Systemic inflammation in xenograft recipients precedes activation of coagulation

Abstract

Background: Dysregulation of coagulation is considered a major barrier against successful pig organ xenotransplantation in non-human primates. Inflammation is known to promote activation of coagulation. The role of pro-inflammatory factors as well as the relationship between inflammation and activation of coagulation in xenograft recipients is poorly understood.

Methods: Baboons received kidney (n=3), heart (n=4), or artery patch (n=8) xenografts from α1,3-galactosyltransferase gene-knockout (GTKO) pigs or GTKO pigs additionally transgenic for human complement-regulatory protein CD46 (GTKO/CD46). Immunosuppression (IS) was based on either CTLA4Ig or anti-CD154 costimulation blockade. Three artery patch recipients did not receive IS. Pro-inflammatory cytokines, chemokines, and coagulation parameters were evaluated in the circulation after transplantation. In artery patch recipients, monocytes and dendritic cells (DC) were monitored in peripheral blood. Expression of tissue factor (TF) and CD40 on monocytes and DC were assessed by flow cytometry. C-reactive protein (C-RP) levels in the blood and C-RP deposition in xenografts as well as native organs were evaluated. Baboon and pig C-RP mRNA in heart and kidney xenografts were evaluated.

Results: In heart and kidney xenograft recipients, the levels of INFγ, TNF-α, IL-12, and IL-8 were not significantly higher after transplantation. However, MCP-1 and IL-6 levels were significantly higher after transplantation, particularly in kidney recipients. Elevated C-RP levels preceded activation of coagulation in heart and kidney recipients, where high levels of C-RP were maintained until the time of euthanasia in both heart and kidney recipients. In artery patch recipients, INFγ, TNF-α, IL-12, IL-8, and MCP-1 were elevated with no IS, while IL-6 was not. With IS, INFγ, TNF-α, IL-12, IL-8, and MCP-1 were reduced, but IL-6 was elevated. Elevated IL-6 levels were observed as early as 2 weeks in artery patch recipients. While IS was associated with reduced thrombin activation, fibrinogen and C-RP levels were increased when IS was given. There was a significant positive correlation between C-RP, IL-6, and fibrinogen levels. Additionally, absolute numbers of monocytes were significantly increased when IS was given, but not without IS. This was associated with increased CD40 and TF expression on CD14+ monocytes and lineage(neg) CD11c+ DC, with increased differentiation of the pro-inflammatory CD14+ CD11c+ monocyte population. At the time of euthanasia, C-RP deposition in kidney and heart xenografts, C-RP positive cells in artery patch xenograft and native lungs were detected. Finally, high levels of both pig and baboon C-RP mRNA were detected in heart and kidney xenografts.

Conclusions: Inflammatory responses precede activation of coagulation after organ xenotransplantation. Early upregulation of C-RP and IL-6 levels may amplify activation of coagulation through upregulation of TF on innate immune cells. Prevention of systemic inflammation in xenograft recipients (SIXR) may be required to prevent dysregulation of coagulation and avoid excessive IS after xenotransplantation.

Keywords: C-reactive protein; coagulation; dendritic cells; inflammation; interleukin-6; monocytes; pig; primate; xenotransplantation.