Suppressed IL-6 Production Research Articles

Convenient preparation of an antigenic oligosaccharide from white kidney bean powder: A useful plant oligosaccharide for synthesis of immunoactive glycopolymer

Plant glycoproteins, especially allergenic glycoproteins such as pollen allergens, often carry antigenic N-glycans with α1-3 fucose and/or β1-2 xylose residue(s) on the trimannosyl core structure. We previously reported that one of such antigenic free-form N-glycans, Man3Xyl1Fuc1GlcNAc2 (M3FX) suppressed IL-4 production from Th2 cells of pollinosis patients. For the molecular-level analysis of this immunoactivity, an effective and convenient procedure for large scale preparation of the immunoactive free-form N-glycan and a synthesis of glycopolymers bearing multivalent M3FX has been required. During the preparation of prebiotic oligosaccharides from several edible beans, we found that the free-form M3FX accumulates in relatively large amounts in white kidney beans. In this report, we describe a new procedure for preparation of M3FX from white kidney bean powders by a combination of ion-exchange method, gel-filtration, and hydrophilic partitioning. The high-purity of M3FX prepared by this procedure was confirmed by MS-analysis and 1H-NMR, suggesting that the free-form M3FX can be used for the synthesis of neoglycopolymer. Using this new procedure, the immunoactive oligosaccharide can be prepared without the chemical method such as hydrazinolysis and other purification steps required to exclude other type of N-glycans.

ILC2s are the predominant source of intestinal ILC-derived IL-10.

Although innate lymphoid cells (ILCs) functionally analogous to T helper type 1 (Th1), Th2, and Th17 cells are well characterized, an ILC subset strictly equivalent to IL-10-secreting regulatory T cells has only recently been proposed. Here, we report the absence of an intestinal regulatory ILC population distinct from group 1 ILCs (ILC1s), ILC2s, and ILC3s in (1) mice bred in our animal facility; (2) mice from The Jackson Laboratory, Taconic Biosciences, and Charles River Laboratories; and (3) mice subjected to intestinal inflammation. Instead, a low percentage of intestinal ILC2s produced IL-10 at steady state. A screen for putative IL-10 elicitors revealed that IL-2, IL-4, IL-27, IL-10, and neuromedin U (NMU) increased IL-10 production in activated intestinal ILC2s, while TL1A suppressed IL-10 production. Secreted IL-10 further induced IL-10 production in ILC2s through a positive feedback loop. In summary, ILC2s provide an inducible source of IL-10 in the gastrointestinal tract, whereas ILCregs are not a generalizable immune cell population in mice.

Extracellular ADP augments microglial inflammasome and NF-κB activation via the P2Y12 receptor.

The NLRP3 inflammasome is a molecular complex that translates signals from pathogens and tissue damage into inflammatory responses, and plays crucial roles in numerous neurological diseases. Activation of the NLRP3 inflammasome leads to caspase-1 dependent cleavage of pro-IL-1β to form mature IL-1β. By acting on the P2X7 purinergic receptor, extracellular ATP is one of the major stimuli that activates the NLRP3 inflammasome. Although microglia express multiple purinergic receptors, their roles in inflammasome-mediated inflammation are largely unknown. We studied the role of the P2Y12 receptor, a metabotropic P2Y receptor enriched in microglia, on inflammation in vitro. Inhibition of the microglial P2Y12 receptor by PSB0739 or siRNA knockdown suppressed IL-1β release. P2Y12 receptor-deficient microglia displayed markedly attenuated IL-1β mRNA expression and release. P2Y12 receptor blockade also suppressed IL-6 production. Both IL-1β and IL-6 responses were augmented by extracellular ADP or ADP-βS and were abrogated by PSB0739. Mechanistically, ADP-βS potentiated NF-κB activation. In addition, ADP altered mitochondrial membrane potential in combination with ATP and increased the number of caspase-1 positive cells through the P2Y12 receptor. These results elucidate a novel inflammatory mechanism by which extracellular ADP acts on the P2Y12 receptor to activate NF-κB and the NLRP3 inflammasome to enhance microglial inflammation.

Bitter Taste Receptors (TAS2Rs) in Human Lung Macrophages: Receptor Expression and Inhibitory Effects of TAS2R Agonists.

