Interleukin-9 protects from microglia- and TNF-mediated synaptotoxicity in experimental multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a progressive neurodegenerative disease of the central nervous system characterized by inflammation-driven synaptic abnormalities. Interleukin-9 (IL-9) is emerging as a pleiotropic cytokine involved in MS pathophysiology. METHODS: Through biochemical, immunohistochemical, and electrophysiological experiments, we investigated the effects of both peripheral and central administration of IL-9 on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. RESULTS: We demonstrated that both systemic and local administration of IL-9 significantly improved clinical disability, reduced neuroinflammation, and mitigated synaptic damage in EAE. The results unveil an unrecognized central effect of IL-9 against microglia- and TNF-mediated neuronal excitotoxicity. Two main mechanisms emerged: first, IL-9 modulated microglial inflammatory activity by enhancing the expression of the triggering receptor expressed on myeloid cells-2 (TREM2) and reducing TNF release. Second, IL-9 suppressed neuronal TNF signaling, thereby blocking its synaptotoxic effects. CONCLUSIONS: The data presented in this work highlight IL-9 as a critical neuroprotective molecule capable of interfering with inflammatory synaptopathy in EAE. These findings open new avenues for treatments targeting the neurodegenerative damage associated with MS, as well as other inflammatory and neurodegenerative disorders of the central nervous system.

The Exposure to Lead (Pb) Exacerbates Immunological Abnormalities in BTBR T+ Itpr3tf/J Mice through the Regulation of Signaling Pathways Relevant to T Cells.

Autism spectrum disorder (ASD) is a common neurodevelopmental illness characterized by abnormal social interactions, communication difficulties, and repetitive and limited behaviors or interests. The BTBR T+ Itpr3tf/J (BTBR) mice have been used extensively to research the ASD-like phenotype. Lead (Pb) is a hazardous chemical linked to organ damage in the human body. It is regarded as one of the most common metal exposure sources and has been connected to the development of neurological abnormalities. We used flow cytometry to investigate the molecular mechanism behind the effect of Pb exposure on subsets of CD4+ T cells in the spleen expressing IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, AhR, IL-10, and Foxp3. Furthermore, using RT-PCR, we studied the effect of Pb on the expression of numerous genes in brain tissue, including IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, AhR, IL-10, and Foxp3. Pb exposure increased the population of CD4+IFN-γ+, CD4+T-bet+, CD4+STAT1+, CD4+STAT4+, CD4+IL-9+, CD4+IRF4+, CD4+IL-22+, and CD4+AhR+ cells in BTBR mice. In contrast, CD4+IL-10+ and CD4+Foxp3+ cells were downregulated in the spleen cells of Pb-exposed BTBR mice compared to those treated with vehicle. Furthermore, Pb exposure led to a significant increase in IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, and AhR mRNA expression in BTBR mice. In contrast, IL-10 and Foxp3 mRNA expression was significantly lower in those treated with the vehicle. Our data suggest that Pb exposure exacerbates immunological dysfunctions associated with ASD. These data imply that Pb exposure may increase the risk of ASD.

MAP kinase inhibitor PD98059 regulates Th1, Th9, Th17, and natural T regulatory cells in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis.

Experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis (MS), provides significant insights into the mechanisms that initiate and drive autoimmunity. MS is a chronic autoimmune disease of the central nervous system, characterized by inflammatory infiltration associated with demyelination. T lymphocyte cells play a crucial role in MS, whereas natural T regulatory (nTreg) cells prevent autoimmune inflammation by suppressing lymphocyte activity. This study sought to investigate the role of PD98059, a selective MAP kinase inhibitor, in Th1, Th9, Th17, and nTreg cells using the SJL/J mouse model of EAE. Following EAE development, the mice were intraperitoneally administered PD98059 (5 mg/kg for two weeks) daily. We evaluated the effects of PD98059 on Th1 (IFN-γ and T-bet), Th9 (IL-9 and IRF4), Th17 (IL-17A and RORγT), and nTreg (FoxP3 and Helios) cells in the spleen using flow cytometry. Moreover, we explored the effects of PD98059 on the IFN-γ, T-bet, IL-9, IRF4, IL-17A, RORγT, FoxP3, and Helios mRNA and protein levels in brain tissues using qRT-PCR and Western blot analyses. PD98059 treatment significantly decreased the proportion of CD4+IFN-γ+, CD4+T-bet+, CD4+IL-9+, CD4+IRF4+, CD4+IL-17A+, CD4+RORγT+, CD4+IL-17A+, and CD4+RORγT+ cells while increasing that of CD4+FoxP3+ and CD4+Helios+ cells. In addition, PD98059 administration decreased the mRNA and protein levels of IFN-γ, T-bet, IL-9, IRF4, IL-17A, and RORγT but increased those of FoxP3 and Helios in the brain tissue of EAE mice. Our findings suggest that PD98059 corrects immune dysfunction in EAE mice, which is concurrent with the modulation of multiple signaling pathways.

