Disease parameters following ocular herpes simplex virus type 1 infection are similar in male and female BALB/C mice.

Sex related differences in the incidence or severity of infection have been described for multiple viruses. With herpes simplex viruses, the best example is HSV-2 genital infection where women have a higher incidence of infection and can have more severe infections than men. HSV-1 causes several types of infections including skin and mucosal ulcers, keratitis, and encephalitis in humans that do not appear to have a strong biological sex component. Given that mouse strains differ in their MHC loci it is important to determine if sex differences occur in multiple strains of mice. Our goal was to answer two questions: Are virus related sex differences present in BALB/C mice and does virulence of the viral strain have an effect? We generated a panel of recombinant HSV-1 viruses with differing virulence phenotypes and characterized multiple clinical correlates of ocular infection in BALB/c mice. We found no sex-specific differences in blepharitis, corneal clouding, neurovirulence, and viral titers in eye washes. Sex differences in neovascularization, weight loss and eyewash titers were observed for some recombinants, but these were not consistent across the phenotypes tested for any recombinant virus. Considering these findings, we conclude that there are no significant sex specific ocular pathologies in the parameters measured, regardless of the virulence phenotype following ocular infection in BALB/c mice, suggesting that the use of both sexes is not necessary for the bulk of ocular infection studies.

METTL3-mediated m6A RNA modification promotes corneal neovascularization by upregulating the canonical Wnt pathway during HSV-1 infection.

BACKGROUND: Corneal neovascularization (CNV) is a symptom of herpes simplex keratitis (HSK), which can result in blindness. The corneal angiogenesis brought on by herpes simplex virus type 1 (HSV-1) is strongly affected by vascular endothelial growth factor A (VEGFA). The N6-methyladenosine (m6A) modification catalyzed by methyltransferase-like 3 (METTL3) is a crucial epigenetic regulatory process for angiogenic properties. However, the roles of METTL3 and m6A in HSK-induced CNV remain unknown. Here, we investigated these roles in vitro and in vivo. METHODS: A PCR array in HSV-1-infected human umbilical vein endothelial cells (HUVECs) was used to screen for METTL3 among the epitranscriptomic genes. Tube formation and scratch assays were conducted to investigate cell migration capacity. The global mRNA m6A abundance was evaluated using a dot blot assay. Gene expression was assessed by RT-qPCR, western blotting, and fluorescence immunostaining. In addition, bioinformatic analysis was conducted to identify the downstream molecules of METTL3 in HUVECs. METTL3 knockdown and STM2457 treatment clarified the specific underlying molecular mechanisms affecting HSV-1-induced angiogenesis in vitro. An acute HSK mouse model was established to examine the effects of METTL3 knockdown or inhibition using STM2457 on pathological angiogenic development in vivo. RESULTS: METTL3 was highly upregulated in HSV-1-infected HUVECs and led to increased m6A levels. METTL3 knockdown or inhibition by STM2457 further reduced m6A levels and VEGFA expression and impaired migration and tube formation capacity in HUVECs after HSV-1 infection. Mechanistically, METTL3 regulated LRP6 expression through post-transcriptional mRNA modification in an m6A-dependent manner, increasing its stability, upregulating VEGFA expression, and promoting angiogenesis in HSV-1-infected HUVECs. Furthermore, METTL3 knockdown or inhibition by STM2457 reduced CNV in vivo. CONCLUSION: Our findings revealed that METTL3 promotes pathological angiogenesis through canonical Wnt and VEGF signaling in vitro and in vivo, providing potential pharmacological targets for preventing the progression of CNV in HSK.

Corneal nerve regeneration is affected by scar location in herpes simplex keratitis: A longitudinal in vivo confocal microscopy study.

