1. Systemic Lupus Erythematosus
Katie Bentivegna
4/13/2016

2. American College of Rheumatology’s “Eleven Criteria of Lupus”
Malar rash (butterfly rash)
Raised, red skin patches
Photosensitivity of skin
Mouth or nose ulcers
Arthritis
Pericarditis or pleuritis
Seizure and/or psychosis
Renal disorder (proteinuria)
Blood disorder
Immunologic disorder (anti-dsDNA, anti-Sm, etc)
Antinuclear antibodies (ANA)
4/11 to diagnosis lupus

3. SLE Difficult to characterize and manifests in multiple forms
Sexual dimorphism – 9:1 (Female:Male)
Autoimmunity and chronic inflammation in multiple tissues or organ systems
Joints, skin, kidney, blood cells, brain, heart, lungs

4. https://www. google. com/url

5. Types of Lupus Systemic Lupus Erythematous Discoid (Cutaneous) Lupus
Neonatal Lupus
Drug-induced Lupus
OCP, HRT

6. Estrogen in Autoimmunity
Females have a more robust humoral immune response compared to males
Females produce higher levels of serum Ig
Estrogen has been shown to increase production of autoantibodies
In SLE mouse models, estrogen has been shown to accelerate disease severity
Administration of testosterone has been shown to reverse these effects
Cutolo et al. “Estrogens and Autoimmune Diseases” Ann. N.Y. Acad. Sci. (2006) 1089: 538–547

7. Aromatase Inhibitor as a Therapeutic Intervention
4-OHA
(4-hydroxyandrostenedione)

8. Effects of an Aromatase Inhibitor on Thymus and Kidney and on Oestrogen Receptors in Female MRL/MP-lpr/lpr Mice
B.D. GREENSTEIN, Y.Y. DHAHER, E. de F. BRIDGES, G. KESER, M.A. KHAMASHTA, J. ETHERINGTON, A.S. GREENSTEIN, P.J. COATES*, P.A. HALL* and G.R.V. HUGHES

9. MRL/MP-lpr/lpr Mice
Homozygous for lymphoproliferation spontaneous mutation (Faslpr)
Mutation in the Fas apoptosis gene

10. Estrogen Receptor

11. Experimental Design MRL/MP-lpr/lpr Female Mice + 4-OHA Pellet
+ Empty Pellet

12. Figure 1 Lower thymus weight of MRL mice when treated with 4-OHA
No significant difference between uterine weights
No significant difference between liver weights (not pictured)
Figure 1. Greenstein et al. “Effects of an Aromatase Inhibitor on Thymus and Kidney and on Oestrogen Receptors in Female MRL/MP-lpr/lpr Mice” Lupus. (1993). vol. 2 no.4 pp

13. Additional Findings
Control group had high levels of plasma cells in thymus
4-OHA group had higher abundance of cells with large nuclei and less cytoplasm (thymocytes)
More severe inflammation of the kidneys in the control group
Leukocyte infiltration is confined to mesangium in the 4-OHA group

14. https://www. google. com/search

15. Scatchard Plot K = affinity constant for ligand binding
Conc. of bound ligand
Conc. of bound ligand to unbound ligand
K = affinity constant for ligand binding
Competitive ligand binding between [H3]moxoestrol and unlabeled steroids
Looked at cytosolic and nuclear fractions

16. Figure 3
Linear plots – one binding site (ligand is binding nicely to the receptor)
Figure 3. Greenstein et al. “Effects of an Aromatase Inhibitor on Thymus and Kidney and on Oestrogen Receptors in Female MRL/MP-lpr/lpr Mice” Lupus. (1993). vol. 2 no.4 pp

17. Table 1
Oestrogen receptors were not detected in thymus cytosol fractions of control
After 4-OHA treatment, receptors were measurable in thymus cytosol
4-OHA treated thymus cytosol fractions had highest Kd value – lower affinity binding
Table 1. Greenstein et al. “Effects of an Aromatase Inhibitor on Thymus and Kidney and on Oestrogen Receptors in Female MRL/MP-lpr/lpr Mice” Lupus. (1993). vol. 2 no.4 pp

18. Figure 4
Greater number of binding sites in uterus nucleus/cytosol compared to other tissues
Lower number of binding sites in liver nucleus of 4-OHA treatment
Figure 4. Greenstein et al. “Effects of an Aromatase Inhibitor on Thymus and Kidney and on Oestrogen Receptors in Female MRL/MP-lpr/lpr Mice” Lupus. (1993). vol. 2 no.4 pp

19. Limitations
No measure of aromatase activity or serum estrogen/testosterone levels after 4-OHA treatment
Only assessed estrogen receptor levels
No assessment of anti-dsDNA antibody production in lupus prone mice

20. Summary
Evidence for decreased thymus weight and decreased inflammation in kidneys in 4-OHA mice
Indirect evidence that decreased estrogen biosynthesis (via 4-OTA) led to the less severe disease phenotype
Effect of estrogen on specific lymphocyte subsets?

