A previous study reported the increased expression of the cytokine TNF in the adipose tissue of genetically obese rodents. To examine this paradigm in humans, we studied TNF expression in lean, obese, and reduced-obese human subjects. TNF mRNA was demonstrated in human adipocytes and adipose tissue by Northern blotting and PCR. TNF protein was quantitated by Western blotting and ELISA in both adipose tissue and the medium surrounding adipose tissue. Using quantitative reverse transcriptase PCR (RT-PCR), TNF mRNA levels were examined in the adipose tissue of 39 nondiabetic subjects, spanning a broad range of body mass index (BMI). There was a significant increase in adipose TNF mRNA levels with increasing adiposity. There was a significant correlation between TNF mRNA and percent body fat (r = 0.46, P < 0.05, n = 23). TNF mRNA tended to decrease in very obese subjects, but when subjects with a BMI > 45 kg/m2 were excluded, there was a significant correlation between TNF mRNA and BMI (r = 0.37, P < 0.05, n = 32). In addition, there was a significant decrease in adipose TNF with weight loss. In 11 obese subjects who lost between 14 and 66 kg (mean 34.7 kg, or 26.6% of initial weight), TNF mRNA levels decreased to 58% of initial levels after weight loss (P < 0.005), and TNF protein decreased to 46% of initial levels (P < 0.02). TNF is known to inhibit LPL activity. When fasting adipose LPL activity was measured in these subjects, there was a significant inverse relationship between TNF expression and LPL activity (r = -0.39, P < 0.02, n = 39). With weight loss, LPL activity increased to 411% of initial levels. However, the magnitude of the increase in LPL did not correlate with the decrease in TNF. Thus, TNF is expressed in human adipocytes. TNF is elevated in most obese subjects and is decreased by weight loss. In addition, there is an inverse relationship between TNF and LPL expression. These data suggest that endogenous TNF expression in adipose tissue may help limit obesity in some subjects, perhaps by increasing insulin resistance and decreasing LPL. (J. Clin. Invest. 1995. 95:2111-2119

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Background Mucosal expression of IFN-γ plays a pivotal role in IBD pathogenesis and IBD-risk regions flank IFNG. The conserved IFNG rs1861494 T/C, introduces a new CpG methylation site, and is associated with disease severity and lack of therapeutic response in other infectious and immune mediated disorders, and is in linkage-disequilibrium with a UC disease severity region. It seems likely that CpG-altering SNPs modify methylation and gene expression. This study evaluated the association between rs1861494 and clinical, serologic and methylation patterns in IBD patients. Methods Peripheral T cells of UC and CD patients were genotyped for rs1861494 and analyzed for allele-specific and IFNG promoter methylation. Serum ANCA and IFN-γ secretion were measured by ELISA and nucleo-protein complex formation by EMSA. Results IFNG rs1861494 T allele carriage in IBD patients was associated with enhanced secretion of IFN-γ. T allele carriage was associated in UC with high levels of ANCA and faster progression to colectomy. In CD, it was associated with complicated disease involving a stricturing/penetrating phenotype. Likewise, IFNG rs1861494 displayed genotype specific modulation of DNA methylation and transcription factor complex formation. Conclusions This study reports the first association of IFNG rs1861494 T allele with enhanced IFN-γ secretion and known IBD clinical parameters indicative of more aggressive disease, as well as serological markers associated with treatment resistance to anti-TNF therapy in IBD patients. These data may be useful prognostically as predictors of early response to anti-TNF therapy to identify IBD patients for improved personalized therapeutics.

Mucosal expression of proinfl ammatory cytokines plays a pivotal role in infl ammatory bowel disease (IBD) pathogenesis. Epigenetic remodeling of chromatin via DNA methylation regulates gene expression. In this study, IFNG DNA methylation was analyzed within the mucosal compartment in both normal and IBD populations and compared to its peripheral counterparts. Overall IFNG methylation (across eight CpG sites) was signifi cantly lower in lamina propria (LP) T cells compared to peripheral blood (PB) T cells. No methylation differences were detected when comparing PB T derived from normal to IBD patients. However, LP T-cell DNA derived from IBD patients displayed different levels of IFNG methylation of the upstream regulatory regions compared to DNA from normal controls. In fact, IFNG DNA promoter methylation levels functionally correlate with IFNG mRNA expression in unstimulated T cells, using quantitative real-time PCR. A 5% decrease in promoter methylation status is associated with nearly a 3-fold increase in IFNG expression. Likewise, methylation of the single −54 bp IFNG SnaB1 site strongly inhibited IFNG promoter expression. These results suggest that the epigenetic methylation status of IFNG may play a mechanistic role in the modulation of cytokine secretion in the mucosa.

SUMMARYTcell activation can lead to local tissueinjury in organ culture studies of human fetal jejunum, either directly throughcytotoxicity or indirectly by the release of cytotoxic cytokines. The goal of this study was to establish in vitro whether cyiotoxic cytokines can be released by isolated colonic T cells and what cytokine interactions are required for killing of human colonic epithelial cells. Cytokinecontaining supernatants were induced by incubating un.separated lamina propria tymphocyies (LPL) or mucosai Tcell subpopulations (separated by indirect panning) wilh anti-C'D3 and/or K562 target cells for 18 hat 37 C. Cytokines were measured by cytotoxicity assays using 1.929 (murine tibroblast) and HT-29 (human colonic tumour) lines as target cells in combination wilh blocking anti-cytokine antibodies. Supernatanls derived from unseparaled, CD4* (>95% pure) and CD8 ' (>90% pure) LPL were cytotoxic to L929 targets (350 U/ml, 230 U/ml and 100 U/m! tumournecrosis factor-alpha, respectively). All or nearly all of the cylotoxicity was due lo the presence of lumour necrosis facioralpha (little or no lumour necrosis factor-bcla was delected). These same supernatanls were cyiotoxic (up to 32% lysis at 1/4 dilution) to HT-29 targets in a 48-h '"In release assay. Recombinant tumour necrosis factor-alpha and interferon-gamma alone produced minimal killing of HT-29. bul together killed the HT-29 target cells. Anii-tumour necrosis factor-alpha or anu-iiucrfcron-pamma alone blocked killing of HT-29 target cells by LPL-derived supernatants. although anti-tumour necrosis factor-beta had no effect upon killing of HT-29. These results demonstrate thai human LPL T cells, triggered by addition of anti-CD3 and target cells, produce tumour necrosis factor-alpha and interferon-gamma, both of which are required for optimal killing of HT-29. Simultaneous release of these cytokines in the vicinity of epithelial cells during immune responses could play an important role in the mucosai damage in chronic inflammatory slates such as inflammatory bowel disca.se.