Understanding Lupus OR Why Rheumatology Is So Cool.

1. Michelle Kahlenberg Senior Talk March 19th/20th 2009 Understanding LupusORWhy Rheumatology Is So Cool.

2. Why is lupus such a mystery to us? Unlike many other autoimmune diseases, it affects many organs with varying disease manifestations over time. This makes it difficult to diagnose: average is 4 years and 3 different doctors There are all those antibodies to know� Treatments are OLD with significant side effects

3. Talk Overview Discuss Lupus diagnostic criteria and clinical manifestations Explore Mechanisms of Lupus (cool!) Defective apoptosis IFN-a TLR signaling B-cell disregulation Pathology of autoantibodies Discuss how research into understanding the mechanisms of SLE is advancing treatment for lupus

4. Lupus Diagnostic Criteria (need 4) 1. Malar Rash: Fixed erythema, flat or raised, over the malar eminences, tending to spare the nasolabial folds 2. Discoid rash: Erythematous raised patches with adherent keratotic scaling and follicular plugging; atrophic scarring may occur in older lesions 3. Photosensitivity: Skin rash as a result of unusual reaction to sunlight, by patient history or physician observation 4. Oral ulcers: Oral or nasopharyngeal ulceration, usually painless, observed by physician

5. Lupus Diagnostic Criteria (need 4) 5. Nonerosive Arthritis: Involving 2 or more peripheral joints, characterized by tenderness, swelling, or effusion 6. Pleuritis or Pericarditis: a) Pleuritis--convincing history of pleuritic pain or rubbing heard by a physician or evidence of pleural effusion OR b) Pericarditis--documented by electrocardigram or rub or evidence of pericardial effusion 7. Renal Disorder: a) Persistent proteinuria > 0.5 grams per day or > than 3+ if quantitation not performed OR b) Cellular casts--may be red cell, hemoglobin, granular, tubular, or mixed 8. Neurologic Disorder: a) Seizures--in the absence of offending drugs or known metabolic derangements; e.g., uremia, ketoacidosis, or electrolyte imbalance OR b) Psychosis--in the absence of offending drugs or known metabolic derangements, e.g., uremia, ketoacidosis, or electrolyte imbalance 9. Hematologic Disorder: a) Hemolytic anemia--with reticulocytosis OR b) Leukopenia--< 4,000/mm3 on = 2 occasions OR c) Lymphopenia--< 1,500/ mm3 on = 2 occasions OR d) Thrombocytopenia--<100,000/ mm3 in the absence of offending drugs 10. Immunologic Disorder: a) Anti-DNA: antibody to native DNA in abnormal titer OR b) Anti-Sm: presence of antibody to Sm nuclear antigen OR c) Positive finding of antiphospholipid antibodies on: an abnormal serum level of IgG or IgM anticardiolipin antibodies, a positive test result for lupus anticoagulant using a standard method, or a false-positive test result for at least 6 months confirmed by Treponema pallidum immobilization or fluorescent treponemal antibody absorption test 11. Positive Antinuclear Antibody: An abnormal titer of antinuclear antibody by immunofluorescence or an equivalent assay at any point in time and in the absence of drugs �

6. Arthritis Painful joints are #1 symptom in 76-100% of patients With or without signs of inflammation, mild effusions if any Usually involve hands, wrists, knees Can be asymmetric Generalized myalgias particularly deltoids and quads <15% with elevations in CPK

7. Nephritis Very closely linked with prognosis 5- and 10-year survival rates are documented as high as 85% and 73%, respectively (used to be about 0% before immunosuppressants) Diagnosed by presence of proteinuria (>500mg/24 hours) Watch for symptoms of edema, puffy eyes, frequent urination. Biopsy helpful to determine whether aggressive treatment is required Associated HTN needs aggressive Rx with ACE/ARB

