Stable expression of the human thrombomodulin transgene in pig endothelial cells is associated with a reduction in the inflammatory response

Abstract

Background: Xenotransplantation is associated with an inflammatory response. The proinflammatory cytokine, TNF-α, downregulates the expression of thrombomodulin (TBM), and induces coagulation dysfunction. Although human (h) TBM-transgenic pigs (p) have been developed to reduce coagulation dysfunction, the effect of TNF-α on the expression of hTBM and its functional activity has not been fully investigated. The aims of this study were to investigate (i) whether the expression of hTBM on pig (p) cells is down-regulated during TNF-α stimulation, and (ii) whether cells from hTBM pigs regulate the inflammatory response.

Methods: TNF-α-producing T, B, and natural killer cells in blood from baboons with pig heart or kidney xenografts were investigated by flow cytometry. TNF-α staining in the grafts was detected by immunohistochemistry. Aortic endothelial cells (AECs) from GTKO/CD46 and GTKO/CD46/hTBM pigs were stimulated by hTNF-α, and the expression of the inflammatory/coagulation regulatory protein, TBM, was investigated.

Results: After pig organ xenotransplantation, there was a trend to increases in TNF-α-producing T and natural killer cells in the blood of baboons. In vitro observations demonstrated that after hTNF-α stimulation, there was a significant reduction in the expression of endogenous pTBM on pAECs, and a significant increase in the expression of inflammatory molecules. Blocking of NF-κB signaling significantly up-regulated pTBM expression, and suppressed the inflammatory response induced by hTNF-α in pAECs. Whereas the expression of pTBM mRNA was significantly reduced by hTNF-α stimulation, hTBM expression on the GTKO/CD46/hTBM pAECs was not affected. Furthermore, after hTNF-α stimulation, there was significant suppression of expression of inflammatory molecules on GTKO/CD46/hTBM pAECs compared to GTKO/CD46 pAECs.

Conclusions: The stable expression of hTBM in pig cells may locally regulate the inflammatory response. This will help suppress the inflammatory response and prevent coagulation dysregulation after xenotransplantation.

Keywords: Inflammation; Pig, genetically-engineered; Thrombomodulin; Xenotransplantation.

Figure 1:. Response of TNF-α in the recipient following xenotransplantation

(A)TNF-α-producing cells in blood were determined by intracellular cytokine staining after pig-to-baboon heart (n=2) or kidney (n=3) xenotransplantation. TNF-α-producing CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells, CD3^-CD8⁺NK cells, and CD3^-CD22⁺B cells were identified by flow cytometry. An increase in TNF-α-producing cells was found in some baboons after organ xenotransplantation. (B) Representative immunohistochemistry (IHC) staining for TNF-α in a naive pig kidney (a) and heart (b) (negative controls). (c) Representative IHC staining for TNF-α in a pig kidney xenograft that underwent hyperacute rejection after transplantation into a baboon (B14115, day 1) (c), a kidney xenograft from a baboon that had developed a systemic infection (B9313, day 136) (d), pig heart grafts in baboons that underwent graft rejection (B18013, day 118) (e) and B17913, day 124) (f) [X20]. Positive staining of TNF-α in the infiltrating mononuclear cells in the xenograft (g) [X40]. (Scale bar = 50 μm [X20])

Figure 2:. Dynamics of surface expression of various molecules on hAECs during TNF-α stimulation

hAECs were stimulated with hTNF-α (10 or 50ng/mL) for 1 to 24 hours. Surface expression of (A) HLA class I, adhesion molecules ICAM-1, CD62E/62P (P-selectin), VCAM-1, and of (B) coagulation-regulatory proteins (TBM, EPCR, CD39, TFPI) on hAECs after hTNF-α stimulation was measured by flow cytometry. (C) Representative data of TBM expression on hAECs after hTNF-α stimulation (by flow cytometry). Isotype control is shown as a dotted line. HLA class I and inflammatory molecules were increased (A), whereas the expression levels of the coagulation-regulatory proteins, TBM and EPCR (but not of TFPI and CD39), were significantly decreased after hTNF-α stimulation (B) (n=4) (*p<0.05, **p<0.01 represent p values for hTNF-α 50ng/mL).

Figure 3:. Increased expression of SLA class I and inflammatory molecules/genes, but downregulation of pTBM expression, in pAECs after stimulation with hTNF-α

GTKO/CD46 pAECs were stimulated with hTNF-α (10 or 50ng/mL) for 1 to 24 hours. (A) Surface expression of SLA class I and E-selectin on pAECs after stimulation with hTNF-α was measured by flow cytometry. mRNA expression of (B) pVCAM-1 and pMCP-1 and (C) pTBM were measured by real-time PCR, and are shown as % expression (control [unstimulated cells]: 100% expression) (n=5) (*p<0.05, **p<0.01, ***p<0.001 represent p values for hTNF-α 50ng/mL). (D) Representative western blot for pTBM protein expression on GTKO/CD46 pAECs after hTNF-α stimulation. The expression levels of both pTBM mRNA and its protein decreased immediately, while the expression levels of SLA class I, and inflammatory molecules/genes were up-regulated after hTNF-α stimulation.

