IL-2 family cytokines IL-9 and IL-21 differentially regulate innate and adaptive type 2 immunity in asthma.

BACKGROUND: Asthma is often accompanied by type 2 immunity rich in IL-4, IL-5, and IL-13 cytokines produced by TH2 lymphocytes or type 2 innate lymphoid cells (ILC2s). IL-2 family cytokines play a key role in the differentiation, homeostasis, and effector function of innate and adaptive lymphocytes. OBJECTIVE: IL-9 and IL-21 boost activation and proliferation of TH2 and ILC2s, but the relative importance and potential synergism between these γ common chain cytokines are currently unknown. METHODS: Using newly generated antibodies, we inhibited IL-9 and IL-21 alone or in combination in various murine models of asthma. In a translational approach using segmental allergen challenge, we recently described elevated IL-9 levels in human subjects with allergic asthma compared with nonasthmatic controls. Here, we also measured IL-21 in both groups. RESULTS: IL-9 played a central role in controlling innate IL-33-induced lung inflammation by promoting proliferation and activation of ILC2s in an IL-21-independent manner. Conversely, chronic house dust mite-induced airway inflammation, mainly driven by adaptive immunity, was solely dependent on IL-21, which controlled TH2 activation, eosinophilia, total serum IgE, and formation of tertiary lymphoid structures. In a model of innate on adaptive immunity driven by papain allergen, a clear synergy was found between both pathways, as combined anti-IL-9 or anti-IL-21 blockade was superior in reducing key asthma features. In human bronchoalveolar lavage samples we measured elevated IL-21 protein within the allergic asthmatic group compared with the allergic control group. We also found increased IL21R transcripts and predicted IL-21 ligand activity in various disease-associated cell subsets. CONCLUSIONS: IL-9 and IL-21 play important and nonredundant roles in allergic asthma by boosting ILC2s and TH2 cells, revealing a dual IL-9 and IL-21 targeting strategy as a new and testable approach.

Evolution of pathologic B-cell subsets and serum environment-specific sIgEs in patients with atopic dermatitis and controls, from infancy to adulthood.

BACKGROUND: While B-cells have historically been implicated in allergy development, a growing body of evidence supports their role in atopic dermatitis (AD). B-cell differentiation across ages in AD, and its relation to disease severity scores, has not been well defined. OBJECTIVE: To compare the frequency of B-cell subsets in blood of 0-5, 6-11, 12-17, and ≥18 years old patients with AD versus age-matched controls. METHODS: Flow cytometry was used to measure B-cell subset frequencies in the blood of 27 infants, 17 children, 11 adolescents, and 31 adults with moderate-to-severe AD and age-matched controls. IgD/CD27 and CD24/CD38 core gating systems and an 11-color flow cytometry panel were used to determine frequencies of circulating B-cell subsets. Serum total and allergen-specific IgE (sIgEs) levels were measured using ImmunoCAP®. RESULTS: Adolescents with AD had lower frequencies of major B-cells subsets (p < .03). CD23 expression increased with age and was higher in AD compared to controls across all age groups (p < .04). In AD patients, multiple positive correlations were observed between IL-17-producing T-cells and B-cell subsets, most significantly non-switched memory (NSM) B-cells (r = .41, p = .0005). AD severity positively correlated with a list of B-cell subsets (p < .05). IL-9 levels gradually increased during childhood, reaching a peak in adolescence, paralleling allergen sensitization, particularly in severe AD. Principal component analysis of the aggregated environmental sIgE data showed that while controls across all ages tightly clustered together, adolescents with AD demonstrated distinct clustering patterns relative to controls. CONCLUSIONS: Multiple correlations between B-cells and T-cells, as well as disease severity measures, suggest a complex interplay of immune pathways in AD. Unique B-cell signature during adolescence, with concurrent allergen sensitization and IL-9 surge, point to a potentially wider window of opportunity to implement interventions that may prevent the progression of the atopic march.

Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Axis in CD4+ T Cells.

