. We now report that mice lacking A20 expression in dendritic cells, A20FL/FL CD11c-Cre mice (or A20dDC mice), spontaneously develop colitogenic intestinal dysbiosis that is evident upon weaning and precedes the onset of colitis. Intestines from A20dDC mice express increased amounts of Reg3β and Reg3γ, but not Ang4. A20 deficient DCs promote gut microbiota perturbation in the absence of adaptive lymphocytes. Moreover, A20 deficient DCs directly induce expression of Reg3β and Reg3γ but not Ang 4 in normal intestinal epithelial cell enteroid cultures in the absence of other cell types. These findings reveal a pathophysiological pathway in which defective expression of an IBD susceptibility gene in DCs drives aberrant expression of anti-bacterial peptides and luminal dysbiosis that in turn confers host susceptibility to intestinal inflammation.

Dendritic cells (DCs), which are known to support immune activation during infection, may also regulate immune homeostasis in resting animals. Here we show that mice lacking the ubiquitin-editing molecule A20 specifically in DCs spontaneously showed DC activation and population expansion of activated T cells. Analysis of DC-specific epistasis in compound mice lacking both A20 and the signaling adaptor MyD88 specifically in DCs showed that A20 restricted both MyD88-independent signals, which drive activation of DCs and T cells, and MyD88-dependent signals, which drive population expansion of T cells. In addition, mice lacking A20 specifically in DCs spontaneously developed lymphocyte-dependent colitis, seronegative ankylosing arthritis and enthesitis, conditions stereotypical of human inflammatory bowel disease (IBD). Our findings indicate that DCs need A20 to preserve immune quiescence and suggest that A20-dependent DC functions may underlie IBD and IBD-associated arthritides.

Psoriasis is a common inflammatory skin disease that affects approximately 1% of the population worldwide. Tumor necrosis factor-alpha-induced protein 3 (TNFAIP3) gene polymorphisms have been strongly associated with psoriasis susceptibility. In this study, we investigate how TNFAIP3, also known as A20, may regulate psoriasis susceptibility. We found that haplo-insufficient A20+/- mice develop severe toll-like receptor (TLR)-induced skin inflammation compared to wild type mice owing to amplified production of interleukin (IL)-17 and IL-23. Examination of TNFAIP3 mRNA expression in skin biopsies from patients with psoriasis revealed reduced expression in both involved and uninvolved skin. Our results demonstrate the clinical importance of reduced dermal expression of A20 in psoriasis and suggest that A20 restriction of the IL-23/17 axis protects against psoriasis.

The IL-17/IL-17R axis has controversial roles in cancer, which may be explained by tumor-specific results. Here, we describe a novel molecular mechanism underlying IL-17RC-controlled tumor-specific proliferation. Triggered by IL-17RC knockdown (KD), B16 melanoma and 4T1 carcinoma cells inversely altered homeostatic tumor proliferation and tumor growth in vitro and in vivo. In contrast to the existing dogma that IL-17RC-dependent signaling activates the JNK pathway, IL-17RC KD in both tumor cell lines caused aberrant expression and activation of different JNK isoforms along with markedly diminished levels of the ubiquitin-editing enzyme A20. We demonstrated that differential up-regulation of JNK1 and JNK2 in the two tumor cell lines was responsible for the reciprocal regulation of c-Jun activity and tumor-specific proliferation. Furthermore, we showed that A20 reconstitution of IL-17RCKD clones with expression of full-length A20, but not a truncation-mutant, reversed aberrant JNK1/JNK2 activities and tumor-specific proliferation. Collectively, our study reveals a critical role of IL-17RC in maintaining baseline A20 production and a novel role of the IL-17RC-A20 axis in controlling JNK isoform-dependent tumor-specific homeostatic proliferation.

LUBAC (linear ubiquitin chain assembly complex) activates the canonical NF-κB pathway through linear polyubiquitination of NEMO (NF-κB essential modulator, also known as IKKγ) and RIP1. However, the regulatory mechanism of LUBAC-mediated NF-κB activation remains elusive. Here, we show that A20 suppresses LUBAC-mediated NF-κB activation by binding linear polyubiquitin via the C-terminal seventh zinc finger (ZF7), whereas CYLD suppresses it through deubiquitinase (DUB) activity. We determined the crystal structures of A20 ZF7 in complex with linear diubiquitin at 1.70-1.98 Å resolutions. The crystal structures revealed that A20 ZF7 simultaneously recognizes the Met1-linked proximal and distal ubiquitins, and that genetic mutations associated with B cell lymphomas map to the ubiquitin-binding sites. Our functional analysis indicated that the binding of A20 ZF7 to linear polyubiquitin contributes to the recruitment of A20 into a TNF receptor (TNFR) signalling complex containing LUBAC and IκB kinase (IKK), which results in NF-κB suppression. These findings provide new insight into the regulation of immune and inflammatory responses.

