METTL14-Mediated m6A Modification of TNFAIP3 Involved in Inflammation in Patients With Active Rheumatoid Arthritis.

OBJECTIVE: The aim of the study was to investigate the role of N6 -methyladenosine (m6A) modification in the progression of rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy controls were collected. The expression of m6A modification-related proteins and m6A levels were detected using polymerase chain reaction (PCR), western blot, and m6A enzyme-linked immunosorbent assay (ELISA). The roles of methyltransferase-like 14 (METTL14) in the regulation of inflammation in RA was explored using methylated RNA immunoprecipitation (MeRIP) sequencing and RNA immunoprecipitation assays. Collagen antibody-induced arthritis (CAIA) mice were used as an in vivo model to study the role of METTL14 in the inflammation progression of RA. RESULTS: We found that m6A writer METTL14 and m6A levels were decreased in PBMCs of patients with active RA and correlated negatively with the disease activity score using 28 joint counts (DAS28). Knockdown of METTL14 downregulated m6A and promoted the secretion of inflammatory cytokines interleukin 6 (IL-6) and IL-17 in PBMCs of patients with RA. Consistently, METTL14 knockdown promoted joint inflammation accompanied by upregulation of IL-6 and IL-17 in CAIA mice. MeRIP sequencing and functional studies confirmed that tumor necrosis factor α induced protein 3 (TNFAIP3), a key suppressor of the nuclear factor-κB inflammatory pathway, was involved in m6A-regulated PBMCs. Mechanistic investigations revealed that m6A affected TNFAIP3 expression by regulation of messenger RNA stability and translocation in TNFAIP3 protein coding sequence. CONCLUSIONS: Our study highlights the critical roles of m6A on regulation of inflammation in RA progression. Treatment strategies targeting m6A modification may represent a new option for management of RA.

Ginsenoside Rh2 Regulates the CFAP20DC-AS1/MicroRNA-3614-3p/BBX and TNFAIP3 Axis to Induce Apoptosis in Breast Cancer Cells.

While a number of coding genes have explained the anticancer activity of ginsenoside Rh2, little is known about noncoding RNAs. This study was performed to elucidate the regulatory activity of long noncoding RNA (lncRNA) CFAP20DC-AS1, which is known to be downregulated by Rh2. MiR-3614-3p, which potentially binds CFAP20DC-AS1, was screened using the LncBase Predicted program, and the binding was verified by assaying the luciferase activity of a luciferase/lncRNA recombinant plasmid construct. The competitive endogenous RNA (ceRNA) relationship of the two RNAs was further validated by quantitative PCR after deregulation of each RNA using siRNA. The effect of miRNA and target genes on the MCF-7 cancer cell growth was determined by monitoring proliferation and apoptosis in the presence of Rh2 after deregulating the corresponding gene. The miRNA decreased the luciferase activity of the luciferase/CFAP20DC-AS1 fusion vector, confirming the binding. SiRNA-based deregulation of CFAP20DC-AS1 attenuated the expression of miR-3614-3p and vice versa. In contrast to CFAP20DC-AS1, miR-3614-3p was upregulated by Rh2, inhibiting proliferation but stimulating apoptosis of the MCF-7 cells. Target genes of miR-3614-3p, BBX and TNFAIP3, were downregulated by Rh2 and the miRNA but upregulated by the lncRNA. Rh2 inhibits CFAP20DC-AS1, which obscures the association of the lncRNA with miR-3614-3p, resulting in the suppression of oncogenic BBX and TNFAIP3. Taken together, the Rh2/CFAP20DC-AS1/miR-3614-3p/target gene axis contributes to the antiproliferation activity of Rh2 in cancer cells.

Mutation analysis of the TNFAIP3 in A20 haploinsufficiency: A case report.

