Role of IL-33/ST2 signaling pathway in fibrosis diseases

Interleukin (IL)-33 is a member of IL-1 family. It is identified as a functional ligand for ST2 which is an IL-1 receptor-like protein. IL-33/ST2 signaling is involved in T-cell-mediated immune responses. Increasing evidence indicates that IL-33 has different roles in different diseases. Recently, some studies have demonstrated that IL-33 may be related to the genesis and development of fibrosis diseases. We review current knowledge of the biological characteristics of IL-33 and the role of IL-33/ST2 signaling pathway in fibrosis diseases.     

Expression of A20 in patients with systemic lupus erythematosus

AIM: To investigate the expression and regulation of A20 in healthy individuals and the patients with systemic lupus erythematosus (SLE). METHODS: The expression levels of A20, NF-κB, MALT1, and MALT1V1 in peripheral blood mononuclear cells (PBMC) of the patients with SLE (including 2 cases with scleroderma, 1 case with rheumatoid arthritis, and 1 case with lymphoma) were analyzed by real-time PCR. RESULTS: A significantly lower A20 expression level was found in the PBMC from SLE group compared with the healthy controls, while the expression levels of MALT1 and NF-κB were also decreased. In addition, no significant correlation between A20 and NF-κB expression levels in healthy group was observed, but a positive correlation was found in SLE group (P<0.05). A significant positive correlation between MALT1 and NF-κB expression levels in healthy group (P<0.05) was observed, and no significant correlation was found in SLE group. The expression level of MALT1V1 in SLE group was significantly lower than that in healthy control group, and there was a positive correlation between A20 and MALT1V1 in healthy volunteers (P<0.01), but that did not exist in SLE group. CONCLUSION: The characteristics of the expression pattern of MALT1-A20-NF-κB in the SLE patients were presented. Lower level of A20 expression was found in the SLE patients, in particular with other autoimmune disease or lymphomas, indicating the lower immune tolerance in SLE. The positive correlation of A20 and NF-κB may relate to positive regulation of MALT1.     

Association of S100B polymorphisms with systemic lupus erythematosus in Guangxi population

AIM: To explore the association of rs9984765, rs2839356 and rs2186358 polymorphisms in S100B gene with the susceptibility to systemic lupus erythematosus (SLE). METHODS: SLE patients (n=313) and age-and sex-matched healthy controls (n=396) were recruited in this study. The genotypes of the 3 sites were determined by single-base extension PCR (SBE-PCR) and DNA sequencing. RESULTS: No difference between the SLE patients and controls in the genotype and allele frequencies of rs9984765 and rs2186358 was observed. However, the frequency distribution of rs2839356 C allele was significantly different in the 2 groups (P=0.040). Stratification analysis showed that the frequency of rs2839356 C allele was higher in the patients with neurologic disorder than the patients without neurologic disorder (P=0.023).CONCLUSION: S100B gene rs9984765 and rs2186358 polymorphisms may not contribute to the susceptibility of SLE in Guangxi population. The rs2839356 C allele might be correlated with the SLE patients with neurologic disorder.     

Effect of pachyman polysaccharides on Th17/Treg balance in systemic lupus erythematosus patients

AIM: To investigate the immunomodulatory effect of pachyman polysaccharides (PPS) on T helper 17 cell (Th17)/regulatory T cell (Treg) balance in the peripheral blood of systemic lupus erythematosus (SLE) patients. METHODS: The CD4⁺ T cells were isolated from the peripheral blood samples obtained from 45 SLE patients and 35 healthy controls enrolled in our study using magnetic bead separation method. The proportions of Th17 and Treg cells were measured by flow cytometry. The CD4⁺ T cells from SLE patients and healthy controls were treated with PPS. The cytoto-xicity of PPS was evaluated by detecting cell viability with MTT assay. The contents of interleukin-17 (IL-17), IL-6, IL-10 and transforming growth factor-β (TGF-β) were measured by ELISA. The expression of retinoid-related orphan receptor γt (RORγt) and forkhead box protein P3 (Foxp3) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTS: The Th17 cells were significantly elevated, while Treg cells were obviously decreased in the SLE patients compared with the healthy control group (P<0.05). Compare with control group, the contents of IL-17 and IL-6 were decreased, while the contents of IL-10 and TGF-β were increased (P<0.05). The expression of RORγt at mRNA and protein levels was down-regulated and the expression of Foxp3 was up-regulated (P<0.05). The ratio of Th17/Treg was decreased in 100 μg/L nontoxic PPS-treated CD4⁺ T cells isolated from the SLE patients (P<0.05). CONCLUSION: PPS treatment inhibits Th17 cells and elevates Treg cells in the CD4⁺ T cells isolated from SLE patients, which may have a therapeutic effect on SLE patients.     