Natural killer (NK) cells protect hosts against viral pathogens and transformed cells. IL-15 is thought to play a critical role in NK cell development, but its role in the regulation of peripheral NK cells is less well defined. We now find that adoptive transfer of normal NK cells into mice lacking the high affinity interleukin (IL)-15 receptor, IL-15Ralpha, surprisingly results in the abrupt loss of these cells. Moreover, IL-15Ralpha-deficient NK cells can differentiate successfully in radiation bone marrow chimera bearing normal cells. Finally, adoptively transferred IL-15Ralpha-deficient NK cells survive in normal but not IL-15Ralpha-deficient mice. These findings demonstrate that NK cell-independent IL-15Ralpha expression is critical for maintaining peripheral NK cells, while IL-15Ralpha expression on NK cells is not required for this function.

Toll-like receptors (TLRs) on host cells are chronically engaged by microbial ligands during homeostatic conditions. These signals do not cause inflammatory immune responses in unperturbed mice, even though they drive innate and adaptive immune responses when combating microbial infections. A20 is a ubiquitin-modifying enzyme that restricts exogenous TLR-induced signals. We show that MyD88-dependent TLR signals drive the spontaneous T cell and myeloid cell activation, cachexia, and premature lethality seen in A20-deficient mice. We have used broad spectrum antibiotics to demonstrate that these constitutive TLR signals are driven by commensal intestinal flora. A20 restricts TLR signals by restricting ubiquitylation of the E3 ligase tumor necrosis factor receptor-associated factor 6. These results reveal both the severe proinflammatory pathophysiology that can arise from homeostatic TLR signals as well as the critical role of A20 in restricting these signals in vivo. In addition, A20 restricts MyD88-independent TLR signals by inhibiting Toll/interleukin 1 receptor domain-containing adaptor inducing interferon (IFN) beta-dependent nuclear factor kappaB signals but not IFN response factor 3 signaling. These findings provide novel insights into how physiological TLR signals are regulated.

Newborns are at high risk of developing neonatal sepsis, particularly if born prematurely. This has been linked to divergent requirements the immune system has to fulfill during intrauterine compared with extrauterine life. By transcriptomic analysis of fetal and adult neutrophils, we shed new light on the molecular mechanisms of neutrophil maturation and functional adaption during fetal ontogeny. We identified an accumulation of differentially regulated genes within the noncanonical NF-κB signaling pathway accompanied by constitutive nuclear localization of RelB and increased surface expression of TNF receptor type II in fetal neutrophils, as well as elevated levels of lymphotoxin α in fetal serum. Furthermore, we found strong upregulation of the negative inflammatory regulator A20 (Tnfaip3) in fetal neutrophils, which was accompanied by pronounced downregulation of the canonical NF-κB pathway. Functionally, overexpressing A20 in Hoxb8 cells led to reduced adhesion of these neutrophil-like cells in a flow chamber system. Conversely, mice with a neutrophil-specific A20 deletion displayed increased inflammation in vivo. Taken together, we have uncovered constitutive activation of the noncanonical NF-κB pathway with concomitant upregulation of A20 in fetal neutrophils. This offers perfect adaption of neutrophil function during intrauterine fetal life but also restricts appropriate immune responses particularly in prematurely born infants.

The generation and efficient maintenance of antigen-specific memory T cells is essential for long-lasting immunological protection. In this study, we examined the role of interleukin (IL)-15 in the generation and maintenance of virus-specific memory CD8 T cells using mice deficient in either IL-15 or the IL-15 receptor alpha chain. Both cytokine- and receptor-deficient mice made potent primary CD8 T cell responses to infection with lymphocytic choriomeningitis virus (LCMV), effectively cleared the virus and generated a pool of antigen-specific memory CD8 T cells that were phenotypically and functionally similar to memory CD8 T cells present in IL-15(+/+) mice. However, longitudinal analysis revealed a slow attrition of virus-specific memory CD8 T cells in the absence of IL-15 signals. This loss of CD8 T cells was due to a severe defect in the proliferative renewal of antigen-specific memory CD8 T cells in IL-15(-/-) mice. Taken together, these results show that IL-15 is not essential for the generation of memory CD8 T cells, but is required for homeostatic proliferation to maintain populations of memory cells over long periods of time.

