Rather, the combined effects of PGE2 and other MSC-associated mediators may be necessary to additionally regulate

the production of Th17-promoting factors by ancillary cell populations such as dendritic cells and monocyte/macrophages 7, 12. In conclusion, this study provides novel evidence that MSC-derived PGE2 is highly induced in Th17-MSC co-cultures and mediates a potent suppressive effect on primary and secondary Th17 induction via the EP4 receptor. We propose that further characterisation of the interactions between Th17 Ibrutinib cells and MSCs, including the nature of the contact-dependent signal responsible for COX-2 up-regulation, will identify SCH772984 nmr additional opportunities for manipulation of the Th17 differentiation program. Furthermore, suppression of IL-17A production by effector-memory Th17 cells derived from a site of “sterile inflammation” indicates the potential for MSCs to ameliorate tissue damage associated with maladaptive acute or chronic Th17 activation if delivered in the correct context. Eight- to 12-wk-old female C57BL/6 (B6) and BALB/c mice were purchased from Harlan Laboratories UK (Bicester, UK) and housed in a specific pathogen-free facility. All animal procedures were carried out under licence

from the Irish Department of Health and Children and approved by the NUI Galway Animal Care Research Ethics Committee. Mouse MSC cultures were carried out in supplemented Iscove’s modified Dulbecco’s medium (see Supplemental Methods for details of media and buffer compositions) (Sigma-Aldrich, St. Louis, USA). Th17 cell culture was carried out in supplemented Dulbecco’s modified Eagle medium. Reagents used included

a range of antibody preparations (see FER Supplemental Methods), recombinant mouse TGF-β1 and IL-6 (Peprotech, Rocky Hill, NJ, USA), mouse CD3/CD28 T-cell expander beads (Dynabeads®, Invitrogen), Indomethacin and PGE2 (Sigma-Aldrich), and COX-2-selective inhibitor (NS-398), selective EP1 antagonist (SC-51322), selective EP2 antagonist (AH 6809), selective EP4 antagonist (L-161,982) and selective EP4 agonist (L-902,688) (all from Cayman Chemicals, Ann Arbor, MI, USA). Mouse MSCs were isolated from bone marrow according to the method described by Peister et al. 41. Tri-lineage differentiation capacity was determined using standard chondrogenic, adipogenic and osteogenic differentiation assays (Supplemental Fig. S1) 18. All experiments were carried out with passage 5–MSCs grown to 80% confluence in T175 tissue culture flasks (Nunc-Fisher Scientific) and detached with trypsin solution (Sigma-Aldrich). Renal cortical fibroblasts were prepared according Alvarez et al. 42.