Antisense-oligonucleotides targeting tumor necrosis factor-alpha in murine herpes simplex virus type 1 retinitis

1. INTRODUCTION

1.1 Acute retinal necrosis (ARN)

ARN syndrome is a well-known clinical entity that was first described in 1971 (Urayama et al., 1971). ARN is a type of retinitis that affects both healthy and immunocompromised patients (Silverstein et al., 1997; Batisse et al., 1996; Nussenblatt et al., 1989). Patients typically experience blurred vision, eye pain, and light sensitivity, in one or sometimes both eyes. Several members of the herpes virus family including herpes simplex virus (HSV)-1, HSV-2, varicella zoster virus (VZV) and, rarely, cytomegalovirus (CMV) are the main causing viruses (Silverstein et al., 1997; Atherton et al., 2001; Lewis et al., 1989; Thompson et al., 1994; Hellinger et al., 1993; Ganatra et al., 2000).

1.1.1 Virology of herpes viruses

They all have a large number of enzymes available that are involved in nucleic acid metabolism, DNA synthesis, and possibly the procession of proteins. The synthesis of DNA and the assembly of the capsid take place in the nucleus. The production of infectious virus particles may lead to the destruction of the infected cell. Furthermore, the herpes viruses establish latent infection in their natural hosts (Roizman et al., 1996). During latency, the herpes virus genomes form closed circular molecules, and only a small number of viral proteins are expressed. There is evidence that selected regulatory genes are active and may maintain latency, but neither the mechanisms to keep the status of latency nor the factors that cause reactivation of viral replication are yet completely known. After reactivation, infectious viruses are transported to peripheral tissues, e.g., by axonal transport in HSV infection (or retrograde transport back to the locus of primary infection). The immune response of the host may determine whether reactivation may result in a symptomatic or asymptomatic course of herpetic disease (Roizman et al., 1996; Roizman & Sears , 1996)

1.1.2 Pathogenesis of HSV-1 induced experimental acute retinal necrosis

The acute stage of ARN is characterized by necrotizing retinitis of all retinal layers. The retinal vessels in the diseased area show fibrinoid necrosis of the vessel wall and vascular occlusion. The retinal pigment epithelium (RPE) shows focal necrosis and is occasionally separated from Bruch’s membrane. The necrotizing retinal cells may reach the overlying vitreous body, where inflammatory cells group around it. The necrotizing retina is mostly sharply demarcated adjacent to the intact retina. In the marginal areas, intranuclear inclusions can be noted histologically, and by electron microscopy, virus particles can be detected in the retinal cells (Minckler et al., 1976; Cibis et al.,1978; Johnson et al.,1977). The adjacent choroid shows severe choroiditis with vascular occlusions. At the same time, optic nerve neuritis and papillitis arises. Inflammatory cells are infiltrating the aqueous humor and the anterior chamber angle. The iris and ciliary body show non-granulomatous and granulomatous cell infiltration and perivasculitis (Culbertson et al., 1982; Naumann et al., 1968; Culbertson et al.,  1986). In the healing phase, the process leads to complete disintegration of the retina and the optic nerve, with reactive metaplasia of the retinal pigment epithelium (Cogan et al., 1964; Rummelt et al., 1992).

Herpes viruses were demonstrated in the retinal lesions and vitreous body in ARN patients by culture methods, histology, electron microscopy, immunohistochemistry, and by polymerase chain-reaction methods (Lewis et al., 1989; Culbertson et al., 1982; Pavan-Langston & Dunkel, 1989; Forster et al., 1990; Culbertson et al., 1986; Pepose & Whittum-Hudson, 1987). After primary infection or reactivation from latency, herpes virus replication follows. From animal experiments (Forster et al., 1990) it is known that viruses migrate through the ipsilateral parasympathetic fibers of the oculomotor nerve that serve the iris and ciliary bodies in the central nervous system. The viral replication within the CNS is fairly well limited to the nucleus of the visual system and to the suprachiasmatic area of the hypothalamus. Viruses than migrate from the brain to the retina via retrograde axonal transport through the optic nerve, along the endocrine-optic path between the retina and the suprachiasmatic nucleus of the hypothalamus.

