Expressions of IL-22 in circulating CD4+/CD8+ T cells and their correlation with disease activity in SLE patients

Recently, Th17 cell-associated responses have received growing attention; however, the role of IL-22 (a cytokines also produced by Th17 cells) in the pathogenesis of systemic lupus erythematosus (SLE) has not been widely explored. In this study, we analyze the frequencies of IL-22-positive CD4+/CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from patients with SLE and their correlations with disease activity and clinical data. Five-color flow cytometry (FCM) was used to assess IL-22 production of CD4+/CD8+ T cells in PBMCs from 31 patients with SLE and 22 healthy control subjects, following stimulation ex vivo with phorbol 12-myristate 13-acetate and ionomycin for 4 h. Results showed that the percentages of IL-22-positive CD4+ T cells were increased in the PBMCs of patients with SLE compared with healthy control subjects, whereas there were no significant differences in the percentages of IL-22-positive CD8+ T cells. There was a strong positive correlation between the proportion of CD4+ T cells expressing IL-22 and SLEDAI score (r _s = 0.65, P < 0.001). Furthermore, the frequencies of IL-22-positive CD4+ T cells were significantly higher in patients with SLE with nephritis than those without nephritis (Z = −2.72, P < 0.01). In conclusion, increased frequencies of IL-22-positive CD4+ T cells in patients with SLE and positive correlation with SLEDAI score and lupus nephritis suggest that this cytokine may be implicated in the pathogenesis of the disease.     

Immunomodulation of human CD19+CD25high regulatory B cells via Th17/Foxp3 regulatory T cells and Th1/Th2 cytokines

Regulatory B (Breg) cells are a special subset of immunoregulatory cells with unique phenotypes and functions. In this study, human CD19⁺CD25^high Breg cells were purified from human peripheral blood. Based on the coculture system of Breg cells and CD4⁺ T cells in vitro, Breg cells were found to promote the increase in regulatory T (Treg) cells while decreasing the number of Th17 cells. Breg cells regulate Treg cells through two processes: cell-cell contact and cytokines. TGF-βsRII, a blocker of transforming growth factor-β (TGF-β), can attenuate the effects of Treg elevation, suggesting that TGF-β is the main cytokine, while Breg cells rather than interleukin-10 (IL-10) regulate the differentiation of Treg cells. However, Th17 cells were mainly regulated by cytokines, without an obvious regulatory effect on cell-cell contacts. Breg cells may regulate Th17 cells by a pathway independent of TGF-β and IL-6. The coculture of Breg cells and CD4⁺ T cells led to changes in the cytokine spectrum, which included significant increases in IL-4, IL-6 and IL-10 but not obvious changes in IL-2, IFN-γ and TNF. The inhibitory effect of Breg cells was weakened by blocking cell-cell contacts in cultures separated with the Transwell chamber because IL-10 decreased while IL-6 increased when compared with cocultured Breg and CD4⁺ T cells. When the IL-10 inhibitor IL-10sRα was added, IL-6 and TNF levels significantly increased, while treatment with the TGF-β inhibitor TGF-βsRII did not result in similar changes, suggesting that IL-10 is an important molecule to inhibit the proinflammatory factors IL-6 and TNF in this culture system.     

Decreased IL-35 levels in patients with immune thrombocytopenia

IL-35 is a novel heterodimeric anti-inflammatory cytokine consisting of Epstein–Barr virus-induced gene 3 (EBI3) and the p35 subunit of IL-12. IL-35 has been shown to possess the potency of inhibiting the CD4+ effector T cells and alleviating autoimmune diseases. In the study we investigated the levels of IL-35 as well as its prospective role in immune thrombocytopenia (ITP).ELISA was adopted to measure plasma IL-35, TGF-β and IL-10 levels. The mRNA expression levels of P35 and EBI3 in peripheral blood mononuclear cells (PBMCs) were studied based on real-time quantitative PCR. The correlation between plasma cytokine levels and clinical parameters was analyzed. Significantly lower plasma IL-35 levels were found in active ITP patients compared with those in remission (p = 0.017) and the healthy controls (p < 0.001). In active ITP patients, the plasma IL-35 levels displayed a significantly positive correlation with platelet counts (r = 0.5335, p < 0.0008). Further, P35 mRNA expression levels were lower in patients with active ITP than patients in remission (p = 0.033) and normal controls (p = 0.016).Thus, for the first time, this research reported a dramatically decreased IL-35 levels in ITP patients, suggesting that IL-35 may be involved in the pathogenesis of ITP.     

