Serum levels of IL-9 and IL-11 serve as predictors for the occurrence of early neurologic deterioration in patients with cerebral infarction.

BACKGROUND AND AIM: Early neurological deterioration (END) is a common complication of cerebral infarction and a significant contributor to poor prognosis. Our study aimed to investigate the predictive value of interleukin-9 (IL-9) and interleukin-11 (IL-11) in relation to the occurrence of END in patients with cerebral infarction. MATERIALS AND METHODS: 102 patients with cerebral infarction and 64 healthy controls were collected. Patients were categorized into two groups based on the development of END following admission: the END group (n = 44) and the non-END group (n = 58). Enzyme-linked immunosorbent assay was used to determine the serum levels of IL-9, IL-11, and BDNF. RESULTS: Serum IL-9 was higher and IL-11 lower in the END group than those in the non-END group (P < 0.01). IL-9 correlated positively with NIHSS score (r = 0.627) and infarction volume (r = 0.686), while IL-11 correlated negatively (r = -0.613, -0.679, respectively). Logistic regression identified age, NIHSS score, and IL-9 as risk factors (P < 0.01), and IL-11 as protective (P < 0.01). Combined IL-9 and IL-11 had an ROC curve area of 0.849. BDNF correlated negatively with IL-9 (r = -0.703) and positively with IL-11 (r = 0.711). CONCLUSION: Serum IL-9 and IL-11 levels can predict the occurrence of END in patient with cerebral infarction and are correlated with serum BDNF levels.

A prospective observational study correlating possible novel biomarkers with disease severity and antihistamine response in chronic spontaneous urticaria.

Background: Role of complement fraction 5a (C5a), interleukin (IL)-9, and apolipoprotein (apo) A-IV as biomarkers of disease severity and antihistamine response in chronic spontaneous urticaria (CSU) remains elusive. Objective: To identify the role of C5a, IL-9, and apo A-IV as potential biomarkers in predicting disease severity and antihistamine response in CSU patients. Methods: This was a prospective observational study of 95 patients and 42 controls. Serum analysis of C5a, IL-9, and apo A-IV was done using enyzme linked immunosorbent assay kits. Also, serum IgE and anti-thyroid peroxidase (TPO) levels were assessed in all patients. All patients were started on oral levocetirizine 5 mg at baseline and dose was titrated upwards to maximum of 20 mg based on response. Patients were categorized into antihistamine responders or nonresponders as per their disease response. Serological markers, serum IgE, and anti-TPO were correlated with baseline disease severity and antihistamine response. Results: C5a levels were significantly higher in cases as compared to controls (P = 0.004). Significantly higher IL-9 levels were observed in antihistamine responders than nonresponders (P = 0.008). Baseline urticaria severity demonstrated a statistically significant positive and negative correlations with IL-9 (ρ = 0.277, P = 0.007) and apo A-IV (ρ = -0.271, P = 0.008) levels, respectively. Levels of serum IgE (P = 0.031) and anti-TPO (P = 0.039) were significantly higher in antihistamine nonresponders compared to responders. Conclusions: IL-9 and apo A-IV might be potential novel biomarkers to predict urticaria severity. Higher IL-9 might be a predictor of antihistamine response. Elevated anti-TPO and serum IgE might predict poor antihistamine response.

Objectively measured prolonged sleep is associated with plasma cytokines in older adults with mild cognitive impairment.

This study aimed to determine whether objective sleep time is associated with the concentrations of various plasma cytokines in older adults with mild cognitive impairment (MCI). In total, 118 adults with MCI (66 women; mean age: 75.7 years) participated in this prospective cohort study. All participants were required to wear a wristband sensor for 7.8 days, on average, every 3 months for 1 year and undergo measurement of 27 plasma cytokines using multiplex immunoassays. After adjusting for potential confounders, the associations of total sleep time with cytokine concentrations were assessed by multiple linear regression analysis. The total sleep time was significantly correlated with plasma interleukin (IL)-9 and macrophage inflammatory protein (MIP)-1ß levels (r = 0.239, p = 0.009, and r = 0.242, p = 0.008, respectively). Moreover, these associations remained significant after adjusting for covariates, including demographic characteristics, lifestyle-related diseases, and apolipoprotein E status (ß = 0.272, 95% confidence interval: 0.095-0.448, p = 0.003, and ß = 0.27, 95% confidence interval: 0.092-0.449, p = 0.003, respectively). Thus, this study is the first to demonstrate the association between objective prolonged sleep and higher plasma IL-9 and MIP-1ß levels in older adults with MCI.

