We observed a significant increase in the proportion of FoxP3+ Tregs in the MLN of 4C12-treated SAMP mice compared with those from the IgG-treated group (1

We observed a significant increase in the proportion of FoxP3+ Tregs in the MLN of 4C12-treated SAMP mice compared with those from the IgG-treated group (1.6-fold increase, 8.0??1.8 vs. Physique S5: DR3 stimulation does not alter innate lymphoid cell group 2 (ILC2s) in SAMP mice. (A) Flow-cytometric analysis of mesenteric lymph Adapalene node (MLN) cells from IgG- or 4C12-treated SAMP mice (10-week-old, test. Data are representative of three impartial experiments. Image_5.TIF (88K) GUID:?56F159D8-A97F-472B-AB91-D337138AF391 Physique S6: DR3 deficiency is associated with constitutive reduced innate lymphoid cell (ILC) number. Flow-cytometric analysis of mesenteric lymph node cell DR3WT and DR3KO mice (10-week-old, access to water and were fed with standard laboratory rodent diet P3000 (Harlan Teklad) throughout the experiments. Mice were genotyped by PCR-based assays of genomic tail DNA. All experimental procedures were approved by the Institutional Animal Care and Use Committee of CWRU and were in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines. All experiments were conducted in a blinded manner, without prior knowledge of treatments and mouse groups by the experimenter. Mice were randomized to different interventions using a progressive numerical number. The code for each mouse was known only to the animal caretaker and was revealed at the end of the study. Treatment Five-week-old SAMP and AKR mice were given intraperitoneal injections of 10?g of 4C12 (or IgG) in 100-L PBS, weekly, for 4?weeks, as previously described elsewhere (26). Mice were sacrificed at the beginning of the fifth week. Histology Mouse ilea were collected, rinsed with phosphate-buffered saline (PBS), fixed in Bouins fixative answer (Fisher Scientific, Pittsburgh, PA, USA), embedded in paraffin, and sectioned. Histological evaluation of inflammation severity was GluA3 decided in hematoxylin and eosin-stained 5-m-thick sections, by using a semi-quantitative scoring system as previously described (42). Briefly, scores ranging from 0 (normal histology) to 3 (maximum severity of histologic changes) were used to evaluate histologic indices for (1) active inflammation (infiltration with neutrophils), (2) chronic inflammation (lymphocytes and plasma cells in the mucosa and submucosa), (3) monocyte inflammation (macrophages in the mucosa and submucosa), (4) villous distortion (flattening and/or widening of normal villus architecture), and (5) transmural inflammation. The total inflammatory index represents the sum of all five individual components. Histological scoring was performed by a single trained pathologist in a blinded fashion. Stereomicroscopy Ileal tissue abnormalities (i.e., cobblestone lesions) and normal mucosa Adapalene were investigated by examining the cellular structural pattern of ileal tissue stereomicroscopy, cm by cm, using a reference catalogue of lesions, as previously described (43). Starting from the distal end, 10?cm of ileum were collected, fixed in Bouins answer overnight, and then transferred to 70% ethanol for stereomicroscopic analysis. Both healthy and cobblestone-like areas were calculated per cm using ImageJ software (NIH, Bethesda, MD, USA). Isolation and Culture of Mesenteric Lymph Node Cells Mesenteric lymph node cells were removed aseptically at the time of sacrifice, and cells were gently dispersed through a 70-m cell strainer to obtain single-cell suspensions. Note that 1??106 resulting cells were cultured in RPMI-1640 with 10% FBS and 1% P/S for 72?h in the presence of 1-g/mL anti-CD3/CD28 monoclonal Ab, as previously described (7). For measurement of IL-17 protein in cell supernatants, MLN cells were placed in a culture medium supplemented with 1-ng/mL TGF-1, 20-ng/mL IL-6, and 20?U/mL IL-2 for 72?h, and then stimulated with 50-ng/mL PMA, 1-g/mL ionomycin, and 1??GolgiStop for 4?h at Adapalene 37C (25). After the incubation period, the cells were collected for flow-cytometry assay, as described below, and supernatants were collected for IL-10, IL-13, IL-17, TNF-, and IFN- analysis by ELISA, according to the manufacturers instructions. Isolation of Lamina Propria Mononuclear Cells Ilea were collected from experimental mice, rinsed in ice-cold PBS, and cut into pieces of approximately 0.5?cm. To remove epithelial cells and intraepithelial lymphocytes, tissues Adapalene were placed in 25-mL Ca2+- and Mg2+-free HBSS supplemented with 5-mM EDTA and 1-mM DTT, and shaken for 30?min.