BackgroundBitter-taste receptors (TAS2Rs) are involved in airway relaxation but are also expressed in human blood leukocytes. We studied TAS2R expression and the effects of TAS2R agonists on the lipopolysaccharide (LPS)-induced cytokine release in human lung macrophages (LMs).MethodsLung macrophages were isolated from patients undergoing surgery for carcinoma. We used RT-qPCR to measure transcripts of 16 TAS2Rs (TAS2Rs 3/4/5/7/8/9/10/14/19/20/31/38/39/43/45 and 46) in unstimulated and LPS-stimulated (10 ng.mL–1) LMs. The macrophages were also incubated with TAS2R agonists for 24 h. Supernatant levels of the cytokines TNF-α, CCL3, CXCL8 and IL-10 were measured using ELISAs.ResultsThe transcripts of all 16 TAS2Rs were detected in macrophages. The addition of LPS led to an increase in the expression of most TAS2Rs, which was significant for TAS2R7 and 38. Although the promiscuous TAS2R agonists, quinine and denatonium, inhibited the LPS-induced release of TNF-α, CCL3 and CXCL8, diphenidol was inactive. Partially selective agonists (dapsone, colchicine, strychnine, and chloroquine) and selective agonists [erythromycin (TAS2R10), phenanthroline (TAS2R5), ofloxacin (TAS2R9), and carisoprodol (TAS2R14)] also suppressed the LPS-induced cytokine release. In contrast, two other agonists [sodium cromoglycate (TAS2R20) and saccharin (TAS2R31 and 43)] were inactive. TAS2R agonists suppressed IL-10 production – suggesting that this anti-inflammatory cytokine is not involved in the inhibition of cytokine production.ConclusionHuman LMs expressed TAS2Rs. Experiments with TAS2R agonists’ suggested the involvement of TAS2Rs 3, 4, 5, 9, 10, 14, 30, 39 and 40 in the inhibition of cytokine production. TAS2Rs may constitute new drug targets in inflammatory obstructive lung disease.

Glycolaldehyde-modified advanced glycation end-products inhibit differentiation of human monocytes into osteoclasts via upregulation of IL-10.

Diabetes patients are at high risk of bone fracture due to accumulation of advanced glycation end products (AGEs) and low bone turnover. Although AGEs inhibit osteoblast functions, little is known about their roles in regulation of human osteoclast differentiation. The aim of this study was to determine the roles of AGEs in regulation of human osteoclast differentiation. Human CD14+ monocytes collected from healthy individuals were stimulated in vitro with conventional cytokines to induce osteoclast differentiation. Simultaneously, glucose-modified AGEs-BSA (Glu-AGEs-BSA) and glycolaldehyde-modified AGEs-BSA (Glyco-AGEs-BSA) were added to analyze their role in regulation of osteoclast differentiation. Human CD14+ cells expressed endogenous receptor for AGE (RAGE). Stimulation with Glyco-AGEs-BSA, but not Glu-AGEs-BSA, reduced the number of tartrate-resistant acid phosphatase-positive cells in a dose-dependent manner and suppressed mRNA expression of nuclear factor of activated T-cells 1 and cathepsin K. Glyco-AGEs-BSA up-regulated pro-inflammatory cytokines and anti-inflammatory cytokine IL-10. The addition of IL-10-neutralizing antibodies abrogated the suppressive effect of Glyco-AGEs-BSA on osteoclast differentiation. Stimulation of Glyco-AGE-BSA resulted in nuclear factor (NF)-κB phosphorylation, and addition of an inhibitor of κB kinase suppressed IL-10 production. We conclude that Glyco-AGEs-BSA inhibited human osteoclast differentiation through induction of IL-10 expression via NF-κB. It can be assumed that AGE bioaccumulation in diabetic patients increases the risk of bone fracture, through inhibition of osteoclast differentiation, reduction of bone turnover, and disruption of bone remodeling.

