Autophagy protects against retinal cell death in mouse model of cytomegalovirus retinitis.

BACKGROUND: Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis (CMV). Our previous results have shown that there is a functional relationship between autophagy and apoptosis during MCMV infection of retinal pigment epithelium (RPE). The purpose of this study was to determine whether autophagy plays a significant role in the death of retinal cells during MCMV retinitis. METHODS: The retinas of adult BALB/c mice were infected with MCMV via supraciliary injection. Rapamycin, a mTOR inhibitor, was injected to MCMV-infected BALB/c mice intraperitoneally. Immunohistochemistry and western blot were performed to observe the spread pattern of virus in retinas and the levels of targeted proteins. Plaque assay was performed to determine the virus titer in different groups. Since Atg5 is a key gene regulating autophagy, we bred Atg5flox/flox; Nestin-Cre mice to deeply elucidate the role of autophagy during MCMV retinitis. Atg5flox/flox; Nestin-Cre mice were genotyped and infected with MCMV. Immunohistochemistry was performed to observe the type of virus-infected cells and apoptosis in retinas during MCMV retinitis. RESULTS: In MCMV mouse model, MCMV infection in outer nuclear layer (ONL) and inner nuclear layer (INL) in the retinas caused cleaved caspase 3 positive apoptosis, which is not co-localized with early antigen (EA) positive virus infected cells in rapamycin treated group. Rapamycin treatment increased the levels of LC3B-II by inhibiting mTOR and decreased the levels of cleaved caspase-3 during MCMV retinitis. However, virus propagation was not affected by rapamycin. In Atg5flox/flox; Nestin-Cre mice, RPE and glial cells were the main targets of viral infection, and number of EA positive retinal cells and TUNEL positive retinal cells was significantly increased compared to Atg5flox/+; Nestin-Cre mice though there was no difference of virus propagation between Atg5flox/flox; Nestin-Cre mice and Atg5flox/+; Nestin-Cre mice. CONCLUSIONS: Autophagy protects retinal cells from MCMV infection induced apoptosis through mTOR-mediated signaling pathway.

Expression of the iron-regulatory protein haemojuvelin in retina and its regulation during cytomegalovirus infection.

Haemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE [HLA-like protein involved in iron (Fe) homoeostasis], transferrin receptor 2, ferroportin, hepcidin and HJV (haemojuvelin). Expression of the first four genes coding for these proteins in retina has been established. Here we report on the expression of HJV. Since infection of retina with CMV (cytomegalovirus) causes blindness, we also investigated the expression of HJV and other iron-regulatory proteins in retina during CMV infection. HJV (HJV gene) mRNA was expressed in RPE (retinal pigment epithelium)/eyecup and neural retina in mouse. In situ hybridization and immunohistochemistry confirmed the presence of HJV mRNA and protein in RPE, outer and inner nuclear layers, and ganglion cell layer. Immunocytochemistry with cell lines and primary cell cultures showed HJV expression in RPE and Müller cells. In RPE, the expression was restricted to apical membrane. Infection of primary cultures of mouse RPE with CMV increased HJV mRNA and protein levels. Under similar conditions, HFE (HFE gene) mRNA levels were not altered, but HFE protein was decreased. Hepcidin expression was, however, not altered. These findings were demonstrable in vivo with CMV-infected mouse retina. The CMV-induced up-regulation of HJV in RPE was independent of changes in HFE because the phenomenon was also seen in HFE-null RPE cells. CMV-infected primary RPE cells showed evidence of iron accumulation and oxidative stress, as indicated by increased levels of ferritin and hydroxynonenal. The observed changes in HJV expression and iron status during CMV infection in retina may have significance in the pathophysiology of CMV retinitis.

Distribution of herpes simplex virus type 1 and varicella zoster virus in ganglia of the human head and neck.

