1. 2012/03/29 Reporter: 牙研所臨床組三年級 郭博仁 Instructor: 傅鍔 江正陽 黃仁勇 教官 Impact factor :2.726

2. Introduction  Porphyromonas gingivalis is strongly associated with the chronic form of periodontitis  P. gingivalis cysteine proteinases (Arg- and Lys- gingipains) play a significant role in host tissue destruction and invasion  Gingipains of P. gingivalis have been shown to mediate the shedding of epithelial cell-surface proteins (ex. syndecan-1, complement regulatory protein) through proteolytic cleavage

3.  Overproduction of different inflammatory mediators may lead to chronic, persistent inflammation and tissue destruction, which is mainly mediated by matrix metalloproteinases (MMPs)  EMMPRIN expression induces tumor progression and invasion by triggering the production of MMPs by fibroblasts and endothelial cells  Both soluble and membrane-anchored forms of EMMPRIN interact with fibroblasts resulting in the expression and secretion of large amounts of several MMPs

4.  Recently, a number of reports have pointed to a link between EMMPRIN and periodontitis.  High levels of soluble EMMPRIN in gingival crevicular fluid have been associated with more severe periodontal inflammation  G. Emingil,et al J. Periodontol. 77 (2006)  Higher levels of EMMPRIN protein and mRNA are found in inflamed gingiva than in healthy gingiva  W. Dong et al J. Periodontal Res. 44 (2009) 125e132.  J. Xiang, et al Quintessence Int. 40 (2009) 683e690  Periodontal therapies that result in improved clinical parameters are associated with reduced levels of EMMPRIN gingival crevicular fluid  G. Emingil et al J. Periodontol. 79 (2008) 469e476 4

5. Aim  i) to investigate the effect of P. gingivalis on the release of soluble EMMPRIN and the expression of the EMMPRIN gene by oral epithelial cells  ii) to evaluate the ability of recombinant EMMPRIN to promote inflammation by inducing the secretion of cytokines and MMPs by human gingival fibroblasts

6. Material and methods  P. gingivalis ATCC 33277  KDP112 :Arg-gingipain mutant (rgpA, rgpB)  KDP129 :Lys-gingipain mutant (kgp)  KDP128 :gingipain-nullmutant (rgpA, rgpB and kgp)  Tosyl-L-lysine chloromethylketone (TLCK) : gingipain inhibitor  Human oral epithelial cell line GMSM-K  Human Gingival fibroblast 6

7. Results

8. human oral epithelial cell line GMSM-K P. gingivalis ATCC 33277 :multiplicities of infection (MOI) of 200 1. P. gingivalis stimulated EMMPRIN shedding by epithelial cells.

9. 9 human oral epithelial cell line GMSM-K P. gingivalis ATCC 33277 at MOIs of 50, 100, and 200 for 24 h To determine whether EMMPRIN was released by epithelial cells in a soluble form or by microvesicle shedding, centrifuged at 150000 g for 1 h to pellet the microvesicles *Significant increase (P < 0.05) compared to untreated control cells 2. The inducible effect of P. gingivalis ATCC 33277 on EMMPRIN shedding was dose-dependent. 24h

10. 10 human oral epithelial cell line GMSM-K P. gingivalis ATCC 33277 : wild type finfipain TLCK : gingipain inhibitor KDP112 is an Arg-gingipain mutant (rgpA, rgpB) KDP129 is a Lys-gingipain mutant (kgp) KDP128 is a gingipain-nullmutant (rgpA, rgpB and kgp) 24h 3. P. gingivalis can mediate the shedding of EMMPRIN from the surface of epithelial cells throughthe activity of gingipains

11. 11 Untreated epithelial cells P. gingivalis ATCC 33277 treated P. Gingivalis KDP128 treated Non-specific staining control 4. The shedding of EMMPRIN from the surface of epithelial cells following the treatment with P. gingivalis was confirmed 24h human oral epithelial cell line GMSM-K

12. 12 human oral epithelial cell line GMSM-K P. gingivalis ATCC 33277 :multiplicities of infection (MOI) of 200 5. P. gingivalis ATCC 33277, in addition to have an effect on cell-surface EMMPRIN, but not modulate EMMPRIN gene expression

13. 13 6. secretion of IL-6 increased dose-dependently when fibroblasts were seeded into wells coated with recombinant EMMPRIN Primary human gingival fibroblasts