The Emerging Role of IL-9 in the Anticancer Effects of Anti-PD-1 Therapy.

PD-1 blockade rescues failing anticancer immune responses, resulting in durable remissions in some cancer patients. Cytokines such as IFNγ and IL-2 contribute to the anti-tumor effect of PD-1 blockade. IL-9 was identified over the last decade as a cytokine demonstrating a potent ability to harness the anticancer functions of innate and adaptive immune cells in mice. Recent translational investigations suggest that the anticancer activity of IL-9 also extends to some human cancers. Increased T cell-derived IL-9 was proposed to predict the response to anti-PD-1 therapy. Preclinical investigations accordingly revealed that IL-9 could synergize with anti-PD-1 therapy in eliciting anticancer responses. Here, we review the findings suggesting an important contribution of IL-9 in the efficacy of anti-PD-1 therapy and discuss their clinical relevance. We will also discuss the role of host factors like the microbiota and TGFß in the tumor microenvironment (TME) in the regulation of IL-9 secretion and anti-PD-1 treatment efficacy.

IL9 Polarizes Macrophages to M1 and Induces the Infiltration of Antitumor Immune Cells via MIP-1 and CXCR3 Chemokines.

Tumor-associated macrophages (TAM) are involved in tumor progression, metastasis, and immunosuppression. Because TAMs are highly plastic and could alter their phenotypes to proinflammatory M1 in response to environmental stimuli, reeducating TAMs has emerged as a promising approach to overcoming the challenges of solid cancer treatment. This study investigated the effect of IL9 on macrophage M1 polarization and verified its antitumor potential to retrain TAMs and promote chemokine secretion. We demonstrated that IL9 stimulated macrophage proliferation and polarized them toward the proinflammatory M1 phenotype in an IFNγ-dependent manner. Tumor-localized IL9 also polarized TAMs toward M1 in vivo and made them release CCL3/4 and CXCL9/10 to recruit antitumor immune cells, including T and natural killer cells, into the tumor microenvironment. Furthermore, peritoneal treatment with recombinant IL9 delayed the growth of macrophage-enriched B16F10 melanoma and 4T1 breast cancer in syngeneic mice, although IL9 treatment did not reduce tumor growth in the absence of macrophage enrichment. These results demonstrate the efficacy of IL9 in macrophage polarization to trigger antitumor immunity. Significance: These findings clarified the effect of IL9 on macrophage M1 polarization and verified its antitumor potential through retraining TAMs and chemokine secretion.

IL-9 stimulates an anti-tumor immune response and facilitates immune checkpoint blockade in the CMT167 mouse model.

