Michael Kruse Meyer

Proteome analysis of rheumatoid arthritis gut mucosa

Rheumatoid arthritis (RA) is an inflammatory joint disease leading to cartilage damage and ultimately impaired joint function. To gain new insight into the systemic immune manifestations of RA, we characterized the colon mucosa proteome from 11 RA-patients and 10 healthy controls. The biopsies were extracted by colonoscopy and analyzed by label-free quantitative proteomics, enabling the quantitation of 5366 proteins. The abundance of dihydrofolate reductase (DHFR) was statistically significantly increased in RA-patient biopsies compared with controls and correlated with the administered dosage of methotrexate (MTX), the most frequently prescribed immunosuppressive drug for RA. Additionally, our data suggest that treatment with Leflunomide, a common alternative to MTX, increases DHFR. The findings were supported by immunohistochemistry with confocal microscopy, which furthermore demonstrated that DHFR was located in the cytosol of the intestinal epithelial and interstitial cells. Finally, we identified 223 citrullinated peptides from 121 proteins. Three of the peptides were unique to RA. The list of citrullinated proteins was enriched in extracellular and membrane proteins and included known targets of anticitrullinated protein antibodies (ACPAs). Our findings support that the colon mucosa could trigger the production of ACPAs, which could contribute to the onset of RA. The MS data have been deposited to ProteomeXchange with identifiers PXD001608 and PXD003082.

AB0020 The Melanocortin System Is Responsive in Disease Driving Immune Cells in Rheumatoid Arthritis and May Offer A Pathway To Curative Treatment

Background Although biological agents may manage rheumatoid arthritis (RA), curative treatment is still the ”holy grail”. Induction of auto-antigen specific immune tolerance might offer a solution and obviate the need for life-long immunosuppression. Melanocortins are small peptides with considerable immune tolerance inducing, inflammation resolving and tissue preserving qualities (1). In animal experimental, autoimmune conditions, melanocortins have been demonstrated to bind differentially to melanocortin receptor (MCR) 1–5 on immune cells and to transform auto-reactive CD4+ T helper (Th) lymphocytes (ly) as well as sensitized CD8+ T cytotoxic (Tc) ly into regulatory T (Treg) ly and thus clear cell-mediated auto-immunity and delayed type hypersensitivity. In contrast to the situation in organ transplantation, clinical application of Treg therapy in human autoimmune disease is still non-existing, primarily because of a lack of in depth understanding. Objectives To explore if the pro-resolving melanocortin system may offer a pathway to immune tolerance induction, we examined whether the melanocortin system is present and responsive in pathogenic immune cell subsets in RA. To this end, we related changes due to TNFα inhibition (I) in MCR1–5 mRNA levels to changes in Th1 signature-, inflammatory and regulatory cytokine mRNA levels. Methods CD4+ Th, CD8+ Tc ly, CD19+ B-ly and CD14+ monocytes from seven patients with definite RA were isolated by Dynabeads before and three months after the start of TNFαI. Total RNA was extracted and mRNAs for MCR1–5 and a panel of disease driving Th1, inflammatory and regulatory cytokines were measured by real-time qRT-PCR. Fold changes in MCR1–5 gene levels were correlated to changes in cytokine gene levels. Results MCR1–5 gene expressions were reduced in al examined cell types, significantly so in CD8+ Tc ly and CD19+ B ly in RA patients responding to TNFαI. In addition, Th1 and inflammatory cytokine mRNA levels were reduced in all cell types in responders. In a non-responding patient MCR1–5 gene expressions as well as Th1 and inflammatory cytokine gene levels increased substantially. The changes in MCR1–5 gene expressions in CD8+ Tc cells correlated significantly to changes in the Th1 cytokine IFNγ gene level in this cell type. Furthermore, we found significant correlations between changes in MCR 1, 3, 5- and change in IL-1β gene levels in CD4+ Th ly. Conclusions Our results point at a responsive melanocortin system in immune cells in RA. Moreover, its regulation seems intimately connected to the disease driving Th1 response, that is IFNγ production by CD8+ Tc ly. Our results are underlined by the recently reported importance of IFNγ producing CD8+ Tc ly in early RA. Thus our findings indicate that the melanocortin pathway lies open to treatment of auto-reactive effector T ly from RA patients with MCR type specific synthetic ligands in vivo or in vitro to induce Treg transformation. Future curative induction of auto-antigen specific immune tolerance in RA may therefore involve the melanocortin system. References Ahmed TJ, Montero-Melendez T, Peretti M, Pitzalis C. Curbing Inflammation through endogenous pathways: Focus on melanocortin peptides. Int J Inflamm 2013;2013:985815. Disclosure of Interest None declared

Melanocortin 2, 3 and 4 receptor gene expressions are downregulated in CD8+ T cytotoxic lymphocytes and CD19+ B lymphocytes in rheumatoid arthritis responding to TNFα inhibition

