Induced IL-6 Secretion Research Articles

Abstract 2877: CB307: A novel selective CD137 agonist for enhancement of immune cell responses to PSMA+ tumors

Abstract Introduction: CD137 (HGNC:TNFRSF9, 4-1BB) is a TNF receptor superfamily costimulatory receptor expressed by T cells and NK cells. Upon engagement, CD137 enhances immune cell survival, proliferation, cytokine production, and memory formation. As such, CD137 agonists have demonstrated promising pre-clinical anti-tumor effects, however their clinical utility has been limited due to dose-limiting hepatic toxicity. PSMA is highly upregulated on tumor cells and tumor neovasculature in prostate cancer and other solid tumors, including lung (NSCLC), kidney (clear cell RCC), bladder and colorectal cancers. CB307 is a half-life extended Humabody® that selectively agonizes CD137 only in the presence of prostate specific membrane antigen (PSMA, HGNC:FOLH1). CB307 is trispecific with VH components selected for optimal binding to PSMA, CD137 and human serum albumin (HSA). We present here the non-clinical pharmacology and toxicology of CB307. Methods: CD137 agonism and immune cell activation were evaluated in vitro using coculture assays with PSMA-expressing tumor cells as assessed by NFκB-driven luciferase and cytokine secretion. CB307 was evaluated as a single agent and in combination with enzalutamide, PD1 or PDL1 inhibitors. In vivo experiments used a syngeneic mouse system transgenic for the human CD137 immune cell target, and human PSMA expressed on mouse tumor cells. Nonclinical toxicology and pharmacokinetics of CB307 were assessed in cynomolgus macaques. Results: In reporter assays and PBMC cocultures, CB307 activated CD137 only in the presence of PSMA-expressing cells leading to enhanced immune cell activation. Enzalutamide pre-treatment of 22Rv1 (PSMA-low) cells induced a 1.5-fold increase in surface PSMA expression leading to enhanced CB307 activity. CB307 or PD1/PDL1 inhibition alone induced IL-2 secretion in PBMC/PSMA-expressing tumor cell cocultures; this effect was synergistically enhanced when CB307 was combined with either PD1 or PDL1 inhibition. In an in vivo pharmacology study in immuno-competent mice, CB307 demonstrated statistically significant inhibition of tumor growth. CB307 was well-tolerated in a GLP-toxicology study in cynomolgus macaques, with an observed half-life supporting weekly clinical administration. Summary: CB307 is a novel CD137 agonist that selectively enhances immune cell activity in the presence of PSMA-positive cells. Our data demonstrates CB307 immunological pharmacology both in vitro and in vivo which has supported the initiation of the phase I ‘POTENTIA’ clinical trial (NCT04839991). Citation Format: Andrew J. Pierce, Phillip M. Brailey, Clare Song, Sophie Archer, Phillip D. Bartlett, Philip Bland-Ward. CB307: A novel selective CD137 agonist for enhancement of immune cell responses to PSMA+ tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2877.

4-OR: IL6R Signaling in Adipogenesis Mediates GLP-1 Analog Effects on Adipose Tissue Beiging

Mechanisms underlying antidiabetes effects of GLP-1 analogs such as liraglutide, collectively termed incretins, remain incompletely understood. While incretins boost insulin secretion, they also modulate the immune system and have anti-obesity effects. We discovered that prediabetic patients treated with incretins have increased thermogenic gene expression in supraclavicular adipose tissue (AT) . Interestingly, incretin-treated patients were found to have elevated levels of IL6 plasma, consistent with the observation that incretin treatment induces IL6 secretion by human monocytes. We hypothesized that incretins induce adipocyte browning via IL6 and used the mouse model to test if IL6 receptor (IL6Ra) signaling underlies the effects of the incretin-IL6 axis. We show that liraglutide transiently increases IL6 in circulation and ILRa signaling in AT while metronomic liraglutide treatment results in AT browning and improved glucose clearance in mice. IL6-blocking antibody treatment inhibited the thermogenic and antidiabetes effects of liraglutide. Based on the observation that IL6 induces thermogenic gene expression in cultured adipocytes, we hypothesized that the incretin-IL6 axis directly induces adipocyte browning. To test this, we generated mice with IL6Ra knockout in Pdgfrb-positive adipocyte progenitors (APC-KO) and in adipocytes (AD-KO) . We show that liraglutide-induced weight loss does not depend on IL6 signaling in the adipocyte lineage. However, both IL6Ra APC-KO and AD-KO mice failed to undergo thermogenic adipocyte browning and glucose utilization increase in response to liraglutide treatment. We conclude that the antidiabetes effects of metronomically administered incretins are mediated by transient upregulation of IL6, which activates canonical ILRa signaling and thermogenesis in adipocytes. Disclosure A.D.Gutierrez: None. Z.Gao: None. Y.Yu: None. M.Kolonin: n/a. Funding National Institutes of Health (NIDDK 5R21DK122234)

