Encephalitis and poor neuronal death-mediated control of herpes simplex virus in human inherited RIPK3 deficiency.

Inborn errors of TLR3-dependent type I IFN immunity in cortical neurons underlie forebrain herpes simplex virus-1 (HSV-1) encephalitis (HSE) due to uncontrolled viral growth and subsequent cell death. We report an otherwise healthy patient with HSE who was compound heterozygous for nonsense (R422*) and frameshift (P493fs9*) RIPK3 variants. Receptor-interacting protein kinase 3 (RIPK3) is a ubiquitous cytoplasmic kinase regulating cell death outcomes, including apoptosis and necroptosis. In vitro, the R422* and P493fs9* RIPK3 proteins impaired cellular apoptosis and necroptosis upon TLR3, TLR4, or TNFR1 stimulation and ZBP1/DAI-mediated necroptotic cell death after HSV-1 infection. The patient's fibroblasts displayed no detectable RIPK3 expression. After TNFR1 or TLR3 stimulation, the patient's cells did not undergo apoptosis or necroptosis. After HSV-1 infection, the cells supported excessive viral growth despite normal induction of antiviral IFN-ß and IFN-stimulated genes (ISGs). This phenotype was, nevertheless, rescued by application of exogenous type I IFN. The patient's human pluripotent stem cell (hPSC)-derived cortical neurons displayed impaired cell death and enhanced viral growth after HSV-1 infection, as did isogenic RIPK3-knockout hPSC-derived cortical neurons. Inherited RIPK3 deficiency therefore confers a predisposition to HSE by impairing the cell death-dependent control of HSV-1 in cortical neurons but not their production of or response to type I IFNs.

RIPK3 and caspase 8 collaborate to limit herpes simplex encephalitis.

Invasion of the brain by herpes simplex virus 1 (HSV1) can lead to the development of herpes simplex encephalitis (HSE) that is often associated with significant morbidity and mortality regardless of therapeutic intervention. Both virus and host immune factors dictate HSE onset and progression. Because programmed cell death pathways including necroptosis are important antiviral defense mechanisms in HSV1-associated peripheral diseases, they might also play critical roles in HSV1 neuropathogenesis. HSV1-encoded ICP6 prevents receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis during infection of human cells, but it also acts as a species-dependent inducer of necroptosis in murine cells and thereby restricts virus replication. We therefore used an established mouse model of HSE to investigate RIPK3-mediated necroptosis impact on HSV1 neuropathogenesis. Following corneal HSV1 inoculation, RIPK3 knockout mice showed increased susceptibility to HSE when compared with wildtype mice indicating RIPK3 helps to limit HSE progression. RIPK3-mediated defense against HSE was found to be independent of the kinase domain necessary to drive necroptosis implicating that a death independent function of RIPK3 protects against HSE. Conversely the pro-necroptotic kinase function RIPK3 served to limit viral replication in corneal tissue implicating a tissue-specific RIPK3 function in limiting HSV1. Further evaluation of the kinase-independent mechanism to restrict HSE revealed that the RIPK3 signaling partner, caspase 8, contributes to limiting HSE neuropathogenesis. Increased HSE susceptibility from loss of caspase 8 and RIPK3 correlated with decreased levels of chemokines, cytokines, and antiviral lymphocytes recruitment to the brain. We conclude that RIPK3 contributes toward host control of HSV1 replication in a tissue-specific fashion. Whereas RIPK3-mediated necroptosis restricts virus replication within the cornea, kinase-independent induction of inflammation by RIPK3 in collaboration with caspase 8 restricts virus replication within the brain during HSE neuropathogenesis.

Programmed Cell Death-Dependent Host Defense in Ocular Herpes Simplex Virus Infection.