PURPOSE: To assess the effect of corneal scar location on corneal nerve regeneration in patients with herpes simplex virus (HSV) keratitis in their affected and contralateral eyes over a 1-year period by in vivo confocal microscopy (IVCM), and to correlate these findings to corneal sensation measured by Cochet-Bonnet Esthesiometer. METHODS: Prospective, longitudinal, case-control study. Bilateral corneal nerve density and corneal sensation were analyzed centrally and peripherally in 24 healthy controls and 23 patients with unilateral HSV-related corneal scars using IVCM. RESULTS: In the central scar (CS) group, total nerve density in the central cornea remained significantly lower compared to controls at follow-up (11.05 ± 1.97mm/mm2, p < 0.001), and no significant nerve regeneration was observed (p = 0.090). At follow-up, total nerve density was not significantly different from controls in the central and peripheral cornea of the peripheral scar (PS) group (all p > 0.05), but significant nerve regeneration was observed in central corneas (16.39 ± 2.39mm/mm2, p = 0.007) compared to baseline. In contralateral eyes, no significant corneal nerve regeneration was observed in central or peripheral corneas of patients with central scars or peripheral scars at 1-year follow-up, compared to baseline (p > 0.05). There was a positive correlation between corneal nerve density and sensation in both central (R = 0.53, p < 0.0001) and peripheral corneas (R = 0.27, p = 0.0004). In the CS group, the corneal sensitivity was <4 cm in 4 (30.8%) and 7 (53.8%) patients in the central and peripheral corneas at baseline, and in 5 (38.5%) and 2 subjects (15.4%) at follow-up, whereas in the PS group only 1 patient (10%) showed a corneal sensation < 4 cm in the central cornea at baseline, and only 1 (10.0%), 3 (30.0%) and 1 (10.0%) patients at follow-up in the central, affected and opposite area of the cornea, respectively. CONCLUSION: The location of HSV scarring in the cornea affects the level of corneal nerve regeneration. Eyes with central corneal scar have a diminished capacity to regenerate nerves in central cornea, show a more severe reduction in corneal sensation in the central and peripheral corneas that persist at follow-up, and have a reduced capability to restore the corneal sensitivity above the cut-off of 4 cm. Thus, clinicians should be aware that CS patients would benefit from closer monitoring for potential complications associated with neurotrophic keratopathy, as they have a lower likelihood for nerve regeneration.

Bilateral interstitial keratitis in a patient with presumed brain tuberculoma.

Interstitial keratitis is an inflammation of the corneal stroma without epithelium or endothelium involvement. The underlying causes are mostly infectious or immune mediated. Brazil has one of the highest incidence rates of tuberculosis in the world. Tuberculosis is considered one of the causes of interstitial keratitis. Malnutrition and anemia are risk factors of the disseminated disease. This is a case report of a 10-year-old child who presented with decreased visual acuity and a clinical diagnosis of bilateral interstitial keratitis and sclero-uveitis. The patient had been treated with topical steroids with partial improvement. Examinations revealed severe iron deficiency anemia, negative serologies for human immunodeficiency virus and syphilis, positivity for cytomegalovirus- and herpes simplex-specific IgG, and purified protein derivative of 17 mm. During the follow-up, the patient presented with tonic-clonic seizures, and magnetic resonance imaging findings suggested a central nervous system tuberculoma. Interstitial keratitis improvement was observed after specific tuberculosis treatment. This is the first case report describing the association of interstitial keratitis and central nervous system tuberculoma.

Sensory Nerve Retraction and Sympathetic Nerve Innervation Contribute to Immunopathology of Murine Recurrent Herpes Stromal Keratitis.

Purpose: Herpes stromal keratitis (HSK) represents a spectrum of pathologies which is caused by herpes simplex virus type 1 (HSV-1) infection and is considered a leading cause of infectious blindness. HSV-1 infects corneal sensory nerves and establishes latency in the trigeminal ganglion (TG). Recently, retraction of sensory nerves and replacement with "unsensing" sympathetic nerves was identified as a critical contributor of HSK in a mouse model where corneal pathology is caused by primary infection. This resulted in the loss of blink reflex, corneal desiccation, and exacerbation of inflammation leading to corneal opacity. Despite this, it was unclear whether inflammation associated with viral reactivation was sufficient to initiate this cascade of events. Methods: We examined viral reactivation and corneal pathology in a mouse model with recurrent HSK by infecting the cornea with HSV-1 (McKrae) and transferring (intravenous [IV]) human sera to establish primary infection without discernible disease and then exposed the cornea to UV-B light to induce viral reactivation. Results: UV-B light induced viral reactivation from latency in 100% of mice as measured by HSV-1 antigen deposition in the cornea. Further, unlike conventional HSK models, viral reactivation resulted in focal retraction of sensory nerves and corneal opacity. Dependent on CD4+ T cells, inflammation foci were innervated by sympathetic nerves. Conclusions: Collectively, our data reveal that sectoral corneal sensory nerve retraction and replacement of sympathetic nerves were involved in the progressive pathology that is dependent on CD4+ T cells after viral reactivation from HSV-1 latency in the UV-B induced recurrent HSK mouse model.