Effects of Gold Nanoparticles with Different Surface Charges on Cellular Internalization and Cytokine Responses in Monocytes

Surface modification of gold nanoparticles (AuNPs) affects the interactions between the AuNPs and biological systems. To use AuNPs in biomedical applications, an investigation of how different surface charges of AuNPs affect the internalization mechanisms and functions of monocytes is needed. In this study, U937 cells were exposed to AuNPs with positive (AuNPs+ve) and negative (AuNPs-ve) surface charges. Cellular responses to the AuNPs, including cell uptake pathways, cell activation and cytokine production, were analysed. The results indicated different responses to AuNPs-ve and AuNPs+ve in outcomes including internalization pathways, cell activation and cytokine production levels. Interestingly, the results suggested that AuNPs+ve tended to enter the cells via macropinocytosis and strongly elicited a cellular response of IL-6 production via NF-kB activation, whereas the AuNPs-ve suppressed IL-6 production. Therefore, these results provide essential information for designing nanoparticles and may contribute to possible future nanomedicine applications, such as drug delivery and vaccination.

A peptide derived from rice inhibits alveolar bone resorption via suppression of inflammatory cytokine production.

Periodontitis is an inflammatory disease that results in alveolar bone resorption due to inflammatory cytokine production induced by bacterial antigens such as lipopolysaccharides (LPS). Here, the preventive effect of the Amyl-1-18 peptide derived from rice in an experimental model of periodontitis and the effect on the anti-inflammatory response were assessed. Alveolar bone resorption, gene transcription of proinflammatory cytokines in the gingiva, and the endotoxin level in the oral cavity were evaluated after oral administration of the Amyl-1-18 peptide for 14 days using a ligature-induced periodontitis model in mice. Additionally, murine macrophages were incubated with LPS of Escherichia coli or Porphyromonas gingivalis in the presence of Amyl-1-18 to analyze the suppressive effects of Amyl-1-18 on the cell signaling pathways associated with proinflammatory cytokine production, including inflammasome activities. Oral administration of Amyl-1-18 suppressed alveolar bone resorption and gene transcription of interleukin (il)6 in the gingiva of the periodontitis model, and decreased endotoxin levels in the oral cavity, suggesting modulation of periodontal inflammation by inhibition of endotoxin activities in vivo. Also, Amyl-1-18 suppressed IL-6 production induced by LPS and recombinant IL-1β in macrophages in vitro but had no effect on inflammasome activity. The Amyl-1-18 peptide from rice inhibited alveolar bone destruction in mouse periodontitis model via suppressing inflammatory cytokine production induced by LPS. It was suggested that Amyl-1-18 peptide has anti-inflammatory property against LPS, not only by neutralization of LPS and subsequent inhibition of nuclear factor-κB signaling but also by inhibition of the IL-1R-related signaling cascade.

Helios predisposes human fetal CD4+ naive T cells towards regulatory T cell differentiation

Abstract Activation of naïve CD4+ T cells by T cell receptor (TCR) stimulation and cytokine cues lead to differentiation into effector T cell populations with distinct pro-inflammatory or regulatory functions. Unlike adult naïve T cells, human fetal naïve CD4+ T cells uniquely differentiate into FOXP3+ regulatory T (Treg) cells upon TCR activation independent of exogenous cytokine signalling. This facility for Treg differentiation is crucial for generating tolerance in utero; however, the mechanisms underlying this ability in fetal naïve cells are largely unknown. Here, we reveal FOXP3-independent transcriptional and epigenetic programs shared between fetal naive T cells and committed adult Treg cells that are inactive in adult naive T cells. We show that a subset of adult Treg-specific super-enhancers is active within fetal naive T cells, including two super-enhancers at Helios, a thymic Treg gene. Helios is expressed in fetal naive T cells, but not in adult naïve T cells, and only fetal-derived induced Treg (iTreg) cells continue to express Helios. Fetal, but not adult iTreg cells, have suppressed IL-2 production, which is regulated by Helios in committed Treg cells. CRISPR-Cas9 ablation of Helios in fetal naive T cells then resulted in increased IL-2 production in fetal iTreg cells. Crucially, the loss of Helios expression in fetal naïve T cells impaired their differentiation into Treg cells upon TCR stimulation, indicating Helios as a critical contributor to the cell-intrinsic predisposition of fetal naive T cells for Treg cell differentiation. Treg-biased transcriptional and epigenetic programs within fetal naive T cells identified here could be utilized to engineer enhanced adult iTreg populations for adoptive cellular therapies.

