TNF Stimulation Research Articles

Regulation and function of apoptosis signal-regulating kinase 1 in rheumatoid arthritis

Despite improved therapy, rheumatoid arthritis (RA) remains an unmet medical need. Previous efforts to validate therapeutic targets in the mitogen-activated protein kinase (MAPK) family have had minimal success. Therefore, we evaluated the potential for targeting an upstream MAPK, namely apoptosis signal-regulating kinase 1 (ASK1), as an alternative approach. ASK1 protein and gene expression were observed in RA and osteoarthritis (OA) synovium as determined by immunohistochemistry (IHC) and qPCR, respectively, particularly in the synovial intimal lining. For RA, but not OA synovium, ASK1 correlated with IL-1β and TNF gene expression. ASK1 was also expressed by cultured fibroblast-like synoviocytes (FLS), with significantly higher levels in RA compared with OA cells. IL-1β and TNF stimulation significantly increased ASK1 expression in a time-and concentration-dependent manner in cultured FLS. ASK1 promoter activity was significantly increased by IL-1β and TNF and was dependent on an upstream RelA binding motif. A selective small molecule ASK1 inhibitor reduced RA FLS invasion, migration and proliferation in vitro and decreased arthritis severity in the rat collagen-induced arthritis (CIA) model. In summary, our findings demonstrate that ASK1 modulates signaling pathways relevant to RA in vitro and in vivo. It is induced by inflammatory cytokines through the activation of NF-κB, which could provide some site- and event specificity. Thus, inhibitors of the upstream MAPK ASK1 could be a novel approach to treating inflammatory arthritis.

The chemokine receptor CCR10 promotes inflammation-driven hepatocarcinogenesis via PI3K/Akt pathway activation

G-protein-coupled receptor (GPCR)-related proteins are dysregulated and the GPCR CC-chemokine receptor 10 (CCR10) is significantly upregulated in inflammation-driven HCC. However, CCR10′s role in inflammation-driven hepatocarcinogenesis remains unknown. The aim of this study was to evaluate the role of CCR10 in inflammation-driven hepatocarcinogenesis. Via a targeted gene expression microarray screening alterations in GPCR family gene expression, we found CCR10 to be significantly upregulated in hepatocytes isolated from inflammation-driven human HCC tumors and matching paracancerous tissues. Tetrachloromethane (CCl4)-induced and diethylnitrosamine (DEN)-induced murine models of inflammatory hepatocarcinogenesis displayed significant hepatocellular TNF and CCR10 upregulation. Exogenous TNF applied to HepG2 and LO2 cell lines as well as wild-type (WT) mice significantly upregulated hepatocellular CCR10 expression, Akt phosphorylation, PCNA expression, and hepatocellular proliferation. Additionally, exogenous TNF significantly upregulated secretion of the natural CCR10 ligand-agonist CCL28 from both cell lines. Transgenic CCR10-knockout (CCR10 KO) in DEN-treated mice significantly increased hepatocellular apoptosis levels and significantly lowered compensatory hepatocellular proliferation but did not affect upstream TNF expression. In addition, DEN-treated CCR10 KO mice showed a significantly lower liver weight/body weight ratio, significantly lower liver tumor incidence, and significantly smaller tumors. Moreover, exogenous CCR10 expression significantly raised xenograft tumor growth in Balb/c nude mice. In vitro, CCR10 transfection or CCL28 treatment in HepG2 and LO2 cell lines significantly increased Akt phosphorylation, PCNA expression, and cell proliferation, while CCR10 silencing or Akt inhibition produced the opposite effects. In vivo, hepatocytes isolated from HCC tumor tissue and matching paracancerous tissue in DEN-treated CCR10 KO mice showed significantly lower Akt phosphorylation and PCNA expression relative to WT hepatocytes. In conclusion, inflammation-induced TNF promotes hepatocellular CCR10 expression and downstream PI3K/Akt-mediated hepatocarcinogenesis. CCR10 appears to function as a linkage between TNF stimulation and downstream PI3K/Akt pathway activation and shows promise as a potential therapeutic target for inflammation-driven HCC.

