Acceleration of Allograft Failure by Cytomegalovirus

Introduction

Human cytomegalovirus (HCMV) is a β-herpesvirus that is a common opportunistic pathogen that persists for the life of the human host after primary infection. Generally, HCMV infection of an immunocompetent host results in sub-clinical disease. By contrast, HCMV infection of immunocompromised individuals may cause serious life-threatening disease including pneumonia, gastrointestinal disease, hepatitis, retinitis, and encephalitis. In addition to direct disease, many groups over the past three decades have implicated HCMV in the acceleration of long term disease processes such as increased graft loss due to acute and chronic rejection (CR) in bone marrow and solid organ transplant (SOT) patients. The best evidence that CMV is involved in acute and CR is based on studies with the anti-HCMV drug ganciclovir in humans and animal models that demonstrate a reduction in allograft failure in a wide range of SOT recipients and in bone marrow transplants [1]. Understanding the mechanisms involved in HCMV acceleration of chronic allograft rejection may provide potential targets for therapeutic intervention.

Association of Human Cytomegalovirus Infections with Increased Allograft Failure

Hematopoietic stem cell transplantation (HSCT) is an important therapeutic tool for malignant and nonmalignant disorders. HCMV reactivation is common in this population of patients due to their immunocompromised state and contributes significantly to HSCT morbidity and mortality [2]. HCMV complications may include pneumonia, gastrointestinal disease, hepatitis, retinitis, and encephalitis. HCMV is also associated with graft failure, which manifests as severe pancytopenia or bone marrow hypoplasia, as well as delayed bone marrow function recovery. Other HCMV-associated problems in this patient population include severe acute graft-versus-host disease and even death from myelosuppression. Prevention and treatment of these HCMV-associated complications has been implemented in the form of preemptive or prophylactic antiviral therapy with ganciclovir or one of its analogs [3,4]. The remainder of this review will focus on the role of HCMV in SOT recipients.

SOT is the current standard of care for end-organ failure from heart, lung, kidney, small intestine, pancreas and liver disease. Risk factors for chronic allograft rejection include donor age, recurrent bouts of acute rejection, HLA mismatches, hypertension, hypercholesterolemia, and HCMV infection [5–7]. Animal and human studies have shown that CMV is involved in accelerating both acute and chronic graft failure in all types of SOT. The HCMV serologic statuses of the donor and recipient are the most important factor in determining the development of severe HCMV disease, and consequently the downstream effect on the transplanted organ. A recent study in kidney allotransplant recipients showed that CMV recipient seropositivity prior to transplantation was a significant risk factor for reduced survival [8]. As expected, the lowest risk for CMV complications occurs in D−/R− patients. The risk of CMV infection/reactivation is highest when the allograft donor (D) is serologically positive (+) and the recipient (R) is serologically negative (−). Intermediate risk occurs in the case of D+/R+, or D−/R+ [9–11]. In D+ patients HCMV genetic material has been detected in the vasculature of the transplanted organ demonstrating that the allograft may be an important source of virus [12]. Similarly, in cardiac transplant recipients, HCMV infection either de novo, or by reactivation in the recipient, nearly doubles the 5-year rate of graft failure due to accelerated transplant vascular sclerosis (TVS), the vascular lesion associated with cardiac allograft CR [13]. Additional proof behind HCMV’s affect on graft vasculopathy comes from the finding of a higher incidence of viral DNA detected in the explant vascular intima of those patients with cardiac allograft TVS than in those explants without vasculopathy [12].