BACKGROUND: Perivascular fibrosis, characterized by increased amount of connective tissue around vessels, is a hallmark for vascular disease. Ang II (angiotensin II) contributes to vascular disease and end-organ damage via promoting T-cell activation. Despite recent data suggesting the role of T cells in the progression of perivascular fibrosis, the underlying mechanisms are poorly understood. METHODS: TF (transcription factor) profiling was performed in peripheral blood mononuclear cells of hypertensive patients. CD4-targeted KLF10 (Kruppel like factor 10)-deficient (Klf10fl/flCD4Cre+; [TKO]) and CD4-Cre (Klf10+/+CD4Cre+; [Cre]) control mice were subjected to Ang II infusion. End point characterization included cardiac echocardiography, aortic imaging, multiorgan histology, flow cytometry, cytokine analysis, aorta and fibroblast transcriptomic analysis, and aortic single-cell RNA-sequencing. RESULTS: TF profiling identified increased KLF10 expression in hypertensive human subjects and in CD4+ T cells in Ang II-treated mice. TKO mice showed enhanced perivascular fibrosis, but not interstitial fibrosis, in aorta, heart, and kidney in response to Ang II, accompanied by alterations in global longitudinal strain, arterial stiffness, and kidney function compared with Cre control mice. However, blood pressure was unchanged between the 2 groups. Mechanistically, KLF10 bound to the IL (interleukin)-9 promoter and interacted with HDAC1 (histone deacetylase 1) inhibit IL-9 transcription. Increased IL-9 in TKO mice induced fibroblast intracellular calcium mobilization, fibroblast activation, and differentiation and increased production of collagen and extracellular matrix, thereby promoting the progression of perivascular fibrosis and impairing target organ function. Remarkably, injection of anti-IL9 antibodies reversed perivascular fibrosis in Ang II-infused TKO mice and C57BL/6 mice. Single-cell RNA-sequencing revealed fibroblast heterogeneity with activated signatures associated with robust ECM (extracellular matrix) and perivascular fibrosis in Ang II-treated TKO mice. CONCLUSIONS: CD4+ T cell deficiency of Klf10 exacerbated perivascular fibrosis and multi-organ dysfunction in response to Ang II via upregulation of IL-9. Klf10 or IL-9 in T cells might represent novel therapeutic targets for treatment of vascular or fibrotic diseases.

Structure-activity relationship studies of pyrogallol as a calcineurin/NFAT signaling suppressor.

Previously, we have shown that pyrogallol alleviated nasal symptoms and suppressed IL-9 gene up-regulation in allergy model rats by inhibiting calcineurin/NFAT signaling. As pyrogallol has antioxidative activity, it may be responsible for inhibiting calcineurin/NFAT signaling-mediated IL-9 gene expression. However, the relationship between antioxidative activity and suppression of IL-9 gene expression has not been elucidated yet. Here, we conducted the structure-activity relationship studies of pyrogallol and its structurally related compounds to understand the mechanism of IL-9 gene suppression by pyrogallol. 2, 2-Diphenyl-1-picrylhydrazyl radical scavenging assay showed that the antioxidative activity of catechol, resorcinol, phloroglucinol, and gallic acid is 60.1%, 10.4%, 18.8%, and 113.5% of pyrogallol, respectively. Catechol, resorcinol, and phloroglucinol did not suppress NFAT dephosphorylation. Gallic acid suppressed dephosphorylation of NFAT. Gallic acid also suppressed ionomycin-induced up-regulation of IL-9 gene expression with the IC50 value of 82.6 µM. However, catechol, resorcinol and phloroglucinol showed no suppressive activity. In addition, using gallic acid-immobilized beads, we isolated and identified Poly(U)-binding-splicing factor 60 (PUF60) as a pyrogallol binding protein. These results suggest that the antioxidative activity of pyrogallol is not likely to be the mechanism of IL-9 gene suppression. Data also suggest that PUF60 is one of its target molecules responsible for the suppression of calcineurin/NFAT signaling by pyrogallol.

Bevacizumab induces ferroptosis and enhances CD8+ T cell immune activity in liver cancer via modulating HAT1 and increasing IL-9.