Herpes simplex virus type 1 (HSV1) remains one of the most ubiquitous human pathogens on earth. The classical presentation of HSV1 infection occurs as a recurrent lesions of the oral mucosa commonly refer to as the common cold sore. However, HSV1 also is responsible for a range of ocular diseases in immunocompetent persons that are of medical importance, causing vision loss that may result in blindness. These include a recurrent corneal disease, herpes stromal keratitis, and a retinal disease, acute retinal necrosis, for which clinically relevant animal models exist. Diverse host immune mechanisms mediate control over herpesviruses, sustaining lifelong latency in neurons. Programmed cell death (PCD) pathways including apoptosis, necroptosis, and pyroptosis serve as an innate immune mechanism that eliminates virus-infected cells and regulates infection-associated inflammation during virus invasion. These different types of cell death operate under distinct regulatory mechanisms but all server to curtail virus infection. Herpesviruses, including HSV1, have evolved numerous cell death evasion strategies that restrict the hosts ability to control PCD to subvert clearance of infection and modulate inflammation. In this review, we discuss the key studies that have contributed to our current knowledge of cell death pathways manipulated by HSV1 and relate the contributions of cell death to infection and potential ocular disease outcomes.

A Mouse Model That Mimics AIDS-Related Cytomegalovirus Retinitis: Insights into Pathogenesis.

With the appearance of the worldwide AIDS pandemic four decades ago came a number of debilitating opportunistic infections in patients immunosuppressed by the pathogenic human retrovirus HIV. Among these was a severe sight-threatening retinal disease caused by human cytomegalovirus (HCMV) that remains today a significant cause of vision loss and blindness in untreated AIDS patients without access or sufficient response to combination antiretroviral therapy. Early investigations of AIDS-related HCMV retinitis quickly characterized its hallmark clinical features and unique histopathologic presentation but did not begin to identify the precise virologic and immunologic events that allow the onset and development of this retinal disease during HIV-induced immunosuppression. Toward this end, several mouse models of experimental cytomegalovirus retinitis have been developed to provide new insights into the pathophysiology of HCMV retinitis during AIDS. Herein, we provide a summary and comparison of these mouse models of AIDS-related HCMV retinitis with particular emphasis on one mouse model developed in our laboratory in which mice with a murine acquired immunodeficiency syndrome (MAIDS) of murine retrovirus origin develops a reproducible and well characterized retinitis following intraocular infection with murine cytomegalovirus (MCMV). The MAIDS model of MCMV retinitis has advanced the discovery of many clinically relevant virologic and immunologic mechanisms of virus-induced retinal tissue destruction that are discussed and summarized in this review. These findings may extend to the pathogenesis of AIDS-related HCMV retinitis and other AIDS-related opportunistic virus infections.

Atypical cytomegalovirus retinal disease in pyroptosis-deficient mice with murine acquired immunodeficiency syndrome.

Pyroptosis is a caspase-dependent programmed cell death pathway that initiates and sustains inflammation through release of pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 following formation of gasdermin D (GSDMD)-mediated membrane pores. To determine the possible pathogenic contributions of pyroptosis toward development of full-thickness retinal necrosis during AIDS-related human cytomegalovirus retinitis, we performed a series of studies using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS). Initial investigations demonstrated significant transcription and translation of key pyroptosis-associated genes within the ocular compartments of MCMV-infected eyes of mice with MAIDS. Subsequent investigations compared MCMV-infected eyes of groups of wildtype MAIDS mice with MCMV-infected eyes of groups of caspase-1-/- MAIDS mice, GSDMD-/- MAIDS mice, or IL-18-/- MAIDS mice to explore a possible contribution of pyroptosis towards the pathogenesis of MAIDS-related MCMV retinitis. Histopathologic analysis revealed typical full-thickness retinal necrosis in 100% of MCMV-infected eyes of wildtype MAIDS mice. In sharp contrast, none (0%) of MCMV-infected eyes of MAIDS mice that were deficient in either caspase-1, GSDMD, or IL-18 developed full-thickness retinal necrosis but instead exhibited an atypical pattern of retinal disease characterized by thickening and proliferation of the retinal pigmented epithelium layer with relative sparing of the neurosensory retina. Surprisingly, MCMV-infected eyes of all groups of deficient MAIDS mice harbored equivalent intraocular amounts of infectious virus as seen in MCMV-infected eyes of groups of wildtype MAIDS mice despite failure to develop full-thickness retinal necrosis. We conclude that pyroptosis plays a significant role in the development of full-thickness retinal necrosis during the pathogenesis of MAIDS-related MCMV retinitis. This observation may extend to the pathogenesis of AIDS-related HCMV retinitis and other AIDS-related opportunistic virus infections.