Melanocortin signalling in leucocyte subsets elicits anti‐inflammatory and immune tolerance inducing effects in animal experimental inflammation. In man, however, the effects of melanocortin signalling in inflammatory conditions have scarcely been examined. We explored the differential reactions of melanocortin 1‐5 receptors (MC1‐5R) gene expressions in pathogenetic leucocyte subsets in rheumatoid arthritis (RA) to treatment with TNF‐α inhibitor adalimumab. Seven patients with active RA donated blood at start and at 3‐month treatment. CD4+ T helper (h) lymphocytes (ly), CD8+ T cytotoxic (c) ly, CD19+ B ly and CD14+ monocytes were isolated, using immunomagnetic beads, total RNA extracted and reverse transcription quantitative polymerase chain reaction (RT‐qPCR) performed. Fold changes in MC1‐5R, Th1‐, inflammatory‐ and regulatory cytokine gene expressions were assessed for correlation. Six patients responded to adalimumab treatment, while one patient was non‐responder. In all lymphocyte subtypes, MC1‐5R gene expressions decreased in responders and increased in the non‐responder. In responders, decrease in MC2R, MC3R and MC4R gene expressions in CD8+ Tc and CD19+ B ly was significant. Fold change in MC1‐5R and IFNγ gene expressions correlated significantly in CD8+ Tc ly, while fold change in MC1R, MC3R and MC5R and IL‐1β gene expressions correlated significantly in CD4+ Th ly. Our results show regulation of MC2R, MC3R and MC4R gene expressions in CD8+ Tc ly and CD19+ B ly. The correlations between fold change in different MCRs and disease driving cytokine gene expressions in CD8+ Tc ly and CD4+ Th ly point at a central immune modulating function of the melanocortin system in RA.

Resistin gene transcription is regulated in adaptive and innate immunity in rheumatoid arthritis

Background Resistin (RETN) was described in 2001 as an adipokine secreted by murine adipocytes resulting in insulin resistance. However, in man, adipocyte synthesis of RETN is controversial, while monocyte RETN production is well established (1). Curiously, RETN synthesis and its regulation in pathogenic immune cell subsets have not been examined in rheumatoid arthritis (RA), although blood RETN levels correlate to disease activity. In RA, -a risk factor for coronary artery disease (CAD), RETN might contribute to atherosclerosis. E.g. macrophage secreted RETN in atheromas, facilitates cholesterol uptake and plaque instability. Considering this, an in depth understanding of the regulation and effects of RETN synthesis in pathogenic immune cell subsets may provide clues to the aetiology of CAD, known to be associated with factors (high CRP, RF and ACPA), characterizing active RA. Objectives The aim of our investigation was to explore whether RETN gene transcription in pathogenic cell subsets of innate and adaptive immunity is detectable and if present, amenable to TNFα inhibition (I) and correlated to important cytokines in RA. Methods We examined the reaction of RETN gene transcription to TNFαI in CD14+ monocytes, CD4+ T helper (Th) lymphocytes (ly), CD8+ T cytotoxic (Tc) ly, and CD19+ B ly in RA patients, responding to adalimumab. Leukocyte subsets from 7 RA patients were isolated by Dynabeads before and 3 months after start of TNFαI. Total RNA was extracted and mRNAs for RETN and a panel of disease driving Th1, inflammatory and regulatory cytokines were measured by real-time qRT-PCR. Results RETN gene transcription was present in al cell subsets and in CD14+ monocytes and CD4+ Th ly responded to TNFαI with a significant decrease. In CD14+ monocytes the RETN gene was transcribed to a significantly higher degree than in lymphocyte subsets of the adaptive immune system both before and during TNFαI. In active RA, prior to TNFαI, RETN and TGFβ mRNA levels correlated significantly in CD4+ Th ly (P=0.03), while in CD14+ monocytes fold change in TGFβ and RETN mRNAs due to TNFαI correlated highly significantly (P=0.01). Furthermore, RETN and IL-8 mRNA levels tended to correlate (P=0.06) in CD14+ monocytes prior to TNFαI. Conclusions We here report regulated RETN gene transcription in human CD4+ Th ly as well as in CD14+ monocytes. Our results point at wider functions for RETN. Thus we found TNFαI regulated RETN gene transcription in CD4+Th ly in RA, indicating a possible immune response directing role. In this aspect, our results are in concert with increased RETN mRNA correlating to CD4+Th17 ly number in human cecum. The idea of RETN pleiotropic actions is further supported by the correlation to TGFβ transcription. RETN and TGFβ share a pathway for their synthesis and fibrogenic ability (2), on top TGFβ also has a prominent role in immune tolerance. The relation to IL-8 indicates influence on neutrophil trafficking (1). References Nagaev I et al. Human resistin is an immune-derived proinflammatory cytokine targeting both leukocytes and adipocytes. PLoS One 2006 Dec 20;1:e30. Chemaly R et al. Differential patterns of replacement and reactive fibrosis in pressure and volume overload are related to the propensity for ischaemia and involve resistin. J Physiol 2013;59:5337–55 Disclosure of Interest None declared