The role of Calprotectin (S100A8/A9) in the pathogenesis of glomerulonephritis and ANCA-associated vasculitis Ruth Jennifer Pepper UCL Thesis submitted for the degree of Doctor of Philosophy 1 Declaration of originality I, Ruth Pepper, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicted in the thesis. 2 Abstract Glomerulonephritis is a common cause of end-stage renal failure and a feature of ANCA- associated vasculitis (AAV). AAV is an example of a small vessel vasculitis, characterised by inflammation of the endothelium and glomeruli in which the interaction between leukocytes and endothelial cells play a crucial role. Macrophages have been demonstrated to have a critical role during the initiation and progression of glomerulonephritis, with the persistence of macrophages in the renal biopsy being associated with a poorer outlook. Calprotectin (also termed S100A8/A9, mrp8/14), is a complex of 2 small calcium binding proteins that is abundantly expressed in neutrophils and monocytes as well as early infiltrating macrophages and is a known ligand of Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end-products (RAGE). There is growing evidence in animal models and in patients with autoimmune diseases, of calprotectin being involved in the inflammatory response, as well as being a marker of activation of innate immunity. I have initially characterised the presence of calprotectin-positive cells in the renal biopsy of patients with focal and crescentic AAV glomerulonephritis, therefore linking the presence of these cells with renal outcome. Patients with active AAV have elevated serum calprotectin levels, as well as significant expression on neutrophils and monocytes, which although decrease during remission, does not normalise. I have demonstrated that mrp14-/- mice are protected from nephrotoxic nephritis, which is abrogated by use of LPS during disease progression. Bone-marrow derived macrophages (BMDMs) release pro-inflammatory cytokines following stimulation with calprotectin; mediated by TLR4. Mrp14-/- BMDMs do not respond to the pro-inflammatory stimulus of S100A8/A9. The co-culture of endothelial cells (EC) and wild-type BMDMs is pro- inflammatory unlike that of mrp14-/- BMDM and ECs. Mrp14-/- mesangial cells also have a decreased pro-inflammatory response. This work demonstrates that calprotectin has a role in experimental glomerulonephritis as well as patients with AAV. This may improve our understanding of the inflammatory response and identify a new novel target for treating patients in the future. 3 Publications Papers S100A8/A9 (calprotectin) is critical for development of glomerulonephritis by mediating pro- inflammatory monocyte-renal cell interactions (in preparation). RJ Pepper, T Vogl, N Hogg, CD Pusey, TH Cook, AD Salama Leukocyte and serum calprotectin (S100A8/S100A9) expression reflect disease activity in ANCA associated vasculitis and glomerulonephritis RJ Pepper, S Hamour, KM Chavele, SK Todd, N Rasmussen, S Flint, PA Lyons, KGC Smith, CD Pusey, HT Cook and AD Salama Kidney International. 2013 Feb 20 (Epub) Intravenous cyclophosphamide and plasmapheresis in dialysis dependent ANCA-associated vasculitis. RJ Pepper, D Chanouzas, R Tarzi, MA Little, A Casian, M Walsh, CD Pusey. L Harper, AD Salama. Clinical Journal of American Society of Nephrology. 2013;8 (2):219-24. Classifying and predicting outcomes in ANCA-associated glomerulonephritis RJ Pepper, AD Salama. Nephrology Dialysis and Transplantation. 2012 Jun;27(6):2135-7 Elevated soluble Flt1 inhibits endothelial repair in PR3-ANCA associated vasculitis. Le Roux S, Pepper RJ (joint 1st), Dufay A, Néel M, Meffray E, Lamandé N, Rimbert M, Josien R, Hamidou M, Hourmant M, Cook HT, Charreau B, Larger E, Salama AD, Fakhouri F. Journal American Society of Nephrology 2012;23(1):155-64. 4 ANCA-stimulated neutrophils release BLyS and promotes B cell survival: a clinically relevant cellular process. Holden NJ, Williams JM, Morgan MD, Challa A, Gordon J, Pepper RJ, Salama AD, Harper L, Savage CO. Ann Rheum Dis. 2011;70(12):2229-33. Pathogenesis of Lupus Nephritis and novel therapies. RJ Pepper, AV Parikh, L Lightstone, BH Rovin. Encyclopaedia of Medical Immunology- Springer. In press. Abstracts Renal Association- Oral presentation 2010 Glomerular macrophage expression of calprotectin and circulating calprotectin levels are increased in patients with ANCA associated vasculitis and promote glomerulonephritis RJ Pepper, S Hamour, KM Chavele, CD Pusey, HT Cook and AD Salama American Society of Nephrology- Poster presentation 2010 Monocyte and glomerular calprotectin in ANCA associated vasculitis RJ Pepper, N Mansfield, S Hamour, R Tarzi, C Pusey, T Cook and AD Salama ANCA workshop- Oral presentation 2011 Glomerular macrophage expression of calprotectin and circulating calprotectin levels are increased in patients with ANCA associated vasculitis and promote glomerulonephritis RJ Pepper, R Tarzi, CD Pusey, HT Cook and AD Salama Renal Association- Oral presentation 2011 Deficiency of calprotectin protects from nephrotoxic nephritis RJ Pepper, R Tarzi, Hsu-Han Wang, CD Pusey, HT Cook, AD Salama American Society of Nephrology- Poster presentation 2011 Calprotectin, an endogenous TLR4 ligand, is critical for induction of glomerulonephritis RJ Pepper, PEH Sharp, G Bhangal, Hsu-Han Wang, R Tarzi, CD Pusey, HT Cook, AD Salama. 