Níveis de Interleucina-35 em Pacientes com Doença Arterial Coronariana Estável / Interleukin-35 Levels in Patients with Stable Coronary Artery Disease

Resumo Fundamento Foi demonstrado que as subunidades de interleucina-35 (IL-35) estão fortemente expressas nas placas ateroscleróticas em humanos. Assim, considera-se que elas têm um papel na aterosclerose. Objetivos Neste estudo, os níveis de IL-35 foram comparados com o grupo controle em pacientes com doença arterial coronariana (DAC) estável, e a associação entre os níveis de IL-35 e o tipo, gravidade e extensão da lesão foram investigadas com o escore Gensini (GS) e o escore Syntax (SS) no grupo de pacientes Métodos Sessenta pacientes (18 mulheres e 42 homens) com DAC, diagnosticados por meio da angiografia coronária, que apresentaram dor no peito típica e teste de esforço não invasivo positivo, e 46 pacientes (18 mulheres e 28 homens) com luminograma normal, foram incluídos no estudo. Tanto o GS quanto o SS foram calculados para o grupo de pacientes, e esses valores foram comparados com os níveis de IL-35. Variáveis com distribuição não normal foram avaliadas com o teste U de Mann-Whitney, enquanto os parâmetros com distribuição normal foram analisados com o teste t de Student. A diferença entre as variáveis categóricas foi avaliada pelo teste de qui-quadrado ou de Fisher. Os valores de p<0,05 foram considerados como estatisticamente sinificativos. Resultados Não foram observadas diferenças significativas entre pacientes e o grupo controle em termos de características demográficas e achados laboratoriais. Em comparação ao grupo controle, os níveis de IL-35 no grupo com DAC foram consideravalmente menores (36,9±63,9 ng/ml vs. 33,2±13,2 ng/ml, p<0,008). Embora não tenha sido estatisticamente significativo, os níveis de IL-35 foram maiores em pacientes com SS mais baixo do que nos com SS mais alto (33,2±13,7 vs. 31,8±8,9, p=0,51). Os valores de IL-35 em pacientes com GS alto foram significativamente mais baixos do que em pacientes com GS baixo (35±17,4 vs. 30,7±8,6, p=0,043). Conclusão Demonstrou-se que os níveis de IL-35 podem ser um novo biomarcador para a DAC estável, e que a IL-35 está associada à extensão da DAC.

Abstract Background It has been shown that interleukin-35 (IL-35) subunits are strongly expressed in atherosclerotic plaques in humans. Therefore, it is considered to play a role in atherosclerosis. Objectives In this study, IL-35 levels were compared with the control group in patients with stable coronary artery disease (CAD), and the association between IL-35 levels and the lesion type, lesion severity and extension was investigated with the Gensini score (GS) and the Syntax score (SS) in the patient group. Methods Sixty patients (18 female and 42 male) with CAD diagnosed by coronary angiography, who presented with typical chest pain and positive noninvasive cardiac stress test, and 46 patients (18 female and 28 male) with normal coronary lumenogram, were included in this study. Gensini and Syntax scores were calculated in the patient group, and these values were compared with IL-35 levels. Non-normally distributed variables were analyzed by the Mann-Whitney U test, whereas normally distributed parameters were assessed by Student's t-test. The difference between categorical variables were evaluated by the Chi-square or Fisher test. P-values<0.05 were considered as statistically significant. Results No significant differences were observed between patients and the control group in terms of demographic characteristics and laboratory findings. Compared to the control group, IL-35 levels of the CAD group were considerably lower (36.9±63.9 ng/ml vs. 33.2±13.2 ng/ml, p<0.008). Although not statistically significant, IL-35 levels were higher in patients with low SS than among those with high SS (33.2±13.7 vs. 31.8±8.9, p=0.51). The IL-35 values of the patients with high GS were significantly lower than in patients with low GS (35±17.4 vs. 30.7±8.6, p=0.043). Conclusion It has been shown that IL-35 levels can be a new biomarker for stable CAD, and IL-35 is associated with the extension of CAD.

Expression of peripheral interleukin-35 and its modulatory effect on regulatory T cell functions in patients with alopecia areata / 中华皮肤科杂志

