Presence Of IL-2 Research Articles

Pan-HDAC inhibitors augment IL2-induced proliferation of NK cells via the JAK2-STAT5B signaling pathway

BackgroundNatural killer (NK) cells are a subtype of lymphocytes with the ability to quickly and efficiently identify and eliminate tumor cells. In the presence of IL2, NK cells can divide rapidly but in limited numbers. According to previous studies, in vivo treatment with histone deacetylase (HDAC) inhibitors did not impair NK-cell function. This study aimed to investigate the effect of HDAC inhibitors on NK-cell proliferation and the underlying regulatory mechanism. MethodsNK92 cells, primary NK (pNK) cells, and CD19-CAR-NK92 cells were treated with low concentrations of pan-HDACi Dacinostat (Dac) and Panobinostat (Pan) in the presence of IL2, and Cell Counting Kit-8 (CCK8), 5-ethynyl-2′-deoxyuridine (EdU), and flow cytometry assays were used to assess cell proliferation and apoptosis. The expression of granzyme B was detected by immunofluorescence, and the expression of CD107a and NKG2D was determined by flow cytometry. The downstream regulatory genes were identified by RNA-seq, and the “JAK-STAT signaling pathway”- and “Cell cycle signaling pathway”-related genes were detected by real-time quantitative polymerase chain reaction (RT–qPCR) and Western blot analysis. The JAK2V617F mouse model was constructed to simulate the upregulation of the JAK2 signaling pathway in vivo, and the NK proliferation was evaluated by flow cytometry. A tumor-bearing nude mouse model was constructed to determine the anti-tumor efficacy of NK92 cells following Dac treatment. ResultsIn the presence of IL2, the proliferation rate of NK92 cells, pNK cells, and CD19-CAR-NK92 cells treated with pan-HDACi Dac and Pan at low nanomolar doses was significantly increased, although cell function was unaffected. Low doses of Dac upregulated the JAK-STAT signaling pathway and enhance the cell cycle via that pathway. In addition, the in vivo experiment in nude mice showed that the capacity of Dac treated NK92 cells to eliminate tumor cells was unaffected. ConclusionLow nanomolar doses of Pan-HDACi enhanced IL2-induced NK cell proliferation without compromising the functioning of NK cells.

TRPA1 as a potential factor and drug target in scleroderma: dermal fibrosis and alternative macrophage activation are attenuated in TRPA1-deficient mice in bleomycin-induced experimental model of scleroderma

BackgroundSystemic sclerosis is a rheumatoid disease best known for its fibrotic skin manifestations called scleroderma. Alternatively activated (M2-type) macrophages are normally involved in the resolution of inflammation and wound healing but also in fibrosing diseases such as scleroderma. TRPA1 is a non-selective cation channel, activation of which causes pain and neurogenic inflammation. In the present study, we investigated the role of TRPA1 in bleomycin-induced skin fibrosis mimicking scleroderma.MethodsWild type and TRPA1-deficient mice were challenged with intradermal bleomycin injections to induce a scleroderma-mimicking disease. Macrophages were investigated in vitro to evaluate the underlying mechanisms.ResultsBleomycin induced dermal thickening and collagen accumulation in wild type mice and that was significantly attenuated in TRPA1-deficient animals. Accordingly, the expression of collagens 1A1, 1A2, and 3A1 as well as pro-fibrotic factors TGF-beta, CTGF, fibronectin-1 and YKL-40, and M2 macrophage markers Arg1 and MRC1 were lower in TRPA1-deficient than wild type mice. Furthermore, bleomycin was discovered to significantly enhance M2-marker expression particularly in the presence of IL-4 in wild type macrophages in vitro, but not in macrophages harvested from TRPA1-deficient mice. IL-4-induced PPARγ-expression in macrophages was increased by bleomycin, providing a possible mechanism behind the phenomenon.ConclusionsIn conclusion, the results indicate that interfering TRPA1 attenuates fibrotic and inflammatory responses in bleomycin-induced scleroderma. Therefore, TRPA1-blocking treatment could potentially alleviate M2 macrophage driven diseases like systemic sclerosis and scleroderma.

Treg-based immunotherapy for antigen-specific immune suppression and stable tolerance induction: a perspective.

FoxP3-expressing regulatory T cells (Tregs), whether naturally generated in the immune system or unnaturally induced from conventional T cells (Tconvs) in the laboratory, have much therapeutic value in treating immunological diseases and establishing transplantation tolerance. Natural Tregs (nTregs) can be selectively expanded in vivo by administration of low-dose IL-2 or IL-2 muteins for immune suppression. For adoptive Treg cell therapy, nTregs can be expanded in vitro by strong antigenic stimulation in the presence of IL-2. Synthetic receptors such as CAR can be expressed in nTregs to equip them with a particular target specificity for suppression. In addition, antigen-specific Tconvs can be converted in vitro to functionally stable Treg-like cells by a combination of antigenic stimulation, FoxP3 induction, and establishment of the Treg-type epigenome. This review discusses current and prospective strategies for Treg-based immune suppression and the issues to be resolved for achieving stable antigen-specific immune suppression and tolerance induction in the clinic by targeting Tregs.