5 Autoimmunity conference 2012- Oral presentation Calprotectin is critical for disease induction in nephrotoxic nephritis and predicts disease activity in ANCA associated vasculitis RJ Pepper, S Hamour, H Wang, CD Pusey, HT Cook and AD Salama Renal Association- Oral presentation 2012 Leukocyte and serum calprotectin expression reflect disease activity in ANCA associated vasculitis and glomerulonephritis RJ Pepper, S Hamour, H Wang, CD Pusey, HT Cook and AD Salama American Society of Nephrology- Poster presentation 2012 Leukocyte and serum calprotectin expression reflect disease activity in ANCA associated vasculitis and glomerulonephritis RJ Pepper, S Hamour, H Wang, CD Pusey, HT Cook and AD Salama ANCA workshop- Oral presentation 2013 Calprotectin amplifies the inflammatory response RJ Pepper, S Hamour, H Wang, CD Pusey, HT Cook and AD Salama 6 Table of Contents Chapter 1 Introduction ....................................................................................21 1.1 Anti neutrophil cytoplasm antibody (ANCA) vasculitis.........................................................21 1.1.1 Epidemiology.................................................................................................................21 1.1.2 Classification, Clinical course and biomarkers..............................................................23 1.1.3 Histological classification and outcome........................................................................25 1.2 Pathogenesis of crescentic glomerulonephritis....................................................................27 1.2.1 Immune complex crescentic glomerulonephritis .........................................................28 1.2.2 Anti-glomerular basement membrane (GBM) or Goodpasture’s disease....................30 1.2.3 Pauci-immune crescentic glomerulonephritis ..............................................................31 1.2.4 Immune mediators in glomerulonephritis....................................................................33 1.3 Animal Models ......................................................................................................................55 1.3.1 Nephrotoxic nephritis ...................................................................................................55 1.3.2 Experimental autoimmune glomerulonephritis ...........................................................56 1.3.3 Vasculitis animal models...............................................................................................57 1.4 Calprotectin, mrp8/14, S100A8/A9 ......................................................................................59 1.4.1 Introduction ..................................................................................................................59 1.4.2 Endogenous TLR4 ligand and RAGE ..............................................................................66 1.4.3 Anti-inflammatory action of S100A8 and S100A9 ........................................................69 1.4.4 Calprotectin and infection ............................................................................................70 1.4.5 Calprotectin in disease..................................................................................................71 1.5 Project hypothesis.................................................................................................................78 Chapter 2 Materials and methods................................................................80 2.1 Materials ...............................................................................................................................80 2.1.1 Animals..........................................................................................................................80 2.2 Methods................................................................................................................................80 2.2.1 Human Studies..............................................................................................................80 7 2.2.2 Animal studies- in vivo