TNF induces neutrophil adhesion via formin-dependent cytoskeletal reorganization and activation of β-integrin function

Although essential for inflammatory responses, leukocyte recruitment to blood vessel walls in response to inflammatory stimuli, such as TNF-α, can contribute to vascular occlusion in inflammatory diseases, including atherosclerosis. We aimed to further characterize the mechanisms by which TNF stimulates adhesive and morphologic alterations in neutrophils. Microfluidic and intravital assays confirmed the potent effect that TNF has on human and murine neutrophil adhesion and recruitment in vitro and in vivo, respectively. Inhibition of actin polymerization by cytochalasin D significantly diminished TNF-induced human neutrophil adhesion in vitro and abolished TNF-induced membrane alterations and cell spreading. In contrast, TNF-induced increases in β2-integrin (Mac-1 and LFA-1) expression was not significantly altered by actin polymerization inhibition. Consistent with a role for cytoskeletal rearrangements in TNF-induced adhesion, TNF augmented the activity of the Rho GTPase, RhoA, in human neutrophils. However, inhibition of the major RhoA effector protein, Rho kinase (ROCK), by Y-27632 failed to inhibit TNF-induced neutrophil adhesion. In contrast, the formin FH2 domain inhibitor, SMIFH2, abolished TNF-induced human neutrophil adhesion and diminished leukocyte recruitment in vivo. SMIFH2 also inhibited TNF-induced cytoskeletal reorganization in human neutrophils and abolished the alterations in β2-integrin expression elicited by TNF stimulation. As such, Rho GTPase/mDia formin-mediated cytoskeletal reorganization appears to participate in the orchestration of TNF-induced neutrophil-adhesive interactions, possibly mediated by formin-mediated actin nucleation and subsequent modulation of β2-integrin activity on the neutrophil surface. This pathway may represent a pharmacologic target for reducing leukocyte recruitment in inflammatory diseases.

The N-terminal ubiquitin-associated domain of Cezanne is crucial for its function to suppress NF-κB pathway.

Cezanne, a deubiquitinating cysteine protease (DUB) belonging to A20 subgroup of ovarian tumor (OTU) protein superfamily, functions as a negative regulator of NF-κB to attenuate NF-κB activation and to restrain pro-inflammatory transcription in response to TNF receptor (TNFR) signaling. It is the first documented OTU DUB that preferably disassembles Lys11-linked polyubiquitin chains and has been shown to regulate multiple cellular events including immune signaling, cell survival and tumor progression. Previous studies showed that in response to TNF stimulation, Cezanne is recruited to the activated TNFR complex to suppress the build-up of polyubiquitinated RIP1 signal by removing Lys63 polyubiquitin from RIP1. However, how is Cezanne recognized and recruited to TNFR complex is not clear yet. In this study, we characterized a ubiquitin-associated (UBA) domain in the N-terminal region of Cezanne and proved its activity to bind Lys63 polyubiquitin chain. By constructing a series of truncated and site-specific point mutants, we further localized the crucial binding sites for Lys63 polyubiquitin chains at Leu9 and Ser10 sites of Cezanne UBA domain. Mutation at these sites disrupted the recruitment of Cezanne to activated TNFR complex and dramatically reduced the inhibition of NF-κB activation by Cezanne. Our study demonstrated that the N-terminal UBA domain is crucial for the function of Cezanne during NF-κB activation. Cezanne is recognized and recruited into activated TNFR complex by specifically binding to polyubiquitinated signaling proteins after TNF stimulation through its N-terminal polyubiquitin binding site. This study sheds light on the molecular mechanism of negative regulation of NF-κB activation by Cezanne.

MK2 phosphorylation of RIPK1 regulates TNF-mediated cell death.