Objective:To investigate changes in the peripheral interleukin-35 (IL-35) level in patients with alopecia areata, and to assess its modulatory effect on regulatory T (Treg) cell activities.Methods:Totally, 81 patients with alopecia areata (alopecia areata group) and 27 healthy volunteers (control group) were enrolled from Shanxi Provincial People′s Hospital between December 2019 and January 2021. Sera and peripheral blood mononuclear cells (PBMCs) were isolated. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the serum IL-35 level, real-time fluorescence-based quantitative PCR to determine the mRNA expression of IL-35 subunits EBI3 and IL-12p35, and flow cytometry to determine the proportion of CD4 + CD25 + CD127 dim/- Treg cells. Sorted Treg cells were stimulated by recombinant human IL-35, ELISA was performed to detect levels of perforin and granzyme B in the culture supernatant, and real-time fluorescence-based quantitative PCR to determine the mRNA expression of EBI3, IL-12p35, and immune checkpoint molecules, such as programmed death protein 1 (PD-1) , T cell immunoglobulin and mucin protein-3 (Tim-3) , cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and lymphocyte activation gene-3 (LAG-3) in Treg cells. IL-35-stimulated or unstimulated Treg cells were co-cultured with autologous PBMCs, and cell counting kit-8 (CCK8) assay was conducted to assess cellular proliferative activity. Measurement data were compared between 2 groups by using t test, comparisons among multiple groups were carried out by using one-way analysis of variance, correlation analysis was carried out by using Pearson correlation analysis, and enumeration data were compared by using chi-square test. Results:Compared with the control group, the alopecia areata group showed significantly decreased IL-35 levels (90.10 ± 11.98 ng/L vs. 100.74 ± 28.71 ng/L, t= 2.71, P= 0.008) , mRNA expression of EBI3 and IL-12p35 in PBMCs (EBI3: 1.06 ± 0.15 vs. 1.25 ± 0.11, t= 6.09, P < 0.001; IL-12p35: 1.00 ± 0.15 vs. 1.38 ± 0.22, t= 10.16, P < 0.001) , and proportions of Treg cells (5.91% ± 1.17% vs. 6.85% ± 1.23%, t= 3.54, P= 0.001) . In the alopecia areata group, the proportion of Treg cells was positively correlated with the serum IL-35 level ( r= 0.25, P= 0.026) , and the mRNA expression of EBI3 and IL-12p35 in PBMCs ( r= 0.31, 0.24, P= 0.004, 0.032, respectively) . Compared with the control group, the unstimulated Treg cells from the alopecia areata group showed significantly decreased supernatant levels of perforin and granzyme B, mRNA expression of EBI3, IL-12p35 and immune checkpoint molecules ( P < 0.05 or 0.001) , as well as weakened inhibitory effect on the proliferative activity of PBMCs ( P= 0.013) . There was no significant difference in the level of perforin or granzyme B between the recombinant human IL-35-stimulated and unstimulated Treg cells from the patients with alopecia areata (both P > 0.05) . However, the mRNA expression of EBI3, IL-12p35 and immune checkpoint molecules was significantly higher in the IL-35-stimulated Treg cells than in the unstimulated Treg cells in the alopecia areata group ( P < 0.05 or 0.001) , and the inhibitory effect on the proliferative activity of PBMCs was also significantly enhanced in the IL-35-stimulated Treg cells compared with the unstimulated Treg cells ( P= 0.037) . Conclusion:The peripheral IL-35 level was significantly decreased in the patients with alopecia areata, which was closely associated with reduced activities of Treg cells, and IL-35 may be involved in the occurrence of alopecia areata.

Anti-Allergic Rhinitis Mechanism of Bikangfu via Regulation of IL-35/STAT3， Inhibition of Eosinophil Activation and Improvement of Immune Function / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of interleukin-35（IL-35）/signal transducer and activator of transcription 3（STAT3）inhibition of eosinophil activation against allergic rhinitis（AR） by Bifukang. MethodOne hundred patients were randomly divided into a control group and a treatment group，50 cases in each group. The control group was given mometasone furoate nasal spray，and the treatment group was given Bifukang by nasal packing. The course of treatment was 28 days. The clinical efficacy，nasal classification and visual analogue scale（VAS） score of the two groups were observed before and after treatment. Enzyme linked immunosorbent assay（ELISA） was used to detect the expression levels of inflammatory factors ［interleukin（IL）-4，IL-10，IL-17，IL-35］ and Eotaxin and CC chemokine receptor-3（CCR3）in serum and nasal secretion of the two groups. The expression levels of STAT1，STAT3 and STAT4 were detected by real-time polymerase chain reaction（Real-time PCR）.The content of immunoglobulin G（IgG） and the ratio of CD4+/CD8+were detected by ELISA and flow cytometry. ResultAfter treatment， compared with before treatment， the levels of IL-4 and IL-17 in serum and nasal secretion in 2 groups were decreased， while the levels of IL-10 and IL-35 were increased （P<0.05， P<0.01）. The expression of STAT1， STAT2 and STAT3 in nasal secretions were significantly decreased （P<0.05）. IgG and CD4+/CD8+ were decreased， and the differences were statistically significant （P<0.05， P<0.01）.After treatment，compared with the control group，the levels of IL-4 and IL-17 in serum and nasal secretions of the treatment group were decreased，while the levels of IL-10 and IL-35 were increased （P<0.05）. The expression of STAT1，STAT3 and STAT4 in the treatment group was significantly inhibited compared with the control group after treatment （P<0.05）. In addition， the post-treatment serum CD4+/CD8+ and immunoglobulin G （IgG） levels were reduced in the treatment group compared with those of the control group （P<0.05， P<0.01）. During the treatment，there were no abnormal changes in heart，liver，kidney function and routine blood and urine tests in the two groups. ConclusionBifukang has a good effect on allergic rhinitis，and its mechanism may be related to the regulation of IL-35/STAT3 pathway，the inhibition of eosinophil activation and the improvement of related immune function.