8. Nephritis Class I Minimal mesangial lupus nephritis Normal glomeruli by light microscopy, but mesangial immune deposits by immunofluorescence Class II Mesangial proliferative lupus nephritis Purely mesangial hypercellularity of any degree or mesangial matrix expansion by lightmicroscopy, with mesangial immune deposits May be a few isolated subepithelial or subendothelial deposits visible by immunofluorescence or electron microscopy, but not by light microscopy Class III Focal lupus nephritisa Active or inactive focal, segmental or global endo- or extracapillary glomerulonephritis involving 50% of all glomeruli, typically with focal subendothelial immune deposits, with or without mesangial alterations Class III (A) Active lesions: focal proliferative lupus nephritis Class III (A/C) Active and chronic lesions: focal proliferative and sclerosing lupus nephritis Class III (C) Chronic inactive lesions with glomerular scars: focal sclerosing lupus nephritis Class IV Diffuse lupus nephritisb Active or inactive diffuse, segmental or global endo- or extracapillary glomerulonephritis involving 50% of all glomeruli, typically with diffuse subendothelial immune deposits, with or without mesangial alterations. This class is divided into diffuse segmental(IV-S) lupus nephritis when 50% of the involved glomeruli have segmental lesions, and diffuse global (IV-G) lupus nephritis when 50% of the involved glomeruli have global lesions. Segmental is defined as a glomerular lesion that involves less than half of the glomerular tuft. This class includes cases with diffuse wire loop deposits but with little or no glomerular proliferation Class IV-S (A) Active lesions: diffuse segmental proliferative lupus nephritis Class IV-G (A) Active lesions: diffuse global proliferative lupus nephritis Class IV-S (A/C) Active and chronic lesions: diffuse segmental proliferative and sclerosing lupus nephritis Active and chronic lesions: diffuse global proliferative and sclerosing lupus nephritis Class IV-S (C) Chronic inactive lesions with scars: diffuse segmental sclerosing lupus nephritis Class IV-G (C) Chronic inactive lesions with scars: diffuse global sclerosing lupus nephritis Class V Membranous lupus nephritis Global or segmental subepithelial immune deposits or their morphologic sequelae by light microscopy and by immunofluorescence or electron microscopy, with or without mesangial alterations Class V lupus nephritis may occur in combination with class III or IV in which case both will be diagnosed Class VI Advanced sclerosis lupus nephritis 90% of glomeruli globally sclerosed without residual activity

9. Nephritis Class I-no Rx Class II-Rx if proteinuria >1000 mg/d Class III and IV at high risk of progression so require aggressive immunosuppressive therapy Class V Rx with steroids Class VI-dialysis or transplant

10. Nervous System Headache is the most common complaint ADD, mood disorders, anxiety, delirium, psychosis, seizures (generalized or partial) Difficult to prove absolute causality Generalized encephalopathies Formal neuropsychiatric testing reveals deficits in 21-67% of patients with SLE Cerebritis Degenerative changes in small vessel walls, often with minimal or no inflammatory infiltrates May be related to immune complex deposition Neuropathy secondary to vasculitis of vasa nervorum (often with dermatomyositis overlap)

11. CV System Pericarditis 6-45% of patients: low likelihood of tamponade or constrictive type. <10% with myocarditis Libman-Sacks endocarditis 1-4 mm vegetations of accumulations of immune complexes and mononuclear cells on mitral, tricuspid or aortic valves Risk of thromboembolism or secondary infective endocarditis (abx prophy) Aortic insufficiency is the most common valvular abnormality. Heart disease Contributes to bimodal pattern of mortality from lupus A study from U of Pittsburgh comparing rates of MI to that of Framingham Offspring Study data showed that risk of MI was 50x higher in woman with lupus ages 35-44 and 2.5-4x higher in older age groups Autopsy data shows CAD in 40% of SLE patients as opposed to only 2% of age matched controls. Atherosclerotic plaque burden (via carotid intima media thickness measurements and by coronary calcium scores) is higher in patients with SLE than in controls �Lupus dyslipoproteinemia� is low HDL, high TG, normal or only slightly elevated LDL, increased lipoprotein(a): this appears to correlate with disease activity Means of prevention focus on risk factor management and inflammation control, but no clear guidelines are available as of yet.

