Innate Cytokine Release Research Articles

MicroRNA23a Overexpression in Crohn’s Disease Targets Tumour Necrosis Factor Alpha Inhibitor Protein 3, Increasing Sensitivity to TNF and Modifying the Epithelial Barrier

Mucosal healing is important in Crohn's disease therapies. Epithelial homeostasis becomes dysregulated in Crohn's, with increased permeability, inflammation, and diarrhoea. MicroRNAs are small non-coding RNAs that regulate gene expression and show changes in inflammatory bowel disease. Tumour necrosis factor alpha [TNFα] inhibitor protein 3 is raised in Crohn's and regulates TNFα-mediated activation of NFκB. We investigated TNFα regulation by microRNA in Crohn's disease [CD], and studied effects on epithelial permeability and inflammation. Colonic epithelium from CD and healthy donor biopsies was isolated using laser capture microdissection, and microRNA was quantified. Tumour necrosis factor alpha inhibitor protein 3 was characterised immunohistochemically on serial sections. Expression effect of microRNA was confirmed with luciferase reporter assays. Functional barrier permeability studies and innate cytokine release were investigated with cell and explant culture studies. MicroRNA23a levels significantly increased in colonic Crohn's epithelium compared with healthy epithelium. Luciferase reporter assays in transfected epithelial cells confirmed that microRNA23a repressed expression via the 3' untranslated region of tumour necrosis factor alpha inhibitor protein 3 mRNA, coinciding with increased NFκB-mediated transcription. Immunohistochemical staining of TNFAIP3 protein in colonic biopsies was reduced or absent in adjacent Crohn's sections, correlating inversely with microRNA23a levels and encompassing some intercohort variation. Overexpression of microRNA23a increased epithelial barrier permeability in a colonic epithelial model and increased inflammatory cytokine release in cultured explant biopsies, mimicking Crohn's disease characteristics. MicroRNA23a overexpression in colonic Crohn's epithelium represses tumour necrosis factor alpha inhibitor protein 3, enhancing sensitivity to TNFα, with increased intestinal permeability and cytokine release.

Abstract 4154: EOS100850 inhibits A2A receptor signaling in human whole blood: Two pharmacodynamic assays to monitor EOS100850 activity in clinical studies

Abstract High levels of extracellular adenosine in the tumor microenvironment promote tumor immune evasion. We and others have shown that adenosine, predominantly through the A2A receptor, suppresses innate and adaptive immune cell responses leading to suppression of antitumor immunity. We therefore developed EOS100850, an A2A receptor antagonist specifically designed as a potent, highly selective, non-brain penetrant, orally administered agent to treat a wide range of tumor types. To monitor A2A receptor engagement by EOS100850 in clinical studies, we developed two pharmacodynamic assays that allow to quantify the inhibition of adenosine signaling in cancer patients dosed with this compound. The assays were developed and optimized to: 1) minimize the amount of patient blood required for each test; 2) minimize sample manipulation; 3) allow marker quantification in cryopreserved samples. The first assay is based on the quantification of phosphorylated cAMP response-element binding protein (pCREB) as a proximal readout of A2A receptor signaling. Whole blood is ex vivo stimulated with the A2A receptor agonist CGS-21608, or with dimethyl sulfoxide or phorbol-12-myristate-13 acetate and ionomycin as controls, and pCREB in CD4+ and CD8+ cells is analyzed by flow cytometry. After having confirmed that pCREB was rapidly induced after incubation with CGS-21680 (median fold-induction in 24 healthy donors was 5.4 and 4.0 in CD4+ and CD8+ cells, respectively), we showed that EOS100850 inhibited CGS-21608-induced pCREB in a dose-dependent manner, with an IC50 of 9.7 nM and 11.2 nM, respectively, for CD4+ and CD8+ T cells, and 83-87% signal inhibition in the presence of 40 nM EOS100850 (data from 10 donors). The second assay is based on the quantification of soluble mediators as a distal readout of A2A receptor signaling. Stimulation of whole blood with lipopolysaccharide (LPS) normally induces the release of innate cytokines and chemokines, but in the presence of the A2A receptor agonist CGS-21680, LPS activity is impaired. In this assay, whole blood is ex vivo stimulated with LPS in the presence or in the absence of CGS-21608 using TruCulture tubes (Myriad RBM), and secreted mediators are quantified using the human multianalyte profile immunoassay platform. After having confirmed that CGS-21680 induced a profound (>50%) alteration of cytokine secretion in LPS-stimulated blood, we showed that EOS100850 restored IFN-γ, IL-8, IL-10 and TNF-α secretion in a dose-dependent manner, with a complete rescue at 40-100 nM of compound (data from 3 donors). Both the assays were qualified in terms of precision, robustness and stability using blood of healthy volunteers, applied to blood of five cancer donors and are now ready to be used for the clinics. Citation Format: Chiara Martinoli, Margreet Brouwer, Erica Houthuys, Marjorie Mercier, Noemie Wald, Bruno Gomes, Stefano Crosignani, Veronique Bodo. EOS100850 inhibits A2A receptor signaling in human whole blood: Two pharmacodynamic assays to monitor EOS100850 activity in clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4154.