Evidence for the involvement of interleukin-1α during development of experimental cytomegalovirus retinitis in immunosuppressed mice.

Interleukin-1α (IL-1α) is an alarmin involved in the recruitment of macrophages and neutrophils during tissue inflammation. IL-1α can undergo cleavage by proteases, such as calpain-1, that enhances IL-1α binding to its receptor, although proteolytic cleavage is not necessary for biological activity. Macrophages and neutrophils are involved in the retinal inflammation associated with development of AIDS-related human cytomegalovirus (HCMV) retinitis. We therefore performed studies to test the hypothesis that IL-1α gene expression is stimulated intraocularly during retinitis development using two mouse models of murine cytomegalovirus (MCMV) retinitis that differ in method of immunosuppression, one by retrovirus-induced immunosuppression (MAIDS) and the other by corticosteroid-induced immunosuppression. MCMV-infected eyes of groups of retinitis-susceptible mice with MAIDS of 10 weeks duration (MAIDS-10 mice) and retinitis-susceptible corticosteroid-treated mice showed significant stimulation of IL-1α mRNA. Western blot analysis confirmed IL-1α protein production within the MCMV-infected eyes of MAIDS-10 mice. Whereas significant intraocular calpain-1 mRNA and protein production were also observed within MCMV-infected eyes of MAIDS-10 mice, the MCMV-infected eyes of retinitis-susceptible corticosteroid-treated mice showed a pattern of mRNA synthesis equivalent to that found within the MCMV-infected eyes of healthy mice that fail to develop retinitis. Our findings suggest a role for the alarmin IL-1α in the pathogenesis of MCMV retinitis in immunosuppressed mice. These findings may extend to the pathogenesis of HCMV retinitis in patients with AIDS or other forms of immunosuppression.

Transcriptional analysis of immune response genes during pathogenesis of cytomegalovirus retinitis in mice with murine acquired immunodeficiency syndrome.

Human cytomegalovirus (HCMV) is an opportunistic human herpesvirus that causes a sight-threatening retinitis in immunosuppressed patients, especially those with AIDS. Using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunodeficiency (MAIDS), we have been attempting to define with greater clarity the immunologic mechanisms that contribute to the progression of AIDS-related HCMV retinitis in the unique immunosuppressive setting of HIV infection. Toward this end, we provide herein a comprehensive assessment of immune response gene expression during the onset and development of MAIDS-related MCMV retinitis employing NanoString nCounter. In so doing, we analyzed and compared the intraocular expressions of 561 immune response genes within MCMV-infected eyes of groups of healthy mice, MCMV-infected mice with MAIDS of 4 weeks' (MAIDS-4) duration, and MCMV-infected eyes of mice with MAIDS of 10 weeks' (MAIDS-10) duration. These animal groups show a progression of retinal disease from absolute resistance to retinitis development in healthy mice to the development of classic full-thickness retinal necrosis in MAIDS-10 mice but through an intermediate stage of retinal disease development in MAIDS-4 mice. Our findings showed that increased susceptibility to MCMV retinitis during the progression of MAIDS is associated with robust upregulation or downregulation of a surprisingly large number of immune response genes that operate within several immune response pathways often unique to each animal group. Analysis of 14 additional immune response genes associated with programmed cell death pathways suggested involvement of necroptosis and pyroptosis during MAIDS-related MCMV retinitis pathogenesis. Use of the NanoString nCounter technology provided new and unexpected information on the immunopathogenesis of retinitis within MCMV-infected eyes of mice with retrovirus-induced immunosuppression. Our findings may provide new insights into the immunologic events that operate during the pathogenesis of AIDS-related HCMV retinitis.