The distribution of the neurotropic alphaherpesviruses-herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella zoster virus (VZV)-was determined in autonomic and sensory ganglia of the head and neck obtained from formalin-fixed human cadavers. HSV-1 and VZV DNA were found in 18 of 58 and 16 of 58 trigeminal, 23 of 58 and 11 of 58 pterygopalatine, 25 of 60 and 14 of 60 ciliary, 25 of 48 and 11 of 48 geniculate, 15 of 50 and 8 of 50 otic, 14 of 47 and 4 of 47 submandibular, 18 of 58 and 10 of 58 superior cervical, and 12 of 36 and 1 of 36 nodose ganglia, respectively. HSV-2 was not detected at any site. Viral DNA positivity and location were independently distributed among autonomic and sensory ganglia of the human head and neck.

Uniocular anterior chamber inoculation of a tumor necrosis factor alpha-expressing recombinant of herpes simplex virus type 1 results in more rapid destruction and increased viral replication in the retina of the uninoculated eye.

Tumor necrosis factor alpha (TNF-alpha) has been shown to have a protective role in the eyes and brains of herpes simplex virus type 1 (HSV-1)-infected mice. To determine whether overexpression of TNF-alpha affected the course of virus infection following uniocular anterior chamber inoculation, a recombinant of HSV-1 that produces TNF-alpha constitutively (KOSTNF) was constructed. BALB/c mice were injected with the TNF-alpha recombinant, a recombinant containing the pCI plasmid, a recombinant rescue virus, or the parental virus. Flow cytometry and immunohistochemistry were used to identify virus-infected cells and to determine the numbers and types of infiltrating inflammatory cells in the uninjected eyes. Virus titers were determined by plaque assay. There were no differences among the groups in virus titers or the route and timing of virus spread in the injected eyes or in the suprachiasmatic nuclei. However, in the uninjected eyes of KOSTNF-infected mice, TNF-alpha expression was increased and there were more viral antigen-positive cells and immune inflammatory cells. There was earlier microscopic evidence of retinal infection and destruction in these mice, and the titers of virus in the uninjected eyes were significantly increased in KOSTNF-infected mice on day 7 postinfection compared with those of KOSpCI-, KOS6beta rescue-, or KOS6beta-infected mice. The results suggest that instead of moderating infection and reducing virus spread, overexpression of TNF-alpha has deleterious effects due to increased inflammation and virus infection that result in earlier destruction of the retina of the uninoculated eye.

Murine cytomegalovirus infection and apoptosis in organotypic retinal cultures.

PURPOSE: An organotypic retinal culture model was used to determine the pattern of murine cytomegalovirus (MCMV) infection and whether apoptosis is induced in MCMV-infected cultured retinas. METHODS: Retinas harvested from C57BL/6 mice were individually cultured at 37 degrees C on 3-microm filter inserts placed in 24-well plates. Some retinas were infected with MCMV (5 x 10(5) PFU/well). At days 4, 7, and 11 after infection (pi), the culture medium and cultured retinas were collected for examination. RESULTS: Replicating virus was recovered and viral early antigen (EA)- and late antigen (LA)-positive cells were observed in the MCMV-infected retinal cultures. Most MCMV-infected cells were glia and horizontal cells. Infection resulted in atrophy of the photoreceptor cells and cytomegaly. Apoptosis of uninfected bystander cells, including photoreceptor cells and horizontal cells, was observed. TNF-alpha was produced by activated microglia during MCMV infection of the retina. Mouse apoptosis microarray studies, caspase activity studies, and RT-PCR studies showed that the genes involved in both the death receptor-mediated apoptotic pathway and the mitochondrial pathway were upregulated. CONCLUSIONS: Many aspects of MCMV infection of retinal cultures parallel those observed during MCMV retinitis in mice. Thus, this in vitro system may be used to explore the role of apoptosis of uninfected retinal cells and the contribution of cytokines and other modulators to the pathogenesis of CMV retinitis.

Depletion of the Receptor-Interacting Protein Kinase 3 (RIP3) Decreases Photoreceptor Cell Death During the Early Stages of Ocular Murine Cytomegalovirus Infection.