TNF is a master proinflammatory cytokine whose pathogenic role in inflammatory disorders can, in certain conditions, be attributedto RIPK1 kinase-dependent cell death. Survival, however, is the default response of most cells to TNF stimulation, indicating that cell demise is normally actively repressed and that specific checkpoints must be turned off for cell death to proceed. We identified RIPK1 as a direct substrate of MK2 in the TNFR1 signalling pathway. Phosphorylation of RIPK1 by MK2 limits cytosolic activation of RIPK1 and the subsequent assembly of the death complex that drives RIPK1 kinase-dependent apoptosis and necroptosis. In line with these in vitro findings, MK2 inactivation greatly sensitizes mice to the cytotoxic effectsofTNF in an acute model of sterile shock caused by RIPK1-dependent cell death. In conclusion, we identified MK2-mediated RIPK1 phosphorylation as an important molecular mechanism limiting the sensitivity of the cells to the cytotoxic effects of TNF.

Ovarian steroids, oxytocin, and tumor necrosis factor modulate equine oviduct function

The oviduct plays important roles in the early reproductive process. The aim of this study was to evaluate gene transcription and protein expression of progesterone receptor (PGR), estrogen receptors 1 (ESR1) and 2 (ESR2); oxytocin receptor (OXTR); prostaglandin F2α synthase (AKR1C3), and prostaglandin E2 synthase (Ptges) in mare oviduct in different estrous cycle stages. Estradiol (E2), progesterone (P4), oxytocin (OXT), and tumor necrosis factor α (TNF) effect on in vitro PGE2 and prostaglandin F2α (PGF2α) secretion by equine oviduct explants or by oviductal epithelial cells (OECs) were also assessed. During the breeding season, oviduct tissue was obtained post mortem from cyclic mares. Protein of ESR1, ESR2, PGR, AKR1C3, and Ptges was present in OECs, whereas OXTR was shown in oviduct stroma. In follicular phase, protein expression of ESR1, ESR2, PGR, and OXTR increased in oviduct explants (P < 0.05), whereas no estrous cycle effect was noted for AKR1C3 or Ptges. In follicular phase, mRNA transcription was upregulated for Pgr but downregulated for Oxtr, Ptges, and Akr1c3 (P < 0.05). Nevertheless, Esr1 and Esr2 mRNA levels did not change with the estrous cycle. In the ampulla, Esr1, Esr2, and Oxtr mRNA transcription increased, but not for Pgr or Ptges. In contrast, Akr1c3 mRNA level was upregulated in the infundibulum (P < 0.05). In follicular phase, E2, P4, and OXT downregulated PGE2 production by OEC (P < 0.05), but no difference was observed in mid-luteal phase. Explants production of PGE2 rose when treated with OXT in follicular phase; with TNF or OXT in early luteal phase; or with TNF, OXT, or P4 in mid-luteal phase. PGF2α production by OEC was downregulated by all treatments in follicular phase but upregulated in mid-luteal phase (P < 0.05). Oviduct explants PGF2α production was stimulated by TNF or OXT in all estrous cycle phases. In conclusion, this work has shown that ESR1, ESR2, OXTR, Ptges, and AKRLC3 gene transcription and/or translation is estrous cycle dependent and varies with oviduct portion (infundibulum vs ampulla) and cell type. Ovarian steroid hormones, OXT and TNF stimulation of PGF2α and/or PGE2 production is also estrous cycle dependent and varies in the different portions of mare oviduct. Differential transcription level and protein localization in various portions of the oviduct throughout the estrous cycle, as well as PG production, suggest coordinated physiologic actions and mechanisms of steroid hormones, OXT, and TNF in the equine oviduct.

Enterotoxigenic Escherichia coli Flagellin Inhibits TNF-Induced NF-κB Activation in Intestinal Epithelial Cells.

Enterotoxigenic Escherichia coli (ETEC) causes childhood diarrhea in developing countries. ETEC strains produce the heat-labile enterotoxin (LT) and/or heat-stable enterotoxins (ST) and encode a diverse set of colonization factors used for adherence to intestinal epithelial cells. We previously found that ETEC secretes a heat-stable protein we designated as ETEC Secreted Factor (ESF) that inhibits the extent of NF-κB activation normally induced by tumor necrosis factor alpha (TNF). Here we fractionated ETEC supernatants using fast protein liquid chromatography (FPLC) and determined that ETEC flagellin was necessary and sufficient to protect IκBα from degradation in response to TNF stimulation. These data suggest a potentially novel mechanism by which ETEC may evade the host innate immune response by down-regulating NF-κB-dependent host responses.