Elevated expression levels of the voltage-gated sodium channel 1.7 and BAFF receptor in peripheral monocytes contribute to activation of monocytes of patients with primary Sjögren’s syndrome

Abstract Background & Purpose We reported that elevated expression of BAFF-receptor (BR3) in peripheral monocytes of patients with primary Sjögren’s syndrome (pSS) resulted in robust increased in IL-6 production by the cells. It has been reported that the pathways via voltage-gated sodium channels, such as Nav1.7, in monocytic lineage are involved in production of inflammatory cytokines, such as IL-6, in inflammatory pain diseases. In this study, we investigated the expression levels of Nav1.7 and BR3 in peripheral monocytes in patients with pSS in an attempt to discriminate the disease from other autoimmune diseases, such as active RA and active SLE. Methods The expression levels of Nav1.7 and BR3 in peripheral monocytes were analyzed by FACS using whole blood samples from patients with pSS (n = 28), active RA (n = 15), active SLE (n = 37) and healthy controls (HC; n = 15). Peripheral monocytes were stimulated with BAFF in the presence or absence of an inhibitor against Nav1.7. The amount of IL-6 in the culture supernatants was measured by ELISA. Results FACS analysis revealed that the expression levels of Nav1.7 and BR3 in pSS monocytes was significantly higher than those of active RA (p = 0.005, 0.003), active SLE (p <0.001, 0.004) and HC (p < 0.001, 0.002). Interestingly, the expression level of Nav 1.7 in pSS monocytes was significantly and positively correlated with that of BR3 in the cells (p = 0.01). Moreover, a specific inhibitor against Nav 1.7 suppressed IL-6 production by sBAFF-stimulated peripheral monocytes in a dose dependent manner. Conclusion Our results strongly suggest that the crosstalk between BAFF signaling and sodium channel is involved in activation of pSS monocytes and can be a therapeutic target for pSS.

Cholecystokinin Downregulates Psoriatic Inflammation by Its Possible Self-Regulatory Effect on Epidermal Keratinocytes.

Cholecystokinin (CCK) is a peptide hormone that functions in digestive organs and the CNS. We previously showed that CCK downregulates peripheral pruritus by suppressing degranulation of mast cells. In this study, we demonstrated that CCK octapeptide (CCK8) was constitutively expressed in the epidermis of normal skin, whereas its expression was lost in acanthotic lesions of psoriasis. In contrast, CCKA receptor (CCKAR), a high-affinity receptor for CCK, was constitutively expressed in the epidermis of psoriatic skin lesions. Expression of CCK was also reduced in skin lesions of an imiquimod (IMQ)-induced psoriatic mouse model. Notably, the expression level of CCK inversely correlated with the severity of epidermal inflammation, raising the possibility that CCK from epidermal keratinocytes suppresses the psoriatic inflammation. To verify this hypothesis, we investigated the effects of sulfated CCK octapeptide (CCK8S) on the development of IMQ-induced psoriatic inflammation. i.p. injection of CCK8S suppressed the IMQ-induced psoriatic inflammation accompanied by reduced mRNA expression of IL-17, IL-22, and IL-6 but not of IL-23. The suppressive effect of CCK8S was completely restored by administration of CCKAR antagonist. In vitro studies showed that exogenous CCK8S suppressed IL-6 production in CCKAR-expressing cultured human keratinocytes, and blocking the endogenous CCK signaling with CCKAR antagonist markedly enhanced IL-6 production. When keratinocytes were stimulated with IL-17, the expression of endogenous CCK was significantly decreased. These findings suggest that CCK physiologically functions as a negative regulator of keratinocyte-based inflammation in an autocrine or paracrine manner, although decreased CCK may pathologically contribute to continuous and aggravated skin lesions such as psoriasis.

Enhanced Anti-Allergic Activity of Milk Casein Phosphopeptide by Additional Phosphorylation in Ovalbumin-Sensitized Mice.