Serum IL-35 and TGF-β1 expression levels in patients with acute anterior uveitis and their clinical significance / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To study the changes of serum IL-35 and TGF-β1 expression levels and the correlation between them in patients with acute anterior uveitis, and to explore the clinical significance of IL-35 and TGF-β1 levels in patients with acute anterior uveitis.<p>METHODS: Thirty patients with acute anterior uveitis confirmed in the Department of Ophthalmology of Gansu Provincial Hospital into 2018-05/2019-05 were selected as the case group, and thirty healthy patients who received physical examination at the Gansu Provincial Hospital during the same period were selected as the control group. Serum IL-35 and TGF-β1 expression levels between the two groups were detected by Elisa. Modified endotoxin-induced uveitis(EIU)clinical standard was used for the severity of acute anterior uveitis. <p>RESULTS: Serum IL-35 and TGF-β1 expression levels in the acute anterior uveitis group were significantly higher than that in the healthy control group(all<i> P</i><0.05), and there was no significant correlation between serum IL-35 and TGF-β1 levels as well as the severity of acute anterior uveitis(<i>r</i>s=0.087, 0.044, all<i> P</i>>0.05). There was a significant positive correlation between serum IL-35 and TGF-β1 expression levels in patients with acute anterior uveitis(<i>r</i>s=0.637, <i>P</i><0.001).<p>CONCLUSION: The expression levels of IL-35 and TGF-β1 in serum are closely related to the occurrence and development of acute anterior uveitis and may play a synergistic role in immunosuppression in acute anterior uveitis.

Relationship between IL-35 level in peripheral blood and disease progression and prognosis in newly diagnosed esophageal cancer patients / 中国医师杂志

Objective:To investigate the relationship between interleukin-35 (IL-35) level in peripheral blood and disease progression and prognosis in newly diagnosed esophageal cancer (EC) patients.Methods:68 newly diagnosed EC patients treated in the People′s Hospital Affiliated to Ningbo University from June 2014 to December 2019 were selected as the observation group and 36 healthy volunteers as the control group. Peripheral blood was collected. The levels of IL-35 were detected by enzyme-linked immunosorbent assay (ELISA). P35 and EBI3 mRNA were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR); the relationship between IL-35 and clinicopathological features and prognosis of newly diagnosed EC patients was analyzed.Results:The levels of IL-35 [(104.46±34.78)pg/ml vs (36.35±16.34)pg/ml], P35 mRNA [(43.79±7.28) vs (20.62±6.76)] and EBI3 mRNA [(18.68±3.86) vs (7.76±2.83)] in newly diagnosed EC patients were significantly higher than those in the control group (all P values <0.05). The level of IL-35 in tumor node metastasis (TNM) stage Ⅲ -Ⅳ patients was significantly higher than that in stage Ⅰ-Ⅱ patients [(115.85±28.85)pg/ml vs (96.19±36.27)pg/ml], with statistically significant difference ( t=2.479, P<0.05); The level of IL-35 in EC patients with lymph node metastasis was significantly higher than that in patients without lymph node metastasis [(119.83±29.74)pg/ml vs (92.23±33.65)pg/ml], and the difference was also statistically significant ( t=3.512, P<0.01); After radical esophagectomy, the level of IL-35 in 28 EC patients decreased significantly [(102.65±15.51)pg/ml vs (43.28±12.93)pg/ml; t=11.551, P<0.01]. The level of IL-35 in EC patients was negatively correlated with overall survival (OS) ( r=-0.45, P<0.01); the OS in EC patients with high level of IL-35 was significantly shorter than that in low level patients ( P<0.01). The results of univariate analysis showed that TNM staging, lymph node metastasis and IL-35 level were the unfavorable factors affecting the OS in EC patients ( P<0.01). Multivariate analysis showed that TNM staging, lymph node metastasis and IL-35 level were independent risk factors affecting OS in EC patients ( P<0.05). Conclusions:IL-35 is highly expressed in patients with EC, which closely related to disease stage and lymph node metastasis.

Immunomodulatory characteristics of mesenchymal stem cells mediated by inflammatory factors / 中国组织工程研究

BACKGROUND: Mesenchymal stem cells possess strong immunomodulatory capacity, mainly involved in the proliferation, differentiation and functional status of immune cells, and the secretion of inflammatory factors. The immunomodulatory capacity of mesenchymal stem cells is regulated by inflammatory factors. As the type and level of inflammatory factors in the microenvironment change, the immune regulation function of mesenchymal stem cells also changes. OBJECTIVE: To review the research progress of inflammatory factor intervention in the immune regulation of mesenchymal stem cells. METHODS: A computer-based retrieval of PubMed, Elsevier and CNKI databases was performed. The keywords were “mesenchymal stem cells, immune regulation, plasticity, interferon gamma, transforming growth factor beta, interleukin-17, interleukin-35, prostaglandins E2” in Chinese and English, respectively. The articles concerning the effects of inflammatory factor intervention on the immune regulation of mesenchymal stem cells were included. RESULTS AND CONCLUSION: After intervention and pre-treatment of interferon-γ, transforming growth factor-β, interleukin-17, interleukin-35 and prostaglandin E2, the biological characteristics of mesenchymal stem cells have also changed. The interventions cannot only reshape the tissue microenvironment and affect the inflammatory response, but also reconstruct the immune balance, which further treats or alleviates the disease progression. Reasonable individualized and differentiated treatments will be performed at different disease stages according to inflammatory and immunological reaction of the disease. All of them are expected to further improve the therapeutic effect of mesenchymal stem cells on immuno-inflammatory diseases.

