Bingjiao Yin

Blockade of Tumor Necrosis Factor (TNF) Receptor Type 1-Mediated TNF-α Signaling Protected Wistar Rats from Diet-Induced Obesity and Insulin Resistance

TNF-alpha plays an important role in the pathogenesis of obesity and insulin resistance in which the effect of TNF-alpha signaling via TNF receptor type 1 (TNFR1) largely remains controversial. To delineate the role of TNFR1-mediated TNF-alpha signaling in the pathogenesis of this disorder, a TNFR1 blocking peptide-Fc fusion protein (TNFR1BP-Fc) was used for the present study. Wistar rats were fed a high-fat/high-sucrose (HFS) diet for 16 wk until obesity and insulin resistance developed. In comparison with increased body weight and fat weight, enlarged adipocytes, and hypertriglyceridemia in the obese state, the subsequent 4-wk treatment with TNFR1BP-Fc resulted in significant weight loss characterized by decreased fat pad weight and adipocyte size and reduced plasma triglycerides. Furthermore, obesity-induced insulin resistance, including hyperinsulinemia, elevated C-peptide, higher degree of hyperglycemia after glucose challenge, and less hypoglycemic response to insulin, was markedly improved, and the compensatory hyperplasia and hypertrophy of pancreatic islets were reduced. Interestingly, treatment with TNFR1BP-Fc markedly suppressed systemic TNF-alpha release and its local expression in pancreatic islets and muscle and adipose tissues. In addition, blockage of TNFR1-mediated TNF-alpha signaling in obese rats significantly enhanced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in the muscle and fat tissues. Our results strongly suggest a pivotal role for TNFR1-mediated TNF-alpha signaling in the pathogenesis of obesity and insulin resistance. Thus, TNFR1BP-Fc may be a good candidate for the treatment of this disease.

Transmembrane TNF-α Promotes Suppressive Activities of Myeloid-Derived Suppressor Cells via TNFR2

It has been reported that TNFR2 is involved in regulatory T cell induction and myeloid-derived suppressor cell (MDSC) accumulation, two kinds of immunosuppressive cells contributing to tumor immune evasion. Because transmembrane TNF-α (tmTNF-α) is the primary ligand for TNFR2, we hypothesized that tmTNF-α is mainly responsible for the activation of MDSCs. Indeed, we found that tmTNF-α, rather than secretory TNF-α (sTNF-α), activated MDSCs with enhanced suppressive activities, including upregulating arginase-1 and inducible NO synthase transcription, promoting secretion of NO, reactive oxygen species, IL-10, and TGF-β, and enhancing inhibition of lymphocyte proliferation. This effect of tmTNF-α was mediated by TNFR2, as TNFR2 deficiency significantly impaired tmTNF-α–induced release of IL-10 and NO and inhibition of T cell proliferation by MDSC supernatant. Furthermore, tmTNF-α caused p38 phosphorylation and NF-κB activation, whereas inhibition of NF-κB or p38 with an inhibitor pyrrolidine dithiocarbamate or SB203580 abrogated tmTNF-α–mediated increased suppression of lymphocyte proliferation by MDSCs. Consistently, our in vivo study showed that ectopic expression of uncleavable tmTNF-α mutant by 4T1 cells significantly promoted tumor progression and angiogenesis, accompanied with more accumulation of MDSCs and regulatory T cells in the tumor site, increased production of NO, IL-10, and TGF-β, as well as poor lymphocyte infiltration. In contrast, enforced expression of sTNF-α mutant by 4T1 cells that only released sTNF-α without expression of surface tmTNF-α markedly reduced MDSC accumulation and induced more lymphocyte infiltration instead, showing obvious tumor regression. Our data suggest that tmTNF-α acts as a potent activator of MDSCs via TNFR2 and reveals another novel immunosuppressive effect of this membrane molecule that promotes tumor immune escape.

Transmembrane TNF-α mediates “forward” and “reverse” signaling, inducing cell death or survival via the NF-κB pathway in Raji Burkitt lymphoma cells