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.

ClinicalTrials.gov Clinical Trial NCT00594880.

Ubiquitylation of the TNFR1 signalling complex (TNF-RSC) controls the activation of RIPK1, a kinase critically involved in mediating multiple TNFα-activated deleterious events. However, the molecular mechanism that coordinates different types of ubiquitylation modification to regulate the activation of RIPK1 kinase remains unclear. Here, we show that ABIN-1/NAF-1, a ubiquitin-binding protein, is recruited rapidly into TNF-RSC in a manner dependent on the Met1-ubiquitylating complex LUBAC to regulate the recruitment of A20 to control Lys63 deubiquitylation of RIPK1. ABIN-1 deficiency reduces the recruitment of A20 and licenses cells to die through necroptosis by promoting Lys63 ubiquitylation and activation of RIPK1 with TNFα stimulation under conditions that would otherwise exclusively activate apoptosis in wild-type cells. Inhibition of RIPK1 kinase and RIPK3 deficiency block the embryonic lethality of Abin-1 -/- mice. We propose that ABIN-1 provides a critical link between Met1 ubiquitylation mediated by the LUBAC complex and Lys63 deubiquitylation by phospho-A20 to modulate the activation of RIPK1.

The high affinity interleukin (IL)-15 receptor, IL-15Ralpha, is essential for supporting lymphoid homeostasis. To assess whether IL-15Ralpha's role in vivo is to trans present IL-15, we generated mixed bone marrow chimera from IL-15Ralpha- and IL-2/15Rbeta-deficient mice. We find that IL-15Ralpha-competent, IL-2/15Rbeta-deficient cells are able to support IL-15Ralpha-deficient natural killer (NK) and memory CD8+ T cells, thus ruling out secondary signals on these cells and demonstrating that IL-15Ralpha-mediated presentation of IL-15 in trans is the primary mechanism by which IL-15Ralpha functions in vivo. Surprisingly, using IL-15- and IL-15Ralpha-deficient mixed chimera, we also find that IL-15 and IL-15Ralpha must be expressed by the same cells to present IL-15 in trans, indicating that IL-15Ralpha is required on a cellular level for the elaboration of IL-15. These studies indicate that IL-15Ralpha defines homeostatic niches for NK and memory CD8+ T cells by controlling both the production and the presentation of IL-15 in trans to NK and CD8+ memory T cells.

Proteins that directly regulate tumour necrosis factor receptor (TNFR) signalling have critical roles in regulating cellular activation and survival. ABIN-1 (A20 binding and inhibitor of NF-kappaB) is a novel protein that is thought to inhibit NF-kappaB signalling. Here we show that mice deficient for ABIN-1 die during embryogenesis with fetal liver apoptosis, anaemia and hypoplasia. ABIN-1 deficient cells are hypersensitive to tumour necrosis factor (TNF)-induced programmed cell death, and TNF deficiency rescues ABIN-1 deficient embryos. ABIN-1 inhibits caspase 8 recruitment to FADD (Fas-associated death domain-containing protein) in TNF-induced signalling complexes, preventing caspase 8 cleavage and programmed cell death. Moreover, ABIN-1 directly binds polyubiquitin chains and this ubiquitin sensing activity is required for ABIN-1's anti-apoptotic activity. These studies provide insights into how ubiquitination and ubiquitin sensing proteins regulate cellular and organismal survival.

Resident microbes in skin and gut predominantly impact local immune cell function during homeostasis. However, colitis-associated neutrophilic skin disorders suggest possible breakdown of this compartmentalization with disease. Using a model wherein neonatal skin colonization by Staphylococcus epidermidis facilitates generation of commensal-specific tolerance and CD4+ regulatory T cells (Tregs), we ask whether this response is perturbed by gut inflammation. Chemically induced colitis is accompanied by intestinal expansion of S. epidermidis and reduces gut-draining lymph node (dLN) commensal-specific Tregs. It also results in reduced commensal-specific Tregs in skin and skin-dLNs and increased skin neutrophils. Increased CD4+ circulation between gut and skin dLN suggests that the altered cutaneous response is initiated in the colon, and resistance to colitis-induced effects in Cd4creIl1r1fl/fl mice implicate interleukin (IL)-1 in mediating the altered commensal-specific response. These findings provide mechanistic insight into observed connections between inflammatory skin and intestinal diseases.