Association of polymorphism of Fc receptor γ chain gene at position-29 in promoter with the susceptibility to systemic lupus erythematosus in southern Chinese

AIM:To detect the association between the polymorphism of Fc receptor γ chain gene at position-29 in promoter and systemic lupus erythematosus(SLE).METHODS:The genotypes at position -29 in promoter of Fc receptor γ chain gene were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in 180 patients with SLE and 140 ethnically matched controls in southern China.RESULTS:The frequencies of TT genotype(33.3%) and T allele (54.4%) at position -29 in patients with SLE were significantly higher than those in controls (17.2% and 42.9%, respectively), whereas, the frequencies of GG genotype (24.4%) and G allele (45.6%) in patients with SLE were remarkably lower than those in controls (31.4% and 57.1%, respectively) (P＜0.05). The TT genotype and T allele at position -29 were not associated with lupus nephritis in SLE patients (P＞0.05).CONCLUSION:Our results indicate that the T allele at position -29 in promoter of Fc receptor gene probably contributes to the susceptibility to SLE, but does not play a role in the occurrence of lupus nephritis.     

Effect of Xingnao enema fluid on brain injury and IL-33/ST2 signaling pathway in rats after cardiopulmonary resuscitation

AIM: To study the effects of Xingnao enema fluid on brain injury and IL-33/ST2 signaling pathway in rats after cardiopulmonary resuscitation, and to explore the brain protective effect and mechanism of Xingnao enema fluid. METHODS: SD rats were randomly divided into sham operation group, model group, low-dose (5 mL/kg), middle-dose (10 mL/kg) and high-dose (20 mL/kg) Xingnao enema liquid groups, and ulinastatin group, with 12 rats in each group. Except for the rats in sham operation group, the rats in other groups were used to establish the model of cardiopulmonary resuscitation and were treated with Xingnao enema fluid and ulinastatin. Seven days later, all rats were scored for neurological deficit. The rats were sacrificed, and the water content of brain tissues was calculated. Hematoxylin-eosin (HE) staining was used to detect the pathological changes of brain tissues of the rats in each group. The levels of super-oxide dismutase (SOD) and malondialdehyde (MDA) were detected in the brain tissue. The levels of S100 calcium bin-ding protein beta subunit (S100β) and neuron-specific enolase (NSE) in serum, and interleukin (IL)-1β and tumor necrosis factor (TNF)-α in brain tissue were measured by ELISA. The expression of interleukin-33 (IL-33) and growth stimulation expressed gene 2 (ST2) in brain tissue was determined by Western blot. RESULTS: Compared with sham operation group, the brain tissue of model group showed tissue disorder, focal hemorrhage, neuronal nucleus contraction, apoptosis and other pathological changes, and the neurological deficit score was increased. The water content of brain tissue, the le-vels of S100β, NSE, MDA, IL-1β, TNF-α, IL-33 and ST2 were significantly increased, and the level of SOD decreased significantly (P<0.05). Compared with model group, the pathological damage of brain tissue in low-, middle- and high-dose Xingnao encma fluid groups and ulinastatin group was reduced, and the neurological deficit score was decreased. The water content of brain tissue, levels of S100β, NSE, MDA, IL-1β, TNF-α, IL-33 and ST2 were significantly decreased, and the level of SOD was increased significantly (P<0.05). There was a dose-dependent relationship in different doses of Xingnao enema fluid groups, and no significant difference between high-dose Xingnao enema fluid group and ulinastatin group was observed (P>0.05). CONCLUSION: Xingnao enema fluid repairs brain injury in rats after cardiopulmonary resuscitation, and down-regulation of IL-33/ST2 signaling pathway may be its mechanism.     

