Ivan Nagaev

Assessment of cytokine mRNA expression profiles in tumor microenvironment and peripheral blood mononuclear cells of patients with high-grade serous carcinoma of the ovary

Objective: Tumor establishment, metastatic spreading and poor survival in ovarian cancer is strongly associated with progressive derangement of the patient’s immune system. Accumulating evidence suggests that immune impairment is influenced by the production and presence of cytokines in the tumor microenvironment. Methods: Cytokine mRNA profiles in tumor tissue and peripheral blood mononuclear cells (PBMC) were analyzed in patients with high grade serous carcinoma (HGSC) of the ovary and compared it to patients with benign ovarian conditions and controls with normal ovaries. Cytokine assessment was done by real-time quantitative RT-PCR and specific primers and probes for 12 cytokines-IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-15, TNF-α, TNF-β/LTA, TGF-β1, and GM-CSF chosen to distinguish between cytotoxic Th1, humoral Th2, regulatory Th3/Tr1 and inflammatory responses. Results: The cytokine mRNA response in the HGSC patients was significantly up regulated compared to patients with benign ovarian conditions and normal ovary controls confirming the immunogenicity of HGSC and implying immune recognition and reaction locally in the tumor microenvironment and systemically in the peripheral blood.There was an up-regulation of inflammatory and inhibitory cytokine mRNA promoting tumor progression, T-regulatory cell priming and T-regulatory cell-mediated immune suppression. In contrast, there was an inability to mount the crucially important IFN gamma response needed for upregulation of the cytotoxic anti-tumor response in the local microenvironment. In addition, systemic IL-4- mediated Th2 response prevailed in the peripheral blood deviating the systemic defense towards humoral immunity. Conclusions: Taken together, these results suggest local and systemic cytokine cooperation promoting tumor survival, progression and immune escape. Our study confirms and extends previous investigations and contributes to the evaluation of potential cytokine candidates for diagnostic cytokine mRNA profiles and for future therapeutic interventions based on cytokine inhibition.

Dynamics of cytokine mRNA expression and fecal biomarkers in school-children undergoing a double-blind placebo-controlled food challenge series

BACKGROUND There is need for prognostic markers for symptomatic food allergy since current diagnostic methods are insufficient and/or time and labor consuming. OBJECTIVE To estimate the cytokine mRNA profiles in peripheral blood mononuclear cells (PBMC) before and after a double-blind placebo-controlled food challenge series in schoolchildren with suspected allergy to milk, egg or cod and in healthy controls. Analyses of fecal inflammatory biomarkers before and after the challenge were included. METHODS Twelve-year-old children from a population-based cohort reporting complete avoidance of milk, egg, cod or wheat due to perceived hypersensitivity were clinically examined and those with suspected food allergy were evaluated with a 3-session double-blind placebo-controlled food challenge (n=18). Seven healthy controls participated in a double-blind challenge with egg. Before and after the challenge series, the cytokine mRNA expression was quantified for 13 cytokines discriminating between humoral Th2-, cytotoxic Th1-, regulatory Th3/Tr1- and inflammatory responses. Fecal calprotectin and eosinophil-derived neurotoxin (EDN) were also analyzed in children with suspected food allergy before and after the challenge series. RESULTS Pre challenge, children with suspected food allergy had higher IL-13andTNF-α expression and lower IFN-γ and IL-15 expression compared to healthy controls (all p<0.05). Children with challenge-proven food allergy had increased IL13andIL-10 expression compared to the levels seen in negative challenges (p<0.05). Post challenge, IL-1β and IL-6 mRNA levels were elevated in the food allergic children compared to controls (p<0.05). Fecal calprotectin and EDN levels were higher in challenge-proven food allergy compared to a negative challenge although not statistically significantly. CONCLUSION & CLINICAL RELEVANCE Increased baseline mRNA levels of the Th2-related cytokine IL-13 and the regulatory cytokine IL-10 predicted a positive food challenge outcome. These cytokines in combination with fecal calprotectin and EDN might serve as future prognostic markers for symptomatic, IgE-mediated food allergy but need further validation in a larger patient cohort.

Lymphocyte profile and cytokine mRNA expression in peripheral blood mononuclear cells of patients with recurrent respiratory papillomatosis suggest dysregulated cytokine mRNA response and impaired cytotoxic capacity

Recurrent respiratory papillomatosis (RRP) is a relatively rare, chronic disease caused by Human Papilloma Virus (HPV) 6 and 11, and characterized by wart‐like lesions in the airway affecting voice and respiratory function. The majority of HPV infections are asymptomatic and resolve spontaneously, however, some individuals are afflicted with persistent HPV infections. Failure to eliminate HPV 6 and 11 due to a defect immune responsiveness to these specific genotypes is proposed to play a major role in the development of RRP.

Resistin gene expression is downregulated in CD4+ T helper lymphocytes and CD14+ monocytes in rheumatoid arthritis responding to TNF-α inhibition

Rheumatoid arthritis (RA) is caused by complex interactions between immune cells and sustained by Th1 response cytokines. Resistin [resistance to insulin; (RETN)] is an inflammatory cytokine, first discovered in murine adipocytes. In man, RETN is mainly secreted by monocytes. The distinct role of RETN in the immune reaction is uncertain; however, RETN has pro‐inflammatory, pro‐fibrotic and possibly tolerogenic properties. The aim was to assess the reaction of RETN gene expression to TNF‐α inhibition (I) in pathogenetic immune cell subsets in RA, in the context of Th1, inflammatory and regulatory cytokine gene expressions. Accordingly, we measured RETN, IFN‐γ, TNF‐β, IL‐1β, TNF‐α, TGF‐β and IL‐10 gene expressions in CD14+ monocytes, CD4+ T helper (Th) lymphocytes (ly), CD8+ T cytotoxic (Tc) ly and CD19+ B ly in active RA before and 3 months after start of TNF‐αI. Leucocyte subsets were separated by specific monoclonal antibody‐covered beads, RNA extracted and levels of RETN, Th1 response, inflammatory and regulatory cytokine mRNAs measured by quantitative reverse transcription‐polymerase chain reaction technique. We found that TNF‐αI caused a significant downregulation of RETN gene expression in CD14+ monocytes and CD4+ Th ly and was unchanged in CD8+ Tc ly and CD19+ B ly. Both in active RA and during TNF‐αI, RETN mRNA levels were significantly higher in CD14+ monocytes than in all other examined cell types. In monocytes, fold change in RETN and TGF‐β gene expressions upon TNF‐αI correlated significantly. Our findings indicate that RETN has pro‐inflammatory as well as proresolving roles in active RA.

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

Enhanced Th1 and inflammatory mRNA responses upregulate NK cell cytotoxicity and NKG2D ligand expression in human pre-eclamptic placenta and target it for NK cell attack

Pre‐eclampsia (PE), a severe human pregnancy disorder, is associated with exaggerated systemic inflammation, enhanced cytokine production, and increased shedding of microvesicles leading to endothelial dysfunction, coagulopathy, and extensive placenta destruction. The cause of PE is still unclear. Evidence suggests that its origin lies in the placenta and that the maternal immune system is involved. A shift in cytokine production in PE pregnancy promotes NK cell activation, suggested to be important in PE pathogenesis. In line with this suggestion, we studied NK cell cytotoxicity in peripheral blood of PE patients and controls and the mRNA expression of cytokines and of the NKG2D receptor and its ligands MICA/B and ULBP1‐3 in PE‐ and normal placenta.

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