Shilpa Deshpande

Contribution of Vascular Endothelial Growth Factor in the Neovascularization Process during the Pathogenesis of Herpetic Stromal Keratitis

ABSTRACT This report analyzes the role of vascular endothelial growth factor (VEGF)-induced angiogenesis in the immunoinflammatory lesion stromal keratitis induced by ocular infection with herpes simplex virus (HSV). Our results show that infection with replication-competent, but not mutant, viruses results in the expression of VEGF mRNA and protein in the cornea. This a rapid event, with VEGF mRNA detectable by 12 h postinfection (p.i.) and proteins detectable by 24 h p.i. VEGF production occurred both in the virus-infected corneal epithelium and in the underlying stroma, in which viral antigens were undetectable. In the stroma, VEGF was produced by inflammatory cells; these initially were predominantly polymorphonuclear leukocytes (PMN), but at later time points both PMN and macrophage-like cells were VEGF producers. In the epithelium, the major site of VEGF-expressing cells in early infection, the infected cells themselves were usually negative for VEGF. Similarly, in vitro infection studies indicated that the cells which produced VEGF were not those which expressed virus. Attesting to the possible role of VEGF-induced angiogenesis in the pathogenesis of herpetic stromal keratitis were experiments showing that VEGF inhibition with mFlt(1–3)-immunoglobulin G diminished angiogenesis and the severity of lesions after HSV infection. These observations are the first to evaluate VEGF-induced angiogenesis in the pathogenesis of stromal keratitis. Our results indicate that the control of angiogenesis represents a useful adjunct to therapy of herpetic ocular disease, an important cause of human blindness.

Bystander Activation Involving T Lymphocytes in Herpetic Stromal Keratitis

Herpes simplex virus infection of mouse corneas can lead to the development of an immunopathological lesion, termed herpetic stromal keratitis (HSK). Such lesions also occur in TCR-transgenic mice backcrossed to SCID (TgSCID) that are unable to mount detectable HSV-specific immune responses. The present study demonstrates that lesion expression in such mice depends on continuous viral replication, whereas in immunocompetent mice, lesions occurred even if virus replication was terminated at 4 days after infection. The continuous replication in TgSCID mice was considered necessary to produce an activating stimulus to CD4+ T cells that invade the cornea. Lesions in TgSCID were resistant to control by cyclosporin A, but were inhibited by treatment with rapamycin. This result was interpreted to indicate that T cell activation involved a non-TCR-mediated cytokine-driven bystander mechanism. Bystander activation was also shown to play a role in HSK lesions in immunocompetent mice. Accordingly, in immunocompetent DO11.10 mice, lesions were dominated by KJ1.26+ OVA-specific CD4+ T cells that were unreactive with HSV. In addition, KJ1.26+ HSV nonimmune cells parked in ocularly infected BALB/c mice were demonstrable in HSK lesions. These results provide insight for the choice of new strategies to manage HSK, an important cause of human blindness.

Lymphotoxin α−/− Mice Develop Functionally Impaired CD8+ T Cell Responses and Fail to Contain Virus Infection of the Central Nervous System

Recent observations have indicated that viral persistence and tumor spreading could occur because of effector function-defective CD8+ T cells. Although chronic exposure to Ag, lack of CD4 help, and epitope dominance are suggested to interfere with CTL differentiation, mechanisms underlying the defective effector function remain obscure. We demonstrate in this report that lymphotoxin α-deficient mice develop CD8+ T cells at normal frequencies when infected with HSV or immunized with OVA Ag but show impaired cytotoxic and cytokine-mediated effector functions resulting in enhanced susceptibility to HSV-induced encephalitis. Although these cells display near normal levels of perforin and Fas ligand, they remain largely at a naive state as judged by high expression of CD62 ligand and failure to up-regulate activation or memory markers. In particular, these CD8+ T cells revealed inadequate expression of the IL-12 receptor, thus establishing a link between CTL differentiation and LTα possibly through regulation of IL-12 receptor. Viruses and tumors could evade immunity by targeting the same pathway.

Herpes Simplex Virus-Induced Keratitis: Evaluation of the Role of Molecular Mimicry in Lesion Pathogenesis

ABSTRACT Viruses are suspected but usually unproven triggering factors in autoimmunity. One favored mechanism to explain the role of viruses in the genesis of autoimmunity is molecular mimicry. An immunoinflammatory blinding lesion called herpetic stromal keratitis (HSK) that follows ocular infection with herpes simplex virus (HSV) is suggested to result from a CD4+ T-cell response to a UL6 peptide of HSV that cross-reacts with a corneal autopeptide shared with the immunoglobulin G2ab (IgG2ab) isotype. The present report reevaluates the molecular mimicry hypothesis to explain HSK pathogenesis. Our results failed to reveal cross-reactivity between the UL6 and IgG2ab peptides or between peptide reactive T cells and HSV antigens. More importantly, animals infected with HSV failed to develop responses that reacted with either peptide, and infection with a recombinant vaccinia UL6 vector failed to cause HSK, in spite of generating UL6 reactivity. Other lines of evidence also failed to support the molecular mimicry hypothesis, such as the failure to affect HSK severity upon tolerization of susceptible BALB/c and B-cell-deficient mice with IgG2ab or UL6 peptides. An additional study system revealed that HSK could be induced in mouse strains, such as the OT2 × RAG1−/− mice (T cell receptor transgenic recognizing OVA323–339) that were unable to produce CD4+ T-cell responses to any detectable HSV antigens. Our results cast doubt on the molecular mimicry hypothesis as an explanation for the pathogenesis of HSK and indicate that if autoimmunity is involved its likely proceeds via a bystander activation mechanism.