Purpose: The purpose of this study was to determine if the receptor-interacting protein kinase 3 (RIP3) plays a significant role in innate immune responses and death of bystander retinal neurons during murine cytomegalovirus (MCMV) retinal infection, by comparing the innate immune response and cell death in RIP3-depleted mice (Rip3-/-) and Rip3+/+ control mice. Methods: Rip3-/- and Rip3+/+ mice were immunosuppressed (IS) and inoculated with MCMV via the supraciliary route. Virus-injected and mock-injected control eyes were removed at days 4, 7, and 10 post infection (p.i.) and markers of innate immunity and cell death were analyzed. Results: Compared to Rip3+/+ mice, significantly more MCMV was recovered and more MCMV-infected RPE cells were observed in injected eyes of Rip3-/- mice at days 4 and 7 p.i. In contrast, fewer TUNEL-stained photoreceptors were observed in Rip3-/- eyes than in Rip3+/+ eyes at these times. Electron microscopy showed that significantly more apoptotic photoreceptor cells were present in Rip3+/+ mice than in Rip3-/- mice. Immunohistochemistry showed that the majority of TUNEL-stained photoreceptors died via mitochondrial flavoprotein apoptosis-inducing factor (AIF)-mediated, caspase 3-independent apoptosis. The majority of RIP3-expressing cells in infected eyes were RPE cells, microglia/macrophages, and glia, whereas retinal neurons contained much lower amounts of RIP3. Western blots showed significantly higher levels of activated nuclear factor-κB and caspase 1 were present in Rip3+/+ eyes compared to Rip3-/- eyes. Conclusions: Our results suggest that RIP3 enhances innate immune responses against ocular MCMV infection via activation of the inflammasome and nuclear factor-κB, which also leads to inflammation and death of bystander cells by multiple pathways including apoptosis and necroptosis.

Tumor necrosis factor-alpha-induced apoptosis in murine cytomegalovirus retinitis.

PURPOSE: Previous results suggest that apoptosis is involved in the pathogenesis of murine cytomegalovirus (MCMV) retinitis. To explore the mechanism underlying retinal apoptosis in MCMV retinitis, this study was initiated to determine whether the tumor necrosis factor receptor (TNFR)1-TNF pathway is involved in apoptosis during MCMV retinitis. METHODS: The left eyes of nonimmunosuppressed (non-IS) BALB/c mice, immunosuppressed (IS) BALB/c mice, TNFR1(-/-) C57BL/6 mice, and wild-type C57BL/6 mice were inoculated with MCMV k181 by way of the supraciliary route. On postinoculation days 3, 7, and 10, injected eyes of non-IS control and IS experimental mice were removed for RT-PCR for TNF-alpha and TNFR1. Protein expression of TNF-alpha, caspase-8, and caspase-3 was determined by staining frozen sections and performing Western blot analysis and quantitative ELISA. Apoptotic cells were identified by TUNEL labeling. RESULTS: In IS BALB/c mice, TNF-alpha mRNA and protein were detected in MCMV-infected eyes throughout the infection. Activation of caspase-3 and caspase-8 was observed. Most of the TNF-alpha-expressing cells were MCMV-infected RPE cells or macrophages derived from RPE cells. TNF-alpha was observed in the area of apoptotic retinal cells, and the level of this cytokine corresponded to the extent of the retinal abnormality and to the number of apoptotic cells. In non-IS MCMV-infected BALB/c mice, TNF-alpha was expressed early in the retinas of MCMV-infected eyes, but its expression was decreased thereafter. TNFR1 mRNA was increased in IS and non-IS BALB/c after MCMV infection. More apoptotic cells were observed in the retinas of non-IS MCMV-infected wild-type C57BL/6 mice than in the retinas of non-IS TNFR(-/-) mice. CONCLUSIONS: These results suggest that the TNFR1-TNF pathway is involved in the induction of apoptosis and the exacerbation of retinal abnormality during MCMV retinitis. Furthermore, because TNF-alpha and TNFR1 were present in IS and non-IS mice, TNF-alpha-induced retinal apoptosis during MCMV infection is not T-cell dependent.

