STAT3 Pathway Research Articles

The Vicious Cycle of Melanoma-Microglia Crosstalk: Inter-Melanoma Variations in the Brain-Metastasis-Promoting IL-6/JAK/STAT3 Signaling Pathway.

Previous studies from our lab demonstrated that the crosstalk between brain-metastasizing melanoma cells and microglia, the macrophage-like cells of the central nervous system, fuels progression to metastasis. In the present study, an in-depth investigation of melanoma-microglia interactions elucidated a pro-metastatic molecular mechanism that drives a vicious melanoma-brain-metastasis cycle. We employed RNA-Sequencing, HTG miRNA whole transcriptome assay, and reverse phase protein arrays (RPPA) to analyze the impact of melanoma-microglia interactions on sustainability and progression of four different human brain-metastasizing melanoma cell lines. Microglia cells exposed to melanoma-derived IL-6 exhibited upregulated levels of STAT3 phosphorylation and SOCS3 expression, which, in turn, promoted melanoma cell viability and metastatic potential. IL-6/STAT3 pathway inhibitors diminished the pro-metastatic functions of microglia and reduced melanoma progression. SOCS3 overexpression in microglia cells evoked microglial support in melanoma brain metastasis by increasing melanoma cell migration and proliferation. Different melanomas exhibited heterogeneity in their microglia-activating capacity as well as in their response to microglia-derived signals. In spite of this reality and based on the results of the present study, we concluded that the activation of the IL-6/STAT3/SOCS3 pathway in microglia is a major mechanism by which reciprocal melanoma-microglia signaling engineers the interacting microglia to reinforce the progression of melanoma brain metastasis. This mechanism may operate differently in different melanomas.

Structural and Biological Features of G-Quadruplex Aptamers as Promising Inhibitors of the STAT3 Signaling Pathway.

In this paper, we investigate the structural and biological features of G-quadruplex (G4) aptamers as promising antiproliferative compounds affecting the STAT3 signalling pathway. Targeting the STAT3 protein through high-affinity ligands to reduce its levels or activity in cancer has noteworthy therapeutic potential. T40214 (STAT) [(G3C)4] is a G4 aptamer that can influence STAT3 biological outcomes in an efficient manner in several cancer cells. To explore the effects of an extra cytidine in second position and/or of single site-specific replacements of loop residues in generating aptamers that can affect the STAT3 biochemical pathway, a series of STAT and STATB [GCG2(CG3)3C] analogues containing a thymidine residue instead of cytidines was prepared. NMR, CD, UV, and PAGE data suggested that all derivatives adopt dimeric G4 structures like that of unmodified T40214 endowed with higher thermal stability, keeping the resistance in biological environments substantially unchanged, as shown by the nuclease stability assay. The antiproliferative activity of these ODNs was tested on both human prostate (DU145) and breast (MDA-MB-231) cancer cells. All derivatives showed similar antiproliferative activities on both cell lines, revealing a marked inhibition of proliferation, particularly at 72 h at 30 µM. Transcriptomic analysis aimed to evaluate STAT's and STATB's influence on the expression of many genes in MDA-MB-231 cells, suggested their potential involvement in STAT3 pathway modulation, and thus their interference in different biological processes. These data provide new tools to affect an interesting biochemical pathway and to develop novel anticancer and anti-inflammatory drugs.

Muscle-derived stem cell exosomes with overexpressed miR-214 promote the regeneration and repair of rat sciatic nerve after crush injury to activate the JAK2/STAT3 pathway by targeting PTEN.