The proteolytic digest of milk casein, known as casein phosphopeptide (CPP-III), exhibits diverse biological activities, including calcium absorption and antioxidant activities. We hypothesized that the additional phosphorylation of this peptide can enhance its immunomodulatory activity such as suppression of allergy-associated cytokine and antigen-specific immune response. This study was conducted to assess whether oral intake of additionally phosphorylated CPP-III (P-CPP) attenuates ovalbumin (OVA)-induced IgE-mediated allergic reactions because of the additional phosphate groups. Female BALB/c mice were intraperitoneally sensitized with OVA twice at intervals of 14 days and then orally fed native CPP-III (N-CPP), P-CPP, and dephosphorylated CPP-III (D-CPP) for 6 weeks. Next, the mice were orally challenged with 50 mg of OVA. Oral administration of P-CPP suppressed total and specific IgE levels in the serum. Mice fed P-CPP exhibited low levels of OVA-specific IgG1 and increased OVA-specific IgG2a. P-CPP also suppressed IL-4 production, while D-CPP showed similar a level compared to that of the control. Further, P-CPP increased the population of the T follicular helper (Tfh) cell in the spleen. These results suggest that additional phosphorylation of CPP can enhance the attenuation of allergen-specific IgE-modulated allergic reactions in a murine food allergy model.

Anti-receptor activator of nuclear factor κB ligand antibody treatment increases osteoclastogenesis-promoting IL-8 in patients with rheumatoid arthritis.

The receptor activator of nuclear factor κB ligand (RANKL) is an important factor for osteoclastogenesis and contributes to the pathology of rheumatoid arthritis (RA); thus, the anti-RANKL antibody (Ab) has been expected to protect joint destruction in RA patients. IL-8 also has osteoclastogenic activity; however, the role of IL-8 in the bone pathology of RA as well as the relation between IL-8 and RANKL remain unclear. In the present study, clinical observation revealed serum IL-8 levels of 611 pg ml−1 in RA patients with anti-RANKL Ab and 266 pg ml−1 in the same patients without anti-RANKL Ab. In vitro assay showed that anti-RANKL Ab induced production of IL-8 from pre-osteoclast-like cells (OCLs), and IL-8 promoted the formation of OCLs from peripheral monocytes even without RANKL activity. We further showed that treatment with FK506 (tacrolimus) possibly inhibits the increase in IL-8 levels in RA patients with anti-RANKL Ab, and in vitro assay confirmed that FK506 suppressed IL-8 production in pre-OCLs. These results suggest that inhibition of RANKL induces the change in osteoclastogenesis-promoting factor from RANKL to IL-8, and FK506 may be a valuable combination drug to support the use of anti-RANKL Ab in treatment of RA.

Novel small molecule IL-6 inhibitor suppresses autoreactive Th17 development and promotes Treg development.

Multiple sclerosis (MS) is the leading cause of non-traumatic neurological disability in the United States in young adults, but current treatments are only partially effective, making it necessary to develop new, innovative therapeutic strategies. Myelin-specific interleukin (IL)-17-producing T helper type 17 (Th17) cells are a major subset of CD4 T effector cells (Teff ) that play a critical role in mediating the development and progression of MS and its mouse model, experimental autoimmune encephalomyelitis (EAE), while regulatory T cells (Treg ) CD4 T cells are beneficial for suppressing disease. The IL-6/signal transducer and activator of transcription 3 (STAT-3) signaling pathway is a key regulator of Th17 and Treg cells by promoting Th17 development and suppressing Treg development. Here we show that three novel small molecule IL-6 inhibitors, madindoline-5 (MDL-5), MDL-16 and MDL-101, significantly suppress IL-17 production in myelin-specific CD4 T cells in a dose-dependent manner in vitro. MDL-101 showed superior potency in suppressing IL-17 production compared to MDL-5 and MDL-16. Treatment of myelin-specific CD4 T cells with MDL-101 in vitro reduced their encephalitogenic potential following their subsequent adoptive transfer. Furthermore, MDL-101 significantly suppressed proliferation and IL-17 production of anti-CD3-activated effector/memory CD45RO+ CD4+ human CD4 T cells and promoted human Treg development. Together, these data demonstrate that these novel small molecule IL-6 inhibitors have the potential to shift the Teff : Treg balance, which may provide a novel therapeutic strategy for ameliorating disease progression in MS.

Overexpression of SOCS3 mediated by adenovirus vector in mouse and human castration-resistant prostate cancer cells increases the sensitivity to NK cells in vitro and in vivo.

