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Common links between Corticosteroid Resistance and Inflammatory Lung Diseases

Common links between Corticosteroid Resistance and Inflammatory Lung Diseases. Ian Adcock National Heart and Lung Institute, Imperial College London. Overview. Steroid resistance occurs in airways disease Inflammatory signalling pathways modulate GR function MAPK, JAK/STAT, NF- k B

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Common links between Corticosteroid Resistance and Inflammatory Lung Diseases

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  1. Common links between Corticosteroid Resistance and Inflammatory Lung Diseases Ian Adcock National Heart and Lung Institute, Imperial College London

  2. Overview • Steroid resistance occurs in airways disease • Inflammatory signalling pathways modulate GR function • MAPK, JAK/STAT, NF-kB • Oxidative stress from cigarette smoke affects GR function • Affects GR nuclear translocation • INFLAMMATORY PATHWAYS REDUCE THE ABILITY OF ACTIVATED GR TO ENTER THE NUCLEUS

  3. Worldwide prevalence of inflammatory lung diseases Reduced steroid responsiveness seen in severe asthma, COPD, CF and IPF – all chronic inflammatory diseases – associated with oxidative stress and – activation of intracellular transcription pathways Donnelly & Rogers 2008

  4. Normal Asthma Cytokines (IL-6, IL-8, etc) Adhesion molecules (e.g. ICAM) inflammatory enzymes iNOS, COX-2 Receptors 80 60 Nuclear staining (%) 40 Normal 20 0 COPD Normal Asthmatic NF-kB is a key mediator of inflammation IL-1 TNF LPS haematopoietic cytokines Oxidative stress UV light TLRs Cigarette Smoke p50 p65 NF-kB is central to inflammatory processes But other TFs also important in cell/stimulus context

  5. TNFa Cytokines Antigen Stimuli IkB Kinase JAK P CaN P STAT IkB NF-ATc P P NF-kB Ub- Proteasome NF-ATc Cytoplasm AP-1 Nucleus NF-kB STAT NF-AT NF-kB and other transcription factors TNFa IL-1b, TLRs Ras MEKK-1 JNKK JNK JUN P FOS P AP-1

  6. Skin Blanching Response to Topical Budesonide SS SR

  7. Severe asthma macrophages have less steroid-suppression of cytokine release Bhavsar et al , Thorax 2008

  8. Mechanisms of steroid action CH2OH C=O OH CH3 OH CH3 CH3 F O Cell membrane Ligand binding CH2OH CH2OH CH2OH CH2OH C=O C=O C=O C=O CH3 CH3 CH3 CH3 OH OH OH OH OH OH OH OH CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 F F F F O O O O Nuclear translocation Co-repressor  IkBa, GILZ X AP-1/ NF-kB DNA binding/co-activator function Co-repressor recruitment/activity

  9. Reduced GR expression and nuclear translocation in ASM cells from severe asthma Chang et al., 2012

  10. MAPKs

  11. IL-2/IL-4 induces dex-insensitivity in PBMCs NS 300 * * * * * * * 200 GM-CSF pg/ml 100 0 - - - + + IL-2, IL-4 + - + + + + PMA/ PHA - Dex - - + - + - Irusen et al., 2002

  12. Nuclear extract GR IP- GR in PBMCs Lamin A - - - - + + IL-2/IL-4 IL-2 + IL-4 + + + + - + - + Dex (10-7M) - - - - + + SB 205380 (1mM) GR nuclear translocation is reduced in IL-2/IL-4 treated U937 cells: role of p38 MAPK Therefore postulated that p38 MAPK drives relative steroid resistance in severe asthma Targets reduced GR nuclear import Yasuo To/Kaz Ito

  13. Enhanced p38 MAPK activity in PBMC and ASMC of severe asthma LPS 10 ng/ml Khorasani et al, 2012 Chang et al, 2012

  14. p38 MAPK sensitivity in BAL macrophages from patients with severe asthma 100 * * * 75 % cytokine release compared to LPS alone 50 25 0 Æ Æ Æ Dex Dex Dex & Dex & Dex & Dex p38 p38 p38 Æ Æ Æ p38 p38 p38 a b IL-6 MIP-1 IL-1 Bhavsar et al., 2008

  15. GW856553 enhances CS mediated suppression of LPS-induced CXCL8 release in severe asthma Bhavsar et al, 2010

  16. Impaired steroid responsiveness of ASM cells in severe asthma Po-Jui Chang

  17. GW856553 enhances dex-mediated suppression of TNFα-induced CCL11 release in severe asthmatics ASM cells Chang et al, 2012

  18. Targeting p38 MAPK restores steroid sensitivity in COPD macrophages MAPK activation in airway disease affects GR nuclear import and function Opportunity for new combination drugs Kent et al., JPET 2009

  19. Other signalling pathways

  20. Cytokine receptors JAK-STAT signalling pathway JAK is an associated tyrosine kinase. Upon receptor activation, it phosphorylates several tyrosine residues on the receptor. An inactive STAT binds the phosphorylated receptor. It is then phosphorylated by JAK and dissociates.