Figure 4:. Suppressive effect of NFκ-B inhibition on pAECs stimulated by hTNF-α

GTKO/CD46 pAECs were incubated without or with hTNF-α (10 ng/mL) for 1 to 24 hours, with/without the NFκ-B inhibitor, parthenolide (2 or 8μM). (A) Surface expression of SLA class I and E-selectin, and (B) mRNA expression of pVCAM-1 and pMCP-1 were measured by flow cytometry and real-time PCR, respectively (n=6) (*p<0.05, **p<0.01, ***p<0.001). The expression of SLA class I and inflammatory molecules/genes in GTKO/CD46 pAECs was significantly suppressed by the NFκ-B inhibitor, in a dose-dependent manner, after hTNF-α stimulation.

Figure 5:. The role of NFκ-B signaling on pTBM expression in pAECs

GTKO/CD46 pAECs were incubated without or with hTNF-α (10 ng/mL) for 1 to 24 hours, with/without the NFκ-B inhibitor, parthenolide (2 or 8μM). (A) Dynamics of mRNA expression of pTBM in pAECs were measured by real-time PCR. The addition of the NFκ-B inhibitor significantly increased the level of pTBM expression in pAECs in a dose-dependent manner (n=3). (B) mRNA expression of pTBM in pAECs was measured 3 hours after stimulation with/without hTNF-α (10ng/mL). The downregulation of expression of pTBM by hTNF-α was significantly reversed by the NFκ-B inhibitor (8μM) (n=3) (*p<0.05, **p<0.01)

Figure 6:. hTBM expression on hTBM-transgenic pAECs

(A) Human TBM constructs of the pTBM promoter and pICAM-2 promoter.With the pTBM promoter, a 12kb fragment containing a porcine TBM promoter driving human TBM was transfected to porcine fetal fibroblasts.With the pICAM-2 promoter, a 6.1kb fragment containing the porcine ICAM-2 enhancer-ICAM-2 promoter driving hTBM cDNA, was used to transfect porcine fetal fibroblasts for subsequent nuclear transfer. (BGHpA = bovine growth hormone polyadenylation signal; SV40 late poly (A) = simian virus 40 late polyadenylation signal.) (B) The expression of hTBM, CD46, CD31 on GTKO/CD46 (control) and GTKO/CD46/hTBM (pTBM promoter) and GTKO/CD46/hTBM (pICAM-2 promoter) transgenic pAECs was measured by flow cytometry. The expression of hTBM on ICAM-2 promoter pAECs was higher than on pTBM promoter pAECs. The results are representative of three independent experiments.

Figure 7:. Stable transgenic-hTBM expression, but downregulation of endogenous pTBM expression, on GTKO/CD46/TBM pAECs during hTNF-α stimulation

Both pTBM promoter and pICAM-2 promoter hTBM-transgenic pAECs were stimulated with hTNF-α. The levels of mRNA of pTBM (A and B [3 hours]) and surface expression of transgenic hTBM (C) on both pAECs were measured by real-time PCR and flow cytometry, respectively. Although the expression of pTBM mRNA was significantly decreased in both pTBM promoter and pICAM-2 promoter hTBM-transgenic pAECs after stimulation with hTNF-α for 6 hours (A), pTBM expression in pICAM-2 promoter hTBM-transgenic pAECs was not significantly decreased until after 3 hours of stimulation with hTNF-α (B) (n=3) (*p<0.05, **p<0.01, ***p<0.001, represent p values for hTNF-α 50ng/mL). (C) Surface expression of transgenic hTBM on both pTBM promoter (n=5) and pICAM-2 promoter (n=2) hTBM-transgenic pAECs was stable.

Figure 8:. Reduction in the expression of E-selectin, pVCAM-1 and pMCP-1 by transgenic expression of hTBM after stimulation with hTNF-α

GTKO/CD46 and GTKO/CD46/hTBM (both pTBM promoter and pICAM-2 promoter) pAECs were stimulated with hTNF-α. (A) Surface expression of E-selectin was measured by flow cytometry. The results are representative of three independent experiments. (B) E-selectin expression (3 hours after hTNF-α stimulation) (measured by flow cytometry) (n=4 to 5), and (C) pVCAM-1 (3 hours after hTNF-α stimulation) and pMCP-1 (12 hours after hTNF-α stimulation) mRNA expression (measured by real-time PCR) (n=3) were significantly reduced when pAECs expressed transgenic hTBM (*p<0.05, **p<0.01)