This study aimed to investigate the effect of muscle-derived stem cell (MDSC) exosomes with overexpressed miR-214 on the regeneration and repair of rat sciatic nerve after crush injury and its molecular mechanism. First, primary MDSCs, Schwann cells (SCs) and dorsal root ganglion (DRG) neurons were isolated and cultured, and the characteristics of MDSCs-derived exosomes were identified by molecular biology and immunohistochemistry. NC mimics and miR-214 mimics were transfected to obtain exo-NC and exo-miR-214. An in vitro co-culture system was established to determine the effect of exo-miR-214 on nerve regeneration. The restoration of sciatic nerve function of rats by exo-miR-214 was evaluated by walking track analysis. Immunofluorescence for NF and S100 was used to detect the regeneration of axon and myelin sheath in injured nerve. The Starbase database was used to analyze the downstream target genes of miR-214. QRT-PCR and dual luciferase reporter assays were used to validate the miR-214 and PTEN interaction relationship. And the expression of the JAK2/STAT3 pathway-related proteins in sciatic nerve tissues were detected by western blot. The above experiments showed that MDSCs-derived exosomes with overexpressed miR-214 was found to promote the proliferation and migration of SCs, increase the expression of neurotrophic factors, promote axon extension of DRG neurons and positively affect the recovery of nerve structure and function. In addition, PTEN was a target gene of miR-214. Exo-miR-214 can significantly inhibit the expression level of PTEN, increase the protein expression levels of p-JAK2 and p-STAT3 and the ratio of p-JAK2/JAK2 and p-STAT3/STAT3, also MDSCs-derived exosomes with overexpressed miR-214 can reduce the occurrence of denervated muscle atrophy. In summary, the MDSCs-derived exosomes with overexpressed miR-214 is involved in peripheral nerve regeneration and repair in rats after sciatic nerve crush injury to activate the JAK2/ STAT3 pathway by targeting PTEN.

Whole exome and transcriptome analysis revealed the activation of ERK and Akt signaling pathway in canine histiocytic sarcoma

Histiocytic sarcoma (HS) is an incurable aggressive tumor, and no consensus has been made on the treatment due to its rare occurrence. Since dogs spontaneously develop the disease and several cell lines are available, they have been advocated as translational animal models. In the present study, therefore, we explored gene mutations and aberrant molecular pathways in canine HS by next generation sequencing to identify molecular targets for treatment. Whole exome sequencing and RNA-sequencing revealed gene mutations related to receptor tyrosine kinase pathways and activation of ERK1/2, PI3K-AKT, and STAT3 pathways. Analysis by quantitative PCR and immunohistochemistry revealed that fibroblast growth factor receptor 1 (FGFR1) is over-expressed. Moreover, activation of ERK and Akt signaling were confirmed in all HS cell lines, and FGFR1 inhibitors showed dose-dependent growth inhibitory effects in two of the twelve canine HS cell lines. The findings obtained in the present study indicated that ERK and Akt signaling were activated in canine HS and drugs targeting FGFR1 might be effective in part of the cases. The present study provides translational evidence that leads to establishment of novel therapeutic strategies targeting ERK and Akt signaling in HS patients.

Ivermectin Protects Against Experimental Autoimmune Encephalomyelitis in Mice by Modulating the Th17/Treg Balance Involved in the IL-2/STAT5 Pathway.

Multiple sclerosis (MS), a T-cell-mediated autoimmune disease that affects the central nervous system (CNS), is characterized by white matter demyelination, axon destruction, and oligodendrocyte degeneration. Ivermectin, an anti-parasitic drug, has anti-inflammatory, anti-tumor, and antiviral properties. However, to date, there are no in-depth studies on the effect of ivermectin on the function effector of T cells in murine experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Here, we conducted in vitro experiments and found that ivermectin inhibited the proliferation of total T cells (CD3+) and their subsets (CD4+ and CD8+ T cells) as well as T cells secreting the pro-inflammatory cytokines IFN-γ and IL-17A; ivermectin also increased IL-2 production and IL-2Rα (CD25) expression, which was accompanied by an increase in the frequency of CD4+CD25+Foxp3+ regulatory T cells (Treg). Importantly, ivermectin administration reduced the clinical symptoms of EAE mice by preventing the infiltration of inflammatory cells into the CNS. Additional mechanisms showed that ivermectin promoted Treg cells while inhibiting pro-inflammatory Th1 and Th17 cells and their IFN-γ and IL-17 secretion; ivermectin also upregulated IL-2 production from MOG35-55-stimulated peripheral lymphocytes. Finally, ivermectin decreased IFN-γ and IL-17A production and increased IL-2 level, CD25 expression, and STAT5 phosphorylation in the CNS. These results reveal a previously unknown etiopathophysiological mechanism by which ivermectin attenuates the pathogenesis of EAE, indicating that it may be a promising option for T-cell-mediated autoimmune diseases such as MS.

Cytokine storm based mechanisms for extra-pulmonary manifestations of SARS-CoV-2 infection.

