T helper cells in IBD

1. T helper cells in IBD Jaimee Doucette

2. Overview • Background Information on T Helper cells • What is known about Th cells in IBD? • What is still unknown about Th cells in IBD? • Paper 1 • Paper 2 • Future studies/specific aims

3. T cells Nature Reviews Immunology 2, 309-322 (May 2002)

4. Activation of naïve t helper cells • T helper cells recognize MHC Class II through their TCR • MHC Class II present exogenous antigen Differentiate into effector T cells by the secretion of cytokines http://www.ncbi.nlm.nih.gov/books/NBK26827/figure/A4531/?report=objectonly

5. https://www.caymanchem.com/app/template/Article.vm/article/2177https://www.caymanchem.com/app/template/Article.vm/article/2177

6. Effector t helper cells http://www.ncbi.nlm.nih.gov/pubmed/19362058

7. Overview • Background Information on Helper T cells • What is known about Th cells in IBD? • What is still unknown about Th cells in IBD? • Paper 1 • Paper 2 • Future studies/specific aims

8. Classically, Crohn’s Disease was thought to be a Th1 driven disease, while ulcerative colitis was thought to be a Th2 driven disease. Th17 cells have gained attention in more recent years as playing a role in IBD pathogenesis http://www.ncbi.nlm.nih.gov/pubmed/19362058

9. There is an apparent increase in IL-17 production in patients with IBD (Fujino, et al.), meaning there must be an increase in Th17 cells • IL-23 has been shown to promote the survival of Th17 cells http://onlinelibrary.wiley.com/doi/10.1002/ibd.20894/pdf

10. IL-23 is a cytokine made up of two subunits • P40 subunit shared with IL-12 • Unique p19 subunit • In IBD, especially in Crohn’s Disease, the role of IL-12 has been focused on • IL-12 promotes the survival of Th1 cells • Since its discovery, many studies of IBD have begun to focus more on IL-23 rather than IL-12 • Therapies have begun to emerge targeting IL-23 for treatment of IBD • I chose to focus on IL-23 and its role in IBD

11. Overview • Background Information on Helper T cells • What is known about Th cells in IBD? • What is still unknown about Th cells in IBD? • Paper 1 • Paper 2 • Future studies/specific aims

12. Questions regarding my topic… • Although IL-23 is clearly important in the pathogenesis of IBD, what exact role does it play? • Does is also affect Th1 cells along with Th17 cells? • With therapeutics to IL-23, how can they be monitored to see if the treatment is making a difference?

13. Overview • Background Information on Helper T cells • What is known about Th cells in IBD? • What is still unknown about Th cells in IBD? • Paper 1 • Paper 2 • Future studies/specific aims

14. Paper 1 • “IL-23 differentially regulates the Th1/Th17 balance in ulcerative colitis and Crohn’s Disease” by Kobayashi, et. al. • Goal: Examine the role of IL-23 in IBD in relation to the balance of Th1 and Th17 cells.

15. Lamina Propria • Found beneath the epithelial cell layer • Thin layer of loose connective tissue • Part of the mucosa • Rich in immune cells http://www.nature.com/nri/journal/v3/n4/fig_tab/nri1057_F1.html

16. Figure 1. Physiological distribution of IL17; highly expressed in LP Conclusion: IL23/IL17 are more prominent and play a more important role in the intestinal mucosal immunity

17. Figure 2. IL17 upregulation in IBD patients, especially UC Mucosal tissue specimens Isolated LP CD4+ T cells

18. Figure 2 (continued). Conclusions: Levels of IL17 and other Th17 related cytokines (IL21 and IL22) tend to be more enhanced in UC than in CD

19. Figure 3. Investigation of IL23R, a marker of Th17 cells, on LP CD4+ Tc Conclusions: Potential Th17 cells exist similarly in both UC and CD patients.

20. Question: Why is IL17 increased in UC intestinal mucosa compared to CD? Figure 4. Expression of IL12 family members in whole mucosa of IBD patients IL23p19 unique to IL23 IL12p35 unique to IL12 IL12p40 shared between IL12 and IL23

21. Figure 5. IL17 production by LP CD4+ Tc after IL23 stimulation Ulcerative Colitis Crohn’s Disease

22. Figure 5 (continued). Conclusions: Th1 cytokines can affect IL17 production, possibly explaining why IL17 levels were higher in UC than in CD

23. Conclusions from paper 1 • Expression levels of IL17 (and other Th17 related cytokines) tend to be higher in IBD patients with UC rather than CD • Potential Th17 cells exist similarly in both UC and CD patients, as shown by the IL23R • There is a positive correlation between IL23 expression and IL17 in UC; there is also a positive correlation between IL23 expression and IFNγ in CD • Exogenous Th1 cytokines (IFNγ and IL12) decrease the production of IL17 Therefore, while CD patients may have an upregulation of Th17 cells as well as UC patients, the characteristic Th1 cytokines may disturb the IL17 production. IL23 may enhance production of distinct cytokines between UC and CD, contributing to the Th1/Th17 balance in IBD.