INTRODUCTION: Haploinsufficiency of A20 (HA20) is a novel genetic disease presented by Zhou et al in 2016. A20 is a protein encoded by TNFAIP3. Loss-of-function mutation in TNFAIP3 will trigger a new autoinflammatory disease: HA20. HA20-affected patients may develop a wide range of clinical manifestations, such as Behcet disease, rheumatoid arthritis, rheumatic fever, juvenile idiopathic arthritis, and systemic lupus erythematosus. HA20 is rarely reported, thus remaining far from thoroughly understood. Sixty-one cases of HA20 have been reported worldwide, among which 29 cases were diagnosed with Behcet disease ultimately. Moreover, 3 cases have been reported in China, which was the first report of HA20 characterized by Behcet disease. A comprehensive understanding of the pathogenic genes of HA20 could help us apply targeted therapy as soon as possible to improve patients' survival rates. PATIENT CONCERNS: A 2-year-old 3-month-old child was presented to our hospital with recurrent infectious enteritis and stomatitis. DIAGNOSIS: Genetic mutations were detected immediately, and a novel pathogenic mutation was found in TNFAIP3. A heterozygous mutation (c.436-437deTC) located at TNFAIP3 was confirmed. The present research indicated that the TNFAIP3 mutation of c.436-437deTC (p.L147Qfs∗7) accounted for familial Behcet-like autoinflammatory syndrome in the child suffering from HA20, while no variation in this locus was found in her parents. INTERVENTIONS: Symptomatic treatments including oral administration of prednisone (12.5 mg/d) and iron supplement were performed, and repeated infection was no longer observed in the child. Pain and activity limitation was found in the knee joints. The treatment regimen was adjusted to oral prednisone (12.5 mg/dose, 2 doses/d) and subcutaneous injection of rhTNFR:Fc (12.5 mg/week).Outcomes: At the last follow-up, the limbs' activities were normal, the inflammatory indicators were reduced or within the normal range. The prednisone dose was reduced to 7.5 mg/d, while the dose of rhTNFR:Fc was not changed. CONCLUSION: We have identified a novel pathogenic HA20 mutation. In this article, 1 case was analyzed in-depth in terms of clinical manifestations of the patient and new sources of such a novel disease, which might improve our understanding of this disease.

Potential of the tumor­derived extracellular vesicles carrying the miR­125b­5p target TNFAIP3 in reducing the sensitivity of diffuse large B cell lymphoma to rituximab.

Diffuse large B­cell lymphoma (DLBCL) is the most common and aggressive form of non­Hodgkin's lymphoma. Extracellular vesicles (EVs) derived from cancer cells are known to modify the tumor microenvironment. The aim of the present study was to investigate the role of miR­125b­3p carried by EVs in DLBCL in vitro and in vivo. TNFAIP3 expression in patient lesions was measured and the upstream miR that regulates TNFAIP3 was predicted using the starBase database. EVs were isolated from DLBCL cells and identified. DLBCL cells were transfected with pcDNA to overexpress TNFAIP3 or inhibit miR­125b­5p expression, incubated with EVs, and treated with rituximab to compare cell growth and TNFAIP3/CD20 expression. DLBCL model mice were administered EVs, conditioned medium, and rituximab to observe changes in tumor size, volume, and weight. TNFAIP3 was downregulated in patients with DLBCL and its levels further decreased in patients with drug­resistant DLBCL. Overexpression of TNFAIP3 in DLBCL cells enhanced the inhibitory effect of rituximab and increased CD20 expression. miR­125b­5p targeted TNFAIP3. Inhibition of miR­125b­5p enhanced the inhibitory effect of rituximab in DLBCL cells. The EV­carried miR­125b­5p reduced the sensitivity of DLBCL cells to rituximab, which was averted by overexpression of TNFAIP3. EVs reduced the sensitivity of DLBCL model mice to rituximab via the miR­125b­5p/TNFAIP3 axis. The study findings indicate that the tumor­derived EVs carrying miR­125b­5p can enter DLBCL cells and target TNFAIP3, thus reducing the sensitivity of DLBCL to rituximab, which may provide a novel therapeutic approach for DLBCL.

Formononetin attenuates atopic dermatitis by upregulating A20 expression via activation of G protein-coupled estrogen receptor.