Bevacizumab is a recombinant humanized monoclonal immunoglobulin (Ig) G1 antibody of VEGF, and inhibits angiogenesis and tumor growth in hepatocellular carcinoma (HCC). Ferroptosis, a new form of regulated cell death function independently of the apoptotic machinery, has been accepted as an attractive target for pharmacological intervention; the ferroptosis pathway can enhance cell immune activity of anti-PD1 immunotherapy in HCC. In this study we investigated whether and how bevacizumab regulated ferroptosis and immune activity in liver cancer. Firstly, we performed RNA-sequencing in bevacizumab-treated human liver cancer cell line HepG2 cells, and found that bevacizumab significantly altered the expression of a number of genes including VEGF, PI3K, HAT1, SLC7A11 and IL-9 in liver cancer, bevacizumab upregulated 37 ferroptosis-related drivers, and downregulated 17 ferroptosis-related suppressors in particular. We demonstrated that bevacizumab triggered ferroptosis in liver cancer cells by driving VEGF/PI3K/HAT1/SLC7A11 axis. Clinical data confirmed that the expression levels of VEGF were positively associated with those of PI3K, HAT1 and SLC7A11 in HCC tissues. Meanwhile, we found that bevacizumab enhanced immune cell activity in tumor immune-microenvironment. We identified that HAT1 up-regulated miR-143 targeting IL-9 mRNA 3'UTR in liver cancer cells; bevacizumab treatment resulted in the increase of IL-9 levels and its secretion via VEGF/PI3K/HAT1/miR-143/IL-9 axis, which led to the inhibition of tumor growth in vivo through increasing the release of IL-2 and Granzyme B from activated CD8+ T cells. We conclude that in addition to inhibiting angiogenesis, bevacizumab induces ferroptosis and enhances CD8+ T cell immune activity in liver cancer. This study provides new insight into the mechanisms by which bevacizumab synergistically modulates ferroptosis and CD8+ T cell immune activity in liver cancer.

Physical activity and circulating inflammatory markers and cytokines during pregnancy: A population-based cohort study.

INTRODUCTION: Physical activity (PA) during pregnancy has numerous benefits, which may be mediated via effects on the immune system. However, supportive evidence is inconsistent and is mainly from studies in high-risk groups. We estimated the effect of PA during pregnancy on systemic inflammatory markers and cytokines in mothers recruited in the Barwon infant study. MATERIAL AND METHODS: The Barwon infant study is a prebirth cohort of 1064 mothers recruited in the Barwon Region of Victoria, Australia. Participants reported their previous week's PA at their 28-week antenatal appointment using the International PA Questionnaire. Women were grouped into low, moderate, and high PA categories based on daily duration and weekly frequency of walking, moderate- or vigorous-intensity PA. Women reporting moderate levels of PA, consistent with current recommendations, served as the comparison group. Markers of systemic inflammation, high-sensitivity C-reactive protein (hsCRP), glycoprotein acetyls (GlycA), and 17 cytokines were measured at 28 weeks gestation and log transformed as appropriate. Regression analyses adjusted for maternal smoking, gestational diabetes mellitus, prepregnancy BMI, and household size were performed. RESULTS: Compared to women in the moderate group (n = 371, 42%), women reporting low PA (n = 436, 50%) had 10.1% higher hsCRP (95% CI (3.7% to 16.6%), p < 0.01) while women in high PA (n = 76, 9%) had a 14% higher hsCRP (95% CI (3.1% to 24.8%), p = 0.01). Women in the high PA category had higher interleukin (IL)-4 (q = 0.03) and IL-9 (q = 0.03) levels compared to those in moderate category. Each vigorous MET minute/week was associated with lower GlycA (ß = -0.004, 95% CI (-0.044 to 0.035); p = 0.03). CONCLUSIONS: Low and high PA are each associated with higher hsCRP than moderate PA, suggesting that undertaking the recommended moderate PA during pregnancy decreases systemic inflammation. High PA affects T cell-associated cytokines during pregnancy. Evidence from our study suggests that PA can modulate the immune responses during pregnancy. Studies are now required to assess whether PA during pregnancy impacts maternal and infant clinical outcomes by modifying inflammatory responses.

Interleukin-9 promotes EMT-mediated PM2.5-induced pulmonary fibrosis by activating the STAT3 pathway.