Regulation of Cytotoxic Immune Effector Function by AJ3 Probiotic Bacteria in Amyotrophic Lateral Sclerosis (ALS).

Our recent studies indicated that amyotrophic lateral sclerosis (ALS) patients suffer from significantly elevated levels of interferon-gamma (IFN-γ) secretion by natural killer (NK) and CD8+ T cells, which may be responsible for the immune-pathologies seen in central nervous system and in peripheral organs of the patients. In order to counter such elevated induction of IFN-γ in patients we designed a treatment strategy to increase anti-inflammatory cytokine interleukin-10 (IL-10) by the use of probiotic strains which significantly increase the levels of IL-10. Therefore, in this paper we demonstrate disease specific functions of Al-Pro (AJ3) formulated for the adjunct treatment of auto-immune diseases including ALS, and compared the function with CA/I-Pro (AJ4) for the treatment of cancer and viral diseases, and NK-CLK (AJ2) for maintenance of immune balance and promotion of disease prevention. The three different formulations of probiotic bacteria have distinct profiles of activation of peripheral blood mononuclear cells (PBMCs), NK, and CD8+ T cells, and their induced activation is different from those mediated by either IL-2 or IL-2 + anti-CD16 monoclonal antibodies (mAbs) or IL-2 + anti-CD3/CD28 mAbs. IL-2 + anti-CD16 mAb activation of PBMCs and NK cells had the highest IFN-γ/IL-10 ratio, whereas IL-2 combination with sAJ4 had the next highest followed by IL-2 + sAJ2 and the lowest was seen with IL-2 + sAJ3. Accordingly, the highest secretion of IFN-γ was seen when the PBMCs and NK cells were treated with IL-2 + sAJ4, intermediate for IL-2 + sAJ2 and the lowest with IL-2 + sAJ3. The levels of IFN-γ induction and the ratio of IFN-γ to IL-10 induced by different probiotic bacteria formulation in the absence of IL-2 treatment remained much lower when compared to those treated in the presence of IL-2. Of note is the difference between NK cells and CD8+ T cells in which synergistic induction of IFN-y by IL-2 + sAJ4 was significantly higher in NK cells than those seen by CD8+ T cells. Based on these results, sAJ3 should be effective in alleviating auto-immunity seen in ALS since it will greatly regulate the levels and function of IFN-γ negatively, decreasing overactivation of cytotoxic immune effectors and prevention of death in motor neurons.

Cytotoxicity of Human Hepatic Intrasinusoidal Gamma/Delta T Cells Depends on Phospho-antigen and NK Receptor Signaling.

We previously showed that human hepatic intrasinusoidal (HI) natural killer (NK) T cells selectively eliminate hepatocellular carcinoma (HCC) cell lines. In this study, we investigated the underlying mechanisms on how HI γδ T cells, expanded with zoledronate, exhibit a superior cytotoxic effect on HI NK-resistant Huh7 HCC cells. γδ T cells were obtained from living liver transplant donors or from peripheral blood mononuclear cells (PBMC) of healthy volunteers and were expanded in the presence of IL-2, IL-15, and zoledronate for 2 weeks. Cytotoxicity was measured using the lactate dehydrogenase (LDH) assay in vitro and by flow cytometry using carboxyfluorescein succinimidyl ester (CFSE) in vivo. The cytotoxicity of expanded HI γδ T cells against Huh7 cells was associated with a higher pyrophosphate expression in Huh7 cells compared to SNU398 cells. In contrast, the cytotoxicity of HI γδ T cells against SNU398 cells depended on NKG2D. HI γδ T cells expressed less PD-1 than PB γδ T cells. The cytotoxicity of HI γδ T cells against Du145 and PC3 prostate cancer cells was also associated with pyrophosphate expression in these cells, as well as NKG2D and DNAM-1. The expression levels of phospho-antigen in tumor cells determined the cytotoxicity of HI γδ T cells, although the NK activating receptors, death ligands, and immune checkpoint molecules also contribute to their cytotoxicity. γδ T cells are attractive candidates for cancer immune cell therapy.

IL-13 and the hydroperoxy fatty acid 13(S)HpODE play crucial role in inducing an apoptotic pathway in cancer cells involving MAO-A/ROS/p53/p21 signaling axis

This study depicted the effect of IL-13 and 13(S)HpODE (the endogenous product during IL-13 activation) in the process of cancer cell apoptosis. We examined the role of both IL-13 and 13(S)HpODE in mediating apoptotic pathway in three different in vitro cellular models namely A549 lung cancer, HCT116 colorectal cancer and CCF52 GBM cells. Our data showed that IL-13 promotes apoptosis of A549 lung carcinoma cells through the involvement of 15-LO, PPARγ and MAO-A. Our observations demonstrated that IL-13/13(S)HpODE stimulate MAO-A-mediated intracellular ROS production and p53 as well as p21 induction which play a crucial role in IL-13-stimulated A549 cell apoptosis. We further showed that 13(S)HpODE promotes apoptosis of HCT116 and CCF52 cells through the up-regulation of p53 and p21 expression. Our data delineated that IL-13 stimulates p53 and p21 induction which is mediated through 15-LO and MAO-A in A549 cells. In addition, we observed that PPARγ plays a vital role in apoptosis as well as in p53 and p21 expression in A549 cells in the presence of IL-13. We validated our observations in case of an in vivo colon cancer tumorigenic study using syngeneic mice model and demonstrated that 13(S)HpODE significantly reduces solid tumor growth through the activation of apoptosis. These data thus confirmed that IL-13 > 15-LO>13(S)HpODE > PPARγ>MAO-A > ROS > p53>p21 axis has a major contribution in regulating cancer cell apoptosis and further identified 13(S)HpODE as a potential chemo-preventive agent which can improve the efficacy of cancer treatment as a combination compound.