ETHNOPHARMACOLOGICAL RELEVANCE: Atopic dermatitis (AD) is a complex skin disease with highly heterogeneous inflammation, which ranks among the largest component of the nonfatal diseases worldwide. The medications currently used to treat AD primarily include antihistamines, vitamin D and anti-inflammatory drugs, etc. But, the usage of these drugs is usually accompanied by various side-effects. Formononetin (FMN), a natural active ingredient of Astragalus membranaceus (Fisch.) Bunge, decreases the AD relapse rate, reduces recurring severity incidence and resists the inflammation in the initial stage of AD. However, the underlying mechanism of FMN on repressing the development of AD is still unknown. AIM OF THE STUDY: To investigate the potential mechanism of FMN on relieving the initial responses of AD and elucidate its possible therapeutic targets in vivo and in vitro. MATERIALS AND METHODS: A fluorescein isothiocyanate (FITC)-induced mouse model of the initial stage of AD was established in vivo. Human keratinocytes (HaCaT) cells were co-stimulated with tumor necrosis factor alpha (TNF-α) and polyinosinic-polycytidylic acid (Poly(I:C)) in vitro. The production of thymic stromal lymphopoietin (TSLP) and immunoglobulin E (IgE) were detected by enzyme-linked immunosorbnent assay (ELISA). The protein expression was measured through immunohistochemistry and western blotting. The mRNA expression was examined by real-time quantitative polymerase chain reaction (RT-qPCR). The impact of TNF-α-induced protein 3 (TNFAIP3/A20) was reflected using its small interfering RNA (siRNA). The role of G protein-coupled estrogen receptor (GPER) was explored using its agonist (G1), antagonist (G15) or siRNA (siGPER) in vitro. RESULTS: We found that FMN upregulated the expression of A20 protein and mRNA in the initial stage of AD model, especially in the epithelial region of ear tissue, and inhibited the production of TSLP simultaneously. Consistently, FMN significantly upregulated A20 protein and its mRNA expression while reduced TSLP protein and its mRNA expression in vitro, and this effect could be antagonized by A20 siRNA (siA20). Moreover, compared with PPT (ERα agonist) and DPN (ERß agonist), G1 could significantly increase the expression of A20. In addition, compared with MPP (ERα antagonist) and PHTPP (ERß antagonist), G15 could markedly reduce the expression of A20. Furthermore, the effects of FMN on A20 were interfered by siGPER and G15 in vitro and in vivo. CONCLUSIONS: These results demonstrated that FMN attenuated AD by upregulating A20 expression via activation of GPER. This new strategy might have effective therapeutic potential for AD and other inflammatory disorders.

ISSAID/EMQN Best Practice Guidelines for the Genetic Diagnosis of Monogenic Autoinflammatory Diseases in the Next-Generation Sequencing Era.

BACKGROUND: Monogenic autoinflammatory diseases are caused by pathogenic variants in genes that regulate innate immune responses, and are characterized by sterile systemic inflammatory episodes. Since symptoms can overlap within this rapidly expanding disease category, accurate genetic diagnosis is of the utmost importance to initiate early inflammation-targeted treatment and prevent clinically significant or life-threatening complications. Initial recommendations for the genetic diagnosis of autoinflammatory diseases were limited to a gene-by-gene diagnosis strategy based on the Sanger method, and restricted to the 4 prototypic recurrent fevers (MEFV, MVK, TNFRSF1A, and NLRP3 genes). The development of best practices guidelines integrating critical recent discoveries has become essential. METHODS: The preparatory steps included 2 online surveys and pathogenicity annotation of newly recommended genes. The current guidelines were drafted by European Molecular Genetics Quality Network members, then discussed by a panel of experts of the International Society for Systemic Autoinflammatory Diseases during a consensus meeting. RESULTS: In these guidelines, we combine the diagnostic strength of next-generation sequencing and recommendations to 4 more recently identified genes (ADA2, NOD2, PSTPIP1, and TNFAIP3), nonclassical pathogenic genetic alterations, and atypical phenotypes. We present a referral-based decision tree for test scope and method (Sanger versus next-generation sequencing) and recommend on complementary explorations for mosaicism, copy-number variants, and gene dose. A genotype table based on the 5-category variant pathogenicity classification provides the clinical significance of prototypic genotypes per gene and disease. CONCLUSIONS: These guidelines will orient and assist geneticists and health practitioners in providing up-to-date and appropriate diagnosis to their patients.

Herbs-partitioned moxibustion improves intestinal epithelial tight junctions by upregulating A20 expression in a mouse model of Crohn's disease.