At this site, the viral invasion can spread out to the contralateral regions, which may explain the involvement of the fellow eye in patients with bilateral acute retinal necrosis (BARN). Along the optic nerve, the viruses may also reach the ganglionic cells of the contralateral retina (Pettit et al., 1965).

The retinal pathology represents a viral-induced cytopathology (Holland et al., 1987; Whittum-Hudson & Pepose, 1987). However, the accompanying immune reactions are decisive for the further inflammatory process that finally results in the development of retinal necrosis (Holland et al., 1987.; Whittum et al., 1984). Local as well as systemic factors come into effect here (Whittum et al., 1984). It has been shown that the retinal HSV infection is under the control of T lymphocytes in experimental herpetic retinitis (Whittum-Hudson et al., 1985). A contribution of T lymphocytes in the pathogenesis of human ARN has been suggested (Verjans et al., 1998).

The severe vascular occlusions lead to ischemia of the retina and choroid and further promote the development of necrosis. The massive breakdown of the blood-retina barrier with the resulting increase of the protein content in the vitreous is associated with a proliferative effect on the pigment epithelium and the fibroblasts.  This may further support the development of proliferative vitreoretinopathy (PVR). The necrotic-related retinal tears and the developing traction from the vitreous space then finally result in the emergence of retinal detachment.

1.2 von Szily model

von Szily was the first scientist who reported about acute retinal necrosis syndrome in rabbits in 1924 (Von Szily, 1924). After injecting replication herpes simplex virus into the right anterior chamber of a rabbit, a rapidly destructive retinitis of the opposite (left) eye developed. The histopathologic analysis of the inoculated eye, however, disclosed that the retina of the injected (right) eye was more or less completely spared of this destructive phenomenon. 

It has been shown that inoculation of the KOS strain of HSV-1 into the anterior chamber of one eye of BALB/c mice induced characteristic retinal changes, including a devastating inflammatory reaction within the posterior segment of the uninoculated eye, resulting in pan-necrosis of the retina 10 to 14 days post inoculation (PI) (Whittum et al., 1984).

Diverse inbred strains of mice have been shown to vary considerably in their resistance and susceptibility to that HSV induced retinal necrosis syndrome. The different strains of BALB/c, C57BL/6 and F1 hybrid had been studied to define the resistance and susceptibility to HSV retinitis. Injected eyes of BALB/c mice showed an anterior uveitis with HSV-1 antigens in the anterior segment and an intact retina that was free of HSV antigens. The retina of the contralateral uninjected eye, in contrast, was necrotic and contained HSV-1 antigens. In both, C57BL/6 and F1 mice, HSV antigens were limited to the structures of the anterior segment in the injected eye, whereas, in contrast to BALB/c mice, the contralateral retina appeared histologically normal and contained no viral antigens. Furthermore, these strains also remained relatively resistant to retinal infection despite being immunosuppressed by radiation (Pepose et al., 1987; Whittum & Pepose, 1988). Another study showed that DBA/2 mice were mildly resistant to HSV-1 retinitis in the uninoculated eye (Kielty et al., 1987).

Although HSV inoculated into anterior chamber of mouse in general induced retinitis, the different strains of HSV induced different courses of that disease. Whereas HSV-1 produces a rapid, explosive retinitis that led to destruction of all cell layers of contralateral retina, HSV-2 induced a retinitis in the ipsilateral eye that was more gradual in onset (Dix et al., 1987).