Viral illnesses like SARS-CoV-2 have pathologic effects on non-respiratory organs in the absence of direct viral infection. We injected mice with cocktails of rodent equivalents of human cytokine storms resulting from SARS-CoV-2 / COVID-19 or Rhinovirus common cold infection. At low doses, COVID-19 cocktails induced glomerular injury and albuminuria in Zhx2 hypomorph and Zhx2+/+ mice to mimic COVID-19 related proteinuria. Common Cold cocktail induced albuminuria selectively in Zhx2 hypomorph mice to model relapse of Minimal Change Disease (MCD), that improved after depletion of TNF-α or sIL-4Rα or IL-6. The Zhx2 hypomorph state increased cell membrane to nuclear migration of podocyte ZHX proteins in vivo (both cocktails) and lowered pSTAT6 activation (COVID-19 cocktail) in vitro. At higher doses, COVID-19 cocktails induced acute heart injury, myocarditis, pericarditis, acute liver injury and acute kidney injury, and high mortality in Zhx2+/+ mice, whereas Zhx2 hypomorph mice were relatively protected, due in part to early asynchronous activation of STAT5 and STAT6 pathways in these organs. Dual depletion of cytokine combinations of TNF-α with IL-2 or IL-13 or IL-4 in Zhx2+/+ mice reduced multiorgan injury and eliminated mortality. Using genome sequencing and CRISPR-Cas9, an insertion upstream of ZHX2 was identified as a cause of the human ZHX2 hypomorph state.

Dysregulation in IFN-γ signaling and response: the barricade to tumor immunotherapy.

Interferon-gamma (IFN-γ) has been identified as a crucial factor in determining the responsiveness to immunotherapy. Produced primarily by natural killer (NK) and T cells, IFN-γ promotes activation, maturation, proliferation, cytokine expression, and effector function in immune cells, while simultaneously inducing antigen presentation, growth arrest, and apoptosis in tumor cells. However, tumor cells can hijack the IFN-γ signaling pathway to mount IFN-γ resistance: rather than increasing antigenicity and succumbing to death, tumor cells acquire stemness characteristics and express immunosuppressive molecules to defend against antitumor immunity. In this review, we summarize the potential mechanisms of IFN-γ resistance occurring at two critical stages: disrupted signal transduction along the IFNG/IFNGR/JAK/STAT pathway, or preferential expression of specific interferon-stimulated genes (ISGs). Elucidating the molecular mechanisms through which tumor cells develop IFN-γ resistance help identify promising therapeutic targets to improve immunotherapy, with broad application value in conjugation with targeted, antibody or cellular therapies.

Acacetin exerts antitumor effects on gastric cancer by targeting EGFR.

Background: Gastric cancer (GC) is a common malignant tumor with a poor prognosis. Combination treatments may prolong the survival of patients with GC. Acacetin, which is a flavonoid, exerts potent inhibitory effects on several types of cancer cells; however, the mechanisms of action remain poorly understood. Methods: Network pharmacology and RNA sequencing were used to predict the targets of acacetin, which were then verified by drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA) and molecular docking. The biological functions of acacetin in MKN45 and MGC803 cells were investigated using TUNEL assays, crystal staining and colony formation assays. The pathways affected by acacetin were verified through reverse experiments. The in vivo antitumor efficacy of acacetin was assessed in a subcutaneous xenotransplanted tumor model. Results: In this study, we identified EGFR from more than a dozen predicted targets as a protein that directly binds to acacetin. Moreover, acacetin affected the level of phosphorylated EGFR. In vitro, acacetin promoted the apoptosis of GCcells. Importantly, EGFR agonists reversed the inhibitory effects of acacetin on the STAT3 and ERK pathways. In vivo, acacetin decreased the protein levels of pEGFR in tumors, resulting in increased GC xenograft tumor regression without obvious toxicity. Conclusion: Our findings highlight EGFR as one of the direct targets of acacetin in GCcells. Acacetin inhibited the phosphatase activity of EGFR in vitro and in vivo, which played a role in the antitumor effects of acacetin. These studies provide new evidence for the use of acacetin as a potential reagent for the treatment of GC.

Pancreatic cancer-derived small extracellular vesical ezrin activates fibroblasts to exacerbate cancer metastasis through STAT3 and YAP-1 signaling pathways.

Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC), play an important role in tumorigenesis, metastasis, and chemoresistance. Tumor-derived small extracellular vesicles (sEVs), which mediate cell-to-cell communication between cancer cells and fibroblasts, are also critical for cancer progression and metastasis. However, it remains unclear how PDAC cell-derived sEVs activate fibroblasts, which contributes to tumor progression. Here, we report that ezrin (EZR) expression in PDAC cell-derived sEVs (sEV-EZR) can activate fibroblasts, resulting in increased migration ability and high expression of α-SMA, PDGFRB, and high production of extracellular matrix in fibroblasts. Reciprocally, sEV-EZR-activated fibroblasts enhanced PDAC cell proliferation, invasion, and metastasis to the liver in animal models. Conversely, fibroblasts treated with PDAC cell-derived sEVs with EZR knockdown resulted in the reduced metastatic ability of PDAC. Mechanistically, we demonstrated that PDAC cell-derived sEV-EZR increases the STAT3 and YAP-1 signaling pathways to induce fibroblast activation, and the activated fibroblasts promote PDAC cell proliferation, invasion, and liver metastasis. Inhibition of the STAT3 and YAP-1 signaling pathways by gene knockdown can abrogate sEV-EZR-induced effects. These findings suggest that targeting the interaction between PDAC cell-derived sEV-EZR and fibroblasts is a potential therapeutic strategy for PDAC.

Triptolide alleviates collagen-induced arthritis in mice by modulating Treg/Th17 imbalance through the JAK/PTEN-STAT3 pathway.

This study aimed to investigate the effects of triptolide (TP) on collagen-induced arthritis (CIA) mice and the related mechanisms. CIA mice were administered TP for 35 days. Mouse ankle joints and serum antibodies and cytokines were examined to assess the therapeutic effects of TP. The ratios of Treg, Th1, and Th17 cells were measured by flow cytometry and RT-qPCR. Reverse docking was used to characterize the binding modes of TP against target proteins. The expression of the STAT3 pathway in CIA mice was evaluated by western blotting and immunofluorescence staining. Mouse spleen lymphocytes were extracted and the expression of the STAT3 pathway after IL-6 stimulation was analyzed. TP could significantly alleviate joint swelling, reduce bone destruction, and downregulate serum inflammation levels. TP improved the imbalance of Treg/Th17 cells in CIA mice. TP could form stable complexes with target proteins. TP significantly inhibited the activation of the JAK/PTEN-STAT3 pathway in mice. Moreover, TP regulated the activation of the JAK1/2-STAT3 signaling pathway in mouse spleen lymphocytes under inflammatory stimulation. TP can inhibit inflammation and alleviate bone destruction in CIA mice. The underlying mechanism is related to the regulation of the imbalance of Treg/Th17 cells through the JAK/PTEN-STAT3 pathway.

Uptake-Dependent and -Independent Effects of Fibroblasts-Derived Extracellular Vesicles on Bone Marrow Endothelial Cells from Patients with Multiple Myeloma: Therapeutic and Clinical Implications.

Extracellular vesicles (EVs) have emerged as important players in cell-to-cell communication within the bone marrow (BM) of multiple myeloma (MM) patients, where they mediate several tumor-associated processes. Here, we investigate the contribution of fibroblasts-derived EVs (FBEVs) in supporting BM angiogenesis. We demonstrate that FBEVs' cargo contains several angiogenic cytokines (i.e., VEGF, HGF, and ANG-1) that promote an early over-angiogenic effect independent from EVs uptake. Interestingly, co-culture of endothelial cells from MM patients (MMECs) with FBEVs for 1 or 6 h activates the VEGF/VEGFR2, HGF/HGFR, and ANG-1/Tie2 axis, as well as the mTORC2 and Wnt/β-catenin pathways, suggesting that the early over-angiogenic effect is a cytokine-mediated process. FBEVs internalization occurs after longer exposure of MMECs to FBEVs (24 h) and induces a late over-angiogenic effect by increasing MMECs migration, chemotaxis, metalloproteases release, and capillarogenesis. FBEVs uptake activates mTORC1, MAPK, SRC, and STAT pathways that promote the release of pro-angiogenic cytokines, further supporting the pro-angiogenic milieu. Overall, our results demonstrate that FBEVs foster MM angiogenesis through dual time-related uptake-independent and uptake-dependent mechanisms that activate different intracellular pathways and transcriptional programs, providing the rationale for designing novel anti-angiogenic strategies.

Leukaemic cells expressing ETV6::FRK are sensitive to dasatinib in vivo.

