Flow cytometric analysis of T lymphocytes and cytokines in aqueous humor of patients with varicella zoster virus-mediated acute retinal necrosis

It keeps difficult to find out the cause of uveitis at early stage for clinicians [1]. However, more and more evidence showed that viruses may be the causes for some uveitis with unknown causes previously, such as Posner-Schlossman syndrome [2‐4] and Fuchs uveitis syndrome [5, 6]. Since the intraocular cytokine environment and molecular mechanisms and the infiltrating intraocular inflammatory cells or cytokines varied remarkably between viral infectious uveitis and non-viral infectious uveitis [7], it may be a potential way to analyzing the intraocular inflammatory cells and cytokines for clinicians to partially confirm the presumed viral-induced uveitis, even when the real-time polymerase chain reaction (PCR) of specific viruses is negative.

Acute retinal necrosis (ARN) is an infectious viral uveitis syndrome which presents itself as a necrotizing retinitis and may lead to a fatal visual outcome. ARN is most commonly caused by varicella zoster virus (VZV), herpes simplex 1 and 2 (HSV-1, HSV-2), cytomegalovirus (CMV), and infrequently, Epstein-Barr virus (EBV) [8]. VZV is the most common cause, followed by HSV (types 1 and 2) [9].

Apart from the direct cytopathic effect of the pathogenic virus on infected retinal cells, the intraocular T cell response to the triggering virus has been indicated to play a part in the ARN’s local inflammatory response [10, 11]. A previous study reported a high number of CD4+ and CD8+ cells in the vitreous cell population in eyes with ARN mediated by HSV-1 or VZV. CD4+ and cytolytic CD8+ T cell responses make great contributions to restraining virus replication [11], but the immune responses that occur in infectious eyes during the acute infection phase have not been analyzed in detail.

The purpose of this study was to undertake a comprehensive detailed investigation of the types and distribution of T lymphocytes present in aqueous humor (AH) and peripheral blood (PB) in patients with confirmed virus-induced uveitis, i.e., ARN, and provide new insights into the immunological mechanisms that may be important in regulating ocular inflammation following VZV infection. We also compared the differences in the cytokine profiles in the AH from both viral infections and non-infectious patients to aid in the elucidation of mechanisms of the disease. The results may be helpful for clinic to find out a new diagnostic way to differentiate the uveitis between viral causes and autoimmune causes.

This study described the immunological features of the AH and PB T lymphocytes population in patients with viral infections or non-infectious uveitis. It also analyzed the mainly inflammatory cytokines of the AH samples of those patients.

The present study reported that there are intrinsic differences in both the T lymphocytes subsets and cytokines within the AH when comparing the VZV mediated ARN patients and the AAU patients. VZV is a double-stranded DNA virus belonging to the herpesviridae family. Among the human herpes viruses, HSV-1 and 2 and VZV are neurotropic, i.e., they establish latent infection in the peripheral nervous system and the viral genome is retained in peripheral sensory ganglia throughout the host’s lifespan [13, 14]. Most ARN patients are immunocompetent, and typical cases of ARN are uncommon in immunocompromised individuals [14]. VZV infection provides stimuli to the antigen-specific cell-mediated immunity essential to recovering from primary infection, to containing VZV reactivation, and to protecting against virus. It was reported that T cells predominate among the inflammatory cells located in AH of ARN patients [15]. The present study described the immunologic profile of AH T lymphocytes in ARN patients characterized by a high percentage of CD8+ T lymphocytes and low CD4/CD8 ratio. It was found that CD8+ T lymphocytes infiltration into the AH was higher in patients with ARN than patients with AAU, and this is consistent with previous reports [16, 17]. In comparison with the non-infectious patients, significant increase of CD8+ cell population and decrease of CD4/CD8 ratio in AH during the acute phase of viral infection were observed in the viral infectious patients. Previous studies found that large immune infiltrates consisted of CD8+ T cells following VZV activation in human ganglia [14, 18]. These results were similar to those found in previous research, providing further support to the hypothesis that changes in T lymphocytes subpopulations, including a higher percentage of CD8+ T cells and a low CD4/CD8 ratio, are associated with viral activation. After antiviral treatment, the symptoms of the ARN patients relieved, manifesting in the form of reduced ocular inflammation and improved visual acuity. The copy number of VZV DNA in AH in the ARN patients significantly decreased after treatment. Correlation analyses showed that the copy number of VZV DNA in AH positively correlated with the percentage of CD8+ T lymphocytes in AH and negatively correlated with the CD4/CD8 ratio in AH. Importantly, this study also showed that after treatment the percentage of CD8+ lymphocytes in the ARN patients was even higher than that in the AAU patients. Similarly, even after antiviral treatment, the ratio of CD4/CD8 was lower in the ARN group than that in the AAU group. Therefore, a high percentage of CD8+ T lymphocytes and a low CD4/CD8 T cell ratio may be a potential biomarker, serving as clinically useful indicators to evaluate the severity of intraocular immune inflammatory response by which the prognosis of ARN can be forecasted. The identification and analysis of T lymphocytes from sites of intraocular inflammation can help investigate the roles of infiltrating T lymphocytes in the pathogenesis of uveitis with different etiologies.

