IL-17A Secretion Research Articles

Curcumin, novel application in reversing myocardial fibrosis in the treatment for atrial fibrillation from the perspective of transcriptomics in rat model

In order to explore the possible mechanism of curcumin in the treatment of AF, we focused on the myocardial fibrosis in the pathogenesis of atrial fibrillation to explore whether curcumin could play a role in the treatment of AF by reducing myocardial fibrosis.Rats were given daily gavage of saline (control and AF groups) or curcumin (4 mL/kg, concentration: 50 mg/mL, curcumin groups) during days 4–28. The rat model of AF was induced by Ach - CaCl2, and evaluate the therapeutic effect of curcumin on the duration of AF rhythm, the degree of myocardial fibrosis and the secretion of inflammatory factors in serum. RNA-seq to explore the possible mechanism of curcumin alleviating myocardial fibrosis of AF. curcumin significantly inhibits the duration of AF and reduces the degree of left atrial fibrosis. ELISA results showed curcumin could significantly reduce the secretion of IL-17A, IL-1β, IL -6 and TGF-β1. Bioinformatics analyses revealed that the IL-17 signaling pathway are involved in the therapeutic mechanism of curcumin. Furthermore, The genes encoding Col1a1, Fasn, Pck1, Bmp10, IL33 and Figf were pivotal and possible key genes for the therapeutic mechanisms of curcumin.Curcumin can reduce the degree of left atrial fibrosis of AF and the secretion of inflammatory factors. The therapeutic effect of curcumin on AF was attributed to its effect on the IL-17 signaling pathway. Besides, COL1A1, FASN, PCK1, BMP10, IL33 and FIGF were the pivotal genes associated with mechanisms of action of curcumin on AF.

Development of a mechanically stable human hair keratin film for cell culture

An easy-to-handle keratin film was successfully fabricated using solely purified hair keratins. Keratin was extracted from human hair by an existing protocol. The extracted keratin was made into a mechanically stable film by solution casting and air-drying at room temperature. The films obtained were characterized for surface morphology, wettability, protein secondary structures, mechanical properties, permeability, and thermal properties. Interestingly, the keratin film showed distinct surface and cross-sectional morphology, and protein secondary structure transformation. In addition, the keratin film exhibited Young’s modulus of 1.05 ± 0.09 GPa when it was dry. In the wet state, the keratin film behaved as viscoelastic material and was highly stretchable at 179 ± 17% strain at break. Permeability test was conducted using 20 kDa-FITC dextran which revealed an anomalous diffusion mechanism through the keratin film. Additionally, the keratin film elicited positive cellular responses by human epidermal keratinocytes (HEKs) in terms of enhanced cell proliferation, viability, keratin 14 expression, and IL-1α secretion, in comparison to collagen I. Taken together, a human hair keratin-based film with its mechanical and thermal stability, and cytocompatibility, presents a promising platform for cell culture applications.

Self-assembled nanoparticles with bilirubin/JPH203 alleviate imiquimod-induced psoriasis by reducing oxidative stress and suppressing Th17 expansion

Psoriasis is a chronic inflammatory skin disease with dysregulation of the immune system, which usually manifests as erythematous scleroma with hyperplasia of the epidermis. Although the pathogenesis remains unclear, oxidative stress and inflammation are inextricably linked during the development of psoriasis. Therefore, scavenging of ROS and suppression of inflammation may be a rational and effective strategy to ameliorate psoriasis and delay its further development. Here, a “Tanghulu”-like nanoplatform (BJN) is developed using the endogenous antioxidant bilirubin and a specific inhibitor (JPH203) of LAT1, an amino acid transporter involved in the activation of mTOR, for treatment of psoriasis by scavenging ROS and interfering with mTOR signaling, consequently blocking Th17 and IL-17 release. The nanoparticles show increased uptake in inflamed keratinocytes and display an efficient ROS scavenging capability. In an in vivo psoriasis model, BJN effectively permeate into the inflamed skin and be retained there, thereby reversing the psoriasis-specific changes in the imiquimod-induced mice as evident from suppression of keratinocyte hyperplasia and decrease in immune cell infiltration. This study shows that BJN can exert a superior anti-psoriasis effect by scavenging ROS, blocking the LAT1-mTOR pathway, inactivating immune reaction, restricting Th17 differentiation, and decreasing IL-17A secretion, offering a promising strategy for psoriasis treatment.

Transcriptional Interactomic Inhibition of RORα Suppresses Th17-Related Inflammation.