The best evidence to support the role of HCMV in the development of CR is the observation that treatment of heart transplant recipients with ganciclovir, a potent inhibitor of viral replication and HCMV disease, delayed the time to development of allograft rejection [14]. In addition, a subsequent study using prophylactic treatment of HCMV seropositive heart transplant recipients with ganciclovir delayed graft rejection in comparison to an untreated HCMV seropositive control group [15]. Subsequently, numerous antiviral protocols, the majority of which use ganciclovir or one of its analogues, were established to improve graft and patient outcomes related to the complications associated with HCMV infection. There is significant controversy in the transplant literature regarding the appropriate timing of therapy, as well as the indication for therapy, i.e. preemptive vs. prophylaxis [1,16–21]. The notion of treating subclinical infection has been studied and it was determined that aggressive prophylaxis post heart transplantation with ganciclovir, valciclovir and CMVIG in D+/R− patients resulted in a reduction in viral levels, acute rejection, and cardiac allograft vascular disease, compared to standard prophylaxis that was used in R+ patients [22]. Furthermore, Sagedal et al. demonstrated that kidney transplant allograft survival was decreased in asymptomatic HCMV infected recipients during the first 100 days post transplantation compared to patients with no HCMV infection suggesting that the presence of HCMV alone, whether asymptomatic or with overt infection, has a negative impact on graft survival [1]. There is a clear advantage to long-term CMV-specific T-cell responses in lung transplant recipients demonstrating that control of CMV infection during transplantation is beneficial [23,24]. Similarly, Tu et al. have reported that early control of subclinical CMV replication after cardiac transplantation by T-cell immunity may reduce allograft vasculopathy and allograft rejection [25–27]. Overall, the use of ganciclovir treatment, or other specific antiviral therapies, has decreased and/or delayed graft loss from CR in addition to decreasing post transplant mortality.

Animal Models of CMV-Accelerated Graft Rejection

While the association of HCMV with human vascular disease is provocative, the most compelling evidence that a herpesvirus infection plays a role in the disease process is exemplified in animal models. In rat heart, kidney, lung and small bowel transplantation models of chronic rejection, it has been shown that acute infection with rat (R)CMV dramatically decreases the mean time to graft failure, and also increases the degree of vasculopathy in the allograft vessels [25,26,28]. Antiviral therapy can prevent or at least reduce the acceleration of rejection in the rat transplant models demonstrating that, similar to the human scenario, active CMV replication is required for these disease processes [29,30]. In the rat heart and small bowel transplant models, using a bone marrow chimera model of tolerance induction, the recipient alloreactive immune response was shown to be required for RCMV-acceleration of graft loss [25]. In a non-tolerant rodent model of transplant CR, chemokine expression was increased in virus-infected recipient grafts compared to uninfected controls [28]. RCMV-infected graft tissues also contained increased numbers of immune cell infiltrates including macrophages, CD4 and CD8 T-cells, and the presence of these cells paralleled the up-regulation of chemokines [28]. Similar findings were seen in a rat CR renal transplant model [27]. Taken together, these findings suggest that the mechanism by which CMV accelerates TVS involves the inflammatory and immune events that accompany the alloreactive response to the donor tissue. Further studies on CMV-accelerated graft loss in rodent models have shown an up-regulation of numerous other proteins associated with inflammation, immune cell trafficking, wound healing and angiogenesis.

Mechanisms of CMV Accelerated TVS and Chronic Rejection

While in vivo animal models have provided solid evidence for the link between CMV and the acceleration of allograft failure, in vitro models allow one to characterize the underlying molecular mechanisms associated with this link. In vivo and in vitro, HCMV productively infects all of the cell types involved in vascular rejection of SOT including smooth muscle cells (SMC), macrophages, endothelial cells, and fibroblasts. The resultant damage to the allograft from CMV infection stems from both direct and indirect effects of the virus. The direct effects are mediated by the result of immune clearance of infected cells and the expression of viral proteins that are pathogenic to the tissues. In cultured cells, HCMV gene expression occurs in three kinetic phases designated as immediate early (IE), early (E), and late (L) [31]. Transcription of IE genes begins shortly following virus entry and does not require de novo protein synthesis. The IE proteins enhance transcription from both host and viral promoters leading to the expression of the E genes, which include genes involved in viral DNA replication. Late viral genes are expressed following viral DNA synthesis and this viral kinetic class of genes is generally involved in viral DNA packaging, assembly, and egress. However, several other classes of viral genes such as G-coupled protein receptors (GPCR) can also be activated during the IE and E phases of replication. Since these genes can be expressed in nonproductively infected cells, they have been implicated as potential mediators in long-term disease processes such as atherosclerosis and TVS. These viral genes are attractive candidates for the acceleration of TVS since latently infected cells can exhibit partial viral expression of these genes without producing virus. Further evidence for this causal link between CMV and CR comes from recent studies in RCMV-infected rat heart allograft recipients. A pattern of virus gene expression was shown to be tissue specific and involve up-regulation of genes involved in host modification of inflammatory, immune and tissue repair processes, all inherent to the CR process, and not those specifically involved in virus replication [32].