Interestingly, some lymphoma cells, expressing high levels of transmembrane (tm)TNF‐α, are resistant to secretory (s)TNF‐α‐induced necrosis but sensitive to tmTNF‐α‐mediated apoptosis. As tmTNF‐α mediates “forward” as well as “reverse” signaling, we hypothesize that a balanced signaling between forward and reverse directions may play a critical role in determining the fate of cells bearing tmTNF‐α. Using Raji cells as a model, we first added exogenous tmTNF‐α on fixed, transfected NIH3T3 cells onto Raji cells to examine tmTNF‐α forward signaling and its effects, showing that constitutive NF‐κB activity and cellular inhibitor‐of‐apoptosis protein 1 transcription were down‐regulated, paralleled with Raji cell death. As Raji cells express tmTNF‐α, an inhibition of their tmTNF‐α expression by antisense oligonucleotide caused down‐regulation of NF‐κB activity. Conversely, increasing tmTNF‐α expression by suppressing expression of TNF‐α‐converting enzyme that cleaves tmTNF‐α led to an enhanced activation of NF‐κB, indicating that tmTNF‐α, but not sTNF‐α, contributes to constitutive NF‐κB activation. We next transfected Raji cells with a mutant tmTNF‐α lacking the intracellular domain to competitively suppress reverse signaling via tmTNF‐α; as expected, constitutive NF‐κB activity was decreased. In contrast, treating Raji cells with sTNFR2 to stimulate reverse signaling via tmTNF‐α ehanced NF‐κB activation. We conclude that tmTNF‐α, when highly expressed on tumor cells and acting as a receptor, promotes NF‐κB activation through reverse signaling, which is helpful to maintain tumor cell survival. On the contrary, tmTNF‐α, when acting as a ligand, inhibits NF‐κB activity through forward signaling, which is inclined to induce tumor cell death.

Targeting Transmembrane TNF-α Suppresses Breast Cancer Growth

TNF antagonists may offer therapeutic potential in solid tumors, but patients who have high serum levels of TNF-α fail to respond to infliximab, suggesting consumption of the circulating antibody and loss of transmembrane TNF-α (tmTNF-α) on tumors by ectodomain shedding. Addressing this possibility, we developed a monoclonal antibody (mAb) that binds both full-length tmTNF-α and its N-terminal truncated fragment on the membrane after tmTNF-α processing but does not cross-react with soluble TNF-α. We documented high levels of tmTNF-α expression in primary breast cancers, lower levels in atypical hyperplasia or hyperplasia, but undetectable levels in normal breast tissue, consistent with the notion that tmTNF-α is a potential therapeutic target. Evaluations in vitro and in vivo further supported this assertion. tmTNF-α mAb triggered antibody-dependent cell-mediated cytotoxicity against tmTNF-α-expressing cells but not to tmTNF-α-negative cells. In tumor-bearing mice, tmTNF-α mAb delayed tumor growth, eliciting complete tumor regressions in some mice. Moreover, tmTNF-α mAb inhibited metastasis and expression of CD44v6, a prometastatic molecule. However, the antibody did not activate tmTNF-α-mediated reverse signaling, which facilitates tumor survival and resistance to apoptosis, but instead inhibited NF-κB activation and Bcl-2 expression by decreasing tmTNF-α-positive cells. Overall, our results established that tmTNF-α mAb exerts effective antitumor activities and offers a promising candidate to treat tmTNF-α-positive tumors, particularly in patients that are nonresponders to TNF antagonists.

Influence of reverse signaling via membrane TNF-α on cytotoxicity of NK92 cells

Membrane tumor necrosis factor-alpha (mTNF-alpha) serves as a receptor transducing signals into mTNF-alpha-bearing cells. Among human peripheral blood mononuclear cells, natural killer (NK) cells have been reported to be the only cell type constitutively expressing mTNF-alpha, which is involved in the cytotoxicity of resting NK cells. Using an IL-2-dependent human NK cell line, NK92, which constitutively expresses mTNF-alpha, we examined the effect of reverse signaling via mTNF-alpha on cellular activities. When the cells were prestimulated with soluble TNFR1 (sTNFR1) which activated mTNF-alpha-mediated reverse signaling, the cytotoxicity of NK92 cells was significantly increased. Further investigation demonstrated that prestimulation with sTNFR1 augmented exocytosis and mRNA transcription of two cytotoxic molecules, perforin and granzyme B, which could serve as underlying molecular mechanisms by which mTNF-alpha-mediated reverse signaling promoted cytotoxicity of NK cells toward K562 cells. On the other hand, pretreatment of NK92 with sTNFR1 boosted the expression of FasL and TNF-alpha, including both the secretory and membrane forms. These molecules also contribute to the NK-mediated cytotoxicity, although K562 cells are Fas-negative and sTNF-alpha-resistant. Interestingly, the mTNF-alpha reverse signaling was found to act synergistically with IL-2 on NK-mediated cytotoxicity. This synergy markedly promoted the production of secretory as well as membrane cytotoxic molecules which may be responsible for the enhanced NK92-mediated cytotoxicity. Our observations suggest that, via reverse signaling, constitutively expressed mTNF-alpha may sensitize NK cells to activating stimuli, such as IL-2, resulting in increased NK-mediated cytotoxicity through promoting the production of multiple cytotoxic effector molecules.