OBJECTIVES: There is mounting evidence that interleukin-9 (IL-9) is associated with various cancers although its function in lung cancer remains elusive. This study aimed to elucidate the role(s) of IL-9 in lung cancer and the mechanisms involved. MATERIALS AND METHODS: Expression of IL-9 receptor (IL-9R) in two murine lung cancer cell lines: CMT167 and Lewis lung carcinoma (LLC) were assessed and syngeneic murine lung cancer models were established. Tumor growth, intratumoral immune responses and downstream signaling pathways in tumor-bearing mice were analyzed upon IL-9 treatment. Human lung cancer cell lines A549 and H1975 were included for in vitro validation. Synergistic effects and immune responses of IL-9 in combination with anti-PD-1 were studied. RESULTS: IL-9R expression was only detected in CMT167 but not LLC cells. IL-9 suppressed CMT167 tumor growth and enhanced anti-tumor T cell responses, both of which were absent in IL-9R-deficient LLC model and lost upon IL-9R knockdown in CMT167 model. In CMT167 tumors, while IL-9 increased CD4+ and CD8+ T cells and dendritic cells, the cytotoxic T subset was the key driver of IL-9-induced tumor suppression. Consistently, in CMT167 and A549 cells, IL-9/IL-9R signaling promoted MHC class I upregulation. Inhibition of ERK signaling abolished IL-9-mediated MHC class I upregulation in CMT167 cells. IL-9 induced expression of PD-1 and PD-L1 on CD8+ T lymphocytes and CMT167 cells respectively. Combined IL-9 treatment with PD-1 blockade further upregulated tumor-infiltrating CD8+ T cell frequencies and synergistically suppressed tumor growth in CMT167 model. CONCLUSION: IL-9 suppresses tumor growth by promoting tumor-derived MHC class I presentation and enhancing cytotoxic T cell immunity. Expression of IL-9R might be used as a biomarker for identification of potential target population susceptible to IL-9 treatment. Our study proposes IL-9 as a promising therapeutic immunomodulatory agent that can be used in combination with PD-1 blockade in lung cancer.

Interferon-γ Stimulates Interleukin-27 Derived from Dendritic Cells to Regulate Th9 Differentiation through STAT1/3 Pathway.

The initiation and progression of allergic asthma (AA) are associated with complex interactions between inflammation and immune response. Herein, we report the specific mechanisms underlying the molecular action of interferon (IFN)-γ in AA regulation. We speculated that IFN-γ inhibits Th9 differentiation by regulating the secretion of interleukin (IL)-27 from dendritic cells (DCs), thereby suppressing airway inflammation in asthma. We constructed a mouse model of ovalbumin-induced AA and overexpressed IFN-γ to evaluate the effect on the IL-27/Th9 axis via the in vitro effect of IFN-γ on IL-27 secretion by DCs and their influence on Th9 differentiation and asthmatic inflammation. IFN-γ overexpression reduced the proportion of Th9 cells and DCs and altered lung morphology and cytokine production in AA-induced mice, thus suppressing the AA phenotype. In addition, exogenous IFN-γ stimulation promoted the secretion of IL-27 and suppressed Th9 differentiation of CD4+ T cells via signal transducer and activator of transcription 1/3 (STAT1/3) signaling in a time-dependent manner. This study aimed to clarify the regulatory effect and mechanism of the IFN-γ/DCs/IL-27/Th9 axis on AA and provide novel insights for effective targeted treatment of asthma.

IL-27 overexpression alleviates inflammatory response in allergic asthma by inhibiting Th9 differentiation and regulating Th1/Th2 balance.

OBJECTIVE: To investigate the effect of IL-27 on Th9 differentiation and Th1/Th2 balance. METHODS: C57BL/6 (B6) mice were treated with ovalbumin to establish an allergic asthma (AA) model and subjected to IL-27 overexpression (OV) and empty vector (EV). Hematoxylin-eosin (HE) staining was performed to observe lung tissue inflammation. Flow cytometry was carried out to evaluate the percentage of Th9, Th1, and Th2 cells. The expression of IL-27, IL-27R, IL-9, T-bet, IFN-γ, and IgE was evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Western blot was conducted to observe the expression of pSTAT-1 and pSTAT-3. RESULTS: Compared with the Model group, the number of Th1 cells in the Model + OV group increased significantly (p < .05), while those of Th9 and Th2 cells decreased significantly (p < .05). The expression of IL-27, IL-27R, and IFN-γ in blood serum was increased (p < .05), and that of IL-9 and IgE was significantly decreased in the Model + OV group compared to the Model (p < .05). Western blot revealed that Model + OV exhibited lower expression of pSTAT-3 than that in the Model and Model + EV groups (p < .05), while pSTAT-1 expression was significantly increased (p < .05). Inflammatory infiltration in the Model + OV group was significantly reduced, and there was no significant difference between the Model and Model + EV groups. CONCLUSIONS: IL-27 OV inhibits Th9 differentiation and regulates the imbalance of Th1/Th2, thereby alleviating inflammatory response in AA. The findings suggest that IL-27 OV may be a potential strategy for clinical treatment of AA.