Lack of iNOS facilitates MCMV spread in the retina.

PURPOSE: The purposes of this study were to identify iNOS-producing retinal cells and to determine whether lack of iNOS facilitates MCMV spread and replication in the retina. METHODS: Immunosuppressed (IS) iNOS(-/-) mice or C57BL/6 (wild-type) mice were inoculated with 5 x 10(4) PFU of MCMV K181 strain (K181) via the supraciliary route. Injected eyes were collected at several times after inoculation and examined by plaque assay for replicating virus, RT-PCR for iNOS RNA, Western blot for iNOS protein and by staining for MCMV early antigen (EA), iNOS, and retinal cell antigens. RESULTS: iNOS mRNA and iNOS proteins were expressed in the MCMV-injected eye of wild-type mice. Most iNOS-producing cells were F4/80-positive, including macrophages, RPE-derived macrophages, and resident microglia. Significantly higher titers of virus were recovered from the injected eyes, and more infected cells were detected in the retina of IS iNOS(-/-) mice than in IS wild-type mice. Retinal necrosis and loss of retinal architecture throughout the retina were noted in IS iNOS(-/-) mice, whereas cytomegalic cells and retinitis were present only in the peripheral retina of IS wild-type mice. CONCLUSIONS: iNOS produced by macrophages, especially resident macrophages including microglia and RPE derived macrophages, plays an important role in limiting spread of MCMV in the retina.

Acute retinal necrosis.

Acute retinal necrosis (ARN) is a rare disease that is usually caused by one of the three neurotropic human herpesviruses - herpes simplex virus type 1(HSV-1), HSV-2 and varicella-zoster virus (VZV). Although much is known about the clinical course of the disease and its treatment and about the viruses that cause it, comparatively little is known about its pathogenesis. This article will review the history of ARN, the typical clinical findings, and methods of diagnosis. Information from studies of the mouse model of ARN including development of anterior chamber-associated immune deviation (ACAID) and routes of spread will be reconsidered, and the combined information from human and mouse studies will be discussed to suggest mechanisms that contribute to the pathogenesis of ARN in human patients. Finally, puzzles and questions about the disease will be considered.

Cytokine profiles and inflammatory cells during HSV-1-induced acute retinal necrosis.

PURPOSE: To investigate infiltrating cells, cytokines, and kinetics of cytokine expression during acute retinal necrosis (ARN) in the uninoculated eye after inoculation of herpes simplex virus (HSV)-1 into the anterior chamber of one eye of BALB/c mice. METHODS: At different time points after inoculation of 2 x 10(4) plaque-forming units (PFU) HSV-1 (KOS strain) or an equivalent volume of Vero cell extract in cell culture medium, the uninoculated eyes were enucleated. RT-PCRs for TNFalpha, IFNgamma, and IL-4 and immunohistochemical staining were performed to identify infiltrating cells and cytokines. Cytometric bead array was used to measure the levels of TNFalpha, IFNgamma, and IL-4 protein. RESULTS: CD4(+) T cells, F4/80(+) macrophages, Gr-1(+) polymorphonuclear cells (PMNs), and CD19(+) B cells were detected in the uninoculated eye of virus-infected mice. Furthermore, RPE65(+) retinal pigment epithelial (RPE) cells and activated Muller cells were also detected in the ARN lesion. TNFalpha, IFNgamma, and IL-4 mRNA and protein were upregulated during the evolution of ARN in HSV-1-infected contralateral eyes compared with levels in control subjects. Immunohistochemistry revealed that cytokines were produced by infiltrating cells as well as by resident retinal cells. CONCLUSIONS: The results of these studies support the idea that T cells and cytokines are actively involved in HSV-1 retinitis. They also suggest that PMNs, B cells, and/or macrophages, as well as resident retinal cells, such as RPE and activated Muller cells, also play a role in the pathogenesis of HSV-1 retinitis.

