STAT3 Pathway Research Articles

Mechanism of miR-125b-5p Carried with Albumin Nanoparticles in Protecting the Injured Nerve Cells in Ischemia-Reperfusion Cerebral Injury

This study assessed the mechanism of miR-125b-5p carried with albumin nanoparticles on injured nerve cells in ischemia-reperfusion cerebral injury. 50 rats were assigned into control set, model set, miR-125b-5p set, IL-6/STAT3 agonist set, and positive control set. The score of neurological impairment, cerebral infarction volume, cerebral water content, apoptosis index of brain cells, synaptic ultrastructure, miR-125b-5p expression, level of IL-6/STAT3 pathway factors, targeting correlation between miR-125b-5p and IL-6 were observed. There was a good dispersibility and uniform size in albumin nanoparticles with an average particle size of 15.5 nanometer. The neurological impairment, cerebral infarction volume, cerebral water content, apoptosis index of brain cells in model set and IL-6/STAT3 agonist set were the highest followed by miR-125b-5p set and positive control set. The level of inflammatory reaction in model set and IL-6/STAT3 agonist set were the highest and oxidative stress were the lowest. miR-125b-5p carried with albumin nanoparticles could directly target on IL-6. The phosphorylation level of IL-6 and STAT3 could be restrained. The activity of IL-6/STAT3 signal pathway could be inhibited, leading to restrained inflammation and oxidative stress in nervous system and reduced cerebral water content, therefore protecting nerve cells. The mechanism might be related with restrained IL-6/STAT3 pathway.

IL-6-Dependent STAT3 Activation and Induction of Proinflammatory Cytokines in Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease with periductal inflammation and fibrosis. Genetic studies suggest inflammatory cytokines and IL-6-dependent activation of transcription factor STAT3 as pivotal steps in PSC pathogenesis. However, details of inflammatory regulation remain unclear. We recruited 50 patients with PSC (36 with inflammatory bowel disease, 14 without inflammatory bowel disease), 12 patients with autoimmune hepatitis, and 36 healthy controls to measure cytokines in the serum, bile, and immune cell supernatant using bead-based immunoassays and flow cytometry and immunohistochemistry to analyze phosphorylation of STATs in immune cells. Finally, we analyzed cytokines and STAT3 phosphorylation of T cells in the presence of JAK1/2 inhibitors. In PSC, IL-6 specifically triggered phosphorylation of STAT3 in CD4 + T cells and lead to enhanced production of interferon (IFN) gamma and interleukin (IL)-17A. Phospho-STAT3-positive CD4 + T cells correlated with systemic inflammation (C-reactive protein serum levels). Combination of immunohistology and flow cytometry indicated that phospho-STAT3-positive cells were enriched in the peribiliary liver stroma and represented CD4 + T cells with prominent production of IFN gamma and IL-17A. JAK1/2 inhibitors blocked STAT3 phosphorylation and production of IFN gamma and IL-6, whereas IL-17A was apparently resistant to this inhibition. Our results demonstrate systemic and local activation of the IL-6/STAT3 pathway in PSC. Resistance of IL-17A to STAT3-targeted inhibition points to a more complex immune dysregulation beyond STAT3 activation.

Abstract 43: Heme Oxygenase 1 Counteracts Pro-Stemness and Pro-Invasive Effect Triggered by Communication With Bone Progenitors in Prostate Cancer Cells

Abstract Purpose: Mortality in prostate cancer (PCa) patients is mostly associated with bone metastasis and aggressive phenotypes promoted by prostate cancer stem cells (PCSCs). We have previously described that heme oxygenase 1 (HO-1), the rate-limiting enzyme in heme catabolism with antioxidant and antiinflammatory properties, presents an antitumoral role in PCa. In this work, we assessed the role of HO-1 as a modulator of stemness and metastasis associated genes in PCa cells conditioned by bone progenitor cells. Methods: An indirect transwell co-culture system of PC3 cells, a PCa cell line, with MC3T3 cells, an osteoblast precursor cell line, was developed as an in vitro model of PCa bone metastasis. Additionally, PC3 cells were pre-treated with hemin, a pharmacological HO-1 inductor. An RNA-seq was performed to analyze the transcriptome of PC3 cells. The expression of a curated list of 87 genes associated with stemness, metastasis and the STAT3 pathway, including PCSC and classic pluripotency markers, together with a gene signature previously described by our group (ADAM15, BCL2L1, LTBR, MBNL2, SPINT1), was assessed. Results were complemented with survival data of PCa patients (TCGA-PRAD, n=565). Results: Our results showed that PC3 cells co-cultured with MC3T3 cells displayed an upregulation of PCSC markers CD44, ALDH1A3 and ALDH1L2, pluripotency markers OCT4 and SOX2, and BCL2L1 and LTBR, two genes included in our signature, compared with non-co-cultured PC3 cells. Further, when comparing co-cultured PC3 cells vs. co-cultured PC3 cells pre-treated with hemin, in the latter group the expression of 71/87 genes associated with stemness, metastasis and STAT3 pathway decreased, including CD44, ALDH1A3, ALDH1L2, OCT4, MBNL2 and ADAM15. Moreover, when evaluating patients’ disease progression free survival, those with high expression of OCT4 showed a lower risk of the event. Conclusion: HO-1 expression modulates stemness and metastasis related genes in PCa, promoting a more differentiated state, counteracting the pro-stemness effect of communication with bone progenitors. This further supports the antitumoral role of HO-1 in PCa pointing out to its potential as a therapeutic target for the disease. Citation Format: Ines Achinelli, Agustina Sabater, Pablo Sanchis, Nicolás Anselmino, Sofía Lage-Vickers, Gaston Pascual, Rocio Seniuk, Pamela Freiria, Javier Cotignola, Elba Vazquez, María Sol Ruiz, Geraldine Gueron, Ayelen Toro. Heme Oxygenase 1 Counteracts Pro-Stemness and Pro-Invasive Effect Triggered by Communication With Bone Progenitors in Prostate Cancer Cells [abstract]. In: Proceedings of the 11th Annual Symposium on Global Cancer Research; Closing the Research-to-Implementation Gap; 2023 Apr 4-6. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(6_Suppl):Abstract nr 43.