Immunofluorescence analysis of human eosinophils.

A prominent inflammatory cell type in allergic diseases is the eosinophil, a granulated white blood cell that releases pro-inflammatory cytokines. Eosinophil-derived cytokines, including interleukin-9 (IL-9) and interleukin-13 (IL-13), can skew the immune response towards an allergic phenotype. Unfortunately, it is challenging to immunolabel and collect quantifiable images of eosinophils given their innate autofluorescence and ability to nonspecifically bind to antibodies. Hence, it is important to optimize permeabilization, blocking, and imaging conditions for eosinophils. Here, we show enhanced protocols to ensure that measured immunofluorescence represents specific immunolabelling. To test this, eosinophils were purified from human blood, adhered to glass coverslips, stimulated with or without platelet-activating factor (PAF), fixed with paraformaldehyde, and then permeabilized with Triton X-100 or saponin. Cells were then blocked with goat serum or human serum and incubated with antibodies labelling cytokines (IL-9 and IL-13) and secretory organelles (CD63 for crystalloid granules and transferrin receptor [TfnRc] for recycling endosomes). Carefully selected isotype controls were used throughout, and cells were imaged using Deltavision super-resolution microscopy. Intensities of fluorescent probes were quantified using Volocity software. Our findings show that permeabilization with saponin, blockage with human serum, and using concentrations of antibodies up to 10 µg/ml allowed us to detect marked differences in fluorescence intensities between isotypes and test antibodies. With the achievement of sufficient qualitative and quantitative measures of increased test probe intensity compared to respective isotypes, these results indicate that our protocol allows for optimal immunolabelling of eosinophils. Using this protocol, future studies may provide further insights into trafficking mechanisms within this important inflammatory cell type.

Th9/IL-9 may participate in the pathogenesis of multiple myeloma.

INTRODUCTION: This research is aimed to evaluate the correlation between Th9-associated cytokine levels in MM patients, clinical features, and therapy. METHODS: Peripheral blood samples were taken in 52 MM patients and 20 healthy volunteers matched by sex and age. The patients with MM were separated into two groups: the untreated group (27) and the remission group (25). An enzyme-linked immunosorbent assay (ELISA) was used to measure the IL-9 plasma levels. The levels of Th9-associated cytokines' mRNA expression (IL-9, PU.1, and IRF4) were measured in RT-qPCR. We also analyzed the correlations between the IL-9 plasma levels and the clinical parameters of newly diagnosed MM patients. RESULTS: The IL-9 plasma levels and the Th9-associated cytokines (IL-9, PU.1, and IRF4) mRNA levels in newly diagnosed MM patients were significantly elevated than those in healthy volunteers and significantly decreased after achieving remission. Moreover, PU.1 and IRF4 had a positive correlation with the IL-9 mRNA expression. Then, we found that the upregulation of IL-9 plasma levels correlates with the severity of anemia and decreased albumin Levels. CONCLUSION: The results demonstrate that Th9/IL-9 may be involved in the pathogenesis of MM and is correlated with worse patient conditions such as lower hemoglobin and serum albumin. More work is necessary to confirm whether they might serve as a useful therapeutic target and prognostic marker for MM.

[Expression and Clinical Significance of Helper T Cells 9 and Its Sytokines Interleukin 9 in Chronic Lymphocytic Leukemia].

