lupus and complement deficiency : insights from knockout mice

1. Marina Botto

2. Disclosures: No Relevant Financial Relationships with Commercial Interests

3. Complement and SLE - objectives To explore the links between complement and break of B cell tolerance To review the links between complement and clearance of dying cells To analyse the role of complement in tissue damage To consider the background strain effects in gene-targeted mice

4. Complement ACTIVATION

5. Complement and SLE Active SLE is associated with hypo-complementaemia (reduced levels of C1q, C2, C4, CH50 and C3) Complement levels increase following treatment Tissue biopsies show deposits of C3 and C4 Complement activation can cause tissue damage

6. Pathogenesis of inflammation in SLE The traditional view Autoantibodies form immune complexes with autoantigens These fix complement Complement causes tissue injury but The clinical evidence doesn’t quite fit!

7. Complement and SLE Clues from clinical observations

8. Complement deficiency and SLE

9. Complement - physiology Interface between innate & adaptive immunity augmentation of antibody responses C3 and C4 fragments bound to antigens and immune complexes C3 receptors on B cells and APCs enhancement of immunological memory C3 and C4 fragments bound to antigens and immune complexes C3 receptors on follicular dendritic cells

10. Importance of complement in immune response

11. The paradox complement deficiency is associated with reduced antibody responses and defective humoral immunity complement deficiency is associated with SLE in which increased antibody responses to self antigens tissue injury mediated by immune complexes

12. Complement FUNCTION

13. Complement and SLE pathogenesis – 3 steps Complement and pathological survival of autoreactive lymphocytes 2) Complement and failure of clearing dying cells 3) Complement and tissue damage

14. Does C1q have a direct role in the maintenance of B cell tolerance ?

15. C1q-deficiency and self tolerance C1q deficient mice bred with hen egg lysozyme (HEL)-transgenic mice expressing: a) Ig H and L chains with high affinity for HEL b) sHEL (10-30ng): sufficient to trigger anergy but not deletion of transgenic B cells

17. Serum anti-HEL antibody levels

18. C1q-deficiency and self tolerance 1) normal “self-tolerance” to sHEL as autoantigen in C1q deficiency 2) C1q deficient mice crossed with anti-ssDNA knock-in transgenic mice (3H9R/Vk8R): regulation of the single specificity anti-ssDNA Anergy induction in both these models was not modified by C1q deficiency

19. SLE pathogenesis – 3 steps Complement and pathological survival of autoreactive lymphocytes 2) Complement and failure of clearing dying cells 3) Complement and tissue damage

21. Does complement contribute to the clearance of dying cells?

22. In vivo binding of C1q to apoptotic keratinocytes

23. C1q deficient mouseGlomerular apoptotic bodies

26. How does C1q contribute to the clearance of apoptotic cells? C1q binds directly to apoptotic cells. C1q bound to apoptotic cells: a) can activate the classical pathway with subsequent C4b and C3b/bi deposition b) is recognised by calreticulin on phagocytes and this in conjunction with CD91 stimulates the uptake of apoptotic cells by pinocytosis C1q binding to apoptotic cells is mediated by IgM antibodies and/or pentraxin and this causes activation of the classical pathway

28. Does the impaired clearance of complement deficiency have an effect on B cells?

29. C1q and B cell tolerance to intracellular antigens Tg mice expressing an intracellular membrane-bound form of HEL (mHEL-KK) that is sequestered in the ER of all MHC I expressing cells mHEL bound on the cell surface induces tolerance by deletion/editing; sHEL induces tolerance by anergy Intracellular mHEL-KK positively selects B1 cells and high titres of IgM autoantibodies. Intracellular self-antigens are immunogenic and can stimulate the conventional B cells that escape negative selection C1q –deficient mice bred with Tg mice expressing mHEL-KK

30. Increased positive selection of IgM autoAb and peritoneal B1 cells in C1q-/- mice

31. Increased numbers of conventional B cells specific for mHEL-KK in C1q-/- mice

32. IgM autoantibody recognise intracellular antigens expressed on apoptotic cells

33. Phagocytosis of non-Tg and mHEL-KK apoptotic thymocytes

34. C1q and B cell tolerance to intracellular antigens - conclusions C1q inhibits the positive selection of B1b B cells and IgM autoantibodies by an intracellular self antigens C1q contributes to the clearance of dying cells that may explain the increased positive selection of auto-reactive B1 cells by intracellular antigens