Bacteroides fragilis Toxin Induces Intestinal Epithelial Cell Secretion of Interleukin-8 by the E-Cadherin/β-Catenin/NF-κB Dependent Pathway.

Enterotoxigenic Bacteroides fragilis (ETBF) has emerged as a gut microbiome pathogen that can promote colitis associated cancer in humans. ETBF secretes the metalloprotease, B. fragilis toxin (BFT), which can induce ectodomain cleavage of E-cadherin and IL-8 secretion through the β-catenin, NF-κB, and MAPK pathways in intestinal epithelial cells. However, it is still unclear whether E-cadherin cleavage is required for BFT induced IL-8 secretion and the relative contribution of these signaling pathways to IL-8 secretion. Using siRNA knockdown and CRISPR knockout studies, we found that E-cadherin cleavage is required for BFT mediated IL-8 secretion. In addition, genetic ablation of β-catenin indicates that β-catenin is required for the BFT induced increase in transcriptional activity of NF-κB, p65 nuclear localization and early IL-8 secretion. These results suggest that BFT induced β-catenin signaling is upstream of NF-κB activation. However, despite β-catenin gene disruption, BFT still activated the MAPK pathway, suggesting that the BFT induced activation of the MAPK signaling pathway is independent from the E-cadherin/β-catenin/NF-κB pathway. These findings show that E-cadherin and β-catenin play a critical role in acute inflammation following ETBF infection through the inflammatory response to BFT in intestinal epithelial cells.

Host immune response mediates changes in cagA copy number and virulence potential of Helicobacter pylori

ABSTRACT Helicobacter pylori is the major risk factor for gastric cancer. H. pylori harboring the type IV secretion system (T4SS) and its effector CagA encoded on the cag pathogenicity Island (cagPAI) increases the risk. H. pylori PMSS1 has a multi-cagA genotype, modulating cagA copy number dynamically from zero to four copies. To examine the effect of the immune response on cagA copy number change, we utilized a mouse model with different immune status. PMSS1 recovered from Rag1−/− mice, lacking functional T or B cells, retained more cagA copies. PMSS1 recovered from Il10 −/− mice, showing intense inflammation, had fewer cagA copies compared to those recovered from wild-type mice. Moreover, cagA copy number of PMSS1 recovered from wild-type and Il10 −/− mice was positively correlated with the capacity to induce IL-8 secretion at four weeks of infection. Since recombination in cagY influences T4SS function, including CagA translocation and IL-8 induction, we constructed a multiple linear regression model to predict H. pylori-induced IL-8 expression based on cagA copy number and cagY recombination status; H. pylori induces more IL-8 secretion when the strain has more cagA copies and intact cagY. This study shows that H. pylori PMSS1 in mice with less intense immune response possess higher cagA copy number than those infected in mice with more intense immune response and thus the multi-cagA genotype, along with cagY recombination, functions as an immune-sensitive regulator of H. pylori virulence.

Polyphyllin I Inhibits Propionibacterium acnes-Induced IL-8 Secretion in HaCaT Cells by Downregulating the CD36/NOX1/ROS/NLRP3/IL-1β Pathway.

