Role And Mechanism Of Th17/Treg Imbalance And Its Interplay With Neutrophils In The Genesis Of Acute Respiratory Distress Syndrome

Objective:Acute respiratory distress syndrome (ARDS) is a life-threatening clinical syndrome of rapid-onset pulmonary failure with high morbidity and mortality in critically ill patients. Despite intense clinical and animal studies, the pathogenesis leading to this devastating disease remains poorly defined. However, activation of multiple inflammatory cells and release of inflammatory mediators play an important role in the development and outcome of ARDS. Local accumulation of polymorphonuclear leucocytes (PMNs) in the lungs and their inhibition of apoptosis or inhibition of clearance of apoptotic PMNs are the primary pathogenesis and resolution of inflammation during ARDS. Most recently, the involvement of lymphocytes, especially CD4+T cells, in the pathogenesis of ARDS has become an active topic of research. The imbalance between T helper (Th)17 cells, a more recently discovered effector subset of CD4+ T cells, and CD4+CD25+Foxp3+ regulatory T (Treg) cells has been found in a number of different inflammation and autoimmune diseases. Moreover, large amounts of inflammatory mediators produced by activated PMNs, are a potential new source of adjusting balance of Th17/Treg. However, both Th17 and Treg cells in turn, participate in the procedure of PMNs apoptosis and their recruitment into the inflammatory site. In this study, we investigated the distribution of Th17 cells in relation to Treg cells and their associated cytokines in ARDS patients, and evaluated the Th17/Treg pattern and its impact on disease severity and outcomes in patients with ARDS. Then by cellular experiments in vitro, after separation and purification of PMNs, Naive CD4+ T cells, Th17 and Treg cells by magnetic cell sorting (MACS), we further investigate how PMNs may influence Th17 and Treg cells differentiation, and elucidate the effect of Th17/Treg imbalance on the PMNs chemotaxis and apoptosis, and the role of cross-talk between PMNs and imbalance of Th17/Treg during the progress of ARDS. In summary, We hope to provide new theoretical basis to improve potential therapeutic strategies for prevention and control of ARDS, and extend our knowledge to better understand the cross-talk between the innate and adaptive immune system involved in the pathogenesis of ARDS.Methods:1. This prospective, observational study enrolled 79 patients who fulfilled the Berlin definition of ARDS and 26 age- and sex-matched healthy controls. Circulation Th17 and Treg cell frequencies were analyzed by flow cytometry (FCM), and the expressions of Th17- and Treg-related cytokines (IL-17, IL-6, IL-10, TGF-β1) in serum were measured by enzyme-linked immunosorbent assay (ELISA). After enrollment, demographics and baseline characteristics such as age, sex, body mass index (BMI), risk factors of ARDS, severity of illness at ICU admission (Acute Physiologic and Chronic Health Evaluation (APACHE) Ⅱ score), Sequential Organ Failure Assessment (SOFA) score, and the Lung Injury Score were calculated. All patients were followed up for 28 days. The main outcomes were mortality at discharge from ICU and hospital at 28 days.2. After separation and purification of PMNs, Naive CD4+T cells, Th17 and Treg cells from ARDS by magnetic cell sorting (MACS), both the purity and viability of these cells assessed by FCM, or by means of trypan blue exclusion, respectively were more than 95%. The effects of PMNs on Naive CD4+ T cells polarization were determined by cell-to-cell contact and transwell co-culture experiments in vitro. The frequencies of Thl7 and Treg cells and surface expression of MHC class Ⅱ molecule on PMNs, the expressions of Th17- and Treg-related cytokines (RORyt mRNA, IL-17, IL-6, and Foxp3mRNA, IL-10, TGF-β1) and the expressions of MHC class Ⅱ molecule in PMNs were analyzed respectively, by FCM, Real-time PCR, ELISA or Western Blot. Chemotaxis assays between PMNs and Th17 cells were performed using 5-um pore polycarbonate filters in the 24-well Transwell chambers, preincubated or not for 30 minutes with Abs neutralizing CCL2, CCL20 or their isotype-related antibodies added to the bottom wells. After a 2-hour incubation, migrated cells were counted. Surface expression of CCR2/CCR6 on Th17 cells were analyzed by FCM, ELISA was used to measure peripheral serum levels of CCL2 and CCL20 in ARDS and controls, or in lysates of their PMNs.3. The abilities of Th17 or Treg cells to directly regulate PMNs life span and function were determined by cell-to-cell contact and transwell co-culture experiments in vitro. In some experiments, preincubated or not for 30min with