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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)-1β 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 (β = 0.272, 95% confidence interval: 0.095-0.448, p = 0.003, and β = 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-1β levels in older adults with MCI., (© 2024 European Sleep Research Society.)

It is not clear whether immunoregulatory cytokines and cells are associated with Disease Activity Score 28 (DAS28) scores and ultrasound grades/scores. Here, we investigated the relationships between immunoregulatory cytokines or cells and different DAS28 scores or ultrasound grades/scores in patients with rheumatoid arthritis (RA). This study enrolled 50 RA patients (with 147 visits) who had remission/low/moderate DAS28-ESR scores (92% in remission and low disease activity) at baseline. Blood was collected and an ultrasound was performed three times in a year. Percentages of regulatory B cells and T regulatory type 1 cells and M2 macrophage numbers in the blood were examined. Plasma levels of 10 immunoregulatory cytokines IL-4, IL-5, IL-9, IL-10, IL-13, IL-27, IL-35, TGF-β1, sTNF-R1, and sTNF-R2 and monocyte chemotactic protein-1 (MCP-1) were assessed using ELISA assay. The correlations of cytokines and cells with different DAS28 scores and ultrasound grades were investigated, and cytokines and cells were compared between different categories of DAS28 scores and ultrasound grades. Plasma TGF-β1 levels were higher in the DAS28-ESR < 2.6 (remission) subgroup than in the DAS28-ESR ≥ 2.6 (nonremission) subgroup ( p = 0.037). However, plasma TGF-β1 levels were higher in the high ultrasound grade subgroup than those in the low ultrasound grade subgroup ( p = 0.007). The number of M2 macrophages was lower in the DAS28-MCP-1 < 2.2 subgroup than in the DAS28-MCP-1 ≥ 2.2 subgroup ( p = 0.036). The levels of TGF-β1, sTNF-R2, IL-10, and IL-27 were higher in patients with high ultrasound grades than in those with low ultrasound grades. IL-27 was also higher in the nonremission DAS28-ESR subgroup than the remission one ( p = 0.025). Moreover, sTNF-R1 levels in the 2011 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) remission subgroup were significantly lower than in the 2011 ACR/EULAR nonremission subgroup ( p = 0.007). This trend was reflected in that lower sTNF-R1 levels correlated with low DAS28-MCP-1 scores (rho = 0.222, p = 0.007). We conclude that high plasma TGF-β1 levels indicate the DAS28-ESR remission (<2.6) subgroup and the high ultrasound grade subgroup. IL-27 probably connects the nonremission DAS28-ESR to high ultrasound grades. Low sTNF-R1 levels probably link low DAS28-MCP-1 scores with the 2011 ACR/EULAR remission subgroup. It suggests that incongruent immuno-inflammatory abnormalities exist between DAS28 scores and ultrasound grades, and are also dissimilar among various DAS28-formula categories. Therefore, this study may provide a basis for further research into individual cytokines and immunoregulatory cells behind each DAS28 formula and ultrasound grades/scores.

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., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Allergic Rhinitis (AR), a prevalent condition in otorhinolaryngology, is mediated by Type 1 hypersensitivity through IgE, characterized by Type 2 inflammatory response and eosinophil infiltration in the nasal mucosa. Since AR disease exhibits significant heterogeneity in symptom severity, an objective assessment of AR severity may facilitate better individualized treatment., Objective: To explore the changes in peripheral blood IL-9, Th9, and BAFF levels of allergic rhinitis (AR) in patients and the clinical significance associated with it., Methods: A retrospective study selected 80 AR patients admitted from January 2022 to October 2022 as the case group, dividing them into mild and moderate-to-severe groups based on symptom scores. Concurrently, 50 patients without AR, who were treated for nasal bone fractures or underwent septoplasty, were selected as the group for comparison. Alterations in the expression levels of peripheral blood IL-9, Th9, and BAFF were analyzed and compared among the different groups. The diagnostic value of serum BAFF for the severity of AR was analyzed using the receiver operating characteristic (ROC) curve., Results: Noticeable variations were observed in clinical variables among the three groups such as, total IgE levels, peripheral blood eosinophil count and proportion, TNSS, and VAS (P< 0.05), while no statistically significant differences were observed in other variables (P> 0.05). The comparison of IL-9, Th9, and BAFF among the three groups revealed statistically significant differences (P< 0.05). Analysis using multivariate logistic regression revealed that IL-9 (OR = 2.365), Th9 (OR = 2.186), BAFF (OR = 2.307) were influencing factors of moderate-to-severe AR (P< 0.05). The ROC curve indicated that the AUC for the diagnosis of moderate-to-severe AR by IL-9, Th9, BAFF were 0.770, 0.734, 0.761, respectively, and the combined detection AUC was 0.888, an area under the curve higher than individual testing., Conclusion: Changes in peripheral blood IL-9, Th9, and BAFF levels in AR patients may function as indicators to assess the level of severity in diagnostic procedures.

Background: Asthma is a chronic inflammatory disease of airways which accounts for a huge economic, morbidity and mortality burden. There are different cytokines that contribute to asthma pathophysiology. Learning about these cytokines leads to attaining novel anti-inflammatory treatments for asthma control., Objectives: The objective of this study is to investigate the association between interleukin-9 serum level and gene polymorphism with asthma susceptibility., Methods: This was a case-control study of 70 asthmatic patients and 77 healthy control adults aged 18-60. Asthma diagnosis and severity were based on physician diagnosis, pulmonary function test (PFT) and 2016 guild line of Global Initiative for Asthma (GINA). Interleukin 9(IL -9) serum level was measured using sandwich enzyme linked immunosorbent assay.  IL9 promoter single nucleotide polymorphism (SNP) (rs2069882) was also assessed using Real-Time PCR System., Results: There was no significant association between IL-9 SNP polymorphism and asthma. IL-9 serum level was significantly associated with asthma susceptibility (p value= 0.016) and absolute eosinophil count (AEC) (P value=0.033) however its corelation with atopic asthma type, asthma sivierity and Immunoglubin E serum level were not statistically significant., Conclusion: Although there was no association between IL-9 SNP and asthma, but IL-9 serum level was significantly correlated with asthma susceptibility and AEC.

Chronic hepatitis B virus (HBV) infection induces dysfunction of immune response and chronic liver damage. However, the mechanisms that account for HBV-related hepatocellular carcinoma (HCC) are poorly understood. The aim of present study was to investigate the modulatory role of interleukin (IL)-35, an immunosuppressive cytokine, to IL-9-secreting T cells in hepatitis B-related HCC. Twenty-two HBV-related HCC patients, twenty-seven chronic hepatitis B (CHB) patients, and eleven controls were enrolled. Serum IL-35 and IL-9 concentration was measured by ELISA. Peripheral and liver-infiltrating non-specific and HBV-specific Th9 and Tc9 cells were assessed by flow cytometry. The regulatory activity of IL-35 to peripheral and liver-infiltrating Th9 cells was assessed in co-culture system between CD8 + T cells and HepG2.2.15 cells. Serum IL-35 was up-regulated, while IL-9 was down-regulated in HBV-related HCC patients compared with in CHB patients and controls. Peripheral non-specific and HBV-specific Th9 cells, but not Tc9 cells, were decreased in HBV-related HCC patients. Liver-infiltrating non-specific and HBV-specific Th9 cells were also reduced in HCC tumor sites. CD8 + T cells from CHB and HBV-related HCC patients revealed decreased cytotoxicity compared with those from controls. Autologous Th9 cells mediated the elevation of CD8 + T cell cytotoxicity, and this process was depending on IL-9 secretion. Recombinant IL-35 stimulation inhibited IL-9 secretion and PU.1 mRNA expression in non-specific and HBV-specific Th9 cells, leading to the suppression of Th9-mediated CD8 + T cell cytotoxicity in CHB and HBV-related HCC patients. Our current data indicated that IL-35 might dampen non-specific and HBV-specific Th9 cells activity in HBV-related HCC patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Yang, Liu, Zhang and Jin.)