A20 regulates NF-κB activation through K48 linked polyubiquitination of NEMO

Abstract Activation of the NF-κB family of transcription factors is related to diverse biological processes, such as development, immune responses, and inflammation. As a result, mutations affecting the NF-κB signaling pathway cause syndromes characterized by immunodeficiency and autoinflammaotory disease. Recently, we identified the C-terminus of NEMO to be a regulator of NF-κB signaling, as a class of NEMO mutations lacking the C-terminus (ΔCT-NEMO) confer gain-of-function properties to the IKK complex due to impaired recruitment of A20 to the TNFR. Here, we investigated details by which A20 recruitment to the TNFR suppresses NF-κB signaling. The C-terminus of NEMO has been previously identified to preferentially recognize K63-linked polyubiquitin that in its unanchored form can mediate NEMO/A20 interaction. We found that the IKK complex containing ΔCT-NEMO forms but not other mutant forms of NEMO exhibit impaired K63 polyubiquitin binding ability. In addition to unanchored K63-lilnked polyubiquitin, we hypothesized that direct polyubiquitination of A20 could enable a functional interaction between A20 and NEMO. Using mass spectroscopy, we identified 4 lysine residues on A20 to be ubiquitination sites. Mutation of these lysines to arginine impaired A20’s ability to suppress TNF induced NF-κB activation. We noted stabilization of NEMO in A20 deficient cells following TNF stimulation. Biochemical studies in Jurkat and 293 cells revealed A20 dependent K48 linked polyubiquitination of NEMO at the TNFR following receptor stimulation. Our data suggest a mechanism of NF-κB regulation in which A20 is directly K63 polyubiquitinated to functionally interacts with NEMO which it targets by K48 linkages proteasomal degradation.

A potential target for methotrexate in macrophages : AMP-activated protein kinase.

Abstract Background Methotrexate (MTX) remains a cornerstone of treatment in multiple forms of inflammatory arthritis. AMPK is a highly conserved protein kinase present in all eukaryotic cells and is activated by an increasing intracellular ADP/ATP levels. Once activated, AMPK will promote ATP production by switching on catabolic and turning off anabolic pathways. We hypothesize that AMPK activation mediates a major portion of the anti-inflammatory effects of MTX. Methods We investigated the role of the anti-inflammatory effect of MTX via AMPK in human monocytes-derived macrophages (MDM) and mouse bone marrow-derived macrophages (BMDM) along with AICAR and A769662 (AMPK activators) and compound C (a selective AMPK inhibitor). AMPK phosphorylation and total AMPK were measured by Western blotting. Cells were then stimulated with LPS or TNF-α, and production of pro-inflammatory cytokines were measured in the supernatant. Results MTX induced AMPK activation with effects comparable to the AMPK activators (A769662, AICAR ) in hMDM and BMDM. MTX-induced AMPK activation was associated with a reduction in the production of IL-6, IL-1 β, and TNF-α in response to LPS and TNF stimulation. Compound C is able to partially reverse the effects of MTX. Conclusions MTX is able to induce AMPK activation in both MDM and BMDM, and suppress pro-inflammatory cytokines in a manner dependent on AMPK activity. These results have been confirmed genetically in macrophages deficient in AMPK subunits and in serum transfer arthritis. Our findings raise the possibility that some anti-inflammatory effects of MTX are mediated by AMPK, suggest that AMPK may be a target for the anti-inflammatory agents and a target for the development of new anti-inflammatory drugs.

Overexpression of RANKL in osteoblasts: a possible mechanism of susceptibility to bone disease in cystic fibrosis.