Inoculation of HSV-1 (KOS) into the anterior chamber of BALB/c mouse eyes produces an intense inflammatory reaction at the inoculation site. Intensity and speed of the inflammatory reaction are dose-dependent over a wide range: 2×10² to 2×10⁵ plaque forming units (PFU) HSV-1. The dose of 2×10⁴ PFU was chosen by the researchers for the following reasons: (1) this particular dose of virus induced anterior chamber-associated immune deviation (ACAID), i.e., recipient mice produced high titers of circulating anti-HSV-1 antibody, but failed to develop T-cell immunity as measured by the capacity to express HSV-1 specific delayed type hypersensitivity (DTH); (2) when inoculated subcutaneously, this dose of virus regularly induced vigorous DTH to HSV-1 as well as humoral anti-HSV-1 responses; and (3) lid vesicles were observed uncommonly after AC inoculation of 2×10⁴PFU HSV, whereas at higher doses lid lesions regularly occurred, raising the possibility that auto-inoculation of virus by infected mice would unduly confound the analysis. Within this dose range, the only visible manifestation of disease in AC-inoculated mice was in the injected eye. None of the mice died or developed signs of disease at other sites (Whittum et al., 1983; Whittum et al.,1984).

In the HSV-1 inoculated eyes, the inflammatory reaction commonly involves the entire anterior segment. The cornea rapidly becomes edematous, and an inflammatory cell infiltration and neovascularization occurs. The central stromal infiltrate peaks by day 7 and resolved completely by day 21. The extensive neovascularization reaches its maximum intensity by day 14. The extensive loss of corneal clarity observed by slit lamp corresponds with the edema and disruption of the stromal architecture. By day 5, the corneal endothelium is destroyed. The anterior chamber of injected eyes contains an increasingly severe cell and flare reaction that is already visible by 1 day after virus inoculation. Over 21 days, the anterior chamber becomes progressively more shallow (loss of form or depth), and it is occupied by a fibrovascular tissue development between day 14 and 21 PI. Iris infiltration and loss of iris integrity are both evident at day 1. Iris atrophy (loss of iris stroma and vessels) occurs by day 3 (Whittum et al., 1984).

In contralateral eye, the inflammatory reaction involves mainly the posterior segment.  The infectious process in the retina of the uninoculated eye has been divided in three phases: acute retinitis, retinal necrosis, and resolution (Cousins et al., 1989). Acute retinitis is observed between days 7 and 9 PI. The retina remains normal until day 7 PI. At this time, small foci of inflammatory cells appear around one or two small vessels in the ganglion cell layer (GCL). These foci are limited generally to one lateral aspect of the GCL per eye, midway between the optic nerve and the anterior edge of the retina (ora serrata). The remainder of the GCL appears normal at this time, as do the outer layers of the retina and underlying choroid.

The retinal necrosis begins on day 10 PI. By day 10, a large necrotic area containing a mixed inflammatory infiltrate extends posteriorly through the entire retina. The choroid underlying the necrotic area also is infiltrated. Inflammatory cells can occasionally be seen around the optic nerve at this time. By day 14 PI, the retina is completely necrotic with inflammatory cells, debris and numerous plasma cells interspersed throughout the disrupted retinal cells. In addition, the choroid is infiltrated extensively.

The resolution phase usually begins on or about day 15 PI. The remnants of the retina are organized into a fibrocellular scar, and the ocular inflammation is gradually resolved (Azumi et al., 1994; Zaltas et al., 1992; Azumi & Atherton, 1998; Cousins et al., 1989).

Following anterior chamber inoculation of HSV-1 into one eye of BALB/c mice, a certain pattern of ocular pathology as well as systemic immune response emerges. It is a suppression of DTH responses to the injected HSV with an intact HSV-1 neutralizing antibody response. This phenomenon is termed anterior chamber associated immune deviation (Whittum et al., 1983). However, compared with the BALB/c strain, the AC infection in C57BL/6 mice was not followed by a contralateral retinitis, while the mice showed a vigorous HSV-1 specific DTH response. Therefore, is has been suggested that the absence of contralateral retinitis might be linked to the induction of virus-specific DTH response. It has been speculated that DTH inducing T-effector cells or other mechanisms might limit the amount of virus that reaches the CNS, which in turn affects the amount of second wave of virus which reaches to uninoculated contralateral eye (Kielty et al., 1987)