Prostate cancer is one of the most common cancers in men. The overactivation of IL-6/JAK/STAT3 signaling and silencing of SOCS3 are frequently observed in prostate cancer. In the present study we undertook to develop Ad-SOCS3 gene therapy for the treatment of prostate cancer and also investigated whether Ad-SOCS3 increased sensitivity to NK cells. We demonstrated that Ad-SOCS3 could significantly inhibit growth of castration-resistant prostate cancer (CRPC) cell lines expressing pSTAT3, DU-145 (at 10, 20, and 40 MOI), and TRAMP-C2 (at 40 MOI), but not the PC-3 CRPC cell line with the STAT3 gene deleted. Ad-SOCS3 (40 MOI) could suppress IL-6 production in DU-145 cells and PD-L1 expression induced by IFN-γ in TRAMP-C2 cells, and increased the NK cell sensitivity of both TRAMP-C2 and DU-145 cells. In the DU-145 mouse xenograft tumor model, intratumoral injections (twice/week for 3 weeks) of 1 × 108 pfu of Ad-SOCS3 significantly inhibited tumor growth and combining the Ad-SOCS3 treatment with intratumoral injections (once/week for 2 weeks) of 1 × 107 human NK cells showed the highest tumor growth inhibitory effect. These results suggested that a combination of Ad-SOCS3 gene therapy and NK cell immunotherapy could be a powerful treatment option for advanced CRPC overexpressing pSTAT3.

Leptin induces IL-6 and IL-8 expression through leptin receptor Ob-Rb in human dental pulp fibroblasts

Objective: Leptin, through binding to its special receptor (Ob-Rb), has potent effects on immunity and inflammation. This study aimed to investigate the expression of leptin receptor Ob-Rb in human dental pulp fibroblasts (HDPFs) and the effects of leptin on the production of proinflammatory cytokines of IL-6 and IL-8 by HDPFs.Methods: Ob-Rb expression was determined by quantitative real-time PCR (real-time PCR), Western blot and immunofluorescence analyses in cultured HDPFs. Small interfering RNA (siRNA) was transfected into HDPFs to down-regulate the expression of Ob-Rb. Real-time PCR and enzyme-linked immunosorbent assay (ELISA) were used to determine the proinflammatory cytokines of IL-6 and IL-8 levels in leptin-stimulated HDPFs. The involved signalling pathways that mediate the leptin-stimulated production of proinflammatory cytokines were investigated using Western blot and specific signalling inhibitor analyses.Results: The expression levels of Ob-Rb mRNA and protein were detected in HDPFs. Leptin could stimulate mRNA and protein expression of IL-6 and IL-8 in HDPFs in a concentration-dependent and time-dependent manner. Transfection with siRNA targeting Ob-Rb resulted in remarkable reduction of IL-6 and IL-8 expressions by HDPFs. In accordance with the enhanced expression of proinflammatory cytokines, leptin stimulation resulted in rapid phosphorylation of STAT3, p38 MAPK, ERK and Akt in HDPFs. Inhibiting JAK2/STAT3, p38 MAPK or PI3K/Akt substantially decreased leptin-induced IL-6 production, whereas blocking ERK and p38 MAPK substantially suppressed IL-8 production from leptin-stimulated HDPFs.Conclusions: Leptin may up-regulate IL-6 and IL-8 production through binding with Ob-Rb in HDPFs via the activation of different intracellular signalling pathways.

IL-15 negatively regulates curdlan-induced IL-23 production by human monocyte-derived dendritic cells and subsequent Th17 response.

OBJECTIVE:In this study, we aimed to assess the effects of long- and short-term IL-15 cytokine exposure of human monocyte-derived curdlan-matured dendritic cells (DCs) on the production of Th17 cell-polarizing cytokine IL-23 and subsequent Th17 cell activation.METHODS:Peripheral blood mononuclear cells (PBMCs) were purified using Ficoll-Paque from healthy donors. Monocytes were magnetically selected using CD14 Miltenyi beads and differentiated into DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for five days in the presence or absence of IL-15 (100ng/ml) for long-term exposure experiments. Then, DCs were matured with peptidoglycan (PGN), or curdlan for 24 hours. For short-term exposure experiments, IL-15 was added only during maturation of DCs. Then, DCs were characterized concerning the expression of MHC II and costimulatory molecules, production of cytokine subunits IL-23p19, IL-12p40, IL-12p35 and cytokine IL-23 via flow cytometry or real-time qPCR or ELISA. Finally, the phosphorylation of signaling molecules after curdlan stimulation was assessed using phospho-flow assays.RESULTS:IL-15 exposure suppressed IL-23 production by DCs. As a result, IL-15-exposed DCs suppressed IL-17 production by allogeneic T cells. Importantly, we observed a reduction in the surface Dectin-1 receptor levels by IL-15-exposed DCs. In line with these observations, curdlan stimulation resulted in reduced phosphorylation of ERK1/2, NF-kB p65 and AKT by human DCs exposed to IL-15 compared with controls. These results may explain why IL-15-exposed DCs produce less IL-23 after maturation with curdlan, which is a ligand of Dectin-1.CONCLUSION:Short- or long-term exposure to IL-15 of human DCs during their differentiation or maturation programs DCs against Th17 cell polarization, which suggests that IL-15 availability may affect CD4+ T cell-mediated protective immunity to fungal infections.