PurposeTh17 cells and their cytokines are implicated in the pathogenesis of various autoimmune diseases. Retinoic acid-related orphan receptor alpha (RORα) is a transcription factor for the differentiation and the inflammatory functions of Th17 cells. In this study, we generated the nucleus-transducible form of transcription modulation domain of RORα (nt-RORα-TMD) to investigate the functional roles of RORα in vitro and in vivo under normal physiological condition without genetic alteration.MethodsThe functions of nt-RORα-TMD were analyzed in vitro through flow cytometry, luciferase assay, ELISA, and transcriptome sequencing. Finally, the in vivo therapeutic effects of nt-RORα-TMD were verified in dextran sulfate sodium (DSS)-induced colitis mice.Resultsnt-RORα-TMD was effectively delivered into the cell nucleus in a dose- and time-dependent manner without any cellular toxicity. nt-RORα-TMD competitively inhibited the RORα-mediated transcription but not RORγt-mediated transcription. Secretion of IL-17A from the splenocytes was suppressed by nt-RORα-TMD without affecting the secretion of Th1- or Th2-type cytokine and T cell activation events such as induction of CD69 and CD25. The differentiation potential of naïve T cells into Th17 cells, not into Th1, Th2, or Treg cells, was significantly blocked by nt-RORα-TMD. Consistently, mRNA sequencing analysis showed that nt-RORα-TMD treatment down-regulated the expression of the genes related to the differentiation and functions of Th17 cells. Treatment of DSS-induced colitis mice with nt-RORα-TMD improved the overall symptoms of colitis, such as body weight change, colon length, infiltration of inflammatory cells, and the level of inflammatory cytokines in the serum. In the mesenteric lymph node (MLN) of the nt-RORα-TMD-treated mice, the population of CD4+IL-17A+ Th17 cells was reduced, and the population of CD4+Foxp3+ Treg cells increased.Conclusionnt-RORα-TMD has a potential to be developed as a novel therapeutic reagent for treating various inflammatory diseases in which Th17 cells are the leading pathological player.

VGLL3 activates inflammatory responses by inducing interleukin-1α secretion.

Vestigial-like family member 3 (VGLL3), a member of the vestigial-like family, is a cofactor of the TEA-domain-containing transcription factor (TEAD). Although elevation in VGLL3 expression is associated with inflammatory diseases, such as inflammatory sarcomas and autoimmune diseases, the molecular mechanisms underlying VGLL3-mediated inflammation remain largely unknown. In this study, we analyzed the relationship between elevated VGLL3 expression and the levels of NF-κB, a transcription factor that plays a pivotal role in inflammation. NF-κB was found to be activated in a cell line stably expressing VGLL3. Mechanistically, VGLL3 was shown to promote the expression and secretion of the potent NF-κB-activating cytokine interleukin (IL)-1α, probably through its association with TEADs. As VGLL3 is a target of transforming growth factor β (TGF-β) signaling, we analyzed IL-1α induction upon TGF-β stimulation. TGF-β stimulation was observed to induce IL-1α secretion and NF-κB activation, and VGLL3 was associated with this phenomenon. The TGF-β transcription factors Smad3 and Smad4 were shown to be necessary for inducing VGLL3 and IL-1α expression. Lastly, we found that VGLL3-dependent IL-1α secretion is involved in constitutive NF-κB activation in highly malignant breast cancer cells. Collectively, the findings suggested that VGLL3 expression and TGF-β stimulation activate the inflammatory response by inducing IL-1α secretion.

In Vitro Cellular and Molecular Interplay between Human Foreskin-Derived Mesenchymal Stromal/Stem Cells and the Th17 Cell Pathway.

Foreskin, considered a biological waste material, has been shown to be a reservoir of therapeutic cells. The immunomodulatory properties of mesenchymal stromal/stem cells (MSCs) from the foreskin (FSK-MSCs) are being evaluated in cell-based therapy for degenerative, inflammatory and autoimmune disorders. Within the injured/inflamed tissue, proinflammatory lymphocytes such as IL-17-producing T helper cells (Th17) may interact with the stromal microenvironment, including MSCs. In this context, MSCs may encounter different levels of T cells as well as specific inflammatory signals. Uncovering the cellular and molecular changes during this interplay is central for developing an efficient and safe immunotherapeutic tool. To this end, an in vitro human model of cocultures of FSK-MSCs and T cells was established. These cocultures were performed at different cell ratios in the presence of an inflammatory setting. After confirming that FSK-MSCs respond to ISCT criteria by showing a typical phenotype and multilineage potential, we evaluated by flow cytometry the expression of Th17 cell markers IL-17A, IL23 receptor and RORγt within the lymphocyte population. We also measured 15 human Th17 pathway-related cytokines. Regardless of the T cell/MSC ratio, we observed a significant increase in IL-17A expression associated with an increase in IL-23 receptor expression. Furthermore, we observed substantial modulation of IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, INF-γ, sCD40, and TNF-α secretion. These findings suggest that FSK-MSCs are receptive to their environment and modulate the T cell response accordingly. The changes within the secretome of the stromal and immune environment are likely relevant for the therapeutic effect of MSCs. FSK-MSCs represent a valuable cellular product for immunotherapeutic purposes that needs to be further clarified and developed.