The virus culture studies have revealed that virus reaches the uninoculated eye in two temporally separate waves after uniocular anterior chamber inoculation. The first wave of virus is detected in the uninoculated eye as early as one day PI, long before virus is found in either of the optic nerves or the brain. The second wave of virus arrives in the uninoculated eye between 7 and 10 days PI (Atherton & Streilein, 1987).

Kahn (1993) investigated the effect of light onto von Szily model. His findings suggested that virus does not reach the contralateral retina in dark-reared mice. He found a decrease in neuronal firing and retrograde axoplasmic flow during darkness (Kahn et al., 1993). Colchicine is known for its axonal-transport-blocking capabilities. It was used to examine whether virus is transported via the optic nerve to the uninoculated eye after anterior chamber inoculation of HSV-1. The results demonstrated that blocking the optic nerve with colchicine prevented the entry of only the second-wave of virus, while the first-wave of virus was not affected. This observation supported the hypothesis that the second-wave of virus reaches the contralateral eye from the central nervous system via the optic nerve (Bosem et al., 1990).

The route of second wave of virus spread was also studied by Vann and Atherton (Vann & Atherton, 1991) in BALB/c mice. The data showed that virus spreads from the injected eye to the central nervous system (CNS) through parasympathetic fibers of the oculomotor nerve that supply the iris and ciliary body. The virus spread in the CNS is limited primarily to the nuclei of the visual system and the suprachiasmatic area of the hypothalamus. Subsequently, the virus is transmitted from the CNS to the retina of the contralateral eye by retrograde axonal transport through the optic nerve along the endocrine-optic pathway between the retina and the suprachiasmatic nucleus (SCN) of the hypothalamus (Fig 1).

1.3 Participation of the immune response to the injected virus

Researchers have concluded that immune response participates in the course of the HSV-1-induced contralateral retinitis (Atherton et al., 1989; Whittum-Hudson et al., 1985). Using flow cytometry and immunohistochemistry, Azumi (Azumi & Atherton, 1998) documented that T cell play a role in the disease. At day 9 PI (acute retinitis), T cells were observed in the uvea but not in the retina of contralateral eye. The CD4⁺ and CD8⁺ T cells were found in the sensory retina coincident with the onset of retinal necrosis (day 11 PI), and CD4⁺ and CD8⁺ T cells were then detected in the remnants of the retina until day 63 PI. The maximum number of infiltrating cell, both of the CD4⁺ and CD8⁺ subgroups were observed at day 21 PI.

Another investigation by the same group of researchers suggested that the CD4⁺ T cell subset contributes to the destruction of the retina, and may accelerate the cellular infiltration and inflammation-induced retinal destruction at day 14 (retinal necrosis phase). At least 2 weeks before inoculation of virus, T-cell-depleted BALB/c mice were injected intravenously with anti-CD4 monoclonal antibody. Indeed, the treatment with an anti-CD4 monoclonal antibody in the von Szily model modified the virus recovery from the posterior segment of the contralateral eye significantly. By day 14 PI, significantly higher titers of virus were recovered from the mice that were depleted of CD4⁺ cells. In conclusion, the CD4⁺ T cells are involved in virus clearance from contralateral eye (Azumi et al., 1994).