Acne vulgaris (AV) is a chronic skin disease involving inflammation of the pilosebaceous units. Propionibacterium acnes (P. acnes) hypercolonization is one pathogenic factor for AV. P. acnes that triggers interleukin-1β (IL-1β) by activating the pyrin domain-containing 3 protein (NLRP3) inflammasome of the NOD-like receptor family in human monocytes. Reactive oxygen species (ROS) acts as a trigger for the production of IL-8 and activates theNLRP3 inflammasome. IL-8 promotes the metastasis and multiplication of different cancerous cells, whereas keratinocyte proliferation and migration contribute to the progression of AV. A steroidal saponin called polyphyllin I (PPI) that is extracted from Paris polyphylla's rhizomes has anti-inflammatory properties. This study investigates the regulatory role of P. acnes in the secretion of IL-8 mediated by the CD36/NADPH oxidase 1 (NOX1)/ROS/NLRP3/IL-1β pathway and the effects of PPI on the CD36/NOX1/ROS/NLRP3/IL-1β/IL-8 pathway and human keratinocyte proliferation and migration. HaCaT cells were cultured and stimulated with 108 CFU/ml of P. acnes for 0, 6, 12, 18, 24, 30, and 36 hours. P. acnes induced IL-8 secretion from HaCaT cells via the CD36/NOX1/ROS/NLRP3/IL-1β pathway. PPI inhibited the CD36/NLRP3/NOX1/ROS/IL-8/IL-1β pathway and HaCaT cell proliferation and migration. PPI alleviates P. acnes-induced inflammatory responses and human keratinocyte proliferation and migration, implying a novel potential therapy for AV.

Immunological Differences in Human Peripheral Blood Mononuclear Cells Treated with Traditional Japanese Herbal Medicines Hochuekkito, Juzentaihoto, and Ninjin'yoeito from Different Pharmaceutical Companies.

Hochuekkito (HET), Juzentaihoto (JTT), and Ninjin'yoeito (NYT) have been used as Hozai, a group of traditional Japanese herbal medicines, to treat physically and mentally weak cancer patients. Their compositions are quite different, and Japanese pharmaceutical companies have been using different types or quantities of herbs for formulations with the same name. Here, we compared the immunological differences between HET, JTT, and NYT with respect to the induced T cell subsets and cytokines. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and treated with 0 (control), 25, 50, 100, 200, or 400 μg/mL HET, JTT, or NYT (manufactured by Tsumura [TJ], Kracie [KR], and Kotaro [KO]). PBMC proliferation, CD4+ T cell, CD8+ T cell, and regulatory T cell (Treg) proportions and interleukin (IL) concentrations (IL-6, IL-10, IL-17A, interferon-γ, tumor necrosis factor-α, and transforming growth factor (TGF)-β) secreted by PBMCs were measured using Cell Counting Kit-8 or flow cytometry bead analysis. PBMC proliferation and CD4+ T cell percentages were similar in the HET, JTT, NYT, and control groups; however, the percentage of CD8+ T cells tended to increase after treatments. Tregs were suppressed by HET, JTT, and NYT, and TJ-JTT significantly decreased Treg numbers (compared with control). The concentrations of all cytokines except TGF-β were increased in a concentration-dependent manner (p < 0.05); particularly, KR-HET induced IL-6 secretion (compared with the control, TJ-HET, and KO-HET; 37-, 7-, and 17-fold, respectively; p < 0.05). The TGF-β concentration was decreased in a concentration-dependent manner by HET, JTT, and NYT (compared with the control). These results suggest that, compared with TJ-HET and KO-HET, KR-HET should be administered with caution. Although HET, JTT, and NYT belong to the same Hozai group and have the same names among companies, their differing effects on immune activity must be considered and they must be administered with caution.

Narrowing down the number of potential plant-based probiotic candidates by successive in vitro, technological and in vivo assays.