AZD0284, a Potent, Selective, and Orally Bioavailable Inverse Agonist of Retinoic Acid Receptor-Related Orphan Receptor C2.

Inverse agonists of the nuclear receptor RORC2 have been widely pursued as a potential treatment for a variety of autoimmune diseases. We have discovered a novel series of isoindoline-based inverse agonists of the nuclear receptor RORC2, derived from our recently disclosed RORC2 inverse agonist 2. Extensive structure-activity relationship (SAR) studies resulted in AZD0284 (20), which combined potent inhibition of IL-17A secretion from primary human TH17 cells with excellent metabolic stability and good PK in preclinical species. In two preclinical in vivo studies, compound 20 reduced thymocyte numbers in mice and showed dose-dependent reduction of IL-17A containing γδ-T cells and of IL-17A and IL-22 RNA in the imiquimod induced inflammation model. Based on these data and a favorable safety profile, 20 was progressed to phase 1 clinical studies.

Effect of Two Unique Nanoparticle Formulations on the Efficacy of a Broadly Protective Vaccine Against Pseudomonas Aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of infections in humans. In addition to its innate antibiotic resistance, P. aeruginosa is very effective in acquiring resistance resulting in the emergence of multi-drug resistance strains and a licensed vaccine is not yet available. We have previously demonstrated the protective efficacy of a novel antigen PaF (Pa Fusion), a fusion of the type III secretion system (T3SS) needle tip protein, PcrV, and the first of two translocator proteins, PopB. PaF was modified to provide a self-adjuvanting activity by fusing the A1 subunit of the heat-labile enterotoxin from Enterotoxigenic E. coli to its N-terminus to give L-PaF. In addition to providing protection against 04 and 06 serotypes of P. aeruginosa, L-PaF elicited opsonophagocytic killing and stimulated IL-17A secretion, which have been predicted to be required for a successful vaccine. While monomeric recombinant subunit vaccines can be protective in mice, this protection often does not transfer to humans where multimeric formulations perform better. Here, we use two unique formulations, an oil-in-water (o/w) emulsion and a chitosan particle, as well as the addition of a unique TLR4 agonist, BECC438 (a detoxified lipid A analogue designated Bacterial Enzymatic Combinatorial Chemistry 438), as an initial step in optimizing L-PaF for use in humans. The o/w emulsion together with BECC438 provided the best protective efficacy, which correlated with high levels of opsonophagocytic killing and IL-17A secretion, thereby reducing the lung burden among all the vaccinated groups tested.

E-cigarette fluids and aerosol residues cause oxidative stress and an inflammatory response in human keratinocytes and 3D skin models

Our goal was to evaluate the effects of EC refill fluids and EC exhaled aerosol residue (ECEAR) on cultured human keratinocytes and MatTek EpiDerm™, a 3D air liquid interface human skin model. Quantification of flavor chemicals and nicotine in Dewberry Cream and Churrios refill fluids was done using GC–MS. The dominant flavor chemicals were maltol, ethyl maltol, vanillin, ethyl vanillin, benzyl alcohol, and furaneol. Cytotoxicity was determined with the MTT and LDH assays, and inflammatory markers were quantified with ELISAs. Churrios was cytotoxic to keratinocytes in the MTT assay, and both fluids induced ROS production in the medium (ROS-Glo™) and in cells (CellROX). Exposure of EpiDerm™ to relevant concentrations of Dewberry Cream and Churrios for 4 or 24 h caused secretion of inflammatory markers (IL-1α, IL-6, and MMP-9), without altering EpiDerm™ histology. Lab made fluids with propylene glycol (PG) or PG plus a flavor chemical did not produce cytotoxic effects, but increased secretion of IL-1α and MMP-9, which was attributed to PG. ECEAR derived from Dewberry Cream and Churrios did not produce cytotoxicity with Epiderm™, but Churrios ECEAR induced IL-1α secretion. These data support the conclusion that EC chemicals can cause oxidative damage and inflammation to human skin.

Bacterial Infection-Mimicking Three-Dimensional Phagocytosis and Chemotaxis in Electrospun Poly(ε-caprolactone) Nanofibrous Membrane.