Considering the immunological impairment in age-related macular degeneration (AMD), we aimed to determine the associations of IL-9 rs1859430, rs2069870, rs11741137, rs2069885, and rs2069884 and IL-10 rs1800871, rs1800872, and rs1800896 polymorphisms and their haplotypes, as well as the serum levels of IL-9 and IL-10 with AMD. 1209 participants were enrolled in our study. SNPs were genotyped using TaqMan SNP genotyping assays by real-time PCR method. IL-9 and IL-10 serum levels were evaluated using ELISA kits. Our study results have shown that haplotypes A-G-C-G-G and G-A-T-A-T of IL-9 SNPs are associated with the decreased odds of early AMD occurrence ( p = 0.035 and p = 0.015, respectively). A set of rare haplotypes was associated with the decreased odds of exudative AMD occurrence ( p = 0.033). Also, IL-10 serum levels were lower in exudative AMD than in controls ( p = 0.049), patients with early AMD ( p = 0.017), and atrophic AMD ( p = 0.008). Furthermore, exudative AMD patients with IL-10 rs1800896 CT and TT genotypes had lower IL-10 serum concentrations than those with wild-type (CC) genotype ( p = 0.048). In conclusion, our study suggests that IL-10 serum levels can be associated with a minor allele at IL-10 rs1800896 and exudative AMD. The haplotypes of IL-9 SNPs were also associated with the decreased odds of early and exudative AMD., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Alvita Vilkeviciute et al.)

Recent studies have described the dichotomous function of IL-9 in various cancer diseases. However, its function has still not been analysed in laryngeal squamous cell carcinoma (LSCC). In the present study, we evaluated five single nucleotide polymorphisms (SNPs) of IL-9 (rs1859430, rs2069870, rs11741137, rs2069885, and rs2069884) and determined their associations with the patients' five-year survival rate. Additionally, we analysed serum IL-9 levels using an enzyme-linked immunosorbent assay. Three hundred LSCC patients and 533 control subjects were included in this study. A significant association between the patients' survival rate and distribution of IL-9 rs1859430 variants was revealed: patients carrying AA genotype had a higher risk of dying ( p = 0.005). Haplotypes A-G-C-G-G of IL-9 (rs1859430, rs2069870, rs11741137, rs2069885, and rs2069884) were associated with 47% lower odds of LSCC occurrence ( p = 0.035). Serum IL-9 levels were found detectable in three control group subjects (8.99 ± 12.03 pg/mL). In summary, these findings indicate that the genotypic distribution of IL-9 rs1859430 negatively influences the five-year survival rate of LSCC patients. The haplotypes A-G-C-G-G of IL-9 (rs1859430, rs2069870, rs11741137, rs2069885, and rs2069884) are associated with the lower odds of LSCC development.

T cells play vital roles in the development and progression of acute coronary syndromes (ACS), including cytotoxicity mediated by CD8 + T cells and immunoregulatory activity mediated by CD4 + T cells. Interleukin (IL)-9-secreting CD4 + T cells (Th9 cells) were recently found to be involved in the onset of ACS. We investigated regulatory role of Th9 cells to CD8 + T cells in patients with stable angina pectoris, unstable angina pectoris, and acute myocardial infarction (AMI). Circulating Th9 cells percentage, plasma IL-9 level, and PU.1 mRNA relative level was up-regulated in AMI patients compared with controls. There was no significant difference of IL-9-secreting CD8 + T cells percentage among groups. CD8 + T cells from AMI patients revealed increased cytotoxicity than those from controls, which presented as enhanced cytotolytic activity to target cells, increased interferon-γ and tumor necrosis factor-α secretion, elevated perforin and granzyme B production, and reduced programmed death-1 and cytotoxic T lymphocyte-associated protein 4. IL-9 stimulation did not affect proliferation, but promoted CD8 + T-cell cytotoxicity from both controls and AMI patients. IL-9-secreting CD4 + T cells were enriched in CD4 + CCR4 - CCR6 - CXCR3 - cells. The enhancement of CD8 + T-cell cytotoxicity induced by CD4 + CCR4 - CCR6 - CXCR3 - cells was dependent on IL-9 secretion. The present results indicated that up-regulation of IL-9-secreting CD4 + T cells may contribute to pathogenesis of AMI through enhancement of CD8 + T-cell cytotoxicity., (© 2020 APMIS. Published by John Wiley & Sons Ltd.)

Cytokine dysregulation is the proposed mechanism for Coronavirus disease 2019 (COVID-19). The aim of this study was to evaluate the serum levels of interferon (IFN)-γ, interleukin (IL)-5, IL-8, Il-9, IL-17, TGF-β and IFN-γ in patients infected with SARS-CoV-2. The study was conducted between 63 adult patients with COVID-19 and compared with 33 age and gender-matched healthy subjects as controls. The age range in both groups was 50-70 years. The patients were classified into mild group (33 patients) and severe group (30 patients). Serum samples were collected from all participants and tested for the cytokine levels by ELISA (enzyme-linked immunosorbent assay) method. Statistical analysis was performed using the one-way ANOVA. The mean serum levels of IFN-γ, TGF-β, IL-17 and IL-8 in the COVID-19 patients were significantly higher than those observed in the control group. A comparison of between the mild and severe groups showed significant differences in TGF-β levels. The mean concentration of serum IL-5 and IL-9 in patients with COVID-19 did not differ from those in the control group. Systemic IL-17 levels correlated positively and significantly with TGF-β in patients with COVID-19. Th1 (IFN-γ), Treg (TGF-β), and Th17 (IL-17) cytokines concentration were increased in COVID-19 patients. Interferon-γ and IL-17 are involved in inducing and mediating proinflammatory responses. Our data suggest that TGF-β can be used as a predictive factor of disease severity in patients with COVID-19., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Psoriasis is a T helper cell-mediated chronic immune-mediated inflammatory disease affecting mainly the skin, although systemic pathological effects are also observed. Cytokine-mediated interaction between T lymphocytes and keratinocytes lead to excessive proliferation of keratinocytes, which in turn leads to formation of a proinflammatory milieu and finally to psoriatic plaque formation., Aim: To measure interleukin (IL)-9, IL-17 and vascular endothelial growth factor (VEGF) levels in patients with psoriasis compared with controls, and to evaluate the effect of methotrexate (MTX) monotherapy on the aforesaid cytokine levels in psoriasis., Methods: This cohort study included 54 patients with psoriasis and 54 age- and sex-matched healthy controls (HCs). IL-9, IL-17 and VEGF levels were measured by using commercially available ELISA kits. Patients with psoriasis who were on MTX monotherapy were followed up for a period of 3 months., Results: Patients with psoriasis had increased levels of IL-9, IL-17 and VEGF at baseline, compared with the HC group. After 3 months of MTX monotherapy, Psoriasis Area Severity Index (PASI), Dermatology Life Quality Index (DLQI) and levels of cytokines (IL-9, IL-17 and VEGF) were significantly decreased compared with baseline. PASI and DLQI at baseline also showed a positive correlation with IL-9, IL-17 and VEGF., Conclusion: Our results suggest the existence of a proinflammatory milieu in psoriasis, with increased levels of IL-9, IL-17 and the proangiogenic growth factor VEGF, showing an increasing trend with increasing disease severity and impaired quality of life (QoL). MTX treatment helps to reduce levels of IL-9, IL-17 and VEGF, thereby limiting disease progression and improving QoL in psoriasis., (© 2020 British Association of Dermatologists.)

