Tumor necrosis factor-α (TNFα), one of the major pro-inflammatory cytokines, plays a key role in an effective immune response. However, the chronic presence of TNFα can lead to several inflammatory disorders like rheumatoid arthritis, psoriasis, Crohn’s disease etc. Inhibition of TNFα by pharmacological inhibitors or antibodies has proven to be effective in palliative treatment to some extent. The aim of this study was to develop an anti-TNFα antibody which may be used as a therapeutic option to inhibit TNFα-mediated cytotoxicity. We characterized several hybridoma clones secreting monoclonal antibodies (mAbs) to human-TNFα. Four mAbs rescued L929 fibroblast cells from TNFα-triggered cell death and one of these, namely C8 was found to have the highest affinity. To gain insights into the mechanism by which mAb C8 inhibits human TNFα-mediated toxicity, the epitope corresponding to the mAb was delineated. The antigenic determinant was found to comprise of the stretch of amino acids 99-120, of which, 102-104 (QRE) form the core epitope. The observation was supported by bioinformatics analyses of an antigen-antibody complex model. In addition, the binding affinity of mAb C8 to TNFα was found to be comparable with that of Infliximab which is a commercially available anti TNFα mAb.

Hepatitis C virus (HCV) core protein is a multifunctional protein that is involved in proliferation, inflammation and apoptosis mechanism of hepatocytes. HCV core protein genetic variability has been implicated in various outcomes of HCV pathology and treatment. In the present study, we aimed to analyze the role of HCV core protein in tumor necrosis factor α (TNFα)-induced death under the viewpoint of HCV genetic variability. Immortalized hepatocytes (IHH), and not the Huh7.5 hepatoma cell line, stably expressing HCV subtype 4a and HCV subtype 4f core proteins showed that only HCV 4a core protein could increase sensitivity to TNFα-induced death. Development of two transgenic mice expressing the two different core proteins under the liver-specific promoter of transthyretin (TTR) allowed for the in vivo assessment of the role of core in TNFα-induced death. Using the TNFα-dependent model of lipopolysaccharide/D-galactosamine (LPS/Dgal) we were able to recapitulate the in vitro results in IHH cells in vivo. Transgenic mice expressing HCV 4a core protein were more susceptible to the LPS/Dgal model while mice expressing HCV 4f core protein had the same susceptibility as their littermate controls. Transcriptome analysis in liver biopsies from these transgenic mice gave insights into HCV core molecular pathogenesis, while linking HCV core protein genetic variability to differential pathology in vivo.

An analysis of the relationship between plasma cytokines and the effectiveness of treatment with TNFαinhibitors was performed in 81 patients with moderate-to-severe psoriasis. Treatment efficacy was assessed by PASI score, and patients were classified into a positive effect (PASI≥75) and no effect group (PASI≤50). A positive effect was reached in 11 (41%) patients for etanercept, 14 (52%) for adalimumab, and 26 (96%) for infliximab. Data analysis did not show  differences in baseline TNFα levels and subsequent treatment effectiveness. The CART algorithm showed that at the baseline level of VEGF ≥ 32 pg/ml and IL17F < 26 pg/ml, there was an 83% probability of a positive effect. Random forest analysis showed the importance of VEGF, ICAM1, sCD40L, IL17F and IL31 baseline levels in the prediction of treatment effectiveness.   Significant differences between the groups before/after the treatment were found only for TNFα: the median values were more than 50 times higher in no effect compared with positive effect group. There were differences before/after therapy in the levels of IL20, ICAM1, IL22, IL23 in the no effect group. The treatment affected the cytokine profile in most cases regardless of the effectiveness of therapy.

Understanding the complex interactions between metabolism and the immune system (“meta-flammation”) is crucial for the identification of key immunomodulatory factors as potential therapeutic targets in obesity and in cardiovascular diseases. Cathelicidin antimicrobial peptide (CAMP) is an important factor of innate immunity and is expressed in adipocytes. CAMP therefore might play a role as an adipokine in metaflammation and adipose inflammation. Toll-like receptor (TLR) 9 is another component of the innate immune system that is expressed and functionally active in adipocytes. The aim of the present study was to investigate the impact of TLR9 signaling on CAMP expression in adipocytes and in adipose tissue. CAMP gene expression in murine 3T3-L1 and human SGBS adipocytes and in murine and human adipose tissues was quantified by real-time PCR. TLR9 knockdown was applied in murine 3T3-L1 adipocytes via siRNA transfection. Adipocyte inflammation was induced in vitro by TNFα stimulation. Serum CAMP concentrations in TLR9 knockout (KO) and in wildtype mice were quantified by ELISA. CAMP gene expression was considerably increased in 3T3-L1 adipocytes during differentiation. TNFα significantly induced CAMP gene expression in mature adipocytes, which was antagonized by inhibitors of NF-κB and PI3K signaling. Cell-free nucleic acids (cfDNA) as endogenous TLR9 ligand significantly impaired CAMP gene expression, whereas synthetic agonistic and antagonistic TLR9 ligands had no effect. Cellular TLR9 knockdown reduced adipocyte CAMP gene expression in vitro and male TLR9 knockout mice exhibited lower systemic CAMP concentrations than wildtype mice. CAMP and TLR9 gene expression were correlated positively in murine and human subcutaneous but not in intraabdominal/visceral adipose tissues. These findings suggest a regulatory role of TRL9 in adipocytic CAMP expression as a novel putative molecular mechanism in adipose tissue innate immunity.