Stable expression of the human thrombomodulin transgene in pig endothelial cells is associated with a reduction in the inflammatory response
Introduction
With the development of genetically-engineered pigs and adequate immunosuppressive therapy, there has been a considerable prolongation of pig xenograft survival in nonhuman primates, which has extended to months or even years [1], [2], [3]. However, coagulation dysregulation and intravascular thrombosis remain obstacles to long‐term xenograft survival [4], [5]. Even in the presence of immunosuppressive therapy, early and/or late xenograft failure is related to the innate immune response, which includes complement cascade activation, inflammation, and dysregulation of coagulation.
Ezzelarab et al. documented a systemic inflammatory response in recipient nonhuman primates after pig organ xenotransplantation [6], [7], [8]. Importantly, this response precedes coagulation dysfunction. The innate immune system is activated at the time of organ transplantation by the release of endogenous inflammatory factors. IL-6 and TNF-α synergistically impair allograft acceptance [9]. Infection abrogates [10], [11], and inflammation impairs, the development of tolerance, and induces acute and chronic rejection [12]. The innate immune system plays a role in achieving a balance between immunity and tolerance [13]. Human inflammatory cytokines, e.g., TNF-α, IL-6, IL-17, and IL-1β, activate pig endothelial cells [14], and promote inflammation and coagulation in vitro [14], [15]. Furthermore, Gao et al, demonstrated that human antibody-mediated cytotoxicity to porcine endothelial cells was increased by human TNF-α [16]. These results indicated that pro-inflammatory cytokines have important pathological effects in the xenografts. We suggest that regulation of inflammation is an important target if long-term xenograft survival is to be achieved [17].
Thrombomodulin (TBM), which plays a role in maintaining an anticoagulant state, also has an anti-inflammatory effect through both coagulation-dependent and coagulation-independent pathways [18]. When cells are exposed to a variety of pathological conditions, e.g., (i) inflammation [e.g., by TNF-α stimulation] [19], (ii) ischemia–reperfusion injury [which triggers sterile inflammation], and (iii) activation of vascular endothelial cells by antibody binding to xenoantigens [4], [20], there is a downregulation of TBM expression, resulting in a loss of TBM activity.
There is molecular incompatibility between human (h) and pig (p) TBM. Although pTBM binds to human thrombin, it only weakly generates activated protein C, which is a strong anticoagulant [21]. Therefore, genetically-engineered pigs that express hTBM, that generate more activated protein C, have been introduced into xenotransplantation [1], [3], [22]. Although pigs transgenic for hTBM help prevent coagulation dysfunction [1], [22], [23], [24], [25], whether (i) expression of hTBM is down-regulated during inflammation (e.g., by hTNF-α), and (ii) hTBM-transgenic pig cells can regulate the inflammatory response, remain unknown.
The aims of the present study were to investigate the expression levels of endogenous pTBM and hTBM on hTBM-transgenic pig cells before and after stimulation with TNF-α, and to determine their anti-inflammatory effects. To our knowledge, this is the first study to demonstrate that, whereas expression of pTBM is downregulated during inflammation, the expression of hTBM remains stable. Transgenic expression of hTBM would therefore appear to be important in maintaining an anticoagulant state and in inhibiting inflammation after pig organ xenotransplantation.
Section snippets
Materials and methods
Human volunteers (who donated blood) gave informed consent per the guidelines of the Institutional Review Board of the University of Pittsburgh (IRB#0608179) and the University of Alabama at Birmingham (UAB) (#300001924). All animal care was in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Research Council (8th edition, revised 2011), and was conducted in an AAALAC-accredited facility. Protocols were approved by the University of Pittsburgh
Frequency of TNF-α-producing cells is increased in immunosuppressed xenograft recipients
To investigate whether inflammatory cytokine-producing cells, especially those that produce TNF-α, which is a well-known proinflammatory cytokine, increase in the recipient after pig-to-nonhuman primate xenotransplantation, intracellular cytokine staining of blood was performed following pig organ (heart or kidney) xenotransplantation in recipient baboons [2], [8], [27] (Supplementary Materials and Methods). Recipient baboons included pig three kidney and two heart recipients (Supplementary
Discussion
TBM (CD141) is an integral membrane protein expressed on endothelial cells that controls thrombosis by converting thrombin from a procoagulant to an anticoagulant, and promoting protein C activation [4], [28]. It consists of five domains [29], [30] – the N‐terminal lectin-like domain (D1), six epidermal growth factor (EGF)‐like domains (D2), an O-glycosylation site-rich domain (D3), a transmembrane domain (D4), and a cytoplasmic tail domain (domain 5).
Many studies have demonstrated that TBM
Authorship
HH participated in research design, the performance of the research, and writing the paper. HH is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. HI and HN participated in the performance of the research, and in review of the manuscript. KK, WE, and CP built the hTBM vector for transgenic pigs, and reviewed the manuscript. DA provided the genetically-engineered
Funding information
Work on xenotransplantation at the University of Alabama at Birmingham is supported in part by NIH NIAID U19 grant AI090959, and in part by a grant to UAB from United Therapeutics, Silver Spring, MD, USA. Anti-CD40 2C10R4 used in these studies was provided by the NIH NHP Reagent Resource funded by NIH grants AI126683 and OD010976. Some of the baboons used in these studies were from the Michale E. Keeling Center. MD Anderson Cancer Center, Bastrop, Tx, which is supported in part by a NIH grant
Declaration of Competing Interest
Kasinath Kuravi, Will Eyestone, Carol Phelps and David Ayares are employees of Revivicor, Blacksburg, VA. The other authors declare no conflicts of interest.
Acknowledgments
We thank Dr. Dezhi Wang (Pathology Core Research Laboratory/Department of Pathology at the University of Alabama at Birmingham) for her dedication to the immunohistochemistry studies.
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