Investigation of the effect of IFN-γ/TNF-α-treated mesenchymal stem cells on Th9- and Treg cell-related parameters in a mouse model of ovalbumin-induced allergic asthma.

OBJECTIVE: Th9- and regulatory T (Treg) cells exert pro- and anti-allergic activity, respectively. Mesenchymal stem cell (MSC)-related immunomodulatory impacts can be enhanced by inflammatory cytokines. Here, the modulatory effects of IFN-γ/TNF-α-induced MSCs on Th9- and Treg cell-related parameters were investigated using an asthma model. METHODS: Allergic asthma was induced in BALB/c mice using sensitized and challenging with ovalbumin (OVA). The asthmatic groups were treated intraperitoneally with PBS, MSCs, IFN-γ-induced MSCs, TNF-α-induced MSCs and 'IFN-γ + TNF-α'-induced MSCs before the challenge phase. The mice were sacrificed 24 h after challenge. The serum IL-9 and IL-35 levels, as well as gene expression of IL-9, PU.1, IL-35-EBI3, and FOXP3 in the lung tissues were assessed using ELISA and real time-PCR, respectively. RESULTS: The differences of Th9 and Treg-related parameters were not significant between untreated asthmatic mice and those treated with non-induced MSCs. In comparison with untreated asthmatic group, treatment with IFN-γ-induced MSCs significantly reduced serum IL-9 levels, reduced lung expression of IL-9 and PU.1, while increasing serum IL-35 levels as well as lung expression of FOXP3; treatment with TNF-α-induced MSCs significantly reduced serum IL-9 levels as well as lung expression of IL-9, and treatment with 'IFN-γ + TNF-α'-induced MSCs, significantly modulated all investigated Th9 and Treg-related parameters. In comparison to mice treated with non-induced MSCs, serum IL-9 levels were remarkably decreased in mice treated with IFN-γ-induced and 'IFN-γ + TNF-α'-induced MSCs. CONCLUSIONS: IFN-γ-and 'IFN-γ + TNF-α' treated MSCs exerted almost comparable impacts, but were more efficient than TNF-α-exposed MSCs. Thus, IFN-γ alone can be sufficient to promote immunomodulatory effects of MSCs.

TRAF6 signaling pathway in T cells regulates anti-tumor immunity through the activation of tumor specific Th9 cells and CTLs.

CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ helper T (Th) cells play a critical role in protective immune responses to tumor cells. Particularly, Th9 cells exert anti-tumor activity by producing IL-9. TNF receptor (TNFR)-associated factor 6 (TRAF6) is an adaptor protein that mediates the signals from both the TNFR superfamily and Toll-like receptors (TLRs). We have previously reported that T cell-specific TRAF6-deficent (TRAF6ΔT) mice spontaneously developed systemic inflammatory diseases. However, the physiological role of TRAF6 in T cells in controlling anti-tumor immune responses remains largely unclear. Here, we found that tumor formation of syngeneic colon cancer cells inoculated in TRAF6ΔT mice was accelerated compared to that in control mice. Although TRAF6-deficient naïve T cells showed enhanced differentiation of Th9 cells in vitro, these T cells produced lower amounts of IL-9 in response to a specific antigen. Moreover, CD4+ tumor-infiltrating lymphocytes (TILs) in tumor-bearing TRAF6ΔT mice expressed lower levels of IL-9 than those in WT mice. Importantly, administration of recombinant IL-9 (rIL-9) strongly suppressed tumor progression in TRAF6ΔT mice. Furthermore, expression levels of the T-box transcription factor Eomesodermin (Eomes) and its target molecules IFN-γ, granzyme B and perforin, as well as cytotoxic activity, were reduced in TRAF6-deficient CD8+ T cells in vitro. TRAF6-deficient T cells were found to express significantly increased levels of immune checkpoint molecules, CTLA-4 and PD-1 on the cell surface. These results demonstrate that the TRAF6 signaling pathway in T cells regulates anti-tumor immunity through the activation of tumor specific Th9 cells and CTLs in a tumor microenvironment.

