IMMUNOTOOLS: EFFECT OF NOTCH-DEFICIENT MACROPHAGES TO AUTOIMMUNE DISEASE WIPAWEE WONGCHANA

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1 IMMUNOTOOLS: EFFECT OF NOTCH-DEFICIENT MACROPHAGES TO AUTOIMMUNE DISEASE WIPAWEE WONGCHANA

2 WHAT DO YOU SEE?

3 Allergy Ref: Ref: Rheumatoid Arthritis Ref: Ref: Immune system Breast cancer Ref: Smallpox Ref:

4 HOW TO DEFENSE AGAINST FOREIGN BODIES? Ref:

5 CELLS MEDIATED IMMUNE RESPONSE

6 CROSSTALK BETWEEN INNATE AND ADAPTIVE IMMUNE RESPONSE DCs, Macrophage, B cell T cell Distinguish between self (body) and non-self (foreign bodies)

7 WHAT HAPPENS WHEN IMMUNE SYSTEM FAIL? Autoimmune disease Recognize and defense against self

8 RESULTS OF AUTOIMMUNE RESPONSE Inflammation Tissue damage

9 FOCUS Macrophage Regulation of cytokine production Co-stimulatory molecule Autoimmune disease Progressive of disease

10 MACROPHAGES Cell line Primary cells Wipawee W. Unpublished data

11 GENERATION OF MACROPHAGE PRIMARY CELLS C57/BL6 mice Wild type Knockout

12 FLOW CYTOMETRY: BIOTECHNOLOGY ANALYSIS OF CELL MARKER

13 PHENOTYPE OF KNOCKOUT MACROPHAGES 100 kda IFNg/LPS 6hr Ctrl N1KO Notch1 100 kda Cleaved Notch1 b-actin

14 CONCEPT OF THE STUDY Transcription Regulation of transcription mrna Translation Expression protein Secreted cytokine Ref:

15 MOLECULAR MECHANISM OF CYTOKINE PRODUCTION Regulation of transcription Notch signaling pathway NF-kB signaling pathway

16 INTERLEUKIN-6

17 DMSO GSI Ctrl N1KO INTERLEUKIN-12

18 CO-STIMULATORY MOLECULE

19 SUMMARY- MACROPHAGES Generate genetic modified macrophages from hematopoietic stem cells Confirm phenotype of the knockout trait Promise data on the reduction of function cytokine production related T cell polarization Decrease in the co-stimulation molecule (CD80) involved in T cell activation

20 MULTIPLE SCLEROSIS Auto reactive T cells; Th1 and Th17 CD4+ T cell DCs and macrophages

21 HOW TO STUDY MULTIPLE SCLEROSIS? Animal model : Use self-peptide, CFA, Pertussis toxin to induce multiple sclerosis in mouse Experimental Autoimmune Encephalomyelitis

22 STUDY STRATEGY C57BL/6 Activated macrophages 1. transfer 2. Induce EAE disease MOG peptide+cfa Pertussis toxin 3. Score at Day 7 after immunization (daily) 4. Sacrify and collect spleen at Day Re- stim splenocyte for 3 day -ELISA -Intracellular cytokine staining for FACS

23 SCORING The progression and severity of disease will be monitored and scored from 0-5 as follows: 0-no disease 1-limp tail 2-hind limb weakness 3-hind limb paralysis 4-hind and fore limb paralysis 5-morbidity and death

24 PROMISING ON PROGRESSIVE OF DISEASE

25 IFNg AND IL-17 PRODUCTION IN RE- STIM-SPLENOCYTES A B

26 SUMMARY- MACROPHAGES IN AUTOIMMUNE DISEASE Delay onset and progressive of EAE after transferring knockout macrophages Transfer knockout macrophage affect Interleukin-17 expression in study model

27 27 OVERVIEW Notch LPS/TLR4 NF-kB MAPKs Gene transcription IL-12p40 IL-6 CD80

28 TAKE HOME MESSAGES

29 PUBLICATIONS Wongchana, W., et al., Impact of Notch1 Deletion in Macrophages on Proinflammatory Cytokine Production and the Outcome of Experimental Autoimmune Encephalomyelitis. J Immunol, (11): p (IF 4.985) Palaga, T., S. Ratanabunyong, T. Pattarakankul, N. Sangphech, W. Wongchana, Y. Hadae, and P. Kueanjinda Notch signaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophages. Cell Mol Immunol 10: (IF 5.193) Boonyatecha, N., N. Sangphech, W. Wongchana, P. Kueanjinda, and T. Palaga Involvement of Notch signaling pathway in regulating IL-12 expression via c-rel in activated macrophages. Mol Immunol 51: (IF 3.375) Wongchana, W., and T. Palaga Direct regulation of interleukin-6 expression by Notch signaling in macrophages. Cell Mol Immunol 9: (IF 5.193) Suwanjunee, S., W. Wongchana, and T. Palaga Inhibition of gammasecretase affects proliferation of leukemia and hepatoma cell lines through Notch signaling. Anticancer Drugs 19: (IF 2.268)

30 ACKNOWLEDGEMENTS

31 THANK YOU