Parthanatos-associated proteins are stimulated intraocularly during development of experimental murine cytomegalovirus retinitis in mice with retrovirus-induced immunosuppression.

The mechanisms that contribute to retinal tissue destruction during the onset and progression of AIDS-related human cytomegalovirus (HCMV) retinitis remain unclear. Evidence for the stimulation of multiple cell death pathways including apoptosis, necroptosis, and pyroptosis during the pathogenesis of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS) has been reported. Parthanatos is a caspase-independent cell death pathway mediated by rapid overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) and distinct from other cell death pathways. Using the MAIDS model of MCMV retinitis, studies were performed to test the hypothesis that intraocular MCMV infection of mice with MAIDS stimulates parthanatos-associated messenger RNAs (mRNAs) and proteins within the eye during the development of retinal necrosis that takes place by 10 days after MCMV infection. MCMV-infected eyes of MAIDS mice exhibited significant stimulation of PARP-1 mRNA and proteins at 3 days after infection but declined thereafter at 6 and 10 days after infection. Additional studies showed the intraocular stimulation of mRNAs or proteins before MCMV retinitis development for two additional participants in parthanatos, polymer of ADP-ribose and poly (ADP-ribose) glycohydrolase. These results provide new evidence for a role for parthanatos during MAIDS-related MCMV retinitis that may also extend to AIDS-related HCMV retinitis.

SOCS and Herpesviruses, With Emphasis on Cytomegalovirus Retinitis.

Suppressor of cytokine signaling (SOCS) proteins provide selective negative feedback to prevent pathogeneses caused by overstimulation of the immune system. Of the eight known SOCS proteins, SOCS1 and SOCS3 are the best studied, and systemic deletion of either gene causes early lethality in mice. Many viruses, including herpesviruses such as herpes simplex virus and cytomegalovirus, can manipulate expression of these host proteins, with overstimulation of SOCS1 and/or SOCS3 putatively facilitating viral evasion of immune surveillance, and SOCS suppression generally exacerbating immunopathogenesis. This is particularly poignant within the eye, which contains a diverse assortment of specialized cell types working together in a tightly controlled microenvironment of immune privilege. When the immune privilege of the ocular compartment fails, inflammation causing severe immunopathogenesis and permanent, sight-threatening damage may occur, as in the case of AIDS-related human cytomegalovirus (HCMV) retinitis. Herein we review how SOCS1 and SOCS3 impact the virologic, immunologic, and/or pathologic outcomes of herpesvirus infection with particular emphasis on retinitis caused by HCMV or its mouse model experimental counterpart, murine cytomegalovirus (MCMV). The accumulated data suggests that SOCS1 and/or SOCS3 can differentially affect the severity of viral diseases in a highly cell-type-specific manner, reflecting the diversity and complexity of herpesvirus infection and the ocular compartment.

Suppressor of Cytokine Signaling 1 (SOCS1) and SOCS3 Are Stimulated within the Eye during Experimental Murine Cytomegalovirus Retinitis in Mice with Retrovirus-Induced Immunosuppression.