The Mac-1 positive cells (macrophages, natural-killer cells, and polymorphonuclear neutrophils) appear in contralateral ciliary body between day 8 and 10 PI. On day 10, a large number of Mac-1 positive cells infiltrate the retina, and mainly the inner retinal layers. Simultaneously, Mac-1 positive cells also infiltrated the choroid. The finding of a predominance of Mac-1 cells in the contralateral retina of susceptible BALB/c mice on day 10 led the researchers to speculate that the cells might be important mediators of necrotizing in contralateral retinal necrosis phase (Zaltas et al., 1992). The DNA microarray results showed that macrophage-related genes were up-regulated in the contralateral eye at day 9 PI. Additional immunohistochemical studies also showed the presence of F4/80-positive cells in the retina (Zheng et al., 2003). In further experiments, an anti-CD11b mAB was injected intravitreally before the first wave of inflammatory cells arrived to the eye. This resulted in a profound suppression of retinal necrosis with significant decrease in both the incidence and the severity of the retinitis, even though herpes simplex viral particles could be detected in the chorioretinal layers of unaffected eyes by indirect immunofluorescence. In agreement with these data, a suppression of retinal necrosis was also noted when macrophages were depleted from mice by the treatment with Cl₂MDP-liposomes. The timing of the anti-CD11b mAb injection appeared to be critical for the inhibition of contralateral retinitis. Suppression of retinal necrosis was effective only when the antibody was administered before the onset of clinical signs. Once the initial clinical signs of contralateral retinitis were observed, anti-CD11b mAb did not alter the course of the disease (Berra et al.,1994). These findings suggested that macrophages are important participants in the effector phase of the inflammatory immune response in HSV-1 induced contralateral retinitis. Macrophages probably play a multifunctional role in the pathogenesis of the disease by direct cytotoxic action and indirectly by releasing chemotactic and immunoregulatory cytokines that finally contribute to retinal destruction. Other possibilities are that they are acting as antigen presenting cells (APC) to increase T-cell function (accessory function of macrophages). They can also provide costimulatory signals to increase effector T-cell function by B7.1/B7.2.

Beside the inflammatory cells, various cytokines and chemokines are actively involved in the course of HSV-1 retinitis. DNA microarray was used to analyze the expression patterns of genes in the uninoculated eye following uniocular anterior chamber inoculation of HSV-1. The most abundant cytokines and chemokines in the contralateral eyes of mice with HSV-1 retinitis were the IFN-family and their receptors, the interleukins IL-1, IL-6, and IL-4 and their receptors, and macrophage inflammatory protein (MIP-1 and MIP-2) and their receptors. Since these cytokine and cytokine-receptor genes were up-regulated in the uninoculated eye at the peak of acute retinal infection, these results suggested that they are likely to be the modulators most closely related to ARN (Zheng et al., 2003). From RT-PCRs assay, the transcriptions of some cytokines were investigated. IFN-g-mRNA was moderately elevated on day 6 PI, increased slightly between days 6 and 8 PI, and then increased slightly again between day 8 and 11 PI. On day 6 and 8 PI, the level of mRNA for IL-4 was only slightly above that observed in the uninjected eyes of the mock-infected mice. Between day 8 and 11 PI, the amount of IL-4 mRNA increased approximately three fold. IFN-γ⁺ and IL-4⁺ cells were observed throughout the retina. Most of CD4⁺, Gr-1⁺, CD19⁺ and F4/80⁺ cells expressed IFN-γ and IL-4 (Zheng et al., 2005).

1.4 Tumor necrosis factor-alpha

The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) originally was identified as a serum factor causing hemorrhagic necrosis of tumors and inducing cachexia. TNF-α is now known to possess many cell-activating and pro-inflammatory activities. TNF-α is produced by many cell types, among them are macrophages, T cells, and natural killer cells. The pro-inflammatory effects include induction of expression or up-regulation of major histocompatibility complex molecules (Sartani et al., 1996; Fong & Lowry, 1990; Fleisher et al., 1990). TNF-α exerts its actions through two distinct receptors: TNF RI (P55) and TNF RII (P75). The TNF-induced cytotoxicity has been attributed in the past to the p55 receptor, and TNF-induced proliferation to the p75 receptor.  However, it has been shown, that p75 can greatly enhance p55-induced cell death (Koizumi et al., 2003; Bigda et al., 1994; Tartaglia et al., 1991 ).