Antibody-based delivery of interleukin-9 to neovascular structures: Therapeutic evaluation in cancer and arthritis.

Interleukin-9 is a cytokine with multiple functions, including the ability to activate group 2 innate lymphoid cells, which has been postulated to be therapeutically active in mouse models of arthritis. Similarly, interleukin-9 has been suggested to play an important role in tumor immunity. Here, we describe the cloning, expression, and characterization of three fusion proteins based on murine interleukin-9 and the F8 antibody, specific to the alternatively spliced EDA domain of fibronectin. EDA is strongly expressed in cancer and in various arthritic conditions, while being undetectable in the majority of healthy organs. Interleukin-9-based fusion proteins with an irrelevant antibody specific to hen egg lysozyme served as negative control in our study. The fusion proteins were characterized by quantitative biodistribution analysis in tumor-bearing mice using radioiodinated protein preparations. The highest tumor uptake and best tumor:organ ratios were observed for a format, in which the interleukin-9 moiety was flanked by two units of the F8 antibody in single-chain Fv format. Biological activity of interleukin-9 was retained when the payload was fused to antibodies. However, the targeted delivery of interleukin-9 to the disease site resulted in a modest anti-tumor activity in three different murine models of cancer (K1735M2, CT26, and F9), while no therapeutic benefit was observed in a collagen induced model of arthritis. Collectively, these results confirm the possibility to deliver interleukin-9 to the site of disease but cast doubts about the alleged therapeutic activity of this cytokine in cancer and arthritis, which has been postulated in previous publications.

Inhibition of Histone H3K27 Acetylation Orchestrates Interleukin-9-Mediated and Plays an Anti-Inflammatory Role in Cisplatin-Induced Acute Kidney Injury.

Nephrotoxicity is a major side effect of cisplatin (CP)- and platinum-related chemotherapy, and inflammation contributes to disease pathogenesis. Interleukin-9 (IL-9) is a pleiotropic cytokine associated with inflammation. Here, we investigated the key role of IL-9 as a regulator of protective mechanisms in CP-induced acute kidney injury (AKI). We observed that IL-9 was decreased not only in a CP-induced AKI mouse model but also in THP-1 and RAW264.7 cell lines. Seventy-two hours post-CP injection, renal dysfunction and tubule injury were significantly attenuated in IL-9 overexpression adeno-associated virus 9 (AAV9)-treated mice. The levels of serum urea, serum creatinine, kidney injury molecule-1 (KIM-1), and histological damage were partially diminished following treatment with IL-9. The renoprotective effects of IL-9 may be attributed to the regulation of cytokines, and we found that IL-9 acted on macrophages in a regulatory manner, promoting an anti-inflammatory phenotype. Furthermore, IL-9 enhanced the suppression of macrophage-driven renal inflammation. Inhibition of H3K27 acetylation orchestrated IL-9-mediated renoprotection in CP-induced AKI. Thus, our findings indicate novel and potent anti-inflammatory properties of IL-9 that confer preservation of kidney function and structure in CP-induced AKI, which may counteract kidney disease procession.

Interleukin 9 prevents immune thrombocytopenia in mice via JAK/STAT5 signaling.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by autoimmune-mediated platelet destruction and impaired platelet production, which can lead to an increased risk of bleeding. The clinical management of ITP currently remains a challenge for hematologists. We explored the role of interleukin-9 (IL-9) in the treatment of CD41-induced ITP, and investigated its underlying mechanisms in a CD41-induced ITP mouse model. IL-9 treatment increased the numbers of mature megakaryocytes (CD41+CD42d+) and CD41+Sca-1+ cells in the bone marrow in these model mice, while IL-9 receptor (IL-9R) small interfering RNA (siRNA) inhibited the process. Moreover, phosphorylated signal transducer and activator of transcription 5 (STAT5), as a downstream molecule of IL-9R, was increased after IL-9 treatment. We next investigated the source of IL-9 in bone marrow, osteoblasts produced the highest level of IL-9. These results confirmed that IL-9 could prevent CD41-induced ITP in BALB/c mice by regulating osteoblasts and activating IL-9R/STAT5 signaling in megakaryocytes, thus providing further evidence for IL-9 as a promising therapeutic agent for the treatment of ITP.

