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The effect of TLR on transplantation and tolerance of the cornea

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

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The effect of TLR on transplantation and tolerance of the cornea

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  1. The effect of TLR on transplantation and tolerance of the cornea Mee Kum Kim M.D1, Jinho Jeong M.D 1, Won Ryang Wee M.D 1, Jin Hak Lee M.D 2 1Department of ophthalmology, Seoul National University Hospital, Seoul, Korea 2Department of ophthalmology, Seoul National University Bundang Hospital, Bundang, Korea The authors have no financial interest in the subject matter of this poster.

  2. 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

  3. Materials and Methods • Mouse cell culture • Group • Group K : keratocytes only : 1x104 cells • Group D : dendritic cells only (DC2.4*) : 1x104 cells • Group M : macrophages only (Raw264.7*) : 1x104 cells • Group K+D : keratocytes + dendritic cells : 0.5x104 + 0.5x104 cells • Group K+M : keratocytes + macrophages : 0.5x104 + 0.5x104 cells 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 *Kindly provided by Kim TJ

  4. 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, 10000 ng/ml • Pam3csk4 : 1, 5, 10, 50, 100 ng/ml TLR

  5. Corneal transplantation • 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

  6. 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

  7. Cytokine secretion after agonist stimulation • After LPS stimulation • Coculture : K + D • Coculture : K + M Markedly increased anti-inflammatory cytokine IL-10 and TGF-β1 in coculture condition K : B6 Keratocyte D : Mouse dendritic cell line (DC 2.4) M : Mouse macrophage cell line (Raw 264.7)

  8. After Pam3csk4 stimulation • Coculture : K + D • Coculture : K + M Markedly increased anti-inflammatory cytokine IL-10 and TGF-β1 in coculture condition K : B6 Keratocyte D : Mouse dendritic cell line (DC 2.4) M : Mouse macrophage cell line (Raw 264.7)

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

  10. At 12 days At 4 weeks 1.90 3.53 1.15 5.49 CD86 Lymph nodes CD 11c CD 11c 2.70 10.64 1.81 2.84 CD80 2.77 1.75 1.57 2.27 CD80 CD 11c Spleens CD 11c 3.48 4.13 3.17 1.95 CD86 C3H  B6 C3H  TLR4KO C3H  B6 C3H  TLR4KO

  11. 0.27 0.31 0.74 0.29 0.60 0.29 0.34 0.52 0.93 C3H  B6 C3H  B6 C3H  TLR4KO

  12. 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

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