ETV6::Fyn-related kinase (FRK), which is a Src family tyrosine-kinase-related fusion gene and firstly identified in our patient with paediatric high risk B cell precursor acute lymphoblastic leukaemia (B-ALL), has no evidence of efficacy of tyrosine kinase inhibitor in vivo. We performed functional analysis of ETV6::FRK to establish molecular targeting therapy and determined that dasatinib could abrogate proliferation activity of ETV6::FRK through the repression of FRK-STAT3/STAT5 pathway in vitro and significantly extended the survival time of the xenografted mice in vivo (p<0.01). Our data support the potential of dasatinib as a therapeutic option for patients with B-ALL harboring FRK rearrangements.

STAT5-regulated autocrine IL-6 signaling dictates IL-10-dependent regulatory functions of neonatal B10 cells

Abstract Neonates have suboptimal immune responses to immunization, resulting in higher susceptibility to microbial infection. B cells have a central role in humoral immunity to immunization, and antigen recognition by BCRs is essential for the activation and differentiation of B cells. Neonatal B cells, however, do not respond to BCR activation as robustly as adult cells do. Our hypothesis is that unique features of neonatal BCR signaling may contribute to suboptimal humoral responses. The aim of this project is to decipher the neonatal BCR signaling pathways and identify those that may be responsible for suboptimal B cell functions. Purified adult and neonatal B cells were stimulated with anti-IgM. RNA-seq analysis revealed that STAT3 and STAT5 signaling pathways were activated in neonatal B cells. STAT3 and STAT5 were phosphorylated in neonatal B cells. BCR-mediated STAT5 phosphorylation induces IL-6 production, which in turn activates STAT3 in an autocrine/paracrine manner. Moreover, IL-6-induced STAT3 activation led to the production of anti-inflammatory cytokine IL-10. Further underscoring the role of IL-6 in IL-10 production, neonatal B cells from IL-6-KO mice did not secrete IL-10. To assess whether the autocrine IL-6 is essential for IL-10-dependent immunosuppression, we incubated peritoneal macrophages with conditioned medium (CM) from adult, wild-type neonatal, or IL-6-KO neonatal B cells. We found that CM from wild-type neonatal B cells suppressed TNF-α production by macrophages in an IL-10-dependent manner, whereas CM from IL-6-KO neonatal B cells or adult B cells did not. Our studies unveiled the essential role for STAT5-induced autocrine IL-6 signaling in IL-10-mediated immunosuppressive functions of neonatal B cells.

The Ikaros zinc finger transcription factor Eos promotes T H2 differentiation and function by propagating IL-2/STAT5 signaling.

Abstract The Ikaros zinc finger transcription factor Eos has largely been associated with chromatin regulation promoting immunosuppressive regulatory T cells. However, Eos’ role in the differentiation and function of pro-inflammatory T cells has remained unclear. Surprisingly, our work reveals that Eos is a positive regulator of CD4 +T helper 2 (T H2) cells—effector T cells that participate in anti-helminthic immunity but are also implicated in inducing allergic asthma. Using in vitro-generated T H2 cells and an in vivo allergic asthma mouse model, we found that Eos-deficient T cells had reduced gene and protein expression of critical T H2 transcription factors (including the lineage-defining transcription factor Gata3), effector cytokines, and differentiation receptors. Among the various T H2-polarizing pathways, the IL-2/STAT5 axis and its downstream T H2 gene targets emerged as one of the most significantly downregulated networks in Eos deficiency. Using in vitro-generated T H2 cells and overexpression of Eos zinc-finger-domain mutants, we discovered that Eos forms a novel complex with and promotes the tyrosine-phosphorylated activation of STAT5. Additionally, we showed that components of the IL-2/STAT5 pathway participate in a feed-forward loop to promote Eos expression in T H2 cells, further supporting Eos’ regulatory connection with the IL-2/STAT5 axis. Together, these data define a novel mechanism whereby Eos mediates IL-2/STAT5 activity to facilitate T H2 differentiation. This work is significant, as its findings reframe our understanding of Eos’ role in T cell differentiation. K.J.O. is supported by a grant from The National Institutes of Health (NIH) AI134972, as well as from The Ohio State University College of Medicine and The Ohio State University Comprehensive Cancer Center. J.A.T. is supported by funding through the Susan Huntington Dean’s Distinguished University Fellowship and the NIH T32 “Interdisciplinary Program in Microbe-Host Biology” pre-doctoral fellowship administered through the OSU Infectious Diseases Institute and OSU Department of Microbial Infection and Immunity. K.M.G. is supported by NIH grant 1R01ES028829-01A1. L.M.C. and K.J.O. were supported in part by the Jeffress Trust Awards Program in Interdisciplinary Research. K.A.R. is supported by funding through The Ohio State University College of Medicine Advancing Research in Infection and Immunity Fellowship Program.