Expression of TNF-α leader sequence renders MCF-7 tumor cells resistant to the cytotoxicity of soluble TNF-α

Transmembrane TNF-α (tmTNF-α) contains a leader sequence (LS) that can be phosphorylated and cleaved at its cytoplasmic portion, inducing IL-12 production. We observed that the breast cancer cell line MDA-MB-231 expressing transmembrane TNF-α (tmTNF-α) at high level was resistant to soluble TNF-α (sTNF-α)-induced cytotoxicity, accompanied by constitutive NF-κB activation. In contrast, MCF-7 cells expressing tmTNF-α at very low level were sensitive to sTNF-α-induced cell death and had no detectable NF-κB activation. Consistently, siRNA-mediated tmTNF-α knockdown blocked NF-κB activation and rendered MDA-MB-231 sensitive. To test our hypothesis that TNF-LS may play an important role in determining the sensitivity of tumor cells to sTNF-α, we stably transfected MCF-7 cells with TNF-LS. We found that transfection of TNF-LS or wild-type TNF-α containing LS constitutively activated NF-κB and conferred the cytotoxic resistance of MCF-7 cells, while transfection of a mutant tmTNF-α lacking the cytoplasmic segment of LS neither activated NF-κB nor affected the sensitivity. However, NF-κB inhibitor PDTC suppressed NF-κB activation and reconstituted sensitivity of TNF-LS/MCF-7 cells. To check whether TNF-LS is required to be cleaved or internalized for NF-κB activation to occur, we used signal peptide peptidase inhibitor (Z-LL)2-ketone and receptor internalization inhibitor MDC to treat cells. Interestingly, both inhibitors increased TNF-LS expression on the cell surface and enhanced NF-κB activation. These results indicate that membrane-anchored TNF-LS contributes to constitutive activation of NF-κB and resistance to sTNF-α-induced cell death. Therefore, TNF-LS appears to be responsible for tmTNF-α-induced resistance in the breast cancer cells.

Blocking TNF-α by combination of TNF-α- and TNFR-binding cyclic peptide ameliorates the severity of TNBS-induced colitis in rats.

Tumor necrosis factor alpha (TNF-α) has been implicated in the pathogenesis of Crohns disease. TNF antagonists are effectively used to treat these patients, although the efficiency of different antagonists varies. In the present study we combined TNF-α binding cyclic peptide (TBCP) and TNFR1 binding cyclic peptide (TRBCP) to treat TNBS-induced colitis in rats for one week. The symptoms of colitis including bloody diarrhea, rectal prolapse, and a profound and sustained weight loss were significantly ameliorated and the colon inflammatory damage, both macroscopic and histological scores, MPO activity, and NO production were markedly decreased in rats by neutralization of TNF-α and blocking TNFR1, as compared with those in rats treated with irrelevant peptide or normal saline (P<0.05). The transcripts of IL-1β and IL-8, and the protein expression of TNF-α in rats treated with both TBCP and TRBCP were also down-regulated (P<0.05), while these proinflammatory cytokines remained unchanged in rats treated with irrelevant peptide or normal saline. These findings suggest that the combination of TNF-α- and TNFR1-binding peptide effectively improves the symptoms of TNBS-induced colitis and alleviates colonic pathological damages in rats. This combination may be a potent candidate for clinical treatment of the inflammatory bowel disease.

Kupffer-cell-expressed transmembrane TNF-α is a major contributor to lipopolysaccharide and D-galactosamine-induced liver injury

Tumor necrosis factor (TNF)-α exists in two bioactive forms, a 26-kDa transmembrane form (tmTNF-α) and a 17-kDa soluble form (sTNF-α). sTNF-α has been recognized as a key regulator of hepatitis; however, serum sTNF-α disappears in mice during the development of severe liver injury, and high levels of serum sTNF-α do not necessarily result in liver damage. Interestingly, in a mouse model of acute hepatitis, we have found that tmTNF-α expression on Kupffer cells (KCs) significantly increases when mice develop severe liver injury caused by lipopolysaccharide (LPS)/D-galactosamine (D-gal), and the level of tmTNF-α expression is positively related to the activity of serum transaminases. Therefore, we hypothesized that KC-expressed tmTNF-α constitutes a pathomechanism in hepatitis and have explored the role of tmTNF-α in this disease model. Here, we have compared the impact of KCstmTNFlow and KCstmTNFhigh on acute hepatitis in vivo and ex vivo and have further demonstrated that KCstmTNFhigh, rather than KCstmTNFlow, not only exhibit an imbalance in secretion of pro- and anti-inflammatory cytokines, favoring inflammatory response and exacerbating liver injury, but also induce hepatocellular apoptosis via tmTNF-α and the expression of another pro-apoptotic factor, Fas ligand. Our data suggest that KCtmTNFhigh is a major contributor to liver injury in LPS/D-gal-induced hepatitis.