This study investigated the impact of PM2.5 on promoting EMT in PM2.5-induced pulmonary fibrosis (PF) development and explored molecular mechanisms of the IL-9/STAT3/Snail/TWIST1 signaling pathway in PF owing to PM2.5. Four groups of male SD rats were formed: control (0 mg/kg.bw), low (1 mg/kg.bw), medium (5 mg/kg.bw), and high-dose (25 mg/kg.bw) PM2.5 groups. Experimental rats were subjected to PM2.5 exposure via intratracheal instillation, given once weekly for 16 weeks. 24 h after the final exposure, blood, BALF, and lung tissues were collected. Pulmonary epithelial cells underwent cultivation and exposure to varying PM2.5 concentrations with/without inhibitors for 24 h, after which total protein was extracted for relevant protein assays. The findings demonstrated that PM2.5 damaged lung tissue to different degrees and led to PF in rats. Rats subjected to PM2.5 exposure exhibited elevated concentrations of IL-9 protein in both serum and BALF, and elevated levels of IL-9 and its receptor, IL-9R, in lung tissues, compared to control counterparts. Furthermore, PM2.5-exposed groups demonstrated significantly augmented protein levels of p-STAT3, Snail, TWIST1, Vimentin, COL-I, and α-SMA, while displaying notably diminished levels of E-Cadherin compared to control group. The same findings were observed in PM2.5-treated cells. In BEAS-2B cells co-treated with Stattic (STAT3 inhibitor) and PM2.5, the opposite results occurred. Similar results were obtained for cells co-treated with IL-9-neutralizing antibody and PM2.5. Our findings suggest PM2.5 mediates PF development by promoting IL-9 expression, leading to STAT3 phosphorylation and upregulation of Snail and TWIST1 expression, triggering EMT occurrence and progression in lung epithelial cells.

Bioenergetic and Inflammatory Alterations in Regressed and Non-Regressed Patients with Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is associated with multiple physiological abnormalities. Current laboratory and clinical evidence most commonly report mitochondrial dysfunction, oxidative stress, and immunological imbalance in almost every cell type of the body. The present work aims to evaluate oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and inflammation-related molecules such as Cyclooxygenase-2 (COX-2), chitinase 3-like protein 1 (YKL-40), Interleukin-1 beta (IL-1ß), Interleukin-9 (IL-9) in ASD children with and without regression compared to healthy controls. Children with ASD (n = 56) and typically developing children (TDC, n = 12) aged 1.11 to 11 years were studied. Mitochondrial activity was examined in peripheral blood mononuclear cells (PBMCs) isolated from children with ASD and from the control group, using a metabolic analyzer. Gene and protein levels of IL-1ß, IL-9, COX-2, and YKL-40 were investigated in parallel. Our results showed that PBMCs of the ASD subgroup of regressed patients (ASD R(+), n = 21) had a specific pattern of mitochondrial activity with significantly increased maximal respiration, respiratory spare capacity, and proton leak compared to the non-regressed group (ASD R(-), n = 35) and TDC. Furthermore, we found an imbalance in the studied proinflammatory molecules and increased levels in ASD R(-) proving the involvement of inflammatory changes. The results of this study provide new evidence for specific bioenergetic profiles of immune cells and elevated inflammation-related molecules in ASD. For the first time, data on a unique metabolic profile in ASD R(+) and its comparison with a random group of children of similar age and sex are provided. Our data show that mitochondrial dysfunction is more significant in ASD R(+), while in ASD R(-) inflammation is more pronounced. Probably, in the group without regression, immune mechanisms (immune dysregulation, leading to inflammation) begin initially, and at a later stage mitochondrial activity is also affected under exogenous factors. On the other hand, in the regressed group, the initial damage is in the mitochondria, and perhaps at a later stage immune dysfunction is involved.

Targeting the serum marker interleukin 9 improves the underlying characterization and immune homeostasis in rheumatoid arthritis.

Introduction: Rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPA) are serological markers used for diagnosing rheumatoid arthritis (RA), an autoimmune disease characterized by inflammatory joint damage. However, there is a subset of RA patients who test negative for both RF and ACPA, known as seronegative rheumatoid arthritis (SNRA). Material and methods: The levels of serum markers were examined in both clinical samples and a rat model of type II collagen-induced RA (CIA). The effect of interleukin 9 (IL-9) on RA was investigated using recombinant rat IL-9 (rrIL-9), anti-rat IL-9 neutralizing monoclonal antibody (mAb), and control IgG antibody in the CIA rat. The severity of arthritis was assessed. Treg and Th17 cells, M1 and M2 macrophages, and inflammatory cytokine levels were analyzed. Results: We observed higher levels of IL-9 in clinical samples from SNRA patients compared to the normal group. Rat models of CIA exhibit increased arthritis scores, weight loss, paw swelling, and severe joint damage. IL-9 was the most sensitive serum marker for the diagnosis of RA in serum assays of CIA rats. IL-9 increased arthritis scores and cartilage damage, whereas treatment with IL-9 inhibitors produced the opposite effect. IL-9 inhibitors promoted Treg/Th17 homeostasis, decreased M1 macrophages, increased M2 macrophages, and decreased levels of inflammatory cytokines in joint tissues. Conclusions: These results suggest that IL-9 has potential as a diagnostic marker for SNRA. Inhibition of IL-9 could reduce the severity of arthritis in CIA rats by ameliorating inflammation and modulating the Treg/Th17 immune balance, M2 and M1 macrophage activation.