Bone fragility and loss are a significant cause of morbidity in patients with cystic fibrosis (CF), and the lack of effective therapeutic options means that treatment is more often palliative rather than curative. A deeper understanding of the pathogenesis of CF-related bone disease (CFBD) is necessary to develop new therapies. Defective CF transmembrane conductance regulator (CFTR) protein and chronic inflammation in bone are important components of the CFBD development. The receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) drive the regulation of bone turnover. To investigate their roles in CFBD, we evaluated the involvement of defective CFTR in their production level in CF primary human osteoblasts with and without inflammatory stimulation, in the presence or not of pharmacological correctors of the CFTR. No major difference in cell ultrastructure was noted between cultured CF and non-CF osteoblasts, but a delayed bone matrix mineralization was observed in CF osteoblasts. Strikingly, resting CF osteoblasts exhibited strong production of RANKL protein, which was highly localized at the cell membrane and was enhanced in TNF (TNF-α) or IL-17-stimulated conditions. Under TNF stimulation, a defective response in OPG production was observed in CF osteoblasts in contrast to the elevated OPG production of non-CF osteoblasts, leading to an elevated RANKL-to-OPG protein ratio in CF osteoblasts. Pharmacological inhibition of CFTR chloride channel conductance in non-CF osteoblasts replicated both the decreased OPG production and the enhanced RANKL-to-OPG ratio. Interestingly, using CFTR correctors such as C18, we significantly reduced the production of RANKL by CF osteoblasts, in both resting and TNF-stimulated conditions. In conclusion, the overexpression of RANKL and high membranous RANKL localization in osteoblasts are related to defective CFTR, and may worsen bone resorption, leading to bone loss in patients with CF. Targeting osteoblasts with CFTR correctors may represent an effective strategy to treat CFBD. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Opposing regulation of the late phase TNF response by mTORC1-IL-10 signaling and hypoxia in human macrophages

Tumor necrosis factor (TNF) is best known for inducing a rapid but transient NF-κB-mediated inflammatory response. We investigated later phases of TNF signaling, after the initial transient induction of inflammatory genes has subsided, in primary human macrophages. TNF signaling induced expression of late response genes, including inhibitors of NF-κB and TLR signaling, with delayed and sustained kinetics 6–24 hr after TNF stimulation. A subset of late phase genes was expressed in rheumatoid arthritis synovial macrophages, confirming their expression under chronic inflammatory conditions in vivo. Expression of a subset of late phase genes was mediated by autocrine IL-10, which activated STAT3 with delayed kinetics. Hypoxia, which occurs at sites of infection or inflammation where TNF is expressed, suppressed this IL-10-STAT3 autocrine loop and expression of late phase genes. TNF-induced expression of IL-10 and downstream genes was also dependent on signaling by mTORC1, which senses the metabolic state of cells and is modulated by hypoxia. These results reveal an mTORC1-dependent IL-10-mediated late phase response to TNF by primary human macrophages, and identify suppression of IL-10 responses as a new mechanism by which hypoxia can promote inflammation. Thus, hypoxic and metabolic pathways may modulate TNF responses during chronic inflammation.

Abstract 2899: Selinexor inhibits NF-κB activity by sequestering IkB-a in the nucleus and blocking IkB-a degradation

