CMV infection in organ transplant patients

In solid organ transplant recipients, CMV is considered to cause both direct and indirect effects. The direct effects reflect the destruction of virus-infected cells by a lytic infection or by the immune system. This scenario is exemplified by prolonged fever and leukopenia (CMV syndrome) and organ invasive disease such as hepatitis, gastrointestinal disease, pneumonitis, pancreatitis, carditis, and retinitis. Since CMV seropositivity and asymptomatic viremia also increase the risk for long-term complications in transplant patients, high viral titers may not be necessary to increase the risk of indirect effects of CMV.

Organ transplant patients who develop CMV infections in the post transplant period are at increased risk for a number of long-term complications after transplantation. These include acute and chronic rejection in the graft, bronchiolitis obliterans, vanishing bile duct syndrome, transplant vascular sclerosis, increased risk of bacterial and fungal infections, cardiovascular disease and myocardial infarction, post-transplant diabetes, and malignancies. Prophylaxis against CMV lowers the incidence of some of these complications, suggesting that the long-term effects are mediated by viral replication. As the virus has been difficult to detect in affected organs by conventional methods, these complications are considered to be indirect effects of CMV. To detect low-grade active CMV infection in transplanted organs, we developed a high-sensitivity immunohistochemistry technique. Using this technique, we found CMV in a majority of kidney grafts with chronic rejection. Thus, active viral replication in these grafts might adversely affect their function.

CMV is reactivated by inflammation

During latency, the virus appears to be silent and causes no clinical symptoms as long as it is kept in balance with the host immune system. The viral DNA in latently infected monocytes may remain in an extrachromosomal circular form, and few, if any, viral proteins are produced during the latency phase. Since immunosuppressed patients develop CMV infection and disease, it was early hypothesized that immunosuppression would lead to reactivation of latent virus. However, we showed that allogeneically stimulated T cells produce inflammatory cyotokines, such as tumor necrosis factor alpha and interferon gamma (that cause monocytes to differentiate into inflammatory macrophages, which can reactivate latent CMV (Cell 1997, J. Clin Invest 1997). Owing to MHC mismatch, virus-specific cytotoxic T cells have a decreased ability to clear CMV infection from the graft. This is most likely why CMV infection is always far more problematic in transplanted organs than in native organs. This scenario would likely take place during episodes of acute rejection or acute graft-versus-host disease in recipients of organ and stem-cell transplants. Immunosuppression will also impair the ability to control the reactivated virus, placing patients at high risk for clinical CMV disease.

 

CMV infection and acute rejection in solid organ transplant recipients

CMV infection has long been associated with acute rejection episodes in organ transplant recipients. Initially it was observed that clinical disease often was associated with acute rejection episodes, but it has been difficult to define whether CMV or rejection represents the egg or the chicken in this process. Clearly, a bidirectional interaction exists between the virus and acute rejection, as exemplified by CMV’s ability to induce and be dependent on inflammation. In more recent studies, detection of CMV DNA by sensitive methods Helanterä et al. correlated with increased creatinine levels in kidney transplant recipients. Thus, CMV may in fact be actively replicating in the kidney, although the virus could be discovered only by sensitive in situ hybridization and PCR techniques.

Recently, we adapted methods for sensitive detection of active CMV infection in tumors for use in tissues from organ transplant recipients. Using high sensitivity immunohistochemistry staining (HSIS) protocols, we can now detect a low-grade active CMV replication in a very high proportion of solid organ transplants diagnosed with acute and chronic rejection (unpublished data). In heart transplant patients, the virus appeared to be detected earlier than rejection, and the viral levels appeared to correlate with the rejection grade over time. Thus, with these new techniques it may be possible to identify patients who are at high risk of rejection and should be offered antiviral treatment. Indeed, aggressive prophylaxis against CMV in heart transplant patients reduces rejection, whereas pre-emptive treatment against CMV prevents CMV disease but not acute rejection.

CMV infection and chronic rejection

Since the first report that CMV infection increases the risk of transplant coronary artery disease in 1989, strong evidence has emerged that the virus contributes to chronic deterioration of transplanted organs. CMV is clearly associated with chronic rejection (i.e., transplant vasculopathy, chronic allograft nephropathy, bronchiolitis obliterans, vanishing bile duct syndrome, and fibrosis) in transplanted organs. In animal models, CMV infection consistently induces earlier and more advanced lesions, which suggests that the virus is a strong co-factor in the development of these diseases. The effect of CMV is linked to rejection, and prophylaxis against CMV and optimal rejection treatment prevents CMV-induced graft damage.

