**3. Discussion**

Chronic, low-grade, local inflammation underlining the OA process has been considered as an essential feature of the disease [6,8,9,35]. Furthermore, the action of an inflamed synovium as a trigger of the OA process has been suggested and points at cells recruited in intra-articular changes [36]. Therefore, the assessment of synovial lesions has been encouraged by both research and clinical practice [37–39].

Previous studies have explored the molecular mechanisms of the cell entry of betaherpesviruses; some authors have designated CD46 as an entry receptor in the case of HHV-6 infection and demonstrated the presence of it on a major target cell—activated T lymphocytes [40]. Alternatively, other authors have explored CD134, which is a receptor specific for HHV-6B, belonging to the TNF receptor superfamily and expressed on activated CD4-positive T cells [41–43].

Human herpesvirus 7, one of the most prevalent viruses in the human population, has also been recognized to target lymphocytes [44]. Furthermore, the activation of infected T lymphocytes leading to the reactivation of HHV-7 has been confirmed [45]. Finally, in HCMV infection, up to 30% of all circulating CD4-positive T lymphocytes become the primary target of the virus in infected elderly individuals. Furthermore, the authors showed that these HCMV-affected T lymphocytes may contribute to significant shifts in the leukocyte composition of peripheral blood and an increase in the number of "effector-memory" T cells [46].

Importantly, the OA synovium has been reported to be a tissue enriched in T cells when compared to the normal synovial membrane [38,39,47–49], and the proportion of CD4-positive T cells and the CD4-positive/CD8-positive ratio found in peripheral blood are recognized as being significantly higher in OA patients when compared to healthy controls [7,50]. Other authors have reported on synovial tissue damage induced by CD4-positive T cells and evidenced, at a later stage of the disease, that activated CD4-positive T cells promote lesions and induce macrophage inflammatory protein-1γ expression and subsequent osteoclast formation in OA patients [51]. Given that the demonstrated overall assessment of CD4-positive T cells is in line with synovial morphological findings, this suggests a clear predominance of OA subjects without histopathologically detectable and low-grade inflammation confirmed in both study groups. In this study, the median number of CD4-positive cells per visual field assessed in the synovial samples of the HHV-7 PCR+ group was significantly higher than that in the HHV-7 PCR- group (*p* < 0.0001). The role of HHV-7 infection in the development and progression of either synovitis or OA remains largely unknown. However, a strong (*r* = 0.6629, *p* = 0.0367) correlation between the number of TNF+ and HHV-7+ lymphocytes has been demonstrated in the samples of the HHV-7 PCR+ group, suggesting the significance of the immune system reaction to a foreign antigen. Furthermore, the vulnerability of vascular endothelial cells, especially in the case of infection reactivation, is shown in the given study. Available serological data suggest that primary herpesvirus infection occurs early in childhood and results in a lifelong infection [24,52–54].

This is consistent with the results of our study. The present study demonstrated the presence of persistent HHV-7 infection in 81.5% of all enrolled OA patients. Similar results were published by Sánchez-Ponce and colleagues conducting studies on beta and gamma human herpesviruses in the case of organ transplantation, and they raised awareness about an increased risk of graft rejection [55].

Our previous studies have pointed out that the eradication of this infection often does not occur, and the virus remains in a latent state [56,57]. Complications associated with virus reactivation involve a wide range of diseases, including joint pathologies; however, the role of HHV-7 has not been completely understood thus far. Other authors have not succeeded in attempts at isolating DNA from herpesviruses in patients with OA, but have showed the presence of DNA from HSV1-2 and VZV in RA and axial spondyloarthritis patients [33]. The strength of our study includes a look at the synovial cellular constituents targeted in the case of latent or active HHV-7 infection. In our study, molecular virology data confirmed that either latent or active HHV-7 infection was coupled to immunohistochemical detection of the viral antigen in the synovial membrane. We observed that HHV-7 latency was characterized by the contribution of inflammatory cells and the presence of CD4-positive cells, establishing a strong, positive correlation with TNF-producers. Simultaneously, we proved that different cellular targets, vascular beds, and their constituents, are more vulnerable and are therefore affected in the case of active HHV-7 infection.

Previous studies have demonstrated that human herpesviruses sustain latency in cells of the hematopoietic lineage, but become reactivated upon immune challenge to cause disease [58]. Synovial macrophages are of great interest in this context since they may act as a reservoir during the period of viral infection latency. This feature has been widely explored, and evidence that even a suppressed viral transcription program exhibits several viral genes that are expressed during latent infection at the protein level has been obtained; many of these have profound effects on the cell and its environment, regulating numerous cellular functions [59,60]. Furthermore, our recent studies provided some insight into the controversial issue related to the chromosomal integration of HHV-7 [61]. In this context, the stimulation of inflammation and oxidative stress are areas of interest when suggesting the contribution of herpesviruses infections to chromosomal telomere attrition [62,63]. Previous studies have proved the role of macrophages in the development of synovial lesions and progression of OA through the production of inflammatory mediators, growth factors, and metalloproteinases, resulting in enhanced cartilage degeneration and osteophyte formation [13]. Furthermore, immunohistochemical studies have produced additional evidence of the presence of TNF, which is the key pro-inflammatory factor released by synovial macrophages, in OA [38,64]. OA synovitis has been recognized as a cytokine-driven disease, especially regarding TNF, even when manifesting with lower levels of pro-inflammatory mediators compared to that in RA [15]. A higher expression of the inflammatory mediators was found in the lining layer, and a lower expression was found in the sublining layer of the lesioned synovium [65,66]. In this study, we found a large number of TNF-labeled synovial lining cells, whereas most of the sublining constituents revealed perivascular localization. Our data are consistent with the results obtained by these authors. Furthermore, more substantial synovial macrophage infiltration demonstrated in patients with early OA, when compared to advanced OA [13], and higher levels of inflammatory cytokines, including IL-6, IL-1, and TNF, were evidenced in early compared to advanced OA [67]. The significance of findings depicting the distribution and functions of synovial macrophages and infiltrating lymphocytes has been pointed out in the given study, without denying other accurate and sensitive tools for visualizing the inflammatory cells in OA [13].

By emphasizing the significance of the immune system reaction to a foreign antigen, one may expect the appearance of system overreaction, causing potentially more harm than the viral agent itself. There is an increasing appreciation of the importance of cellular plasticity on the one hand, and the diversity of strategies and molecular mechanisms for escaping detection by the immune system evolved by HHV-7 on the other [68], suggesting the necessity for further research.
