1. Pathogenesis of Sjogren’s Syndrome: Translating Basic Science from “Bench to Bedside”

2. Sjogren’s Syndrome Increased mortality risk, particularly due to lympho-proliferative complications Quality of life- equated with moderate angina “Disability” predominantly due to fatigue and cognitive “Limitations”: –dry eyes (limits work- especially computer) –dry mouth (limits sleep and social interactions around eating) –Expense of artificial tears and dental decay

3. Background-1 Sjogren’s syndrome represents the interface of: a)Immune and exocrine secretory functions (dryness) b)Immune and neural function (neuropathy/cognitive) c)Immune and hypothalamic-adrenal axis (autonomic) d) Autoimmune proliferation and lymphoma e) Lupus like features of vasculitis and immune complex

4. Background-2 The Danger Signal When we get “flu symptoms” of arthralgia, fatigue, cognitive dysfunction— it is a result of the cytokines/neurotransmitters released by the innate immune system When these reactions persist due to a vicious cycle perpetuated in genetically predisposed individual by the acquired immune system, the result is autoimmune disease.

5. Pathogenesis: Take Home Lessons-1 1.Innate and Acquired Immune System are targets for current therapy—including TNF, BAFF and IL-6 inhibitors, steroids, traditional DMARD’s and new oral agents (Jak and syk inhibitors) 2.Functional circuit that controls immune and neural function are the new “frontier” for therapy from “fibromyalgia to depression.”The functional circuit is the link between cytokines and symptoms.

6. Take Home Lesson - 2 The two arms of the immune system mutually interact in the initiation and perpetuation of Sjogren’s Syndrome (Adaptive, immediate) - HLA independent Dendritic cells Cytokines-particularly Type I interferon Interferon-gamma BAFF, IL-6, IL-17 Complement, CRP Sensors of the innate system Toll receptors (TLR)-pathogen motiffs DAMP (damage recognition patterns)-apoptosis RIG-1 (retinoid inducible genes) NOD/Card receptors-more than in colitis (HLA-DR)-memory Traditional T-cell and B-cell and their cytokines HLA-DR association with autoantibody production Target of drugs such as DMARDs and certain biologics Acquired System Innate System

7. Take Home Lesson 3: The Functional Circuit (Cytokines are not enough) Control of tear or saliva flow is a complex process that involves both afferent nerve pathways that go to the midbrain and efferent nerves that modulate glandular function. The midbrain signals are influenced by the cortical outflow and the hypothalamic axis.

8. Normal tearing or salivation secretion requires a functional unit Nerves on mucosal 6. Stimulation of gland 2. Midbrain of central nervous system 5. Stimulation of blood vessel Afferent nerves 3. Cortical Outflow Tracts And HPA Lacrimatory or salivatory nuclei water mucin protein water nutrients hormones 1.Ocular or oral surface irritation

9. In Sjogren’s syndrome, the release of Ach and VIP by efferent nerves to the glands (and the response of the glands to neural transmitters) is impaired by lymphocytes that enter the gland and release inflammatory factors ocular and oral dryness Focal lymphocytic infiltrates in the glands lymphocytes Gland dysfunction Autoantibodies (anti-muscarinic antibody) Cytokines (type I IFN,  -IFN) Metalloproteinases (outside-inside signalling molecules)

10. In Sjogren’s, only 50% of the acini and ducts are destroyed. Despite their retension of neural innervation, the residual glands do not function as a result of the inflammatory environment Sjogren’s Normal Foci of lymphs

11. In Sjogren’s syndrome The residual glandular cells are paralyzed by the local immune reaction. Even though the acini/ducts are 50% present, their innervation and their receptors for neurotransmitters are present.

12. Thus, the interesting question is: Why are the residual glandular elements not working? This fundamental question of how immune and neural systems interact will be the “holy grail” of neuroscience for the next decade.

13. Pathogenesis Take Home Lesson- 2 Although many complex interactions take place in the salivary gland, a characteristic type I interferon gene signature is noted repeatedly. The relationship of autoantibody to SS-A/SS-B and type I interferon signature has recently been suggested. This links our blood tests (SS-A) and clinical features.