Effects of interleukin-35 on the balance of helper T cell 17/regulatory T cell in peripheral blood of patients with oral lichen planus / 中华口腔医学杂志

Objective@#To investigate the effects of exogenous interleukin (IL)-35 on the balance of helper T cell 17 (Th17) and regulatory T cell (Treg) in peripheral blood of patients with oral lichen planus (OLP).@*Methods@#Totally 12 peripheral blood samples of OLP patients (OLP group, one male and 11 female, 26-68 years old; four cases of reticular OLP and eight cases of erosive OLP) were collected from patients of Department of Oral Mucosal Specialist of the Affiliated Hospital of Guizhou Medical University from October to December 2016. During the same period, thirteen normal peripheral blood samples were collected from the Physical Examination Center of the Affiliated Hospital of Guizhou Medical University (normal control group, one male and 12 female, 20-68 years old). The peripheral blood mononuclear cells (PBMC) were extracted in sterile condition and CD4+ T cells were sorted by flow cytometry (FCM). Quantitative real-time PCR (qPCR) technique was used to detect the mRNA expression levels of retinoid-related orphan nuclear γt (RORγt) and forkhead box3 (Foxp3). The CD4+ T cells were divided into experimental group and control group. The CD4+ T cells of experimental group were cultured in vitro by adding rhIL-35, and the CD4+ T cells of control group were cultured with the same volume of phosphate buffered saline (PBS). After the completion of the culture, the cells were collected. The expression levels of the same factors were detected by qPCR.@*Results@#The expression [M(Q25, Q75)] of Foxp3 [0.15 (0.09, 0.30)] and RORγt mRNA [1.04 (0.45, 2.15)] in the CD4+ T cells of OLP were significantly higher than those in normal control group [0.04 (0.02, 0.06), 0.10 (0.05, 0.11)] (Z=-4.134, P<0.01; Z=-3.699, P<0.01). The ratio of ROR γt/Foxp3 mRNA in OLP group [6.22(3.67, 15.34)] was higher than that in normal control group [2.50 (1.24, 5.23)] (Z=-2.665, P=0.007). In the CD4+ T cells of OLP patients, the expression of Foxp3 mRNA in the experiment group [0.40 (0.21, 1.22)] was higher than that in the control group [0.15 (0.11, 0.26)](Z=-2.510, P=0.012), and the expression of ROR γt mRNA between two groups showed no significant difference (P>0.05). The ROR γt/Foxp3 mRNA ratio [3.44 (1.55, 8.16)] of the experiment group was lower than that in the control group [6.22 (4.43, 12.21)] (Z=-2.746, P=0.006).@*Conclusions@#There was a Th17/Treg imbalance with predominated by Th17 cells in the peripheral blood of patients with OLP. Exogenous rhIL-35 had an immunomodulatory effect on the balance of Th17/Treg.

The regulatory activity of interleukin-35 to CD4+ and CD8+ T cells in sepsis patients / 中华急诊医学杂志

Objective@#To investigate plasma interleukin-35 (IL-35) expression in sepsis patients, and to assess the regulatory activity of IL-35 on CD4+ and CD8+ T cells in sepsis patients.@*Methods@#A prospective study was conducted. Forty-one sepsis patients and nineteen healthy controls were enrolled in this study. According to the survival outcome on 28 day after admission, sepsis patients were further divided into the survival group (n=30) and death group (n=11). Peripheral blood samples were collected within one hour of visit. Plasma IL-35 level was measured by enzyme linked immunosorbent assay (ELISA), while CD3+, CD4+, and CD8+ T cell counts were measured by flow cytometry. The correlation between IL-35 expression/T cell counts and sequential organ failure assessment (SOFA) score was also assessed. Peripheral CD4+ and CD8+ T cells from sepsis patients were purified and stimulated with recombinant human IL-35 for 48 h. mRNA relative levels of T-bet, GATA-3, FoxP3, and RORγt in CD4+ T cells and perforin, granzyme B, and FasL in CD8+ T cells were measured by real-time PCR. Interferon-γ (IFN-γ), IL-4, IL-10, IL-17, and tumor necrosis factor-α (TNF-α) levels in the cultured supernatants were measured by ELISA. Transcription factor level and cytokine expression was compared prior to and post IL-35 stimulation.@*Results@#Plasma IL-35 was significantly elevated in sepsis patients when compared with controls [(76.76±10.33) pg/mL vs (27.53±8.31) pg/mL, P<0.01], however, it did not correlate with SOFA score (r=0.172, P=0.281). Plasma IL-35 was also notably increased in the death group when compared with the survival group [(83.18±6.48) pg/mL vs (74.40±10.55) pg/mL, P=0.014]. Plasma IL-35 level in the death and survival groups were analyzed by receiver operating characteristic curve with an area under curve of 0.770 (P=0.009). There were no remarkable differences of CD3+, CD4+, and CD8+ T cell counts between sepsis patients and controls, or between the death and survival groups (all P>0.05). Moreover, T cell counts were negatively correlated with SOFA score (P<0.01). IL-35 stimulation resulted in the reduction of mRNA relative level of Th1 transcription factor T-bet and Th17 transcription factor RORγt in CD4+ T cells from sepsis patients (P<0.01), while elevation in regulatory T cell transcription factor FoxP3 mRNA (P<0.01). However, IL-35 stimulation did not affect Th2 transcription factor GATA-3 mRNA in CD4+ T cells (P=0.745). IFN-γ and IL-17 production by CD4+ T cells was significantly decreased in response to IL-35 stimulation (P<0.05), while IL-10 was increased (P<0.01) and IL-4 was comparable (P=0.536) between CD4+ T cells with and without IL-35 stimulation. IL-35 stimulation led to the down-regulation of mRNA relative level of perforin and granzyme B (P<0.01). However, FasL mRNA was comparable between CD8+ T cells with and without IL-35 stimulation (P=0.795). IFN-γ and TNF-α secretion was also reduced in response to IL-35 stimulation (P<0.05).@*Conclusion@#Elevated plasma IL-35 level is an important indicator for poor prognosis of sepsis patients. IL-35 inhibits the activity of CD4+ and CD8+ T cells in sepsis patients, and might take part in the pathogenesis of sepsis.

