1. Academic Trainees Meeting – 5 th May, 2011 Interesting aspects of complement regulation…… Matthew Pickering Wellcome Trust Senior Fellow in Clinical Science Consultant Rheumatologist

2. Complement activation protein deficiency Terminal pathway Recurrent Neisseria infections C3 Infection Classical pathway SLE-like illness Recurrent infection with encapsulated bacteria e.g. pneumococci, Haemophilus influenzae Vasculitis, glomerulonephritis

3. Complement dysregulation Terminal pathway dysregulation Paroxysmal nocturnal haemoglobinuria C1 inhibitor deficiency [classical pathway dysregulation] Hereditary angioedema Alternative pathway dysregulation Dense deposit disease, Atypical haemolytic uraemic syndrome renal thrombotic microangiopathy

4. Disorders of complement Activation protein deficiency ‘too little’ complement Tell us what might happen if we therapeutically inhibit complement Regulatory protein deficiency ‘too much’ complement’ Provide diseases in which complement inhibiting therapies ought to be effective

5. C3b C3 C3b FOREIGN SURFACE C3b ‘C3b amplification loop’ Complement activation lectin pathway Bacterial Carbohydrate, ficolins C4b2a classical pathway immune complexes C3bBb alternative pathway ‘always on’ C5 activation MAC C5a MAC = membrane attack complex

6. C3b C3 Complement regulation lectin pathway C4b2a classical pathway C3bBb alternative pathway MAC = membrane attack complex iC3bC3b CR1CD46CD59 MAC Factor I DAF (CD55) C1 inhibitor C4bp C1 inhibitor Factor H Factor I iC3b Factor H

7.  Physiological control of complement activation REGULATORS Complement dysregulation and disease: ACTIVATORS Loss of functionGain of function The balance is influenced by mutations (extreme) and and/or polymorphisms (‘fine tuning’)

8. What does factor H do?  Critical negative regulator of the alternative pathway and C3b amplification loop  What happens to C3 levels in individuals with complete genetic deficiency of CFH? Uncontrolled spontaneous activation of the alternative pathway and secondary consumption of C3

9. Why is factor H important?  It is associated with human disease: Dense deposit disease mutations rare ‘protective’ and ‘at risk’ polymorphisms common

10. Dense deposit disease  Electron-dense transformation of the glomerular basement membrane Glomerular C3 staining in DDD DDD retinopathy

11. Dense deposit disease C3b C3 C3bBb Factor H B, D C3 nephritic factor Anti-factor H  Associated with plasma C3 activation:

12. Dense deposit disease  Animal models : Spontaneous porcine factor H deficiency and gene-targeted factor H- deficient mice  Profound plasma C3 depletion – 5% of normal C3 levels  Spontaneous renal disease – ‘murine/porcine DDD’ Factor H deficiency Wild-type C3 staining wild-type 0 200 400 600 Cfh -/- Plasma C3 - mg/l

13. Dense deposit disease  What have the animal models taught us? The renal disease does not develop if activation of C3 is blocked The renal disease does develop if C5 activation is blocked  Dense deposits still develop  Glomerular inflammation reduced but not absent  Murine dense deposit disease is dependent on the ability to activate C3 but not C5 Glomerular basement membrane deposits in mice with combined deficiency of factor H and C5 Pickering MC, et al. PNAS 2006 103(25):9649-54.

14. Human complement deficiency Deficiency State: Plasma C3: Factor I lowabsent C3 Associations: Recurrent infection immune complex-mediated renal disease e.g. MPGN type I Factor H low Dense deposit disease C3b iC3b, C3d Pickering MC, Cook HT. Clin Exp Immunol. 2008 51(2):210-30.

15. Plasma C3 regulation  Continuous activation of C3 occurs in plasma through the C3 ‘tick-over’pathway C3b C3 C3bBbFactor H C3b Factor I iC3b C3dC3c Factor B Factor D

16. Dense deposit disease  Administration of factor I to mice with combined deficiency of H and I restores GBM C3 staining Rose KL et al. J Clin Invest. 2008 118(2):608-18.

17. Why is factor H important?  It is associated with human disease: Dense deposit disease Atypical haemolytic uraemic syndrome mutations rare ‘protective’ and ‘at risk’ polymorphisms common

18. Atypical haemolytic uraemic syndrome Atypical Haemolytic uraemic syndrome renal thrombotic microangiopathy Alternative pathway dysregulation Associated with: COMPLEMENT MUTATIONS Loss of function mutations in regulators Factor H Mutations Hybrid gene (copy number variation) Factor I MCP (CD46) Gain of function mutations in activation proteins C3 Factor B ACQUIRED COMPLEMENT DYSREGULATION Anti-factor H autoantibodies

19. Atypical Haemolytic uraemic syndrome – factor H mutations Factor I iC3b C3b C3 B, D C3bBb HOST SURFACE RENAL ENDOTHELIUM C3b C5 activation MACC5a CD46 C3 regulation Surface recognition