Background: Neuregulin 4 (Nrg4) is a novel adipocytokine that has been proposed to play a role in modulating energy metabolism and pathogeneses of atherosclerosis. However, no published research is available about the mechanisms underlying the anti-atherosclerotic effect of Nrg4. Regarding the close link between adipocytokines and the immune system, we wonder whether there is a relation between Nrg4 and athero-protective cytokines. The aim of this study was to investigate the relationship between serum Nrg4 levels and type 2 cytokines in Iranian patients with coronary artery disease (CAD)., Methods: In this case-control study, 125 CAD patients were compared to 55 healthy controls. The serum concentrations of Nrg4, IL-5, IL-9, and IL-13 were measured using ELISA. The associations of circulating Nrg4 and IL-5, IL-9, IL-13 were assessed using linear regression analyses., Results: Serum concentration of IL-9 was significantly higher in patients compared to the healthy controls (317.9±139 versus 228.3±99.1, P= ˂0.0001). IL-13 and Nrg4 were significantly lower in patients compared to the healthy controls (4.3±3.7 versus 6.1±3.9, P=0.01 and 0.5 versus 1.3, P=0.001 respectively). Multiple linear regression analyses revealed that IL-9 was negatively correlated with Nrg4 (β= -0.3, P=0.009)., Conclusion: Our study, for the first time, provides the clinical evidence revealing that circulating Nrg4 concentrations are inversely correlated with IL-9 in Iranian patients with CAD, suggesting that the protective role of Nrg4 on atherosclerosis may be, in part, mediated by the proatherogenic cytokine, IL-9., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Malaria strongly predisposes to bacteremia, which is associated with sequestration of parasitized red blood cells and increased gastrointestinal permeability. The mechanisms underlying this disruption are poorly understood. Here, we evaluated the expression of factors associated with mast cell activation and malaria-associated bacteremia in a rodent model. C57BL/6J mice were infected with Plasmodium yoelii yoelli 17XNL, and blood and tissues were collected over time to assay for circulating levels of bacterial 16S DNA, IgE, mast cell protease 1 (Mcpt-1) and Mcpt-4, Th1 and Th2 cytokines, and patterns of ileal mastocytosis and intestinal permeability. The anti-inflammatory cytokines (interleukin-4 [IL-4], IL-6, and IL-10) and MCP-1/CCL2 were detected early after P. yoelii yoelii 17XNL infection. This was followed by the appearance of IL-9 and IL-13, cytokines known for their roles in mast cell activation and growth-enhancing activity as well as IgE production. Later increases in circulating IgE, which can induce mast cell degranulation, as well as Mcpt-1 and Mcpt-4, were observed concurrently with bacteremia and increased intestinal permeability. These results suggest that P. yoelii yoelii 17XNL infection induces the production of early cytokines that activate mast cells and drive IgE production, followed by elevated IgE, IL-9, and IL-13 that maintain and enhance mast cell activation while disrupting the protease/antiprotease balance in the intestine, contributing to epithelial damage and increased permeability., (Copyright © 2020 Céspedes et al.)

Immune regulation status may indicate immunological remission in rheumatoid arthritis (RA). This cross-sectional study aimed to determine the Regulatory T cell (Treg) properties, together with 14 plasma cytokines levels between active RA and clinical remission patients. Peripheral blood (PB) Foxp3+ Treg was collected from RA patients for determination of Treg inhibitory activity using a co-culture system. Other PB T cell types and plasma cytokines were determined by flow-cytometry. The Treg results were analyzed according to the disease activity score-28 (DAS28). Then sensitivity and specificity were calculated for the indication of the remission status. The number and inhibitory activity of Treg are higher in the clinical remission as compared to the active RA (p value < 0.0001). Also, Treg: CD4+CD25+CD127+ cell ratio demonstrates the similar result (p value < 0.05). Treg inhibitory activity is inversely correlated with the DAS28. Specificity and positive likelihood ratio of inhibitory activity for indicating remission status are 92.31% (95% CI 63.97-99.81) and 11.14 (95% CI 1.67-74.14), respectively. Treg inhibitory activity is a promising prognostic marker and probably represents the immunological remission status in RA.

Objective: The contributions of intervertebral disc disease and subject-specific covariates to systemic inflammation in low back pain are unknown. We examined the effects of symptomatic disc herniation (DH) and MRI herniation severity on serum cytokine levels in clinical subjects., Design: Cytokine levels from lumbar DH subjects (N = 78) were compared to control subjects (N = 57) accounting for effects of DH, age, body mass index (BMI) and gender. Effect of DH severity on cytokine levels was analyzed on subsets of subjects with acute or chronic pain. Serum cytokines were also analyzed in a subset of patients between pre- and 3 months post-surgery., Results: Cytokine levels were elevated in the serum of patients with symptomatic DH, and the covariates age, BMI and gender significantly contributed to levels of some cytokines. Severity of herniation was a significant contributor to pain intensity (VAS), serum levels of HMGB1, PDGFbb, and IL-9. The relationship between DH severity and cytokine levels was confirmed in subjects with chronic, but not acute symptoms. Serum levels of macrophage migration inhibitory factor (MIF) decreased, whereas levels of CCL3, CCL11, CXCL1, and CXCL10 were significantly elevated post surgery., Conclusions: This study is the first to show that DH severity is coordinately associated with changes in serum levels of inflammatory cytokines in chronic pain subjects. HMGB1, PDGFbb and IL-9 are novel mediators of increasing DH severity, indicative of cellular damage, neuro-inflammation and angiogenesis. Resolution of inflammation was observed with decrease in MIF post surgery. However, elevated chemokine levels indicate ongoing remodeling and wound healing at 3-month time point., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The aim of the study was to determine the levels of selected cytokines and chemokines in the serum of multiple myeloma (MM) patients treated with bortezomib-based regimens. A total of 71 MM patients were examined: 41 with primary refractory disease (17) or early relapse (28), and 30 who were bortezomib sensitive with no progression for at least six months. Patients who demonstrated CR or PR after bortezomib-based therapies longer than six months after treatment discontinuation were designated bortezomib sensitive. Serum cytokine levels were assayed with Bio-Rad Bio-Plex Pro Human Cytokine 27-Plex Assay on the MAGPIX Multiplex Reader and the Bio-Plex® 200 System (Bio-Rad). Higher levels of MIP-1 α and lower levels of MIP-1 β and IL-9 were associated with better responses to bortezomib-based treatment, and higher levels of IL-1ra and IL-8 were associated with bone involvement. MCP-1 was elevated in patients with hemoglobin < 10 g/dl compared to those without anemia. The levels of IL-8, MIP-1 α , and TNF- α were significantly higher in patients with renal insufficiency. Only MIP-1 α was elevated in patients with hypercalcemia compared to patients with normal calcium levels. In conclusion, distinct cytokines are involved in the pathogenesis of MM and may play a prominent role in the prediction of treatment response. However, a single measurement of serum cytokines should be interpreted with caution and further studies are needed., Competing Interests: The authors have no conflicts of interest that are directly relevant to the content of this article., (Copyright © 2020 Paweł Robak et al.)

Background: Multiple interleukin (IL) family members were reported to be closely related to hypertension. We aimed to investigate whether IL-9 affects angiotensin II- (Ang II-) induced hypertension in mice., Methods: Mice were treated with Ang II, and IL-9 expression was determined. In addition, effects of IL-9 knockout (KO) on blood pressure were observed in Ang II-infused mice. To determine whether the effects of IL-9 on blood pressure was mediated by the signal transducer and activator of the transcription 3 (STAT3) pathway, Ang II-treated mice were given S31-201. Furthermore, circulating IL-9 levels in patients with hypertension were measured., Results: Ang II treatment increased serum and aortic IL-9 expression in a dose-dependent manner; IL-9 levels were the highest in the second week and continued to remain high into the fourth week after the treatment. IL-9 KO downregulated proinflammatory cytokine expression, whereas it upregulated anti-inflammatory cytokine levels, relieved vascular dysfunction, and decreased blood pressure in Ang II-infused mice. IL-9 also reduced smooth muscle 22 α (SM22 α (SM22., Conclusions: IL-9 KO alleviates inflammatory response, prevents phenotypic transformation of smooth muscle, reduces vascular dysfunction, and lowers blood pressure via the STAT3 pathway in Ang II-infused mice. IL-9 might be a novel target for the treatment and prevention of clinical hypertension., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Yunzhao Yang et al.)

Background: Extremely low-frequency electromagnetic fields (ELF-EMFs) are abundantly produced in modern societies. In recent years, interest in the possible effects of ELF-EMFs on the immune system has progressively increased., Objective: To examine the effects of ELF-EMFs with magnetic flux densities of 1, 100, 500, and 2000 μT on the serum levels of interleukin (IL)-9, IL-10, and tumor necrosis factor-alpha (TNF-α)., Methods: 80 adult male rats were exposed to ELF-EMFs at a frequency of 50 Hz for 2 h/day for 60 days. The serum cytokines were measured at two phases of pre- and post-stimulation of the immune system by human serum albumin (HSA)., Results: Serum levels of IL-9 and TNF-α, as pro-inflammatory cytokines, were decreased due to 50 Hz EMFs exposure compared with the controls in the pre- and post-stimulation phases. On the contrary, exposures to 1 and 100 μT 50 Hz EMFs increased the levels of antiinflammatory cytokine, and IL-10 only in the pre-stimulation phase. In the post-stimulation phase, the mean level of serum IL-10 was not changed in the experimental groups., Conclusion: The magnetic flux densities of 1 and 100 μT 50 Hz EMFs had more immunological effects than EMFs with higher densities. Exposure to 50 Hz EMFs may activate anti-inflammatory effects in rats, by down-modulation of pro-inflammatory cytokines (IL-9 and TNF-α) and induction of the anti-inflammatory cytokine (IL-10).