14. IFN type I in salivary gland suggests a role in Sjogren’s Syndrome SS SG biopsy with type I IFN SS SS SG biopsy with type I IFN gene profile NML Non-SS sicca

15. Take home lesson-3 Homing receptors determine both glandular and extraglandular features 1.Salivary glands normally lack lymphocytes, so their mere presence in an extraglandular tissues imply a lymphocyte aggressive process. 1.Homing to the gland tissue is due to specific receptors/ligands controlled by chemokines/cytokines. 2.Retention of lymphocytes in the tissue is due to specific ligands. 3.Their apoptosis or expansion is regulated through Fas pathways that are modulated by cytokines and bcl-2

16. Pathogenesis: Take Home Lessons-4 1.Extraglandular manifestations are determined by lymphocyte homing to tissues, factors that govern their retention in tissues and their apoptosis, 2.Factors governing their clonal expansion and lympho-proliferation lead to lymphoma-derived from B-cells themselves, T-cells, and dendritic cells.

17. 1. Tissue Homing/Retention of lymphocytes is the key process for accumulation of glandular infiltrates, as virtually no mitotic cells are seen in the gland. 2. Subsequent migration from gland into efferent lymphatic defines re-circulating memory lymphocyte pool.

18. AB Homing Receptors are up-regulated on high endothelial venules in Sjogren’s Lip Biopsy Peripheral Lymph Node Addressin (PNA-d) (peanut agglutinin) Chemokine receptor CCL21 Ref 63

19. B The endothelial cells attract T-cells by ICAM’s and Chemokines Sjogren’s Lip Biopsy

20. AB The endothelial cells release B-cell chemo-attractants Ref 63

21. Endothelial cells attract dendritic cells to home to the gland.

22. The interesting point is that the homing receptors expressed by salivary glands for T-cells, B-cells, and dendritic cells occur in NOD.scid mice so that they are independent of cytokines released from the lymphocytes. Thus, the story of Sjogren’s syndrome is not a poor salivary gland that is “beaten up” by the lymphocytes-- but that the glands participate in the homing and pathogenesis of inflammatory cells and subsequent inflammation.

23. Pathogenesis Take Home Lesson 4 SS has lymphoproliferative properties— it lies on the border between autoimmunity and lymphoma.

24. Sjogren’s Syndrome – with parotid enlargement indicates lymphoproliferative tendency

25. In patients whose minor salivary glands develop germinal centers, there is increased chance of lymphoma The T-cells and dendritic cells drive B-cell clonal expansion, particularly driven by BAFF, until a B-cell clone escapes to become a lymphoma.

26. This provides a rational of understanding for 1. anti-CD20 (rituximab) 2. anti-BAFF and anti-TACI 3. anti-CD22 antibody therapies

27. Overview of the steps in pathogenesis that help explain role of sex (TLR receptors) autoantibodies (anti-SS A) interferon-type I signature HLA-DR association

28. SS: Hormonal Factors (SS predominantly in women) X-chromosome location of Toll receptor; X-linked genes for apoptosis; X-linked genes for transcription promoter of pro-inflammatory loci including NF-K; X-linked control of metalloproteinase release under prolactin hormonal regulation.

29. Time course of autoimmune response* 1. Environmental stress is interpreted in context of genetic factors. 2. Antibodies precede disease. 3. Presence of antibody does not mean disease. Genetic Factors (including sex) (HLA-DR) Genetic Factors (including sex) (HLA-DR) Genetic Factors (including sex) (HLA-DR) Genetic Factors (including sex) (HLA-DR) Auto- antibodies Acquired Immune system (HLA-DR) T/B-cells Disease Manifestations Time period of years Innate Immune system (Toll receptor) Genetic Factors (including sex) (HLA-DR) Environmental Stress (virus-such as EBV ) (apoptotic fragment) Type I IFN Immune complex Ref. 32-33

30. Genetic Predisposition in SS to Type I Interferon In genome wide screens, association of IRF5 alleles and Stat 4, with predisposition to development of SS* * Refs 36-38

31. Other Factors in Pathogenesis Gender - SS is a predominantly a disease of women. Onset and increase of dryness with menopause. Increased risk of Klinefelter (XXY) in male SS* —Toll receptor translocation (BXB model). Aromatase knockout mouse gets SS. RbAp48--estrogen dependent apoptosis. DHEA and CRISP-role in glandular processing. * Refs 34-40

32. Summary 1. Ability to stimulate saliva or tears remains inadequate involves a complex pathway. 2. Extraglandular manifestations reflect homing pathways, as well as factors that influence tissue retention and apoptosis. 3. Neuro-endocrine manifestations (cognitive impairment and fatigue) remain the frontier of research for the next decade.