Targeted blockade of interleukin 9 inhibits tumor growth in murine model of pancreatic cancer.

BACKGROUND: Interleukin 9 (IL-9) has been implicated in the pathogenesis of several tumor types, but the role of anti-IL-9 in pancreatic cancer remains unclear. OBJECTIVES: We aimed to explore the mechanism and effects of blockading IL-9 in a pancreatic cancer mouse model. MATERIAL AND METHODS: Panc02 cells were injected subcutaneously into mice to establish a mouse model. The mice were randomly categorized into 3 groups - the control group, the immunoglobulin G (IgG) group and the anti-IL-9 group - corresponding to intravenous tail injection of phosphate-buffered saline (PBS), IgG isotype antibody and anti-IL-9 antibody, respectively. Then, the expression of IL-9, interleukin-9 receptor (IL-9r), Janus kinase 1 (Jak1), Jak3, and signal transducer and activator of transcription 3 (Stat3) mRNA was tested with quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Interleukin 9 in the tumor tissue was detected using enzyme-linked immunosorbent assay (ELISA). Western blotting and immunocytochemistry were performed to detect STAT3 and phosphorylation signal transducers and activators of transcription-3 (pSTAT3). Matrix metalloproteinase 2 (MMP2), MMP9 and vascular endothelial growth factor (VEGF) levels were assessed using immunocytochemistry. RESULTS: Tumor weight in the anti-IL-9 group was significantly lower than in the other groups (p < 0.05). There was a remarkable survival benefit in the anti-IL-9 group compared to the other groups (p < 0.05). The concentration of IL-9 in tumor tissue was significantly downregulated in the anti-IL-9-treated mice (p < 0.05). The expression of Jak1 and Jak3 mRNA and pSTAT3, MMP2 and MMP9 proteins in the anti-IL-9 group was lower than that of the PBS or IgG groups (p < 0.05), but the STAT3 and VEGF protein levels showed no significant difference (p < 0.05). CONCLUSIONS: Anti-IL-9 antibody could effectively restrain the growth of pancreatic cancer in mice, and this effect may partly occur by blocking the STAT3 pathway.

Neutralization of interleukin-9 ameliorates experimental stroke by repairing the blood-brain barrier via down-regulation of astrocyte-derived vascular endothelial growth factor-A.

Astrocytes mediate the destruction of the blood-brain barrier (BBB) during ischemic stroke (IS). IL-9 is a pleiotropic cytokine that we previously found to be highly expressed in peripheral blood mononuclear cells from patients with IS, and the presence of IL-9 receptors on astrocytes has been reported in the literature. Here, we detected the effect of IL-9 on astrocytes using an anti-IL-9-neutralizing antibody to treat rats with experimental stroke. Supernatants from astrocytes treated with or without oxygen-glucose deprivation and/or IL-9 were incubated with bEnd.3 cell monolayers after blocking the IL-9 receptor on the endothelium. Immunofluorescence staining and Western blot analyses were conducted to observe the change in tight junction proteins (TJPs) in bEnd.3 cells as well as the level of VEGF-A and possible signal pathways in astrocytes. We also applied middle cerebral artery occlusion (MCAO) models to determine the effect of anti-IL-9-neutralizing antibodies on IS. As a result, astrocyte-conditioned medium treated with IL-9 aggravated the disruption of the BBB accomplished by the degradation of TJPs in endothelial cells. In addition, IL-9 increased the level of VEGF-A in astrocytes, and blocking the effect of VEGF-A reversed the breakdown of the BBB. In the MCAO model, anti-IL-9-neutralizing antibody reduced the infarct volume and BBB destruction. Mechanistically, the anti-IL-9-neutralizing antibody repaired the damaged TJPs (zonula occludens 1, occludin, and claudin-5) and induced a decrease in VEGF-A expression in ischemic lateral brain tissue. In contrast, a local injection of recombinant murine IL-9 to the brain resulted in a marked up-regulation of VEGF-A in the striatum. In conclusion, anti-IL-9-neutralizing antibody can reduce the severity of IS partially by alleviating the destruction of the BBB via down-regulation of astrocyte-derived VEGF-A. This finding suggests that targeting IL-9 or VEGF-A could provide a new direction for the treatment of IS.-Tan, S., Shan, Y., Lin, Y., Liao, S., Zhang, B., Zeng, Q., Wang, Y., Deng, Z., Chen, C., Hu, X., Peng, L., Qiu, W., Lu, Z. Neutralization of IL-9 ameliorates experimental stroke by repairing the blood-brain barrier via down-regulation of astrocyte-derived vascular endothelial growth factor-A.