Repurposing ketotifen as a therapeutic strategy for neuroendocrine prostate cancer by targeting the IL-6/STAT3 pathway.

Neuroendocrine prostate cancer (NEPC), a highly aggressive subtype of prostate cancer displaying resistance to hormone therapy, presents a poor prognosis and limited therapeutic options. Here, we aimed to find novel medication therapies for NEPC and explore the underlying mechanism. A high-throughput drug screening utilizing an FDA-approved drug library was performed and ketotifen, an antihistamine agent, was identified as a potential therapeutic candidate for NEPC. The whole-transcriptome sequencing analysis was conducted to explore mechanism of ketotifen inhibitory in NEPC. Multiple cell biology and biochemistry experiments were performed to confirm the inhibitory effect of ketotifen in vitro. A spontaneous NEPC mice model (PBCre4:Ptenf/f;Trp53f/f;Rb1f/f)was used to reveal the inhibitory effect of ketotifen in vivo. Our in vitro experiments demonstrated that ketotifen effectively suppressed neuroendocrine differentiation, reduced cell viability, and reversed the lineage switch via targeting the IL-6/STAT3 pathway. Our in vivo results showed that ketotifen significantly prolonged overall survival and reduced the risk of distant metastases in NEPC mice model. Our findings repurpose ketotifen for antitumor applications and endorse its clinical development for NEPC therapy, offering a novel and promising therapeutic strategy for this formidable cancer subtype.

ROS Scavenging Nanozyme Modulates Immunosuppression for Sensitized Cancer Immunotherapy.

Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), two immunosuppressive myeloid components within the tumor microenvironment (TME), represent fundamental barriers in cancer immunotherapy, whereas current nanomedicines rarely exert dual modulatory roles on these cell types simultaneously. Reactive oxygen species (ROS) not only mediates MDSC-induced immunosuppression but also triggers differentiation and polarization of M2-TAMs. Herein, an ROS scavenging nanozyme, Zr-CeO, with enhanced superoxide dismutase- and catalase-like activities for renal tumor growth inhibition is reported. Mechanistically, intracellular ROS scavenging by Zr-CeO significantly attenuates MDSC immunosuppression via dampening the unfolded protein response, hinders M2-TAM polarization through the ERK and STAT3 pathways, but barely affects neoplastic cells and cancer-associated fibroblasts. Furthermore, Zr-CeO enhances the antitumor effect of PD-1 inhibition in murine renal and breast tumor models, accompanied with substantially decreased MDSC recruitment and reprogrammed phenotype of TAMs in the tumor mass. Upon cell isolation, reversed immunosuppressive phenotypes of MDSCs and TAMs are identified. In addition, Zr-CeO alone or combination therapy enhances T lymphocyte infiltration and IFN-γ production within the TME. Collectively, a promising strategy to impair the quantity and function of immunosuppressive myeloid cells and sensitize immunotherapy in both renal and breast cancers is provided.

Investigating in vitro NF-κB and STAT3 activation in MOGAD (S40.010)