IL-9 promotes methicillin-resistant Staphylococcus aureus pneumonia by regulating the polarization and phagocytosis of macrophages.

In this study, we examined the effect of Il9 deletion on macrophages in methicillin-resistant Staphylococcus aureus (MRSA) infection. MRSA-infected mice were employed for the in vivo experiments, and RAW264.7 cells were stimulated with MRSA for the in vitro experiments. Macrophage polarization was determined by flow cytometry and quantitative real-time PCR; macrophage phagocytosis was assessed by flow cytometry and laser scanning confocal microscopy; cell apoptosis was assessed by flow cytometry and western blotting. Il9 deletion markedly elevated macrophage phagocytosis and M2 macrophages in MRSA infection, which was accompanied by elevated expression of Il10 and Arg1 and reduced expression of Inos, tumor necrosis factor-α (Tnfα), and Il6. Il9 deletion also inhibited macrophage apoptosis in MRSA infection, which was manifested by elevated B-cell lymphoma 2 (BCL-2) protein level and reduced protein levels of cleaved cysteine protease 3 (CASPASE-3) and BCL2-Associated X (BAX). Both the in vivo and in vitro experiments further showed the activation of phosphoinositide 3-kinase (PI3K)/AKT (also known as protein kinase B, PKB) signaling pathway in MRSA infection and that the regulation of Il9 expression may be dependent on Toll-like receptor (TLR) 2/PI3K pathway. The above results showed that Il9 deletion exhibited a protective role against MRSA infection by promoting M2 polarization and phagocytosis of macrophages and the regulation of Il9 partly owing to the activation of TLR2/PI3K pathway, proposing a novel therapeutic strategy for MRSA-infected pneumonia.

Mass cytometry analysis identifies T cell immune signature of aplastic anemia and predicts the response to cyclosporine.

Aplastic anemia (AA) is an auto-activated T cell-mediated bone marrow failure. Cyclosporine is often used to treat non-severe AA, which demonstrates a more heterogeneous condition than severe AA. The response rate to cyclosporine is only around 50% in non-severe AA. To better predict response to cyclosporine and pinpoint who is the appropriate candidate for cyclosporine, we performed phenotypic and functional T cell immune signature at single cell level by mass cytometry from 30 patients with non-severe AA. Unexpectedly, non-significant differences of T cell subsets were observed between AA and healthy control or cyclosporine-responder and non-responders. Interestingly, when screening the expression of co-inhibitory molecules, T cell trafficking mediators, and cytokines, we found an increase of cytotoxic T lymphocyte antigen 4 (CTLA-4) on T cells in response to cyclosporine and a lower level of CTLA-4 on CD8+ T cells was correlated to hematologic response. Moreover, a decreased expression of sphingosine-1-phosphate receptor 1 (S1P1) on naive T cells and a lower level of interleukin-9 (IL-9) on T helpers also predicted a better response to cyclosporine, respectively. Therefore, the T cell immune signature, especially in CTAL-4, S1P1, and IL-9, has a predictive value for response to cyclosporine. Collectively, our study implies that immune signature analysis of T cell by mass cytometry is a useful tool to make a strategic decision on cyclosporine treatment of AA.

Cytokine Kinetics during Progression of COVID-19 in Rwanda Patients: Could IL-9/IFNγ Ratio Predict Disease Severity?

For effective treatments and preventive measures against severe COVID-19, it is essential to determine early markers of disease severity in different populations. We analysed the cytokine kinetics of 129 COVID-19 patients with mild symptoms, 68 severe cases, and 20 healthy controls for the first time in Rwanda. Pro-inflammatory (IFNγ, IL-6, TNFα), Treg (IL-10, TGFß1, TGFß3), Th9 (IL-9), Th17 (IL-17), and Th2 (IL-4, IL-13) cytokines, total IgM and IgG, as well as gene expressions of FoxP3, STAT5+, IFNγ-R1, and ROR alpha+, were measured at day 1, day 7, day 14, day 21, and day 28 post-infection. Severe cases showed a significantly stronger increase than mild patients in levels of all cytokines (except IL-9) and all gene expression on day 1 of infection. Some cytokine levels dropped to levels comparable to mild cases at later time points. Further analysis identified IFNγ as a marker of severity throughout the disease course, while TGFß1, IL-6, and IL-17 were markers of severity only at an early phase. Importantly, this study revealed a striking low IL-9 level and high IFNγ/IL-9 ratio in the plasma of patients who later died compared to mild and severe cases who recovered, suggesting that this could be an important biomarker for predicting the severity of COVID-19 and post-COVID-19 syndrome.