Recently, using the HSIS technique, we examined kidney biopsies from 28 transplanted patients with chronic rejection. Active CMV microinfection was detected in all biopsies, but not in kidneys from CMV-seronegative subjects (unpublished data). Similar findings were obtained in preliminary studies of recipients of lung and liver transplants. Importantly, active viral infection is often detected in areas of the graft with clear disease pathology involving vascular changes and fibrosis. Since CMV has been strongly linked to chronic rejection, it is important to determine whether a low-grade CMV infection in the graft is a cause of rejection or simply an epiphenomenon of inflammation. Solid evidence from transplant patients favors the hypothesis that the virus causes or is a co-factor in many long-term complications, suggesting that it contributes to disease pathogenesis at the molecular level. However, it is extremely difficult to link a particular virus to a disease that may not produce clinical symptoms for several years. Therefore, in vitro models of isolated cellular phenomena as well as animal models have been useful to further investigate the specific roles of the virus in disease development.

Can CMV affect the development of fibrosis?

Fibrosis is a general feature of inflammation, and the end stage of chronic rejaction. No clear specific mechanisms can explain why fibrosis develops as consequence of inflammation. In a rat transplant model, CMV infection increased the expression of both type I and type III collagens and the accumulation of myofibroblasts, which correlated with enhanced interstitial fibrosis in chronic renal allograft rejection. Human CMV upregulates MMP-2 activity in smooth muscle cells. We found that CMV infection of macrophages specially shuts off MMP-9 expression but upregulates TIMP-1 (J. Virol. 2009). These observations suggest that perhaps CMV infection directly affects the composition of the extracellular matrix by influencing the synthesis and degradation of extracellular matrix components. This process may be affected differently depending on which cell types are locally infected, and may lead to instability of an atherosclerotic plaque or to increased fibrosis. TGF-b is induced by CMV infection and results in increased collagen synthesis and fibrosis. Consistent with this possibility, we recently detected CMV-infected cells in areas of fibrosis in transplanted heart explants with severe fibrosis.

Opportunistic infections

Clinical evidence suggests that CMV infection increases the risk of concomitant infections in transplant recipients. In meta-analyses, prophylaxis against CMV reduced HSV and VZV infections by 73%, but also reduced bacterial infections by 35% and protozoal infections by 69%. Several lines of evidence suggest that CMV is immunosuppressive, which is probably why CMV-infected patients are at higher risk for other infections. CMV controls specific immune functions through the action of viral proteins. For example, the CMV proteins US2, US3, US6, and US11 can in different ways inhibit the presentation of HLA class I molecules on infected cells; this would lead to an inability to present microbial peptides to cytotoxic T cells. At least three viral mechanisms control the expression of HLA class II molecules, which would severely impair activation of cytotoxic T cells, B cells and NK cells by a lack of T helper cell produced cytokines. NK cells, an important part of the innate immune response, are the first line of defense against viral infections and are also targeted by CMV. Several CMV proteins inhibit the activation of NK cells, and we demonstrated that UL16 mediates protection against cytolytic proteins released from cytotoxic T cells and NK cells (J. Virol 2003). As a result, infected cells are protected against killin..

Antigen-presenting cells such as monocytes/macrophages and dendritic cells are also directly influenced by the virus. We demonstrated that CMV inhibits the differentiation of monocytes into both macrophages and myeloid dendritic cells and impairs their ability to take up and present peptides to T cells (J. Virol 2004, J. Immunol 2004) and to migrate in response to inflammatory chemokines (J Leuk. Biol. 2006). Maturation of immature into mature dendritic cells represents a central phenomenon in dendritic cell biology, and is required before these cells can migrate to lymph nodes. We found that CMV affects the ability of dendritic cells to mature, and when mature myeloid dendritic cells become infected, they rapidly release RANTES, macrophage inflammatory proteins 1a and 1b, which bind to their receptors and internalize the receptor complex; as a result, the cells lack CCR1 and CCR5 on the cell surface and their ability to migrate is severely impaired (J Leuk. Biol. 2006). In heart transplant patients, we found that CMV infection leads to a reduction or a complete loss of dendritic cells in peripheral blood and a severely impaired T-cell response (Transplantation 2006). Similar findings were reported in immunocompetent individuals with CMV mononucleosis, suggesting that the virus has immunomodulatory effects in the absence of immunosuppressive drugs (J Leuk. Biol. 2005). Although this scenario should improve the immune response to bacterial infections, clinical observations demonstrate increased bacterial infections in CMV-infected transplant patients. We found that neutophils are activated by CMV, and apoptosis is delayed (Microbes Infect 2006). However their specific function against bacteria is still unknown. Thus, undefined specific mechanisms may explain why CMV-infected patients are more susceptible to bacterial infections.

In this research project, we focus on understanding the molecular mechanisms of CMVs indirect effects in transplant recipients. Mainly, we study immune dysfunctions predisposing for opportunistic infections at the same time as the virus induces inflammation and is involved in acute and chronic rejection.