OBJECTIVE: To investigate the expression and clinical significance of T helper cell 9 (Th9) and its cytokine interleukin 9(IL-9) in peripheral blood of patients with chronic lymphocytic leukemia(CLL). METHODS: A total of 43 newly diagnosed patients with chronic lymphocytic leukemia in the First Affiliated Hospital of Xinjiang Medical University from June 2021 to June 2022 were selected as the case group. The patients were divided into Binet A group (13 cases), Binet B group (20 cases) and Binet C group (10 cases) by Binet staging system, and 20 healthy volunteers who underwent physical examinationin in our hospital in the same period served as control group. The proportion of Th9 cells in peripheral blood was detected by flow cytometry, the expression level of Th9 specific transcription factors PU.1 and IRF4 was detected by Western blot, and the expression level of serum cytokine IL-9 was detected by ELISA. The proportion of Th9, the expression of PU.1, IRF4 and IL-9 in each group were compared, and the correlation between the proportion of Th9, IL-9 and clinicopathological indexes of CLL patients was analyzed. RESULTS: The proportion of Th9, the expression of PU.1, IRF4 and IL-9 in CLL group were significantly higher than those in control group (P<0.05), the proportion of Th9 and the expression of IL-9 in Binet B and C group were higher than those in Binet A group (P<0.05), but there was no significant difference in the proportion of Th9 cells between Binet B group and C group (P>0.05). The expression of IL-9 in Binet C group was significantly higher than that in Binet B group (P<0.05) . The proportion of Th9 cells and IL-9 were highly expression in patients with ß2 microglobulin abnormality, IGHV unmutation, P53 abnormality and hepatosplenic lymph node enlargement(P<0.05), but not related to age and sex (P>0.05). The results of Spearman correlation analysis showed that the proportion of Th9 in patients with CLL was negatively correlated with the lymphocytic account and lymphocyte proportion(rs=-0.32，rs=-0.34). The proportion of Th9 and IL-9 were positively correlated with Binet stage, Rai stage and CLL-IPI Scoring (rs=0.79，rs=0.54，rs=0.58； rs=0.72，rs=0.63，rs=0.45), but not with WBC, CD4+ T cells and CD8+T cells (P>0.05). The proportion of Th9 was positively correlated with IL-9 (rs=0.53). CONCLUSION: Th9 cells and IL-9 are abnormally highly expressed in CLL, which is related to the poor prognosis of CLL.

Characterization of chicken interleukin-9 receptor alpha chain.

Interleukin-9 receptor alpha chain (IL-9Rα) is the ligand-binding subunit of IL-9R that plays roles in IL-9-mediated allergy, inflammation, infection, and tumor immunity. While mammalian IL-9Rαs have been extensively investigated, avian IL-9Rα has not yet been identified and characterized. In this study, we cloned chicken IL-9Rα (chIL-9Rα) and performed a phylogenetic analysis, analyzed its tissue distribution, characterized the expression form of natural chIL-9Rα. Phylogenetic analysis showed that chIL-9Rα has less than 25% amino acid homology with mammalian IL-9Rαs. The chIL-9Rα mRNA was abundantly detected only in heart and mitogen-activated peripheral blood mononuclear cells. Furthermore, 4 monoclonal antibodies (mAbs) against chIL-9Rα were generated using prokaryotic recombinant chIL-9Rα (rchIL-9Rα). Using anti-chIL-9Rα mAbs, natural chIL-9Rα expressed on the splenocytes of chickens was observed by indirect immunofluorescence assay (IFA), and its molecular weight of 51 kDa was identified by Western blotting. Overall, our study reveals for the first time the presence of IL-9Rα in birds, and provides immunological tools for further investigating the roles of chIL-9 in diseases and immunity.

Higher IL-9 Level is Associated with Psoriasis Vulgaris Complicated by Metabolic Syndrome.

Purpose: The underlying pathophysiology linking psoriasis vulgaris (PV) and metabolic syndrome (MetS) is not fully understood. The present study aimed to investigate the serum level of interleukin (IL)-9 and tissue levels of IL-9 and its receptor in PV patients with MetS and analyze the correlation of IL-9 levels with psoriasis disease severity and MetS. Methods: This study enrolled 75 PV patients with MetS, 57 PV patients without MetS, 20 healthy blood donors, and 7 healthy skin donors. Clinical, socio-demographic, and anthropometric data were obtained from all individuals. Fasting blood glucose, insulin, lipid profile levels, and serum levels of IL-9 and IL-17A were measured. The expression of IL-9 and its receptor in skin specimens in PV patients and healthy controls was determined using immunohistochemistry. Normal human epidermal keratinocytes were stimulated with five pro-inflammatory cytokines (tumor necrosis factor-α, oncostatin M, IL-22, IL-17A, and IL-1α) to establish a psoriatic keratinocyte model and subsequently treated with IL-9. Their mRNA levels of antimicrobial peptides and chemokines were measured using quantitative real-time polymerase chain reaction. Results: Serum level of IL-9 and tissue levels of IL-9 and its receptor were upregulated in PV patients with MetS. IL-9 level was positively correlated to IL-17A level; however, no significant correlation of IL-9 level with psoriasis area severity index was observed. IL-9 level had a positive correlation with the presence of MetS and its components. Correspondingly, IL-9 level positively correlated with waist circumference, body mass index, homeostasis model assessment-insulin resistance, blood pressure, and triglyceride level and negatively correlated with high-density lipoprotein cholesterol level. Additionally, IL-9 stimulated the expression of antimicrobial peptides and chemokines in a psoriatic keratinocyte model. Conclusion: Our findings confirmed that higher IL-9 level is associated with PV complicated by MetS, suggesting that IL-9 may be a link between PV and MetS.