24. Overview • Background Information on Helper T cells • What is known about Th cells in IBD? • What is still unknown about Th cells in IBD? • Paper 1 • Paper 2 • Future studies/specific aims

25. Paper 2 • Since IL23 may be important in upregulating specific IBD related cytokine production, anti-IL23 therapies have been implemented • “Biomarkers of Therapeutic Response in the IL-23 Pathway in Inflammatory Bowel Disease” by Cayatte, et. al. • Goal: Evaluate the effects of mAbs to IL23R, and to identify biomarkers for anti-IL23 therapy in Crohn’s Disease

26. Mouse models • Anti-CD40 IBD Mouse model, T cell independent; direct activation of inflammatory dendritic cells and macrophages with a CD40 agonist that mimics activated T cells • C57BL/6 Rag2 -/- • 1 day before induction, mice were injected subcutaneously with anti-IL23R mAbs or IgG control antibody • At day 0, mice were administered intravenously with 50 ug of anti-CD40 to induce IBD • C3H/SCID mice, T cell dependent mouse model of IBD • Cecal bacterial antigen (CBA) activated CBA-specific T cells • 1 x 106 CBA specific T cells were injected into mice. • Adpotive transfer of CBA-specific Adoptive transfer of anti-CD3 Bir14 CD4+ T cells activated T cells

27. Figure 1. Candidate biomarker gene expression/protein concentration in mouse colon tissue or feces

28. Figure 1 (continued). Anti-CD40 induced IL22, IL17, and TNF alpha  treatment with mAbs to IL23R blocked these increases. Shotgun proteomics analysis identified 57 proteins modulated in disease; 7 were chosen for validation. Selection criteria: up or down regulation in disease, control levels upon IL23R mAb treatment, known association with IBD or IL23, availability of reagents.

29. Figure 1 (continued). Conclusions: IL23R therapies can return IBD associated proteins to near control levels

30. Figure 2. Expression of candidate biomarkers in Tc-dependent mouse model • Preventative Treatment: mAbs given on same day as cell transfer and weekly thereafter

31. Figure 2 (continued). • Therapeutic Treatment: mAbs given 4 weeks after disease onset and continued for 4 weeks

32. Figure 3. Regulation of biomarkers by IL17A, IL22, and TNF-alpha in HT-29 cells • Human colonic epithelial cells responsive to IL17 and IL22 stimulation • Absence of IL23R Conclusion: S100A8/9, PAP, LCN2, DMBT1, and CCL20 are regulated by IL17A, IL22, and TNF-alpha in human colonic epithelial cells

33. Figure 4. Expression of candidate biomarkers in patients with Crohn’s Disease

34. Figure 4 (continued).

35. Paper 2 conclusions • Inactivation of the IL-23 receptor blocks IBD • Consistent with previous studies blocking p19 itself • IL23 therapeutic effect shows the potential value in monitoring anti-IL23 therapies • Calprotectin (S100A8/9), PAP (REG3β/γ), and MIF are elevated in CD • Also, LCN2 and CCL20 • PAP and LCN2 are upregulated in feces of CD patients • Measuring these markers in serum and stool can serve as a tool for monitoring anti-IL23 therapies

36. Overview • Background Information on Helper T cells • What is known about Th cells in IBD? • What is still unknown about Th cells in IBD? • Paper 1 • Paper 2 • Future studies/specific aims

37. Future studies • Traditionally, Crohn’s Disease is associated with Th1 cells based on the cytokines made • Recently, IL-23/Th17 pathway has shown to be important for the manifestation of CD as well • According to a study in Therapeutic Advances in Gastroenterology, therapies using a p40 mAb show great success in CD patients SPECIFIC AIM: To determine biomarkers for Crohn’s Disease therapies involving a monoclonal antibody to the p40 subunit of IL-23 and IL-12, representative cytokines of Th17 and Th1 cells, respectively. Therap Adv Gastroenterol. Mar 2010; 3(2): 99–106.

38. references Abraham, C., Cho, J. Interleukin-23/Th17 Pathways and Inflammatory Bowel Disease. Inflamm Bowel Dis 2009; 15: 1090-1100 Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Brock, T. G. (2014). www.caymanchem.com. Retrieved March 11, 2014, from Cayman Chemical: https://www.caymanchem.com/app/template/Article.vm/article/2177 Cayatte, C., et al. Biomarkers of Therapeutic Response in the IL-23 Pathway in Inflammatory Bowel Disease. Clinical and Translational Gastroenterology (2012) 3. Fujino, S., Andoh, A., Bamba, S., Ogawa, A., Hata, K., Araki, Y., Bamba, T., Fujiyama, Y. Increased expression of interleukin 17 in inflammatory bowel disease. Gut. 2003;52:65-70 Germain, Ronald N. T-cell development and the CD4–CD8 lineage decision. Nature Reviews Immunology 2, 309-322 (May 2002) Kobayashi, T., et al. IL23 differentially regulates the Th1/Th17 balance in ulcerative colitis and Crohn's disease. Gut. 2008 Dec;57(12):1682-9 Mowat, A. Anatomical basis of tolerance and immunity to intestinal antigens. Nature Reviews Immunology 3, 331-341 (April 2003) Murphy, K. M. (2011). Janeway'sImmunobiology, 8th Edition. Garland Science. Reenaers, C. Current directions of biologic therapies in inflammatory bowel disease. TherapAdvGastroenterol. Mar 2010; 3(2): 99–106. Zenewicz LA, Antov A, Flavell RA. CD4 T-cell differentiation and inflammatory bowel disease. Trends MolMed. 2009 May;15(5):199-207.