20. Murine model of factor H-associated atypical haemolytic uraemic syndrome  Gene-targeted factor H-deficient mice transgenically expressing a mutant mouse factor H protein (FH  16-20) Cfh -/-  FH16-20 Cfh -/- 0 25 50 75 100 Plasma C3 - mg/l wild-type mouse CFH Mutated mouse FH 16-20 Renal histology in Cfh -/-.FH16-20

21. Murine model of factor H-associated atypical haemolytic uraemic syndrome  Use this model to determine contribution of C5 activation to renal injury  Spontaneous renal disease does not occur in C5-deficient Cfh -/-  FH16-20 animals

22. Murine model of factor H-associated atypical haemolytic uraemic syndrome  Cfh -/-  FH16-20 animals are hypersensitive to experimentally triggered renal injury – this injurious response is C5 dependent C3 C9

23. Atypical haemolytic uraemic syndrome - therapy  C5 inhibition successful in case reports – examples: Eculizumab for aHUS – N. Engl. J. Med. 2009 360:5 pp542-543 Eculizumab for congenital aHUS – N. Engl. J. Med. 2009 360:5 pp544-6  Open Label Controlled Trial of Eculizumab in Adult Patients With Plasma Therapy-sensitive / -resistant Atypical Hemolytic Uremic Syndrome (aHUS) Successful outcomes announced in ASN 2010 meeting http://clinicaltrials.gov/ct2/results?term=eculizumab

24. Why is factor H important?  It is associated with human disease: Dense deposit disease Atypical haemolytic uraemic syndrome mutations rare ‘protective’ and ‘at risk’ polymorphisms common

25. Factor H and Age-related macular degeneration

26. Factor H and AMD – the ‘Y402H’ polymorphism From Sofat et al., Atherosclerosis 213 (2010) 184-90

27. Alternative pathway dysregulation Age-related macular degeneration Factor H and Age-related macular degeneration Ocular drusen Associated with: Polymorphic variants in: Regulators Factor H Y402H ‘at risk’ V62I ‘protective’ activation proteins C3C3FF ‘at risk’ Factor BBf32Q ‘protective’

28. Age-related macular degeneration Functional differences in the Valine62Isoleucine CFH polymorphism 62Isoleucine more efficient at preventing red cell lysis 14nM vs. 22.6nM at 50% lysis 62Valine 62Isoleucine Factor H and Age-related macular degeneration

29. Age-related macular degeneration Complement dysregulation and eye disease – age-related macular degeneration Dense deposit disease mutations alternative pathway activation Ocular drusen ‘at risk’ polymorphisms ‘protective’ polymorphisms DDD retinopathy Factor H 402H* Factor H 62V Factor B 32R C3F Factor H 402Y* Factor H 62I Factor B 32Q C3S CFHR1/3 deletion* *functional consequences not understood Factor H null alleles C3 3923∆DG

30. Why is factor H important?  It is associated with human disease: Dense deposit diseaseAtypical haemolytic uraemic syndrome mutations rare ‘protective’ and ‘at risk’ polymorphisms common Age-related macular degeneration Meningococcal sepsis

31. Factor H and susceptibility to meningococcal infection Meningococcal sepsis

32. The factor H family

33. Why are the factor H-related proteins important?  They are associated with human disease: mutations rare ‘protective’ and ‘at risk’ polymorphisms common

34. The factor H family: copy number variation CFHCFHR3CFHR1CFHR4CFHR2CFHR5 Most frequent CFH-CFHR allele Deletion homozygotes:African American 16% Hageman et al, Ann. Medicine 2006 European Americans4.7% CFHCFHR4CFHR2CFHR5 CFHR1-3 deletion allele polymorphism (common) Others (uncommon - <1%) CFHCFHR1CFHR4CFHR2CFHR5CFHCFHR3CFHR4CFHR2CFHR5CFHCFHR3CFHR2CFHR5CFHCFHR3CFHR1CFHR3CFHR1CFHR4CFHR2CFHR5CFHCFHR3CFHR1 CFHR4CFHR2CFHR5

35. Why are the factor H-related proteins important?  They are associated with human disease: ‘protective’ and ‘at risk’ polymorphisms common Age-related macular degeneration CFHR1-3 deletion allele polymorphism associated with protection against AMD Mol Immunology 44 (2007):3921.

36. Complement therapeutics Pathologies in which complement is activated

37. Complement therapeutics Examples of the many complement inhibitors in development Eric Wagner and Michael Frank Nature Reviews 2010, vol. 9, 43-56.

38. Thanks  Elena Goicoechea de Jorge  Katherine Vernon  Mitali Patel  Kirsten Rose  Talat Malik  Sharmal Narayan  Marieta Ruseva  Tamara Montes  Lola Sanchez-Nino  Danielle Paixao-Cavalcante  Fadi Fakhouri  Terence Cook  Marina Botto  Santiago Rodriguez de Cordoba  Veronique Fremeaux -Bacchi  Patrick Maxwell  Danny Gale