Soluble factors from TLR4- or TCR-activated cells contribute to stability of the resting phenotype and increase the expression of CXCR4 of human memory CD4 T cells.

It has been proposed that cytokines can induce activation of resting T cells in an antigen-independent manner. However, experimental conditions have included the use of fetal serum and nanogram concentrations of added cytokines. To evaluate the effect of cytokines and chemokines generated by activated immune cells on the phenotypic profile of human memory CD4 T cells, the cells were cultured in FBS-free conditions in the presence of IL-15 and 5% of hAB serum and incubated with conditioned medium (CM) obtained from PBMC activated through the TCR using anti-CD3/CD28/CD2 antibodies (TCR-CM) or through TLR4 using bacterial LPS (TLR4-CM). Cytokines and chemokines present in the CMs were evaluated by ProcartaPlex immunoassay. Cell viability, proliferation, and surface markers were determined by flow cytometry on day 2, 5, and 8 of culture. Cell viability was maintained by TLR4-CM plus IL-15 for 8days but decreased in the presence of the TCR-CM plus IL-15. In combination with IL-15, the TLR4-CM, but not the TCR-CM, maintained the expression of CD3 and CD4 stable. Both conditions stabilized the expression of CD45RO and CCR5. Thus, the TLR4-CM better supported the viability and stability of the memory phenotype. None of the CMs induced proliferation or expression of activation markers; however, they induced an increased expression of CXCR4. This study indicates that resting memory CD4 T cells are not activated by, but may be sensitive to soluble factors produced by antigen or PAMP-stimulated cells, which may contribute to their homeostasis and favor the CXCR4 expression.

Effects of combination of Cryptococcus gattii and IFN-γ, IL-4 or IL-27 on human bronchial epithelial cells

BackgroundAirway epithelial cells are crucial for the establishment of cryptococcosis. In experimental cryptococcosis, the Th2 immune response is associated with host susceptibility, while Th1 cells are associated with protection. The absence of IL-27 receptor alpha in mice favor the increase Cryptococcus neoformans burden in the lung. Here, we evaluated the effects of the combination of IL-4, IFN-γ or IL-27 with C. gattii on human bronchial epithelial cells (BEAS-2B). MethodsBEAS-2B were stimulated with IL-4, IFN-γ or IL-27 (100 ng/mL) and/or live yeast forms of C. gattii (multiplicities of infection (MOI) of 1–100) and vice-versa, as well as with heat-killed cells of C. gattii for 24 h. ResultsNone of the C. gattii MOIs had cytotoxic effects on BEAS-2B when compared to control. The cells stimulated by cytokines (IL-4, IFN-γ or IL-27) followed by live yeast forms of C. gattii (MOI of 100) infection and vice-versa demonstrated a reduction in IL-6, IL-8 and/or CCL2 production and activation of STAT6 (induced by IL-4) and STAT1 (induced by IL-27 or IFN-γ) when compared to cells stimulated with C. gattii, IL-4, IFN-γ or IL-27. In the combination of cytokines and heat-killed cells of C. gattii, no inhibition of these inflammatory parameters was observed. The growth of C. gattii was increased while the phagocytosis of live yeast forms of C. gattii in the BEAS-2B were reduced in the presence of IL-4, IFN-γ or IL-27.ConclusionThe association of live yeast forms, but not heat-killed yeast forms, of C. gattii with IL-4, IFN-γ or IL-27 induced an anti-inflammatory effect.

The Novel Bispecific Innate Cell Engager (ICE®) AFM28 Efficiently Directs Allogeneic NK Cells to CD123+ Leukemic Stem- and Progenitor Cells in AML

