1. Introduction
The β-herpesviruses, which include cytomegalovirus (HCMV), human herpesvirus 6A (HHV6A), 6B (HHV6B), and 7 (HHV7), as well as the γ-herpesviruses, Epstein–Barr virus (EBV) and Kaposi sarcoma-associated virus (KSHV), are human viruses known for causing lifelong persistent infections. Particularly, β- and γ-herpesviruses target immune cells for infection, establishing host cell lifelong reservoirs in differentiated lymphoid and myeloid cells or hematopoietic progenitors.
A significant proportion of the global population carries at least three of these herpesviruses, which is a prevalence largely attributed to their biphasic life cycle, encompassing latent and lytic phases. The latent phase is characterized by low to no expression of viral genes, enabling evasion of the immune system and persistence in the host. In contrast, during the lytic phase, there is a heightened expression of viral genes leading to the production of new viral infectious particles [
1]. The transition from the latent to the lytic phase is known as reactivation. While the majority of infected hosts remain asymptomatic, in cases of associated diseases, the reactivation of these herpesviruses is typically observed and measured as detectable viral loads in peripheral blood.
In individuals with compromised immune systems, β- and γ-herpesvirus emerge as a substantial contributor to morbidity and mortality, as exemplified in solid organ transplant recipients undergoing pharmacologic immunosuppression. The intersection of the need for immunosuppression, with consequently herpesvirus reactivation, jeopardizes the success of the transplant, leaving patients vulnerable to clinical complications, such as organ rejection, post-transplant lymphoproliferative syndrome (PTLD) and HCMV-disease [
2,
3,
4,
5,
6]. This delicate balance requires optimal clinical management with a focus on the monitoring of viral loads and the implementation of preemptive strategies to mitigate the impact of the herpesviruses.
In addition to their individual effects, the simultaneous detection of multiple herpesviruses has been reported in transplanted patients with some studies supporting an association between co-detection and worse clinical outcomes [
7]. The tropism for immune cells and reliance on similar mechanisms for reactivation support the notion that β- and γ-herpesviruses mutually influence their biological cycles, collectively impacting the survival of the transplanted organ and the transplanted patient [
7]. Cytokines are likely important mediators of mutual connection, since all β- and γ-herpesviruses have evolved immunomodulatory genes not only to block antagonizing host responses but also to influence the activation, survival, differentiation, and expansion of the immune cells that act as reservoirs for their persistent infections. Furthermore, β- and γ-herpesviruses encode their own set of functional homologs of immune-related genes, including virokines [
8,
9,
10].
A previous analysis of this pediatric cohort revealed associations between specific herpesviruses and rejection. Notably, HCMV and HHV6 alone appeared to be associated with rejection, while EBV contributed to rejection in co-detection events, suggesting interactions between EBV and the β-herpesviruses. In this study, searching for potential channels of herpesvirus interactions, we analyzed the concentrations of 17 cytokines in the plasma of post-transplant patients over a one-year follow-up period. We correlated these cytokine concentrations with the loads of β- and γ-herpesviruses and with graft rejection. This investigation aims to deepen our understanding of the intricate relationship between immunosuppression, herpesvirus infection, and clinical outcomes in solid organ transplant recipients.
4. Discussion
β- and γ-herpesviruses have evolved over hundreds of millions of years in close association with our immune system [
14]. The capacity to alternate between latent and lytic states endows herpesviruses with a remarkable ability to achieve high fitness within hosts. Despite lifelong persistence, they are generally undetectable, indicating a homeostatic state in which latency is most probably the prevalent viral cycle. However, latency can be disturbed by molecules targeting immune cells, including cytokines, interferons, and bacterial and parasite products that stimulate pattern recognition receptors [
15,
16,
17,
18,
19]. To modulate the immune system to lessen its antiviral effects and foster viral latency, approximately 30% of the β- and γ-herpesvirus genomes encode proteins or non-coding transcripts that target immune cells or immune-related processes [
8,
20].
There is evidence suggesting that one herpesvirus can influence the biological cycle of another herpesvirus, although such evidence is limited and has primarily been observed through in vitro experimentation. For instance, HHV6 has been shown to reactivate both EBV and KSHV; HCMV can reactivate KSHV, and HHV7 can reactivate HHV6 [
21,
22,
23,
24]. In addition to our study [
11], multiple other studies support the association of elevated DNAemia of the β- and γ-herpesviruses with post-transplant complications, including EBV and HCMV [
25], as well as different combinations of the β-herpesviruses [
26,
27,
28,
29,
30,
31,
32,
33,
34]. In these studies, the co-detection of more than one herpesvirus is usually associated with a higher risk for an unfavorable clinical outcome. For instance, in a kidney transplant study, the co-detection of EBV and HCMV correlated with graft damage (
p = 0.035, RR = 2.1). The detection of HHV6 and/or HHV7 often precedes HCMV detection and HCMV disease [
28,
34,
35,
36], potentially implying cross-reactivation mechanisms. In a solid organ transplant study, HCMV reduced the number of EBV-directed NK cells, increasing the risk of EBV-associated PTLD [
37]. However, some studies have not found significant associations between herpesvirus co-detection and enhanced risk for rejection [
38,
39].