Interleukin 26 Induces Macrophage IL-9 Expression in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease with chronic inflammation, bone erosion, and joint deformation. Synovial tissue in RA patients is full of proinflammatory cytokines and infiltrated immune cells, such as T help (Th) 9, Th17, macrophages, and osteoclasts. Recent reports emphasized a new member of the interleukin (IL)-10 family, IL-26, an inducer of IL-17A that is overexpressed in RA patients. Our previous works found that IL-26 inhibits osteoclastogenesis and conducts monocyte differentiation toward M1 macrophages. In this study, we aimed to clarify the effect of IL-26 on macrophages linking to Th9 and Th17 in IL-9 and IL-17 expression and downstream signal transduction. Murine and human macrophage cell lines and primary culture cells were used and stimulated by IL26. Cytokines expressions were evaluated by flow cytometry. Signal transduction and transcription factors expression were detected by Western blot and real time-PCR. Our results show that IL-26 and IL-9 colocalized in macrophage in RA synovium. IL-26 directly induces macrophage inflammatory cytokines IL-9 and IL-17A expression. IL-26 increases the IL-9 and IL-17A upstream mechanisms IRF4 and RelB expression. Moreover, the AKT-FoxO1 pathway is also activated by IL-26 in IL-9 and IL-17A expressing macrophage. Blockage of AKT phosphorylation enhances IL-26 stimulating IL-9-producing macrophage cells. In conclusion, our results support that IL-26 promotes IL-9- and IL-17-expressing macrophage and might initiate IL-9- and IL-17-related adaptive immunity in rheumatoid arthritis. Targeting IL-26 may a potential therapeutic strategy for rheumatoid arthritis or other IL-9 plus IL-17 dominant diseases.

Genetic association analysis between IL9 and coronary artery disease in a Chinese Han population.

Interleukin-9 (IL-9) plays important role in coronary artery disease (CAD). However, the exact relationship between them is not explored yet. Here, four tag SNPs covering IL9 (rs31563, rs2069868, rs2069870 and rs31564) were selected to conduct case-control association analyses in a total of 3704 individuals from Chinese Han population (1863 CAD vs 1841 control). Results showed that: first, rs2069868 was associated with CAD combined with hypertension (Padj = 0.027); second, IL9 haplotype (CGAT) was associated with CAD (Padj = 0.035), and the combination genotype of "rs31563_CC/rs31564_TT" would remarkably decrease the risk of CAD (Padj = 0.001); third, significant associations were found between rs2069870 and decreased LDL-c levels and decreased total cholesterol levels, and between rs31563 and increased HDL-c levels (Padj < 0.05). Therefore, we conclude that IL9 might play a causal role in CAD by interacted with CAD traditional risk factors, which might confer a new way to improve the prevention and treatment of CAD.

The absence of IL-9 reduces allergic airway inflammation by reducing ILC2, Th2 and mast cells in murine model of asthma.

Allergic asthma is an allergic inflammatory disease of the airways, in which numerous cell types and cytokines have been shown to contribute to pathogenesis of the disease. Although increased expression of IL-9 has been shown to influence the activity of structural as well as eosinophils and mast cells in asthma, the influence of IL-9 on function of ILC2 and Th2 cells remains unclear. This study therefore aimed to elucidate the role of IL-9 on ILC2 and Th2 cells using a murine model of asthma. A murine model of asthma was established using wild type (WT) and IL-9-deficient (Il9-/-) transgenic mice sensitized to house dust mite (HDM). Bronchoalveolar lavage fluid (BALF) and lung tissues were collected, and analysed for inflammatory cells (eosinophils, mast cells, Th2 cells and ILC2 cells), histopathological changes, and several cytokines. HDM challenge significantly increased accumulation of ILC2 cells, Th2 cells and mast cells, as well as goblet cell hyperplasia, and the expression of cytokines IL-4, IL-5 and IL-13, but not IFN-γ, in WT mice compared to saline-challenged control group. In contrast, all pathological changes, including infiltration of ILC2 cells, Th2 cells and mast cells, were significantly attenuated in HDM-challenged Il9-/- mice. Furthermore, the number of Ki67+ILC2 cells, Ki67+Th2 cells and Ki67+mast cells were significantly reduced in the absence of IL-9 signalling. These data suggest that IL-9 promotes the proliferation and type 2 cytokine production of type 2 cells in the murine models of asthma, and therefore might be a potential therapeutic target for asthma treatment.