Introduction: Acute myeloid leukemia (AML) is a common hematologic malignancy characterized by expansion of undifferentiated myeloid cells in the bone marrow (BM) and peripheral blood. Although AML can be treated with curative intent, residual leukemic stem cells (LSCs) are frequently reservoirs for relapse, which is associated with a poor prognosis. Moreover, especially elderly patients are often not eligible for intensive treatment regimen. Therefore, there is a high need for novel treatment strategies, which are capable of eliminating LSCs in order to induce long-term remissions, suppress measurable residual disease (MRD) and prevent relapse. A promising emerging approach is the adoptive transfer of allogeneic natural killer (NK) cells, which was shown to be clinically active and could induce complete remissions in relapsed/refractory (R/R) AML. Innate Cell Engagers (ICE®) may further augment the activity of NK cell therapies, in particular against LSCs and hold the potential to increase frequency and durability of responses. The tetravalent bispecific CD123/CD16A ICE®, AFM28, directs the cytotoxic activity of CD16A-expressing NK cells towards CD123-expressing (CD123+) AML blasts and LSCs. In this study, we evaluated the efficacy of AFM28 in pre-clinical models of AML. Methods: Antibody-dependent cell-mediated cytotoxicity (ADCC) data were obtained by a 4h calcein release assay using AML cell lines and primary human NK cells (E:T ratio 2.5:1) in the presence of titrated antibodies. CD64 and CD123 expression levels on AML cell lines were evaluated by flow cytometry. Intracellular FACS was used to detect STAT5/pSTAT5. Proliferation of TF-1 cells in the presence of IL-3 or GM-CSF was measured via CellTiter Glo Assay (Promega). Using primary BM samples of n=6 AML patients and n=4 healthy donors, ADCC assays and colony-forming unit (CFU) assays were performed. BM-MNC (ADCC assay) or enriched CD34+ cells (CFU assay) and allogeneic NK cells (E:T ratio 1:1) were treated with AFM28 or an Fc-enhanced anti-CD123 antibody comparator at different concentrations of AFM28 (0-500 or 0/10/100/1000 pM, respectively) for 24h. Subsequently, ADCC assays were analyzed by flow cytometry and CFU assays were evaluated after 7-14 days. Systemic anti-tumor activity of AFM28 in vivo was assessed using in-life imaging (BLI) in a T cell-depleted huFcgR-C57BL6 transgenic mouse model against intravenously administered luciferase-transfected murine AML cells expressing human CD123 as tumor antigen. Results: Pre-clinical in vitro models using a panel of AML cell lines showed efficacious ADCC against CD123+ tumor cells by allogeneic NK cells in the presence of AFM28. ADCC induction by AFM28 was independent of leukemic cell mutational profiles and even targeted cells with low levels of CD123 surface expression. Of note, expression of the high-affinity IgG receptor CD64 on AML cell lines completely abolished ADCC induction by an Fc-enhanced anti-CD123 antibody whereas AFM28 also showed high ADCC efficacy against all tested CD64+ cell lines. In addition, AFM28 exerted an NK cell-independent inhibitory effect on the IL-3-induced phosphorylation of STAT5 and abolished proliferation of CD123+ TF-1 cells, indicating an antagonistic effect on IL-3R signaling. In ex vivo models using primary AML patient cells, AFM28 demonstrated strong ADCC mediated by allogeneic NK cells. CFU assays of patient-derived AML BM samples performed subsequent to incubation with allogeneic NK cells with and without AFM28 showed significantly reduced numbers of outgrowing colonies in AFM28 treated samples, indicating that AML LSCs and progenitor cells were eliminated. In the in vivo efficacy study with huFcgR-C57BL6 mice, treatment with AFM28 at 15 mg/kg b.w. led to complete inhibition of tumor growth in 5/5 animals throughout a treatment period of 42 days. In contrast, all untreated control mice (6/6) developed systemic disease. Conclusion: AFM28 induced highly potent and selective lysis of CD123+ leukemic cells, including LSCs and progenitor cells, by allogeneic NK cells in vitro and in vivo. The capacity to eradicate LSCs holds the promise for durable responses and the potential for long-term remissions in patients with R/R AML. AFM28 is currently being prepared for a first-in-human clinical investigation as monotherapy and in combination with allogeneic NK cells.

BCMA CAR-T Cell Phenotype and Functionality Is Affected By Disease Stage of Multiple Myeloma Patients