Infection of retinal neurons during murine cytomegalovirus retinitis.

PURPOSE: Previous results suggest that retinal neurons are infected early during murine cytomegalovirus (MCMV) infection of the inner retina. The purposes of this study were to identify which retinal neurons are infected and to determine the routes by which MCMV spreads in the retina. METHODS: Immunosuppressed (IS) BALB/c mice were inoculated with 5 x 10(3) PFU of MCMV (k181) through the supraciliary route. Injected eyes were collected at several times after inoculation, sectioned, and examined by electron microscopy and by staining for retinal cell antigens and for MCMV early (EA) or late (LA) antigen. RESULTS: MCMV-infected cells were observed in the choroid and RPE by day 3 after infection (PI) and in the inner retina beginning at day 5 PI. At this time, many horizontal and bipolar cells were MCMV-antigen-positive but only rare MCMV-infected amacrine cells (glycine positive or gamma-aminobutyric acid [GABA] positive) or MCMV-infected ganglion cells (NF positive) were observed in the inner retina. At day 10 PI, most virus-infected cells were glial fibrillary acidic protein (GFAP)- and GABA-positive glia. Virions were observed by electron microscopy in the choroid, RPE, and inner nuclear layer of the retina. Although virions were observed in the endothelium of the retinal vessels and the nearby retinal cells, the endothelial cell lining of the retinal vessels remained intact. Both apoptotic cells and necrotic cells were seen in the inner retina. CONCLUSIONS: In the inner retina, horizontal and bipolar cells were the early (< or = day 7 PI) targets of MCMV infection. Virus spread from the RPE and the photoreceptor layer to the inner retina through infected Muller cells and within the inner retina horizontally through infected horizontal cells.

Ocular reactivation of MCMV after immunosuppression of latently infected BALB/c mice.

PURPOSE: The purpose of this study was to identify the site(s) of MCMV latency and reactivation in the eye. METHODS: Three months after supraciliary inoculation of 5 x 10(2) PFU of MCMV, BALB/c mice underwent immunosuppression with methylprednisolone and antibodies specific for CD4 T cells, CD8 T cells, and NK cells or with methylprednisolone alone. Control mice were infected but did not receive the immunosuppressants. After 2 or 3 weeks of immunosuppression, the mice were killed. Replicating virus and viral antigen were detected in the injected eyes, peripheral blood leukocytes (PBLs), and extraocular tissues by plaque assay and by staining for early antigen (EA) and beta-galactosidase (beta-gal), respectively. RESULTS: In latently infected, nonimmunosuppressed control mice, replicating-virus-and viral-antigen-positive cells were not detected in the injected eyes or extraocular tissues. After immunosuppression with methylprednisolone and antibodies, EA and beta-gal were detected, and replicating virus was recovered from the injected eye and from several extraocular sites, including liver, lungs, salivary glands, and kidneys. No virus was recovered from PBLs. beta-Gal- or EA-positive cells were observed in the RPE of most mice, and a few virus-infected cells were also observed in the nuclear layers and ganglion cells. Microscopic changes, including retinal folding and detachment, photoreceptor atrophy, macrophage infiltration, and a few EA-positive cytomegalic cells, were observed in the injected eye of immunosuppressed mice. CONCLUSIONS: After immunosuppression, MCMV reactivates in the injected eye and extraocular tissues, and RPE cells are the initial site of MCMV ocular reactivation in the eye. The timing of virus recovery from all sites suggests that MCMV observed in the injected eye is from in situ reactivation of virus and not from spread of virus from extraocular sites via infected PBLs.

Infiltration of T-lymphocytes in the brain after anterior chamber inoculation of a neurovirulent and neuroinvasive strain of HSV-1.