21. Estrogen up-regulates Bcl-2 and blocks tolerance induction of naive B cells
Margaret S. Bynoe, Christine M. Grimaldi, and Betty Diamond

22. B Cell Tolerance

23. Mouse Model
Female BALB/c mice transgenic for the R4A-γ2b heavy (H) chain of nephritogenic anti-DNA antibody
Ability to tolerize high-affinity autoreactive B cells
3 populations of B cells:
Nontolerized B cells secrete Abs with low affinity for dsDNA
Anergic B cells secrete Abs with high affinity for dsDNA after LPS stimulation
Deleted B cells - High affinity B cell population

24. Figure 1
Increased serum anti-dsDNA antibody in E2 treated mice compared to placebo at 2 and 5 months of age
Figure 1. Bynoe et al. “Estrogen up-regulates Bcl-2 and blocks tolerance induction of naïve B cells” Proc Natl Acad Sci U S A. (2000) vol. 97 no. 6 pp 2703–2708

25. Figure 2 Increased glomerular Ig deposition in E2 treated mice
No glomerular Ig deposition in Placebo treated mice
Figure 2. Bynoe et al. “Estrogen up-regulates Bcl-2 and blocks tolerance induction of naïve B cells” Proc Natl Acad Sci U S A. (2000) vol. 97 no. 6 pp 2703–2708

26. Figure 3
Increased B cells expressing the y2b heavy chain in the E2 treated mice
Increased high-affinity anti-dsDNA-secreting B cells in the spleen in E2 treated me
Figure 3. Bynoe et al. “Estrogen up-regulates Bcl-2 and blocks tolerance induction of naïve B cells” Proc Natl Acad Sci U S A. (2000) vol. 97 no. 6 pp 2703–2708

27. Table 1
E2 treatment maintains high-affinity anti-DNA B cells in hybridomas that normally undergo deletion/anergy induction in non-autoimmune mice
Table 1. Bynoe et al. “Estrogen up-regulates Bcl-2 and blocks tolerance induction of naïve B cells” Proc Natl Acad Sci U S A. (2000) vol. 97 no. 6 pp 2703–2708

28. Figure 5 Increased Bcl-2 expression in spleens of E2 treated mice
Figure 5. Bynoe et al. “Estrogen up-regulates Bcl-2 and blocks tolerance induction of naïve B cells” Proc Natl Acad Sci U S A. (2000) vol. 97 no. 6 pp 2703–2708

29. Summary
E2 treatment led to a rise of anti-DNA serum titers and Ig deposition in glomeruli
E2 treated mice express high-affinity anti-DNA antibody secreting B cells in the spleen
E2 rescues these high-affinity anti-DNA B cells from tolerance induction
E2 could be increasing the risk of autoimmunity in lupus through these mechanisms

30. Background and Significance
Estrogen promotes autoimmunity:
In non-autoimmune mice, estrogen interferes with tolerance induction of naive autoreactive B cells. The presence of these B cells in the periphery is associated with increased Bcl-2 activation (Bynoe et al., 2014)

31. Background and Significance
Aromatase Inhibitors may reduce autoimmunity
In one epidemiologic study, there was a trend for aromatase inhibitors to reduce the incidence of lupus (Chen et al., 2014)
In lupus mice, aromatase inhibitors may reduce the production of plasma cells in the thymus, and inflammation of the kidney. This finding suggests that blocking estrogen production may be useful for lupus (Greenstein et al., 1993)

32. Objectives
Given that estrogen promotes autoimmunity and there is some limited evidence that aromatase may reduce autoimmunity, the objective of this study is:
To determine whether an aromatase inhibitor can reduce specific markers of autoimmunity (especially dsDNA) in lupus prone mice

33. Hypothesis
Treatment with aromatase inhibitor (4-OHA) will reduce estrogen levels and consequently reduce autoimmunity seen in lupus-prone mice.

34. Bibliography
Bynoe, M. S., C. M. Grimaldi, and B. Diamond. "Estrogen Up-regulates Bcl-2 and Blocks Tolerance Induction of Naive B Cells." Proceedings of the National Academy of Sciences 97.6 (2000): Web.
Cell Death Pathways: Apoptosis, Autophagy and Necrosis Metabolism and Cancer Progression: Meeting Abstract Book. Silverthorne: Keystone Symposia, Print.
Chen, J. Y., and S. P. Ballou. "The Effect of Antiestrogen Agents on Risk of Autoimmune Disorders in Patients with Breast Cancer." The Journal of Rheumatology 42.1 (2014): Web.
Cutolo, M., Capellino, A., Serioli, B., Secchi, M., Villaggio, B., Straub, R. “Estrogens and Autoimmune Diseases” Ann. N.Y. Acad. Sci (2006): 538–547. Web.
Greenstein, B.d., Y.y. Dhaher, E. D. F. Bridges, G. Keser, M.a. Khamashta, J. Etherington, A.s. Greenstein, P.j. Coates, P.a. Hall, and G.r.v. Hughes. "Effects of an Aromatase Inhibitor on Thymus and Kidney and on Oestrogen Receptors in Female MRL/MP-lpr/lpr Mice." Lupus 2.4 (1993): Web.
"History of Lupus." St.Thomas' Lupus Trust (symptoms, Advice, Research and News). N.p., n.d. Web. 13 Apr
"Lupus Research Institute." Lupus Diagnosis. N.p., n.d. Web. 13 Apr
"MRL/MpJ-Faslpr/J." N.p., n.d. Web. 13 Apr