Optical coherence tomography angiography in herpetic leucoma.

BACKGROUND: Herpes simplex virus (HSV) keratitis remains a leading infectious cause of blindness worldwide. Although all forms of HSV keratitis are commonly recurrent, the risk is greatest in stromal keratitis, which is the most likely to result in corneal scarring, thinning, and neovascularization. Recent studies showed the ability of Optical Coherence Tomography Angiography (OCTA) to detect and study vascular abnormalities in the anterior segment, including abnormal corneal vessels. This study intends to investigate the potential of OCTA device to image and describe quantitatively the vascularization in eyes diagnosed with herpetic leucoma and to discuss and review the usefulness of this technique in this pathology. METHODS: A Cross-sectional study was made, including 17 eyes of 15 patients with leucoma secondary to herpetic keratitis. All eyes underwent anterior segment Slit-Lamp photography (SLP), and OCTA with en-face, b-scans and c-scans imaging. The vessel density (VD) was analyzed in the inferior, nasal and temporal corneal margin in all patients, and in the central area, in eyes with central corneal neovascularization (CoNV). The measurements were calculated after binarization with ImageJ software, using OCTA scans with 6 × 6 mm in a depth of 800 µm. RESULTS: Patients included had a mean age 53.267 ± 21.542 (years ± SD). The mean total vessel area was 50.907% ± 3.435%. VD was higher in the nasal quadrant (51.156% ± 4.276%) but there were no significant differences between the three analyzed areas (p = 0.940). OCTA was able to identify abnormal vessels when SLP apparently showed no abnormal vessels; OCTA was able to distinguish between larger and smaller vessels even in central cornea; OCTA scans allowed the investigation of several corneal planes and the relation of them with clinical findings. CONCLUSIONS: OCTA can be useful in both qualitative and quantitative follow-up of patients and may become a non-invasive alternative to objectively monitor treatment response in eyes with corneal vascularization due to herpetic infection.

Herpes Simplex Virus 2 Blepharokeratoconjunctivitis.

PURPOSE: To retrospectively review the clinical characteristics of patients with herpes simplex virus type 2 (HSV-2) blepharokeratoconjunctivitis. METHODS: Laboratory-proven HSV-2 blepharokeratoconjunctivitis cases were reviewed between 1995 and 2021. RESULTS: Ten of 725 (1.4%) patients had HSV-2 infection. Data were available for nine patients. Associated conditions included neonatal herpes (1/9, 11%), severe atopy (1/9, 11%), genital herpes (2/9, 22%), and systemic immune disorders (2/9, 22%). The most common presenting finding was pain and blurred vision (55.5%). Two patients (22%) had dendritic lesions and one patient (11%) had reduced corneal sensation. Complete resolution was reported in four patients (44.5%). Recurrence was noted in four patients (44.5%) despite antiviral prophylaxis. Corneal complications included scarring and neovascularization. The visual acuity at the last follow-up was 20/40 or worse in four patients (44.5%). CONCLUSIONS: HSV-2 is an uncommon cause of keratitis. Dendrites and loss of corneal sensation were uncommon. Recurrence was noted despite antiviral prophylaxis.

Intramuscular Vaccination With the HSV-1(VC2) Live-Attenuated Vaccine Strain Confers Protection Against Viral Ocular Immunopathogenesis Associated With γδT Cell Intracorneal Infiltration.

Herpes simplex virus type-1 (HSV-1) ocular infection is one of the leading causes of infectious blindness in developed countries. The resultant herpetic keratitis (HK) is caused by an exacerbated reaction of the adaptive immune response that persists beyond virus clearance causing substantial damage to the cornea. Intramuscular immunization of mice with the HSV-1(VC2) live-attenuated vaccine strain has been shown to protect mice against lethal ocular challenge. Herein, we show that following ocular challenge, VC2 vaccinated animals control ocular immunopathogenesis in the absence of neutralizing antibodies on ocular surfaces. Ocular protection is associated with enhanced intracorneal infiltration of γδ T cells compared to mock-vaccinated animals. The observed γδ T cellular infiltration was inversely proportional to the infiltration of neutrophils, the latter associated with exacerbated tissue damage. Inhibition of T cell migration into ocular tissues by the S1P receptors agonist FTY720 produced significant ocular disease in vaccinated mice and marked increase in neutrophil infiltration. These results indicate that ocular challenge of mice immunized with the VC2 vaccine induce a unique ocular mucosal response that leads into the infiltration of γδ T cells resulting in the amelioration of infection-associated immunopathogenesis.