Interleukin-9 rescues class switching of Memory B cells derived from Common variable immunodeficiency patients.

Impaired class switch memory (CSM) B cell formation is the hallmark of common variable immunodeficiency (CVID). Various T cell abnormalities have been observed in CVID patients indicating inadequate T-cell help to B cells. A major setback in understanding its pathogenesis is due to diverse clinical presentation. Therefore, we performed extensive immunological investigation in a cohort of CVID patients with similar clinical findings in order to unravel the T cell dysfunction and its influence on the defective humoral immune response. All recruited CVID patients exhibited B cells in the normal range, but reduced CSM B cells. However, patients showed reduced T cell proliferation, reduced level of serum Interleukin-9 (IL-9) and frequency of IL-9 expressing CD4 (Th-9) cells. IL-9 supplementation along with CD40 engagement was effective in inducing in vitro CSM B cells formation in CVID patients. Thus, IL-9 supplementation has the potential to restore impaired CSM B cell formation in CVID.

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.

Characteristics of Th9 cells in Schistosoma japonicum-infected C57BL/6 mouse mesenteric lymph node.

Interleukin 9 (IL-9) is an effective cytokine secreted by newly defined Th9 cells, which is involved in allergic and infectious diseases. In this study, lymphocytes were isolated from mesenteric lymph node (MLN), spleen, liver, lung, and Peyer's patches (PP) of C57BL/6 mice 5-6 weeks after S. japonicum infection, intracellular cytokine staining was done to detect the percentage of IL-9-producing CD4+ T cells. The qPCR and ELISA were used to verify the content of IL-9 in MLN. The population of IL-9-producing lymphocyte subset was identified by FACS. In addition, the dynamic changes and cytokine profiles of Th9 cells in the MLN of infected mice were detected by FACS. ELISA was used to detect IL-9 induced by soluble egg antigen (SEA) from isolated lymphocytes in mouse MLN. The results showed that the percentage of IL-9-secreting Th9 cells in the MLN of the infected mouse was higher than that in the spleen, liver, lung, or PP. Though CD8+ Tc cells, NKT cells, and γδT cells could secrete IL-9, CD4+ Th cells were the main source of IL-9 in S. japonicum-infected C57BL/6 mice (P < 0.05). The percentage of Th9 cells in MLN of infected mouse increased from week 3-4, and reached a peak at week 5-6, then began to decrease from week 7-8 (P < 0.05). Moreover, Th9 cells could also secrete a small amount of IL-4, IFN-γ, IL-5, and IL-10. Our results suggested a higher percentage of Th9 cells was induced in the MLN of S. japonicum-infected mice, which might play an important role in the early stage of S. japonicum-induced disease.

Patients with Chronic Spinal Cord Injury and a Long Period of Evolution Exhibit an Altered Cytokine Production by CD4 and CD8 T Cell Populations.

