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Identifying New Therapeutic Targets for Severe Asthma: The S100A8/A9-RAGE Axis

Identifying New Therapeutic Targets for Severe Asthma: The S100A8/A9-RAGE Axis Andrew J. Halayko, PhD Canada Research Chair in Airway Cell & Molecular Biology. *. Epi. ASM. Mucous gland. Airway Remodeling in Asthma. TGF 1, EGF. (Myo)fibroblasts. Blood vessels. Smooth muscle.

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Identifying New Therapeutic Targets for Severe Asthma: The S100A8/A9-RAGE Axis

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  1. Identifying New Therapeutic Targets for Severe Asthma: The S100A8/A9-RAGE Axis Andrew J. Halayko, PhD Canada Research Chair in Airway Cell & Molecular Biology

  2. * Epi ASM Mucous gland Airway Remodeling in Asthma TGF1, EGF (Myo)fibroblasts Blood vessels Smooth muscle eotaxin, IL-8, IL-6, collagen Mucous gland INFLAMMATION, TISSUE DAMAGE & REPAIR TH-2 / Th-1 cytokines & chemokines Halayko & Ghavami Can J Physiol Pharmacol 87:743-755, 2009 Eosinophil Neutrophil Lymphocyte Mast cell

  3. Neutrophils & Airway Inflammation • Steroid refractory asthma and COPD (smoking): neutrophilic inflammation (+/- eosinophils) & airway remodeling • Need to identify neutrophil-released factors that circumvent steroid therapy “priming” • Degranulation • Oxidative burst (ROS) • Proteases (elastase) • Cytokines (IL-1β, 6, 8, TNF, GMCSF) • Lipid mediators (LTB4) • Other? (S100A8 & S100A9) “activation”

  4. Ca2+ Ca2+ S100 Protein Family (Calgranulins) • >20 acidic Ca2+-binding proteins (1q21gene cluster) • Cell, tissue & species-specific expression S100A8 • Intracellular role: • Ca2+ signaling; microtubule-associated reg. of migration • Extracellular role (a.k.a. alarmins; endokines): • Pro-inflammatory Damage-Associated Molecular Pattern proteins (eg. HMGB1, S100s, IL-33) • Receptors include TLRs & RAGE

  5. Ca2+ Ca2+ S100A8 (MRP8) & S100A9 (MRP9) • >40% of soluble cytoplasmic proteins in neutrophils & macrophages • Heterodimer secretion induced in phagocytes, endothelium, and epithelium (eg. foreign surface contact; LPS) S100A8 monomer • Pro-inflammatory (leukocyte • recruitment) & innate immunity regulation (inhibit dendritic • cell differentiation via MDSC) S100A8 corticosteroid refractory in LPS-induced neutrophilic lung inflammation in mice (Bozinovski et al. J Proteome Res 4: 136-45, 2005 )

  6. sRAGE intracellular S100A8/A9 Receptor: RAGE Receptor for Advanced Glycation End Products • Multi-ligand immunoglobulin family receptor: • (AGEs; β-amyloid protein; HMGB1 S100s incl. S100A12, S100A8/A9) • Multiple variants & signaling (cell, tissue & environment specific profile) • Low expression in “healthy” tissue EXCEPT lung • RAGE KO mice resistant to bleomycin lung fibrosis

  7. Healthy Human BALF 3000 2500 S100A8/A9 (ng/mL) P<0.05 500 Asthma 250 Healthy Severe Asthma S100A8/A9 in Human Asthma

  8. S100A8 S100A9 HMGB1 RAGE TLR4 GAPDH 650 bp 300 bp 200 bp 100 bp S100-Receptor Axis in Human Airway Smooth Muscle Cells

  9. - + - - + BSA (10μM) - - - + - AGE-BSA (10μM) - + + - - S100A8/A9 (2.5μg/mL) Collagen (130kDa) p-ERK Total ERK GAPDH RAGE: A Functional S100A8/A9 Receptor on Human ASM

  10. S100A8/A9 Induces Proliferation: Human ASM PROLIFERATION (BrdU % Control)

  11. * * * * Eotaxin-1 (ng/mL) Eotaxin-1 * IL-8 IL-8 (ng/mL) * 2 10 20 Control TNF (10ng/mL) S100A8/A9 (g/mL) Pro-Inflammatory Role of S100A8/A9: Cultured Human ASM

  12. “Cell Associated” S100A8/A9 (2 µg/ml) - + + + - - - S100A8/A9 (10 µg/ml) - - - - + + + Time (hrs) 0 24 48 72 24 48 72 Collagen 1 (140 kD) RAGE (42 kD) GAPDH (37 kD) “Secreted” S100A8/A9 (2 µg/ml) - + + + - - - S100A8/A9 (10 µg/ml) - - - - + + + Time (hrs) 0 24 48 72 24 48 72 Collagen 1 (140 kD) Mature collagen 1 (70 kD) Pro-Fibrotic Role of S100A8/A9: Cultured Human ASM