The suppression of cervical cancer ferroptosis by macrophages: The attenuation of ALOX15 in cancer cells by macrophages-derived exosomes.

Induction of cancer cell ferroptosis has been proposed as a potential treatment in several cancer types. Tumor-associated macrophages (TAMs) play a key role in promoting tumor malignant progression and therapy resistance. However, the roles and mechanisms of TAMs in regulating tumor ferroptosis is still unexplored and remains enigmatic. This study shows ferroptosis inducers has shown therapeutic outcomes in cervical cancer invitro and invivo. TAMs have been found to suppress cervical cancer cells ferroptosis. Mechanistically, macrophage-derived miRNA-660-5p packaged into exosomes are transported into cancer cells. In cancer cells, miRNA-660-5p attenuates ALOX15 expression to inhibit ferroptosis. Moreover, the upregulation of miRNA-660-5p in macrophages depends on autocrine IL4/IL13-activated STAT6 pathway. Importantly, in clinical cervical cancer cases, ALOX15 is negatively associated with macrophages infiltration, which also raises the possibility that macrophages reduce ALOX15 levels in cervical cancer. Moreover, both univariate and multivariate Cox analyses show ALOX15 expression is independent prognostic factor and positively associated with good prognosis in cervical cancer. Altogether, this study reveals the potential utility of targeting TAMs in ferroptosis-based treatment and ALOX15 as prognosis indicators for cervical cancer.

Inhibition of FGF23 is a therapeutic strategy to target hematopoietic stem cell niche defects in β-thalassemia.

Clinical evidence highlights a relationship between the blood and the bone, but the underlying mechanism linking these two tissues is not fully elucidated. Here, we used β-thalassemia as a model of congenital anemia with bone and bone marrow (BM) niche defects. We demonstrate that fibroblast growth factor 23 (FGF23) is increased in patients and mice with β-thalassemia because erythropoietin induces FGF23 overproduction in bone and BM erythroid cells via ERK1/2 and STAT5 pathways. We show that in vivo inhibition of FGF23 signaling by carboxyl-terminal FGF23 peptide is a safe and efficacious therapeutic strategy to rescue bone mineralization and deposition in mice with β-thalassemia, normalizing the expression of niche factors and restoring hematopoietic stem cell (HSC) function. FGF23 may thus represent a molecular link connecting anemia, bone, and the HSC niche. This study provides a translational approach to targeting bone defects and rescuing HSC niche interactions, with potential clinical relevance for improving HSC transplantation and gene therapy for hematopoietic disorders.

Norcantharidin potentiates sorafenib antitumor activity in hepatocellular carcinoma rat model through inhibiting IL-6/STAT3 pathway

In hepatocellular carcinoma (HCC), sorafenib (Sora) efficacy is limited by primary and/or acquired resistance. Emerging evidence shows that the inflammatory factor interleukin 6 (IL-6) plays a role in Sora resistance. Norcantharidin (NCTD), a derivative of cantharidine, was identified as a potent IL-6 inhibitor. Thus, in this study, we evaluated NCTD ability to improve the Sora efficacy in HCC and its underlying molecular mechanisms. Male Sprague Dawely rats were administered NCTD (0.1 mg/kg/day; orally) or Sora (10 mg/kg day; orally) or combination for 6 weeks after HCC induction using thioacetamide (200 mg/kg; ip; 2 times/wk) for 16 weeks. Our results showed that NCTD greatly enhanced Sora activity against HCC and potentiated Sora-induced oxidative stress. NCTD enhanced Sora-induced tumor immunity reactivation by decreasing both fibrinogen-like protein 1 level and increasing both tumor necrosis factor-α gene expression along with CD8+ T cells number. Also, NCTD augmented Sora attenuation activity against TAA-induced angiogenesis and metastasis by decreasing VEGFA, HIF-1α, serum lactate dehydrogenase enzyme, and vimentin levels. The combined use of NCTD/Sora suppressed drug resistance and stemness by downregulating ATP-binding cassette subfamily G member 2, neurogenic locus notch homolog protein, spalt-like transcription factor 4, and CD133. NCTD boosted Sora antiproliferative and apoptotic activities by decreasing Ccnd1 and BCL2 expressions along with increasing BAX and caspase-3 expressions. To our knowledge, this study represents the first study providing evidence for the potential novel therapeutic use of NCTD/Sora combination for HCC. Moreover, no previous studies have reported the effect of NCTD on FGL1.

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.