12. Lungs and Pleura Over 30% will have pleuritis or an effusion over the course of their disease Fluid is exudative, normal glucose, high protein, WBC <10,000 (neutrophilic or lymphocytic), decreased complement Can have pneumonitis, pulmonary hemorrhage (rare but often fatal), PE, pulmonary HTN Pulmonary HTN more likely to be associated with Raynaud�s

13. Hematologic System Chronic anemia is present in up to 80% of patients Leukopenia is present in up to 50% of patients (lymphopenia more common than neutropenia). Thrombocytopenia ranges from modest to severe with bleeding complications May reflect disease activity May be first sign of SLE; predating other signs and symptoms by years. Associated with the presence of anti-platelet antibodies Secondary APS seen in about 40% of patients with SLE

14. BUT WHAT CAUSES ALL OF THESE SYMPTOMS?

15. Defective clearance of apoptotic cells One common theme is defects in clearance of apoptotic cells resulting in autoantibody production Phagocytes from lupus patients engulf far less during a 7 day period in vitro than phagocytes from healthy patients

16. Defective clearance of apoptotic cells There may be a genetic component to defective apoptosis. Concordance is 25% among monozygotic twins but only 2% among dizygotic twins suggesting a genetic component HLA-DR2 and HLA-DR3 confer relative risk of 2-5. C1q deficiency results in high likelihood of developing SLE Complement C4a deficiency: 80% of people with SLE have at least one null allele Can lead to decreased clearance of apoptotic cells and increased inflammation and presentation of self antigens Patients with SLE may also develop autoantibodies against adaptor molecules which facilitate phagocytosis of apoptotic cells (C1q, MBL) resulting in defective clearance, classical pathway complement activation, and recruitment of inflammatory cells.

17. Defective clearance of apoptotic cells Delayed or defective apoptosis then allows for prolonged exposure of intracellular antigens, �inflammatory cell death phenotype,� inflammatory cell recruitment and presentation of normally protected intracellular components as antigens allowing for autoantibody production

18. The importance of IFN-a IFN-a is able to activate APCs after uptake of self material as well as promote B cell differentiation into plasma cells IFN-a levels appear to correlate with disease severity and levels of anti-DS DNA in SLE Patients with non-autoimmune diseases treated with IFN-a can develop positive ANA, anti-DS DNA abs and occasionally SLE. Conditions that naturally increase IFN-a levels (sunburn, viral infections) can induce SLE flares. IFN-a regulated genes are expressed at higher levels in the blood of SLE patients Plasmacytoid DCs are the major producers of IFN-a. SLE patients have 50-100 fold fewer in circulation as they have migrated to lymph tissues where they remain activated SNPs in interferon signaling related genes (Tyk2 and interferon regulatory factor 5) also confer increased likelihood of developing lupus SLE susceptibility polymorphism in STAT4 results in increased sensitivity to IFN-a signaling.

19. The Innate Immune System May Also Play a Role Toll-Like Receptors recognize molecular patterns (double stranded RNA, DNA, LPS etc) in order to provide rapid response to invading pathogens. They use defined signaling pathways to result in production of inflammatory cytokines and initiate inflammatory reactions. TLR7 and 9 are selectively expressed on PDCs Regulation in endosomes may regulate control of NFkB vs. IRF7 activation in human plasmacytoid dendritic cells

21. TLRs are thought to also recognize self antigens in the context of inflammatory diseases All TLRs (except 5 and 10) have been shown to be activated by endogenous molecules in the context of cell death TLR7 and 9 are expressed only in endosomes to decrease the chance of coming in contact with endogenous RNA or DNA TLR7 and 9 are activated by DNA/anti-DNA IgG complexes resulting in IFN-a and autoantibody production. However, immune complexes are taken up by cells with FcgRIIa and taken to the endosome where they can activate TLR 7 and 9. This Results in signaling cascade activation that increases production of IFN-a.