The regulatory activity of interleukin-35 to CD4+and CDS+T cells in sepsis patients / 中华急诊医学杂志

Objective To investigate plasma interleukin-35(IL-35)expression in sepsis patients,and to assess the regulatory activity of IL-35 on CD4+and CD8+T cells in sepsis patients.Methods A prospective study was conducted.Forty-one sepsis patients and nineteen healthy controls were enrolled in this study.According to the survival outcome on 28 day after admission,sepsis patients were further divided into the survival group(n=30)and death group(n=11).Peripheral blood samples were collected within one hour of visit.Plasma IL-35level was measured by enzyme linked immunosorbent assay(ELISA),while CD3+,CD4+,and CD8+T cell counts were measured by flow cytometry.The correlation between IL-35 expression/T cell counts and sequential organ failure assessment(SOFA)score was also assessed.Peripheral CD4+and CD8+T cells from sepsis patients were purified and stimlated with recombinant human IL-35 for 48 h.mRNA relative levels of T-bet,GATA-3,FoxP3,and RORγt in CD4+T cells and perforin,granzyme B,and FasL in CD8+T cells were measured by real-time PCR.Interferon-γ(IFN-γ,IL-4,IL-10,IL-17,and tumor necrosis factor-α(TNF-α)levels in the cultured supernatants were measured by ELISA.Transcription factor level and cytokine expression was compared prior to and post IL-35 stimulation.Results Plasma IL-35 was significantly elevated in sepsis patients when compared with controls [(76.76±10.33)pg/mL vs(27.53±8.31)pg/mL,P＜0.01],however; it did not correlate with SOFA score(r=O.172,P=0.281).Plasma IL-35 was also notably increased in the death group when compared with the survival group [(83.18±6.48)pg/mL vs(74.40±10.55)pg/mL,P=0.014].Plasma IL-35lvel in the death and survival groups were analyzed by receiver operating characteristic curve with an area under curve of 0.770(P=0.009).There were no remarkable differences of CD3+,CD4+,and CD8+T cell counts between sepsis patients and controls,or between the death and survival groups(all P＞0.05).Moreover,T cell counts were negatively correlated with SOFA score(P＜0.01).IL-35 stimulation resulted in the reduction of mRNA relative level of Th1 transcription factor T-bet and Th17 transcription factor RORγt in CD4+T cells from sepsis patients(P＜0.01),while elevation in regulatory T cell transcription factor FoxP3 mRNA(P＜0.01).However,IL-35 stimulation did not affect Th2 transcription factor GATA-3 mRNA in CD4+T cells(P=0.745).IFN-γ and IL-17 production by CD4+T cells was significantly decreased in response to IL-35 stimulation(P＜0.05),while IL-10 was increased(P＜0.01)and IL-4 was comparable(P=0.536)between CD4+T cells with and without IL-35 stimulation.IL-35 stimulation led to the down-regulation of mRNA relative level of perforin and granzyme B(P＜0.01).However,FasL mRNA was comparable between CD8+T cell with and without IL-35 stimulation(P=0.795).IFN-γ and TNF-α secretion was also reduced in response to IL-35 stimulation(P＜0.05).Conclusion Elevated plasma IL-35 level is an important indicator for poor prognosis of sepsis patients.IL-35 inhibits the activity of CD4+and CD8+T cells in sepsis patients,and might take part in the pathogenesis of sepsis.

Expression and clinical significance of the novel inhibitory cytokine IL-35 in patients with sepsis and multiple organ dysfunction syndrome / 中华急诊医学杂志

Objective To investigate the expression of interleukin-35 (IL-35) in patients with sepsis and multiple organ dysfunction syndrome (MODS),and to explore its effect on disease prognosis.Methods A prospective study was conducted.Twenty-two patients with sepsis and MODS were selected as study group,and 20 healthy volunteers were selected as controls.Blood samples of the patients and the volunteers were taken within 1 hour of the patient's visit,and cytokines,such as IL-35,IL-4,IL-10,IL-17,INF-γ,and TGF-β,were simultaneously detected by enzyme-linked immunosorbent assay (ELISA).The expression level of CD4+CD25+FOXP3+ Treg cells was detected by flow cytometry,and the acute physiology and chronic health status scoring system Ⅱ (APACHE Ⅱ) was calculated.According to the survival outcome at 28 days after admission,the expression levels of IL-35 in the survival group (12 cases)and the death group (8 cases) were compared.The Spearman method was used to analyze the correlation between IL-35 level and the.above indicators in patients with sepsis and MODS.Results Compared with the control group,the IL-35 levels were significantly increased in the study group (P＜0.05),and the IL-35 levels began to increase gradually on the first day of the disease,and significantly higher on the 7th day (P＜0.01).The number of CD4+CD25+FOXP3+regulatory T cells increased in the study group,especially on the 4th day (P＜0.01),and on the first day of the disease,the IL-35 levels were positively correlated to the number of CD4+CD25+FOXP3+Treg cells (r=0.60,P＜0.05).Compared with the control group,the levels of IL-10,IL-4,IL-17,INF-γ and TGF-β in the study group increased gradually with the course of disease,and were significantly higher on the 7th day than those on the 1st day and the 4th day (P＜0.05).The IL-35 levels in the study group were positively correlated with INF-γ and TGF-β cytokines (P＜0.05),whereas there was no significant relationship between the levels of IL-10,IL-4,and IL-17 (P＞0.05).The IL-35 level in the death group was significantly lower than that in the survival group (P＜0.05).IL-35 levels in the survival and death groups were analyzed by ROC curve with a AUC of 0.78 (P=0.03).The ~ IL-35 level was negatively correlated with the APACHE-Ⅱ score in the study group (r=-0.78,P＜0.01).Conclusions The IL-35 levels in patients with sepsis and MODS is significantly higher than that in healthy controls,and negatively correlated with APACHE Ⅱ score.The level of IL-35 has an important implication for the prognosis for patients with sepsis.