<h3>Objective:</h3> To investigate the in vitro activation of NF-κB (nuclear factor kappa B) and STAT3 pathways in central memory CD4+ T (CD45RACD62L+, Tcm) cells of MOGAD patients after antigen and cytokine stimulation or glucocorticoid treatment compared to age-matched healthy controls (HC). <h3>Background:</h3> There is increasing evidence that T cells play a key role in MOGAD. Proinflammatory cytokines IL6 and TNFα, activators of STAT3 and NF-κB pathways, respectively, are reported to increase in CSF of MOGAD patients and increase during infections, which commonly precede MOGAD relapses, We previously found decreased A20, a negative regulator of NF-κB, serum levels at relapse. <h3>Design/Methods:</h3> 4 pediatric MOGAD remission samples (untreated) and 4 sex and age-matched HC were studied. 8×10⁴ PBMCs were plated per well. Cells were stimulated with MOG peptides, anti-CD3/CD28, TNFα, IL6, or PMA/Ionomycin. A20, IκBα (inhibitor kappa B-alpha), p-p65 (phospho-p65), and p-STAT3 (phospho-STAT3) expression levels (MFI, mean fluorescence intensity) were determined by flow cytometry and MFI levels in Tcm. <h3>Results:</h3> Ex vivo expression levels of A20, IκBα, p-p65, and p-STAT3 levels were not significantly different in MOGAD compared to HC in Tcm. Upon short IL6 or TNFα (0–15/7–75 minutes) stimulation, we observed similar NF-κB and STAT3 activation in the Tcm, while STAT3 activation was significantly higher at 75 minutes in MOGAD. MOG peptide stimulation for 24 hours decreased A20 and IκBα levels and increased p-STAT3 and p-p65 levels in both MOGAD and HC. When co-incubated with either MOG peptide or CD3, prednisolone decreased NF-κB and STAT3 activation. <h3>Conclusions:</h3> MOG peptide stimulation activates STAT3 and NF-κB in CD4+Tcm cells from MOGAD and HC, while prednisolone treatment had the opposite effect. IL6 and TNFα also activate Tcm similarly in MOGAD and HC, while STAT3 activation was more extended in MOGAD. Modulating the NF-κB-A20 or IL6/STAT3 pathway in T cells may have therapeutic potential in MOGAD. <b>Disclosure:</b> Mr. Corbali has nothing to disclose. Shrishti Saxena has nothing to disclose. Dr. Patel has nothing to disclose. Dr. Chitnis has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Novartis. Dr. Chitnis has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Roche-Genentech. Dr. Chitnis has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Academic CME. The institution of Dr. Chitnis has received research support from Novartis. The institution of Dr. Chitnis has received research support from Sanofi. The institution of Dr. Chitnis has received research support from Octave.

CD73 mediated host purinergic metabolism in intestine contributes to the therapeutic efficacy of a novel mesenchymal-like endometrial regenerative cells against experimental colitis.

The disruption of intestinal barrier functions and the dysregulation of mucosal immune responses, mediated by aberrant purinergic metabolism, are involved in the pathogenesis of inflammatory bowel diseases (IBD). A novel mesenchymal-like endometrial regenerative cells (ERCs) has demonstrated a significant therapeutic effect on colitis. As a phenotypic marker of ERCs, CD73 has been largely neglected for its immunosuppressive function in regulating purinergic metabolism. Here, we have investigated whether CD73 expression on ERCs is a potential molecular exerting its therapeutic effect against colitis. ERCs either unmodified or with CD73 knockout (CD73-/-ERCs), were intraperitoneally administered to dextran sulfate sodium (DSS)-induced colitis mice. Histopathological analysis, colon barrier function, the proportion of T cells, and maturation of dendritic cells (DCs) were investigated. The immunomodulatory effect of CD73-expressing ERCs was evaluated by co-culture with bone marrow-derived DCs under LPS stimulation. FACS determined DCs maturation. The function of DCs was detected by ELISA and CD4+ cell proliferation assays. Furthermore, the role of the STAT3 pathway in CD73-expressing ERCs-induced DC inhibition was also elucidated. Compared with untreated and CD73-/-ERCs-treated groups, CD73-expressing ERCs effectively attenuated body weight loss, bloody stool, shortening of colon length, and pathological damage characterized by epithelial hyperplasia, goblet cell depletion, the focal loss of crypts and ulceration, and the infiltration of inflammatory cells. Knockout of CD73 impaired ERCs-mediated colon protection. Surprisingly, CD73-expressing ERCs significantly decreased the populations of Th1 and Th17 cells but increased the proportions of Tregs in mouse mesenteric lymph nodes. Furthermore, CD73-expressing ERCs markedly reduced the levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) and increased anti-inflammatory factors (IL-10) levels in the colon. CD73-expressing ERCs inhibited the antigen presentation and stimulatory function of DCs associated with the STAT-3 pathway, which exerted a potent therapeutic effect against colitis. The knockout of CD73 dramatically abrogates the therapeutic ability of ERCs for intestinal barrier dysfunctions and the dysregulation of mucosal immune responses. This study highlights the significance of CD73 mediates purinergic metabolism contributing to the therapeutic effects of human ERCs against colitis in mice.