The interactions between herpesviruses appear to be significantly mediated by cytokines. For instance, it has been reported that IL-4, induced by parasites and HSV1, can reactivate γ-herpesviruses [
40,
41]. In vitro studies have documented that myeloid cells produce IL-1β and TNF-α in response to HHV6 infection [
42], and TNF-α induces HCMV reactivation [
43]. In this study, we observed an association between the cytokines IL-4, IL-8, and IL-10 with graft rejection with detection of the β-herpesviruses correlating with the upregulation of these cytokines. Notably, HCMV and HHV6 correlated with high cytokine levels whether detected individually or in conjunction with other herpesviruses. In contrast, elevated cytokine levels associated with EBV and HHV7 were predominantly observed when these viruses were co-detected with other herpesviruses. Overall, our findings suggest two potential mechanisms for graft rejection: (i) the sole deregulation of HCMV and HHV6, which alone can lead to high cytokine levels, and (ii) the simultaneous deregulation of EBV and HHV7. This latter mechanism was associated with graft rejection primarily through IL-8.
There is a high heterogeneity in the literature concerning which cytokines are important markers of graft rejection [
44]. IL-4 has been associated with liver allograft rejection [
45], and the blocking of IL-4 has been proposed to improve long-term grafted kidney preservation [
46]. IL-8 has also been related to deterioration of the transplanted liver and proposed as a predictive marker of acute rejection in liver transplantation [
47,
48]. Several studies have also analyzed IL-10 levels after transplantation, yielding conflicting results. Low levels of IL-10 have been found in chronic kidney rejection [
49,
50], and IL-10-positive blood cells quantified through ELISPOT were found significantly diminished in acute and chronic kidney rejection [
51]. On the contrary, elevated IL-10, IL-17 and IP-10 (interferon gamma-inducible protein 10) had an estimated 94% sensitivity and 97% specificity to predict graft rejection [
52]. Elevated levels of IL-10 have also been proposed as a marker for an enhanced risk of HCMV disease in kidney or liver allograft recipients [
53]. Importantly, EBV and HCMV secrete BCRF1 and UL111A/cmvIL-10, respectively, which are IL-10 homologous proteins that modulate the host immune system [
7]. These viral IL-10s have been shown to inhibit the synthesis of several proinflammatory cytokines, such as IL-1α, IL-6, IL-12, IFN-γ, and TNF-α [
9,
10].
EBV appeared to display an immunomodulatory capacity, as its single detection was associated with decreased cytokine levels, including those linked with rejection. While there is abundant information about the capacity of EBV to establish an immunosuppressive environment in EBV-associated neoplasms, there is a general lack of information for other pathological conditions. For instance, EBV can downregulate the capacity of HHV6 to trigger TNF-α secretion in infected blood mononuclear cells [
54]. Remarkably, the pattern of cytokine downregulation changed when EBV was co-detected with the β-herpesviruses, leading to elevated levels of the cytokines. A noteworthy observation was that EBV, when detected in isolation, was preferentially found in the cellular fraction of peripheral blood, suggesting a predominant latent state. However, in co-detection with β-herpesviruses, it was found in plasma, suggesting viral reactivation. These observations support a scenario where latent EBV can downregulate cytokine levels, and this balance is disrupted when other β-herpesviruses are upregulated. In such events, EBV is observed in plasma, hinting at a crosstalk between the herpesviruses that may trigger EBV reactivation. Conversely, β-herpesviruses were consistently associated with the upregulation of cytokines in both single and multiple detections as well as in both latent and reactivation states.
An important limitation of this study is that it is an associative study, and in this complex interplay of virus, cytokines and graft rejection, we cannot conclude which one comes first, triggering the others. For instance, IL-10 is considered the prototype of anti-inflammatory cytokines. It is conceivable that IL-10 enhanced levels may arise as a graft-protective mechanism rather than an instrument for graft damage; in other words, the upregulation of IL-10 serves as a compensatory mechanism to counterbalance graft deterioration. In support of this scenario, experimental rat models with an exogenous expression of IL-10 have demonstrated extended graft survival [
55,
56,
57,
58]. A similar protective role has been proposed for IL-4 in rat experimental models [
59,
60]. Another limitation of this study is that picks of viral loads and the frequency of simultaneous detections may only be reflective of the degree of host immunosuppression or the donor–receptor previous exposure to the herpesvirus of interest. We were surprised by the low levels of cytokines found in patients, but they may also be reflective of the patients′ immunosuppressive state. Collectively, the limitations of this study underscore the fundamental challenge inherent in observational research: the difficulty of inferring causality from temporal associations alone. While the findings presented here offer valuable insights and generate hypotheses regarding causality, they do not definitively establish causal relationships.
Whether they act as the cause or consequence, our study underscores the importance of monitoring of EBV, HCMV, HHV6, and HHV7, along with IL-4, IL-8, and IL-10, as markers indicating an increased risk of graft rejection during clinical follow-up after transplantation. Regrettably, current worldwide recommendations do not advocate for the consistent monitoring of HHV6 and HHV7, despite HHV6´s strong association with graft rejection. Moreover, there is a need for a standardized method of quantifying herpesviruses to establish clear thresholds of viral loads that strongly indicate unfavorable clinical outcomes. These insights are crucial for advancing our understanding of the significance of herpesvirus detection in clinical outcomes, guiding targeted therapeutic interventions, and developing refined preventive strategies. Ultimately, these efforts aim to improve the overall prognosis and enhance the quality of life for transplanted patients.