AIDS-related human cytomegalovirus retinitis remains the leading cause of blindness among untreated HIV/AIDS patients worldwide. To study mechanisms of this disease, we used a clinically relevant animal model of murine cytomegalovirus (MCMV) retinitis with retrovirus-induced murine AIDS (MAIDS) that mimics the progression of AIDS in humans. We found in this model that MCMV infection significantly stimulates ocular suppressor of cytokine signaling 1 (SOCS1) and SOCS3, host proteins which hinder immune-related signaling by cytokines, including antiviral type I and type II interferons. The present study demonstrates that in the absence of retinal disease, systemic MCMV infection of mice without MAIDS, but not in mice with MAIDS, leads to mild stimulation of splenic SOCS1 mRNA. In sharp contrast, when MCMV is directly inoculated into the eyes of retinitis-susceptible MAIDS mice, high levels of intraocular SOCS1 and SOCS3 mRNA and protein are produced which are associated with significant intraocular upregulation of gamma interferon (IFN-γ) and interleukin-6 (IL-6) mRNA expression. We also show that infiltrating macrophages, granulocytes, and resident retinal cells are sources of intraocular SOCS1 and SOCS3 protein production during development of MAIDS-related MCMV retinitis, and SOCS1 and SOCS3 mRNA transcripts are detected in retinal areas histologically characteristic of MCMV retinitis. Furthermore, SOCS1 and SOCS3 are found in both MCMV-infected cells and uninfected cells, suggesting that these SOCS proteins are stimulated via a bystander mechanism during MCMV retinitis. Taken together, our findings suggest a role for MCMV-related stimulation of SOCS1 and SOCS3 in the progression of retinal disease during ocular, but not systemic, MCMV infection.IMPORTANCE Cytomegalovirus infection frequently causes blindness in untreated HIV/AIDS patients. This virus manipulates host cells to dysregulate immune functions and drive disease. Here, we use an animal model of this disease to demonstrate that cytomegalovirus infection within eyes during retinitis causes massive upregulation of immunosuppressive host proteins called SOCS. As viral overexpression of SOCS proteins exacerbates infection with other viruses, they may also enhance cytomegalovirus infection. Alternatively, the immunosuppressive effect of SOCS proteins may be protective against immunopathology during cytomegalovirus retinitis, and in such a case SOCS mimetics or overexpression treatment strategies might be used to combat this disease. The results of this work therefore provide crucial basic knowledge that contributes to our understanding of the mechanisms of AIDS-related cytomegalovirus retinitis and, together with future studies, may contribute to the development of novel therapeutic targets that could improve the treatment or management of this sight-threatening disease.

Reduced frequency of murine cytomegalovirus retinitis in C57BL/6 mice correlates with low levels of suppressor of cytokine signaling (SOCS)1 and SOCS3 expression within the eye during corticosteroid-induced immunosuppression.

AIDS-related human cytomegalovirus retinitis remains a leading cause of blindness worldwide. We compared two C57BL/6 mouse models of experimental murine cytomegalovirus (MCMV) retinitis for intraocular expression of suppressors of cytokine signaling (SOCS)1 and SOCS3, host proteins that are inducible negative feedback regulators of cytokine signaling. These mouse models differed in method of immune suppression, one by retrovirus-induced immune suppression (MAIDS) and the other by corticosteroid-induced immune suppression. Following subretinal injection of MCMV to induce retinitis, intraocular SOCS1 and SOCS3 were only mildly stimulated, and often without significance, within MCMV-infected eyes during the progression of MCMV retinitis in corticosteroid-immunosuppressed mice, contrary to MCMV-infected eyes of mice with MAIDS that showed significant high stimulation of SOCS1 and SOCS3 expression in agreement with previous findings. Frequency and severity of retinitis as well as amounts of intraocular infectious MCMV in corticosteroid-immunosuppressed mice were also unexpectedly lower than values previously reported for MAIDS animals during MCMV retinitis. These data reveal a major difference between two mouse models of experimental MCMV retinitis and suggest a possible link between the amplitude of SOCS1 and SOCS3 stimulation and severity of disease in these models.

Murine cytomegalovirus infection of mouse macrophages stimulates early expression of suppressor of cytokine signaling (SOCS)1 and SOCS3.