Following anterior chamber (AC) inoculation of BALB/c mice with the KOS strain of herpes simplex virus type 1 (HSV-1), or with H129, a neuroinvasive and neurovirulent strain of HSV-1, both strains of virus spread from the injected eye through the brain to cause retinitis. However, KOS-infected mice develop retinitis in the uninoculated eye only, whereas H129-infected mice develop bilateral retinitis. Previous studies have shown that infiltrating T-cells in the suprachiasmatic nuclei (SCN) of the hypothalamus of KOS-infected mice concomitant with or before virus protect KOS-infected mice from ipsilateral retinitis. To determine the timing of T cell infiltration and cytokine production in the brain of H129-infected mice, adjacent, frozen sections of the brain were immunostained for virus, T-cells, IL-2, TNF-alpha or IFN-gamma. T-cells infiltrated the brains of H129-infected mice and cytokines were produced in infected tissues. However, virus spread to the optic nerve and retina of both the inoculated and uninoculated eye before T-cells and cytokines were detected in the SCN of H129-infected mice. These results suggest that infiltrating T-cells in the SCN of H129-infected mice may arrive too late to prevent the spread of virus into the optic nerves and retinas and thus prevent development of bilateral retinitis in infected mice.

Detection of herpes simplex virus type 1 in human ciliary ganglia.

PURPOSE: To determine whether herpes simplex virus type 1 (HSV-1) DNA is present in the ciliary ganglion (CG). METHODS: Fifty CG and 47 trigeminal ganglia (TG) were resected from 63 formalin-fixed cadavers between 56 and 98 years of age that had been embalmed within 12 hours of death. The donors had no known active HSV infection at the time of death. DNA was extracted from all ganglia by proteinase-K digestion (TG) or digestion by a mild lysis buffer (CG). DNA was amplified by polymerase chain reaction for sequences from human chromosome 18, D18S1259 (positive control), and from the HSV-1 DNA polymerase gene, U(L)30. The amplified DNA was separated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with the appropriate digoxigenin-labeled probe that was detected by alkaline phosphatase-conjugated monoclonal antibody. RESULTS: The D18S1259 sequence was amplified from 47 TG and 30 CG samples. Of these samples, 32 (68.0%) of the 47 TG samples and 20 (66.6%) of the 30 CG samples were positive for the UL(30) HSV-1 sequence. CONCLUSIONS: Using amplification of HSV-1 DNA as a surrogate marker of latency, the finding that the frequency of HSV-1 in the CG was approximately the same as that of the TG suggests that the CG may be an additional site of HSV-1 latency in humans. Active infection in or reactivation of HSV-1 from non-TG sites may explain why this virus is able to infect sites, such as the retina, that have no direct connections to the trigeminal nerve.

Delayed spread and reduction in virus titer after anterior chamber inoculation of a recombinant of HSV-1 expressing IL-16.

PURPOSE: The timing of T-cell infiltration of the hypothalamus is crucial in the prevention of bilateral retinitis in mice inoculated with HSV-1 through the anterior chamber (AC). In H129-infected mice, T-cells are recruited to the suprachiasmatic nuclei of the hypothalamus too late to protect infected mice from development of bilateral retinitis. The purpose of these studies was to determine whether alteration of T-cell recruitment to the hypothalamus would affect the timing and pattern of virus spread after AC inoculation. METHODS: A recombinant of the H129 strain of HSV-1 expressing IL-16, a cytokine with lymphocytic and monocytic chemoattractant properties, was constructed, and mice were inoculated in the AC with H129wt, H129wt and H129/IL-16, or H129wt and H129/pGal10 (a recombinant virus containing vector only). RESULTS: AC inoculation of BALB/c mice with H129wt and H129/IL-16 resulted in a delay of virus spread to the hypothalamus and the contralateral retina, and this delay correlated with decreased virus titers in infected tissues, compared with mice infected with H129wt or mice infected with H129wt and H129/pGal10. Although the number of infiltrating T-cells in the brains of mice infected with H129wt, H129wt and H129/IL-16, or H129wt and H129/pGal10 was similar, more Mac-1-positive cells were detected early (postinoculation day 2) in the injected eyes of mice infected with H129wt and H129/IL-16 than in mice infected with H129wt and/or H129wt and H129/pGal10. CONCLUSIONS: These results suggest that early recruitment of Mac-1-positive cells to the injected eye may play a role in delaying virus spread in mice infected with H129wt and the IL-16-expressing recombinant virus. IL-16 delivery vectors could be exploited to prevent or delay HSV-1 infection of the hypothalamus, allowing development of the antiviral immune response and subsequent inhibition of virus spread into the optic nerve and retina.

