1. S jogren’s S yndrome: Pathogenesis and New Directions for Therapy Robert I. Fox, M.D., Ph.D. Carla M. Fox, R.N. Scripps Memorial Hospital Scripps Memorial/XiMED La Jolla, California RobertFoxMD@mac.com

2. Goals-1 Recognize that: Sjogren’s has benign symptoms (dry eyes/mouth), fatigue, myalgias and cognitive impairment. These symptoms are are the largest factor in patient “disability”– although not well treated by either local or biologic therapy.

3. Goals-2 Systemic manifestations (rash, arthritis, myositis, lymphoproliferative, biomarkers) These respond much better to systemic therapy. Targets correspond to genes identified in genomic screens. Characteristic cytokine, mRNA and gene methylation/acetylation maps.

4. Goals-3 The ability to improve extraglandular manifestations but not “pain/cognitive” indicates that we are missing an important nociceptive pathway. New advances in neuroprotection in Multiple Sclerosis provide new opportunities to target microglial cells

5. Roadblocks to Treatment of Dry Eyes and Dry Mouth Problems with study design Poor correlation with objective clinical and laboratory values Concept of “functional circuit” not recognized by immunologists or Pharma— while it is the basis for neurologists and pain therapy

6. Goals for Therapy Fatigue, Myalgias and Cognitive Impairment This is the “holy grail” of neuro-immunology to solve in the next decade. Flu-like symptoms, “ jet lag,” or after treatment of hepatitis C with IFN. We have animal models from duloxetene (now for back pain) or modafinil (jet lag) Molecular targets such as mTOR and AKT need to be considered.

7. Fatigue and Cognitive Impairment Much more common in SS or SLE than in RA - What is that telling us about pathogenesis? Involves the neuro-endocrine-immune axis associated with “stress” response (hypothalamic-adrenal axis) which we must add to innate and acquired responses as targets

8. Background-1 The functional Circuit To understand “benign symptoms” and develop better therapies—we must review the concept of the functional circuit in SS the interaction of immune activation on microglial cells and associated neurons New targets include mTor and AKT pathways

9. Background-2 The functional circuit in SS 1. Mucosal Surface (inflammatory cytokines and metalloproteinase) 1. Mucosal Surface (inflammatory cytokines and metalloproteinase) 2. Midbrain Vth Nucleus (lymphocytes and glial cells) 2. Midbrain Vth Nucleus (lymphocytes and glial cells) 4. Gland (lymphs, cytokines, metalloproteinase) 4. Gland (lymphs, cytokines, metalloproteinase) 3. Vascular (iNOS, CAMs, Chemokines) 3. Vascular (iNOS, CAMs, Chemokines) Brain Cortex Nociception (pain) glial cells and corticcal neurons Brain Cortex Nociception (pain) glial cells and corticcal neurons These sites and their cytokines correlate with systemic manifestations We must understand these sites to treat “benign” symptoms

10. Does this apply to Sjogren’s syndrome? Patients with early SS had corneal pain that decreased completely with topical anesthesia* Patients with chronic SS showed only a partial (30% decrease) in eye pain after topical anesthetic* Functional MRI (fMRI) showed nocioceptive pattern—called phantom pain amplification *Rosenthal et al

11. To study the mechanism of neurogenic or nociceptive pain we must use animal model-1 The thrombospondin (-/-) mouse (TSP null) or the TGF-  receptor mutation both develop SS like disease The mouse develops both oral and ocular lesions The mouse develops ANA and SS-A antibodies Thrombospondin is a matrix protein that plays a role in activation of latent TGF-  Activated TGF-  promotes Treg and inhibits Th-17 (IFN-  Thus, TSP (null) has high levels of Th-17, IL-17 and IFN- 

12. Thrombospondin (-/-) mouse model of SS 4 wks Lacrimal gland biopsies The mouse has ANA+, SS-A+ TSP null can not activate TGF-  In absence TGF-  continuous Th-17 TGF-  and cytokine activation stimulates mTor/AKT WTWT Tsp-/- 24 wks

13. At the level of the Vth nerve (Tsp -/- mouse) Microglial cells translate inflammatory signals that go to nociceptive cortex WT TSP (-/-) mTor and AKT activated in response to “lower stimuli” in the tsp (-/-) mouse

14. Thrombospondin (-/-) Mouse at 24 wks Where a trivial stimuli Causes pain response Wild type A pain stimuli that is innocuous in Wild Type does cause nociceptive pain in tsp (-/-) mouse model The Pain Threshold is Lowered in the Tsp (-/-) mouse Ocular chemical stress model of nociceptive pain Le Bars D, Animal models of Nociception. Pharmacological reviews 2001;53:597- 652.

15. Moulton et*. Al used fMRI in SS patients with chronic ocular pain using fMRI of nociceptive pain have been studied Cortical regions that activate with ocular pain signal at “benign stimuli levels” occur only in chronic SS patients with severe pain *Moulton EA, Becerra L, Rosenthal P, Borsook D. An Approach to Localizing Corneal Pain Representation in Human Primary Somatosensory Cortex. PloS one 2012;7:e44643.