The interest on plant-based fermented food is in raise in Western countries. The aim of this study was to select interleukin (IL)-10 inducing strains for the development of potential probiotic plant-based fermented foods. Departing from a collection of 52 lactic acid bacteria (LAB) strains derived from plant material, in vitro co-culture with murine macrophages allowed us to narrow down the number of candidates to 21 strains able to induce IL-10 secretion. 14 of these strains were able to promote the production of tumour necrosis factor-α too. The capacity to induce IL-6 was used to further reduce the number of strains to 4, from which Lactiplantibacillus plantarum subsp. plantarum LpAv was selected to ferment oat and carrots. L. plantarum LpAv was able to ferment oat and carrots until reaching counts of ca. 108 and 109 cfu/ml. Fermented oat and carrots were orally administered to mice for 10 consecutive days and challenged with a single infective dose of Salmonella enterica serovar. Typhimurium. Counts of L. plantarum LpAv in fermented carrots were 9.23±0.05 cfu/ml and 9.27±0.01 cfu/ml, at day 1 and 10 of the feeding period. Fermented carrots were able to confer enhanced protection (80% of survival) against infection, when compared to control mice (less than 25% of survival). However, L. plantarum LpAv administered as pure culture was not able to confer protection against Salmonella infection. L. plantarum LpAv was selected among 52 plant-derived LAB and it was able to ferment oat and carrots, being only fermented carrots able to confer enhanced protection against Salmonella infection. A succession of in vitro to in vivo tests is proposed as a tool to narrow down the number of candidates when searching for potential novel probiotics from a collection of autochthonous strains.

IRE1α Expedites the Progression of Castration-Resistant Prostate Cancers via the Positive Feedback Loop of IRE1α/IL-6/AR.

Castration-resistant prostate cancer (CRPC) is the lethal form of prostate cancer (PCa), and the underlying molecular mechanism has not been fully elucidated. Inositol requiring enzyme 1 alpha (IRE1α), a key regulator of unfolded protein response (UPR), is intimately associated with PCa progression. However, whether IRE1α is implicated in CRPC development remains unknown. Here, we showed that IRE1α expression was significantly increased in CRPC tissues and high-grade PCa tissues. Overexpression of IRE1α promoted PCa cell proliferation under the androgen deficiency condition in vitro and in vivo. Mechanistically, increased IRE1α expression induced IL-6 secretion via the IRE1α/XBP-1s signal pathway. IRE1α-induced IL-6 activated androgen receptor (AR), and the activation of AR by IL-6, in turn, promoted IRE1α expression. IRE1α formed a positive feedback loop with IL-6 and AR to promote prostate cancer cell proliferation under the androgen-deficient condition. In clinical PCa samples, high IRE1α expression correlated with elevated IL-6 and increased PSA expression. Our findings demonstrated a novel mechanism of CRPC progression and suggest targeting IRE1α may be a potential target for the prevention and treatment of CRPC.

CagL polymorphisms between East Asian and Western Helicobacter pylori are associated with different abilities to induce IL-8 secretion.

Helicobacter pylori colonizes human gastric mucosa. Its infection is associated with gastric diseases including gastric cancer. CagA is one of the most important toxins produced by H. pylori. It is related to gastric cancer which can be injected into host cells via a type IV secretion system (T4SS). CagL is a structural component of T4SS apparatus, which triggers host cell signaling pathway. It has been reported that CagL polymorphisms may influence the severity of disease development. To explore the contribution of CagL polymorphisms between East Asian and Western H. pylori in pathogenesis, cagL gene in G27 H. pylori was swapped by K74 cagL which is identical to East Asian CagL consensus sequence and by Western 26695 H. pylori, resulting in G27 ΔcagL/cagLK74 and G27 ΔcagL/cagL26695, respectively. Intriguingly, G27 ΔcagL/cagLK74 showed significantly less ability of IL-8 induction than G27 ΔcagL/cagL26695 while displayed similar abilities of CagA phosphorylation, and cell elongation. Taken together, this study suggests that the CagL polymorphism may influence IL-8 induction, and K74 CagL has less ability to induce IL-8 secretion than G27 or 26695 CagL. Further research should address how the different capabilities of IL-8 induction between intraspecies-CagL are associated with the large differences of the incidence of gastric cancer between East Asian and Western countries.

Glycated Albumin Triggers an Inflammatory Response in the Human Airway Epithelium and Causes an Increase in Ciliary Beat Frequency.