33. Thank you for your time and attention I would be happy to entertain any questions now or later. The slides are available to you for your use. RobertFoxMD@mac.com

35. How does the process start? There may be many different triggers in the genetically predisposed individual… 1.Defective apoptosis of glandular cells and clearance of these autoantigens; 2.Viral infection including EBV (in Caucasion and Japanese) and Coxsackie (in Greek patients); 3.Other viral infections (examples of Hep C, HIV and HTLV-1) can mimic SS; 4.Activation of endogenous retroviral fragments. Ref 1

36. Role of Autoantibody: Anti-SS A SS-A Antibody to SS-A hYRNA (ds RNA) To the innate immune system (dendritic cells), hYRNA is a double-stranded RNA and looks like a viral RNA that binds to a specific Toll receptor. Anti-SS A antibody (associated with HLA-DR3) binds to SS-A which is complexed to hYRNA

37. Salivary gland dendritic cells bind to the Fc  receptor to internalize the immune complexes containing SS-A/hYRNA Plasmacytoid Dendritic Cell SS-A 1. Immune complex antibody to SS-A hYRNA (ds RNA) 4. IFN Type 1 3. Toll 3 receptor is in located in the cytoplasm 2. Fc-  R

38. --The Vicious Cycle -- of innate and acquired leads to IFN type I (links genetic and autoantibody response) 4. Dendritic Cell with Toll Receptor and Fc-g Receptor 6. B-cell Anti-body response Anti-SS-A in HLA-DR3 pre-disposed female 5. IFN-a 1.Apoptotic Cell 2. Immune Complex containing… ______ 3. Toll receptor Fc-gamma R SS-A/hYRNA

40. Typical Clinical Features of dry eyes, dry mouth and swollen glands

41. Dryness results in the clinical appearance of keratoconjunctivitis sicca (KCS) characteristic of Sjogren’s syndrome The upper lid literally sticks to the surface epithelial surface and pulls surface mucin layers off. The Rose Bengal dye retention is like “rain water pooling in a street pothole” This test can be done at bedside and allows “triage” and rapid referral of patients to Ophthalmology

42. The Functional Circuit involves Known neural connections to the brain The Functional Circuit involves Known neural connections to the brain *Pflugfelder SC, et. al. Dry Eye and Ocular Surface Disorders. NY: Dekker, 2004.

43. This provides rationale for new therapies that interfere with homing 1. T- and B-cells have surface “homing receptors” when generated in node or marrow. CD4+ B cell Blood 3. When the homing receptor encounters vascular adhesive molecules, the lymphocyte enters tissue. 4. Pearl: Failure to bind to homing receptor in 72 hours leads to obligate apoptosis of the lymphocyte. This is why we do not become one large lymph node. 2. Lymphs migrate through blood to tissues.

44. For example IL-17 plays a key role in decreased secretion of water, proteins and mucin required in tears and saliva

45. Severe Xerostomia with dry tongue

46. Sjogren’s Syndrome- Cervical Dental Caries

47. Background 2 “The Danger Signal” The immune system is the 6 th sense of the brain. Lymphocytes are sent out to detect foreign pathogens and clean up debris from dead (apoptotic) cells. Lymphocytes report back to the brain in the form of neurokines and cytokines.

48. The Body’s 2 Distinct But Interconnected Immune Systems ACQUIRED Lymphyocytes (Type 2 interferon signature) Beutler B et al. Blood Cells Mol Dis. 1998;24:216-230. HLA-DR4–dependent: T cells respond to peptide antigens and generate memory cells INNATE Dendritic Cells (Type 1 interferon signature) HLA-DR–independent: Dendritic cells respond to specific structures found on bacteria and apoptotic Products (Toll receptors)

49. AB Homing Receptors are up-regulated on high endothelial venules in Sjogren’s Lip Biopsy Peripheral Lymph Node Addressin (PNA-d) (peanut agglutinin) Chemokine receptor CCL21 Ref 63