BACKGROUND: To investigate effects moxibustion exerts on A20 expression and regulation of intestinal epithelial tight junctions via the TNF-α-NF-κB-MLCK pathway in Crohn's disease (CD). METHODS: C57BL/6 wild type (WT) and A20IEC-KO mice (48 each) were randomly divided into normal control (NC), model control (MC), mesalazine (MESA) and herbs-partitioned moxibustion (HPM) groups (12 mice per group). An experimental model of CD was established using 2, 4, 6 trinitrobenzene sulfonic acid. MESA and HPM mice were treated with MESA and HPM (at Tianshu (ST25) and Qihai (CV6)), respectively. In HPM group, moxa cones (0.5 cm in diameter and 0.3 cm in height) made of refined mugwort floss were placed on herbal cakes (medicinal formula dispensing [radix] Aconiti praeparata, [cortex] Cinnamomi, etc.) at Tianshu (ST25) and Qihai (CV6) and ignited. The moxa cones were ignited, and two moxa cones were used for each treatment once daily for 10 days. In MESA group, mice were fed MESA, which was prepared at a proportion of 1:0.0026, twice daily for 10 days. RESULTS: Intestinal epithelial ultrastructure of WT HPM mice improved more than A20IEC-KO HPM mice compared to MC mice. WT HPM mice exhibited greater expression of A20 compared with MC mice (P < 0.01). TNF-α, NF-kB p65, MLCK, MLC, TRAF6 and RIP1 levels in A20IEC-KO and WT HPM mice were all decreased compared to MC mice (Pall < 0.01). NF-κB p65、MLCK and TRAF6 levels were increased in A20IEC-KO HPM mice as compared to WT HPM mice (Pall < 0.05). Intestinal epithelial levels of occludin, claudin-1, ZO-1 and F-actin increased in all HPM mice (Pall  < 0.01-0.05), while occludin, claudin-1, and ZO-1 levels were lower in A20 IEC-KO HPM mice (P < 0.05, P < 0.01, P < 0.01). CONCLUSION: HPM downregulates abnormal activation of the TNF-α-NF-κB-MLCK pathway by upregulating expression of A20 in a mouse model of CD, thereby protecting intestinal epithelial tight junctions and repairing the damage CD causes to the intestinal epithelial barrier.

Zinc inhibited LPS-induced inflammatory responses by upregulating A20 expression in microglia BV2 cells.

BACKGROUND: Our previous studies have proved that zinc supplement effectively alleviate depression symptoms in mice, but the mechanisms are still uncertain. Neuroinflammation is considered as an important aspect in pathogenesis of depression. To elucidate the role of zinc on neuroinflammation, in this study, we investigated effects of zinc on lipopolysaccharide (LPS)-induced inflammation in BV2 microglia cells, a kind of innate immune cells in central nervous system. METHODS: BV2 cells were treated by 100 ng/ml LPS to induce inflammatory responses and the effects of zinc sulfate (ZnSO4) addition on LPS-induced inflammation were observed. Besides, through culturing HT-22 hippocampus cells by using medium transferred from zinc-intervened BV2 cells, the protective roles of zinc on hippocampus cells were identified. RESULTS: LPS treatment up-regulated expressions of CD11b, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) and level of reactive oxygen species (ROS). Meaningfully, zinc was capable of blocking ROS generation and reducing expressions of the above inflammatory cytokines at both 10 µM and 30 µM. In addition, it was proved that zinc intervention to BV2 cells could increase the viabilities of hippocampal HT-22 cells cultured by medium of BV2 cells. Furthermore, the zinc-finger protein A20, an anti-inflammation factor, was increased by zinc supplement, while levels of p65, p-IκB and p-p65 were significantly decreased. LIMITATIONS: More compelling proofs were needed to ensure roles of A20 in anti-inflammatory effects of zinc. CONCLUSIONS: The present results suggested that zinc inhibits inflammatory responses mediated by microglia cells via upregulation of zinc-finger A20. It was proposed that this anti-inflammatory action might be underlying mechanism of previously observed anti-depressive effects of zinc.

IL-17R deletion predicts high-grade colorectal cancer and poor clinical outcomes.