Background: Refractory or relapsed (R/R) multiple myeloma (MM) is nowadays an incurable disease. CAR-T immunotherapy against BCMA has proven its efficacy with complete response rates of 83-85%. However, despite the initial favorable response, most patients relapse with a median progression free survival of 8-24 months. This lack of long-term response could be related to the fitness and the functionality of the CAR-T cells. Thus, we hypothesize that CAR-T cells generated from patients at early stages of the disease would present improved phenotype and functionality. In this work we performed phenotypic and functional characterization of CAR-T generated from healthy donors and MM patients at different disease stage. Methodology: Second generation CAR-T cells targeting BCMA and co-expressing BFP as a reporter gene (modified from ARI0002h CAR-T sequence1), were generated by lentiviral transduction of aCD3/aCD28 activated T cells, from each group of donor/patients, and expanded in the presence of IL-7/IL-15. Phenotypic analyses were performed by flow cytometry. Cytotoxic activity was measured by standard luciferase-based procedures and cytokine production was quantified by ELISA and Luminex. In vivo antitumoral efficacy was evaluated in xenogeneic tumor models in NSG mice. RNA-seq analysis was performed following stablished protocols2. Results: We were able to generate CAR-T cells from young (n=5) and senior healthy donors (n=5), as well as patients with MGUS (n=5), SMM (n=5), MM at diagnosis (n=3), MM after 1st line of treatment (n=5) and MM after >2 lines of treatment (n=4). CAR-T cells presented increasing variability in CAR transduction and cell expansion along disease progression. Phenotypic analysis revealed a decreasing memory phenotype, of both stem cell memory and central memory CAR-T cells, with significantly increasing number of effector cells during disease progression. This phenotype was already observed in T cells used for CAR-T cell generation, suggesting that initial phenotype strongly affects final CAR-T cell features. However, no significant differences were observed in activation (HLA-DR, CD137) and exhaustion markers (PD1, LAG3, TIM3, TIGIT). Functional in vitro characterization of CAR-T cells revealed a reduced cytotoxicity against MM cell lines once MM is diagnosed, that was associated with lower production of effector cytokines (IFNg, Granzyme A, MIP1a), especially in CAR-T cells form MM patients with >2 lines of treatment. Moreover, after continuous exposure to tumoral cells, CAR-T cells from these MM patients presented a significant upregulation of exhaustion markers (PD1, LAG3, TIM3, TIGIT), suggesting that long-term functionality could be compromised. In contrast, these phenotypic and functional differences observed in vitro were not reflected in the in vivo antitumoral response, that showed a similar efficacy between CAR-T cells from the different groups. This lack of differences could be probably due to the xenogeneic tumor model in NSG mice, that would require stringent conditions to observe differences in antitumor efficacy. Transcriptomic analyses revealed that CAR-T cells did not cluster according to disease stage due to the high variability between patients. We focus our analysis on CAR-T cells from MM patients at diagnosis (MMd) and after >2 lines of treatment (MM>2Lines), using age-matched senior healthy donors (SHD) as control. Major differences were observed between MM>2Lines and MMd/SHD, with few differences between these last groups. Gene ontology analysis of differentially expressed genes in MM>2Lines group revealed enrichment in pathways related to T cell activation, cell-cell adhesion, and lymphocyte differentiation. Moreover, deeper characterization of CAR-T cells from MM>2Lines patients showed decreased score of proliferation and polyfunctional gene sets, with enrichment in genes related to terminally effector features, corroborating functional results previously observed. Conclusions: Our results would indicate that CAR-T generated from patients at later stages of the disease present a more differentiated phenotype, with reduced functionality and increased exhaustion features, that could compromise antitumor efficacy. Transcriptomic analysis allows the identification of molecular mechanisms that could be modulated for the development of improved therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Production of Stem Cell Antigen 1 Sca-1/Ly-6A/E by Freshly Isolated NK Cells Cultured with Relevant Cytokines.

Activated NK cells in appropriate conditions are known to express stem cell antigen 1 (Sca-1/Ly-6A/E). To investigate its production, NK cells isolated from mouse spleens were incubated ex vivo in the presence of different combinations of cytokines (IL-12, IL-15, IL-18, and IFNγ). Expression of Sca-1 was found to be considerably higher in NK cells incubated in the presence of IL-18, IL-15, and IL-12 than in those treated with IL-15 and IL-18 only. To test the hypothesis that the effect of IL-12 was due to stimulation of IFNγ production, we replaced IL-12 with IFNγ in some samples and added specific anti-IFNγ antibody to some samples cultured with IL-15/IL-18+IL-12. In the subpopulations incubated in the presence of IL-15/IL-18 with added IFNγ instead of IL-12, the expression of Sca-1 was not increased. By contrast, in samples treated with IL-15/IL-18+IL-12 and anti-IFNγ antibody, the expression of Sca-1 was activated to a similar extent as in those stimulated by IL-15/IL-18+IL-12 combination without the antibody. The obtained data suggest that IL-12 activates the production of Sca-1 by NK cells through an IFNγ-independent mechanism.

Neuromedin U Induces Activation of Peripheral Group 2 Innate Lymphoid Cells through the ERK Pathway in Allergic Rhinitis Patients

Introduction: In allergic diseases, group 2 innate lymphoid cells (ILC2s) play critical roles. Neuromedin U (NMU), a highly conserved multifunctional neuropeptide, is secreted by cholinergic neurons and involved in asthma pathogenesis by amplifying lung inflammation driven by ILC2s. However, the precise effects of NMU on ILC2s in allergic rhinitis (AR) and related diseases remain unclear. Methods: A total of 15 patients with persistent AR and 8 healthy controls (HCs) were enrolled in the study. Visual analog scale (VAS) scores are used to assess the severity of clinical symptoms in AR patients. The percentages of ILC2s in peripheral blood mononuclear cells (PBMCs) were enumerated using flow cytometry. The soluble or intracellular cytokines (IL-5 and IL-13) in PBMCs or sorted ILC2s were assessed in response to various stimuli with IL-33, NMU, IL-33 combined with extracellular signal-related kinase (ERK) inhibitor or NMU combined with ERK inhibitor in the presence of IL-2. Results: We confirmed the proportion of circulating ILC2s was significantly higher in AR patients than in HCs. ILC2s levels were found to be positively related to VAS scores. We also discovered that the release of IL-5 and IL-13 in AR patients’ PBMCs stimulated by NMU (p < 0.0001 and p < 0.0001, respectively) or IL-33 (p = 0.002; p = 0.044, respectively) was significantly higher than in HCs. In AR patients, NMU stimulated PBMCs or ILC2s to generate greater inflammatory factors IL-5 and IL-13 compared to IL-33. Furthermore, we observed that NMU-promoted ILC2s activation and proliferation functions were restricted when the ERK pathway was inhibited. Conclusion: NMU effectively activated ILC2s in AR patients to produce Th2-type cytokines, and this activation can be prevented by ERK pathway inhibitors. Our findings shed new light on the neuro-immune mechanism of AR and offer new insights into its prevention and treatment.

