1. Cytokines and signaling pathways in healthy and disease DENT516 Ge Jin, Ph.D. Email: ge.jin@case.edu Phone: 3683791 Office: DO3570, School of Dental Medicine March 26, 2012

2. Learning Objectives: 1. What are cytokines? 2. understand that NF  B and STAT proteins are transcription factors that modulate immune responses 3. understand that cytokines play important roles in PD

3. Immune cells and the mediators they produce

4. Cytokines  small, secreted, non-antibody proteins  produced by cells involved in both innate & adaptive immunity  mediate and regulate immunity, inflammation, and hematopoiesis

5. Cytokines  Lymphokine: made by activated lymphocytes, especially T H cells, e.g. IL-2  Monokine: made by mononuclear phagocytes, e.g. Mig/CXCL9  Chemokine: chemotactic activity, e.g. IL-8, CXCL12  Interleukin: interaction between leukocytes IL-1, IL-8, IL-10, IL-13……  named by activity: T umor N ecrosis F actor  (TNF  ), C olony S timulation F actor (CSF), T ransforming G rowth F actor  (TGF  )….

6. Properties of Cytokines  produced in response to immune stimuli -- not store pre-formed -- synthesis: DNA  mRNA  protein  secretion -- slow cellular response  can act on the cells that produce them (autocrine action)  can act on nearby cells (paracrine action)  can act on distance cells (endocrine action)

7. Properties of Cytokines  can be produced by many cell types and act on many cell types (pleiotropic)  different cytokines can have similar actions (redundant)

8. Properties of Cytokines  can modulate synthesis of other cytokines - cascades: e.g. TNF   IL-1  IL-6, IL-8… - enhance or suppress production of other cytokines: positive or negative  influence the action of other cytokines - antagonistic - additive - synergistic  short half life, low plasma concentration, bind to receptor with high affinity

9. Immunoglobulin superfamily: IL1R, TLRs… Cytokine Receptors (grouped by structures into families) Toll-IL-1 Receptor domain (TIR) Toll-like receptors (TLRs) leucine-rich repeats immunoglobulin domain IL-1 receptor cell membrane

10. TNFR family: TNF , FasL, CD40L… Cytokine Receptors

11. GM-CSFR  IL-3R IL-5R    GM-CSF IL-2 IL-3 IL-5 IL-15IL-7IL-9   IL-2R  IL-2  IL-15R  IL-2  IL-7RIL-9R class I receptor family: hematopoietin family gp130 CNTFR cell membrane GM-CSF: Granulocyte macrophage colony-stimulating factor IL: interleukin gp130: glycoprotein 130 (m.w. 130 kDa) IL-6IL-11 CNTF LIF/OSM

12. -- share receptors -- defect in a unique cytokine have little effect -- defect in a share component (common receptor) can have profound effects e.g. IL-2R  defect X- linked SCID (Severe Combined Immunodeficiency)

13. class II receptor family: Interferon , , and  Cytokine Receptors IFNARI IFNAR2c IFNAR2b IFN  R  IFN  R  type I IFN receptor type II IFN receptor cell membrane type I IFN: IFN  and IFN , type II: IFN 

14. chemokine receptor family: CCR1-5, CXCR1-4 NH 3 COOH binding of a ligand to the receptor Cytokine Receptors cell membrane chemokine receptor family: CCR1-5, CXCR1-4 G-Protein-Coupled Receptor (GPCR)

15. Cytokine Signaling cytokines membrane receptors phosphorylation cascades gene transcription, Ca++ influx… signal transduction binding activation

16. Cytokine Signaling  NF  B signaling pathways  JAK/STAT signaling pathways  Chemokine/GPCR signaling pathways  Cross-talk between pathways

17. NF  B Signaling NF  B signaling inducers:  cytokines: IL-1, TNF ,…  microbial cell components: Fn cell wall, lipopolysacchrides (LPS),…  virus: retroviruses, double-stranded RNA (dsRNA),… Receptors:  IL-1 receptors  Toll-like receptors (TLRs, innate immunity)  TNF  receptors Nuclear Factor  B (NF  B):  dimeric transcription factors: p65/RelA, c-Rel, RelB, p105/p50, p100/p52  sequestered by inhibitory I  B proteins and retained in cytosol in resting cells