One important indirect effect of CMV infection resulting in acceleration of CR is the increase in the host immune response to the allograft and the virus resulting in recruitment of inflammatory cells, and inflammatory effectors such as chemokines and cytokines including IFN-γ, TNF-α, IL-4, IL-18, RANTES, MCP-1, MIP-1α, IL-8, and IP-10 [27,28,31,33–52]. CMV also encodes CC and CXC chemokine homologues that recruit a multitude of host cellular infiltrates [53,54]. In addition, CMV modifies a number of cellular functions that then mediate viral spread, persistence and immune evasion. For instance, CMV also induces cellular factors involved in angiogenesis and wound repair processes including adhesion molecules (ICAM-1, VCAM-1, VAP-1, and E-selectin,) and growth factors and receptors (TGF-β, PDGF-AA, VEGF, and PDGFR) [37,52,55–65]. In addition, increased matrix metalloproteinase (MMP)-2 activity is observed in HCMV infected cells in conjunction with a reduction in matrix gene expression, resulting in a malleability to SMC migration, an alteration in vessel remodeling which promotes a vasculopathy [49,50]. In aggregate, these events result in a tip in the balance to a pro-fibrotic state, vessel neointimal hyperplasia and vessel narrowing, the end product of which is CR and graft failure (Figure 1).

SMC migration from the media into the neointimal space and subsequent proliferation is the hallmark of vascular lesion formation during CR suggesting that pathogen-mediated acceleration of vascular disease likely involves enhanced accumulation of SMC in the lesion. A reduction in apoptosis caused by HCMV infection of SMC could lead to accumulation of these cells at sites of vascular injury. HCMV encodes at least two potent inhibitors of cell death: viral inhibitor of caspase 8 activation (vICA) and viral mitochondrial-localized inhibitor of apoptosis (vMIA) [58]. Furthermore, the HCMV immediate early proteins (IE1) bind and cytoplasmically sequester the tumor suppressor gene p53, which contributes to blocking apoptosis [59]. A mechanism of cellular accumulation occurs through increased SMC proliferation at the site of vascular injury. For example, HCMV infection of endothelial cells increases expression of fibroblast growth factor (EGF) and platelet derived growth factor (PDGF-BB), which are potent stimuli in SMC proliferation [60]. HCMV infection of rat SMC increases the expression of PDGF receptor, which may induce both proliferation and migration of these cells [61]. It has been proposed that HCMV infection enhances SMC migration preferentially towards sites of vascular injury due to expression in infected SMC of the virally-encoded chemokine receptor US28 and the binding of the CC-chemokines RANTES or MCP-1 [37]. The resulting SMC accumulation in the vessel intima leads to neointimal hyperplasia and vessel narrowing. MCMV and RCMV induce SMC migration that is mediated by virally encoded chemokine receptors M33 and R33, respectively [62,65]. Furthermore, infection with a recombinant strain of RCMV containing an insertional deletion of R33 significantly increases the time to CR, and slows the kinetics of TVS in the rat heart transplant model suggesting that this viral chemokine receptor plays an important role in RCMV acceleration of CR [65].

Conclusion

The main obstacles to the success of SOT and HSCT remain CR and graft failure, the development of which CMV plays a significant role. CMV infection modulates the host inflammatory and immune responses promoting CR through the up-regulation of host mediators as well as through the expression of pathogenic viral genes. Future studies aimed at determining the specific up-stream gene targets that would enable prevention or abrogation of the CMV-associated vasculopathy and CR are ongoing and will expand the therapeutic armamentarium for this very important clinical problem.