The role of inflammation in airway epithelial cells and its regulation are important in several respiratory diseases. When disease is present, the barrier between the pulmonary circulation and the airway epithelium is damaged, allowing serum proteins to enter the airways. We identified that human glycated albumin (GA) is a molecule in human serum that triggers an inflammatory response in human airway epithelial cultures. We observed that single-donor human serum induced IL-8 secretion from primary human airway epithelial cells and from a cystic fibrosis airway cell line (CF1-16) in a dose-dependent manner. IL-8 secretion from airway epithelial cells was time dependent and rapidly increased in the first 4 h of incubation. Stimulation with GA promoted epithelial cells to secrete IL-8, and this increase was blocked by the anti-GA antibody. The IL-8 secretion induced by serum GA was 10–50-fold more potent than TNFα or LPS stimulation. GA also has a functional effect on airway epithelial cells in vitro, increasing ciliary beat frequency. Our results demonstrate that the serum molecule GA is pro-inflammatory and triggers host defense responses including increases in IL-8 secretion and ciliary beat frequency in the human airway epithelium. Although the binding site of GA has not yet been described, it is possible that GA could bind to the receptor for advanced glycated end products (RAGE), known to be expressed in the airway epithelium; however, further experiments are needed to identify the mechanism involved. We highlight a possible role for GA in airway inflammation.

Inhibitory effects of α-Mangostin on T cell cytokine secretion via ORAI1 calcium channel and K+ channels inhibition.

BackgroundAs one of the main components of mangosteen (Garcinia mangostana), a tropical fruit, α-mangostin has been reported to have numerous pharmacological benefits such as anti-cancer, anti-inflammatory, and anti-allergic effects through various mechanisms of action. The effects of α-mangostin on intracellular signaling proteins is well studied, but the effects of α-mangostin on ion channels and its physiological effects in immune cells are unknown. Generation of intracellular calcium signaling is a fundamental step for T cell receptor stimulation. This signaling is mediated not only by the ORAI1 calcium channel, but also by potassium ion channels, which provide the electrical driving forces for generating sufficient calcium ion influx. This study investigated whether α-mangosteen suppress T cell stimulation by inhibiting ORAI1 and two kinds of potassium channels (Kv1.3 and KCa3.1), which are normally expressed in human T cells.MethodsThis study analyzed the inhibitory effect of α-mangostin on immune cell activity via inhibition of calcium and potassium ion channels expressed in immune cells.Resultsα-mangostin inhibited ORAI1 in a concentration-dependent manner, and the IC50 value was 1.27 ± 1.144 µM. Kv1.3 was suppressed by 41.38 ± 6.191% at 3 µM, and KCa3.1 was suppressed by 51.16 ± 5.385% at 3 µM. To measure the inhibition of cytokine secretion by immune cells, Jurkat T cells were stimulated to induce IL-2 secretion, and α-mangostin was found to inhibit it. This study demonstrated the anti-inflammatory effect of α-mangostin, the main component of mangosteen, through the regulation of calcium signals.

KLHDC7B-DT aggravates pancreatic ductal adenocarcinoma development via inducing cross-talk between cancer cells and macrophages.

Tumor microenvironment (TME) exerts key roles in pancreatic ductal adenocarcinoma (PDAC) development. However, the factors regulating the cross-talk between PDAC cells and TME are largely unknown. In the present study, we identified a long noncoding RNA (lncRNA) KLHDC7B divergent transcript (KLHDC7B-DT), which was up-regulated in PDAC and correlated with poor survival of PDAC patients. Functional assays demonstrated that KLHDC7B-DT enhanced PDAC cell proliferation, migration, and invasion. Mechanistically, KLHDC7B-DT was found to directly bind IL-6 promoter, induce open chromatin structure at IL-6 promoter region, activate IL-6 transcription, and up-regulate IL-6 expression and secretion. The expression of KLHDC7B-DT was positively correlated with IL-6 in PDAC tissues. Via inducing IL-6 secretion, KLHDC7B-DT activated STAT3 signaling in PDAC cells in an autocrine manner. Furthermore, KLHDC7B-DT also activated STAT3 signaling in macrophages in a paracrine manner, which induced macrophage M2 polarization. KLHDC7B-DT overexpressed PDAC cells-primed macrophages promoted PDAC cell proliferation, migration, and invasion. Blocking IL-6/STAT3 signaling reversed the effects of KLHDC7B-DT on macrophage M2 polarization and PDAC cell proliferation, migration, and invasion. In conclusion, KLHDC7B-DT enhanced malignant behaviors of PDAC cells via IL-6-induced macrophage M2 polarization and IL-6-activated STAT3 signaling in PDAC cells. The cross-talk between PDAC cells and macrophages induced by KLHDC7B-DT represents potential therapeutic target for PDAC.