Modulation of Zinc Transporter Expressions by Additional Zinc in C2C12 Cells Cultured in a High Glucose Environment and in the Presence of Insulin or Interleukin-6.

Zn status has been related to various chronic diseases presenting oxidative stress and inflammation, such as type 2 diabetes. Zn supplementation has been suggested to be a potential coadjuvant in the management of this condition. Zn transporters constitute a key component in the maintenance of Zn homeostasis. Our aim was to evaluate the modulatory effect of additional Zn (10 or 100µM; as a ZnSO4*7H20) on the mRNA relative expression of selected Zn transporters (ZnT1, ZnT5, ZnT7, ZIP6, ZIP7, ZIP10, ZIP14), in myoblast (C2C12) cells cultured in normal (10mM) and high glucose (30mM), and in the absence or presence of insulin (1nM), and interleukin-6 (IL-6; 5nM) for 24h. The main findings of our study were that in high glucose conditions in absence of insulin or IL-6, additional Zn increased ZnT1 and ZIP6, and decreased ZnT5 and ZIP7 expressions. However, this situation is modified by insulin, where incremental Zn induced increased expressions of ZnT1, ZnT5, and all the ZIP transporters studied. In high glucose conditions and in the presence of IL-6, additional Zn caused increased expressions of ZnT7, ZIP7, and ZIP14, compared with results in the absence of IL-6. This study provides preliminary evidence for the differential expression of selected Zn transporters in C2C12 cells subjected to high glucose and incremental Zn, suggesting that important changes in intracellular Zn distribution take place in response to inflammatory and high-insulin environments. Further study is necessary to understand the implications of these findings.

Ionic liquid functionalized Fe3O4 core–shell nanoparticles: A magnetically separable Brønsted acid catalyst for the synthesis of polythioamides

In these days, development of multicomponent‐based polymerization is gaining much attraction. Keeping this point in view, we synthesized fluorescent conjugated and nonconjugated polythioamide in one‐pot using dialdehyde, diamines, and elemental sulfur in presence of IL1–IL2@Fe3O4NPs as catalyst via Willgerodt–Kindler reaction. Herein, ionic liquid‐coated over magnetized iron nanoparticles heterogenous catalyst were synthesized. Catalyst was characterized by Brunauer–Emmett–Teller (BET), X‐ray photoelectron spectroscopy (XPS), powder X‐ray diffraction (PXRD), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy. Polymerization of multicomponent reaction proceeds smoothly in the presence of catalyst at ambient temperature with excellent yield. As magnetic catalyst recovered easily from the reaction mixture, this method shows excessive potential for efficient polymerization approach toward structurally diversified polythioamides. By the development of new magnetic catalyst, we overawed the limitations of multicomponent polymerization such as the poor solubility of polymer, low molecular weight, stoichiometric balance, easy removal of catalyst, and complicated workup. The prepared polythioamides were characterized with 1H‐NMR, 13C‐NMR, FTIR spectroscopy, and gel permeation chromatography (GPC).

Interferon Gamma Targeted Therapy: Is It Justified in Primary Sjögren's Syndrome?

Background: The pathomechanism of primary Sjögren syndrome (pSS) is multifactorial. Many cytokines take part in this process, including interferon. The study aimed to quantify certain cytokines involved in the pathomechanism of primary Sjögren syndrome (IL2, IL5, IL6, IL10, IL13, TNFα, IFNγ) and determine their common clinical correlation. On this basis, we discuss the potential use of anti-cytokine drugs in pSS therapy. Methods: The study group consisted of adult patients with a confirmed diagnosis of pSS. Results: The most frequently detected cytokines were IFNγ (82% of patients), TNFα (70%), IL6 (50%), and IL2 (42.5%). In all patients, except for one patient, IFNγ was found in the presence of other specific cytokines. There was no difference in clinical symptoms, age, and laboratory test results between the group of patients with IL-6 + TNFα + IFNγ positive cytokine, and the group of patients in whom they were not detected. There was no correlation between the presence of IL5, IL13, IL2, IL6, IL10, TNFα and musculoskeletal symptoms, skin lesions, glandular domains, pulmonary neurological, lymphadenopathy, biological and hematological domains in ESSDAI (p > 0.05). Conclusions: IFNγ most likely plays a central role in the pathomechanism of the disease. We have not noticed a clinical correlation between the three most common cytokines (IL6, IFNγ and TNFα), preliminary research results open up the possibility of searching for new treatments for pSS. The lower percentage of patients with detectable levels of TNFα and IL6 may explain the ineffectiveness of drugs targeting cytokines in clinical trials to date.