The IL-17 receptor (IL-17R) has a perplexing role in cancer, which may be explained by its yin-yang signaling pathways. Recently, the critical role of IL-17R in maintaining basal levels of A20-a key negative regulator of NF-κB and JNK-c-Jun pathways has been demonstrated in cancer cell lines. Cross-cancer analyses of somatic copy number alterations in IL-17RA, IL-17RC and A20 genes reveal that IL-17RA-deletion is common in colorectal cancer (CRC) patients, representing 24, 26, 37 and 49% of stage I, II, III and IV of patients, respectively, and mutually exclusive with patients displaying microsatellite instability. Importantly, patients with IL-17R-deletion or concurrent deletions of A20 show significantly reduced overall survival. Analysis of multiple published microarray studies confirms that IL-17RA expression is significantly reduced in CRC samples compared to normal counterparts, and its level is closely associated with A20 expression. Analyses of RNAseq data indicate that tumors with IL-17R-deletion express strong molecular markers of tumor invasion, growth and metastasis. Notably, approximately 20 genes responsible for protein synthesis and mitochondrial metabolism are inversely correlated with both IL-17RA and A20. Immunohistochemistry staining in human colorectal tissue arrays further reveals that high-grade tumors have significantly reduced IL-17RA staining compared to low-grade tumors. Thus, collective evidence strongly supports a previously unrecognized CRC-promoting mechanism triggered by IL-17RA-deletion and highlights its utility as a prognostic marker in CRC.

Zinc Inhibits Phosphate-Induced Vascular Calcification through TNFAIP3-Mediated Suppression of NF-κB.

Background The high cardiovascular morbidity and mortality of patients with CKD may result in large part from medial vascular calcification, a process promoted by hyperphosphatemia and involving osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Reduced serum zinc levels have frequently been observed in patients with CKD, but the functional relevance of this remains unclear.Methods We performed experiments in primary human aortic VSMCs; klotho-hypomorphic (kl/kl), subtotal nephrectomy, and cholecalciferol-overload mouse calcification models; and serum samples from patients with CKD.Results In cultured VSMCs, treatment with zinc sulfate (ZnSO4) blunted phosphate-induced calcification, osteo-/chondrogenic signaling, and NF-κB activation. ZnSO4 increased the abundance of zinc-finger protein TNF-α-induced protein 3 (TNFAIP3, also known as A20), a suppressor of the NF-κB pathway, by zinc-sensing receptor ZnR/GPR39-dependent upregulation of TNFAIP3 gene expression. Silencing of TNFAIP3 in VSMCs blunted the anticalcific effects of ZnSO4 under high phosphate conditions. kl/kl mice showed reduced plasma zinc levels, and ZnSO4 supplementation strongly blunted vascular calcification and aortic osteoinduction and upregulated aortic Tnfaip3 expression. ZnSO4 ameliorated vascular calcification in mice with chronic renal failure and mice with cholecalciferol overload. In patients with CKD, serum zinc concentrations inversely correlated with serum calcification propensity. Finally, ZnSO4 ameliorated the osteoinductive effects of uremic serum in VSMCs.Conclusions Zinc supplementation ameliorates phosphate-induced osteo-/chondrogenic transdifferentiation of VSMCs and vascular calcification through an active cellular mechanism resulting from GPR39-dependent induction of TNFAIP3 and subsequent suppression of the NF-κB pathway. Zinc supplementation may be a simple treatment to reduce the burden of vascular calcification in CKD.

TNF phase III signalling in tolerant cells is tightly controlled by A20 and CYLD.

Following the acute phase of an inflammatory reaction, a strictly controlled resolution of inflammation is necessary. A dysregulation of this process leads to hyperinflammation, chronic inflammatory disease, or immune paralysis. Different mechanisms participate in the coordinated termination of the inflammatory process, e.g. the expression of antiinflammatory molecules and different forms of tolerance. To better understand the processes which mediate resolution of TNF-dependent inflammation and induce tolerance, it is necessary to characterize the signal transduction quality during TNF long-term (pre)incubation. Within a time frame from 12 to 48h, designated as phase III of the TNF response, we measured an ongoing, constitutive activation of TNFR1/NF-κB-dependent pathways in monocytic cells. Phase III signalling which was also named "constitutive signaling in TNF tolerant cells" induces the expression of low- and high-sensitive target genes including A20 which is differentially regulated by transcriptional and proteolytic events. A20 strictly controls TNF long-term constitutive signalling in an IκB kinase complex- and partially RIP-dependent manner supported by adjuvant ABIN1. In addition, CYLD proteins participate in the regulation of this late-phase signal transduction, whereas downstream molecules such as Bcl3 and p50 are not involved. A20 and CYLD are expressed with different mRNA kinetics resulting in a strong or only a modest increase in protein levels, respectively. The identification of mechanisms which contribute to the termination of inflammation will provide additional diagnostic and therapeutic aspects to specifically diagnose certain aspects of inflammation and specifically modulate them.