  21. Airway epithelial cells (BEAS2B, primary cells) • IFNγ-induced CXCR3 chemokines (CXCL9, CXCL10, CXCL11) • (relatively steroid ineffective) IFNγ-induced CXCL10 release Dexa JAK inhibitor • JAK inhibitor more effective with IFNγ+TNFα (X-talk with NF-κB signalling) • No effect on IL-1-induced IL-6 (steroid responsive)

  22. STAT6 activation in severe asthma Normal Mild asthma STAT6 STAT6 Severe asthma Mild asthma STAT6 control Mullings et al., J Allergy Clin Immunol. 2001

  23. STAT4 activation in COPD Di Stefano et al., ERJ 2004

  24. Viral infection activates STAT signalling Human Epithelial cells EGFR mutant (K721R) kinase inactive defective STAT activation Kong et al., BBRC 2003 Liu et al., JBC 2008

  25. RV16 infection attenuates steroid function

  26. RV16 infection reduces GR nuclear import:reversed by NF-kB and JNK inhibitors

  27. LY reverses Bud-insensitivity induced by IL-17

  28. Smoking and oxidative stress

  29. Smokers (n=17) 0 0 Smoking and steroid responses in asthma Non-smokers (n=21) 30 ** 25 Placebo 20 Cigarette smoking inhibits the inflammatory response to corticosteroids in asthma Fluticasone (1mg/day) 15 Change in morning PEF (%) 10 5 0 -5 Sputum Eos 0 -1.8* % change Chalmers GW et al: Thorax 2002

  30. 3000 # NAC (-) NAC (+) 2000 † IL-8 (pg/ml) * 1000 0 - - + + IL-1b - - + + H2O2 Oxidative stress enhances IL-1b-stimulated inflammatory gene expression 3000 600 # # IL-8 GM-CSF # 2000 400 ** ** IL-8 (pg/m) GM-CSF (pg/m) 1000 200 0 0 - - + IL-1b + - - + + H2O2 A-549 cells

  31. Oxidative stress reduces corticosteroid activity ? Importance in COPD, smoking asthma and severe asthma BUD FP Amir Hakim 2012

  32. Oxidatives stress reduces GR nuclear translocation BUD FP Nuclear GR TATA BP * * # # Due to effects on Importin 7/Ran GTP system * p < 0.05 vs baseline # p < 0.05 vs. corticosteroid Amir Hakim 2012

  33. Summary • Chronic inflammation is associated with relative corticosteroid insensitivity • Linked to activation of key transcription pathways • NF-kB • p38 MAPK • JAK/STAT activation • Viral infection and exacerbation • Oxidative stress • Affects NF-kB-dependent transcription • ROS stress also affects GR function • GR nuclear import/expression, HDAC2 • The current drivers for excessive pathway activation are unknown in disease – not affect therapeutic strategies

  34. Acknowledgements Fan Chung Peter J. Barnes Kazuhiro Ito Pank Bhavsar Mark Hew John Matthews Elvis Irusen John Marwick Karina Enesa Paul Evans Amir Hakim Elen Jazrawi Nadia Khorasani Po-Jui Chang Louise Donnelly Alberto Papi Gaetano Caramori Antonino Di Stefano Funded by: NIH-RO1 grant HL-69155 Imperial College Trust

  35. Is There Anything Common between Corticosteroid Resistance and Inflammatory Lung Diseases? 30 mins

  36. 4 r= -0.76 p<0.05 Smoker 3 2 1 COPD 0 0 25 50 75 100 125 150 FEV1 (% pred.) Increased oxidative stress in COPD 4-HNE staining Kinnula et al., ERJ 2007 Brindicci et al., Chest 2007 Irfan Rahman

  37. Altered glutathione homeostasis in children with severe asthma: ELF measurements 31 6 31 25 Fitzpatrick et al., JACI 2009

  38. Sputum 8-isoprostane in severe asthma Wood et al., AJRCCM 2005

  39. Inflammatory mediators in respiratory disease Cytokines, Cigarette smoke, infectious agents Epitheium Endothelium IL-6 IL-8 GM-CSF TNF PDGF PGE2 Proteases IL-1 IL-8 GM-CSF Adhesion Macrophage Neutrophil IL-1 IL-6 IL-8 GM-CSF Eicosanoids Migration TNF- INF- PDGF Eosinophil T-lymphocyte IL-3 IL-4 IL-5 IL-6 IL-3 IL-5 IL-6 GM-CSF Migration Adhesion TNF- TGF-/- IL-2 INF- GM-CSF Migration Mast Cell IL-3 IL-4 IL-5 IL-6 TNF-

  40. Clinical phenotypes of asthma Fixed obstruction Reduced steroid responsiveness in severe asthma, COPD, CF and IPF – all chronic inflammatory diseases – associated with oxidative stress and – activation of intracellular transcription pathways Exacerbation prone Severe Eosinophilic steroid-responsive Allergic Bel EH, Curr Opin Pulm Med 2004;10:44-50 Wenzel S, Lancet 2006;386:804-813

  41. PNAS 2012

  42. 600 +IL-1b 500 400 Duration of p65-dsRed nuclear localisation (min) 300 200 100 0 0 100 200 H2O2 (mM) Oxidative stress prolongs NF-B activity by suppressing its nuclear export HeLa cells

  43. IL-1b + H2O2 Non-stim IL-1b 60 mins Acetylated H4 IP NF-kB p65 IP INPUT IL-1b - - + + - + H2O2 - + - + - - TSA - - - - + + ROS stress does not enhance rate of p65 nuclear entry: stimulates histone acetylation

  44. Cigarette smoke reduces HDAC activity in macrophages Y146 - activity Y253 - expression

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