Campylobacter coli strains from Brazil can invade phagocytic and epithelial cells and induce IL-8 secretion.

Campylobacter spp. have been a predominant cause of bacterial foodborne gastroenteritis worldwide, causing substantial costs to public healthcare systems. This study aimed to assess the invasion and pro-inflammatory cytokine production capacity of Campylobacter coli strains isolated in Brazil. A total of 50 C. coli isolated from different sources in Brazil were analyzed for their capacity of invasion in Caco-2 and U-937 cell lines. The production of pro-inflammatory cytokines was quantitatively measured in response to C. coli. All the strains studied showed invasion percentage ≥ 40% in polarized Caco-2 cells. In U-937 cells assay, 35 of 50 C. coli strains studied showed invasion percentage ≥ 50%. A significant increase in IL-8 production by infected U-937 cells was observed for 17.5% of the C. coli isolates. The high percentages of invasion in Caco-2 and U-937 cells observed for all studied strains, plus the increased production of IL-8 by U-937 cells against some strains, highlighted the pathogenic potential of the C. coli studied and bring extremely relevant data since it has never been reported for strains isolated in Brazil and there are a few data for C. coli in the literature.

Role of c-Myc haploinsufficiency in the maintenance of HSCs in mice

AbstractThis study was conducted to determine the dosage effect of c-Myc on hematopoiesis and its distinct role in mediating the Wnt/β-catenin pathway in hematopoietic stem cell (HSC) and bone marrow niche cells. c-Myc haploinsufficiency led to ineffective hematopoiesis by inhibiting HSC self-renewal and quiescence and by promoting apoptosis. We have identified Nr4a1, Nr4a2, and Jmjd3, which are critical for the maintenance of HSC functions, as previously unrecognized downstream targets of c-Myc in HSCs. c-Myc directly binds to the promoter regions of Nr4a1, Nr4a2, and Jmjd3 and regulates their expression. Our results revealed that Nr4a1 and Nr4a2 mediates the function of c-Myc in regulating HSC quiescence, whereas all 3 genes contribute to the function of c-Myc in the maintenance of HSC survival. Adenomatous polyposis coli (Apc) is a negative regulator of the Wnt/β-catenin pathway. We have provided the first evidence that Apc haploinsufficiency induces a blockage of erythroid lineage differentiation through promoting secretion of IL6 in bone marrow endothelial cells. We found that c-Myc haploinsufficiency failed to rescue defective function of Apc-deficient HSCs in vivo but it was sufficient to prevent the development of severe anemia in Apc–heterozygous mice and to significantly prolong the survival of those mice. Furthermore, we showed that c-Myc–mediated Apc loss induced IL6 secretion in endothelial cells, and c-Myc haploinsufficiency reversed the negative effect of Apc-deficient endothelial cells on erythroid cell differentiation. Our studies indicate that c-Myc has a context-dependent role in mediating the function of Apc in hematopoiesis.

Fusobacteria modulate oral carcinogenesis and promote cancer progression

ABSTRACT Background: Evidence suggest periodontal bacterial infection can contribute to oral cancer initiation and progression. Aim: To investigate the effects of periodontal bacteria on oral cancer cell behavior using a cell-based system and a mouse carcinogenesis model. Methods: Oral cancer cell lines were polyinfected with four periodontal bacteria. Cytokine levels and relative changes in oncogene mRNA expression were determined post-infection. Oral tumours in mice induced by 4-nitroquinoline-1-oxide (4NQO) were compared with and without administrating periodontal bacteria. Results: Polyinfected oral cancer cells had upregulated MMP1, MMP9, and IL-8. The expression of cell survival markers MYC, JAK1, and STAT3 and epithelial-mesenchymal transition markers ZEB1 and TGF-β were also significantly elevated. Monoinfections showed F. nucleatum alone had comparable or greater effects than the four bacteria together. Fusobacterial culture supernatant, primarily LPS, was sufficient to induce IL-8 secretion, demonstrating that direct contact of live Fusobacteria with cancer cells might not be required to exert changes in cancer cell behaviour. In the 4NQO-induced oral tumour model, mice infected with bacteria developed significantly larger and more numerous lesions compared to those not infected. Conclusion: This study demonstrated that Fusobacteria could potentially enhance cancer cell invasiveness, survival, and EMT when presented in the oral tumour microenvironment. Abbreviations: 4NQO, 4-nitroquinoline-1-oxide; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial–mesenchymal transition; IL-8, interleukin-8; JAK1, Janus kinase 1; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; OSCCs, oral squamous cell carcinomas; PK, proteinase K; PMB, Polymyxin B; qRT-PCR, quantitative real-time polymerase chain reaction; STAT3, signal transducer and activator of transcription 3; TGF-β, transforming growth factor beta; ZEB1, zinc finger E-Box binding homeobox 1