In this study, we developed a three-dimensional (3D) in vitro infection model to investigate the crosstalk between phagocytes and microbes in inflammation using a nanofibrous membrane (NM). Poly(ε-caprolactone) (PCL)-NMs (PCL-NMs) were generated via electrospinning of PCL in chloroform. Staphylococcus aureus and phagocytes were able to adhere to the nanofibers and phagocytes engulfed S. aureus in the PCL-NM. The migration of phagocytes to S. aureus was evaluated in a two-layer co-culture system using PCL-NM. Neutrophils, macrophages and dendritic cells (DCs) cultured in the upper PCL-NM layer migrated to the lower PCL-NM layer containing bacteria. DCs migrated to neutrophils that cultured with bacteria and then engulfed neutrophils in two-layer system. In addition, phagocytes in the upper PCL-NM layer migrated to bacteria-infected MLE-12 lung epithelial cells in the lower PCL-NM layer. S. aureus-infected MLE-12 cells stimulated the secretion of tumor necrosis factor-α and IL-1α in 3D culture conditions, but not in 2D culture conditions. Therefore, the PCL-NM-based 3D culture system with phagocytes and bacteria mimics the inflammatory response to microbes in vivo and is applicable to the biomimetic study of various microbe infections.

Pirfenidone ameliorates silica-induced lung inflammation and fibrosis in mice by inhibiting the secretion of interleukin-17A.

Silicosis is a global occupational disease characterized by lung dysfunction, pulmonary inflammation, and fibrosis, for which there is a lack of effective drugs. Pirfenidone has been shown to exert anti-inflammatory and anti-fibrotic properties in the lung. However, whether and how pirfenidone is effective against silicosis remains unknown. Here, we evaluated the efficacy of pirfenidone in the treatment of early and advanced silicosis in an experimental mouse model and explored its potential pharmacological mechanisms. We found that pirfenidone alleviated silica-induced lung dysfunction, secretion of inflammatory cytokines (TNF-α, IL-1β, IL-6) and deposition of fibrotic proteins (collagen I and fibronectin) in both early and advanced silicosis models. Moreover, we observed that both 100 and 200 mg/kg pirfenidone can effectively treat early-stage silicosis, while 400 mg/kg was recommended for advanced silicosis. Mechanistically, antibody array and bioinformatic analysis showed that the pathways related to IL-17 secretion, including JAK-STAT pathway, Th17 differentiation, and IL-17 pathway, might be involved in the treatment of silicosis by pirfenidone. Further in vivo experiments confirmed that pirfenidone reduced the production of IL-17A induced by silica exposure via inhibiting STAT3 phosphorylation. Neutralizing IL-17A by anti-IL-17A antibody improved lung function and reduced pulmonary inflammation and fibrosis in silicosis animals. Collectively, our study has demonstrated that pirfenidone effectively ameliorated silica-induced lung dysfunction, pulmonary inflammation and fibrosis in mouse models by inhibiting the secretion of IL-17A.

Imbalanced inflammatory response in subchronic arsenic-induced liver injury and the protective effects of Ginkgo biloba extract in rats: Potential role of cytokines mediated cell-cell interactions.

Arsenic is a well-known environmental toxicant and carcinogen, which has been epidemiologically proved related to the increased hepatic disorders. Researches have shown that aseptic inflammation and abnormal immune response are associated with arsenic-induced liver injury. However, the immunotoxic effects of liver have not been extensively characterized. Ginkgo biloba extract (GBE), a natural products of G. biloba leaves with proven anti-inflammatory and potential immunoregulatory activities, was used as intervention agent to explore its protective effects on arsenic-induced hepatotoxicity. Thus, the underlying mechanism of the immunotoxic effects on arsenic-induced liver injury were investigated in 2.5, 5.0, and 10.0mg/kg NaAsO2 of Wistar rats for 16 weeks. Subsequently, GBE was used as intervention agent in 50 mg/kg for 6 weeks after cessation of arsenic exposure. The ratio of Th17 to Treg cells in peripheral blood as well as the secretion of inflammatory cytokines IL-17A, IL-6, TGF-β1, and IL-10 in serum and liver were detected. Meanwhile, the notable activation of aseptic inflammation-related molecule TLR4 and its downstream targets MyD88 and NF-κB in the liver were observed. In this work, we confirmed that subchronic exposed to arsenic triggered the infiltration of inflammatory cells in rat liver, coupled with obvious histopathological changes and aberrant hepatic serum biochemical parameters. Meanwhile, imbalanced immune response was verified by the notable abnormal ratio of Th17 to Treg cells in peripheral blood as well as the secretion of inflammatory cytokines IL-17A, IL-6, TGF-β1, and IL-10 in serum and liver of arsenic exposed rats. Further, the level of TLR4, MyD88, and NF-κB in liver both transcription and translation activity were raised. Subsequently, GBE markedly mitigated arsenic-induced liver injury, most impressively, post treatment with GBE prominently suppressed the overactivated inflammatory-related TLR4-MyD88-NF-κB pathway and evidently decreased the secretion of inflammation cytokines. Meanwhile, the disturbance of pro- and anti-inflammatory response was reversed. We concluded that the disruption of pro- and anti-inflammatory T-cells balance caused by cytokines mediated cell-cell interactions may be one of the mechanisms underlying arsenic-induced liver injury and that GBE intervention exerts an evidence protective effects, which might be closely associated with the suppression of inflammatory-related TLR4 pathway.