Abstract Background: Selinexor is a small-molecule therapeutic that inhibits XPO1 mediated nuclear export, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) and subsequently cancer cell death while sparing normal cells. It has been previously demonstrated that the inhibitor of NFκB, IκB-, is localized to the cytoplasm by XPO1 in several cancer cell lines and that treatment of cancer cells with selinexor reduces NF-κB transcriptional activity. The mechanism of NF-κB inhibition by selinexor, however, is not fully understood. We hypothesized that nuclear retention of IκB- and down-regulation of IκB- kinase (IKK) in response to selinexor treatment would inhibit NF-κB transcriptional activity. Methods: U2OS (Osteosarcoma) cells were treated with selinexor in the presence or absence of TNF stimulation and whole protein lysates were analyzed by Western blotting. IκB- localization was evaluated by immunofluorescence microscopy and NF-κB transcriptional activity by ELISA (Thermo Scientific). Results: TNFα induced the phosphorylation of NF-κB p65 subunit on serine 536 and IκB- on serine 32/36 through IKK. This resulted in the dissociation of IκB-α from NF-κB and led to IκB-α degradation via the 26S proteasome. Free NF-κB could now migrate into the nucleus and initiate transcriptional activation supporting tumorigenicity and inflammation. The IKK kinase is a complex made of two kinases (IKKα and IKKβ) and one regulatory subunit, NEMO/IKKγ. We found that selinexor treatment blocked IKK activity through the down regulation of the IKK (IKKγ) gamma subunit protein levels. This inhibition was dose dependent and prevented IκB- phosphorylation, thereby protecting IκB- from degradation. The protection of intact IκB- from degradation and its forced nuclear accumulation through XPO1 inhibition enabled inhibition of NF-κB transcriptional activity even in the presence of TNFα. Selinexor did not alter the protein levels of IKK or IKK. Conclusions: Selinexor blocks TNFα induced degradation of IκB- by reducing the levels of IKK. In addition, selinexor increased nuclear IκB- levels through the inhibition of nuclear export. This blocked NF-κB activity and enhanced cancer cell death. We are currently investigating the beneficial effects of combining selinexor with proteasome inhibitors, which are known to prevent IκB- degradation. Citation Format: Trinayan Kashyap, Christian Argueta, Boris Klebanov, TJ Unger, Benjamin Link, Maxwell Werman, Margaret Lee, Sharon Shacham, Yosef Landesman. Selinexor inhibits NF-κB activity by sequestering IkB-a in the nucleus and blocking IkB-a degradation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2899.

Abnormal PTPN11 enhancer methylation promotes rheumatoid arthritis fibroblast-like synoviocyte aggressiveness and joint inflammation.

The PTPN11 gene, encoding the tyrosine phosphatase SHP-2, is overexpressed in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) compared with osteoarthritis (OA) FLS and promotes RA FLS invasiveness. Here, we explored the molecular basis for PTPN11 overexpression in RA FLS and the role of SHP-2 in RA pathogenesis. Using computational methods, we identified a putative enhancer in PTPN11 intron 1, which contained a glucocorticoid receptor- binding (GR-binding) motif. This region displayed enhancer function in RA FLS and contained 2 hypermethylation sites in RA compared with OA FLS. RA FLS stimulation with the glucocorticoid dexamethasone induced GR binding to the enhancer and PTPN11 expression. Glucocorticoid responsiveness of PTPN11 was significantly higher in RA FLS than OA FLS and required the differentially methylated CpGs for full enhancer function. SHP-2 expression was enriched in the RA synovial lining, and heterozygous Ptpn11 deletion in radioresistant or innate immune cells attenuated K/BxN serum transfer arthritis in mice. Treatment with SHP-2 inhibitor 11a-1 reduced RA FLS migration and responsiveness to TNF and IL-1β stimulation and reduced arthritis severity in mice. Our findings demonstrate how abnormal epigenetic regulation of a pathogenic gene determines FLS behavior and demonstrate that targeting SHP-2 or the SHP-2 pathway could be a therapeutic strategy for RA.

Loss of intestinal epithelial autophagy leads to catastrophic susceptibility to acute T. gondii-mediated inflammation