Expression and significance of interleukin-35 in lesion tissue of oral lichen planus patients / 中华口腔医学杂志

Objective@#To investigate the expression of Epstein-Barr virus-induced gene 3 (EBi3) and interleukin-12p35 (IL-12p35) in two subunits of interleukin-35 (IL-35) in oral lichen planus (OLP) lesions, and to explore the role of IL-35 played in the formation and development of OLP lesions.@*Methods@#Totally 41 samples of OLP lesions and 15 samples of normal tissues were collected from patients of the Department of Periodontology and Oral Medicine, Affiliated Stomatological Hospital of Guizhou Medical University from October 2010 to December 2016. The expression levels of EBi3 mRNA and IL-12p35 mRNA in the samples were detected by quantitative real-time PCR (qPCR) and the distribution and expression of protein EBi3 and IL-12p35 were detected by immunohistochemistry. The potential relationship between IL-35 and clinicopathological features of OLP was analyzed.@*Results@#The expression [M(Q25, Q75)] of EBi3 [3.38 (1.63, 11.25)] and IL-12p35 mRNA [6.39 (2.55, 14.30)] in OLP lesion tissues were significantly higher than those in normal control group [1.41 (0.33, 3.16), 2.47 (1.10, 5.14)] (Z=-2.806, P=0.005; Z=-2.276, P=0.023), respectively. The positive expression rates of EBi3 and IL-12p35 were 66% (27/41) and 39% (16/41), respectively, were significantly higher in OLP lesion tissues comparing with that in normal oral mucosa tissues [0%(0/15)] (P<0.05). The relative expressions of EBi3 and IL-12p35 were positively correlated (r=0.404, P=0.009). A significant correlation was found between EBi3 protein over expression and the degeneration of base cells in OLP lesions (χ2=9.172, P=0.010). The positive expression rate of IL-12p35 protein in erosive type lesions was higher than that in non-erosive type lesions (χ2=7.220, P=0.007). The positive expression rate of IL-35 protein in OLP lesions [34% (14/41)] was higher than that in normal control group (χ2=6.829, P=0.009). The expression rate of IL-35 in erosive type lesions (10/20) was significantly higher than that in eruption type lesions (4/21) (χ2=4.364, P=0.037).@*Conclusions@#The expression of IL-35 in OLP localized lesions was up-regulated, suggesting that IL-35 might play an important role in OLP lesion formation.

IL-35-producing regulatory T cells regulate intestinal inflammatory immune response / 中华微生物学和免疫学杂志

Objective To evaluate the function of interleukin-35 (IL-35)-producing regulatory T cells (IL-35-Treg) in regulating intestinal inflammatory immune response. Methods The percentages and characteristics of IL-35-Treg in the intestinal lamina propria of transgenic mice expressing IL-35 were ana-lyzed by flow cytometry. A mouse model of inflammatory bowel disease ( IBD) was established by giving 1. 5％ DSS in drinking water. Influences of IL-35-Treg depletion on mouse weight, pathological injury and the secretion of IFN-γ were analyzed. Results IL-35-Treg were enriched in the intestinal lamina propria, and mainly derived from thymic Treg (tTreg). Intestinal IL-35-Treg expressed high levels of programmed death 1 (PD-1). Depletion of IL-35-Treg in mice with DSS-induced IBD resulted in an aggravation through up-regulating the expression of IFN-γ. Conclusion IL-35-Treg might play an important role in the regula-tion of intestinal inflammatory immune response.

Advances in the mechanism of IL-35 regulating the development of malignant tumors / 中国肿瘤临床

Interleukin-35 (IL-35) produced by regulatory T cells is a newly discovered inhibitory cytokine. It's a heterodimer consisting of IL-27βchain Epstein-Barr virus-induced gene 3 and IL-12αchain p35. In recent years, many studies have found that T cells, B cells, and tumor cells can secrete IL-35, which has a major inhibitory effect on the immune system. IL-35 plays an important role in tumor im-mune evasion and promotion of tumor progression and metastasis, and is an important factor which promotes the occurrence and de-velopment of tumors. Reducing IL-35 secretion may improve disease control, and it is very important to make a thorough inquiry into the role of IL-35 in regulating the activities of tumor cells. This review summarizes the molecular structure, biological functions, and regulatory roles of IL-35 in the development of malignant tumors. It is hoped that it can provide a novel perspective on the judgment of tumor prognosis and tumor targeted molecular therapy in the future.