Human cytomegalovirus (HCMV) is a species-specific ß-herpesvirus that infects for life up to 80% of the world's population and causes severe morbidity in at-risk immunocompromised populations. Suppressors of cytokine signaling (SOCS)1 and SOCS3 are host proteins that act as inducible negative feedback regulators of cytokine signaling and have been implicated in several ocular diseases and viral infections. We recently found in our mouse model of experimental cytomegalovirus retinitis that subretinally-injected murine cytomegalovirus (MCMV) stimulates ocular SOCS1 and SOCS3 during retrovirus-induced immune suppression of murine AIDS (MAIDS), and that infiltrating macrophages are prominent cellular sources of retinal SOCS1 and SOCS3 expression. Herein we investigate possible virologic mechanisms whereby MCMV infection may stimulate SOCS1 and/or SOCS3 expression in cell culture. We report that infection of IC-21 mouse macrophages with MCMV propagated through the salivary glands of BALB/c mice, but not from tissue culture in C57BL/6 fibroblasts, transiently stimulates SOCS1 and SOCS3 mRNA transcripts, but not SOCS5 mRNA. Viral tegument proteins are insufficient for this stimulation, as replication-deficient UV-inactivated MCMV fails to stimulate SOCS1 or SOCS3 in IC-21 macrophages. By contrast, infection of murine embryonic fibroblasts (MEFs) with either productive MCMV or UV-inactivated MCMV significantly stimulates SOCS1 and SOCS3 mRNA expression early after infection. Treatment of MCMV-infected IC-21 mouse macrophages with the antiviral drug ganciclovir significantly decreases MCMV-stimulated SOCS3 expression at 3 days post-infection. These data suggest cell type-specific, different roles for viral immediate early or early gene expression and/or viral tegument proteins in the early stimulation of SOCS1 and SOCS3 during MCMV infection. Furthermore, putative biphasic stimulation of SOCS3 during late MCMV infection of IC-21 mouse macrophages may occur by divergent virologic mechanisms.

Viral forensic genomics reveals the relatedness of classic herpes simplex virus strains KOS, KOS63, and KOS79.

Herpes simplex virus 1 (HSV-1) is a widespread global pathogen, of which the strain KOS is one of the most extensively studied. Previous sequence studies revealed that KOS does not cluster with other strains of North American geographic origin, but instead clustered with Asian strains. We sequenced a historical isolate of the original KOS strain, called KOS63, along with a separately isolated strain attributed to the same source individual, termed KOS79. Genomic analyses revealed that KOS63 closely resembled other recently sequenced isolates of KOS and was of Asian origin, but that KOS79 was a genetically unrelated strain that clustered in genetic distance analyses with HSV-1 strains of North American/European origin. These data suggest that the human source of KOS63 and KOS79 could have been infected with two genetically unrelated strains of disparate geographic origins. A PCR RFLP test was developed for rapid identification of these strains.

Murine cytomegalovirus downregulates interleukin-17 in mice with retrovirus-induced immunosuppression that are susceptible to experimental cytomegalovirus retinitis.

Interleukin-17 (IL-17), a pro-inflammatory cytokine produced by CD4+ Th17 cells, has been associated with the pathogenesis of several autoimmune diseases including uveitis. The fate of IL-17 during HIV/AIDS, however, remains unclear, and a possible role for IL-17 in the pathogenesis of AIDS-related diseases has not been investigated. Toward these ends, we performed studies using a well-established animal model of experimental murine cytomegalovirus (MCMV) retinitis that develops in C57/BL6 mice with retrovirus-induced immunosuppression (MAIDS). After establishing baseline levels for IL-17 production in whole splenic cells of healthy mice, we observed a significant increase in IL-17 mRNA levels in whole splenic cells of mice with MAIDS of 4-weeks (MAIDS-4), 8-weeks (MAIDS-8), and 10-weeks (MAIDS-10) duration. In contrast, enriched populations of splenic CD4+ T cells, splenic macrophages, and splenic neutrophils exhibited a reproducible decrease in levels of IL-17 mRNA during MAIDS progression. To explore a possible role for IL-17 during the pathogenesis of MAIDS-related MCMV retinitis, we first demonstrated constitutive IL-17 expression in retinal photoreceptor cells of uninfected eyes of healthy mice. Subsequent studies, however, revealed a significant decrease in intraocular levels of IL-17 mRNA and protein in MCMV-infected eyes of MAIDS-10 mice during retinitis development. That MCMV infection might cause a remarkable downregulation of IL-17 production was supported further by the finding that systemic MCMV infection of healthy, MAIDS-4, or MAIDS-10 mice also significantly decreased IL-17 mRNA production by splenic CD4+ T cells. Based on additional studies using IL-10 -/- mice infected systemically with MCMV and IL-10 -/- mice with MAIDS infected intraocularly with MCMV, we propose that MCMV infection downregulates IL-17 production via stimulation of suppressor of cytokine signaling (SOCS)-3 and interleukin-10.