Vaccine Composition Formulated with a Novel Lactobacillus-Derived Exopolysaccharides Adjuvant Provided High Protection against Staphylococcus aureus.

A vaccine that effectively targets methicillin-resistant Staphylococcus aureus (MRSA) is urgently needed, and has been the focus of studies by numerous research groups, but with limited success to date. Recently, our team found that exopolysaccharides derived from probiotic Lactobacillus casei strain WXD030 as an adjuvant-formulated OVA could upregulate IFN-γ and IL-17 expression in CD4+ T cells. In this study, we developed a vaccine (termed rMntC-EPS) composed of S. aureus antigen MntC and Lactobacillus casei exopolysaccharides, which conferred high levels of protection against S. aureus infection. Methods: Six–eight-week-old female mice were vaccinated with purified rMntC-EPS30. The immune protection function of rMntC-EPS30 was assessed by the protective effect of rMntC-EPS30 to S. aureus-induced pulmonary and cutaneous infection in mice, bacterial loads and H&E in injury site, and ELISA for inflammation-related cytokines. The protective mechanism of rMntC-EPS30 was assessed by ELISA for IgG in serum, cytokines in the spleen and lungs of vaccinated mice. In addition, flow cytometry was used for analyzing cellular immune response induced by rMntC-EPS30. For confirmation of our findings, three kinds of mice were used in this study: IL-17A knockout mice, IFN-γ knockout mice and TCRγ/δ knockout mice. Results: rMntC-EPS30 conferred up to 90% protection against S. aureus pulmonary infection and significantly reduced the abscess size in the S. aureus cutaneous model, with clearance of the pathogen. The rMntC-EPS vaccine could induce superior humoral immunity as well as significantly increase IL-17A and IFN-γ production. In addition, we found that rMntC-EPS vaccination induced robust Th 17/γδ T 17 primary and recall responses. Interestingly, this protective effect was distinctly reduced in the IL-17A knockout mice but not in IFN-γ knockout mice. Moreover, in TCRγ/δ knockout mice, rMntC-EPS vaccination neither increased IL-17A secretion nor provided effective protection against S. aureus infection. Conclusions: These data demonstrated that the rMntC formulated with a novel Lactobacillus-derived Exopolysaccharides adjuvant provided high protection against Staphylococcus aureus. The rMntC-EPS vaccine induced γδ T cells and IL-17A might play substantial roles in anti-S. aureus immunity. Our findings provided direct evidence that rMntC-EPS vaccine is a promising candidate for future clinical application against S. aureus-induced pulmonary and cutaneous infection.

GARP and GARP-Treated tDC Prevented the Formation of Atherosclerotic Plaques in ApoE-/- Mice.

PurposeThis study aims to clarify the specific mechanism by which GARP affects the atherosclerotic plaques in ApoE−/- mice and the effect of GARP-tDC on atherosclerosis.MethodsThe mice were randomly divided into three groups: the control group, the GARP-overexpressed group and the GARP-inhibited group. After 12 weeks, all the mice were euthanized, and the specimens were collected. In vitro, experiments were conducted to observe the effect of GARP on DC phenotype and the changes of the proportion of CD4+CD25+Foxp3+ Treg cells when GARP-tDCs were co-cultured with CD4+ T cells. Furthermore, adoptive transmission of GARP-tDCs was used to observe the effect on atherosclerotic plaque in mice.ResultsThe GARP-overexpressed group enhanced the biological activity of Foxp3+ CD4+CD25+ Tregs and resulted in increased expression of LAP in T cells. In addition, the GARP-overexpressed group significantly suppressed the function of Th1 and Th17, and decreased the secretion of INF-γ and IL-17A. Thus, GARP had a protective effect on atherosclerosis. In vitro, we found that GARP-tDC had a tolerance-inducing phenotype, and GARP-tDC also had the ability to induce tolerance when co-cultured with CD4+ T cells. More importantly, adoptive transmission of GARP-tDCs reduced the size of atherosclerotic plaques.ConclusionGARP and the GARP-tDC play protective roles in atherosclerosis. The protective effect of GARP on atherosclerosis is achieved by increasing CD4+CD25+Foxp3+ Treg cells and inhibiting the production of IFN-γ and IL-17A.