Interleukin-6 induces the generation of IL-10-producing Tr1 cells and suppresses autoimmune tissue inflammation

Compared with its pro-inflammatory function, the mechanisms underlying the anti-inflammatory effect of IL-6 are poorly understood. IL-6 can cooperate with TGF-β to induce IL-10 production in Th17 cells in vitro. However, the effect of IL-6 on generation of Tr1 cells and the in vivo importance of this effect are mostly uncharacterized. In this study, we showed that in vitro, IL-6 can induce the generation of IL-10-producing Tr1 cells from naïve CD4 T cells, independently of IL-27 and TGF-β. IL-6 induces IL-21 production in CD4 T cells and IL-10-inducing effect of IL-6 requires both IL-21 and IL-2. Although IL-6 cannot induce IL-10 production in CD8 T cells in a cell-autonomous manner, it can do so indirectly through promoting CD4 T cell IL-21 production. The IL-10-producing T cells induced by IL-6 have phenotypic, genetic and functional traits of Tr1 cells and can suppress LPS-induced in vivo inflammatory response in an IL-10-dependent fashion. Blockade of IL-6 in two autoimmune inflammation models, induced respectively by anti-CD3 antibody or Treg-depletion, led to reduction in IL-10-producing T cells and exacerbated inflammation of lung and intestine. Thus, we delineated critical pathways involved in IL-6-induced generation of Tr1 cells and demonstrated the importance of this event in restraining autoimmune tissue inflammation.     

Selected biologic markers of inflammation and activity of Crohn’s disease

Abstract

The study aimed to compare the accuracy of selected biologic markers in assessing the disease activity in patients with Crohn’s disease (CD). The analysis included serum IL-2, IL-6, IL-17, TNF-α, IFN-γ, hsCRP, peripheral CD4?+?CD25?+?FOXP3?+?regulatory T cells, as well as fecal calprotectin and lactoferrin. A group of 55 adults with CD was enrolled to the study. Disease activity was assessed using Crohn’s Disease Endoscopic Index of Severity (CDEIS), which currently represents the gold standard for the evaluation of endoscopic activity. For clinical activity scoring, the Crohn’s Disease Activity Index (CDAI) was used. Concentrations of investigated markers were estimated by means of flow cytometry and enzyme-linked immunosorbent assays, and the results were correlated with both indices. The study demonstrated that both fecal markers, i.e. calprotectin (r?=?0.827, p?<?0.001) and lactoferrin (r?=?0.704, p?<?0.001), correlate closely with CDEIS score, and might be used to evaluate the severity of CD in clinical setting. The correlation of those markers with CDAI was also significant, with r?=?0.742 for calprotectin (p?<?0.001) and r?=?0.675 for lactoferrin (p?<?0.05). As for the other investigated markers, only hsCRP (r?=?0.672, p?<?0.001) and IL-17 (r?=?0.296, p?<?0.005) correlated closely with CDEIS. The correlation of the markers with CDAI was also significant, though weaker, with r?=?0.518 for hsCRP (p?<?0.001) and r?=?0.296 for IL-17 (p?<?0.05). The study showed that IL-17, despite its vague role in the pathogenesis of CD, might be a useful marker, comparable with hsCRP, in assessing the activity of the disease.     