Apoptosis in the retina during MCMV retinitis in immunosuppressed BALB/c mice.

BACKGROUND: Cytomegalovirus (CMV) retinitis is the most common opportunistic ocular infection observed in immunosuppressed (IS) adult and pediatric patients. Due to the species restriction of the cytomegaloviruses, mice infected with murine CMV (MCMV) have been used to study the pathogenesis of CMV retinitis. OBJECTIVES: The objectives of this study were to determine if retinal glial cells are the targets of MCMV infection and to determine which cells in the retina become apoptotic following inoculation of MCMV via the supraciliary route. STUDY DESIGN: Adult female BALB/c mice were IS with methylprednisolone; one half of the mice were injected with MCMV and one half of the mice were injected with an equivalent volume of tissue culture medium via the supraciliary route. Animals were sacrificed and frozen sections of eyes were stained for MCMV early antigen, RPE65, CD45 or TUNEL; additional slides were double stained with combinations of the above reagents. RESULTS AND CONCLUSIONS: The results indicate that most apoptotic cells in the retina were not virus infected, most apoptotic cells were not infiltrating CD45 positive leukocytes, and retinal glial cells were infected with MCMV but only late in infection. Together, these results suggest that retinal cells that undergo apoptosis during MCMV infection are neurons and that apoptosis of uninfected bystander cells is an important component of the pathogenesis of CMV retinitis.

DNA microarray analysis of the uninoculated eye following anterior chamber inoculation of HSV-1.

PURPOSE: To use DNA microarray to analyze the expression patterns of genes in the uninoculated eye following uniocular anterior chamber inoculation of HSV-1. METHODS: On Day 9 following inoculation of 2 x 10( 4) PFU of HSV-1 (KOS strain) or an equivalent volume of tissue culture medium into one anterior chamber of BALB/c mice, the uninoculated eyes were enucleated, pooled, and total RNA was isolated. cDNA was synthesized from the total RNA. The gene expression patterns were inferred based on the hybridization intensities of the probes on the cDNA array. The hybridization signals were globally normalized and filtered. The data were analyzed using hierarchical and gene tree clustering algorithms. Additional uninoculated eyes collected on Day 9 p.i. were stained for F4/80 and CD19. RESULTS: Compared with the uninoculated eye of control mice, 3800 genes were upregulated at least twofold in the contralateral eye of HSV-1-infected mice. Among the 10 most upregulated genes, T cell-specific protein, MHC II antigen A, and MHC II k region locus 2 were upregulated 179-, 164-, and 162-fold, respectively. Ten T-cell receptor-related genes, 61 cytokine and chemokine genes, and 16 MHC genes were upregulated. Furthermore, 11 immunoglobulin and B cell genes and 11 macrophage-related genes were also upregulated. F4/80+ and CD19+ cells were observed on Day 9 p.i. CONCLUSIONS: The DNA microarray results support the idea that T cells and immunomodulatory factors (cytokines, chemokines) are likely to be involved in HSV-1 retinitis. These results also suggest that B cells and/or macrophages play a role in the pathogenesis of HSV-1 retinitis.

Decrease of murine cytomegalovirus-induced retinitis by intravenous delivery of immediate early protein-3-specific siRNA.