Abstract The protozoan parasite Toxoplasma gondii triggers severe small intestinal immunopathology characterized by inflammation, Paneth cell loss and bacterial dysbiosis. Paneth cells are the predominant secretory epithelial cells which reside at the base of the crypt and release antimicrobial peptides that maintain intestinal homeostasis. T. gondii-triggered IFN-γ is a major inducer of Paneth cell death. However, mechanisms of IFN-γ-mediated Paneth cell death are unknown. We recently observed that under steady state conditions Paneth cells undergo active autophagy, which is both microbiota- and IFN-γ-dependent. To study autophagy’s role in T. gondii–induced Paneth cell loss we used mice deficient in the critical autophagy gene Atg5 in the intestinal epithelium (Villin-Cre × ATGfl/fl mice, E-ATG5−/−). Infected E-ATG5−/− mice exhibited catastrophic inflammation, characterized by the complete destruction of the intestinal crypts, establishing ATG5’s protective role during intestinal inflammation. To determine ATG5’s protective mechanism we generated ATG5-deficient intestinal organoids and observed that lack of ATG5 increased sensitivity to the TNF-induced cell death. Organoid death was further exacerbated when TNF stimulation occurred in combination with IFN-γ. Reexamining our in vivo model showed significantly elevated levels of TNF receptor 2 in infected E-ATG5−/− mice, indicating these mice are prone to TNF-triggered small intestinal immunopathology. Our results reveal that ATG5 expression within Paneth cells plays a highly protective role against cytokine-mediated immunopathology in the intestine during acute T. gondii infection.

NLRX1 Acts as an Epithelial-Intrinsic Tumor Suppressor through the Modulation of TNF-Mediated Proliferation.

The mitochondrial Nod-like receptor protein NLRX1 protects against colorectal tumorigenesis through mechanisms that remain unclear. Using mice with an intestinal epithelial cells (IEC)-specific deletion of Nlrx1, we find that NLRX1 provides an IEC-intrinsic protection against colitis-associated carcinogenesis in the colon. These Nlrx1 mutant mice have increased expression of Tnf, Egf, and Tgfb1, three factors essential for wound healing, as well as increased epithelial proliferation during the epithelial regeneration phase following injury triggered by dextran sodium sulfate. In primary intestinal organoids lacking Nlrx1, stimulation with TNF resulted in exacerbated proliferation and expression of the intestinal stem cell markers Olfm4 and Myb. This hyper-proliferation response was associated with increased activation of Akt and NF-κB pathways in response to TNF stimulation. Together, these results identify NLRX1 as a suppressor of colonic tumorigenesis that acts by controlling epithelial proliferation in the intestine during the regeneration phase following mucosal injury.

Recruitment of A20 by the C-terminal domain of NEMO suppresses NF-κB activation and autoinflammatory disease

Receptor-induced NF-κB activation is controlled by NEMO, the NF-κB essential modulator. Hypomorphic NEMO mutations result in X-linked ectodermal dysplasia with anhidrosis and immunodeficiency, also referred to as NEMO syndrome. Here we describe a distinct group of patients with NEMO C-terminal deletion (ΔCT-NEMO) mutations. Individuals harboring these mutations develop inflammatory skin and intestinal disease in addition to ectodermal dysplasia with anhidrosis and immunodeficiency. Both primary cells from these patients, as well as reconstituted cell lines with this deletion, exhibited increased IκB kinase (IKK) activity and production of proinflammatory cytokines. Unlike previously described loss-of-function mutations, ΔCT-NEMO mutants promoted increased NF-κB activation in response to TNF and Toll-like receptor stimulation. Investigation of the underlying mechanisms revealed impaired interactions with A20, a negative regulator of NF-κB activation, leading to prolonged accumulation of K63-ubiquitinated RIP within the TNFR1 signaling complex. Recruitment of A20 to the C-terminal domain of NEMO represents a novel mechanism limiting NF-κB activation by NEMO, and its absence results in autoinflammatory disease.

TNF Regulates Essential Alternative Complement Pathway Components and Impairs Activation of Protein C in Human Glomerular Endothelial Cells.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy with severe renal injury secondary to an overactive alternative complement pathway (AP). aHUS episodes are often initiated or recur during inflammation. We investigated gene expression of the surface complement regulatory proteins (CD55, CD59, CD46, and CD141 [thrombomodulin]) and AP components in human glomerular microvascular endothelial cells (GMVECs) and in HUVECs, a frequently used investigational model of endothelial cells. Surface complement regulatory proteins were also quantified by flow cytometry. All experiments were done with and without exposure to IL-1β or TNF. Without cytokine stimulation, we found that GMVECs had greater AP activation than did HUVECs. With TNF stimulation, THBD gene expression and corresponding CD141 surface presence in HUVECs and GMVECs were reduced, and gene expression of complement components C3 (C3) and factor B (CFB) was increased. Consequently, AP activation, measured by Ba production, was increased, and conversion of protein C (PC) to activated PC by CD141-bound thrombin was decreased, in GMVECs and HUVECs exposed to TNF. IL-1β had similar, albeit lesser, effects on HUVEC gene expression, and it only slightly affected GMVEC gene expression. To our knowledge, this is the first detailed study of the expression/display of AP components and surface regulatory proteins in GMVECs with and without cytokine stimulation. In aHUS patients with an underlying overactive AP, additional stimulation of the AP and inhibition of activated PC-mediated anticoagulation in GMVECs by the inflammatory cytokine TNF are likely to provoke episodes of renal failure.

