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Background and objectives MiR-31 is upregulated in repeatedly activated Th1 and in effector/memory Th lymphocytes isolated from the synovial fluid of patients suffering from rheumatic joint diseases. Several respective miR-31 targets are downregulated and essential for the cytoskeletal rearrangement of repeatedly activated Th1 cells. This suggests that miR-31 regulates the motility and immobilises proinflammatory Th1 cells in inflamed tissues. Materials and methods Assuming that Th cells involved in chronic inflammation have a history of repeated stimulation by autoantigens, we have in vitro generated four times activated Th1 cells. We have determined the transcriptomes and the migratory behaviour of these cells after Antagomir mediated miR-31 knockdown by using microarrays and in vitro transwell migration assays. For the identification of potential direct targets we have applied the target prediction algorithm TargetScan. In order to determine the putative function of miR-31 in proinflammatory Th1 cells a gene set enrichment (GSE) analysis of the obtained transcriptome data after miR-31 knockdown was performed. Results Specific inhibition of miR-31 in repeatedly activated Th1 cells promoted their migration up to 30% in a transwell migration assay. In sum, we have identified 283 differentially expressed genes in repeatedly activated Th1 cells after treatment with a miR-31 specific or an unspecific Antagomir for 36, 48 and 72 h. The GSE analysis revealed an enrichment of genes, which regulate cell-intrinsic and extrinsic pathways involved in motility and tissue localization. Among the putative direct targets we found actin binding LIM Protein 1 (Ablim1), Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Epsilon (Ywhae), actin gamma 1 (Actg1), actin beta (Actb), Lamellipodin (Raph1), T-cell lymphoma invasion and metastasis-inducing protein 1 (TIAM1) and Ras homolog gene family, member A (RhoA). From these genes, we could verify the upregulation of Actg1, Actb, Raph1 and RhoA after 48 to 72 h of Antagomir-31 treatment from 1.2 to 3 fold using quantitative RT-PCR. Conclusion We show that miR-31 restricts the motility of repeatedly activated Th1 cells in vitro most likely by repressing genes involved in cytoskeletal rearrangement. MiR-31 might represent a molecular switch important for the persistence of proinflammatory Th cells in inflamed tissues.

Background and objectives T helper type 1 (Th1) cells are involved in rheumatic diseases such as Crohn´s disease or rheumatoid arthritis and have a history of chronic autoantigenic stimulation. We have shown that Th1 cells adapt to chronic inflammation by upregulating the expression of the microRNA (miR)-148a. MiR-148a promotes the survival of chronically activated Th1 cells by regulating the expression of the proapoptotic protein Bim. Thus, we tested the suitability of miR-148a as a therapeutic target for the selective elimination of proinflammatory Th1 cells in a preclinical model of colitis. Methods Chronically activated Th1 cells were transferred into Rag1-deficient mice to induce colitis. Then, mice were intravenously injected with antagomirs that specifically target the microRNA-148a (antagomir-148a) or with control antagomirs (antagomir-scr). For assessing the function of T cell-intrinsic expression of miR-148a in colitis, we transduced Th1 cells with inducible microRNA sponges prior to transfer into Rag1-deficient mice. Colitic inflammation was determined by the weight-to-length ratios of colons. Colonic Th cells were sorted by FACS to measure miR-148a expression by qRT-PCR. The expression of Bim and Bcl-2 as well as numbers of viable Th1 cells, were determined by flow cytometry. Results Systemic inhibition of miR-148a by antagomirs alleviated colitis in mice as measured by reduced weight-to-length ratios of their colons. The numbers of viable Th1 cells were reduced up to 50% in mice that were treated with antagomir-148a when compared to mice that were injected with antagomir-scr. Antagomir-148a injections were efficient and resulted in a 30% reduction of miR-148a in colonic Th1 cells. The expression of the proapoptotic protein Bim was increased up to 30% in Th cells from antagomir-148a treated mice, while the anti-apoptotic protein Bcl-2 was unchanged. Th cell specific inhibition of miR-148a by miR-sponges during colitis led to a 30% reduction of Th cells in the colons of colitic mice and reduced the weight-to-length ratio. Conclusions We suggest that miR-148a controls the survival of proinflammatory Th1 cells in chronic inflammation by inhibiting Bim expression in a Th cell intrinsic fashion. Thus, miR-148a might represent a suitable target for the selective depletion of proinflammatory Th1 cells in chronic inflammation.

Background Proinflammatory T helper (Th) cells are critically involved in the initiation and perpetuation of chronic inflammatory diseases (CID). In these diseases, proinflammatory Th cells persist in inflamed tissues and are refractory to current immunosuppressive therapies. The molecular factors regulating the survival of proinflammatory Th cells are not fully understood. Recently, we have shown that proinflammatory Th1 cells adapt to chronic inflammation by upregulating the expression of the microRNA (miRNA, miR)-148a 1 . MiR-148a promotes the survival of proinflammatory Th1 cells by regulating the expression of the pro-apoptotic protein Bim. Memory Th cells isolated from inflamed synovia of patients with rheumatoid arthritis induce the expression of miR-148a, presumably enhancing their survival. Therefore we hypothesize, that miR-148a could represent a therapeutic target for the selective elimination of proinflammatory Th1 cells in CID. Objectives To test the suitability of miR-148a as a therapeutic target in a preclinical murine model of transfer colitis. Methods We have generated a retroviral based inducible miR-148a inhibitor, containing complementary binding sites for miR-148a, termed “miR-148a sponge”. We transduced transgenic Th cells with the miR-148a sponge or a scrambled (scr) control sponge. Induction of sponge expression was initiated by tamoxifen treatment. Expression of miRNA sponges was detected by GFP reporter gene expression and the functionality of miR148a inhibition was determined by real-time PCR for the miR-148a target Bim. MiR-148a sponge-transduced CD4 + Th1 cells were FACS-sorted and transferred into Rag1 -/- mice. After manifestation of colitis, mice were sacrificed and T cells were re-isolated from the spleens and colons for determining their phenotype and number by FACS analysis. Results Expression of miR-148a sponges resulted in a 2 fold increase of Bim expression in activated Th1 cells in vitro . In addition, we found reduced numbers of miR-148a-sponge expressing Th cells as compared to scr-sponge expressing Th cells in the spleens and colons of colitic mice. Finally, miR-148a sponge-expressing Th cells from spleens and colons of colitic mice showed higher levels of Bim expression per cell than scr-sponge expressing cells. Conclusions Our data suggest that miR-148a controls the survival of pro-inflammatory Th1 cells in chronic inflammation by inhibiting Bim expression. Thus, therapeutic targeting of miR-148a probably is suitable for the selective depletion of proinflammatory Th1 cells in chronic inflammation. References Haftmann, C. et al. miR-148a is upregulated by Twist1 and T-bet and promotes Th1-cell survival by regulating the proapoptotic gene Bim. European journal of immunology, doi:10.1002/eji.201444633 (2014). Acknowledgements Patrick Maschmeyer is supported by EUTRAIN, a FP7 Marie Curie Initial Training Network for Early Stage Researchers funded by the European Union. Disclosure of Interest None declared