Effect of protease inhibitor bortezomib on treatment of rheumatoid arthritis by affecting IL-33/ST2 signaling pathway

AIM To investigate the effect of bortezomib, a protease inhibitor, on the treatment of rheumatoid arthritis （RA） and it mechanism, based on interleukin-33 （IL-33）/suppression of tumorigenicity 2 （ST2） signaling pathway. METHODS A total of 40 Wistar rats were randomly divided into 4 groups： control group, model group, and low- and high-dose bortezomib groups, with 10 rats in each group. In addition to control group, the rats in other groups were used to construct RA model. Bortezomib was given intraperitoneally at 0.2 mg/kg and 0.5 mg/kg in low- and high-dose bortezomib groups, respectively, while the rats in control group and model group were injected with the same amount of saline, once a day for 21 d. The general situation of the rats in each group was observed, the swelling degree of the foot was calculated, and the inflammation score was evaluated. HE staining was used to observe the pathological changes of ankle joint. The automatic biochemical analyzer was used to detect blood hemoglobin content, the total number of platelets （PLT）, serum creatinine （SCr） level and blood urea nitrogen （BUN） level. The serum levels of IL-6, tumor necrosis factor-α （TNF-α）, IL-33 and ST2 were measured by ELISA. The protein expression of IL-33 and ST2 in ankle tissues of each group was determined by Western blot. RESULTS On the 7th, 14th and 21th days after modeling, compared with control group, the degree of paw swelling in model group was significantly increased （P<0.05）. Compared with model group, the swelling degree of paw in low- and high-dose groups was decreased （P<0.05）. At the end of administration, compared with control group, the synovial cells in model group were increased and in disorder, with a lot of inflammatory exudates in the articular cavity, and the inflammatory score, the levels of PLT, SCr and BUN, the serum levels of IL-6, TNF-α, IL-33 and ST2, and the protein expression of IL-33 and ST2 in ankle tissues were significantly increased （P<0.05）. Compared with model group, the inflammatory exudates in the articular cavity of the rats in low- and high-dose bortezomib groups were decreased, and the inflammatory score, the levels of PLT, SCr and BUN, the serum levels of IL-6, TNF-α, IL-33 and ST2, and the protein expression of IL-33 and ST2 in ankle tissues were decreased （P<0.05）. CONCLUSION Bortezomib may reduce the inflammation and swelling of the joints in RA rats by regulating the IL-33/ST2 signaling pathway.     

IL-33-modified BMSCs attenuate acute kidney injury induced by sepsis in rats through MyD88

AIM To investigate the effect of interleukin-33 (IL-33)-modified bone marrow mesenchymal stem cells (BMSCs) on sepsis-induced acute kidney injury (AKI) in rats and the expression of myeloid differentiation factor 88(MyD88). METHODS A septic rat model was established by cecal ligation and puncture. The SD rats (n=80) were randomly divided into control group, model group, negative transfection group (transplanting untransfected BMSCs) and IL-33 transfection group (transplanting BMSCs transfected with IL-33), with 20 in each group. Survival rates of the rats within 72 h in the 4 groups were compared. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured before, and 24, 48 and 72 h after transplantation. The kidney pathological damage was observed by HE staining, and the apoptosis of renal cells was detected by TUNEL method 72 h after transplantation. Western blot was used to detect the protein expression levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), Toll-like receptor 4 (TLR4) and MyD88. RESULTS The survival rate of the rats in model group was significantly lower than that in control group (P<0.05). The survival rate of the rats in IL-33 transfection group was higher than that in model group and negative transfection group (P<0.05). The levels of SCr and BUN in model group were higher than those in control group (P<0.05). The levels of SCr and BUN in IL-33 transfection group were significantly reduced after transplantation, and were lower than those in model group and negative transfection group (P<0.05). The renal tissue pathological injury score in model group was significantly higher than that in control group (P<0.05). Compared with model group and negative transfection group, the renal tissue pathological injury score in IL-33 transfection group was significantly reduced (P<0.05). The proportion of apoptotic cells in the kidney tissues in model group were higher than that in control group (P<0.05). Compared with model group and negative transfection group, the proportion of apoptotic cells in the kidney tissues in IL-33 transfection group was significantly reduced (P<0.05). The protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in model group were significantly higher than those in control group (P<0.05). Compared with model group and negative transfection group, the protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in IL-33 transfection group were significantly decreased (P<0.05). CONCLUSION IL-33 gene-modified BMSCs significantly improve the renal function of AKI rats with sepsis. The mechanism may be related to IL-33 regulating TLR4/MyD88 signaling pathway and inhibiting renal inflammatory response.     