Activation of aryl hydrocarbon receptor in Langerhans cells by a microbial metabolite of tryptophan negatively regulates skin inflammation

BackgroundSkin commensal bacteria play important roles in skin homeostasis. Langerhans cells (LCs) are epidermis-resident dendritic cells that sense environmental stimuli and are critical in the induction of immune tolerance to allergen and bacterial skin flora. However, response of LCs to the metabolites of the skin microbiota is not clear. ObjectiveTo explore the effects of the skin microbial metabolites on LCs activation. MethodsLCs derived from CD34+ hematopoietic stem cells in the cord blood were treated with a microbial metabolite of tryptophan, indole-3-aldehyde (IAId). Activation aryl hydrocarbon receptor (AhR) signaling, production of IL-10, and expression of receptor activator of NF-κB (RANK) / receptor activator of NF-κB ligand (RANKL) in LCs or keratinocytes were analyzed using quantitative PCR, western blotting and flow cytometry. LCs maturation induced by IAId and CD4+ T cell response induced by IAId-conditioned LCs were also investigated. ResultsIAId induced the production of indoleamine 2,3-dioxygenase (IDO) and IL-10 in LCs through the activation of AhR. IAId promoted the expression of RANK and RANKL on LCs and keratinocytes in an AhR-dependent manner respectively, which might result in activation of NF-κB signaling and production of IL-10. Moreover, a mature phenotype of LCs was induced by IAId, and IAId-activated LCs inhibited CD4+ T cell proliferation and induced IL-10 secretion. ConclusionsOur study revealed a negatively regulatory function of a tryptophan metabolite on LCs through the activation of AhR, and the microbial metabolites could be utilized in future treatment for inflammatory skin diseases.

The impact of the level and distribution of methyl-esters of pectins on TLR2-1 dependent anti-inflammatory responses

Pectins have anti-inflammatory effects via Toll-like receptor (TLR) inhibition in a degree of methyl-esterification-(DM)-dependent manner. However, pectins also vary in distribution of methyl-esters over the galacturonic-acid (GalA) backbone (Degree of Blockiness - DB) and impact of this on anti-inflammatory capacity is unknown. Pectins mainly inhibit TLR2-1 but magnitude depends on both DM and DB. Low DM pectins (DM18/19) with both low (DB86) and high DB (DB94) strongly inhibit TLR2-1. However, pectins with intermediate DM (DM43/DM49) and high DB (DB60), but not with low DB (DB33), inhibit TLR2-1 as strongly as low DM. High DM pectins (DM84/88) with DB71 and DB91 do not inhibit TLR2-1 strongly. Pectin-binding to TLR2 was confirmed by capture-ELISA. In human macrophages, low DM and intermediate DM pectins with high DB inhibited TLR2-1 induced IL-6 secretion. Both high number and blockwise distribution of non-esterified GalA in pectins are responsible for the anti-inflammatory effects via inhibition of TLR2-1.

Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients.