Modulating glutamine metabolism to control viral immuno-inflammatory lesions.

Infection of the cornea with HSV results in an immune-inflammatory reaction orchestrated by proinflammatory T cells that is a major cause of human vision impairment. The severity of lesions can be reduced if the representation of inflammatory T cells is changed to increase the presence of T cells with regulatory function. This report shows that inhibiting glutamine metabolism using 6-Diazo-5-oxo-l-norleucine (DON) administered via intraperitoneal (IP) starting 6 days after ocular infection and continued until day 15 significantly reduced the severity of herpetic stromal keratitis lesions. The therapy resulted in reduced neutrophils, macrophages as well proinflammatory CD4 Th1 and Th17 T cells in the cornea, but had no effect on levels of regulatory T cells. A similar change in the representation of inflammatory and regulatory T cells occurred in the trigeminal ganglion (TG) the site where HSV infection establishes latency. Glutamine metabolism was shown to be required for the in-vitro optimal induction of both Th1 and Th17 T cells but not for the induction of Treg that were increased when glutamine metabolism was inhibited. Inhibiting glutamine metabolism also changed the ability of latently infected TG cells from animals previously infected with HSV to reactivate and produce infectious virus.

An intact complement system dampens cornea inflammation during acute primary HSV-1 infection.

Corneal transparency is an essential characteristic necessary for normal vision. In response to microbial infection, the integrity of the cornea can become compromised as a result of the inflammatory response and the ensuing tissue pathology including neovascularization (NV) and collagen lamellae destruction. We have previously found complement activation contributes to cornea pathology-specifically, denervation in response to HSV-1 infection. Therefore, we investigated whether the complement system also played a role in HSV-1-mediated neovascularization. Using wild type (WT) and complement component 3 deficient (C3 KO) mice infected with HSV-1, we found corneal NV was accelerated associated with an increase in inflammatory monocytes (CD11b+CCR2+CD115+/-Ly6G-Ly6Chigh), macrophages (CD11b+CCR2+CD115+Ly6G-Ly6Chigh) and a subpopulation of granulocytes/neutrophils (CD11b+CCR2-CD115+Ly6G+Ly6Clow). There were also increases in select pro-inflammatory and pro-angiogenic factors including IL-1α, matrix metalloproteinases (MMP)-2, MMP-3, MMP-8, CXCL1, CCL2, and VEGF-A that coincided with increased inflammation, neovascularization, and corneal opacity in the C3 KO mice. The difference in inflammation between WT and C3 KO mice was not driven by changes in virus titer. However, viral antigen clearance was hindered in C3 KO mouse corneas suggesting the complement system has a dynamic regulatory role within the cornea once an inflammatory cascade is initiated by HSV-1.

Healing of Ocular Herpetic Disease Following Treatment With an Engineered FGF-1 Is Associated With Increased Corneal Anti-Inflammatory M2 Macrophages.

Herpes simplex virus 1 (HSV-1) infects the cornea and caused blinding ocular disease. In the present study, we evaluated whether and how a novel engineered version of fibroblast growth factor-1 (FGF-1), designated as TTHX1114, would reduce the severity of HSV-1-induced and recurrent ocular herpes in the mouse model. The efficacy of TTHX1114 against corneal keratopathy was assessed in B6 mice following corneal infection with HSV-1, strain McKrae. Starting day one post infection (PI), mice received TTHX1114 for 14 days. The severity of primary stromal keratitis and blepharitis were monitored up to 28 days PI. Inflammatory cell infiltrating infected corneas were characterized up to day 21 PI. The severity of recurrent herpetic disease was quantified in latently infected B6 mice up to 30 days post-UVB corneal exposure. The effect of TTHX1114 on M1 and M2 macrophage polarization was determined in vivo in mice and in vitro on primary human monocytes-derived macrophages. Compared to HSV-1 infected non-treated mice, the infected and TTHX1114 treated mice exhibited significant reduction of primary and recurrent stromal keratitis and blepharitis, without affecting virus corneal replication. The therapeutic effect of TTHX1114 was associated with a significant decrease in the frequency of M1 macrophages infiltrating the cornea, which expressed significantly lower levels of pro-inflammatory cytokines and chemokines. This polarization toward M2 phenotype was confirmed in vitro on human primary macrophages. This pre-clinical finding suggests use of this engineered FGF-1 as a novel immunotherapeutic regimen to reduce primary and recurrent HSV-1-induced corneal disease in the clinic.