Evidence for multiple cell death pathways during development of experimental cytomegalovirus retinitis in mice with retrovirus-induced immunosuppression: apoptosis, necroptosis, and pyroptosis.

AIDS-related human cytomegalovirus (HCMV) retinitis remains a major ophthalmologic problem worldwide. Although this sight-threatening disease is well characterized clinically, many pathogenic issues remain unresolved, among them a basic understanding of the relative roles of cell death pathways during development of retinal tissue destruction. Using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS), we initially investigated MCMV-infected eyes for evidence of apoptosis-associated molecules in mice with MAIDS of 4 weeks' (MAIDS-4) and 10 weeks' (MAIDS-10) duration, which were resistant and susceptible to retinal disease, respectively, but which harbored equivalent amounts of infectious MCMV. Whereas MCMV-infected eyes of MAIDS-4 mice showed little evidence of apoptosis-associated molecules, MCMV-infected eyes of MAIDS-10 mice showed significant amounts of tumor necrosis factor alpha (TNF-α), TNF receptors 1 and 2, active caspase 8, active caspase 3, TNF-related apoptosis-inducing ligand (TRAIL), TRAIL-R(DR5), Fas, and Fas ligand mRNAs and/or proteins, all detected at peak amounts prior to development of most severe retinal disease. Immunohistochemical staining showed macrophages, granulocytes (neutrophils), Müller cells, and microglial cells as TNF-α sources. Remarkably, quantification of apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay suggested that apoptosis contributed minimally to retinal disease in MCMV-infected eyes of MAIDS-10 mice. Subsequent studies demonstrated that MCMV-infected eyes of MAIDS-10 mice, but not MAIDS-4 mice, showed evidence of significant increases in molecules associated with two additional cell death pathways, necroptosis (receptor-interacting protein 1 [RIP1] and RIP3 mRNAs) and pyroptosis (caspase 1, interleukin 1ß [IL-1ß], and IL-18 mRNAs). We conclude that apoptosis, necroptosis, and pyroptosis participate simultaneously during MAIDS-related MCMV retinitis, and all may play a role during AIDS-related HCMV retinitis.

Macrophage activation associated with chronic murine cytomegalovirus infection results in more severe experimental choroidal neovascularization.

The neovascular (wet) form of age-related macular degeneration (AMD) leads to vision loss due to choroidal neovascularization (CNV). Since macrophages are important in CNV development, and cytomegalovirus (CMV)-specific IgG serum titers in patients with wet AMD are elevated, we hypothesized that chronic CMV infection contributes to wet AMD, possibly by pro-angiogenic macrophage activation. This hypothesis was tested using an established mouse model of experimental CNV. At 6 days, 6 weeks, or 12 weeks after infection with murine CMV (MCMV), laser-induced CNV was performed, and CNV severity was determined 4 weeks later by analysis of choroidal flatmounts. Although all MCMV-infected mice exhibited more severe CNV when compared with control mice, the most severe CNV developed in mice with chronic infection, a time when MCMV-specific gene sequences could not be detected within choroidal tissues. Splenic macrophages collected from mice with chronic MCMV infection, however, expressed significantly greater levels of TNF-α, COX-2, MMP-9, and, most significantly, VEGF transcripts by quantitative RT-PCR assay when compared to splenic macrophages from control mice. Direct MCMV infection of monolayers of IC-21 mouse macrophages confirmed significant stimulation of VEGF mRNA and VEGF protein as determined by quantitative RT-PCR assay, ELISA, and immunostaining. Stimulation of VEGF production in vivo and in vitro was sensitive to the antiviral ganciclovir. These studies suggest that chronic CMV infection may serve as a heretofore unrecognized risk factor in the pathogenesis of wet AMD. One mechanism by which chronic CMV infection might promote increased CNV severity is via stimulation of macrophages to make pro-angiogenic factors (VEGF), an outcome that requires active virus replication.