22. B-cell Disregulation

23. Mechanism Summary Defects in clearance of apoptotic cells can lead to exposure of intracellular immunogenic components which can be taken up by DC and presented to autoreactive B cells (made this way during random somatic hypermutation). In the right genetic environment, these B cells may become activated to produce autoantibodies. Polymorphisms or mutations in genes in numerous steps of B-cell regulation or IFN-a responsiveness can predispose to SLE (FcgRIIa, IRF5, STAT4, BLK)

24. Mechanism Summary Once autoantibodies (particularly anti-DS DNA) are present, they can complex with DNA exposed on dying cells and then bind to the FcgRIIa on PDCs, activate TLR 7 and 9, and result in high levels of IFN-a production. IFN-a encourages a feed-forward mechanism of continued plasma cell activation to produce increased amounts of autoantibodies and encourage further disease progression and tissue destruction.

25. Autoantibodies ANA: against targets in the nucleus, but only those which have intrinsic immunological activity: i.e.. They can activate the innate immune system via Toll-like receptors Anti DS-DNA in particular recognizes DNA in complex with nucleosome components (histone-derived peptides in particular) Can correlate with nephritis Immune complexes with anti-DNA ab/DNA can increase the expression of IFN-a via plamacytoid dendritic cells Anti-Sm: detects ribonucleoproteins involved in processing of mRNA; doesn�t track with disease, specific for lupus SSA/Ro and SSB/La: detect ribonucleoproteins, associated with SICCA syndrome and photosensitivity Anti NMDA to subunits NR2a and NR2b may be associated with neuropsychiatric symptoms �Antiphospholipid� antibodies are ab against phospholipid-binding proteins or phospholipids that are prothrombogenic. Ex: lupus anticoagulant, anticardiolipin, and anti beta2-glycoprotein I

26. Why are autoantibodies so bad? Renal disease IgA, IgM, IgG and complement deposition in the mesangium and subendothelial and subepithelial of the GBM that results in complement activation and recruitment of inflammatory cells that result in tissue destruction. Cross reactivity of anti-DS DNA antibodies with a-actinin may also result in a direct focusing of complement activation Skin disease Inflammation and breakdown of the dermal-epidermal junction. UV exposure can worsen because it promotes apoptosis in the skin resulting in autoantibody binding and tissue injury via complement activation or inflammatory cell activation Anti-Ro antibodies are associated with skin flares

27. Why are autoantibodies so bad? In the CNS, vasculitis is rare Anti-NMDA receptor antibodies may contribute to cerebral lupus phenotypes See more microinfarcts and degeneration or proliferative changes in blood vessels, thought to be related to IC deposition Antiphospholipid abs may contribute to thrombotic events anywhere in the body aPLs bind to endothelial cells, monocytes, neutrophils and platelets causing inflammation and procoagulant release This process is dependent on complement activation

28. How Understanding the Mechanism of SLE will influence therapy Currently, general immunosuppressants and antimalarials are the therapy of choice for lupus (steroids, plaquenil) and lupus nephritis (cyclophosphamide �cellcept may become an approved option) Current therapies are limited by side effects No new FDA approved drug for lupus have surfaced in 40 years! Research into the underlying mechanisms will allow for more directed therapies that may provide better control of SLE with fewer side effects

29. How Understanding the Mechanism of SLE will influence therapy Removal of B cells may improve disease control Open label trials of rituximab (anti CD-20) have shown up to 80% response, 50% with sustained response after 12 months. A recent RCT (EXPLORER) did not show a benefit with rituximab but patients were very sick and both control and study patients received high doses of steroids which may have undercut the benefit seen in patients given rituximab Trials of anti-IFN-a antibodies are underway Preliminary trials of inhibitory DNA sequences to block immune complex binding to TLRs suggest that preventing aberrant TLR 7 and 9 activation decreases IFN-a levels and disease flares Anti-IL-10 trials are ongoing. Preliminary trials suggest improvement in skin and joint symptoms Trials are also ongoing to block other B-cell stimulating signals (anti-BlyS=Belimumab, atacicept (soluble fusion protein that inhibits Blys ligand) and to block helper T cell activation.

30. Summary Lupus is a disease of autoantibody formation that results in varied clinical manifestations Disregulation of apoptosis, B-cell survival and proliferation and IFN-a production appear to be the major inciting events Ongoing research into the mechanisms which lead to SLE will hopefully provide us with novel effective therapies with improved side effect profiles

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32. Kids!!!