16. Emotional Physiological Similar pattern of Fos-ir in PVH neurons in response to distinct stressors

17. Neuroinflammation plays a prominent role in the pathophysiology of several neurodegenerative disorders, including Multiple Sclerosis and Sjogren’s syndrome. Microglia contribute to initiating and maintaining brain inflammation, and once activated release pro-inflammatory mediators potentially cytotoxic, like nitric oxide (NO)

18. mmt mTor and AKT pathway have multiple targets for drug screening in response to cytokines screening

19. mTOR inhibitors reduced NOS activity and NOS2 expression induced by cytokines, but not those induced by LPS. In conclusion, mTOR selectively controls microglial activation in response to pro- inflammatory cytokines and appears to play a crucial role in microglial viability

20. Review of Sjogren’s Systemic From the point of view of rheumatologist

21. EYE DRYNESS results in the clinical appearance of keratoconjunctivitis sicca (KCS) characteristic of Sjogren’s Syndrome The upper lid literally sticks to the Epithelial surface and pulls surface mucin layers off. The Rose Bengal dye retention test 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

23. Severe “Xerostomia” (dry mouth) with dry tongue Angular cheilitis (candida)

24. Therapy of “benign” symptoms a. dry eyes b. painful eyes c. dry mouth d. painful mouth e. fatigue f. myalgias Symptomatic and objective findings are poorly correlated.

25. For this audience, I will not review the myriad of artificial tears and salivas. I review these on my website (in downloadable files for patients) to facilitate providing information to patients. robertfoxmd.com

26. Simple Reminders: a. artificial salivas or secretagogues will not work until you treat the oral candida first. b. artificial tears with preservatives cannot be used more than 4 times a day, and will not work until you treat blepharitis

27. Cortical Map for Corneal Pain Figure 3

28. Hypothalamic Axis We also know from the high frequency of autonomic dysfunction, that we have not yet influenced the secondary effects on the hypothalamic axis. We know that prednisone works, but we can not therapeutically obtain the same benefit in SS from other medications.

29. Figure 4

30. Extraglandular Manifestations May be Lupus-like (immune complex)- pleurisy, hemolytic anemia, ITP, vasculitis May be lymphocytic infiltrate- Interstitial pneumonitis, RTA, lymphoma, neuropathy (central nervous or peripheral) Measured by ESSDAI scale (weighted by importance)

31. Therapy for Extraglandular Manifestations Manifestations correlate with acute phase reactants and biomarkers Manifestations respond to systemic therapy such as rituximab, abatacept, belumimab, anti-CD22 (epratazumab)

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

36. Scleritis (vasculitis)

37. But in today’s “super speed medicine” Not everything is so simple Caution that it is not a simple systemic manifestation of SS

38. Ulcerative keratitis (in patient given broad spectrum antibiotic plus anesthetic)

39. Not all rashes are disease flares

40. Patient with NSIP who developed pneumocystis on therapy

41. Steps in Pathogenesis Homing to specific tissues (glands) Production of autoantibodies Pathogenesis of salivary gland lesions

42. A key “target” in Sjogren’s is that lymphocytes “home” to the glands 1. Lymphocytes 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. 2. Lymphs migrate through blood to tissues.

43. Interfere with homing (obligate apoptosis if not bind “addressin”) Natalizumab (Tsabri)- cell adhesion a4-integrin Odulimomab (ICAM, CD54, LFA-1)- adhesion and migration Fingolimod (Gilenya)- sphingosine-1 receptor CD22 (Epratazumab)

44. In Sjogren’s Syndrome, many acini and ducts are spared Sjogren’sNormal

45. Gene expression profiling of minor salivary glands clearly distinguishes primary Sjögren's syndrome patients from healthy control subjects Arthritis & Rheumatism Volume 52, Issue 5, pages 1534-1544, 5 MAY 2005 DOI: 10.1002/art.21006 http://onlinelibrary.wiley.com/doi/10.1002/art.21006/full#fig1 Volume 52, Issue 5, http://onlinelibrary.wiley.com/doi/10.1002/art.21006/full#fig1

46. Time course of autoimmune response* 1. Genetic factors predispose to Sjogren’s 2. Environmental factors such as a viral infection may lead to formation of autoantibodies. 2. Antibodies precede disease. 3. However, presence of antibody does not necessarily 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 Factor (virus-such as EBV ) (apoptotic fragment) Type I IFN Immune complex Ref. 32-33

47. The main cytokine targets match those identified in genome wide screens HLA-DR (T-cell), CTLA and IFN-  NF-K /IkB Homing receptor (CXCR5) Type I IFN –IRF5, STAT4, TLR3/7/9 and pkR (cytoplasmic sensor) B-cell activation –BLK, BAFF, IL12, and A20 (TNFAIP3)

48. Previously Studied in SS Anti-CD20 –glandular and extraglandular Anti-CD22-epratazumab BAFF (Blys)-ACR 2012 abstracts* Abatacept (CD40 L)-ACR 2012* Allogeneic mesenchymal cells-ACR 2012 abstracts and article in Blood www.rheumatology.org/wren/acrsearch.asp?zoom_query=acr%20abstracts%2 02012&st=nocache&actn=search&dt=12/24/2012%202:29:59%20P

49. Limited Success with antibodies to type I IFN in SS In animal and early clinical trials, little benefit after the disease is established— although may help delay onset. Novel new target IRF8 and SLAC4A, molecules that links TLR and IFN-type 1, appear more promising

50. Other Inhibitors of IFN a. Initial trials of anti-type 1 IFN had infusion reactions and only modest efficacy. b. Medi 546 (type 1 IFN-R antagonists) now in phase 1 (scleroderma) and juvenile SLE phase 2 trial.

51. Now we have methylation maps

52. These methylation maps show different targets

53. Summary 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 (endocrine) d) Autoimmune proliferation and lymphoma e) Lupus-like features of vasculitis and immune complex

54. Thank You It is an honor to visit with you today The slides are available on my website RobertFoxMD.com