18. NF  B IRAK TRAF6 p p IRAK4 pellino1 IBIB IBIB p p TF IRAK p p p p u u degradation u TAK1 activation u u u u p p IBIB IBIB ubiquitination& degradation u u TRAF6 p TAK1 TAB1 TAB3 TAB2 Ubc13/Uev1A complex III IRAK TRAF6 p p IRAK4 pellino1 TAK1 TAB1 TAB3 TAB2 p complex II NEMO IKK  IKK  p IKK activation p u ubiquitination phosphorylation IL-1 receptor IRAK TollipMyD88 IRAK4 TRAF6 p p complex I IL-1 nucleus cell membrane TNFa IL-6 IL-8 hBD-2 …. innate & adaptive immunity p NF  B activation NF  B p nuclear translocation p TF NF  B p

19. JAK/STAT Signaling Pathway  JAK (Janus Kinases) : a family of tyrosine kinases, JAK1-3, Tyk2  STAT (Signal Transducers and Activators of Transcription) : transcription factors, STAT1-6,…  Cytokines: IFN  / , IFN- , Epo, GM-CSF, IL-6, IL-13…  in resting cells, non-phosphorylated, monomeric STATs reside in cytoplasm.

20. SJ Baker et al, Oncogene (2007) 26, 6724–6737. cdc25a, cyclin D1~3, c-myc, cyclin E, MMP9 p21, caspase 3, 1, 8…

21. Chemokine Signaling  2 major families: the CCL (C-C motif ligand) family and CXCL (C-x-C motif ligand) family  CCL1~27, CXCL1~14  receptors: G-protein coupled 7 transmembrane receptor (GPCR)

22.    GiGi SDF    PI-3KMAPKPLC target genes Adhesion, polarization, chemotaxis CCL12 (SDF) signaling p p GRK  -arrestin recycling degradation SDF

23. pathogens/infection bacteria, virus inflammation chemokine production, recruitment of phagocytes…. increased blood supply, capillary permeability, leukocyte migration killing of pathogens neutralizing toxins, limiting pathogen spread tissue repair phagocytosis of debris, pathogens, dead cells; cells growth resolution elimination of a pathogen, disappearance of leukocytes, full regeneration of tissue function

24. Failure of acute inflammation resolution leads to chronic inflammation and autoimmune disorders Examples: periodontal diseases (PD) chronic inflammation after dental treatment

25. Cytokines and Periodontal Diseases (PD) the most common diseases in the world and the leading cause of tooth loss, a chronic infection that slowly attacks and destroys the gums and bone that support the teeth. caused by bacterial (~200 species) infection followed by a persistent immune response resulted in tissue damage

26. bacteria A. actinomycetemcomitans, P. gingivalis, B. forsythus, T. denticola, T. socranskii, P. intermedia cell wall components (lipopolysaccharides, LPS) exotoxinshydrolytic enzymes inhibit antimicrobial peptides (lysozymes) activity toxic to phagocytes (macrophages) tissue damage/bone resorption stimulate cytokine production of immune cells/chronic inflammation

27. Bacteria LPS, exotoxin, etc. binding to Toll-like receptors (TLRs) activation of the NFkB signaling pathway in immune cells and epithelial cells TNF , IL-1, IL-6, IL-8, IFN  / , chemokines… perodontitis (autoimmune disorder) neutrophils macrophages dendritic cells (DCs) T cells B cells phagocytosis H2O2, NO AMP

28. Tumor Necrosis Factor  (TNF  )  produced by activated macrophages and T cells  most important mediator of acute inflammation in response to microbes, such as LPS  produced by the LPS-membrane bound Toll-like receptors (TLRs)  mediates recruitment of neutrophils and microphages to site of inflammation  stimulates endothelial cells and macrophages to produce chemokines  a potent pyrogen causing fever by direct action or via IL-1  promotes production of acute phase proteins, such as CRP  roles in rheumatoid arthritis, psoriasis, tuberculosis, …

29. TNF  and PD high in diseased periodontal tissues influences leukocyte migration corrected with RANKL and matrix metalloproteinase (MM) production stimulates IL-1, IL-8, IL-6 proinflammatory cytokine production central role in inflammatory reaction, alveolar bone resorption, and the loss of connective tissue attachment

30. Interleukin 1 (IL-1)  Produced by activated macrophages, stimulated lymphocytes, keratinocytes, fibroblasts  Activates the NF  B signaling pathway (similar effects to TNF)  Helps activate T cells  Can be induced by inflammation, injury, and infection

31. Summary (PD and cytokines) TNF  and IL-1 play critical roles in amplifying the inflammatory response, leading to production of lytic enzymes and proinflammatory cytokines in PDs. TNF  and IL-1 antagonists block the progression of the inflammatory cell infiltrate and prevent the periodontal lesions

32. Cytokines properties, categories, signaling, function What are cytokine? Interleukines, chemokines, monokines, … cytokine receptors NFkB, JAK/STATA, chemokine signaling pathways role of cytokines in immunomodulation cytokines and oral diseases (PD)