IL-35 Is Involved in the Pathogenesis of Guillain–Barré Syndrome Through Its Influence on the Function of CD4+ T Cells

CD4+ T cells and many cytokines play critical roles in the pathogenesis of Guillain–Barré Syndrome (GBS), an immune-mediated inflammatory disease. However, the role of IL-35, a novel member of the IL-12 cytokine family, in this kind of disease has not yet been elucidated. In this study, we investigated the functional changes of CD4+ T cells from GBS patients with IL-35 treatment in vitro. This study involved 21 GBS patients and an equal number of healthy controls (HCs). The results indicated that the average concentration of IL-35 in the plasma of GBS patients was lower than that of healthy controls (HCs). Increased levels of STAT1, STAT3 and STAT4 proteins and T-bet, ROR γt, IFN-γ and IL-17A mRNA were observed in CD4+ T cells from GBS patients. In contrast, the levels of STAT5 and STAT6 proteins and GATA3, Foxp3, IL-4 and TGF-β1 mRNAs were decreased in GBS patients in comparison with those of HCs. In addition, treatment of CD4+ T cells from GBS patients with IL-35 upregulated IL-35, STAT5 and STAT6 protein and T-bet, GATA3, Foxp3, IFN-γ, IL-4, IL-17A and TGF-β1 mRNA while inhibited levels of STAT3 and STAT4 protein and RORγt and IL-17A mRNA. These results indicate that IL-35 might play a potential role in GBS pathogenesis. Further studies are required in order to evaluate its role in GBS.     

Antigen-independent IL-17A production by bystander-activated CD4+IL-1R1+ cells in patients with multiple sclerosis

Multiple sclerosis (MS) is a demyelinating disease caused by auto-antigen recognizing CD4⁺ T cells. However, IL-17A-producing CD4⁺ T cells that are bystander-activated by IL-1β and IL-23, and T cell receptors independently, could contribute to experimental autoimmune encephalomyelitis. Here, we studied the differences in the frequency and function of bystander-activated CD4⁺ T cells in patients with MS. A significantly higher frequency of CD4 + IL-1Rl + T cells was found in memory than in naïve CD4⁺ T cells and in Th17/Th17.1 than in Th1/Th2 subtypes in both MS and healthy controls (HC). Following IL-1β and IL-23 stimulation, IL-1Rl expression was markedly increased in both memory and Th17/Th17.1 cells, and their IL-17A-production was increased after bystander-activation, which was significantly higher in MS compared with HC. Our study suggests a potential role of IL-17A-producing bystander-activated CD4⁺IL-1Rl⁺ T cells in MS.     

Differential Expression of CD30 on CD3 T Lymphocytes in Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune systemic disease caused as a result of an imbalance of Th1‐/Th2‐type cytokines. The soluble form of CD30 (CD30s) released from peripheral blood cells has been described as a marker of active disease in Th2‐type immune response as in SLE. However, the expression of CD30 on CD3 T lymphocytes from patients with SLE has not been studied yet. Therefore, we have addressed our study to attempt this issue, studying CD30 expression by flow cytometry on CD3 T lymphocytes and CD4/CD8 subsets in samples from SLE patients mainly with lupus nephritis. In parallel, we have determined the production of the cytokines IL‐4 (Th2), IFNγ (Th1), IL‐10 and TGFβ by intracellular staining. Differences between positive CD30 T cells in healthy controls and patients with SLE were found, with a higher percentage of CD30‐expressing T cells in patients with SLE (P = 0.001). In contrast to healthy controls, CD30 was mainly expressed on CD8 T cells from patients with SLE. The intracellular cytokine staining showed that TGFβ is the main cytokine expressed in CD3 T cells from patients with SLE. In addition to this, we have found a positive correlation between CD30‐expressing T cells and IL‐4, IFNγ, and immunosuppressive cytokines (IL‐10 and TGFβ) (P < 0.05). These results suggest that CD30 could play a role in the pathogenesis of SLE and its expression on CD3 T lymphocytes is not restricted only to Th2‐type response.     