The role of Th9 CD4+ T cells and IL-9 during primary Sjogren's syndrome.

OBJECTIVE: The objective of this study is to investigate the expression levels of Th9 CD4+ T cells and IL-9 secretion in peripheral blood mononuclear cells of patients with primary Sjogren's syndrome. Further, this study aimed to investigate the role of Th9 cells in the occurrence and development of pSS. METHODS: A total of 20 pSS patients and 20 healthy people, matched with age and gender, were selected as the experimental and control group, respectively. Flow cytometry and ELISA were used to detect the expression of Th9 cytokines in peripheral blood mononuclear cells and IL-9 in serum, respectively. These factors were then correlated to other clinical indicators. RESULTS: The levels of Th9 CD4+ T cells and IL-9 of pSS patients were significantly higher than those of the control group. Th9 CD4+ T cells and IL-9 levels in peripheral blood of pSS patients were negatively correlated with salivary flow rate, while IL-9 level was positively correlated with globulin. The transcription levels of IL-9 and immune-related genes including IL-4, IL-7, IL-17, SMAD3, STAT5 and JAK3 were dramatically increased in serum of pSS patients. CONCLUSION: The expression levels of Th9 in peripheral blood and serum IL-9 of patients with pSS were significantly increased, which were correlated with clinical immunological indexes. Together, these data suggest that Th9 cells and IL-9 may be involved in the pathogenesis of pSS.

IL-4-BATF signaling directly modulates IL-9 producing mucosal mast cell (MMC9) function in experimental food allergy.

BACKGROUND: This study group has previously identified IL-9-producing mucosal mast cell (MMC9) as the primary source of IL-9 to drive intestinal mastocytosis and experimental IgE-mediated food allergy. However, the molecular mechanisms that regulate the expansion of MMC9s remain unknown. OBJECTIVES: This study hypothesized that IL-4 regulates MMC9 development and MMC9-dependent experimental IgE-mediated food allergy. METHODS: An epicutaneous sensitization model was used and bone marrow reconstitution experiments were performed to test the requirement of IL-4 receptor α (IL-4Rα) signaling on MMC9s in experimental IgE-mediated food allergy. Flow cytometric, bulk, and single-cell RNA-sequencing analyses on small intestine (SI) MMC9s were performed to illuminate MMC9 transcriptional signature and the effect of IL-4Rα signaling on MMC9 function. A bone marrow-derived MMC9 culture system was used to define IL-4-BATF signaling in MMC9 development. RESULTS: Epicutaneous sensitization- and bone marrow reconstitution-based models of IgE-mediated food allergy revealed an IL-4 signaling-dependent cell-intrinsic effect on SI MMC9 accumulation and food allergy severity. RNA-sequencing analysis of SI-MMC9s identified 410 gene transcripts reciprocally regulated by IL-4 signaling, including Il9 and Batf. Insilico analyses identified a 3491-gene MMC9 transcriptional signature and identified 2 transcriptionally distinct SI MMC9 populations enriched for metabolic or inflammatory programs. Employing an in vitro MMC9-culture model system showed that generation of MMC9-like cells was induced by IL-4 and this was in part dependent on BATF. CONCLUSIONS: IL-4Rα signaling directly modulates MMC9 function and exacerbation of experimental IgE-mediated food allergic reactions. IL-4Rα regulation of MMC9s is in part BATF-dependent and occurs via modulation of metabolic transcriptional programs.