Upregulation of neuronal zinc finger protein A20 expression is required for electroacupuncture to attenuate the cerebral inflammatory injury mediated by the nuclear factor-kB signaling pathway in cerebral ischemia/reperfusion rats.

BACKGROUND: Zinc finger protein A20 (tumor necrosis factor alpha-induced protein 3) functions as a potent negative feedback inhibitor of the nuclear factor-kB (NF-kB) signaling. It exerts these effects by interrupting the activation of IkB kinase beta (IKKß), the most critical kinase in upstream of NF-kB, and thereby controlling inflammatory homeostasis. We reported previously that electroacupuncture (EA) could effectively suppress IKKß activation. However, the mechanism underlying these effects was unclear. Therefore, the current study further explored the effects of EA on A20 expression in rat brain and investigated the possible mechanism of A20 in anti-neuroinflammation mediated by EA using transient middle cerebral artery occlusion (MCAO) rats. METHODS: Rats were treated with EA at the "Baihui (GV20)," "Hegu (L14)," and "Taichong (Liv3)" acupoints once a day starting 2 h after focal cerebral ischemia. The spatiotemporal expression of A20, neurobehavioral scores, infarction volumes, cytokine levels, glial cell activation, and the NF-kB signaling were assessed at the indicated time points. A20 gene interference (overexpression and silencing) was used to investigate the role of A20 in mediating the neuroprotective effects of EA and in regulating the interaction between neuronal and glial cells by suppressing neuronal NF-kB signaling during cerebral ischemia/reperfusion-induced neuroinflammation. RESULTS: EA treatment increased A20 expression with an earlier peak and longer lasting upregulation. The upregulated A20 protein was predominantly located in neurons in the cortical zone of the ischemia/reperfusion. Furthermore, neuronal A20 cell counts were positively correlated with neurobehavioral scores but negatively correlated with infarct volume, the accumulation of pro-inflammatory cytokines, and glial cell activation. Moreover, the effects of EA on improving the neurological outcome and suppressing neuroinflammation in the brain were reversed by A20 silencing. Finally, A20 silencing also suppressed the ability of EA to inhibit neuronal NF-kB signaling pathway. CONCLUSIONS: Ischemia/reperfusion cortical neurons in MCAO rats are the main cell types that express A20, and there is a correlation between A20 expression and the suppression of neuroinflammation and the resulting neuroprotective effects. EA upregulated neuronal A20 expression, which played an essential role in the anti-inflammatory effects of EA by suppressing the neuronal NF-kB signaling pathway in the brains of MCAO rats.

Rewired NFκB signaling as a potentially actionable feature of activated B-cell-like diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive lymphoma in the Western world and remains a clinical challenge. Two types of DLBCL are distinguishable, namely a germinal center B-cell-like phenotype (GCB) and an activated B-cell-like phenotype (ABC). Particularly ABC-DLBCL is difficult to treat, as this subentity typically displays resistance against frontline chemo-immune therapy. Through the availability of novel experimental technologies, such as next-generation sequencing and cutting-edge mouse models, we recently caught an unprecedentedly detailed glimpse at the genomic and biological features of ABC-DLBCL. Currently, a picture is emerging which suggests that ABC-DLBCL critically depends on sustained activity of the NFκB pathway, which, among others, is achieved through numerous distinct genetic aberrations, including CD79A/B-, CARD11-, and MYD88 mutations. Further genomic aberrations include amplifications of BCL2 and inactivating mutations in PRMD1. These molecular insights have spurred the development of novel autochthonous mouse models that faithfully mimic the biology and genetics of human ABC-DLBCL and could serve as preclinical platforms in future experiments. Furthermore, our genomic understanding of the disease now enables us to develop and validate novel targeted therapeutic intervention strategies that aim at decapitating non-physiological NFκB activity and repressing anti-apoptotic BCL2 signaling. In this review, we highlight these recent developments and make suggestions for further tool development and the design and stratification of future clinical trials.

RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury.

Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia-reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia-reperfusion injury model, the high-dose TNF model, and in A20(-/-) mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia-reperfusion injury model and no benefit in A20(-/-) mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.