IL-35 promoted STAT3 phosphorylation and IL-10 production in B cells, but its production was reduced in patients with coronary artery diseases

Interleukin (IL)-35 is a heterodimeric cytokine composed of the IL-12A subunit and the Epstein-Barr virus induced gene 3 (EBI3) subunit. Binding of IL-35 with IL-12 receptor subunit beta 2 (IL-12RB2) and IL-6 signal transducer (IL-6ST) occupies the binding sites of IL-6, IL-12, and IL-27 and prevents their signal transduction. IL-35 is also shown to promote the development of regulatory T cells (Tregs) and regulatory B cells (Bregs). In this study, we investigated B cell-mediated IL-35 production in patients with coronary artery disease (CAD). The expression levels of IL-35 subunits and IL-10 were significantly lower in B cells from CAD patients than in B cells from healthy control individuals. Exogenous IL-35 could effectively increase the IL-10 production by B cells in a concentration-dependent manner. IL-35 promoted the phosphorylation of STAT1 and STAT3 in B cells, and the inhibition of STAT3 phosphorylation suppressed IL-10 production. Raising the IL-35 concentration in cell culture eliminated the difference in IL-10 expression between CAD B cells and healthy B cells. We also demonstrated that B cells from CAD patients presented lower capacity to suppress interferon gamma (IFNG) and tumor necrosis factor (TNF) expression by T cells than B cells from healthy controls. Exogenous IL-35 could significantly improve the suppressive capacity of B cells in both healthy controls and CAD patients. Together, these results demonstrated that a reduction in IL-35 production was associated with Breg defects in CAD patients. IL-35 and IL-35 targets may serve as therapeutic candidates in the treatment of CAD and related diseases.

Biochemical Properties of TAK-828F, a Potent and Selective Retinoid-Related Orphan Receptor Gamma t Inverse Agonist

Background/Aims: Retinoid-related orphan receptor gamma t (RORγt) is a master regulator of T helper 17 cells that plays a pivotal role in the production of inflammatory cytokines including interleukin (IL)-17. Therefore, RORγt has attracted much attention as a target receptor for the treatment of inflammatory diseases including rheumatoid arthritis, multiple sclerosis, inflammatory bowel diseases, and psoriasis. This study aims to characterize TAK-828F, a potent and selective RORγt inverse agonist. Methods: The biochemical properties of TAK-828F were evaluated using Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay, surface plasmon resonance (SPR) biosensor assay, cofactor recruitment assay, reporter assay, and IL-17 expression assay. Results: TR-FRET binding assay and SPR biosensor assay revealed rapid, reversible, and high affinity binding of TAK-828F to RORγt. The cofactor recruitment assay showed that TAK-828F inhibited the recruitment of steroid receptor coactivator-1 to RORγt. Furthermore, TAK-828F inhibited the transcriptional activity of human and mouse RORγt with selectivity against human RORα and RORβ. TAK-828F also suppressed IL-17 production in Jurkat cells, overexpressing human RORγt. Conclusion: These favorable properties will be of advantage in the evaluation of TAK-828F in clinical studies for inflammatory diseases. Furthermore, these findings demonstrate that TAK-828F could serve as a pharmacological tool for further studies of RORγt and inflammatory diseases.

Correction: Downregulation of miR-200a-3p, Targeting CtBP2 Complex, Is Involved in the Hypoproduction of IL-2 in Systemic Lupus Erythematosus-Derived T Cells.