334.8: Extracellular Vesicles as Mediators of Infectious Tolerance in Human Alloreactive Regulatory Cells

Introduction: We have developed a method for ex vivo expansion of donor antigen specific T regulatory cell enriched lines (ASTRLs) from stable human kidney transplant recipients (KTRs). Rat derived ASTRLs induced prolonged graft survival and donor specific tolerance in a rat kidney transplant model. Human ASTRLs are suppressive in their phenotype and function. We examined the various mechanisms of ASTRL mediated suppression including extracellular vesicle (EV)-mediated suppression. Method: ASTRLs were expanded ex vivo from PBMCs of stable KTRs by stimulation with donor allopeptides in presence of IL-2. ASTRLs were characterized for conventional Treg markers by flow cytometry. Functional characterization of ASTRLs were performed using standard suppression assays. Extracellular ATP hydrolysis was determined by malachite green assay. EVs were isolated from the ASTRL expansion media via differential ultracentrifugation and characterized by liquid chromatography-mass spectrometry. Results: The suppressive mechanism of ASTRLs was alloantigen-specific and dependent on cell-cell contact as shown in Figure 1 (top panel). ASTRLs upregulated the expression CD39 and CD73, ectonucleotidases associated with the adenosinergic pathway, and ASTRLs demonstrated increased extracellular ATP hydrolysis (eATP) that was inhibited by POM1, a CD39 specific inhibitor (Figure 1 left bottom panel) which also abrogated the suppressive effect of ASTRLs. In KTRs ASTRLs specific for one donor alloantigen were able to suppress an effector response against other donor alloantigens demonstrating bystander/ linked suppression and inhibit effector T cell proliferation in response to both direct and indirect allorecognition (Figure 1 bottom right panel). EVs isolated from ASTRLs enhanced the suppressive ability of ASTRLs and hydrolyze eATP independently, which was inhibited by POM1 (Fig. 2) and increase production of IL-4, IL-13, and IL-1RA by alloantigen stimulated T cells, indicating a biasing towards a regulatory response (Fig. 2). Global proteomics identified 1,709 unique proteins in the large EV fraction and 1,356 unique proteins in the small EV fraction. Pathway analysis revealed that both EV fractions under stimulation were enriched for proteins involved in T cell receptor (TCR) signaling and Treg differentiation (Fig. 2) with 145 differentially enriched proteins (DEPs) in the large and 162 DEPs in the small EVs (q<0.05). Pathway analysis of DEPs will be presented. Conclusion: ASTRLs expanded from stable KTRs suppress donor antigen specific effector responses predominantly via the adenosinergic pathway and demonstrate bystander/linked immunosuppression. ASTRL-EVs enhance the suppressive capacity of ASTRLs and exhibit an increase in CD39 function. These findings indicate that human ASTRLs may be useful as autologous regulatory cell therapy in transplant recipients and that ASTRL related EVs may be useful adjuncts to ASTRL cell therapy. Saxena Transplantation Fund.

The ketone body β-hydroxybutyrate mitigates ILC2-driven airway inflammation by regulating mast cell function.

Ketone bodies are increasingly understood to have regulatory effects on immune cell function, with β-hydroxybutyrate (BHB) exerting a predominantly anti-inflammatory response. Dietary strategies to increase endogenous ketone body availability such as the ketogenic diet (KD) have recently been shown to alleviate inflammation of the respiratory tract. However, the role of BHB has not been addressed. Here, we observe that BHB suppresses group 2 innate lymphoid cell (ILC2)-mediated airway inflammation. Central to this are mast cells, which support ILC2 proliferation through interleukin-2 (IL-2). Suppression of the mast cell/IL-2 axis by BHB attenuates ILC2 proliferation and the ensuing type 2 cytokine response and immunopathology. Mechanistically, BHB directly inhibits mast cell function in part through GPR109A activation. Similar effects are achieved with either the KD or 1,3-butanediol. Our data reveal the protective role of BHB in ILC2-driven airway inflammation, which underscores the potential therapeutic value of ketone body supplementation for the management of asthma.

Pattern recognition receptor ligand-induced differentiation of human transitional B cells

B cells represent a critical component of the adaptive immune response whose development and differentiation are determined by antigen-dependent and antigen-independent interactions. In this study, we explored the effects of IL-4 and pattern-recognition receptor (PRR) ligands on B cell development and differentiation by investigating their capacity to drive the in vitro maturation of human transitional B cells. In the presence of IL-4, ligands for TLR7/8, TLR9, and NOD1 were effective in driving the in vitro maturation of cord blood transitional B cells into mature, naïve B cells as measured by CD23 expression, ABCB1 transporter activation and upregulation of sIgM and sIgD. In addition, several stimulation conditions, including TLR9 ligand alone, favored an expansion of CD27+ IgM memory B cells. Transitional B cells stimulated with TLR7/8 ligand + IL-4 or TLR9 ligand, with or without IL-4, induced a significant subpopulation of CD23+CD27+ B cells expressing high levels of sIgM and sIgD, a minor B cell subpopulation found in human peripheral blood. These studies illustrate the heterogeneity of the B cell populations induced by cytokine and PRR ligand stimulation. A comparison of transitional and mature, naïve B cells transcriptomes to identify novel genes involved in B cell maturation revealed that mature, naïve B cells were less transcriptionally active than transitional B cells. Nevertheless, a subset of differentially expressed genes in mature, naïve B cells was identified including genes associated with the IL-4 signaling pathway, PI3K signaling in B lymphocytes, the NF-κB signaling pathway, and the TNFR superfamily. When transitional B cells were stimulated in vitro with IL-4 and PRR ligands, gene expression was found to be dependent on the nature of the stimulants, suggesting that exposure to these stimulants may alter the developmental fate of transitional B cells. The influence of IL-4 and PRR signaling on transitional B cell maturation illustrates the potential synergy that may be achieved when certain PRR ligands are incorporated as adjuvants in vaccine formulations and presented to developing B cells in the context of an inflammatory cytokine environment. These studies demonstrate the potential of the PRR ligands to drive transitional B cell differentiation in the periphery during infection or vaccination independently of antigen mediated BCR signaling.