Abs neutralizing IL-8, GM-CSF and IFN-γ or their isotype-related antibodies added to the bottom wells. After a 1-hour incubation, migrated cells were counted. ELISA was used to measure levels of IL-8, GM-CSF and IFN-γ in lysates of Th17 or Treg cells from ARDS and controls, surface expression of CD11b on PMNs and their apoptosis were analyzed by FCM.Results:1. Within 24 hours after the onset of ARDS, the changes of peripheral circulating Th17 and Treg cell frequencies gradually increased from mild to severe ARDS, leading to the Th17/Treg imbalance toward a proinflammatory Th17 response when compared to health controls. IL-6 and IL-17 were significantly higher compared with those in the control group, and both of these two cytokines were associated with the severity or progression of ARDS. While IL-10 levels was increased in ARDS patients when compared to those in the control group, there was no significant difference in the TGF-β1 levels among all groups. Th17/Treg ratio was positively correlated with APACHE II score, SOFA score, and Lung Injury Score, while negatively correlated with PaO2/FiO2. The areas under the receiver operating characteristic (AUC) curves of Th17/Treg ratio for predicting 28-day mortality in ARDS patients was higher than that of APACHE Ⅱ score, SOFA score, Lung injury score, as well as PaO2/FiO2. Using a Th17/Treg ratio cutoff value of >0.79 for predicting 28-day mortality in patients with ARDS, the sensitivity and specificity was 87.5% and 68.1%, respectively, and the positive and negative likelihood ratios were 2.74 and 0.18. Multivariate logistic regression showed Th17/Treg ratio >0.79 (odds ratio = 8.68, P = 0.002) was the independent predictor for 28-day mortality in patients with ARDS. Finally, cumulative survival rates at 28-day follow-up also differed significantly between patients with Th17/Treg ratio >0.79 and ≤0.79 (Log-rank test, P＜0.001).2. After cell-to-cell contact co-culture with Naive CD4+T cells, surface expression of MHC class Ⅱ molecule on PMNs was significantly increased. Th17 cell frequencies and the expressions of Th17-related cytokines (RORyt mRNA, IL-17, IL-6) was also higher than that of the controls. However, all of these changes was greatly suppressed by specific MHC Ⅱ-neutralizing antibodies preincubated with PMNs. Meanwhile, PMNs are unable to modulate Treg cells differentiation. In addition, both of CCR2 and CCR6 expression on Th17 cells surface, and the expressions of CLL2 and CCL 20 in ARDS patients and their PMNs were higher than that of in the controls. When preincubated with Abs neutralizing CCL2 and CCL20, the chemotactic effect of PMNs on Th17 cell can be greatly suppressed.3. The expressions of IL-8, GM-CSF and IFN-y was higher in Th17 cells from ARDS patients than that from the controls. Remarkably, the use of neutralizing antibodies against IL-8 completely suppressed the chemotactic effects of Th17 cells on PMNs. Both in cell-to-cell contact and transwell co-culture experiments, Th17 cells could significantly stimulate the increased CDllb expression on PMNs, and reduce their apotosis, both of which could be significantly suppressed when preincubated with Abs neutralizing GM-CSF and IFN-y to Th17 cells. Furthermore, Treg cells have no chemotactic effect on PMNs. Treg cells, incubated in direct contact but not transwell co-culture with PMNs, down-regulated expression of CD11b on PMNs and promoted a significant increase in PMN apoptosis.Conclusions:1. In patients with early ARDS, the peripheral circulating Th17 and Treg cells and their associated cytokines increased. The Th17/Treg ratio towards Th17 cell subset contributes to the pathogenesis of ARDS, and higher Th17 to Treg cell ratio may be associated with poorer prognosis. Moreover, strategies designed to restoring the Th17/Treg balance may be a novel and effective therapeutic approach in many inflammation and autoimmune diseases including ARDS.2. Cell-to-cell contact is required for PMNs to promote differentiation of Th17 cells in vitro from Naive CD4+ T cells in a MHC Ⅱ-dependent manner. PMNs can also recruit Th17 cells via chemokine CCL2/CCL20 release into the lungs, leading to the Th17/Treg imbalance toward a proinflammatory Th17 cells response.3. Th17 cells not only promoted a IL-8-dependent chemotactic effect on PMNs, but also increased PMNs activity and delayed their apotosis through GM-CSF and IFN-y release, and ultimately creating a pathogenic proinflammatory loop to amplify proinflammatory responses in patients with ARDS.4. Although having no chemotactic effect on PMNs, Treg cells can through cell-to-cell contact, decrease PMNs activity and promote their apotosis to counteract Th17 cell-mediated responses.