Kliniken & Institute … Institute Zentrum für… Medizinische… Forschung Research Heeg

Differentiation of DC and B cells: role of microRNAs

Exogenous or endogenous Toll-like receptor (TLR) ligands influence differentiation and maturation of myeloid cells and B lymphocytes. DCs are important sentinels of the innate immune system monitoring the presence of infectious micro-organisms; their unique feature is to activate naïve cells of the adaptive immune system. DCs are able to initiate the primary immune response including the polarization of developing T cells subsets.

B lymphocytes isolated from peripheral blood responded to TLR ligands. The TLR9 ligand CpG-DNA was found to directly activate this B cell population and induced a long lasting activation of the ZAP70-dependent signal pathway leading to PKB activation and mTOR dependent signaling. Following CpG-activation B cells gained the capability to produce IL-10, indicating that these B cells might act as "regulatory" B cells. Further analyses revealed that IL-10 induction was dependent on the kinase syk and sensitive to the calcineurin inhibitors Cyclosporin A (CsA) and FK506. This observation might be important for transplantation immunology since according to our results CsA therapy might not only suppress the activation of T effector cells but also prevent the induction of tolerogenic B cells and thus hamper tolerance induction. The pathway leading to IL-10 induction was further characterized. In contrast to other CsA-sensitive signaling pathways, NFAT activation was not involved.

We have analysed the differentiation of DC from monocytes in vivo in the presence of TLR-agonists. We could show that signaling induced by TLR agonist alters this differentiation process in quantitative and qualitative aspects. Most important we found, that TLR agonist induce APC which were able to induce differentiation of naïve CD4 T cells into FoxP3+ T regulatory cells (Treg). One goal of the project is the analyses of the mechanisms of Toll-like receptor (TLR)-driven induction of tolerogenic antigen presenting cells (TLR-APC) and IL-10-producing regulatory B cells.

We have defined respective signal pathways and identified several proteins involved in tolerogenic function by mRNA expression analyses and proteomics. We found that TLR-APCs showed a prolonged activation and phosphorylation of STAT3 while STAT5 signals were reduced. STAT3 phosphorylation was due to paracrine or autocrine secretion of IL-6 and IL-10. Chromatin immunoprecipitation (CHIP) analyses further revealed that phosphorylated STAT3 directly binds to the PD-L1 promoter, thus explaining the upregulation of PD-L1.

Moreover, we found characteristic micro-RNA (miR) expression profiles in tolerogenic APC suggesting that miR at least in part control the functional phenotype of these cells. We have developed a the new method (hybrid oligonucleotides) to down-regulate effectively and specifically miRs (Hybrid-antagomiR). This method can be also utilized to silence gene expression with siRNA constructs (Hybrid-siRNA).

In the future we will further define the mechanisms of tolerogenic function initially concentrating on the proteins already identified. Using hybrid-antagomiRs and differential gel electrophoresis we will further identify new targets for miRs and thus new potential proteins contributing to tolerogenic function. Hybrid-ODN (either antagomiR or siRNA) allow us to directly target and manipulate APC function. Using this approach we will determine the rules governing activation/tolerization mediated by miR in vitro.

Selected publications

  1. Ziegler, S., K. Gartner, U. Scheuermann, T. Zoeller, J. Hantzschmann, B. Over, S. Foermer, K. Heeg, and I. Bekeredjian-Ding. 2014. Ca(2+) -related signaling events influence TLR9-induced IL-10 secretion in human B cells. Eur. J. Immunol. 44: 1285-1298
  2. Ziegler, S., M. E. Eberle, S. J. Wölfle, K. Heeg, and I. Bekeredjian-Ding. 2013. Bifunctional Oligodeoxynucleotide/AntagomiR Constructs: Evaluation of a New Tool for MicroRNA Silencing. Nucleic Acid Therapeutics 23: 427–434.
  3. Parcina, M., M. A. Miranda-Garcia, S. Durlanik, S. Ziegler, B. Over, P. Georg, S. Foermer, S. Ammann, D. Hilmi, K.-J. Weber, M. Schiller, K. Heeg, W. Schneider-Brachert, F. Götz, and I. Bekeredjian-Ding. 2013. Pathogen-Triggered Activation of Plasmacytoid Dendritic Cells Induces IL-10–Producing B Cells in Response to Staphylococcus aureus. J Immunol 190: 1591–1602.
  4. Over, B., S. Ziegler, S. Foermer, A. N. R. Weber, K. A. Bode, K. Heeg, and I. Bekeredjian-Ding. 2013. IRAK4 turns IL-10+ phospho-FOXO+ monocytes into pro-inflammatory cells by suppression of protein kinase B. European Journal of Immunology 43: 1630–1642.
  5. Wölfle, S. J., J. Strebovsky, H. Bartz, A. Sähr, C. Arnold, C. Kaiser, A. H. Dalpke, and K. Heeg. 2011. PD-L1 expression on tolerogenic APCs is controlled by STAT-3. Eur. J. Immunol. 41: 413–424.
  6. Bekeredjian-Ding, I., A. Doster, M. Schiller, P. Heyder, H. M. Lorenz, B. Schraven, U. Bommhardt, and K. Heeg. 2008. TLR9-activating DNA up-regulates ZAP70 via sustained PKB induction in IgM+ B cells. J.Immunol. 181: 8267.
  7. Bartz, H., N. M. Avalos, A. Baetz, K. Heeg, and A. H. Dalpke. 2006. Involvement of suppressors of cytokine signaling in toll-like receptor-mediated block of dendritic cell differentiation. Blood 108: 4102–4108.