Low-Dose Daily Subcutaneous Interleukin-2 in Combination with Highly Active Antiretroviral Therapy in HIV + Patients: A Randomized Controlled Trial

Abstract

Purpose: Previous studies with intermittent interleukin-2 (IL-2) therapy using intermediate and high levels of IL-2 have demonstrated significant increases in the CD4 + T cell count in HIV-infected patients. Intermittent regimens are amenable to outpatient use, but severe adverse events are frequently experienced with intermediate- and high-dose levels of IL-2. Therefore in this study, the effect of daily, subcutaneous low-dose IL-2 therapy on safety and immunological endpoints was investigated to determine whether immunological benefit could be achieved without toxicity in HIV-infected patients also receiving highly active antiretroviral therapy (HAART). Method: A total of 115 patients were enrolled in the trial. Fifty-six asymptomatic HIV-infected patients who had CD4 + T cell counts less than 300 cells/μL at screening and a stable HIV viral load received low-dose IL-2 (1.2 million IU [MIU]/m 2 beginning dose) once daily in conjunction with HAART (IL-2 group). Fifty-nine patients received HAART alone (control group). Results: A dramatic effect of IL-2 on the natural killer (NK) cell population was observed with mean increases of 156 cells/μL in the IL-2 group compared to 19.93 cells/μL in the control group (p < .001). Additionally, IL-2-treated patients experienced a statistically significant increase in the mean percentage of CD4 + T cells (3.52% increase) when compared to control patients (1.33% increase) (p < .001). The expanded CD4 + T cell population was primarily of the naive phenotype, with mean increases of 4.53% for the IL-2 group and 0.31% for the control group (p < .001 for between-group difference). In addition, a higher proportion of IL-2-treated patients (67%) compared to control patients (33%) achieved increases of greater than 50% in the CD4+ T cell count (p = .08). Adverse events of grade 3 or grade 4 toxicity were infrequent in the current study and were substantially lower by comparison to those in studies of intermittent dose IL-2 therapy. Also, negligible changes in the HIV viral load from baseline to final measurement were observed in both groups. A trend toward a reduced number of modifications of antiretroviral therapy was apparent in the IL-2 group when compared to control patients. Conclusion: Daily, low-dose subcutaneous IL-2 therapy in conjunction with HAART is safe and well tolerated and is effective in expanding lymphocyte cell types including NK cells and naive T cells in individuals who have <300 CD4+ T cells.     

High-throughput Methods for Measuring Autoantibodies in Systemic Lupus Erythematosus and other Autoimmune Diseases

The transforming growth factor-beta (TGF-β) protein family is highly evolutionarily conserved and they have been implicated in many biological processes. Also, TGF-β can exert pivotal functions in the immune system. It is widely accepted that regulatory T cells (Treg cells) play an important role in the maintenance of the immune homeostasis, but the underlying molecular mechanisms through which they can gain and/or perform suppressive functions in an active way remains to be defined. Though the engagement of TGF-β in the Treg cells has been discounted for a period of time, an emerging body of data has established a close link between Treg cells and TGF-β, as TGF-β has been demonstrated to induce the expression of Foxp3, which acts as a master regulator for the development and function of Treg cells. We will, herein, focus on the crucial role of TGF-β signaling in Treg cell biology and summarize the current studies regarding TGF-β in the generation and function of CD4⁺CD25⁺Treg cells both in vivo and in vitro.     

Alterations of lymphocyte subpopulations and TGF-β in children with transient or persistent cow’s milk allergy

The aim of the study was to evaluate changes in surface receptor expression of B and T lymphocytes and concentration of TGF-β in children who either developed tolerance to cow’s milk protein (CMP) or manifested persistent cow’s milk allergy (CMA). The study involved 30 patients with CMA who underwent an open food challenge after 12 months of milk-free diet. After the milk challenge, decreased concentration of CD19⁺CD23+ was observed in children who acquired tolerance to CMP, in comparison with the test before cow’s milk (CM) challenge (42.2% vs. 29.1%, p?=?.006). The same group demonstrated lower concentration of TGF-β than patients with persistent allergy (median 37.9 pg/ml vs. 52.8 pg/ml, p?=?.003, respectively). Moreover, before CM challenge, higher percentage of CD3+CD8+CD28+CD152+ cells (median 2.88% vs. 1.2%, p?=?.03) and CD3+CD4⁺CD25⁺CD62L⁺ (median 42.3% vs. 13.4%, p?=?.032) was noted in children who acquired tolerance to CMP, in comparison with subjects who remained allergic to CMP.     