E069 Antagonism of IL-27 bioactivity

Abstract Background/Aims Rheumatoid arthritis (RA) is a chronic autoimmune disease that results in joint damage and bony destruction. The inflammatory process is central in the pathology of RA, with cytokines mediating the inflammatory response. Cytokines, in particular IL-6, have been targeted as treatments for RA. The IL-27 cytokine is part of the IL-6 family, and acts via the same signalling pathway. IL-27 can have the same function as IL-6, but can also antagonise IL-6. The presence of ectopic lymphoid structures (ELSs) is associated with more severe disease and worse joint damage, and is also associated with low IL-27 levels. Hence, targeting IL-27 may aid in severe RA and offer a novel treatment option. Aims: 1) To generate a recombinant IL-27 antagonist, 2) to determine the concentration and bioactivity of IL-27 antagonist, 3) to test the bioactivity of IL-27 antagonist as an inhibitor of IL-27 activity. Methods Chinese hamster ovarian (CHO) cells were transduced with adenovirus to express the IL-27 antagonist. The concentration of these IL-27 antagonists was determined using ELISA. To determine the bioactivity of the IL-27 antagonist, both acute leukaemia cell lines (THP1 cells) and cells from C57BL/6J mice were treated with human IL-27 and IL-27 antagonists and STAT3 activation determined using flow cytometry and ELISA. Results THP1 cells treated with both 5ng/ml and 15ng/ml of human recombinant IL-27 showed STAT3 activation, with 29.4% and 30.7% of cells being STAT3 positive respectively. Cells that remained untreated only showed STAT3 activation in 2.54% of cells, indicating IL-27 has a role in the activation of the STAT3 pathway. STAT3 activation was not witnessed in CD4 cells from C57BL/6J mice treated with human recombinant IL-27. Furthermore, the recombinant IL-27 antagonist was not seen to be able to antagonise the actions IL-27 and alter STAT3 activation. Conclusion In conclusion, this study has demonstrated that IL-27 treatment of cells can cause increased STAT3 activation, however we were unable to demonstrate that the IL-27 antagonist was able to inhibit the actions of IL-27. IL-27 shows great potential for use as a novel therapy, since both its pro and anti-inflammatory functions can be targeted, however there needs to be much more information about the action of IL-27 as this is still largely unknown. Future studies could aim to optimise the protocol in order to understand the bioactivity of the IL-27 antagonist, thus understanding its role in altering STAT3 activation. Disclosure L. Green: None.

Plasticity between type 2 innate lymphoid cell subsets and amphiregulin expression regulates epithelial repair in biliary atresia.

Although a dysregulated type 1 immune response is integral to the pathogenesis of biliary atresia, studies in both humans and mice have uncovered a type 2 response, primarily driven by type 2 innate lymphoid cells. In nonhepatic tissues, natural type 2 innate lymphoid cell (nILC2s) regulate epithelial proliferation and tissue repair, whereas inflammatory ILC2s (iIlC2s) drive tissue inflammation and injury. The aim of this study is to determine the mechanisms used by type 2 innate lymphoid cell (ILC2) subpopulations to regulate biliary epithelial response to an injury. Using Spearman correlation analysis, nILC2 transcripts, but not those of iILC2s, are positively associated with cholangiocyte abundance in biliary atresia patients at the time of diagnosis. nILC2s are identified in the mouse liver through flow cytometry. They undergo expansion and increase amphiregulin production after IL-33 administration. This drives epithelial proliferation dependent on the IL-13/IL-4Rα/STAT6 pathway as determined by decreased nILC2s and reduced epithelial proliferation in knockout strains. The addition of IL-2 promotes inter-lineage plasticity towards a nILC2 phenotype. In experimental biliary atresia induced by rotavirus, this pathway promotes epithelial repair and tissue regeneration. The genetic loss or molecular inhibition of any part of this circuit switches nILC2s to inflammatory type 2 innate lymphoid cell-like, resulting in decreased amphiregulin production, decreased epithelial proliferation, and the full phenotype of experimental biliary atresia. These findings identify a key function of the IL-13/IL-4Rα/STAT6 pathway in ILC2 plasticity and an alternate circuit driven by IL-2 to promote nILC2 stability and amphiregulin expression. This pathway induces epithelial homeostasis and repair in experimental biliary atresia.