Major depressive disorder (MDD) has a prevalence of 5% in adolescents. Several studies have described the association between the inflammatory response and MDD, but little is known about the relationship between MDD and growth factors, such as IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF. It must be appointed that there are scarce reports on growth factors in adolescents with MDD and even fewer with a clinical follow-up. In this work, we evaluated the levels of growth factors (IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF) in MDD adolescents and the clinical follow-up during eight weeks of treatment with fluoxetine. Methods . All patients were diagnosed according to the DSM-IV-TR, and the severity of the symptoms was evaluated using the Hamilton Depression Rating Scale (HDRS). Growth factors IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF were quantified by cytometric bead array using serum samples from 22 adolescents with MDD and 18 healthy volunteers. Results . All patients showed clinical improvement since the fourth week of pharmacological treatment according to the HDRS. Considerably higher levels of IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF were detected in MDD adolescents as compared to healthy volunteers. A significant but temporal decrease was detected in basic FGF, G-CSF, and GM-CSF at week four of fluoxetine administration. Conclusions . To the best of our knowledge, this is the first report to show alterations in the levels of growth factors, such as IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF in MDD adolescents during eight weeks of clinical follow-up. These disturbances might be involved in the physiopathology of MDD since such growth factors have been proven to participate in the neural development and correct functioning of the CNS; therefore, subtle alterations in it may contribute to MDD., Competing Interests: The authors declare no conflicts of interest associated with the present manuscript., (Copyright © 2019 Enrique Becerril-Villanueva et al.)

Background: Multiple myeloma (MM) is one of the most common types of hematological malignancies, but its pathogenesis is poorly understood. Interleukin-9 (IL-9) is a growth factor, mainly produced by helper T cells. A series of observations suggested that IL-9 might act as a factor promoting oncogenesis. This study was aimed at detecting the serum concentrations of IL-9 in patients with MM, and to investigate its potential clinical significance., Methods: The serum IL-9 levels in 34 patients with MM and 15 normal controls were quantified by using the Enzyme Linked Immunosorbent Assay (ELISA)., Results: Our results showed that the serum IL-9 concentration in MM patients was significantly higher than that in the controls ( p <0.0001). Interestingly, the IL-9 level in serum was found to be negatively associated with the hemoglobin concentration among the newly diagnosed MM patients ( p =0.0108, r =-0.5850). Moreover, MM patients with renal dysfunction showed a significant increase in serum IL-9 concentration over those with normal renal function ( p =0.0395)., Conclusion: These findings may imply a novel role of IL-9 in anemia and/or renal dysfunction development in MM., (© 2019 by the Association of Clinical Scientists, Inc.)

Introduction: Considering the role of interleukin (IL)-9 and IL-9-producing Th9 cells in pathogenesis of autoimmune diseases, this study aimed to evaluate serum IL-9 levels in patients with Behçet's disease (BD) compared to healthy subjects and to assess whether there is an association between serum IL-9 levels and disease characteristics in BD., Methodology: In this cross-sectional study, 32 BD patients according to the International Criteria for BD (ICBD) and 56 age-matched healthy controls were included. In patients, clinical examination was performed and Behcet's Disease Current Activity Form (BDCAF), Iranian Behcet's Disease Dynamic Activity Measure (IBDDAM) and Total Inflammatory Activity Index (TIAI) were assessed. Serum IL-9 level was measured using ELISA kit., Results: The mean ± SD age of patients and controls was 39.06 ± 9.86 and 38.64 ± 8.40 years, respectively and 41% of patients and 66% of controls were males. The most common clinical symptoms in BD patients were oral aphthous ulcers, ocular involvement, and genital ulcers, respectively. The median (Min-Max) of BDCAF, IBDDAM and TIAI in patients were 2 (0-4), 1.3 (0-7), and 2 (0-22), respectively. There was no significant difference in serum IL-9 levels between BD patients (47.12 ± 7.34 mg/dL) and healthy controls (48.61 ± 7.76 mg/dL) (P > 0.05). There were no significant correlations between serum IL-9 levels with BD clinical characteristics as well as with disease severity (P > 0.05)., Conclusion: Our study revealed no significant difference in serum IL-9 between BD patients and healthy controls as well as no significant correlation between serum IL-9 with clinical characteristics and disease severity. Further studies are certainly needed, but on a wider cohort of BD patients to identify IL-9 involvement in BD pathogenesis., (Copyright © 2019 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

The trend for improved more precise diagnostics and management of disease heavily relies on the measurement of panels of biomarkers in physiological samples of patients. Ideally, the ultimate goal would be to detect as many clinically relevant biomarkers as possible in a single drop of blood, achieving quick, sensitive, reproducible, and affordable detection in small volume physiological samples. Bioluminescent (BL) proteins provide many of the desired characteristics required for such labels, including detection at extremely low concentrations, no interference from physiological fluids leading to excellent detection limits, and compatibility with many miniaturized systems. However, to date the use of BL proteins has been restricted by their limited multiplexing capabilities. BL proteins typically exhibit a single emission profile and decay kinetics making the simultaneous detection of multiple analytes difficult. Recent progresses in this area include the use of two different engineered luminescent proteins to achieve resolved signals via one-dimensional time resolution. This approach, however, to date only lead to a dual analyte detection. Herein, we have demonstrated that using a two-dimensional approach that combines both temporal and spatial resolution, we can expand the multiplexing capabilities of bioluminescent proteins. To that end, the photoprotein aequorin (AEQ) has been employed for the simultaneous detection of three separate analytes in a single well, differentiated through the use of three discrete time/wavelength windows. Through a combination of site-specific mutations and synthetic coelenterazines "semi-synthetic" AEQ variants have been developed with altered emission profiles and decay kinetics. In this study, two AEQ mutant proteins were genetically conjugated to three pro-inflammatory cytokines (tumor necrosis factor alpha, interleukins 6 and 8) resulting in AEQ-labeled cytokines. These fusion proteins were combined with synthetic coelenterazines resulting in proteins with differing emission maxima and half-lives to allow for the simultaneous detection of all three cytokines in a single sample. The validity of the assay was demonstrated in serum by employing human physiological samples and comparing our results with commercially available individual tests for each of the three cytokines.

Plant lipid transfer proteins and homologues of the main birch pollen allergen Bet v 1 are involved in the development of allergic reactions of varying severity to plant foods and pollen. In this study, the sera from patients with tree and weed pollen allergies in the Moscow region were examined. The levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-17A, IFNγ, TNFα, and TNFβ cytokines were determined in the sera of patients with specific IgE antibodies to Bet v 1 and Pru p 3 allergens. It was confirmed that patients with pollen allergy are often characterized by Th2 response of the immune system, though other mechanisms of allergy development occurred in some cases. The data obtained demonstrate the necessity of detailed analysis of the individual mechanism of allergic reactions and patient-centered approach to the personalized allergy treatment.

Ulcerative colitis (UC) is a chronic condition in which the overreacting immune system may play an important role. It has been confirmed that the interleukin (IL) 9 (IL-9) participates in the pathogenesis of UC but the molecular mechanism is not fully illustrated. Here, we show that levels of peripheral blood cytokines IL-9, IL-8, IL-10, IL-6, IL-1β, IL-12, and tumor necrosis factor (TNF) were higher in patients with UC than normal control, and serum and local IL-9 levels were positively correlated with the disease activity grade. Moreover, IL-9 stimulation inhibited suppressor of cytokine signaling 3 (SOCS3) expression and wound healing ability in colonic epithelial cells and promoted the phosphorylation level of signal transducers and activators of transcription 3 (STAT3). And IL-9 stimulation promoted claudin-2 expression while inhibited claudin-3 and occludin expression. Furthermore, SOCS3 overexpression rescued the IL-9-induced effects. Altogether, IL-9 participates in the pathogenesis of UC through STAT3/SOCS3 signaling pathway and has the potential to serve as a possible therapeutic candidate in patients with UC., (© 2018 The Author(s).)