Detection of IL-35-expressing monocytes in mice infected with BCG and its clinical significance / 中国免疫学杂志

Objective:To detect IL-35 in mononuclear cells of peripheral blood and lung tissue with BCG infected mouse,and to analyze its effect on the tuberculosis immune mechanism and pathology.Methods: Mycobacterium tuberculosis infecting animal model was made by BCG injection in wild type C57BL/6 mouse.Then,mice were sacrificed in different time points.The peripheral blood and lung tissue were isolated.The bacterial load and histopathological analysis in lung tissue were performed,and the expression of IL-35 subunits P35 and EBI3 in monocytes were detected by flow cytometry.Furthermore,the correlation of IL-35-expressing monocytes with the load of bacterium was analyzed.Results:The expression of P35 and EBI3 in monocytes in peripheral blood and lung tissue of BCG-infected mice was significantly higher than that of the control group,and the peripheral blood of the experimental group was significantly increased at 4 weeks after infection.The expression of EBI3 in lung tissue diaplayed significant rising trend.The correlation analysis showed that the P35 expression was positively correlated with the expression of EBI3 in monocyte,and the IL-35-ex-pressing monocyte in lung tissue was negatively correlated with the load of bacteria.The IL-35-expressing monocyte in peripheral blood and lungs increased significantly at 2-week and 4-week,while it increased in lung tissue at 8 week,significantly.Conclusion: BCG-infects mouse monocytes with high expression of IL-35,it may participate in anti-tuberculosis immune regulation.

Study on expression and mutual regulation of serum IL-35 and miR-21 in septic patients / 国际检验医学杂志

Objective To detect the expression level and correlation of interleukin 35(IL-35) and micro RNA-21(miR-21) in septic patients and to explore the role of their mutual regulation in sepsis .Methods The serum was collected from the patients with sepsis and healthy controls .The expressions of serum IL-35 and miR-21 were detected by ELISA and real-time PCR ,and their correlation was analyzed ;mice were intraperito-neally injected by lipopolysaccharide(LPS) to establish the sepsis model ,then the mice were given anti-IL-35 p35 antibody ,IgG ,sepsis miR-21 mimics(agomir-21) and sepsis miR-21 negative control(agomir-21 NC) by in-traperitoneal injection ,after that ,the serum miR-21 expression level in each group was detected by real-time PCR and IL-35 ,TNF-αand IL-6 levels were detected by ELISA .Results The expression levels of serum IL-35 and miR-21 in sepsis patients were significantly increased compared with the healthy control group ,the difference was statistical significance (P<0 .05) ,moreover their expression levels showed the positive correla-tion (P<0 .05);after septic mice antagonizing to IL-35 ,the serum miR-21 expression was significantly in-creased ,meanwhile the expressions of inflammatory factor TNF-α and IL-6 were increased obviously ,the difference was statistical significance (P<0 .05) .After treating the septic mice by agomir-21 ,the serum IL-35 level was decreased significantly ,while the expression of inflammatory factors TNF-αand IL-6 were increased significantly ,the difference was statistical significance(P<0 .05) .Conclusion Serum IL35 and miR-21 in the patients with sepsis are jointly participate in the septic inflammatory response by mutual regulation .

Detection of peripheral blood Treg cells and secreted IL-35 level in patients with rheumatoid arthritis and its clinical significance / 重庆医学

Objective To detect the number of peripheral blood Treg cells and secreted IL-35 expression level in the patients with rheumatoid arthritis(RA) and to explore their correlation with RA occurrence.Methods Peripheral blood was collected from 45 cases of RA,22 cases of osteoarthritis(OA) and 26 persons undergoing healthy physical examination(control group).The number of CD4+ CD25+ foxp3+ regulatory T cells was determined by flow cytometry,while plasma IL-35 level was determined by ELISA.Then the relationship between Treg and expressed IL-35 with clinical indicators was analyzed.Results The percentage of peripheral blood Treg ceils to total CD4+T cells in the RA group was (5.65 ± 2.33)％,which was significantly increased compared with (4.12 ± 1.75) ％ in the control group(P＜0.05);the difference between the OA group and RA group was not statistically significant(P=0.086).The average fluorescence intensity of foxp3 had no statistical difference among 3 groups(P＞0.05).The higher the DAS28 score,the lower the peripheral blood Treg cells number and foxp3 fluorescence intensity.The plasma IL-35 level in the RA group [(34.22± 14.35)ng/L] was significantly lower than that in the OA group[(78.63± 24.58)ng/L] and control group [(67.56±25.43)ng/L],the difference was statistically significant(P＜0.05).The Treg number in RA patients was negatively correlated with ESR and DAS28 score(r=-0.223,-0.343,P=0.023,0.011),but had no correlation with rheumatoid factor,C-reactive protein and anti-CCP antibody.Conclusion The peripheral blood Treg cells number in RA patients is elevated,while the IL-35 level is decreased,the negative regulation ability in the patients with Treg cell function deficit is attenuated.