Bystander activation of CD4(+) T cells can represent an exclusive means of immunopathology in a virus infection.

Herpetic stromal keratitis (HSK) is an immunopathological lesion involving herpes simplex virus (HSV) infection and CD4+ T cells of the Th1 phenotype, but the nature of the target antigens which drive HSK remains uncertain. In the present report we show that ovalbumin TCR‐transgenic mice backcrossed to SCID mice unable to recognize HSV show clinical signs of HSK but die of viral encephalitis before the lesions become severe. However, passive transfer of anti‐HSV serum at 24 h clears virus and affords protection from both HSK lesions and death. Adoptive transfer of CD8+ T cells at 72 h usually conferred protection but animals developed severe corneal pathology by 3 weeks post infection. At this time viral antigens were not demonstrable in the cornea and the T cells in the inflammatory lesions were CD4+KJ1‐26.1 idiotype positive, i.  e. OVA peptide specific. These results indicate bystander activation of CD4+ T cells in a virus‐induced inflammatory milieu. This mechanism of immunoinflammation may represent an important component of any lesion which involves CD4+ T cells.

Herpetic eye disease: immunopathogenesis and therapeutic measures.

Infection of the cornea with herpes simplex virus (HSV) can result in a chronic disease called herpetic stromal keratitis (HSK). The disease represents one of the leading causes of infectious blindness in the Western world. Immune-mediated cellular damage is suspected in the pathogenesis of human HSK. The murine model has been pivotal in further establishing HSK as an immunopathological disease. This article reviews understanding of HSK, both in humans and in the mouse model, with an emphasis on possible future therapeutic strategies to counteract this blinding immunoinflammatory disease.

Mechanisms of pathogenesis in herpetic immunoinflammatory ocular lesions.

This article reviews possible mechanisms by which ocular infections with herpes simplex virus result in a blinding immunoinflammatory lesion in the cornea. We conclude that this immunoinflammatory response involves multiple immune mechanisms including autoimmunity.

Herpetic stromal keratitis in the absence of viral antigen recognition

Herpetic stromal keratitis (HSK), resulting from ocular infection with herpes simplex virus (HSV), is thought to represent a T cell mediated immunopathologic lesion. Antigens recognized by the inflammatory T cells remain unresolved and non-TCR mediated activation of T cells (bystander activation) is considered as also involved. This report documents further evidence for the bystander activation mechanisms using three T cell transgenic RAG-/- mouse strains. Accordingly HSK occurred in PCC RAG-/-, P14 RAG-/-, and OT-1 RAG-/- mice. In none of the models could HSV specific T cell reactivity be demonstrated and animals were unprotected from lesion development by immunization prior to HSV ocular infection. The results support the role of bystander activation as a mechanism of T cell mediated immunopathology and show that CD8(+) as well as CD4(+) T cells can participate in HSK lesion development.

IL-12 suppresses the expression of ocular immunoinflammatory lesions by effects on angiogenesis

Topical application of plasmid DNA encoding IL‐12 to the cornea of mice prior to ocular infection with Herpes simplex virus type 1 (HSV) results in diminished corneal immunoinflammatory lesions. Such herpetic stromal keratitis (HSK) reactions in humans represent an important cause of blindness. The effect of IL‐12 pretreatment acted via inhibitory effects on corneal neovascularization rather than by inhibiting viral replication or the function of CD4+ T cells that mediate HSK. The antiangiogenesis induced by IL‐12 DNA application was mediated indirectly via the cytokine IFN‐γ and one or both of two chemokine molecules, IP‐10 and MIG. Thus IL‐12 DNA administration lacked modulatory effects on HSK in GKO mice, indicating the necessary involvement of IFN‐γ induction for antiangiogenesis. In contrast, exposure of GKO mice to IP‐10 DNA did suppress the severity of HSK. Furthermore, treatment with specific antisera to IP‐10 and MIG in HSV‐infected mice abrogated the IL‐12‐induced inhibitory effect on lesion severity. Taken together, our data indicate that the HSV‐induced ocular immunoinflammatory lesions can be modulated by IL‐12 and that this effect results from chemokine inhibition of angiogenesis. The use of antiangiogenesis therapy might represent a useful control measure against HSK.

Pathogenesis of Herpes Simplex Virus-Induced Ocular Immunoinflammatory Lesions in B-Cell-Deficient Mice

ABSTRACT The role of B cells and humoral immunity in herpes simplex virus (HSV) ocular infections was studied in immunoglobulin μ chain gene-targeted B-cell-deficient mice (μK/O). At doses of virus well tolerated by immunocompetent mice, heightened susceptibility of μK/O mice to herpetic encephalitis as well as to herpetic stromal keratitis (HSK) was observed. An explanation was sought for the increased severity of HSK in the μK/O mice. First, the lack of antibody responses in μK/O mice resulted in longer viral persistence and dissemination to the corneal stroma, the site of inflammation. Prolonged virus expression in the corneal stroma was suggested to cause bystander activation of Th1-type CD4+ T cells, further contributing to the severity of HSK lesion expression in μK/O mice. Second, μK/O mice generated minimal Th2 cytokine responses compared to wild-type mice. Such responses might serve to downregulate the severity of Th1-mediated HSK lesions.