35. C1q, clearance of dying cells and SLE: hypothetical model

36. SLE pathogenesis – 3 steps Complement and pathological survival of autoreactive lymphocytes 2) Complement and failure of clearing dying cells 3) Complement and tissue damage

37. What is the role of complement in glomerulonephritis?

38. C1q deficiency, alternative pathway deficiency and nephritis

39. Spontaneous glomerulonephritis and complement deficiency

40. C1q-deficient mice -conclusions C1q-deficient mice developed a lupus-like disease and had a defect in the clearance of dying cells that may explain the association between complement deficiency and SLE C1q inhibits the positive selection of B1 B cells and IgM autoantibodies by an intracellular self antigens

41.

43. Do lupus-prone strains of mice have an impaired clearance of apoptotic cells?

46. Is a defective clearance of dying cells sufficient to induce SLE ?

49. Background strain effects on autoimmunity in knockout models of SLE Our lab is working on SLE (Systemic Lupus Erythematosus, or Lupus as it is commonly known). This is a multi-system autoimmune disease with a complex genetic basis. Murine models of SLE have proved useful in attempting to identify the genes involved. Three major mouse models of the disease have been described: NZB/NZW hybrid, MRL lpr and BXSB. Targeted disruptions of genes of immune system proteins have also been used (gene knockouts) and these seem to have a surprisingly high frequency of autoimmune phenotype associated with them. Our lab has generated several knockouts of immune system genes. Today I am presenting some interesting findings which have come out recently after several years research. I will introduce briefly the genetic basis of making a knockout as this is relevant to our findings. Our lab is working on SLE (Systemic Lupus Erythematosus, or Lupus as it is commonly known). This is a multi-system autoimmune disease with a complex genetic basis. Murine models of SLE have proved useful in attempting to identify the genes involved. Three major mouse models of the disease have been described: NZB/NZW hybrid, MRL lpr and BXSB. Targeted disruptions of genes of immune system proteins have also been used (gene knockouts) and these seem to have a surprisingly high frequency of autoimmune phenotype associated with them. Our lab has generated several knockouts of immune system genes. Today I am presenting some interesting findings which have come out recently after several years research. I will introduce briefly the genetic basis of making a knockout as this is relevant to our findings.

50. The phenotype of Apcs (SAP) or C1qa deficiency in different mouse strains This is a table showing some of the results from our initial results using two of the gene knockouts generated in our lab: C1q and Apcs. There are distinct strain differences in the amount of autoimmunity developed in these animals after one year. In the Apcs -/- mice the (129 x BL/6) cohort developed high titres of autoantibody against the nucleus (ANA) and considerable autoimmune glomerulonephritis (GN) in the kidney. The C57BL/6 Apcs-/- cohort developed even higher ANA and GN, but the 129Sv cohort did not develop any autoimmunity at all. In the C1q-/- cohorts, the only strain which developed any autoimmunity was the (129 x BL/6) cohort. Cohorts of C1q -/- in BL/6 and 129Sv were negative. However the most surprising result for us was the wild type mice in (129 x BL/6), which spontaneously developed autoimmunity!This is a table showing some of the results from our initial results using two of the gene knockouts generated in our lab: C1q and Apcs. There are distinct strain differences in the amount of autoimmunity developed in these animals after one year. In the Apcs -/- mice the (129 x BL/6) cohort developed high titres of autoantibody against the nucleus (ANA) and considerable autoimmune glomerulonephritis (GN) in the kidney. The C57BL/6 Apcs-/- cohort developed even higher ANA and GN, but the 129Sv cohort did not develop any autoimmunity at all. In the C1q-/- cohorts, the only strain which developed any autoimmunity was the (129 x BL/6) cohort. Cohorts of C1q -/- in BL/6 and 129Sv were negative. However the most surprising result for us was the wild type mice in (129 x BL/6), which spontaneously developed autoimmunity!