Abstract 1910: Augmented secretion of IL-1α from mouse oral squamous cell carcinoma (OSCC) cells caused by the serum deprivation and hypoxia promotes immune-suppressive activity of mesenchymal stromal cells

Abstract Objectives; We have already reported that mouse oral squamous carcinoma (OSCC), Sq-1979-1, cells produce IL-1α which specifically enhances the immunosuppressive activity of co-cultured mesenchymal stromal, 10T1/2, cells (Morimoto-Ito, Open Dent J. 2019). In this report, we evaluated conditions promoting the expression of IL-1α of Sq-1979-1 cells to further enhance immunosuppressive function of the 10T1/2 cells, and also examined the expression in OSCC tissues. Methods; Expression of IL-1α in Sq-1979-1 cells was evaluated by RT-PCR, western blots and enzyme-linked immune sorbent assay (ELISA). Conditioned mediums (CMs) from Sq-1979-1 cells were co-cultured with 10T1/2 cells and anti-CD3 antibody-stimulated mouse spleen cells. The production of interferon (IFN)- γ and IL-1α was evaluated using ELISA. The function of IL-1α in CM was examined by anti- IL-1α antibody. Immunohistochemical analysis of OSCC tissues was performed. Results; The production of IL-1α from Sq-1979-1 cells was specifically enhanced in lower serum (0.5% or 1.0%FBS), or under hypoxia (1.0% oxygen) compared to that in the standard growth condition supplemented with 10% FBS under normoxia. Compared to standard CM, the CMs prepared in 0.5% and 1.0% FBS, or under hypoxia significantly reduced IFN-γ production from stimulated spleen cells co-cultured with 10T1/2 cells. These functions of the CMs completely abolished by anti- IL-1α antibody. Expression of IL-1α in OSCC tissues was prominent in the midst of carcinomatous cellular lesion, or nearby necrotic lesion, where the supply deficiency could be occurred. Conclusions; The promotion of immunosuppressive activity of mesenchymal cells by Sq-1979-1 cells was specifically augmented in lower serum and hypoxic conditions, mediated by increased IL-1α production. Citation Format: Nobuo Kondoh, Akihiro Matsunami, Masako Mizuno-Kamiya, Naoki Umemura, Eiji Takayama, Harumi Kawaki, Kyouhei Ueno, Hiroe Morimoto-Ito, Yasunori Muramatsu, Shinichiro Sumitomo. Augmented secretion of IL-1α from mouse oral squamous cell carcinoma (OSCC) cells caused by the serum deprivation and hypoxia promotes immune-suppressive activity of mesenchymal stromal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1910.

Intestinal Microbiota Play a Critical Role in Neutrophil Engraftment Posttransplant and Recovery after Chemotherapy By Stimulating T Cell Production of IL-17A