The TNF/TNF-R family plays a key role in the activation, differentiation and effector responses of T-cells. Subsequent analysis of TNF-R expression revealed that antigen activation was required for the up-regulation of p75 and to a lesser extent p55 TNF-R and the acquisition of TNF responsiveness by different T-cell subsets in vitro, but more importantly at sites of inflammation (Ware et al., 1991; Brennan et al.,1992; Cope et al.,1995). Both in vitro and in vivo co-stimulatory effects may arise through different mechanisms, including activation and differentiation of antigen-presenting cells, such as dendritic cells, as well as antigen-presenting function (Sallusto et al., 1995). The treatment with rabbit anti-TNF-α serum in experimental autoimmune uveoretinitis (EAU) during the afferent stage significantly reduced the autoantigen specific lymphocyte proliferation and DTH (Sartani et al., 1996). Greiner and co-workers investigated 15 patients with posterior segment intraocular inflammation (PSII) refractory to conventional immunosuppressive therapy and who then received a single infusion of a recombinant protein generated by fusing the p55 TNF-α receptor with human IgG1. Interestingly, the authors found that the anti-TNF-α agent induced an up-regulation of IL-10-expression in peripheral blood CD4⁺ T cells and an alteration in the ratio of IL-10- and IFN-γ-producing CD4⁺ T cells (Greiner et al., 2004).

Macrophages are versatile cells that are intimately involved in diverse aspects of the immune response and inflammation. The cytotoxic macrophages and production of nitric oxide (NO) or reactive oxygen species (ROS) might cause cell membrane peroxidation and destruction. Classic macrophage activation after stimulation with IFN-γ and TNF refers to the ability of a macrophage to express nitric oxide synthase (NOS2) and generate nitrite, peroxynitrites, and superoxides, which in turn induce lipid peroxidation of cell membranes and cell death (Robertson et al., 2002; Erwing et al., 1998; Li & Verma, 2002; van Strijp et al., 1991). In experimental cutaneous leishmaniasis, the blockade of the TNF activity resulted in a reduced NOS2 expression from draining lymph nodes and macrophages (Engwerda et al., 2002; Fonseca et al., 2003). With a treatment of mice with sTNFr-IgG in the EAU model, infiltrating macrophages reduced expression nitrite production at the height of disease, and the level of apoptosis within the retina was reduced too (Robertson, 2003).

In the von Szily model, TNF-α mRNA and protein was up-regulated during the evolution of ARN (from day 6 to 14 PI) in the contralateral eyes compared with levels in control subjects (Zheng et al., 2005). The DNA microarray results showed that among the most up-regulated cytokines and chemokines in contralateral eye of mice with HSV-1 retinitis was the TNF family cytokines and their receptors (Zheng et al., 2003). The immunohistochemical stainings revealed that TNF-α was produced by infiltrating cells such as CD4⁺, Gr-1⁺, CD19⁺, and F/80⁺ cells. Approximately one third of the RPE cells produced TNF-α at day 9 PI. In addition, a smaller number (4-14%) of Müller cells also produced TNF-α at the same time (Zheng et al., 2005).

Latently infected trigeminal ganglia (strain KOS) were excised and placed in vitro. TNF-α was added daily. The reactivation replication rate in the TNF-α treated group was higher than the control group. These experiments demonstrated that TNF-α enhanced the reactivation frequency and replication of HSV (Walev et al., 1995). Also, the number of TNF-α producing cells was investigated during the acute replication phase of HSV in trigeminal ganglia. The appearance of TNF-α producing cells in the trigeminal ganglia was correlated to virulence and replication of HSV dependent on the time of appearance. The higher the virulence the earlier commenced replication and the more increased the number of TNF-α- producing cells (Walev et al., 1995).

In conclusion, it appears that TNF-α is an one of the most important pro-inflammatory cytokines in ARN (Oettinger & D’Szouza, 2003).

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