The Rheumatoid Arthritis Pathogenesis Centre of Excellence [RACE]

The role of micro-RNAs in the anti-inflammatory actions of glucocorticoids and interleukin 10

The role of micro-RNAs in the anti-inflammatory actions of glucocorticoids and interleukin 10

Lead supervisor - Prof Andy Clark, University of Birmingham

Additional supervisor - Dr Mariola Kurowska-Stolarska, University of Glasgow

Aims

To determine how changes in expression of micro-RNAs contribute to the negative regulation of pro-inflammatory gene expression in macrophages.

Project overview

Activated macrophages secrete pro-inflammatory factors that have powerful, pleiotropic effects on several different cell types. In the rheumatoid synovium they are the main sources of TNF, and disease activity is strongly correlated with their number (1). Macrophage pro-inflammatory activity is normally limited by a number of intrinsic negative feedback loops, which switch off signalling pathways and terminate expression of inflammatory mediators. Examples include the dual specificity phosphatase DUSP1, which inactivates MAPKs (2), and tristetraprolin (TTP), which destabilises pro-inflammatory mRNAs (3). In addition, endogenous anti-inflammatory factors such as glucocorticoids and interleukin 10 converge on the same negative feedback loops, enhancing or prolonging the expression of DUSP1, TTP and other feedback regulators in order to downregulate the inflammatory response. In the rheumatoid synovium such negative feedback mechanisms appear ineffective. For example, TTP is expressed at high levels yet fails to downregulate its pro-inflammatory mRNA targets (4). IL-10 is present, but anti-inflammatory responses to this cytokine are impaired (5). We hypothesise that failure of negative feedback mechanisms for the constraint of macrophage activity contribute to state of chronic inflammation.

In order to identify novel anti-inflammatory mechanisms we have carried out microarray analyses of gene expression in primary human and mouse macrophages treated with lipopolysaccharide (LPS) with or without IL-10 or the glucocorticoid dexamethasone (dex). Amongst differentially expressed transcripts a number of micro RNA (miR) precursor transcripts were found to be strongly regulated by the anti-inflammatory agonists. We hypothesise that these contribute to the suppression of inflammatory responses of macrophages. A few examples are given here

  1. Dex and LPS cooperated to induce the expression of miR-21, which was previously shown to mediate switching of macrophages from pro-inflammatory to pro-resolution phenotype (6, 7). 
  2. IL-10 inhibited the expression of miR-221 and -222, which are positive regulators of NF-kB function, putative negative regulators of TTP expression, and elevated in RA synovium (8). 
  3. IL-10 and LPS cooperated to induce expression of miR-302b, which targets IRAK-4 to negatively regulate TLR-induced cell signalling (9). 
  4. Dex inhibited expression of miR-155, a micro-RNA we previously demonstrated to play a key pathogenic role in RA (10). 
  5. Dex inhibited the expression of miR-887, a putative negative regulator of 11b-HSD1 expression. This is a potential mechanism for enhanced responsiveness to anti-inflammatory effects of endogenous glucocorticoids (11).

Techniques

  1. qPCR will be used to confirm dex- and IL10-induce changes in expression of mature miRs and their precursors. We will test the miRs listed above and approximately 12 other interesting candidates that have emerged from microarray analyses. The remainder of the project will focus on the dex- or IL-10-mediated effects that are strongest and most statistically robust.
  2. Parallel microarray databases will be determine whether dex- or IL-10-induced changes of miR expression are conserved between species.
  3. LysM-Cre mice will be crossed with mice containing a floxed Dicer gene. Macrophages deficient in miR maturation will be generated and challenged with LPS in the absence or presence of dex or IL-10 to test the contribution of miRs to the effects of the anti-inflammatory agonists. Readouts will be cytokine and chemokine expression measured by multiplex bead capture assay and qPCR.
  4. Antagomirs, miR mimetics and miR sponges will be transfected into primary human macrophages (using techniques established in our labs) to test whether anti-inflammatory effects of IL-10 or dex are mediated by specific miRs. Readouts as above.
  5. Mechanisms of action of miRs will be assessed by interrogating mRNA profiles of IL-10 and dex treated macrophages with predictive micro RNA target algorithms (Targetscan, Targetminer, MiRTAR..). Selected targets will be validated using luciferase reporter constructs, qPCR and appropriate readout of target protein expression (eg W blot, ELISA, flow cytometry, kinase assay).
  6. To extend findings to clinical context, expression of relevant miRs in RA synovial biopsies will be examined using methods we have established.

References

  1. McInnes, I. B., and G. Schett. 2011. The pathogenesis of rheumatoid arthritis. N Engl J Med 365: 2205-2219.
  2. Abraham, S. M., and A. R. Clark. 2006. Dual-specificity phosphatase 1: a critical regulator of innate immune responses. Biochem Soc Trans 34: 1018-1023.
  3. Brooks, S. A., and P. J. Blackshear. 2013. Tristetraprolin (TTP): Interactions with mRNA and proteins, and current thoughts on mechanisms of action. Biochim Biophys Acta 1829: 666-679.
  4. Brooks, S. A., J. E. Connolly, R. J. Diegel, R. A. Fava, and W. F. Rigby. 2002. Analysis of the function, expression, and subcellular distribution of human tristetraprolin. Arthritis Rheum 46: 1362-1370.
  5. Antoniv, T. T., and L. B. Ivashkiv. 2006. Dysregulation of interleukin-10-dependent gene expression in rheumatoid arthritis synovial macrophages. Arthritis Rheum 54: 2711-2721.
  6. Perdiguero, E., Y. Kharraz, A. L. Serrano, and P. Munoz-Canoves. 2012. MKP-1 coordinates ordered macrophage-phenotype transitions essential for stem cell-dependent tissue repair. Cell Cycle 11.
  7. Quinn, S. R., and L. A. O'Neill. 2011. A trio of microRNAs that control Toll-like receptor signalling. Int Immunol 23: 421-425.
  8. Pandis, I., C. Ospelt, N. Karagianni, M. C. Denis, M. Reczko, C. Camps, A. G. Hatzigeorgiou, J. Ragoussis, S. Gay, and G. Kollias. 2012. Identification of microRNA-221/222 and microRNA-323-3p association with rheumatoid arthritis via predictions using the human tumour necrosis factor transgenic mouse model. Ann Rheum Dis 71: 1716-1723.
  9. Zhou, X., X. Li, Y. Ye, K. Zhao, Y. Zhuang, Y. Li, Y. Wei, and M. Wu. 2014. MicroRNA-302b augments host defense to bacteria by regulating inflammatory responses via feedback to TLR/IRAK4 circuits. Nat Commun 5: 3619.
  10. Kurowska-Stolarska, M., S. Alivernini, L. E. Ballantine, D. L. Asquith, N. L. Millar, D. S. Gilchrist, J. Reilly, M. Ierna, A. R. Fraser, B. Stolarski, C. McSharry, A. J. Hueber, D. Baxter, J. Hunter, S. Gay, F. Y. Liew, and I. B. McInnes. 2011. MicroRNA-155 as a proinflammatory regulator in clinical and experimental arthritis. Proc Natl Acad Sci U S A 108: 11193-11198.
  11. Chapman, K. E., A. E. Coutinho, Z. Zhang, T. Kipari, J. S. Savill, and J. R. Seckl. 2013. Changing glucocorticoid action: 11beta-hydroxysteroid dehydrogenase type 1 in acute and chronic inflammation. J Steroid Biochem Mol Biol 137: 82-92.