Introduction: Delay in recovery or engraftment of neutrophils is a risk for infection and associated with poor prognosis after cancer chemotherapy or hematopoietic stem cell transplantation (SCT). Recent studies demonstrate that intestinal microbiota plays a critical role in the development of bone marrow (BM) granulopoiesis in infants after birth (Deshmukh, et al. Nat Med 2014). Here, we addressed the impact of microbiota on neutrophil recovery after chemotherapy and engraftment after syngeneic SCT.Method: Wild type (WT) or RAG1-/- B6 mice were given a combination of ampicillin 1g/L, streptomycin 1g/L, and vancomycin 1g/L in drinking water daily from day -7 or left untreated.Recipient WT, RAG1-/-, or IL-17A-/- B6 mice were lethally irradiated and transplanted with 7,500 purified lineage-sca-1+c-kit+ cells plus 20,000 granulocyte-macrophage progenitors (GMPs) purified from congenic B6-CD45.1 mice on day 0. For in vivo T cell depletion, mice were i.p. injected with 200 μg of anti-CD90 monoclonal antibodies (αCD90) thrice a week from day -7. To assess reactive granulopoiesis after chemotherapy, B6 mice were i.p. injected with 5-fluorouracil (5-FU) at a dose of 200 mg/kg.Results: To address if gut microbiota could play a role in neutrophil engraftment, gut decontamination using the antibiotics was performed from day -7 of SCT. We found that gut decontamination significantly delayed engraftment of neutrophils, suggesting that microbiota promotes neutrophil engraftment after SCT (Figure A). Plasma levels of G-CSF and a G-CSF-inducing cytokine, IL-17A were significantly elevated 14 days after SCT, while the antibiotics completely abrogated upregulation of these cytokines. IL-17A expression in host αβ and γδ T cells was significantly suppressed by the antibiotic treatment after SCT, suggesting that microbiota stimulates host T cells to produce IL-17A after SCT. Next, we studied the role of IL-17A in neutrophil engraftment using IL-17A-/- mice as recipients. IL-17A deficiency phenocopied the antibiotic treatment, with lower plasma levels of G-CSF and delayed neutrophil engraftment compared to WT recipients. When recipients were devoid of host T cells by administration of αCD90 mAbs or by using RAG1-/- mice as recipients, plasma levels of both IL-17A and G-CSF were significantly decreased, suggesting a critical role of host T cells in IL-17A secretion in response to microbiota (Figure B and data not shown). Neutrophil engraftment was delayed in RAG 1 -/- mice, while gut decontamination did not impact on neutrophil engraftment in RAG 1 -/- recipients, ruling out direct toxicity of the antibiotics on engraftment and further emphasizing a critical role of host T cells in granulopoiesis after SCT. Flow cytometric analysis demonstrated that the numbers of GMPs in the BM of IL17A-/- recipients were significantly less than those in WT controls, suggesting that IL-17A promoted granulopoiesis in the BM. Emergence of BrdU+ neutrophils in the peripheral blood after BrdU injection on day +18 after SCT was delayed in IL17A-/- recipients compare to WT controls, indicating that IL-17A not only stimulates granulopoiesis but also facilitates neutrophil egress from the BM to peripheral blood (Figure C). Finally, we found that neutrophil recovery after chemotherapy with 5-FU was delayed in the antibiotic-treated mice or IL-17A-/- mice, suggesting that neutrophil recovery after chemotherapy was also dependent on IL-17A secretion in response to microbiota (Figure D). Plasma levels of IL-17A and T-cell IL-17A expression were significantly lower in naïve MyD88-/- mice than those in WT mice, suggesting that IL-17A production in T cells were dependent on MyD88 signaling.Conclusion: Our study uncovers a novel role of gut microbiota in neutrophil engraftment after SCT and neutrophil recovery after chemotherapy; gut microbiota enhances granulopoiesis and neutrophil egress from the BM by stimulating T cells to produce IL-17A.Figure: (A) Gut decontamination with antibiotics delayed neutrophil engraftment after SCT. (B) T cells were responsible to IL-17 production after SCT. (C) Neutrophil egress from BM was impaired in IL17A-/- recipients. (D) IL-17 played a critical role in reactive granulopoiesis after chemotherapy. [Display omitted] DisclosuresTeshima:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Kyowa-Hakko Kirin: Research Funding; Pfeizer: Research Funding; Chugai: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Augmented secretion of IL-1α from mouse oral squamous cell carcinoma (OSCC) vcells caused by serum deprivation and hypoxia promotes immune suppressive activity of mesenchymal stromal cells

ObjectivesWe have previously reported that mouse oral squamous carcinoma (OSCC), Sq-1979-1, produces interleukin (IL)-1α, which specifically enhances the immunosuppressive activity of co-cultured mesenchymal stromal 10T1/2 cells. This study assessed the conditions promoting the production of IL-1α in Sq-1979-1 cells, which could further enhance the immunosuppressive function of 10T1/2 cells, and evaluated its expression in OSCC tissues. MethodsThe expression of IL-1α was examined by RT-PCR, western blotting, and enzyme-linked immune sorbent assay (ELISA). The interferon (IFN)- γ-producing capability of anti-CD3 antibody-stimulated mouse spleen cells co-cultured with 10T1/2 cells and conditioned medium (CM) from Sq-1979-1 cells was examined by ELISA. The function of IL-1α was examined using an anti-IL1α antibody. Immunohistochemical analysis of the OSCC tissues was performed. ResultsThe production of IL-1α from Sq-1979-1 cells was synergistically enhanced in lower serum (0.5% or 1.0% FBS) at the transcriptional level, and under hypoxia (1.0% oxygen) at the release level compared to that in the control medium supplemented with 10% FBS under normoxia. The IFN-γ-producing capability of stimulated spleen cells co-cultured with 10T1/2 cells was significantly reduced in the CMs prepared with the lower serum or under hypoxia. These functions of CMs were completely abolished by the anti-IL-1α antibody. The expression of IL-1α in OSCC tissues was prominent in the midst of a carcinomatous cellular lesion or a nearby necrotic lesion, where a supply deficiency could occur. Conclusions: IL-1α production by Sq-1979-1 cells was synergistically augmented under low serum and hypoxic conditions, which could promote the immunosuppressive activity of mesenchymal cells.

Myricetin Inhibits SARS-CoV-2 Viral Replication by Targeting Mpro and Ameliorates Pulmonary Inflammation.