Th2 Cytokines (Interleukin-5 and -9) Polymorphism Affects the Response to Anti-TNF Treatment in Polish Patients with Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is an inflammatory disease that belongs to the spondyloarthritis family. IL-5 and IL-9 belong to the group of Th2 cytokines of anti-inflammatory nature. Polymorphisms in their coding genes have been so far associated with various inflammatory diseases, but there are no reports regarding their involvement in AS pathogenesis to date. The purpose of the study was to investigate relationships between IL5 and IL9 genetic variants with AS susceptibility, clinical parameters as well as response to therapy with TNF inhibitors. In total 170 patients receiving anti-TNF therapy and 218 healthy controls were enrolled in the study. The genotyping of IL5 rs2069812 (A > G) and IL9 rs2069885 (G > A) single nucleotide polymorphisms was performed using the Real-Time PCR method based on LightSNiP kits assays. The present study demonstrated significant relationships between IL5 rs2069812 and IL9 rs2069885 polymorphisms and response to anti-TNF therapy. Presence of the IL5 rs2069812 A allele in patients positively correlated with better response to treatment (p = 0.022). With regard to IL9 rs2069885, patients carrying the A allele displayed better outcomes in anti-TNF therapy (p = 0.046). In addition, IL5 rs2069812 A and IL9 rs2069885 A alleles were associated with lower CRP and VAS values. The obtained results may indicate a significant role for IL-5 and IL-9 in the course of AS and response to anti-TNF therapy.

IL-9 blockade attenuates inflammation in a murine model of mechanical ventilation-induced lung injury by inhibiting the NLRP3 inflammasome pathway.

OBJECTIVES: Ventilation-induced lung injury (VILI) causes a huge economic and social burden, and its prevention and treatment have gained increasing attention in recent years. IL-9 is an important inflammatory factor, but its potential role in VILI remains unclear. This study intended to explore whether blocking IL-9 could alleviate VILI and explore its underlying mechanism. METHODS: Lung injury was induced by mechanical ventilation (MV) in C57BL/6 mice. Changes in inflammatory factors and NLRP3-related proteins were assessed using quantitative reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. Subsequently, Nlrp3-/- mice were used to further elucidate the underlying mechanism. RESULTS: The percentage of Th9 cells in the peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissues of MV mice was increased compared to those of control mice. Treatment with anti-IL-9 mAb significantly alleviated the changes in lung histopathology, wet/dry lung proportion, total protein content, and neutrophil content in BALF induced by VILI. Additionally, administering anti-IL-9 mAb significantly downregulated the expression levels of inflammatory factors in BALF and lung tissues of mice with VILI. In addition, administering anti-IL-9 mAb inhibited NLRP3 inflammasome activation, as evidenced by the observed downregulation of NLRP3, ASC, cleaved caspase-1, and GSDMD-N. Additionally, NLRP3-deficient mice had lower lung injury induced by VILI than wild-type mice. Furthermore, the anti-IL-9 mAb only partially inhibited VILI in Nlrp3-/- mice. CONCLUSIONS: In MV mice, the anti-IL-9 mAb alleviated lung injury and reduced the secretion and expression of inflammatory factors partly by inhibiting the NLRP3 inflammasome pathway.

Dexmedetomidine-up-regulated microRNA-381 exerts anti-inflammatory effects in rats with cerebral ischaemic injury via the transcriptional factor IRF4.

Dexmedetomidine (Dex) possesses analgesic and anaesthetic values and reported being used in cerebral ischaemic injury therapeutics. Accumulating studies have determined the effect of microRNAs (miRNAs) on the cerebral ischaemic injury. Thus, the present study aimed to unravel the molecular mechanism of miR-381 and Dex in cerebral ischaemic injury. For this purpose, the cerebral ischaemic injury rat model was established by induction of middle cerebral artery occlusion (MCAO) and expression of miR-381 and IRF4 was determined. Thereafter, MCAO rats were treated with Dex, miR-381 mimic, miR-381 inhibitor and oe-IRF4 respectively, followed by evaluation of neurological function. Furthermore, neuron cells were isolated from the hippocampus of rats and subjected to oxygen-glucose deprivation (OGD). Then, OGD-treated neuron cells and primary neuron cells were examined by gain- and loss-of-function assay. Neuron cell apoptosis was detected using TUNEL staining and flow cytometry. The correlation between interferon regulatory factor 4 (IRF4) and interleukin (IL)-9 was detected. Our results showed down-regulated miR-38 and up-regulated IRF4 in MCAO rats. Besides, IRF4 was targeted by miR-381 in neuron cells. Dex and overexpressed miR-381, or silenced IRF4 improved the neurological function and inhibited neuron cell apoptosis in MCAO rats. Additionally, in MCAO rats, Dex was found to increase the miR-381 expression and reduced IRF4 expression to decrease the IL-9 expression, which suppressed the inflammatory response and cell apoptosis both in vivo and in vitro. Importantly, our study demonstrated that Dex elevated the expression of miR-381, which ultimately results in the inhibition of inflammation response in rats with cerebral ischaemic injury.