In the uveitis associated with systemic and intraocular infections, it is unclear whether the T lymphocytes enter the eye as part of a peripheral immune response, or there is a passive extravasation of T lymphocytes enter the eye during a breakdown of the blood-ocular barrier. In line with previously published data, the percentages of CD4+ and CD8+ T lymphocytes in AH were higher than those found in PB [11]. In this study, compared with the non-infectious patients, ARN manifests peculiar focal lymphocytosis characterized by an elevated percentage of CD8+ T lymphocytes and a corresponding decreased CD4/CD8 ratio in AH. However, the changes of CD8+ T lymphocytes and CD4/CD8 patterns were observed only in AH but not in PB. The results suggest that there may be a particular mechanism attracting T lymphocytes into the eye, or the blood-aqueous barrier may channel the selective infiltration of activated T cells into uveitis patients’ ocular tissues and AH. Another possible explanation for this is that the ARN patients with ocular inflammation only, but without other systemic symptoms, are too constrained to enable detection of whatever alterations in the blood’s cell phenotypes. PB is not the perfect sample for research on immune abnormalities in intraocular inflammation in that it cannot represent the immune inflammatory processes taking place within the eye. Further detailed studies are necessary to investigate the immune function of intraocular T lymphocyte subsets and immunologic abnormalities in viral infectious uveitis, or to study whether there is a characteristic T lymphocyte population in the AH of viral infection patients.

CD4 + CD25+ T cells have been shown to be one distinct subset of regulatory T cells for the prevention of autoimmunity [19]. Several studies have shown that regulatory CD4 + CD25+ T cells contribute much to controlling immune homeostasis, to maintaining self-tolerance, and to preventing autoimmunity, as impaired CD4 + CD25+ T cell activity can cause autoimmune disease [20, 21]. There was predominant CD4 + CD25+ T cell enrichment in the inflammatory sites during the disease course and their accumulation correlated with disease resolution [22]. In this study, the CD25+ T lymphocytes comprised 20.5% of the CD4+ T lymphocytes in AH in the AAU patients, who had significantly increased CD4 + CD25+ T lymphocytes in AH compared with PB. All the AAU patients had coexisting autoimmune disorder or serum autoantibodies, and the anterior uveitis of those patients was occurring in association with systemic diseases. In the AAU patients, the enrichment of regulatory CD4 + CD25+ T lymphocytes in AH may suppress the activation and proliferation of T cells to regulate local inflammatory responses [23]. However, there were collaborative roles for several mechanisms requiring both cell-cell contact and cytokines production in the identification of the quantity and function of CD4 + CD25+ T cells in the inflammatory sites and peripheral immune sites [24]. Further mechanism research is needed to investigate the regulatory mechanism and immunosuppressive activity of regulatory CD4 + CD25+ T lymphocytes.

Chemokines are unnegligible inflammatory mediators excreted by inflammatory and immune cells during ocular inflammation [25]. The intraocular concentration of IL-10 is very useful for etiology diagnosis of uveitis, especially in intraocular lymphoma (IOL) [26]. Previous studies have also reported that IL-10 was highly elevated in herpetic and noninfectious uveitis [7, 27]. IL-10 is an immunosuppressive regulatory cytokine and can be produced by T lymphocytes. It has been suggested that IL-10 downregulates the immune responses in order to moderate T cell mediated immune reaction and regulate the extent of tissue damage [28, 29]. The high expression of intraocular IL-10 in the ARN patients was in keeping with previous research [7, 26]. However, the high IL-10 level in AH of the ARN patients seems paradoxical regarding the severe course of the intraocular inflammation. One possible reason is that the expression of IL-10 is not sufficient to suppress the harmful inflammatory immune response. Alternatively, in addition to the immune responses downregulated by IL-10, other potential inflammatory response mechanisms may contribute in combination to the damage. Because the cytokine and chemokine expression patterns are constituents of a very complex regulatory network that depends on many environmental stimuli, they are likely to vary remarkably over the progression of the disease. Therefore, the effect of cytokines on the T lymphocytes necessitates thorough investigation if cytokine immunology therapy is to be employed in future. Combined with changes of intraocular cytokines, immune cells profiles in AH seem to provide insights into the severity and stage of the inflammatory process. Our results show that through cytometric analysis of AH, the immunologic information of this disease was useful for the differential diagnosis of intraocular viral infection. It helps improve our ability to classify viral infection from other uveitis, which could enable an early and less-invasive diagnosis.

Undeniably, this study has its limitations. First, the number of the ARN and AAU patients enrolled in the study was not sufficient, and therefore future research needs to further enlarge the sample size to analyze the changes of T lymphocyte subsets in these diseases. Further studies will provide detailed information on immunopathologic mechanisms of lymphocytes in AH from different types of uveitis. Second, this study focused on the distribution of T lymphocytes subsets in AH and PB of ARN and AAU patients, but did not analyze the function of those cells. Further thorough studies are necessitated to investigate the detailed T lymphocyte subsets and functional properties of intraocular infiltrating T lymphocytes in uveitis patients in order to elucidate underlying immunopathogenic mechanisms of this disorder. Third, the follow-up period for the patients with antiviral treatment was not long enough, but future longitudinal follow-up studies in ARN patients are expected to overcome this shortcoming, and hopefully, they may provide precious insights into the progression of immune inflammation in ARN.

In conclusion, the patients with ARN caused by VZV in this study had a higher percentage of CD8+ T lymphocytes and a lower CD4/CD8 ratio in AH compared with the control subjects suffering non-infectious uveitis. The percentage of the CD8+ T lymphocyte subset and the ratio of the CD4/CD8 in AH from the ARN patients significantly correlated to the aqueous VZV load during the course of disease treatment. AH cell profiles might be discriminative for the diagnosis of viral-infectious and immune-mediated uveitis, and AH cell examination has the potential to become a useful research tool for investigating various uveitis entities.