IL-6 is involved in thoracic ossification of the ligamentum flavum.

Thoracic ossification of the ligamentum flavum (TOLF) is a heterotopic ossification of spinal ligaments. TOLF is the major cause of thoracic spinal canal stenosis and myelopathy, and its underlying mechanisms are not clear. Bone formation is a complex developmental process involving the differentiation of mesenchymal stem cells to osteoblasts, and regulated by BMP2, RUNX2, Osterix (OSX), etc. In this study, we continue to further characterize properties of TOLF. Our immunohistochemistry experiments showed that expressions of osteoblastic factors such as BMP2 and RUNX2 increased in TOLF. According to flow cytometry analysis the proportion of S phase of cell cycle in primary TOLF cells was 9% higher than the control. Alizarin red staining and ALP staining observations were consistent with immunohistochemistry results. It was also observed that inflammatory cytokine IL-6 level dramatically increased in the culture supernatant of primary TOLF cells. We propose the hypothesis that IL-6 is involved in TOLF. To testify the hypothesis, we examined the effect of IL-6. Our results showed that IL-6 was able to activate expressions of osteoblastic factors such as BMP2, RUNX2, OSX, OCN and ALP, and that expressions of cell proliferation factors cyclin D1 and cyclin C increased in the presence of IL-6. Moreover, IL-6-induced BMP2 expression was inhibited by p38 inhibitor SB203580, indicating that IL-6 regulated the osteogenic BMP2 activation through p38 MAPK pathway. These data suggest that IL-6 is involved in TOLF.

Effect of Baricitinib on the epithelial-mesenchymal transition of alveolar epithelial cells induced by IL-6

ObjectiveInterstitial lung disease (ILD) is one of the common complications of Connective tissue disease (CTD). Epithelial-mesenchymal transition (EMT) is one of the main pathological mechanisms of ILD. IL-6 may induce ILD through the JAK/STAT pathway. Therefore, exploring the mechanism of IL-6 on the EMT of alveolar epithelial cells and inhibition JAK/STAT pathway with Baricitinib on the EMT of alveolar epithelial cells is helpful in revealing the pathogenesis of CTD-ILD and guiding treatment. MethodsElectrochemiluminescence was applied to detect the changes in serum IL-6 levels before and after treatment in 37 patients with anti-synthetase syndrome-associated ILD; A549 cells (a human AEC cell line) were incubated with IL-6, Baricitinib, or both IL-6 and Baricitinib, and changes in EMT-related markers levels were measured using real-time PCR, western blotting and fluorescence microscopy. The related proteins in the JAK/STAT signaling pathways were examined by western blot. The level of Connective tissue growth factor (CTGF) and Hydroxyproline (Hyp) in cell supernatants was measured by ELISA. ResultsSerum IL-6 level in patients with anti-synthetase syndrome-associated ILD was significantly higher than that in health (6.78(4.19, 16.14)pg/ml vs. 2.10(1.43, 5.18)pg/ml, p < 0.01). The level of IL-6 in the improvement group of ASS-ILD was considerably decreased than that before treatment(before(7.48(4.54, 22.76) pg/mL vs. 5.00(3.46, 11.32)pg/mL, p < 0.01), p < 0.01), and the level of IL-6 in the progressive group of ASS-ILD was significantly higher than that before treatment(before(7.49(6.77, 35.80) pg/mL vs. 30.02(8.01, 82.98) pg/mL, p < 0.05). IL-6 increased the expression of epithelial phenotypic marker E-cadherin and inhibited mesenchymal phenotypic markers, including vimentin and N-cadherin in A549 cells. Moreover, IL-6-induced EMT was attenuated by Baricitinib. Furthermore, we found that IL-6 activated the phosphorylation of JAK1/2, STAT3, and Baricitinib, partially inhibiting these changes in this process. Baricitinib reduced the secretion of CTGF and Hyp in A549 cells. ConclusionThe significant higher level of IL-6 in patients with anti-synthase syndrome-associated ILD may be related to disease activity and recurrence. Our results suggest that Baricitinib attenuates epithelial-mesenchymal transition in alveolar epithelial cells in the presence of IL-6 through the JAK/STAT signaling pathway.