Spinal cord injury (SCI) is a disabling neurological condition coursing with serious multisystem affections and morbidities. Changes in immune cell compartments have been consistently reported in previous works, representing a critical point of study for understanding the pathophysiology and progression of SCI from acute to chronic stages. Some relevant variations in circulating T cells have been noticed in patients with chronic SCI, although the number, distribution, and function of these populations remain to be fully elucidated. Likewise, the characterization of specific T cell subpopulations and their related cytokine production can aid in understanding the immunopathological role of T cells in SCI progression. In this sense, the objective of the present study was to analyze and quantify the total number of different cytokine-producers T cells in the serum of patients with chronic SCI (n = 105) in comparison to healthy controls (n = 38) by polychromatic flow cytometry. Having this goal, we studied CD4 and CD8 lymphocytes as well as naïve, effector, and effector/central memory subpopulations. SCI patients were classified according to the duration of the lesion in chronic SCI with a short period of evolution (SCI-SP) (comprised between 1 and 5 years since initial injury), early chronic phase (SCI-ECP) (between 5 and 15 years since initial injury) and late-chronic phase (SCI-LCP) (>15 years since initial injury). Our results show that patients with chronic SCI exhibited an altered immune profile of cytokine-producer T cells, including CD4/CD8 naïve, effector, and memory subpopulations in comparison to HC. In particular, IL-10 and IL-9 production seems to be importantly altered, especially in patients with SCI-LCP, whereas changes in IL-17, TNF-α, and IFN-γ T cell populations have also been reported in this and other chronic SCI groups. In conclusion, our study demonstrates an altered profile of cytokine-producer T cells in patients with chronic SCI, with marked changes throughout the course of the disease. In more detail, we have observed significant variations in cytokine production by circulating naive, effector, and effector/central memory CD4 and CD8 T cells. Future studies should be directed to explore the possible clinical consequences of these changes or develop additional translational approaches in these groups of patients.

Mechanistic insight of interleukin-9 induced osteoclastogenesis.

Interleukin (IL)-9 is an emerging player in the pathogenesis of various chronic inflammatory diseases including bone disorders like rheumatoid arthritis (RA) and psoriatic arthritis. Recently, IL-9 was shown to enhance the osteoclast formation and their function in RA. However, the mechanisms by which IL-9 influences osteoclastogenesis are not known. Therefore, in this study we aimed to unravel the direct and indirect ways by which IL-9 can influence osteoclast formation. We used mouse bone marrow precursor cells for checking the effect of IL-9 on osteoclast differentiation and its function. Next, IL-9 induced signalling pathway were checked in the process of osteoclastogenesis. T cells play an important role in enhancing osteoclastogenesis in inflammatory conditions. We used splenic T cells to understand the impact of IL-9 on the functions of T effector (Teff) and regulatory T (Treg) cells. Furthermore, the effect of IL-9 mediated modulation of the T cell response on osteoclasts was checked using a coculture model of T cells with osteoclast precursors. We showed that IL-9 enhanced osteoclast formation and its function. We found that IL-9 activates STAT3, P38 MAPK, ERK1/2, NFκB and we hypothesize that it mediates the effect on osteoclastogenesis by accelerating mitochondrial biogenesis. Additionally, IL-9 was observed to facilitate the functions of pro-osteoclastogenic IL-17 producing T cells, but inhibits the function of anti-osteoclastogenic Treg cells. Our observations suggest that IL-9 can influence osteoclastogenesis directly by modulating the signalling cascade in the precursor cells; indirectly by enhancing IL-17 producing T cells and by reducing the functions of Treg cells.

Thrombopoietin-independent Megakaryocyte Differentiation of Hematopoietic Progenitor Cells from Patients with Myeloproliferative Neoplasms.

Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient-derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders. This protocol was validated in: EMBO Rep (2022), DOI: 10.15252/embr.202152904 Graphical abstract Schematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation.

Combined in vitro and cell-based selection display method producing specific binders against IL-9 receptor in high yields.

We combined cell-free ribosome display and cell-based yeast display selection to build specific protein binders to the extracellular domain of the human interleukin 9 receptor alpha (IL-9Rα). The target, IL-9Rα, is the receptor involved in the signalling pathway of IL-9, a pro-inflammatory cytokine medically important for its involvement in respiratory diseases. The successive use of modified protocols of ribosome and yeast displays allowed us to combine their strengths-the virtually infinite selection power of ribosome display and the production of (mostly) properly folded and soluble proteins in yeast display. The described experimental protocol is optimized to produce binders highly specific to the target, including selectivity to common proteins such as BSA, and proteins potentially competing for the binder such as receptors of other cytokines. The binders were trained from DNA libraries of two protein scaffolds called 57aBi and 57bBi developed in our laboratory. We show that the described unconventional combination of ribosome and yeast displays is effective in developing selective small protein binders to the medically relevant molecular target.