STAT6 inhibitory peptide reduces dendritic cell migration to the lymph nodes to control Th2 adaptive immunity in the mouse lung

Type 2 immunity in the lung is promoted through the release of innate cytokines, including TSLP, from lung structural cells. These cytokines drive Type 2 immunity in part through upregulation of OX40L on dendritic cells (DCs). DCs expressing OX40L are potent inducers of Th2 differentiation. We have shown previously that STAT6 inhibitory peptide (STAT6-IP), a cell penetrating peptide designed to inhibit the STAT6 transcription factor, reduces the induction of Th2 adaptive immunity in murine models of respiratory syncytial virus infection. Here we show that intranasal administration of STAT6-IP at the time of antigen priming with ovalbumin (OVA), in conjunction with the Nod2 agonist, MDP, reduced frequencies of CD11b+ lung DCs expressing OX40L. Consistent with these reductions, fewer activated DCs were localized to the lung draining lymph nodes in STAT6-IP-treated mice. Upon OVA challenge four weeks later, mice treated with STAT6-IP at the time of OVA/MDP priming did not develop airway hyperresponsiveness (AHR) and had reduced influx of eosinophils into the airways, mucus production, and serum OVA-specific IgE levels. Our findings provide evidence that the long-lasting inhibitory effects of STAT6-IP are due in part to inhibition of DC responses that drive maladaptive Th2 adaptive immunity and allergic airways disease.

Alternatively activated brain-resident macrophages rapidly acquire inflammatory properties following CNS infection (INC6P.301)

Abstract Classic (M1) and alternative (M2) polarization of macrophages has been broadly studied in vitro but its relevance in the context of meningeal inflammation is unknown. The meningeal and perivascular spaces of the central nervous system (CNS) are inhabited by specialized macrophages. To gain novel insights into these cells, we analyzed the naive and virally infected brain using intravital two-photon microscopy (TPM) and flow cytometry. Examination of the naive brain by TPM revealed that meningeal and perivascular macrophages are highly dynamic and constantly survey their immediate surroundings similar to microglia. Under steady state conditions, we uncovered that they are maintained in a M2 state, which likely facilitates brain homeostasis. Interestingly, during the development of fatal viral meningitis induced by lymphocytic choriomeningitis virus, these cells were directly engaged by infiltrating virus-specific CD8+ T cells following acquisition of viral antigen and conversion into an inflammatory M1 phenotype. Mechanistically, the M2 to M1 transition occurred in the absence of CD8+ T cells and relied instead on innate cytokine release. These data indicate that meningeal and perivascular macrophages are highly plastic cells that can rapidly convert from a M2 to M1 phenotype and participate in the antiviral defense against an invading pathogen. This rapid inflammatory transition may explain why most CNS immune responses first develop in the meningeal and perivascular spaces.

Damage-associated molecular pattern and innate cytokine release in the airways of competitive swimmers.

Daily intensive exercise by elite athletes can result in exercise-induced asthma especially in elite swimmers and this may be linked to epithelial damage. To study airway epithelial damage and release of damage-associated molecular patterns (DAMPs) after intensive exercise in elite athletes and controls. We recruited competitive swimmers (n=26), competitive indoor athletes (n=13) and controls (n=15) without any history of asthma. Lung function was measured before, immediately after and 24h after a 90-min intensive exercise protocol. Sputum induction was performed at baseline and 24h after exercise. Exercise-induced bronchoconstriction (EIB) was assessed by the eucapnic voluntary hyperventilation test. Baseline sputum uric acid, high mobility group box-1, CXCL8 mRNA, sputum neutrophils and serum Clara cell protein-16 (CC-16) were significantly higher in competitive swimmers compared with controls. Intensive swimming for 90min resulted in an increase of sputum IL-1β, IL-6 and TNF mRNA in competitive swimmers, and of sputum IL-6 mRNA and sputum neutrophils in controls. Although all participants were asymptomatic, seven competitive swimmers, one indoor athlete and one control met the criteria for EIB. Our findings show that the intensive training combined with exposure to by-products of chlorination induces airway epithelial damage in competitive swimmers. This is associated with increased damage-associated molecular patterns, innate cytokine release and neutrophilic airway inflammation.

Sepsis induces an early increased spontaneous release of hepatocellular stimulatory factor (interleukin-6) by Kupffer cells in both endotoxin tolerant and intolerant mice

Previous studies have shown that hepatocellular function is significantly depressed early during sepsis and that this is associated with a marked increase in the circulating levels of the hepatocellular stimulatory factor (IL-6). It remains unknown, however, whether or not Kupffer cells (KC) are activated during sepsis and whether these cells are the major contributors to the increased circulating levels of this cytokine. The objectives of this study were, therefore, to determine whether or not during sepsis: (1) KC are stimulated in vivo to release IL-6, as compared to other cytokines; (2) KC differ from splenic macrophages (SMφ) in their ability to release cytokines; and (3) there is a difference in macrophage (Mφ) cytokine release between endotoxin (ET)-tolerant (C3H/HeJ) and ET-intolerant (C3H/HeN) mice. To assess this, KC and SMφ were harvested at 1 or 24 hr from mice which had been subjected to polymicrobial sepsis by cecal ligation and puncture (CLP) or sham-operation. Following depletion of the nonadherent cells, KC and SMμ cultures were incubated for 24 hr in the presence or absence of 10 μg of ET/ml, and the levels of interleukin (IL)-1, IL-6, and TNF release were determined by bioassays. Sepsis induced an early (at 1 hr) in vivo stimulation of KC but not SMφ IL-6 release, irrespective of ET-tolerance/intolerance. However, the release of IL-1 or TNF was not markedly different for either CLP or sham KC. KC harvested from mice 24 hr post-CLP no longer showed an increase in innate cytokine release, whereas SMφ from these same animals exhibited a marked reduction in cytokine release both in the presence and in the absence of added ET. These findings support the notion that during early sepsis KC are selectively activated to release IL-6, irrespective of the animals' sensitivity to ET. Furthermore, the enhanced localized release of IL-6 by KC induced by CLP may contribute to hepatocellular dysfunction observed during early sepsis.