14. Discussion  EMMPRIN was naturally shed from the surface of uninfected epithelial cells in a time-dependent manner  P. gingivalis, involved in the chronic form of periodontitis, can mediate the shedding of EMMPRIN from the cell surface of oral epithelial cells.  MMP-2, and MMP-9 have been reported to be involved in the release of a shorter form of EMMPRIN.  K. Hanata et al Arch. Histol. Cytol. 70 (2007) 267e277.  EMMPRIN inhibition leads to decreased IL-6 cytokine production in vitro and in vivo  N.R. Dean et al Clin. Cancer Res. 15 (2009) 4058e4065

15. 15 The CsA and control groups showed similar expression of all three of these antigens. Neurothelin (CD147): Neurothelin was also very strongly (++++) expressed on basal epithelium cells

16.  P. gingivalis mediates the shedding of epithelial cell membrane-anchored EMMPRIN, which in turn has the potential to induce pro-inflammatory cytokine and MMP secretion by gingival fibroblasts. 16 Summary

17. Marcrophage U937 Fibroblast(HGF) MMP-2 ↑ MMPs ↑ sEMMPRIN MMP-9 ↑ Microenvironment/positive feedback EMMPRIN(CD147)

18. 18

19. Reporter: 牙研所臨床組二年級 郭博仁 Instructor: 傅鍔 江正陽 邱賢忠 教官

20. Introduction  Atherosclerosis (AS) is recognized as an inflammation-driven pathology of the vascular wall  Reactive oxygen species (ROS) is a critical driver to the inflammatory cascade.  Oxidized lipoprotein  Mechanical forces (pressure,shear stress)  Growth factors (angiotensin II,PDGF)

21.  Cyclophilin A (CyPA) as a major Secreted Oxidative stress induced Factors (SOXF) in atherosclerosis and several inflammatory diseases, including sepsis and rheumatoid arthritis  CyPA would be deleterious only after binding to extracellular matrix metalloproteinase inducer (EMMPRIN)

22. Aim  Is EMMPRIN (CD147) the signal receptor of CyPA that mediates the pro-inflammatory effect in monocytes and macrophages?

23. Results

24. CyPA was a strong monocyte chemoattractant and induced MMP-9 and IL-6 expression in monocytes (THP-1)

25. CyPA ↑ EMMPRIN(CD147) ERK JUK P38 Nf-kB IL-6 ↑ MMP9 ↑ Migration ↑ Results 1

26. CyPA activated NF-kappaB via the ERK pathway Cytoplasmic Nuclear

27. CyPA activated NF-kappaB via the ERK pathway PD98059 : ERK inhibitor

28. CyPA ↑ EMMPRIN(CD147) ERK JUK P38 Nf-kB ↑ IL-6 ↑ MMP9 ↑ Migration ↑ Results 2a CsA

29. CyPA ↑ EMMPRIN(CD147) ERK ↑ JUK ↑ P38 ↑ Nf-kB ↑ IL-6 ↑ MMP9 ↑ Migration ↑ Results 2b ERK inhibitor

30. EMMPRIN was required for mediating the effect of CyPA EMMPRIN monoclonal antibody (UM8D6, Ancell)

31. EMMPRIN was required for mediating the effect of CyPA

34. CyPA ↑ EMMPRIN(CD147) ERK ↑ JUK ↑ P38 ↑ Nf-kB ↑ IL-6 ↑ MMP9 ↑ Migration ↑ Results 3 EMMPRIN siRNA or mAb Data no shown

35. Summary  CyPA induced migration of monocytes and the expression of MMP-9, IL-6 and TNF-alpha  CyPA activated NF-kappaB by ERK1/2 pathway  Blocking EMMPRIN in monocytes, chemoattractant migration, activation of MAPK/NF-kappaB and cytokines releasing were significantly inhibited  CyPA simulation had no effect on EMMPRIN expression in monocytes  CyPA–EMMPRIN interaction is one of the key pro- inflammatory signaling pathways in monocytes

36. Fibroblast (HGF) Marcrophage (U937) Co-culture LPS MMP activities ↑ Collagen degradation CD147 ↑ CsA Pro-inflammtion cytokine ↑ IL-6, IL-1α,TNF-β Co-culture system ?? 36 CyPA ?

37. 37