In TNF-stimulated cells, RIPK1 promotes cell survival by stabilizing TRAF2 and cIAP1, which limits induction of non-canonical NF-κB and activation of caspase-8.

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-κB, but also regulates caspase-8 activation and cell death. Although overexpression of RIPK1 can cause caspase-8-dependent cell death, when RIPK1(-/-) cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities. To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1(-/-) cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1(-/-) cells caused TRAF2 and cIAP1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-κB2 to p52, a decrease in levels of cFLIP(L), and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.

Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease.

Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity1. Herein we describe a new syndrome caused by high penetrance heterozygous germline mutations in the NFκB regulatory protein TNFAIP3 (A20) in six unrelated families with early onset systemic inflammation. The syndrome resembles Behçet’s disease (BD), which is typically considered a polygenic disorder with onset in early adulthood2. A20 is a potent inhibitor of the NFκB signaling pathway3. TNFAIP3 mutant truncated proteins are likely to act by haploinsufficiency since they do not exert a dominant-negative effect in overexpression experiments. Patients’ cells show increased degradation of IκBα and nuclear translocation of NFκB p65, and increased expression of NFκB-mediated proinflammatory cytokines. A20 restricts NFκB signals via deubiquitinating (DUB) activity. In cells expressing the mutant A20 protein, there is defective removal of K63-linked ubiquitin from TRAF6, NEMO, and RIP1 after TNF stimulation. NFκB-dependent pro-inflammatory cytokines are potential therapeutic targets for these patients.

TNF up-regulates Pentraxin3 expression in human airway smooth muscle cells via JNK and ERK1/2 MAPK pathways.

BackgroundLong pentraxin 3 (PTX3) is a novel candidate marker for inflammation in many chronic diseases. As a soluble pattern recognition receptor, PTX3 is involved in amplification of inflammatory reactions and regulation of innate immunity. Previously, we demonstrate that human airway smooth muscle cells (HASMC) express constitutively PTX3 and upon TNF stimulation. However, very little is known about the mechanism governing its expression in HASMC. We sought to investigate the mechanism governing TNF induced PTX3 expression in primary HASMC.MethodsHASMC were stimulated with TNF in the presence of transcriptional inhibitor actinomycin D (ActD) or MAPKs pharmacological inhibitors. PTX3 mRNA and protein expression were analyzed by Real-time RT-PCR and ELISA, respectively. PTX3 promoter activity was determined using luciferase assay.ResultsPTX3 mRNA and protein are expressed constitutively by HASMC and significantly up-regulated by TNF. TNF-induced PTX3 mRNA and protein release in HASMC were inhibited by transcriptional inhibitor actinomycin D. TNF induced significantly PTX3 promoter activation in HASMC. MAPK JNK and ERK1/2 specific inhibitors (SP600125 and UO126), but not p38, significantly down regulates TNF induced PTX3 promoter activity and protein release in HASMC. Finally, TNF mediated PTX3 promoter activity in HASMC was abolished upon mutation of NF-κβ and AP1 binding sites.ConclusionsOur data suggest that TNF induced PTX3 in HASMC at least via a transcriptional mechanism that involved MAPK (JNK and ERK1/2), NF-κβ and AP1 pathways. These results rise the possibility that HASMC derived PTX3 may participate in immune regulation in the airways.