  13. Current State of Knowledge The existence of a link between S100 protein expression and asthma symptoms, pathogenesis and severity is unresolved. Murine models of allergic airway inflammation that lead to asthma-like symptoms and pathology hold promise to asses this link. S100A8/A9 is casually linked with airway inflammation, hyperresponsiveness & remodeling in murine models of “asthma” Hypothesis

  14. S100A8 S100A9 HMGB1 TLR4 RAGE GAPDH 300 bp 200 bp 100 bp S100-Receptor Axis in Murine Lung

  15. Intranasal HDM Challenge Chronic Acute Week 1 Week 2 Weeks 3 - 7 35L HDM (0.7mg/mL) 35L HDM (0.7mg/mL) 35L HDM (0.7mg/mL) D1 D3 D5 D1 D1 D2 D2 D3 D3 D4 D4 D5 D5 Respiratory Mechanics & BALF, Serum Acute & Chronic HDM Exposure Mouse Model D. pteronnyssinus

  16. ** Eosinophils Cells/mL BALF ** Airway Resistance ** ** Raw (cmH2O.s/mL) * ** Cells/mL BALF Neutrophils ** ** * * Methacholine (mg/mL) 8 24 48 72 Naive Mid-wk Time after HDM (hrs) Acute HDM Challenge: Temporal Pattern of Neutrophil & Eosinophil Recruitment

  17. … & tracks with neutrophil infiltration Eosinophils Neutrophils Acute HDM Challenge Induces S100A8/A9 Accumulation in Airways S100A8/A9 (ng/mL) BALF 8 24 48 72 Naive Mid-wk Time after HDM (hrs) **

  18. Acute HDM Challenge Induces Airway S100A8/A9 Accumulation Naïve Balb/c HDM Challenge (8hrs post)

  19. HDM Challenge Induces RAGE and Associated Downstream Signaling Whole lung lysate – Balb/c mice Chronic Acute Control HDM Control HDM RAGE Total-p38 phospho-p38 Total-ERK1/2 phospho-ERK1/2 β-Actin

  20. Naïve Balb/c ASM Acute HDM Challenge Induces Airway RAGE Expression HDM Challenge (8hrs post)

  21. Airway Resistance H & E collagen sm--actin Chronic HDM Naive Naive 1.8 Raw (cmH2O.s/mL) 1.5 1.2 0.9 0.6 7 weeks HDM 0.3 0 Saline 3 6 12 25 50 Methacholine (mg/mL) Chronic HDM Challenge Induces Airway Remodeling & AHR

  22. Eosinophils Neutrophils ** ** ** BALF cells/mL ** * * HDM HDM HDM HDM HDM HDM Naive Naive Naive Naive Naive Naive 5 Wks 7 Wks 9 Wks 5 Wks 7 Wks 9 Wks Chronic HDM Challenge: Sustained Airway Eos and Neutrophils

  23. S100A8/A9 (ng/mL) S100A8/A9 (ng/mL) 3000 900 BALF Serum 2500 2000 600 1500 1000 300 500 0 0 Naive Naive Acute Acute Chronic (9 wks) Chronic (9 wks) Chronic (5 WKS) Chronic (5 WKS) HDM Challenge HDM Challenge Chronic HDM Challenge: Sustained BALF & SERUM S100A8/A9

  24. Chronic HDM Challenge Sustains Elevated Lung S100A8/A9 Naïve Balb/c epi 5 Weeks HDM epi

  25. Naïve Balb/c 5 Weeks HDM Chronic HDM Challenge Induced Sustained RAGE Expression Chronic Acute Control HDM Control HDM RAGE

  26. ASM Summary • S100A8/A9 increased in asthmatic airway (tissue and BALF) & RAGE induced by allergic inflammation • S100A8/A9, via RAGE, promotes remodeling responses in airway mesenchymal cells • Allergen airway challenge induces sustained increase in RAGE and S100A8/A9 in airways and serum • S100A8/A9 accumulation in airways tracks with neutrophil recruitment and development of airway remodeling

  27. ASM Unanswered Questions • S100A8/A9 & RAGE causally linked to airway remodeling and AHR? (S100A9-/- & RAGE-/- mice) • S100A8/A9 role in mucosal (innate) immunity in the airways • Source of S100A8/A9 in allergic inflammation? Selective suppression? • Is S100A8/A9 expression linked with asthma phenotype & severity? (biomarker; steroid response; exacerbation trigger [RTI])

  28. Contributors Jamila Chakir, Laval Michel Laviolette, Laval Johannes Roth, Muenster Thomas Vogl, Muenster John Gordon, Saskatchewan Abdel Soussi Gounni Helmut Unruh Sujata Basu Karen Detillieux Saeid Ghavami Aruni Jha Hessam Kashani Andrea Kroeker Mark Mutawe Dedmer Schaafsma Pawan Sharma Jacquie Schwartz Gerald Stelmack Soma Tripathi Behzad Yaganeh Canada Research Chairs Program Canadian Institutes of Child Health Canada Foundation for Innovation Man. Inst. Child Health

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