Systemic lupus erythematosus (SLE) damages multiple organs by producing various autoantibodies. In this study, we report that decreased microRNA (miR)-200a-3p causes IL-2 hypoproduction through zinc finger E-box binding homeobox (ZEB)1 and C-terminal binding protein 2 (CtBP2) in a lupus-prone mouse. First, we performed RNA sequencing to identify candidate microRNAs and mRNAs involved in the pathogenesis of SLE. We found that miR-200a-3p was significantly downregulated, whereas its putative targets, ZEB2 and CtBP2, were upregulated in CD4+ T cells from MRL/lpr-Tnfrsf6lpr mice compared with C57BL/6J mice. ZEB1 and ZEB2 comprise the ZEB family and suppress various genes, including IL-2 by recruiting CtBP2. IL-2 plays a critical role in immune tolerance, and insufficient IL-2 production upon stimulation has been recognized in SLE pathogenesis. Therefore, we hypothesized that decreased miR-200a-3p causes IL-2 deficit through the ZEB1-CtBP2 and/or ZEB2-CtBP2 complex in SLE CD4+ T cells. Overexpression of miR-200a-3p induced IL-2 production by downregulating ZEB1, ZEB2, and CtBP2 in EL4 cell lines. We further revealed that miR-200a-3p promotes IL-2 expression by reducing the binding of suppressive ZEB1-CtBP2 and ZEB2-CtBP2 complexes on negative regulatory element A in the IL-2 promoter in EL4 cells. Interestingly, the ZEB1-CtBP2 complex on negative regulatory element A was significantly upregulated after PMA/ionomycin stimulation in lupus CD4+ T cells. Our studies have revealed a new epigenetic pathway in the control of IL-2 production in SLE whereby low levels of miR-200a-3p accumulate the binding of the ZEB1-CtBP2 complex to the IL-2 promoter and suppress IL-2 production.

PO-294 BRAFV600E/PTEn-loss status is associated with interleukin (IL)−8 expression in preclinical models of colorectal cancer (CRC)

IntroductionMutational status in CRC is a strong predictor for overall survival; unfortunately, tumour microenvironment (TME) and tumor-stroma interactions (TSI) also increase cancer cells’ drug resistance, leading to an urgent need to better understand the molecular mechanisms of acquired tumor-resistance, which remains crucial to determine overall patient benefit.Material and methodsProduction of IL-8 and vascular endothelial growth factor (VEGF) was determined by ELISA under standardised culture conditions. Modulation of cytokine production after exposure to selective inhibitors of the MAPK and PI3K pathways was assessed, by both ELISA assay and real time-PCR. BRAF, MEK1, ERK1 and ERK2 expression were modulated using siRNAs specifically targeting these genes.Results and discussionsCRC cell lines harbouring both BRAFV600E and PTEN-loss expressed the highest levels of IL-8 and a ROC curve-based prediction algorithm based on these two mutations had 68% accuracy in predicting IL-8 production (p=0.002); on the other hand, VEGF levels inversely correlated with KRAS mutational status. IL-8 is tightly and transcriptionally controlled by activation of the MEK/ERK pathway, as the MEK inhibitor trametinib downregulated ERK Thr202/Tyr204 phosphorylation in the nuclear compartment and profoundly suppressed IL-8 production regardless of the genetic background; conversely, the selective BRAF inhibitor dabrafenib downregulated IL-8 only in BRAFV600E contexts, but upregulated its production in parallel with ERK Thr202/Tyr204 and STAT3 Ser727 phosphorylation in the nuclear compartment of BRAF-wt CRC cells. PI3K/mTOR inhibitors did not significantly modulate IL-8 production, confirming that the IL-8 release is downstream the BRAF/MEK/ERK pathway.In order to better investigate the role of MAPK pathway in IL-8 regulation, components of the MAPK-signalling pathways (BRAF, MEK, ERK1 and ERK2) were silenced by siRNA: IL-8 production was significantly downregulated after BRAF, MEK, or ERK2 silencing, regardless of the BRAF mutational status; conversely, no effects on IL-8 expression were observed after ERK1 silencing.ConclusionThese results showed that, in preclinical models of CRC, IL-8 expression is regulated by a BRAF/MEK/ERK2/STAT3 axis, and differential modulation of IL-8 after pharmacological treatments is dependent on BRAF mutational status. Such evidence allows better understanding of the molecular mechanisms of chemokine regulation, which can, in turn, contribute to the development of new targeted therapies in CRC.