Purpose: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has consistently demonstrated clinical efficacy in metastatic melanoma. Recent widespread use of checkpoint blockade has shifted the treatment landscape, raising questions regarding impact of these therapies on response to TIL and appropriate immunotherapy sequence. Patients and Methods: Seventy-four metastatic melanoma patients were treated with autologous TIL and evaluated for clinical response according to irRC, overall survival, and progression-free survival. Immunologic factors associated with response were also evaluated. Results: Best overall response for the entire cohort was 42%; 47% in 43 checkpoint-naïve patients, 38% when patients were exposed to anti-CTLA4 alone (21 patients) and 33% if also exposed to anti-PD1 (9 patients) prior to TIL ACT. Median overall survival was 17.3 months; 24.6 months in CTLA4-naïve patients and 8.6 months in patients with prior CTLA4 blockade. The latter patients were infused with fewer TIL and experienced a shorter duration of response. Infusion of higher numbers of TIL with CD8 predominance and expression of BTLA correlated with improved response in anti-CTLA4 naïve patients, but not in anti-CTLA4 refractory patients. Baseline serum levels of IL9 predicted response to TIL ACT, while TIL persistence, tumor recognition, and mutation burden did not correlate with outcome. Conclusions: This study demonstrates the deleterious effects of prior exposure to anti-CTLA4 on TIL ACT response and shows that baseline IL9 levels can potentially serve as a predictive tool to select the appropriate sequence of immunotherapies. Clin Cancer Res; 24(18); 4416-28. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Osteonecrosis of the femoral head (ONFH) often causes severe symptoms in young people and limits the mobility of the hip joint. Interleukin-9 (IL-9) is a multi-functional inflammatory factor that participates in lumbar disk herniation and arthritis and has been reported in many studies. However, the correlation between IL-9 and ONFH is unclear. The present study aimed to determine the role of IL-9 in the pathogenetic mechanism of osteonecrosis. To assess IL-9 expression in ONFH and femoral neck fracture patients, cartilage tissue was examined through western blot analysis and immunohistochemistry. Human primary chondrocytes were stimulated with IL-9, and inflammation-related cytokines and cartilage matrix-degrading enzymes were assessed via real-time PCR. After being treated with IL-9, Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling were tested through western blot analysis. Our results showed a significant increase in the expression of IL-9 in ONFH patients. IL-9 raised the level of inflammation-related cytokines and cartilage matrix-degrading enzymes and enhanced the activation of JAK-STAT signaling. Furthermore, blocking the JAK-STAT signaling pathway reduced the secretion of inflammation-related cytokines and cartilage matrix-degrading enzymes and markedly alleviated the degradation of the cartilage matrix. These findings provide new insights into the role that IL-9 plays in the pathogenetic mechanism of osteonecrosis and also provide a potential treatment for ONFH., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs) exposure was reported to be associated with childhood asthma. However, the quantitative relationship between PAHs exposure and adult asthma and possible inflammatory pathways are less clear. We aimed to investigate potential associations between urinary PAHs metabolites and adult asthma. We enrolled 507 adult asthma cases and 536 matched controls. The concentrations of 12 urinary PAHs metabolites and plasma cytokines of interleukin (IL)-9 and eotaxin were measured. Potential associations between urinary PAHs metabolites and adult asthma were analyzed by logistic regression. The relationships between urinary PAHs metabolites and plasma cytokines were determined by generalized linear regression. After adjusted for covariates, each 1-unit-increase in natural log-transformed concentrations of 2-hydroxyfluorene (2-OHFLU), 4- hydroxyphenanthrene (4-OHPHE), 1-OHPHE, 2-OHPHE, 1-Hydroxypyrene (1-OHPYR) and ∑OH-PAHs were significantly associated with elevated risk of adult asthma with odds ratios of 2.04, 2.38, 2.04, 1.26, 2.35 and 1.34, respectively. And the associations were more pronounced in the subjects who were female, younger than 45 years, smoker and had history of occupational dust exposure. No associations were observed between urinary PAHs metabolites levels and expressions of IL-9 and eotaxin. Our results demonstrated that elevated urinary PAHs metabolites levels were associated with increased risk of asthma in adults.

Background: Chronic hepatitis B (CHB) is one of the major infectious diseases in which CD4+ T helper (Th) cells play a crucial role. Th9 cells are a distinct subset of CD4+ Th cells with important physiological functions., Objective: The study aimed to assess the potential involvement of Th9 cells in the inadequate immune response leading to chronic HBV infection., Patients and Methods: Peripheral blood samples were collected from 22 CHB patients and 16 healthy controls (HC). The frequencies of circulating Th9, Tc9, Th1, and Tc1 cells were determined by flow cytometry. Serum levels of IL-9 and IL-10 were analyzed by ELISA. Serum biochemical indices of liver function were measured using an automated analyzers. Serum HBV DNA loads were analyzed by real-time PCR. The potential association of the frequency of Th9, Tc9, Th1 or Tc1 cells with clinical parameters was assessed., Results: The frequency of circulating Th9 cells was significantly lower in CHB patients than those in HC. However, no significant differences in the frequency of Tc9, Th1 or Tc1 cells were observed between the two groups. The percentages of Th9 cells, but not Tc9 cells, were negatively correlated with HBV DNA loads, whereas the percentages of Tc1 cells were positively correlated with viral loads in CHB patients. Moreover, there was a positive correlation between serum levels of IL-9 and IL-10 and HBV DNA loads in patients with chronic HBV infection., Conclusion: The depletion of Th9 cells is associated with the development of chronic HBV infection., (© 2017 Wiley Periodicals, Inc.)

Interleukin-9 (IL-9) has been shown to be upregulated in rheumatoid arthritis (RA). The exact role of IL-9 has not yet been effectively studied. Mesenchymal stem cells (MSCs) have shown a promising immunomodulatory role towards repairing cartilage and restoring joint function. One of the key problems influencing the therapeutic efficacy of stem cell therapy is the poor cell survival following transplantation. This is attributed to oxidative and inflammatory stresses at the injured sites. Hesperidin (Hsd), a flavanone present in citrus fruits, has been studied as potential therapeutic agents that have anti-oxidant and anti-inflammatory activities. The objective of this study is to evaluate the therapeutic paracrine action of bone marrow MSCs on the IL-9 level in adjuvant-induced arthritis (AIA) and the enhancement effect of Hsd on transplanted MSCs. Articular tissue inflammation and cartilage damage were assessed by histological scoring. Antinuclear autoantibodies, tumour necrosis factor-alpha (TNF-α), IL-9, IL-4, interferon gamma (IFN-δ), and transforming growth factor-beta1 (TGF-β1), as well as malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) levels, were assessed in spleen tissue homogenates after treatment with MSCs either alone or combined with Hsd for 4 weeks in an AIA rat model. Results of this study confirmed that MSCs decreased IL-9 levels in AIA and provide novel insights into the application of Hsd on MSC-based treatments. Highlights Adjuvant-induced arthritis (AIA) is one of the most widely used models that has a great similarity to rheumatoid arthritis (RA). Few studies in recent years have estimated IL-9 in rheumatic diseases and it remains an understudied cytokine. For the first time, bone marrow mesenchymal stem cells (MSCs) therapy has a vital role in splenocytes IL-9 level and further studies are required. Combined therapy of MSCs with antioxidants as hesperidin (Hsd) can alleviate oxidative stress and enhance stem cells immunomodulatory action.

T helper 9 (Th9) cells and interleukin (IL)-9 are involved in the pathogenesis of several autoimmune diseases. The exact role of IL-9 and Th9 cells in patients with systemic sclerosis (SSc) have not yet been studied adequately. IL-9, IL-9R, transcription factor PU.1 (PU.1), IL-4, thymic stromal lymphopoietin (TSLP) and transforming growth factor (TGF)-β expression were assessed in skin and kidney biopsies of SSc patients and healthy controls (HC) by immunohistochemistry (IHC). The cellular source of IL-9 was also analysed by confocal microscopy analysis. Peripheral IL-9-producing cells were also studied by flow cytometry. The functional relevance of IL-9 increased expression in SSc was also investigated. Our results demonstrated a strong expression of IL-9, IL-9R, IL-4, TSLP and TGF-β in skin tissues of patients with both limited and diffuse SSc. IL-9 expression was observed mainly in the context of skin infiltrating mononuclear cells and keratinizing squamous epithelium. IL-9 over-expression was also observed in renal biopsies of patients with SSc. IL-9 producing cells in the skin were identified as Th9 cells. Similarly, Th9 cells were expanded and were the major source of IL-9 among SSc peripheral blood mononuclear cells (PBMC), their percentage being correlated directly with the modified Rodnan skin score. Infiltrating mononuclear cells, mast cells and neutrophils expressed IL-9R. In in-vitro studies stimulation with rIL-9 significantly induced NET (neutrophil extracellular traps) release by dying cells (NETosis) in neutrophils, expansion of mast cells and increase of anti-systemic scleroderma 70 (Scl70) production by B cells. Our findings suggest that Th9 cells and IL-9 could be implicated in the pathogenesis of SSc., (© 2017 British Society for Immunology.)

