EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 3343-3351, 2020 CCL26 regulates the proportion of CD4+CD25+FOXP3+ Tregs and the production of inflammatory factors in peripheral blood mononuclear cells following acute ischemic stroke via the STAT5 pathway ZHIQIANG DONG1*, LIMEI CAO1*, LAN GUO2, YUAN HONG2, JINXIU CAO2 and XU CHEN1 1Department of Neurology, Shanghai Eighth People's Hospital; 2Geriatric Nursing Ward, Shanghai Eighth People's Hospital, Shanghai 200233, P.R. China Received January 20, 2020; Accepted May 20, 2020 DOI: 10.3892/etm.2020.9046 Abstract. Acute ischemic stroke (AIS) is the most common of inflammatory factors in PBMCs following AIS via the type of stroke. Recent studies have found that AIS is closely STAT5 pathway. involved in the immune regulation function of regulatory T cells (Tregs). C-C motif chemokine ligand 26 (CCL26) is a Introduction member of the chemokine family that plays an essential role in cell activation, cell differentiation, lymphocyte homing, At present, stroke has become the second most leading cause and inflammatory and immune responses. The present study of mortality worldwide and is also one of the chief factors aimed to investigate the role of CCL26 in the regulation of causing long-term disability among middle-aged and older Tregs in AIS. Peripheral blood mononuclear cells (PBMCs) individuals (1,2). There are approximately 17 million newly were incubated with a CCL26-neutralizing antibody. The diagnosed stroke cases each year worldwide. In Western proportion of cluster of differentiation (CD)4+CD25+ forkhead countries, stroke is the most common lethal disease ranked box P3 (FOXP3)+ Tregs was increased, and the expression of after cancer and acute myocardial infarction, with acute isch- FOXP3, phosphorylated signal transducer and activator of emic stroke (AIS) accounting for approximately 80% of all transcription 5 (p-STAT5), and that of the immunosuppressive stroke cases (3,4). Recent studies have found that the severity factors, interleukin (IL)-10 and transforming growth factor of damage in ischemic stroke cases is closely related to the (TGF)-β1, was upregulated. Conversely, the expression of immune response elicited by the time and extent of ischemia, immune-promoting factors, such as tumor necrosis factor particularly regulatory T cells (Tregs)-induced immune (TNF)-α and IL‑6 was significantly downregulated. Further responses; Tregs are a type of T cells with immunoregulatory experiments using CCL26 recombinant protein-treated PBMCs function (5,6). Related studies have demonstrated that Tregs revealed a decreased proportion of CD4+CD25+FOXP3+ are of utmost importance to the regulation of the pathogenesis Tregs and the downregulated expression of FOXP3, of immune diseases. Furthermore, studies have demonstrated p-STAT5, TGF-β1 and IL-10. Moreover, the expression that the proportion of Tregs in peripheral blood is signifi- of immunostimulatory factors, such as CX3C chemokine cantly lower in patients with ischemic stroke than in healthy receptor 1, TNF-α and IL‑6 was significantly upregulated. On controls (7,8) and that the decreased proportion of Tregs the whole, these results demonstrate that CCL26 regulates the induces the alleviation of its inhibitory effect on the inflam- proportion of CD4+CD25+FOXP3+ Tregs and the production matory response, which further results in the occurrence of inflammation, finally causing an imbalance of autoimmune inhibition and promoting the occurrence of ischemic stroke. Chemotactic cytokines are a group of secreted small-molecule proteins with cell chemotaxis that are responsible for cell directionality. At present, >50 types of Correspondence to: Dr Xu Chen, Department of Neurology, chemokines have been identified, and they are mainly divided Shanghai Eighth People's Hospital, 8 Caobao Road, Xuhui, Shanghai 200233, P.R. China into 4 categories, CXC, CC, C and CX3C. Chemokines play a E-mail: [email protected] critical role in cell activation, cell differentiation, immune and inflammatory responses, and lymphocyte homing based on *Contributed equally their regulatory functions in leukocyte migration. C-C motif chemokine ligand 26 (CCL26), whose receptor is CCR3, is a Key words: acute ischemic stroke, C-C motif chemokine ligand 26, typical CC class chemokine. It is also known as macrophage regulatory T cells, signal transducer and activator of transcription 5 inflammatory protein 4α, and it primarily plays a role in the regulation of eosinophils and T cells (9,10). Studies have confirmed that CCL26 can block the CCL2 response and 3344 DONG et al: CCL26 TARGETS Tregs that specific molecules, such as pro‑inflammatory cytokines, Japan) at 490 nm. The absolute concentrations of IL-10, including interleukin (IL)-4, tumor necrosis factor (TNF)-α, TGF-β1, TNF-α and IL-6 were calculated with reference to IL-1β and interferon-γ can promote the synthesis of CCL26 the standard curve. in human monocytes (11-13). Thus, CCL26 is of utmost importance to the regulation of inflammatory responses. Cell supernatant assay. PBMCs were isolated from peripheral The present study investigated the role of CCL26 in the blood samples collected from each subject in tubes containing pathogenesis of AIS by isolating peripheral blood mono- K2 ethylenediamine tetraacetic acid as the anticoagulant, as nuclear cells (PBMCs) from patients with AIS and treating previously described (16). They were treated with various them with a CCL26-neutralizing antibody or recombinant concentrations of recombinant human CCL26 (ab243255, protein. Thereafter, the proportion of Tregs and the expres- Abcam) (Rep CCL26; 0, 5, 25 and 100 ng/ml) or with 200 mg/l sion levels of phosphorylated signal transducer and activator anti-CCL26 neutralizing antibody (ab109612, Abcam) in the of transcription 5 (p-STAT5), forkhead box P3 (FOXP3), absence or presence of 1 µM the STAT5 inhibitor for 24 h, transforming growth factor (TGF)-β1, IL-10, TNF-α and IL-6 STAT5-IN-1 (CAS: 285986-31-4, EMD Millipore). Following were determined. treatment, the PBMC culture medium was centrifuged at 1,000 x g/min, and the supernatant was collected to perform Materials and methods ELISA, as mentioned above. Patients. Peripheral blood was collected from 35 patients Flow cytometric analysis. PBMCs were treated with various (20 males and 15 males) with acute ischemic middle cerebral concentrations of anti-CCL26 neutralizing antibody (ab109612, artery stroke who were aged between 54 and 69 years at Abcam) (0, 100, 200 and 500 mg/l), Rep CCL26 (0, 5, 25, and Shanghai Eighth People's Hospital from 2016 to 2018. The 100 ng/ml), or 200 mg/l of anti-CCL26 neutralizing antibody present study also included a control group that comprised 35 in the absence or presence of 1 µM STAT5‑IN‑1. Following healthy subjects (18 males and 17 males) aged between 53 and treatment, the PBMCs were counted, diluted to 1x106 cells/ml 66 years. The diagnosis of stroke was made according to the with PBS, and then incubated for 30 min at 4˚C in darkness World Health Organization criteria (14). Computed tomography with anti-human cluster of differentiation CD4 (11-0048-42, was performed upon admission, and intracerebral hemorrhage Ebioscience), anti-human CD25 (17-0257-42, Ebioscience) and and other possible causes of focal neurological symptoms were anti-human Foxp3 antibodies, according to the instructions excluded in each patient. Patients with diabetes were excluded provided with the Anti-Human Foxp3 Staining kit (560133; from the study, and other exclusion criteria were as follows: BD Biosciences). Appropriate isotype controls-human IgG1 Abnormal urinalysis findings (hematuria, leukocyturia and kappa antibody (ab206198, Abcam) were included in each proteinuria of 1,300 mg/24 h); clinical or laboratory signs experiment. Subsequently, the fluorescence intensities were or symptoms of complications following stroke; renal or determined using the BD FACSCalibur flow cytometry system hepatic failure; glomerulonephritis; and known concomitant (BD Biosciences). neoplastic disease. Investigators recorded data on sex, age and the presence of risk factors for stroke (hypertension, Western blot analysis. Following experimental treatment, ischemic heart disease and cigarette smoking) for each total protein was extracted from the PBMCs using patient. Neurological deficit was estimated using the 58‑point radioimmunoprecipitation assay buffer (JRDUN Biotech Scandinavian stroke scale (15). Blood samples were obtained Co., Shanghai, China). The total protein was quantified using upon admission, and serum and PBMCs were separated for BCA protein assay kit (23227, Thermo Fisher Scientific, Inc.). performing enzyme-linked immunosorbent assay (ELISA) And protein expression was detected using an enhanced and for the detection of the proportion of Tregs. The present chemiluminescence system (35055, Thermo Fisher Scientific, study was approved by the Ethics Committee of Shanghai Inc.) according to the manufacturer's instructions. Equal Eighth People's Hospital, and written informed consent was amounts of protein (35 µg) were separated on a 12% gel using obtained from each subject. sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (JRDUN Biotech ELISA Co.). After blocking with 5% skimmed milk, the membranes Patient serum processing. Serum was dissolved at 37˚C were incubated overnight with anti-CCL26 antibody (ab190669, and commercial ELISA kits were used to determine the Abcam) at 4˚C and followed by secondary antibody anti‑IgG