Establishment and Characterization of a Murine Mucosal Mast Cell Culture Model.

Accumulating evidence suggests that mast cells play critical roles in disruption and maintenance of intestinal homeostasis, although it remains unknown how they affect the local microenvironment. Interleukin-9 (IL-9) was found to play critical roles in intestinal mast cell accumulation induced in various pathological conditions, such as parasite infection and oral allergen-induced anaphylaxis. Newly recruited intestinal mast cells trigger inflammatory responses and damage epithelial integrity through release of a wide variety of mediators including mast cell proteases. We established a novel culture model (IL-9-modified mast cells, MCs/IL-9), in which murine IL-3-dependent bone-marrow-derived cultured mast cells (BMMCs) were further cultured in the presence of stem cell factor and IL-9. In MCs/IL-9, drastic upregulation of Mcpt1 and Mcpt2 was found. Although histamine storage and tryptase activity were significantly downregulated in the presence of SCF and IL-9, this was entirely reversed when mast cells were cocultured with a murine fibroblastic cell line, Swiss 3T3. MCs/IL-9 underwent degranulation upon IgE-mediated antigen stimulation, which was found to less sensitive to lower concentrations of IgE in comparison with BMMCs. This model might be useful for investigation of the spatiotemporal changes of newly recruited intestinal mast cells.

Analysis of Long Noncoding RNA and mRNA Expression Profiles in IL-9-Activated Astrocytes and EAE Mice.

BACKGROUND/AIMS: Multiple sclerosis (MS) is an autoimmune disease in the central nervous system associated with demyelination and axonal injury. Astrocyte activation is involved in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. This study was designed to find potential lncRNAs in EAE mice and activated astrocytes. METHODS: we performed microarray analysis of lncRNAs from the brain tissues of EAE mice and primary mouse astrocytes treated with IL-9(50 ng/ml). 12 lncRNAs were validated through real-time PCR. Gene ontology and KEGG pathway analysis were applied to explore the potential functions of lncRNAs. RESULTS: Differentially expressed 3300 lncRNAs and 3250 mRNAs were in the brain tissues of EAE mice, and 3748 lncRNAs and 3332 mRNAs were in activated astrocytes. Notably, there were 2 co-up-regulated lncRNAs and 3 co-down-regulated lncRNAs both in the brain tissues of EAE mice and in activated astrocytes, including Gm14005, Gm12478, mouselincRNA1117, AK080435, and mouselincRNA0681, which regulate the ER calcium flux kinetics, zinc finger protein and cell apoptosis. Similarly, there were 7 mRNAs co-up-regulated and 2 mRNAs co-down-regulated both in vivo and in vitro. Gene ontology and KEGG pathway analysis showed that the biological functions of differentially expressed mRNAs were associated with metabolism, development and inflammation. The results of realtime PCR validation were consistent with the data from the microarrays. CONCLUSIONS: Our data uncovered the expression profiles of lncRNAs and mRNAs in vivo and in vitro, which may help delineate the mechanisms of astrocyte activation during MS/EAE process.

[Interleukin-9 promotes pancreatic cancer cell proliferation and migration via activation of STAT3 pathway].