Autism spectrum disorder (ASD) gradually develops predominantly neurodevelopmental disorders, which are socially diagnosed in early childhood. Though the etiopathology of ASD is not clear, immune alteration has been suggested as autism's pathophysiological mechanism. Previous studies found that several cytokines and transcription factor activation pathways were significantly increased in ASD. IL-9 has been confirmed to play a significant role in the central nervous system (CNS). The aim of the present study was to investigate the understudied role of pro- and anti-inflammatory cytokines and the JAK-STAT signaling pathway in ASD. We examined the IL-1β, IL-4, IFN-γ, and IL-9 positive immunostaining in all cells, and CD4 + T cells, in ASD and normally developing control children (TD), on peripheral blood mononuclear cells (PBMCs), using flow cytometry. We explored PBMC mRNA expression levels for IL-1β, IL-4, IFN-γ, IL-9, JAK1, and STAT5, by using real-time PCR (RT-PCR). We also explored PBMC protein expression levels for IL-1β, IL-4, IL-9, pJAK1, and pSTAT5 by using western blotting. We found that the children with ASD had increased IL-1β, IL-4, IFN-γ, and IL-9 positive immunostaining in all cells, and in CD4 + cells, relative to the TD controls. The mRNA and protein expression for IL-1β, IL-4, IFN-γ, IL-9, JAK1, pJAK1, STAT5, and pSTAT5 were also significantly elevated in ASD relative to TD controls. These results suggested that cytokines and JAK-STAT activation signaling have an essential role in immune dysfunction in ASD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Interleukin (IL)-9 drives gut inflammation, but its role in Crohn's disease (CD) is unclear. We aimed to analyze correlations between serum IL-9 levels and disease severity and to evaluate their predictive value in relation to the clinical efficacy of infliximab (IFX) in patients with CD., Methods: Between January 2013 and December 2015, 100 consecutive patients with active CD and 50 age- and sex-matched control individuals were recruited from a tertiary center. Their serum IL-9 levels were measured using an enzyme-linked immunosorbent assay. Correlations between the serum IL-9 levels and disease severity were examined. The serum IL-9 level was explored as a predictor of clinical remission and mucosal healing at week 30 in 50 patients for whom IFX therapy was administered., Results: The serum IL-9 levels were significantly higher in the patients with active CD (22.0 pg/mL) than in the control individuals (6.3 pg/mL) (P < 0.001); they differed according to disease severity (moderate-to-severe CD: 29.1 pg/mL versus mild CD: 12.9 pg/mL) (P < 0.001), and they correlated well with the clinical activity of CD. IFX lowered the serum IL-9 level in patients who achieved efficacy at week 30. The areas under the curves for the IL-9 levels at weeks 14 and 30 that could predict clinical remission and mucosal healing at week 30 were 0.803 and 0.752 and 0.746 and 0.781, respectively., Conclusions: Serum IL-9 levels correlate with disease severity and the clinical efficacy of IFX in patients with CD, and IL-9 may be a promising novel biomarker for CD monitoring.

Although allergic inflammation is characterized by a T helper (Th) 2-dominant immune response, the discovery of a role for new T cell subsets in inflammatory diseases has added an additional layer of complexity to the understanding of the pathogeneses of allergic diseases. We evaluated plasma cytokine profiles in infants with cows' milk allergy (CMA), who were being treated with an elimination diet. In a prospective, randomized and controlled study, infants (aged 8.4 ± 3.9 months) with CMA were treated with an elimination diet for 120 days, which replaced cows' milk with a hydrolysed soy protein formula ( n = 26) or a free amino acid formula ( n = 20). Blood samples were collected before treatment during active disease (T0) and after 120 days, when symptoms were absent (T1). Plasma cytokine concentrations were measured. Infants with CMA had higher plasma concentrations of interleukin (IL)-4 and IL-13 and lower concentrations of IL-9, IL-17A and interferon-γ, compared with healthy breast-fed infants. At T0, there was a positive correlation between blood eosinophil numbers and plasma concentrations of IL-4, IL-9, IL-17A and IL-22. Treatment with a cows' milk elimination diet resulted in a decrease in plasma IL-4, IL-9, IL-13 and IL-22 and an increase in plasma IL-17A. We conclude that IL-4 and IL-13 are elevated in active CMA. The association of IL-9 and IL-22 with eosinophilia, and the decrease in these two cytokines with cows' milk elimination, suggests that they both play a role in the symptoms observed in CMA and may be important targets for future interventions., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

We aimed at investigating whether the frequency and function of T helper 17 (Th17) and regulatory T cells (Treg) are affected by a restriction of dietary sodium intake in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We enrolled RA and SLE patients not receiving drugs known to increase urinary sodium excretion. Patients underwent a dietary regimen starting with a restricted daily sodium intake followed by a normal-sodium daily intake. The timepoints were identified at baseline (T0), after 3 weeks of low-sodium dietary regimen (T3), after 2 weeks of normal-sodium dietary regimen (T5). On these visits, we measured the 24-hour urinary sodium excretion, the frequency and function of Th17 and Treg cells in the peripheral blood, the serum levels of cytokines. Analysis of urinary sodium excretion confirmed adherence to the dietary regimen. In RA patients, a trend toward a reduction in the frequencies of Th17 cells over the low-sodium dietary regimen followed by an increase at T5 was observed, while Treg cells exhibited the opposite trend. SLE patients showed a progressive reduction in the percentage of Th17 cells that reached a significance at T5 compared to T0 (p = 0.01) and an increase in the percentage of Treg cells following the low-sodium dietary regimen at both T1 and T3 compared to T0 (p = 0.04 and p = 0.02, respectively). No significant apoptosis or proliferation modulation was found. In RA patients, we found a reduction at T5 compared to T0 in serum levels of both TGFβ (p = 0.0016) and IL-9 (p = 0.0007); serum IL-9 levels were also reduced in SLE patients at T5 with respect to T0 (p = 0.03). This is the first study investigating the effects of dietary sodium intake on adaptive immunity. Based on the results, we hypothesize that a restricted sodium dietary intake may dampen the inflammatory response in RA and SLE patients.

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., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Th1, Th2, Th17, Treg and Tfh cells play important roles in schistosomiasis. Th9 cells secrete IL-9 as a signature cytokine and contribute to several classes of inflammatory disease. However, the effects of Th9 cells in schistosomiasis are unknown. We aimed to explore the dynamic changes and potential roles of Th9 cells in the pathogenesis of hepatic egg granulomatous inflammation in mice infected with Schistosoma japonicum., Methods: Twenty mice with S. japonicum infection and five normal controls (NC) were used as models. The average areas of egg granulomas were estimated by hematoxylin-eosin (H & E) staining. Hepatic IL-9 and transcription factor PU.1 levels were detected by immunohistochemistry. Flow cytometry techniques were used to analyze the proportions of Th9 cells. With the help of ELISA, serum levels of IL-9 were examined., Results: The egg granulomas began to form from four weeks after infection and continued to develop. In parallel with the development of egg granulomas, the hepatic levels of IL-9 and PU.1 increased very slowly during the first four weeks post-infection and increased rapidly thereafter. Moreover, the proportions of splenic Th9 cells and levels of serum IL-9 had similar developmental trends with the egg granulomas., Conclusion: The proliferation of Th9 cells and levels of IL-9 were significantly higher in S. japonicum-infected mice compared to NC. In addition, dynamic changes of Th9 and IL-9 were synchronous with the developmental trend of hepatic egg granulomatous inflammation, suggesting that Th9 cells might be a new subset in the pathogenesis of schistosomiasis.