Systemic reduction of interleukin-4 or interleukin-10 fails to reduce the frequency or severity of experimental cytomegalovirus retinitis in mice with retrovirus-induced immunosuppression.

Interleukin-4 (IL-4) and interleukin-10 (IL-10) are key cytokines whose increased production during systemic HIV infection has been associated with decreased cellular immunity during AIDS. We examined whether HIV-induced stimulation of IL-4 or IL-10 production leads to increased susceptibility to AIDS-related human cytomegalovirus retinitis. It was confirmed that there were increased amounts of IL-4 and IL-10 mRNA levels in mice with MAIDS of 10 weeks duration when most susceptible to MCMV retinitis. Surprisingly, however, MCMV-infected eyes of IL-4 -/- and IL-10 -/- mice with MAIDS of 8 weeks duration exhibited retinitis and infectious virus equivalent to that observed in MCMV-infected eyes of wild-type mice with MAIDS. We conclude that neither IL-4 nor IL-10 alone play a role in increased susceptibility to MAIDS-related MCMV retinitis, but may work collectively with other retrovirus-induced immunosuppressive factors to allow for retinal disease.

Nicotine treatment alters NF-kappaB expression in human cytomegalovirus-infected ARPE-19 cells.

PURPOSE: To test if nicotine counteracts the dampening effect of human cytomegalovirus (HCMV) infection of NF-kappaB in retinal pigment epithelial (RPE) cells, thereby increasing the permissiveness of RPE cells for HCMV replication. METHODS: Human ARPE-19 cells were transfected with NF-kappaB luciferase DNA, inoculated with HCMV at 24 hr post-transfection, and maintained in the absence or presence of a physiologic dose of nicotine at 1 hr prior to HCMV inoculation. RESULTS: Whereas HCMV-infected ARPE-19 cells without nicotine treatment showed a dramatic decrease in NF-kappaB levels, nicotine treatment reduced this decrease but did not abolish it completely. Nicotine treatment of uninfected ARPE-19 cells had no effect on baseline NF-kappaB levels. CONCLUSIONS: Treatment of HCMV-infected ARPE-19 cells with nicotine at a physiologic dose dampened the downregulation of NF-kappaB observed in HCMV-infected ARPE-19 cells without nicotine treatment. We conclude that nicotine can serve as a cofactor to stimulate productive, lytic replication of HCMV.

Interleukin-2 immunotherapy and AIDS-related cytomegalovirus retinitis.

Cytokines are small proteins produced by T lymphocytes that mediate immune responses. Those produced by the CD4+ Th1 subset induce cell-mediated immunity, whereas those produced by the CD4+ Th2 subset are more efficient at stimulating immunoglobulin production. The goal of cytokine immunotherapy is prevention or reduction of disease progression through stimulation of cell-mediated immunity (i.e., immune reconstitution) by administration of an exogenous Th1 cytokine such as interleukin-2 (IL-2). Cytokine immunotherapy has its origins in cancer immunobiology where IL-2 has been used successfully to manage several human cancers (metastatic melanoma, acute myelogenous leukemia, and metastatic renal cell carcinoma). More recent work has demonstrated cytokine immunotherapy to be effective at improving immune responses in patients with HIV-1 disease. To explore cytokine immunotherapy for sight-threatening AIDS-related human cytomegalovirus (HCMV) retinitis, we developed a mouse model of experimental murine cytomegalovirus (MCMV) retinitis that employs mice with MAIDS, a retrovirus-induced immunodeficiency syndrome. Systemic cytokine immunotherapy with IL-2, but not with interleukin-12 (IL-12), provides absolute protection against MAIDS-related MCMV retinitis by stimulation of the perforin-mediated pathway of cytotoxicity used by natural killer cells and cytotoxic CD8+ T cells to kill virus-infected cells. Our findings warrant additional studies on the use of cytokine immunotherapy for management of HCMV retinitis (and possibly other opportunistic infections) during HIV-1-induced immunodeficiency. We envision systemic cytokine immunotherapy as an altemative or adjunct to traditional antiviral chemotherapy for optimal management of AIDS-related HCMV retinitis.