51. Is the hybrid 129 x C57BL/6 genetic background sufficient to induce SLE?

52. Background strain effects on autoimmunity in knockout models of SLE Here I have given a diagrammatic representation of the chromosomes of a wild type (129 x B6) F2 mouse. Due to crossing over the entire genome contains random areas of 129 and B6 DNA. However please note that although the rest of the genome of the Apcs-/- mouse is randomised, chromosome 1 contains an area around the Apcs knocked out gene which is ES cell DNA on both alleles i.e. it is permanently fixed as 129Sv DNA.Here I have given a diagrammatic representation of the chromosomes of a wild type (129 x B6) F2 mouse. Due to crossing over the entire genome contains random areas of 129 and B6 DNA. However please note that although the rest of the genome of the Apcs-/- mouse is randomised, chromosome 1 contains an area around the Apcs knocked out gene which is ES cell DNA on both alleles i.e. it is permanently fixed as 129Sv DNA.

53. Unsorted data for ANA at 12 months of age. A significant statistical difference is found between wild type controls and Apcs-/- cohorts. (Each small dot is a single mouse, the large dot is a group of mice, the number of which is in brackets alongside) Unsorted data for ANA at 12 months of age. A significant statistical difference is found between wild type controls and Apcs-/- cohorts. (Each small dot is a single mouse, the large dot is a group of mice, the number of which is in brackets alongside)

54. Background strain effects on autoimmunity in knockout models of SLE Here I have given a diagrammatic representation of the chromosomes of a wild type (129 x B6) F2 mouse. Due to crossing over the entire genome contains random areas of 129 and B6 DNA. However please note that although the rest of the genome of the Apcs-/- mouse is randomised, chromosome 1 contains an area around the Apcs knocked out gene which is ES cell DNA on both alleles i.e. it is permanently fixed as 129Sv DNA.Here I have given a diagrammatic representation of the chromosomes of a wild type (129 x B6) F2 mouse. Due to crossing over the entire genome contains random areas of 129 and B6 DNA. However please note that although the rest of the genome of the Apcs-/- mouse is randomised, chromosome 1 contains an area around the Apcs knocked out gene which is ES cell DNA on both alleles i.e. it is permanently fixed as 129Sv DNA.

55. ANA data wild type controls sorted for fixed 129 interval. The difference disappears!ANA data wild type controls sorted for fixed 129 interval. The difference disappears!

56. QTL ANALYSIS CHROMOSOME 1

57. Background strain effects on autoimmunity- Congenic strains

58. We aged three cohorts for 12 months. One cohort was a pure BL/6 control, one had a non-functional Apcs gene on an otherwise pure BL/6 background containing the 129Sv KO region on distal Chr1, and the third was a wild type congenic mouse containing a similar (slightly larger) 129Sv segment on Chr 1 with the Apcs gene functional.We aged three cohorts for 12 months. One cohort was a pure BL/6 control, one had a non-functional Apcs gene on an otherwise pure BL/6 background containing the 129Sv KO region on distal Chr1, and the third was a wild type congenic mouse containing a similar (slightly larger) 129Sv segment on Chr 1 with the Apcs gene functional.

59. Phenotypes at 12 months of age were analysed. ANA titre showed that the congenic and the Apcs KO cohort were the same! The control BL/6 mice had a much lower number of positives and were clearly significantly different from either of the other two cohorts.Phenotypes at 12 months of age were analysed. ANA titre showed that the congenic and the Apcs KO cohort were the same! The control BL/6 mice had a much lower number of positives and were clearly significantly different from either of the other two cohorts.

60. CHROMOSOME 1 – SLE candidate genes

61. Gene-targeted mice have contributed to characterise effector mechanisms involved in disease pathogenesis There are important epistatic interactions between genes from 129 and C57BL/6 mice on the development of SLE The phenotype of knockout mice can be influenced not only by the disruption of the targeted gene but ALSO by their genetic background Genes located on telomeric portion of chromosome 1 are involved in SLE pathogenesis

62. Thanks to

63. SLE – pathogenesis Disease pathway Putative disease mechanisms Dysregulation of the immune system Abnormal antigen clearance Uncontrolled inflammation

64. Physiological uptake of apoptotic cells

66. 129 SLE loci on chromosome 1

67. C1q-deficient mice -conclusions C1q inhibits the positive selection of B1 B cells and IgM autoantibodies by an intracellular self antigens C1q contributes to the clearance of dying cells that may explain the association between complement deficiency and SLE

69. GLOMERULONEPHRITIS

71. SLE - complement, anti-DNA and disease activity

72. Antinuclear antibodies

73. Immune complexes and complement are found in inflamed tissues