JAIMEE DOUCETTE T HELPER CELLS IN IBD

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

T CELLS Nature Reviews Immunology 2, (May 2002)

ACTIVATION OF NAÏVE T HELPER CELLS Differentiate into effector T cells by the secretion of cytokines  T helper cells recognize MHC Class II through their TCR  MHC Class II present exogenous antigen

Main Role in Adaptive Immunity Activate infected macrophages Provide help for B cell Ab production; switching to IgE Increase neutrophil response Suppressor T cells

EFFECTOR T HELPER CELLS

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

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

 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

 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

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

 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? Questions regarding my topic…  With therapeutics to IL-23, how can they be monitored to see if the treatment is making a difference?

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

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.

Lamina Propria  Found beneath the epithelial cell layer  Thin layer of loose connective tissue  Part of the mucosa  Rich in immune cells

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

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

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

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.

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 IL12p40 shared between IL12 and IL23 IL23p19 unique to IL23 IL12p35 unique to IL12

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

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

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.

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

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

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 10 6 CBA specific T cells were injected into mice. Adpotive transfer of CBA-specificAdoptive transfer of anti-CD3 Bir14 CD4+ T cellsactivated T cells

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

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.

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

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

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

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

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

Figure 4 (continued).

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

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

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.

REFERENCES Abraham, C., Cho, J. Interleukin-23/Th17 Pathways and Inflammatory Bowel Disease. Inflamm Bowel Dis 2009; 15: Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; Brock, T. G. (2014). Retrieved March 11, 2014, from Cayman Chemical: 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, (May 2002) Kobayashi, T., et al. IL23 differentially regulates the Th1/Th17 balance in ulcerative colitis and Crohn's disease. Gut Dec;57(12): Mowat, A. Anatomical basis of tolerance and immunity to intestinal antigens. Nature Reviews Immunology 3, (April 2003) Murphy, K. M. (2011). Janeway's Immunobiology, 8th Edition. Garland Science. Reenaers, C. Current directions of biologic therapies in inflammatory bowel disease. Therap Adv Gastroenterol. Mar 2010; 3(2): 99–106. Zenewicz LA, Antov A, Flavell RA. CD4 T-cell differentiation and inflammatory bowel disease. Trends Mol Med May;15(5):