PURPOSE: Retinitis induced by both human and murine cytomegaloviruses following immunosuppression is characterized by progressive loss of retinal architecture, due to necrosis of virus-infected cells as well as widespread apoptosis of uninfected bystander cells. Because small inhibitory RNA molecules (siRNA) can reduce murine cytomegalovirus (MCMV) gene expression and thereby inhibit virus replication in vitro, we tested siRNAs directed against MCMV immediate early protein-3 (IE-3) to determine if MCMV-induced retinitis could be alleviated in vivo. METHODS: Immunosuppressed Balb/c mice (2.0 mg methylprednisolone acetate every 3 days beginning on day -2) were infected with 5 × 10(3) pfu of the K181 strain of MCMV via the supraciliary route. At day 2 post infection, mice were treated with various doses of IE-3-specific siRNA ranging from 0.1 nmol to 10 nmol, in a volume of 20 µL PBS via tail vein injection. Injected eyes were collected at various times post inoculation and subjected to plaque assay for virus titer, MCMV antigen staining, H&E staining, TUNEL assay, and Western blot for MCMV IE-3 protein. RESULTS: Small but significant amounts of fluorescently labeled IE-3-specific siRNA localized to the RPE layer 48 hours after intravenous injection. IE-3-specific siRNA significantly reduced virus titers at all concentrations tested (ranging from 0.1 nmol to 10 nmol), but the most potent effect of siRNA was observed at a dose of 1 nmol. We also observed that IE-3-specific siRNA produced a substantial decrease in MCMV titers and a substantial reduction in bystander cell apoptosis over the time course of virus infection. CONCLUSIONS: Systemic administration of IE-3-specific siRNA could alleviate MCMV retinitis by inhibiting virus replication and subsequent death of uninfected retinal cells.

Role of Bax in death of uninfected retinal cells during murine cytomegalovirus retinitis.

PURPOSE: Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis. Our previous results have shown that caspase 3-dependent and -independent pathways are involved in death of uninfected bystander cells during murine cytomegalovirus (MCMV) retinitis and also that Bcl-2, an important inhibitor of apoptosis via the Bax-mediated mitochondrial pathway, is downregulated during this process. The purpose of this study was to determine whether Bax-mediated mitochondrial damage has a significant role in the death of uninfected retinal cells. METHODS: BALB/c mice, Bax(-/-) mice, or Bax(+/+) mice were immunosuppressed with methylprednisolone and infected with 5 × 10(3) plaque-forming units (PFU) of the K181 strain of MCMV via the supraciliary route. Injected eyes were analyzed by plaque assay, electron microscopy, hematoxylin and eosin (H&E) staining, TUNEL assay, Western blot (for caspase 3, caspase 12, Bax, receptor interacting protein-1 [RIP1] and receptor interacting protein-3 [RIP3]), as well as immunohistochemical staining for MCMV early antigen and cleaved caspase 3. RESULTS: Significantly more Bax was detected in mitochondrial fractions of MCMV-infected eyes than in mitochondrial fractions of mock-infected control eyes. Furthermore, the level of cleaved caspase 3 was significantly lower in MCMV-infected Bax(-/-) eyes than in MCMV-infected Bax(+/+) eyes. However, more caspase 3-independent cell death of uninfected bystander retinal cells and more cleaved RIP1 were observed in Bax(-/-) than in Bax(+/+) eyes. CONCLUSIONS: During MCMV retinitis, Bax is activated and has an important role in death of uninfected bystander retinal cells by caspase 3-dependent apoptosis. Although the exact mechanism remains to be deciphered, active Bax might also prevent death of some types of uninfected retinal cells by a caspase 3-independent pathway.

Virus spread and immune response following anterior chamber inoculation of HSV-1 lacking the Beclin-binding domain (BBD).

The autophagy response induced by HSV-1 infection is antagonized by the Beclin-binding domain (BBD). The purpose of this study was to determine if lack of the BBD affects viral spread and immune response in the eyes and brain. Our results showed that lack of the BBD increases autophagy response and activation of NLRP3 inflammasome, which in turn induces a more rapid innate immune response mediated by macrophage/microglia and NK cells in the injected eye, limiting virus replication and retinal damage. We conclude that autophagy plays a role in controlling HSV-1 infection by more rapid induction of the innate immune response.