Aim: To evaluate circulating IL9 in inflammatory bowel disease and disease-associated anemia/cachexia and assess its potential as a mucosal healing marker., Methods: Serum IL9 as well as other cytokines (IL1β, IL6, IL13, IFNγ, TNFα, and VEGF-A) were determined in 293 individuals: 97 patients with Crohn's disease (CD) and 74 with ulcerative colitis (UC) and in 122 apparently healthy controls. The clinical activity of CD and UC was expressed in terms of the Crohn's Disease Activity Index (CDAI) and the Mayo Scoring System (MDAI), respectively, and the severity of bowel inflammation in UC patients was assessed using Mayo endoscopic score. Cytokine concentrations were measured by a flow cytometry-based method using Luminex xMAP ® technology. High-sensitive C-reactive protein concentrations (hsCRP) were determined in CD and UC patients using the enhanced immunoturbidimetric method., Results: Systemic IL9 was significantly lower in healthy individuals [9 pg/mL (95%CI: 8.2-10)] than in patients with inflammatory bowel disease (IBD): both inactive [14.3 pg/mL (11.9-19.9)] and active [27.6 pg/mL (24.5-32), P < 0.0001]. Cytokine concentrations were significantly higher in active CD [27.4 pg/mL (23.4-32.2)] and in active UC [32.7 pg/mL (27-38.9)] compared to inactive diseases [15.9 pg/mL (10.8-23.4) in CD and 19.4 pg/mL (13.9-27.1) in UC, P = 0.001]. IL9 correlated weakly with CDAI ( ρ = 0.32, P = 0.003) and MDAI ( ρ = 0.35, P = 0.002) and strongly with endoscopic inflammation in UC ( ρ = 0.74, P < 0.0001). As a negative marker of mucosal healing (MH), IL9 had an accuracy superior to hsCRP and IL6 [97% ( P < 0.0001), 67% ( P = 0.071), and 55% ( P = 0.525), respectively]. IL9 was significantly higher in cachectic IBD patients [30.25 pg/mL (24.4-37.5) vs 21.88 pg/mL (18-26.5), P = 0.026] and negatively correlated with hemoglobin concentrations ( ρ = -0.27, P < 0.001). Multiple regression showed IL1β and IL13 to be the independent predictors of circulating IL9 in healthy individuals, IFNγ or IL6 in active and inactive UC, respectively, and IL13 and VEGF-A in both active and inactive CD., Conclusion: The systemic IL9 level is higher in IBD and corresponds with endoscopic inflammation, suggesting its possible application as a negative marker of mucosal healing in UC., Competing Interests: Conflict-of-interest statement: We have no competing interest to declare.

Liver granulomatous inflammation and fibrosis were the primary pathological changes observed during Schistosoma japonicum (S. japonicum) infection. In the present study, the characteristics of IL-9 were investigated in the liver of S. japonicum infection C57BL/6 mice. Immunofluorescence, qRT-PCR, and ELISA results demonstrated that the expression of IL-9 significantly increased after infection (P < 0.01). FACS results indicated that the peak of IL-9 + Th9 cells in the liver mononuclear cells appeared at the early phase of infection (week 5), except that Th9 cells, CD8 + Tc cells, NKT and γδT cells could secrete IL-9 in this model. Although IL-9 neutralization has a limited effect on liver granulomatous inflammation, it could decrease the level of fibrosis-associated factor, PC-III, in the serum of infected mice (P < 0.05). Taken together, our results indicated that IL-9 was an important type of cytokine involved in the progression of S. japonicum infection-induced hepatic damage.

Currently, therapy for squamous cancer (SqC) is unsatisfactory. Staphylococcal enterotoxin B (SEB) has strong immune regulatory activity. This study tests the hypothesis that SEB enforces the effect of immunotherapy on SqC growth in a mouse model. C3H/HeN mice and the SqC cell line squamous cell carcinoma VII were used to create an SqC mouse model. Immune cell assessment was performed by flow cytometry. Real-time RT-PCR and western blotting were used to evaluate target molecule expression. An apoptosis assay was used to assess the suppressive effect of T helper-9 (Th9) cells on the SqC cells. The results showed that immunotherapy consisting of SEB plus SqC antigen significantly inhibited SqC growth in the mice. The frequency of Th9 cells was markedly increased in the SqC tissue and mouse spleens after treatment. SEB markedly increased the levels of signal transducer and activator of transcription 5 phosphorylation and the expression of histone deacetylase-1 (HDAC1) and PU.1 (the transcription factor of the interleukin 9 (IL-9) gene) in CD4 + T cells. Exposure to SqC-specific Th9 cells markedly induced SqC cell apoptosis both in vitro and in vivo. In conclusion, the administration of SEB induces Th9 cells in SqC-bearing mice, and theseTh9 cells inhibit SqC growth.

Hepatocellular carcinoma (HCC) is a common and aggressive human malignancy. An imperative demand is present for a better understanding of the functions and regulations of the immune system and how they affect HCC pathogenesis. Recently, a group of interleukin 9 (IL-9)-producing CD4 + T cells, termed Th9 cells, has been described in mice and humans with both tumour-inhibiting as well as tumour-promoting effects. The specific roles of Th9 cells in human HCC are not entirely understood. Here, we examined the frequencies and functions of IL-9-producing Th9 cells in HCC patients. We found that the frequencies of circulating IL-9-producing Th9 cells were significantly higher in HCC patients compared to in healthy individuals. In HCC patients, the frequencies of IL-9-producing Th9 cells were significantly higher in peritumour and tumour tissues than in unaffected liver tissues. Interestingly, HCC patients with higher tumour-infiltrating Th9 frequency had significantly shorter disease-free survival period after resection. Previously, high expression of CCL20 was associated with poor prognosis in HCC. CCL20 also induced epithelial-mesenchymal transition-like changes in HCC cells. We found that incubation of primary HCC cells with autologous Th9 significantly elevated the CCL20 production from tumour cells, which could be partially inhibited by suppressing STAT3. Together, this study suggested a tumour-promoting role of Th9 cells in HCC., (© 2016 John Wiley & Sons Australia, Ltd.)

Increased free radical production had been documented in group A ( β -hemolytic) streptococcus infection cases. Comparing 71 erysipelas patients to 55 age-matched healthy individuals, we sought for CAT, SOD1, and SOD2 single polymorphism mutation (SNPs) interactions with erysipelas' predisposition and serum cytokine levels in the acute and recovery phases of erysipelas infection. Whereas female patients had a higher predisposition to erysipelas, male patients were prone to having a facial localization of the infection. The presence of SOD1 G7958, SOD2 T2734, and CAT C262 alleles was linked to erysipelas' predisposition. T and C alleles of SOD2 T2734C individually were linked to patients with bullous and erythematous erysipelas, respectively. G and A alleles of SOD1 G7958A individually were associated with lower limbs and higher body part localizations of the infection, respectively. Serum levels of IL-1 β , CCL11, IL-2R α , CXCL9, TRAIL, PDGF-BB, and CCL4 were associated with symptoms accompanying the infection, while IL-6, IL-9, IL-10, IL-13, IL-15, IL-17, G-CSF, and VEGF were associated with predisposition and recurrence of erysipelas. While variations of IL-1 β , IL-7, IL-8, IL-17, CCL5, and HGF were associated with the SOD2 T2734C SNP, variations of PDFG-BB and CCL2 were associated with the CAT C262T SNP.

Objectives . To investigate the potential association of a set of serum cytokines with the severity of coronary artery disease (CAD). Methods . A total of 201 patients who underwent coronary angiography for chest discomfort were enrolled. The concentrations of serum IFN- γ , TNF- α , IL-2, IL-4, IL-6, IL-10, IL-9, and IL-17 were determined by xMAP multiplex technology. The CAD severity was assessed by Gensini score (GS). Results . The serum levels of TNF- α , IL-6, IL-9, IL-10, and IL-17 were significantly higher in high GS group (GS ≥ 38.5) than those in low GS group (GS < 38.5). Positive correlations were also found between these cytokines and the severity of CAD. After adjustment for other associated factors, three serum cytokines (IL-6, IL-9, and IL-17) and two clinical risk factors (creatinine and LDL-C) were identified as the independent predictors of increased severity of CAD. ROC curve analysis revealed that the logistic regression risk prediction model had a good performance on predicting CAD severity. Conclusions . Combinatorial analysis of serum cytokines (IL-6, IL-9, and IL-17) with clinical risk factors (creatinine and LDL-C) may contribute to the evaluation of the severity of CAD and may help guide the risk stratification of angina patients, especially in primary health facilities and in the catheter lab resource-limited settings., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Takayasu's arteritis (TAK) is a type of large vessel vasculitis which involves the aorta and its major branches. Interleukin (IL)-9 or IL-9-producing Th9 cells were found to be involved in pathogenesis of autoimmune arteritis such as giant cell arteritis, but IL-9 or Th9 cells in TAK were not well known. Here, this study aims to analyze the levels of serum IL-9 and their major source Th9 cells in TAK. With the help of cytometric bead array (CBA), a total of 21 patients with TAK were examined for serum levels of cytokines IL-4, IL-6, IL-8, IL-17, IL-10, TNF-α, IFN-γ, and IL-9. Flow cytometry techniques were used to examine the frequencies of Th9 cells from peripheral blood mononuclear cells (PBMCs) for 11 patients with active TAK and 10 healthy controls. Higher serum levels of serum IL-6 (P < 0.05), TNF-α (P < 0.05), and IL-9 level (P < 0.05) were observed in TAK patients compared to those of healthy controls. Higher frequencies of CD4 + IL-9 + T cells and CD4 + PU.1 + T cells in PBMCs and IL-9 + PU.1 + T cells in CD4 + T cells were observed in active TAK patients than those in healthy controls (all P < 0.01). The levels of IL-9 had a positive correlation with ESR (r = 0.975, P = 0.015) in these cases. Our data suggested that Th9 cells and IL-9 could possibly be involved in the pathogenesis of TAK.