Transmembrane tumor necrosis factor-α promotes the recruitment of MDSCs to tumor tissue by upregulating CXCR4 expression via TNFR2.

&NA; Myeloid‐derived suppressor cells (MDSCs) accumulated in tumor sites promote immune evasion. We found that TNFR deficiency‐induced rejection of transplanted tumor was accompanied with markedly decreased accumulation of MDSCs. However, the mechanism(s) behind this phenomenon is not completely understood. Here, we demonstrated that TNFR deficiency did not affect the amount of MDSCs in bone marrow (BM), but decreased accumulation of Gr‐1+CD11b+ MDSCs in the spleen and tumor tissues. The chemotaxis of Tnfr−/− MDSCs was prominently decreased in response to both tumor cell culture supernatants and tumor tissue homogenates from Tnfr−/− and wild‐type mice, indicating an effect of TNFR signaling on chemokine receptor expression in MDSCs. We used real‐time PCR to detect gene expression for several chemokine receptors in MDSCs from BM and found that CXCR4 was the most affected molecule at the transcriptional level in Tnfr−/− MDSCs. Neutralizing CXCR4 in wild‐type MDSCs by a specific antibody blocked their chemotactic migration. Interestingly, it was tmTNF‐&agr;, but not sTNF‐&agr;, that induced CXCR4 expression in MDSCs. This effect of tmTNF‐&agr; was totally blocked in TNFR2−/− but not in TNFR1−/− MDSCs, and partially inhibited by PDTC or SB203580, an inhibitor of NF‐&kgr;B or p38 MAPK pathway, respectively. Adoptive transfer of wild‐type MDSCs restored MDSCs accumulation in tumors of Tnfr−/− mice, but this could be partially blocked by treatment with a CXCR4 inhibitor AMD3100. Our data suggest that tmTNF‐&agr; upregulates CXCR4 expression that promotes chemotaxis of MDSCs to tumor, and give a new insight into a novel mechanism by which tmTNF‐&agr; facilitates tumor immune evasion. HighlightsTNFR deficiency decreases MDSC accumulation in blood, spleen and tumor tissues.TNFR deficiency reduces CXCR4 expression in MDSCs and thus impaired their chemotaxis.tmTNF‐&agr; induces CXCR4 expression in MDSCs via TNFR2 by NF‐&kgr;B and p38 pathways.

The involvement of β-actin in the signaling of transmembrane TNF-α-mediated cytotoxicity.

Actin cytoskeleton has been shown to play a regulating role in several signaling pathways, and disruption of actin filament has been reported to increase sTNF‐α‐induced cell death. However, whether actin is involved in tmTNF‐α‐mediated cytotoxicity remains unclear. Here, we demonstrated that pretreatment of HL‐60 with CytD or LatA to depolymerize actin significantly suppressed tmTNF‐α‐mediated apoptosis. Interestingly, tmTNF‐α increased the actin immunoprecipitated by anti‐TNFR2 but not anti‐TNFR1 antibody, and disruption of the actin filament totally blocked this effect. In addition, TNFR1 knockdown by siRNA did not affect tmTNF‐α‐mediated cytotoxicity and the inhibitory effect of CytD, suggesting that the involvement of actin in the tmTNF‐α‐induced apoptosis is linked to the TNFR2 pathway. Our results revealed further that tmTNF‐α signaled the inhibition of IκB degradation and NF‐κB activity by recruiting RIP1 to and uncoupling TRAF2 from the TNFR2 complex. Nevertheless, CytD totally reversed the tmTNF‐α signaling and activated NF‐κB by recruiting TRAF2 to and dissociating RIP1 from the TNFR2 complex. Furthermore, tmTNF‐α led to activation of caspase‐8 by dissociation of cFLIP from TNFR2 and inhibition of the cFLIP expression. Activated caspase‐8 cleft RIP1 to suppress NF‐κB activity and also mediated tmTNF‐α‐induced apoptosis. However, CytD blocked the tmTNF‐α‐induced uncoupling of cFLIP from TNFR2 and prevented caspase‐8 activation and the resulting cleavage of RIP1, converting the signaling for tmTNF‐α‐mediated apoptosis into one for activating NF‐κB to survive. These results suggest that the actin cytoskeleton functions in transmitting signals via TNFR2 to mediate tmTNF‐α‐induced apoptosis.