Objective To investigate the impact of interleukin-9 (IL-9) on proliferation, invasion and migration of pancreatic cancer cells and its mechanism. Methods PANC-1 cells were cultured in vitro and treated with IL-9 at different concentrations (5, 10, 20 ng/mL) for 24 hours. The level of IL-9R mRNA was analyzed by quantitative real-time PCR. CCK-8 assay was used to test the proliferation of the cells and flow cytometry to detect the cell apoptosis. TranswellTM assay was employed to determine the invasion and migration of PANC-1 cells. Western blotting was used to detect the STAT3 and p-STAT3 protein expression levels. After PANC-1 cells were treated with different concentrations of STAT3 pathway inhibitor AG490, followed by IL-9 treatment, the STAT3 and p-STAT3 protein expressions, as well as the proliferation of the cells were detected again. Results The level of IL-9R mRNA and the proliferation rate of PANC-1 cells were enhanced with the increase of IL-9 concentration, and the capacities of cell invasion and migration were promoted significantly. The relative protein expression of p-STAT3 increased greatly in PANC-1 cells after the treatment of IL-9, but STAT3 were not changed significantly compared with the ones without IL-9 treatment. The proliferation-promoting effect of IL-9 on AG490-pretreated PANC-1 cells was induced, and the p-STAT3 protein expression level was notably inhibited. Conclusion The activation of STAT3 pathway is strongly associated with the process that IL-9 mediates the promotion of proliferation, invasion and migration in pancreatic cancer cells.

Germinal-center development of memory B cells driven by IL-9 from follicular helper T cells.

Germinal centers (GCs) support high-affinity, long-lived humoral immunity. How memory B cells develop in GCs is not clear. Through the use of a cell-cycle-reporting system, we identified GC-derived memory precursor cells (GC-MP cells) that had quit cycling and reached G0 phase while in the GC, exhibited memory-associated phenotypes with signs of affinity maturation and localized toward the GC border. After being transferred into adoptive hosts, GC-MP cells reconstituted a secondary response like genuine memory B cells. GC-MP cells expressed the interleukin 9 (IL-9) receptor and responded to IL-9. Acute treatment with IL-9 or antibody to IL-9 accelerated or retarded the positioning of GC-MP cells toward the GC edge and exit from the GC, and enhanced or inhibited the development of memory B cells, which required B cell-intrinsic responsiveness to IL-9. Follicular helper T cells (TFH cells) produced IL-9, and deletion of IL-9 from T cells or, more specifically, from GC TFH cells led to impaired memory formation of B cells. Therefore, the GC development of memory B cells is promoted by TFH cell-derived IL-9.

Exacerbation of oxygen-glucose deprivation-induced blood-brain barrier disruption: potential pathogenic role of interleukin-9 in ischemic stroke.

Interleukin (IL)-9 exerts a variety of functions in autoimmune diseases. However, its role in ischemic brain injury remains unknown. The present study explored the biological effects of IL-9 in ischemic stroke (IS). We recruited 42 patients newly diagnosed with IS and 22 age- and sex-matched healthy controls. The expression levels of IL-9 and percentages of IL-9-producing T cells, including CD3+CD4+IL-9+ and CD3+CD8+IL-9+ cells, were determined in peripheral blood mononuclear cells (PBMCs) obtained from patients and control individuals. We also investigated the effects of IL-9 on the blood-brain barrier (BBB) following oxygen-glucose deprivation (OGD) and the potential downstream signaling pathways. We found that patients with IS had higher IL-9 expression levels and increased percentages of IL-9-producing T cells in their PBMCs. The percentages of CD3+CD4+IL-9+ and CD3+CD8+IL-9+ T cells were positively correlated with the severity of illness. In in vitro experiments using bEnd.3 cells, exogenously administered IL-9 exacerbated the loss of tight junction proteins (TJPs) in cells subjected to OGD plus reoxygenation (RO). This effect was mediated via activation of IL-9 receptors, which increased the level of endothelial nitric oxide synthase (eNOS), as well as through up-regulated phosphorylation of signal transducer and activator of transcription 1 and 3 and down-regulated phosphorylated protein kinase B/phosphorylated phosphatidylinositol 3-kinase signaling. These results indicate that IL-9 has a destructive effect on the BBB following OGD, at least in part by inducing eNOS production, and raise the possibility of targetting IL-9 for therapeutic intervention in IS.