Serum levels of IL-9 and IL-17 cytokines were evaluated in patients in the acute, chronic phases and clinical forms of human schistosomiasis and in different classifications of periportal fibrosis. No significant differences between the groups of the disease with serum levels of cytokine were found. However, this study discusses the results of some cytokines that have not fully defined roles in the pathology of human schistosomiasis. Furthermore, an examination was made of subjects in the acute phase. This is an important group that is difficult to identify in areas where the disease is endemic. More studies are being undertaken to study the role of IL-9 and IL-17 in human Schistosoma mansoni infection and their relationship with the immunopathogenesis of disease., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Objective To investigate the expression and clinical significance of interleukin 9 (IL-9) in patients with primary biliary cirrhosis (PBC). Methods The study enrolled 80 PBC patients and 100 healthy subjects. Levels of serum IL-9, interferon γ (IFN-γ), IL-6, tumor necrosis factor α (TNF-α), IL-10 and IL-17 were determined by cytometric beads array (CBA); level of IL-9 mRNA in peripheral blood mononuclear cells (PBMCs) was detected by quantitative real-time PCR; the expression and location of IL-9 in liver tissues were detected by immunohistochemistry. Results Serum levels of IL-9, TNF-α, IL-6, IL-17, IFN-γ and IL-10 significantly increased in PBC patients in comparison with healthy controls, and a positively correlation between IL-9 and IgG was observed in PBC patients. In addition, the expression of IL-9 mRNA in PBC groups increased. Immunohistochemical results suggested that positive cells appeared in the PBC liver tissue, mainly in the portal area. Conclusion Expression of IL-9 remarkably increases in peripheral blood and liver tissues in patients with PBC.

The role of adaptive immune cytokines in the pathogenesis of type-1 Diabetes Mellitus (T1DM) is well known. Even though reports on the serum levels of both Th-1 and Th-2 cytokines are available, those on newly described T cell cytokines such as IL-17, IL-33 and IL-9 in T1DM are scarce. We therefore measured the serum levels of both T cell polarizing (IL-33 and IL-12) and T cell effector (IFN-γ, IL-4, IL-10, IL-17 and IL-9) cytokines in T1DM subjects with and without microvascular (retinopathy and nephropathy) complications (MVC). All the tested cytokines were significantly elevated in T1DM subjects except for IFN-γ (which failed to attain statistical significance) with no significant difference between those with and without MVC. From the serum cytokine analysis, no apparent Th polarization could be determined for the T1DM subjects., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The aim of this study was to determine the prevalence of T helper 9 (Th9) lymphocytes in rheumatoid arthritis (RA) patients and to identify a possible association between the percentage of Th9 and the discontinuation of a biological treatment with an anti-tumor necrosis factor (TNF) (infliximab). We collected peripheral blood mononuclear cells (PBMCs) from 55 consecutive RA outpatients and 10 healthy controls. Among RA patients, 15 were not receiving any immunosuppressive drug, 20 were successfully treated with infliximab and 20 discontinued infliximab because of adverse events or inefficacy and were treated with other biological agents. PBMCs were cultured with/without infliximab 50 mg/L for 18 h, and the percentage of Th9 cells was assessed by means of flow cytometry. Th9 lymphocytes were identified as interferon gamma, interleukin (IL)4-, IL17-, IL9-secreting cluster of differentiation 4 (CD4)+ T cells. Cytometric analysis revealed no significant decrease in the percentage of Th9 cells after infliximab exposure in any of the groups, although it was lower in healthy controls than RA patients either before and after the infliximab stimulation assay. Th9 cells are IL-9-secreting T helper lymphocytes whose role in RA is still poorly known. IL-9 levels are increased in RA patients, in whom this cytokine plays a crucial role. Th9 cells are the major producers of IL-9, and their prevalence is higher in RA patients than in healthy subjects; however our experiment in vitro does not demonstrate an association between Th9 lymphocytes and the response to infliximab. Further studies are required to evaluate the real involvement of Th9 population in the immunogenicity of anti-TNF agents.

Objective: To investigate the changes in the expression levels of peripheral blood T helper 9 (Th9) cells and cytokine interleukin-9 (IL-9) in children in the acute stage of Kawasaki disease (KD) and their clinical significance., Methods: A total of 45 children in the acute stage of KD who were treated from April 2014 to July 2015 were enrolled, and the children were followed up in the recovery stage. Another 45 healthy children who underwent physical examination were enrolled as the control group. Flow cytometry was used to measure the percentage of peripheral blood Th9 cells, and ELISA was used to measure the serum level of IL-9., Results: The children in the acute stage of KD showed a significantly higher percentage of Th9 cells and a significantly higher serum level of IL-9 compared with those in the recovery stage and the control group (P<0.05). The percentage of Th9 cells and serum level of IL-9 showed no significant differences between the children in the recovery stage and those in the control group (P>0.05). In the acute stage, the percentage of Th9 cells was positively correlated with the levels of IL-9, C-reactive protein (CRP), procalcitonin (PCT), erythrocyte sedimentation rate (ESR), platelet count (PLT), and globulin (r=0.624, 0.324, 0.402, 0.382, 0.467, and 0.386 respectively, all P<0.05), but negatively correlated with serum albumin (r=-0.306, P<0.05). The serum level of IL-9 was positively correlated with the levels of CRP, PCT, ESR, PLT, and globulin (r=0.365, 0.456, 0.403, 0.423, and 0.453 respectively, all P<0.05), but negatively correlated with serum albumin (r=-0.343, P<0.05)., Conclusions: The children in the acute stage of KD show significant increases in the percentage of peripheral Th9 cells and serum cytokine IL-9 level, which return to normal in the recovery stage. In the acute stage of KD, the expression levels of Th9 and IL-9 are closely correlated with laboratory markers. The results suggest that Th9 cells and IL-9 play important roles in the pathogenesis and outcome of KD.

The purpose of this study was to examine the roles of T helper 9 (Th9) cells and the serum interleukin (IL)-9 level in the pathogenesis of osteoarthritis (OA). The numbers of IL-9(+) CD4(+) CD8(-) T cells, interferon (IFN)-γ+ CD4(+) CD8(-) T cells, IL-4(+) CD4(+) CD8(-) T cells, and IL-17A(+) CD4(+) CD8(-) T cells in 25 OA patients and 13 healthy controls (HC) were examined by flow cytometry. The serum concentrations of IL-9, IL-4, IL-17A, and IFN-γ were also determined. The numbers of CD4(+) CD45RO(+) T cells, Th9 cells, Th1 cells, and Th17 cells in OA patients were significantly higher than those in HCs. Furthermore, serum IL-9, IL-17A, and IFN-γ levels in OA patients were higher than those in HCs. The number of Th9 cells was positively correlated with the number of Th17 cells in OA patients. Furthermore, greater numbers of Th9 cells were positively associated with elevated C-reactive protein, and both Th9 cells and IL-9 levels were positively correlated with the Western Ontario and McMaster Universities Osteoarthritis index in OA patients. Th9 cell numbers and IL-9 levels are correlated with OA patient symptoms and joint functionality and may be a marker of disease activity., (© 2016 John Wiley & Sons Australia, Ltd.)