Expression and Clinical Significance of Helper T Cells 9 and Its Sytokines Interleukin 9 in Chronic Lymphocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression and clinical significance of T helper cell 9 (Th9) and its cytokine interleukin 9(IL-9) in peripheral blood of patients with chronic lymphocytic leukemia(CLL).@*METHODS@#A total of 43 newly diagnosed patients with chronic lymphocytic leukemia in the First Affiliated Hospital of Xinjiang Medical University from June 2021 to June 2022 were selected as the case group. The patients were divided into Binet A group (13 cases), Binet B group (20 cases) and Binet C group (10 cases) by Binet staging system, and 20 healthy volunteers who underwent physical examinationin in our hospital in the same period served as control group. The proportion of Th9 cells in peripheral blood was detected by flow cytometry, the expression level of Th9 specific transcription factors PU.1 and IRF4 was detected by Western blot, and the expression level of serum cytokine IL-9 was detected by ELISA. The proportion of Th9, the expression of PU.1, IRF4 and IL-9 in each group were compared, and the correlation between the proportion of Th9, IL-9 and clinicopathological indexes of CLL patients was analyzed.@*RESULTS@#The proportion of Th9, the expression of PU.1, IRF4 and IL-9 in CLL group were significantly higher than those in control group (P<0.05), the proportion of Th9 and the expression of IL-9 in Binet B and C group were higher than those in Binet A group (P<0.05), but there was no significant difference in the proportion of Th9 cells between Binet B group and C group (P>0.05). The expression of IL-9 in Binet C group was significantly higher than that in Binet B group (P<0.05) . The proportion of Th9 cells and IL-9 were highly expression in patients with β2 microglobulin abnormality, IGHV unmutation, P53 abnormality and hepatosplenic lymph node enlargement(P<0.05), but not related to age and sex (P>0.05). The results of Spearman correlation analysis showed that the proportion of Th9 in patients with CLL was negatively correlated with the lymphocytic account and lymphocyte proportion(rs=-0.32，rs=-0.34). The proportion of Th9 and IL-9 were positively correlated with Binet stage, Rai stage and CLL-IPI Scoring (rs=0.79，rs=0.54，rs=0.58； rs=0.72，rs=0.63，rs=0.45), but not with WBC, CD4+ T cells and CD8+T cells (P>0.05). The proportion of Th9 was positively correlated with IL-9 (rs=0.53).@*CONCLUSION@#Th9 cells and IL-9 are abnormally highly expressed in CLL, which is related to the poor prognosis of CLL.

Comparison of Serum Levels of Interleukin 17 and Interleukin 9 in Leprosy Patients with and without Lepra Reaction: A Cross-Sectional Analytical Study from Central India.

New pathways of host defence have emerged in leprosy, such as T helper (Th) -17, Th-9, T regulatory cells, and other factors like transforming growth factor-beta, etc. Interleukin (IL) 17 produced by Th17 cells has been found to be elevated in lepra reaction, especially type 2 lepra reaction (T2R). Role of IL-9 has not been studied widely in leprosy reactions so far. The study aimed to compare serum levels of IL-17 and IL-9 in leprosy patients with and without lepra reaction. This was a cross-sectional analytical study including untreated adult leprosy patients with and without lepra reaction. A total of 65 patients were included in the study with 30 leprosy patients without reaction and 35 with lepra reaction. Serum levels of IL-17 and IL-9 were measured in these patients using direct enzyme-linked immunosorbent assay and were compared. Borderline tuberculoid (BT) leprosy with type 1 and Lepromatous (LL) leprosy with T2R patients showed significantly higher levels of IL-17 than BT and LL leprosy patients without lepra reaction, respectively. LL patients with T2R showed significantly lower levels of IL-9 than lepromatous cases without reaction. IL-9 levels were higher in BT patients with T1R as compared to BT patients without reaction but the difference was not significant. We found evidence in support of role of IL-17 in the pathogenesis of T2R, which might serve as useful serum markers for the same. IL-17 might have a role in BT leprosy with T1R. IL-9 seems to have a protective role in T2R as opposed to IL-17, working in synergism with Th1 cytokines.