Abnormal activity of ROCK contributes to increased adhesion and migration of peripheral blood T cells from patients with systemic lupus erythematosus

AIM: To investigate the role of Rho kinase (ROCK) in the regulation of adhesion and migration of the T cells from systemic lupus erythematosus (SLE) patients. METHODS: The T cells were isolated by RosettSep T cell purification kit. ROCK activity was assessed by Western blotting. T cell migration was examined by Transwell chambers. RESULTS: Compared with the T cells from healthy controls and rheumatoid arthritis patients, the activity of ROCK in ex vivo T cells from SLE patients was significantly increased. In vitro, the adhesion and migration of the T cells from SLE patients were also increased. Furthermore, the adhesion and migration of the T cells from SLE patients were inhibited by a specific ROCK inhibitor Y27632. CONCLUSION: The results indicate that ex vivo T cells from SLE patients exhibit increased activity of ROCK. Alteration of ROCK activity may contribute to the abnormal adhesion and migration of T cells from SLE patients.     

Effects of lipoxin A4 on proliferation and IL-2/IL-2 receptor expression in Jurkat T cells

AIM: To investigate the effect of lipoxin A4 on the proliferation and IL-2 production in Jurkat T cells. METHODS: Jurkat T cells were activated in vitro with anti-CD3 (2 mg/L) and anti-CD28 (2 mg/L) antibodies in the absence or presence of lipoxin A4 (0.1 nmol/L-100 nmol/L) for 24 h, then ［3H］-TdR was added into the medium and radioactivities were measured by scintillation counting. The concentrations of IL-2 in the supernants were determined by ELISA. Cells were harvested and the expression of CD25 was assessed by FCM. For analysing the cell cycle, the cells were stained with PI and DNA contents were detected by FCM. RESULTS: Lipoxin A4 suppressed the proliferation of anti-CD3 and anti-CD28 antibodies activated Jurkat cells in a dose-dependent manner, which was associated with reduced proportion of S phase cells. Furthemore, lipoxin A4 significantly inhibited the production of IL-2 but had no obvious effect on CD25 expression. CONCLUSION: Lipoxin A4 can suppress proliferation of activate Jurkat cells and IL-2 production, through which lipoxin A4 might negatively regulate immune response.     

Study on COX activity and its mRNA expression,and PGE2 release from cerebral microvascular endothelial cells stimulated by IL-1β

AIM: To study the cyclooxygenase(COX) activity and its mRNA expression, and PGE₂ release from rats cerebral microvascular endothelial cells (rCEMC) stimulated by IL-1β(30 μg/L) at different times. METHODS: rCMEC were cultured, and identified by immunohistochemistry for von Willebrand factor (Ⅷ factor, a marker for all endothelial cells) in cytoplasm of the cells. After rCEMC grew to confluency, they were stimulated with IL-1β for 0.5, 1, 2, 4, 8, 12 and 24 h, respectively. Activity of COX-1 and COX-2 in rCEMC and production of PGE₂ in the conditioned media were detected by ELISA. COX-1 and COX-2 mRNA expressions were measured by real-time quantity PCR. The amplification product was tested by melting curve and identified by electrophoretic gel. RESULTS: ① Positive immunostaining for Ⅷ factor was present diffusely in the cytoplasm in more than 90% rCMEC. ② Compared to the cells without IL-1β stimulation, the production of PGE₂ increased significantly (P<0.05) at 4 h after rCEMC were incubated with IL-1β and reached the top level at 12 h (P<0.01), then declined thereafter at 24 h (P<0.05). ③ There was no significant difference on COX-1 activity between IL-1β group and non-IL-1β group. COX-2 activity increased significantly compared with those in non-IL-1β (P<0.05) at 8 h after rCEMC were incubated with IL-1β and reached the top level at 12 h (P<0.01), then declined thereafter at 24 h (P<0.05). ④ There was no significant difference on COX-1 mRNA expression between IL-1β group and non-IL-1β group. COX-2 mRNA was induced and became detectable at 1 h, and reached the top level at 4 h, then declined thereafter at 8 h and became undetectable by 12 h and 24 h after incubation with IL-1β. The melting curve showed there was no nonspecific amplification and electrophoretic gel showed the lengths of amplification products accorded with the predicted lengths. CONCLUSION: While rCEMC are stimulated by IL-1β, the excretion of PGE₂ increases and reaches the top level at 12 h, which is related with its induction on COX-2 mRNA expression and COX-2 activity.     

