The effect of TLR on transplantation and tolerance of the cornea Mee Kum Kim M.D 1, Jinho Jeong M.D 1, Won Ryang Wee M.D 1, Jin Hak Lee M.D 2 1 Department.

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Presentation transcript:

The effect of TLR on transplantation and tolerance of the cornea Mee Kum Kim M.D 1, Jinho Jeong M.D 1, Won Ryang Wee M.D 1, Jin Hak Lee M.D 2 1 Department of ophthalmology, Seoul National University Hospital, Seoul, Korea 2 Department of ophthalmology, Seoul National University Bundang Hospital, Bundang, Korea The authors have no financial interest in the subject matter of this poster.

Purpose To investigate the role of toll-like receptor (TLR)  On the MHC mismatched allograft rejection in corneal transplantation  On the immune tolerance of the cornea

Materials and Methods Mouse cell culture  Group Group K : keratocytes only : 1x10 4 cells Group D : dendritic cells only (DC2.4*): 1x10 4 cells Group M : macrophages only (Raw264.7*): 1x10 4 cells Group K+D : keratocytes + dendritic cells: 0.5x x10 4 cells Group K+M : keratocytes + macrophages: 0.5x x10 4 cells * Kindly provided by Kim TJ Mouse primary corneal keratocyte DMEM/F12 (1:1) with 10% FBS and 1%PS Mouse DC cell line: DC2.4 * RPMI1640 with 10%FBS and 1%PS Mouse macrophage cell line: Raw264.7 * DMEM with 10%FBS and 1%PS

 TLR stimulation with agonist LPS (TLR4 agonist) Pam3csk4 (TLR2 agonist)  Change in TLR expression and its products of TLR signaling pathway TLR2 and TLR4 expression  RT-PCR  Stimulation for 6 and 24 hours with LPS or Pam3csk4 Phospho-IkappaB-alpha (p-IκB-α)  Western blot  Stimulation for 2, 6, and 24 hours with LPS or Pam3csk4 Cytokine assay  ELISA  Candidate cytokines : IL-6, TNF-α, TGF-ß, IL-10  Stimulation for 24 hours with various concentration  LPS : 1, 10, 100, 1000, ng/ml  Pam3csk4 : 1, 5, 10, 50, 100 ng/ml TLR

 Group (Donor  Recipeint) C57BL/6 (B6)  B6: Autograft (n=10) C3H/HeN (C3H)  B6: MHC mismatched allograft (n=11) C3H  TLR4 knockout (TLR4KO): (n=13)  Transplantation procedure Donor /recipient : 3.0 / 2.0 mm trephine 8 to 10 interrupted 10-0 Nylon sutures  Clinical evaluation Analysis of graft survival using Slit-lamp biomicroscopy  Definition of rejection (end point) : 100% graft edema  Kaplan-Meier survival analysis (Log rank (Mantel-Cox) test)  Immune cell assay Cervical lymph node and spleen 12 days & 4 weeks after transplantation Flow cytometry (FACSCalibur) and FlowJo software  CD11c, CD80/CD86: CD4/8, CD44/69: CD4/8, IFN  TLR4 knock-out mice on a C57BL6 (B6 background) were bred at a SPF animal facility and were kindly provided by Dr. S-Y Seong at the Department of Microbiology and Immunology, Seoul National University College of Medicine Corneal transplantation

Results TLR expression after agonist stimulation P-IκB-α expression after agonist stimulation  TLR4 in M, D, and K  TLR2 in D and K  P-IκB-α in M, D, and K  P-IκB-α in M,and D

Cytokine secretion after agonist stimulation  After LPS stimulation Coculture : K + D Coculture : K + M K : B6 Keratocyte D : Mouse dendritic cell line (DC 2.4) M : Mouse macrophage cell line (Raw ) Markedly increased anti-inflammatory cytokine IL-10 and TGF-β1 in coculture condition

 After Pam3csk4 stimulation Coculture : K + D Coculture : K + M K : B6 Keratocyte D : Mouse dendritic cell line (DC 2.4) M : Mouse macrophage cell line (Raw ) Markedly increased anti-inflammatory cytokine IL-10 and TGF-β1 in coculture condition

Corneal allograft survival and immunologic profiling  Median survival time : C3H  B6 > C3H  TLR4KO  IFN-γ secreting CD8 T cell : C3H  B6 < C3H  TLR4KO CD8 IFN-  in L/N at 12 days P<0.05, Mann Whitney U testP<0.05, Log Rank test

CD86 CD80 CD 11c C3H  B6 C3H  TLR4KO C3H  B6 C3H  TLR4KO Lymph nodes Spleens CD80 CD86 CD 11c At 12 daysAt 4 weeks

C3H  B6 C3H  TLR4KO C3H  B6

Conclusions TLR4 is likely to be involved in immune modulation of MHC mismatched corneal allograft rejection Fibroblast seems to be involved in immune modulation through TLR signaling