The coronavirus disease 2019 (COVID-19) has spread widely around the world and has seriously affected the human health of tens of millions of people. In view of lacking anti-virus drugs target to SARS-CoV-2, there is an urgent need to develop effective new drugs. In this study, we reported our discovery of SARS-CoV-2 Mpro inhibitors. We selected 15 natural compounds, including 7 flavonoids, 3 coumarins, 2 terpenoids, one henolic, one aldehyde and one steroid compound for molecular docking and enzymatic screening. Myricetin were identified to have potent inhibit activity with IC50 3.684 ± 0.076 μM in the enzyme assay. The binding pose of Myricetin with SARS-CoV-2 Mpro was identified using molecular docking method. In the binding pocket of SARS-CoV-2 Mpro, the chromone ring of Myricetin interacts with His41 through π-π stacking, and the 3’-, 4’- and 7-hydroxyl of Myricetin interact with Phe140, Glu166and Asp187 through hydrogen bonds. Significantly, our results showed that Myricetin has potent effect on bleomycin-induced pulmonary inflammation by inhibiting the infiltration of inflammatory cells and the secretion of inflammatory cytokines IL-6, IL-1α, TNF-α and IFN-γ. Overall, Myricetin may be a potential drug for anti-virus and symptomatic treatment of COVID-19.

USP4 is pathogenic in allergic airway inflammation by inhibiting regulatory T cell response

AimsAsthma is characterized by chronic inflammation and airway hyperresponsiveness (AHR). It is controllable, but not curable. Ubiquitin-specific peptidase 4 (USP4) has been verified as a regulator of regulatory T (Treg) cells and Th17 cells in vitro. In this study, we aim to investigate whether USP4 could serve as a therapeutic target for asthma. Main methodsAge-matched USP4 wild-type and knockout mice received an intraperitoneal injection of 100 μg ovalbumin (OVA) mixed in 2 mg aluminum hydroxide in 1 × PBS on days 0, 7 and 14. On days 21 to 27, the mice were challenged with aerosolized 1% OVA in 1 × PBS for 30 min. Tissue histology, ELISA and flow cytometry were applied 24 h after the last OVA challenge. Key findingsUSP4 deficiency protected mice from OVA-induced AHR and decreased the production of several inflammatory cytokines in T cells in vivo. Compared to the lung cells isolated from WT mice, Usp4−/− lung cells decreased secretion of IL-4, IL-13 and IL-17A upon stimulation in vitro. Meanwhile, the percentage of CD4+Foxp3+ Treg cells was elevated, with more CCR6+Foxp3+ Treg cells accumulating in the lungs of OVA-challenged USP4 deficient mice than in their wild-type counterparts. Treatment with the USP4 inhibitor, Vialinin A, reduced inflammatory cell infiltration in the lungs of OVA-challenged mice in vivo. SignificanceWe found USP4 deficiency contributes to attenuated airway inflammation and AHR in allergen-induced murine asthma, and Vialinin A treatment alleviates asthma pathogenesis and may serve as a promising therapeutic target for asthma.

The PI-3-Kinase P110α Catalytic Subunit of T Lymphocytes Modulates Collagen-Induced Arthritis.

The phosphatidylinositol 3-kinase (PI3K) family of enzymes plays a determinant role in inflammation and autoimmune responses. However, the implication of the different isoforms of catalytic subunits in these processes is not clear. Rheumatoid arthritis (RA) is a chronic, systemic autoimmune inflammatory disease that entails innate and adaptive immune response elements in which PI3K is a potential hub for immune modulation. In a mouse transgenic model with T-cell-specific deletion of p110α catalytic chain (p110α−/−ΔT), we show the modulation of collagen-induced arthritis (CIA) by this isoform of PI3K. In established arthritis, p110α−/−ΔT mice show decreased prevalence of illness than their control siblings, higher IgG1 titers and lower levels of IL-6 in serum, together with decreased ex vivo Collagen II (CII)-induced proliferation, IL-17A secretion and proportion of naive T cells in the lymph nodes. In a pre-arthritis phase, at 13 days post-Ag, T-cell-specific deletion of p110α chain induced an increased, less pathogenic IgG1/IgG2a antibodies ratio; changes in the fraction of naive and effector CD4+ subpopulations; and an increased number of CXCR5+ T cells in the draining lymph nodes of the p110α−/−ΔT mice. Strikingly, T-cell blasts in vitro obtained from non-immunized p110α−/−ΔT mice showed an increased expression of CXCR5, CD44 and ICOS surface markers and defective ICOS-induced signaling towards Akt phosphorylation. These results, plus the accumulation of cells in the lymph nodes in the early phase of the process, could explain the diminished illness incidence and prevalence in the p110α−/−ΔT mice and suggests a modulation of CIA by the p110α catalytic chain of PI3K, opening new avenues of intervention in T-cell-directed therapies to autoimmune diseases.