Correlation of serum interleukin-9 and platelet-activating factor levels with total IgE levels, disease severity and disease course in patients with chronic spontaneous urticaria / 中华皮肤科杂志

Objective:To explore the relationship of serum interleukin (IL) -9 and platelet-activating factor (PAF) levels with serum total immunoglobulin E (IgE) levels, disease severity and disease course in patients with chronic spontaneous urticaria (CSU) .Methods:A total of 60 patients with active CSU were collected from Department of Dermatology, the First Affiliated Hospital of Hebei North University from March 2018 to March 2019 (active CSU group), and divided into mild group, moderate group and severe group according to 7-day urticaria activity score (UAS7). After 28-day standard antihistamine therapy, the patients whose condition became stable were included in the stable CSU group. During the same period, 30 health examinees were included in the healthy control group. Peripheral venous blood samples were collected from the subjects in each group, enzyme-linked immunosorbent assay (ELISA) was performed to detect serum levels of IL-9 and PAF, and immunoturbidimetric assay to detect the serum total IgE level. Correlations of serum IL-9 and PAF levels with serum total IgE levels, UAS7 scores and disease courses were analyzed in patients with CSU. One-way analysis of variance was used for comparisons among multiple groups, least significant difference- t test for multiple comparisons, and Pearson correlation analysis for correlation analysis. Results:Totally, 28 males and 32 females were included in the active CSU group, their age ranged from 11 to 68 years (34.68 ± 8.62 years), and the disease duration ranged from 2 months to 7 years (1.42 ± 0.41 years). In the healthy control group, 14 were males and 16 were females, and their age ranged from 10 to 70 years (35.06 ± 7.89 years). According to UAS7, 12, 26, and 22 patients were diagnosed with mild, moderate and severe CSU respectively, and 22 were included in the stable CSU group after standard treatment. The levels of serum IL-9, PAF and total IgE significantly differed among the active CSU group, stable CSU group and healthy control group (IL-9: 144.34 ± 23.19 vs. 109.25 ± 20.77 vs. 107.23 ± 19.23 pg/ml; PAF: 362.45 ± 51.45 vs. 223.18 ± 32.46 vs. 221.23 ± 28.38 pg/ml; total IgE: 168.12 ± 32.48 vs. 24.04 ± 7.04 vs. 21.76 ± 5.95 IU/ml; F = 38.80, 148.38, 499.12, respectively, all P < 0.001), and were significantly higher in the active CSU group than in the stable CSU group and healthy control group (all P < 0.05), while there was no significant difference between the stable CSU group and healthy control group (all P > 0.05). Pearson correlation analysis showed that the serum IL-9 and PAF levels were positively correlated with serum total IgE levels and UAS7 scores (all P < 0.05), but not correlated with the disease course (both P > 0.05) . Conclusion:Serum IL-9 and PAF levels in patients with active CSU were markedly elevated along with the increase in disease severity, and closely correlated with serum total IgE levels.

[Interleukin-9 promotes the activation of hepatic stellate cells in mice infected with Schistosoma japonicum].

OBJECTIVE: To investigate the effects of interleukin-9 (IL-9) in the activation of hepatic stellate cells (HSCs) in mice infected with Schistosoma japonicum. METHODS: Primary HSCs were isolated from mice 7 weeks post-infection with S. japonicum using the in situ liver perfusion and density gradient centrifugation, and cultured in vitro. HSCs were randomly assigned to the PBS control group and IL-9 stimulation group (stimulation with 20 ng/mL IL-9). HSCs were harvested 48 h and 72 h poststimulation, and the expression of α-smooth muscle actin (α-SMA), type I collagen (Col I) and type III collagen (Col III) was determined in HSCs using Western blotting. RESULTS: Following stimulation with 20 ng/mL IL-9 for 48 h, the expression of α-SMA [(0.87 ± 0.02) vs. (0.69 ± 0.01); t = 17.39, P < 0.01], Col I [(0.74 ± 0.02) vs. (0.65 ± 0.01); t = 9.56, P < 0.01] and Col III [(0.94 ±0.04) vs. (0.75 ± 0.03); t = 6.15, P < 0.01] was significantly greater in HSCs in the IL-9 stimulation group than in the PBS control group. Following stimulation with 20 ng/mL IL-9 for 72 h, the expression of α-SMA was significantly greater in HSCs in the IL-9 stimulation group than in the PBS control group[(0.76 ± 0.02) vs. (0.58 ± 0.02); t = 12.52, P < 0.01]; however, there were no significant differences between the two groups in terms of Col I [(0.68 ± 0.02) vs. (0.66 ± 0.02); t = 1.15, P > 0.05] or Col III expression [(0.75 ± 0.01) vs. (0.72 ± 0.02); t = 2.22, P > 0.05]. CONCLUSIONS: IL-9 promotes the activation of HSCs in mice infected with S. japonicum.

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.