Initially underestimated as platelet dust, extracellular vesicles are continuously gaining interest in the field of inflammation. Various studies addressing inflammatory diseases have shown that microvesicles (MVs) originating from different cell types are systemic transport vehicles carrying distinct cargoes to modulate immune responses. In this study, we focused on the clinical setting of multiple trauma, which is characterized by activation and dysfunction of both, the fluid-phase and the cellular component of innate immunity. Given the sensitivity of neutrophils for the complement anaphylatoxin C5a, we hypothesized that increased C5a production induces alterations in MV shedding of neutrophils resulting in neutrophil dysfunction that fuels posttraumatic inflammation. In a mono-centered prospective clinical study with polytraumatized patients, we found significantly increased granulocyte-derived MVs containing the C5a receptor (C5aR1, CD88) on their surface. This finding was accompanied by a concomitant loss of C5aR1 on granulocytes indicative of an impaired cellular chemotactic and pro-inflammatory neutrophil functions. Furthermore, in vitro exposure of human neutrophils (from healthy volunteers) to C5a significantly increased MV shedding and C5aR1 loss on neutrophils, which could be blocked using the C5aR1 antagonist PMX53. Mechanistic analyses revealed that the interaction between C5aR1 signaling and the small GTPase Arf6 acts as a molecular switch for MV shedding. When neutrophil derived, C5a-induced MV were exposed to a complex ex vivo whole blood model significant pro-inflammatory properties (NADPH activity, ROS and MPO generation) of the MVs became evident. C5a-induced MVs activated resting neutrophils and significantly induced IL-6 secretion. These data suggest a novel role of the C5a-C5aR1 axis: C5a-induced MV shedding from neutrophils results in decreased C5aR1 surface expression on the one hand, on the other hand it leads to profound inflammatory signals which likely are both key drivers of the neutrophil dysfunction which is regularly observed in patients suffering from multiple traumatic injuries.

Dexmedetomidine promotes the progression of hepatocellular carcinoma through hepatic stellate cell activation

Dexmedetomidine (DEX) is an anesthetic that is widely used in the clinic, and it has been reported to exhibit paradoxical effects in the progression of multiple solid tumors. In this study, we sought to explore the mechanism by which DEX regulates hepatocellular carcinoma (HCC) progression underlying liver fibrosis. We determined the effects of DEX on tumor progression in an orthotopic HCC mouse model of fibrotic liver. A coculture system and a subcutaneous xenograft model involving coimplantation of mouse hepatoma cells (H22) and primary activated hepatic stellate cells (aHSCs) were used to study the effects of DEX on HCC progression. We found that in the preclinical mouse model of liver fibrosis, DEX treatment significantly shortened median survival time and promoted tumor growth, intrahepatic metastasis and pulmonary metastasis. The DEX receptor (ADRA2A) was mainly expressed in aHSCs but was barely detected in HCC cells. DEX dramatically reinforced HCC malignant behaviors in the presence of aHSCs in both the coculture system and the coimplantation mouse model, but DEX alone exerted no significant effects on the malignancy of HCC. Mechanistically, DEX induced IL-6 secretion from aHSCs and promoted HCC progression via STAT3 activation. Our findings provide evidence that the clinical application of DEX may cause undesirable side effects in HCC patients with liver fibrosis.

312 Activation of AhR in Langerhans cells by a microbial metabolite of tryptophan maintains skin homeostasis

Skin commensal bacteria play important roles in maintenance of skin homeostasis, and Langerhans cells (LCs) are critical in induction of peripheral tolerance; however, the role of LCs' response to the skin microbiota, specifically the microbial metabolites, in skin homeostasis is not clear. In the present study, LCs that derived from CD34+ hematopoietic stem cells in the cord blood cells were utilized, and we found that a microbial metabolite of tryptophan (Trp), indole-3-aldehyde (IAId), induced the production of indoleamine 2,3-dioxygenase and IL-10 in LCs through activation of aryl hydrocarbon receptor (AhR). Moreover, IAId promoted the expression of receptor activator of NF-κB and receptor activator of NF-κB ligand on LCs and keratinocytes in an AhR-dependent manner respectively, which might result in the activation of NF-κB signaling and production of IL-10. Additionally, despite a mature phenotype of LCs induced by IAId, IAId-activated LCs inhibited CD4+ T cell proliferation and induced IL-10 secretion. Our study revealed a negatively regulatory function of a Trp microbial metabolite on LCs through activation of AhR, and microbial metabolites could be developed as a new strategy for the treatment of inflammatory skin diseases.