Circulating lymphocyte subsets and regulatory T cells in patients with postpartum thyroiditis during the first postpartum year

The lymphocyte subsets and the percentages of activated T cells or regulatory T cells were observed during the course of postpartum thyroiditis (PPT). Heparin anticoagulant vein bloods were collected at 3, 6, 9 and 12 months postpartum consequently from 27 PPT patients and 23 normal postpartum subjects. Lymphocyte surface antigen CD3, CD4, CD8, HLA-DR and CD25 were stained in appropriate combination and detected with flurorescence-activated cell sorter analysis. The percentage of CD4 was significantly lower in both PPT groups with biphasic diseases and isolated hypothyroidism at 3 months postpartum as compared to control postpartum women separately (both P < 0.05). Then, decreased CD4/CD8 ratios were also appeared in these groups. Patients with both positive TPOAb and TgAb had higher percentage of activated T (HLA-DR⁺CD3⁺) cells compared to control postpartum women at 3 months postpartum (P < 0.05). The percentage of activated T cells correlated with a raised percentage of CD8⁺ T cell subset (P < 0.001) and a decreased percentage of regulatory T (CD25⁺CD4⁺) cells (P < 0.01). The percentages of regulatory T cells were significantly higher in control postpartum women at 3, 6 and 9 months postpartum compared with non-postpartum women (P < 0.05). However, it was lower in PPT patients at 3 months compared to itself at 6 and 9 months postpartum (P < 0.05). In the early postpartum period of PPT patients, a reduced helper/inducer T cell subset, an increased percentage of activated T cells and a reduced percentage of regulatory T cells were reported, indicating that T cells may play a key role in the pathogenesis of PPT.     

A TGF-β mediated regulatory mechanism modulates the T cell immune response to rotavirus in adults but not in children

Children with acute RV-gastroenteritis (GE) had low or undetectable levels of circulating IFN-γ⁺, IL-13⁺, IL-2⁺, IL-10⁺ or IL-17⁺ RV-T cells. IFN-γ⁺ T cells and low frequencies of IL-10⁺ and IL-2⁺ CD4⁺ T cells were found in adults with RV-GE during acute and convalescence phases, respectively. Circulating single IFN-γ⁺ > double IFN-γ⁺/IL-2⁺ > single IL-2⁺RV-CD4⁺T cells were observed in healthy adults. In this group, frequencies of IFN-γ⁺ RV-T cells increased after removing CD25⁺cells, blocking TGF-β with its natural inhibitor, LAP, or inhibiting TGF-βRI signalling pathway with ALK5i. The frequencies of IFN-γ⁺ RV-T cells were also incremented in PBMC depleted of CD25⁺cells and treated with ALK5i, suggesting that TGFβ inhibition may be independent of Treg cells. The ALK5i effect was observed in adults but not in children with RV-GE, who had normal numbers of TGF-β+ Treg cells. Thus, a TGF-β-mediated regulatory mechanism that modulates RV-T cells in adults is not evident in children.     

呼吸道合胞病毒毛细支气管炎患儿外周血CD4~+CD25~+调节性T细胞与Th17细胞功能变化及意义

目的:探讨呼吸道合胞病毒(RSV)毛细支气管炎患儿外周血CD4+CD25+调节性T细胞和Th17细胞及其分泌细胞因子IL-10、TGF-β、IL-17水平变化与RSV毛细支气管炎发病的关系。方法:收集2010-09/2011-04在滨州医学院附属医院儿科住院的33例RSV毛细支气管炎患儿、28例做为阳性对照的非RSV感染性肺炎患儿(肺炎组)及26例正常对照组的健康体检儿外周血,采用流式细胞术(FCM)检测外周血CD4+CD25+调节性T细胞、Th17细胞百分率,酶联免疫吸附(ELISA)法检测血浆IL-10、TGF-β、IL-17的水平。结果:RSV毛细支气管炎患儿外周血CD4+CD25+调节性T细胞、IL-10、TGF-β水平显著低于肺炎患儿及健康体检儿(P<0.05),而Th17、IL-17水平则显著高于肺炎患儿与健康体检儿(P<0.05)。结论:RSV毛细支气管炎患儿外周血存在CD4+CD25+调节性T细胞与Th17细胞表达失衡,可能是RSV毛细支气管炎发病机制之一。