Electroacupuncture improves spatial learning and memory ability of rats with chronic cerebral hypoperfusion and signal regulation of hippocampal IL-6/JAK2/STAT3

AIM:To observe the effect of electroacupuncture (EA) on the inflammatory response and hippocampal JAK2/STAT3 signaling pathway in the rats with chronic cerebral hypoperfusion (CCH), and to explore the mechanism of EA attenuating the spatial learning and memory impairment induced by CCH. METHODS:Adult male Sprague-Dawley rats were randomly divided into sham group, model group and EA group (n=10). Modified permanent bilateral common carotid artery occlusion was used to establish animal model. The rats in EA group were stimulated at "Baihui" and "Dazhui" acupoints by 2/15 Hz frequency (30 min/d for 4 weeks), while the rats in the other 2 groups received balanced treatment. The spatial learning and memory ability and regional cerebral blood flow (rCBF) were detected by the methods of Morris water maze and laser Doppler flowmetry. The concentrations of interleukin (IL)-6 and IL-1β, the mRNA expression of JAK2 and STAT3, and the phosphorylated JAK2 and STAT3 protein levels in the hippocampus were determined by ELISA, RT-PCR and Western blot. The pathological changes of the hippocampus were observed with HE staining. RESULTS:In EA group, the rCBF, the average escape latency at every time point, and the original platform quadrant residence time were better than those in model group (P<0.01 or P<0.05). The level of IL-1β in EA group was significantly lower than that in model group (P<0.05), and the level of IL-6 was significantly increased (P<0.05). The mRNA expression of JAK2 and STAT3, and the protein levels of p-JAK2 and p-STAT3 in EA group were significantly higher than those in model group (P<0.05). The impairment of nerve cells in the hippocampal CA1 region was reduced. CONCLUSION:Electroacupuncture inhibits inflammatory response, and alleviates the hippocampal damage and the cognitive disorder by regulating IL-6/JAK2/STAT3 signaling pathways.     

Proportion of CD14+CD16+ monocytes in peripheral blood from patients with type 2 diabetic mellitus and effect of LPS and IL-15 on expression of STAT5 in monocytes

AIM: To characterize the proportion of CD14⁺CD16⁺ monocytes in peripheral blood from type 2 diabetes (T2DM) patients and to observe the response of CD14⁺CD16⁺ monocytes to lipopolysaccharide (LPS) and interleukin-15 (IL-15) for further exploring the potential mechanism of inflammatory immune response in the pathogenesis of T2DM. METHODS: Twenty-eight patients with T2DM and 20 healthy volunteers were enrolled in the study. The peripheral blood was collected for determining the percentage of CD14⁺CD16⁺ monocytes by flow cytometry. The peripheral blood mononuclear cells (PBMC) were isolated and subject to stimulation with LPS and IL-15 for 4 h. The protein expression of STAT5 was detected by Western blotting and the phosphorylated (p)-STAT5 was determined by Western blotting and immunofluorescence. Serum levels of 25-hydroxyvitamin D₃ and IL-6, and the concentrations of IL-6 and monocyte chemoattractant protein-1(MCP-1) in the culture supernatants were assessed by ELISA. Serum level of C-reactive protein (CRP) was measured by immunoturbidimetry. RESULTS: There were positive correlations between the quantity of CD14⁺CD16⁺ monocytes and serum levels of CRP and IL-6 (r=0.394, P<0.05 and r=0.741, P<0.01), while serum 25 (OH) D₃ was negatively correlated with the quantity of CD14⁺CD16⁺ monocytes (r=-0.409, P<0.01), serum CRP(r=-0.479,P<0.01) and serum IL-6 (r=-0.774,P <0.01). After stimulated with LPS and IL-15, PBMC showed significant up-regulation of p-STAT5 protein expression, and significant increases in the supernatant levels of IL-6 and MCP-1 were observed (P<0.05). The expression of p-STAT5 existed in the nucleus.CONCLUSION: These findings suggest that the functional disturbance in monocytes occurs in T2DM, which may be related to insufficiency of vitamin D₃. The aberrant activation of STAT5 signaling pathway underlies the functional abnormalities of the monocytes in T2DM.