Study on the relationship between anti-inflammatory cytokine IL-35 and delayed renal graft function / 器官移植

Objective To investigate the relationship between the interleukin (IL)-35 and the recovery of renal graft function. Methods Clinical data of 45 recipients receiving renal transplantation from donation after cardiac death (DCD) were retrospectively analyzed. According to the presence of delayed graft function (DGF) after renal transplantation, all recipients were divided into the immediate graft function (IGF) group (n=32) and DGF group (n=13). The serum creatinine (Scr) level and estimated glomerular filtration rate (eGFR) in the recipients were statistically compared between two groups at 1, 2, 3, 7, 14, 28 d and 3, 6 and 12 months after renal transplantation. The IL-35 levels in the serum and urine samples of the recipients were statistically compared between two groups at 1, 2, 3, 7, 14, 28 d following renal transplantation. Results In the DGF group, the renal function was restored slowly. Compared with the IGF group, the Scr level was significantly higher, whereas the eGFR was considerably lower in the DGF group at postoperative 7 d (both P<0.05). At 1 year after surgery, there was no significant difference in the Scr level between two groups. Compared with the IGF group, the eGFR in the DGF group was significantly lower at postoperative 1 year (P<0.05). At 1, 2, 3, 7, 14 d after operation, the serum levels of IL-35 in the DGF group were evidently lower than those in the IGF group (all P<0.05). Compared with the IGF group, the serum level of IL-35 in the DGF group was significantly increased at postoperative 28 d (P<0.05). At postoperative 1, 2, 3, 7 d, the IL-35 levels in the urine samples in the DGF group were significantly lower than those in the IGF group (all P<0.05). At postoperative 14 and 28 d, the IL-35 levels in the urine samples did not significantly differ between two groups (both P>0.05). Conclusions The low levels of IL-35 in the serum and urine of recipients after renal transplantation are associated with the incidence of DGF to certain extent, prompting that excessively weak systemic and local anti-inflammatory responses early after renal transplantation and uncontrolled excessive inflammatory response are probably the pivotal causes of DGF.

Modulation of CD4+CD25+CD127 dim/-regulatory T cells by interleukin-35 in the pathogenesis of vent- ilator-associated pneumonia / 中华微生物学和免疫学杂志

Objective To investigate the changes in CD4+CD25+CD127 dim/- regulatory T cells ( Tregs) and interleukin ( IL)-35 in peripheral blood and bronchoalveolar lavage fluid ( BALF) samples from patients with ventilator-associated pneumonia ( VAP) . Methods A total of 23 patients with VAP and 11 normal controls ( NCs) were enrolled in this study. Peripheral blood mononuclear cells, serum and BALF samples were isolated and collected. Levels of IL-35 were measured by enzyme linked-immunosorbent assay. Percentages of CD4+CD25+CD127dim/-Tregs were measured by flow cytometry. CD4+CD25+CD127dim/-Tregs in BALF samples were isolated and purified, which were then stimulated with recombinant human IL-35 and co-cultured with autologous CD4+CD25-T cells. Cells and supernatants were harvested for analysis of cell proliferation and cytokine secretion. Results No significant difference in peripheral Tregs and serum IL-35 was found between patients with VAP and NCs. The percentages of Tregs and the levels of IL-35 in BALF samples collected from infectious sites were remarkably higher than those collected from non-infectious sites in patients with VAP. Moreover, there was a positive correlation between Tregs and IL-35 in BALF ( r=0. 441, P=0. 035). Tregs and IL-35 in BALF samples as well as Treg-secreted IL-35 were significantly re-duced in patients who had good response to therapy. However, no significant change in these parameters was observed in patients who had poor response to therapy. Besides, suppression of cell proliferation and IL-10 secretion that were related to Tregs were inhibited in patients whose condition was improved as compared with those in patients who had no response to therapy. Stimulation with recombinant human IL-35 enhanced the immunosuppressive function of purified Tregs that were separated from BALF of treatment-na?ve patients with VAP, which was mainly marked by suppressed cell proliferation, increased secretion of inhibitory cytokines (IL-35 and IL-10), and decreased secretion of proinflammatory cytokines (IFN-γ and TNF-α). However, IL-35 had little effect on the activities of Tregs that were separated from patients with VAP who responded to therapy. Conclusion Both IL-35 and Tregs are increased in BALF of patients with VAP and IL-35 en-hances the immunosuppressive function of Tregs, which indicates that IL-35-mediated modulation of Tregs might take part in the pathogenesis of VAP.

Important role of interleukin-35 in infectious diseases and its significance / 中华危重病急救医学

Infectious diseases are resulted from the invasion of an organism's body tissues by multiple disease-causing agents. It has been demonstrated that the occurrence and development of infectious diseases are closely associated with the functional status of immune system. Cytokines play significant roles in modulating the host immune response to the clearance of pathogenic microorganisms and maintaining immune homeostasis. Interleukin-35 (IL-35), as a newly identified member of IL-12 family, exerts suppressive effect on immune response by means of a specific pattern. With the progress of research in recent years, IL-35 might serve as an essential contributor in the immunopathogensis of vast infectious diseases, including hepatitis B, sepsis, tuberculosis and parasite infection, which simultaneously appear to be closely related to the severity, progression as well as prognosis of the illness. Apparently, IL-35 is regarded as a potent and promising anti-inflammatory cytokine in clinical application; its potential value may shed light on the therapeutic strategies for infectious diseases. Herein, we mainly review the potential role and its mechanism of IL-35 in the pathogenesis of infectious diseases.