Bilateral Limbal Stem Cell Alterations in Patients With Unilateral Herpes Simplex Keratitis and Herpes Zoster Ophthalmicus as Shown by In Vivo Confocal Microscopy.

Purpose: The purpose of this study was to investigate the limbal changes in the palisades of Vogt (POV) in patients with herpes simplex keratitis (HSK) and herpes zoster ophthalmicus (HZO) with the application of in vivo confocal microscopy (IVCM). Methods: We enrolled 35 eyes of 35 consecutive patients with HSK and 4 patients with HZO in this observational study. Thirty-five participants were also recruited from a healthy population as the control group. All subjects were examined by IVCM in addition to routine slit-lamp biomicroscopy. The IVCM images of the corneal basal epithelial cells, corneal nerve, and the corneoscleral limbus were acquired and then were analyzed semiquantitatively. Results: The rate of absent and atypical POV was significantly higher in the affected eyes of patients with HSK than in the contralateral eyes and eyes of controls (88.57% vs. 65.71% vs. 17.14%, P < 0.01). In the HZO group, the rate of absent and atypical POV was 100% in the affected eyes and 50% in the contralateral eyes. When compared to the contralateral unaffected eyes and control eyes, the average density of the central basal epithelial cells and the sub-basal nerve plexus density and the total number of nerves in the central area of the affected eyes were significantly lower in the HSK group (1541 ± 704.4 vs. 2510 ± 746.8 vs. 3650 ± 746.1 cells/mm2, P < 0.0001). Spearman's rank correlation showed that the presence of absent and atypical POV had a significant negative correlation with central corneal basal epithelial cells (rs = -0.44979, P < 0.0001), the density of total nerves (rs = -0.49742, P < 0.0001), and the total nerve numbers (rs = -0.48437, P < 0.0001). A significant positive correlation was established between the presence of absent and atypical POV and HSK severity in affected eyes in the superior, inferior, nasal, and temporal quadrants (rs = 0.68940, rs = 0.78715, rs = 0.65591, and rs = 0.75481, respectively, P < 0.0001) and the contralateral eyes (rs = 0.51636, rs = 0.36207, rs = 0.36990, rs = 0.51241, correspondingly, P < 0.0001). Conclusions: Both eyes of patients with unilateral HSK and HZO demonstrated a profound and significant loss of limbal stem cells, which may explain the fact that HSK and HZO are risk factors for limbal stem cell deficiency (LSCD) in both eyes. The loss of LSCs was strongly correlated with the sub-basal nerve plexus and central basal epithelial cell alterations as shown by IVCM.

Polluted Air Exposure Compromises Corneal Immunity and Exacerbates Inflammation in Acute Herpes Simplex Keratitis.

Air pollution is a serious environmental issue worldwide in developing countries' megacities, affecting the population's health, including the ocular surface, by predisposing or exacerbating other ocular diseases. Herpes simplex keratitis (HSK) is caused by the herpes simplex virus type 1 (HSV-1). The primary or recurring infection in the ocular site causes progressive corneal scarring that may result in visual impairment. The present study was designed to study the immunopathological changes of acute HSK under urban polluted air, using the acute HSK model combined with an experimental urban polluted air exposure from Buenos Aires City. We evaluated the corneal clinical outcomes, viral DNA and pro-inflammatory cytokines by RT-PCR and ELISA assays, respectively. Then, we determined the innate and adaptive immune responses in both cornea and local lymph nodes after HSV-1 corneal by immunofluorescence staining and flow cytometry. Our results showed that mice exposed to polluted air develop a severe form of HSK with increased corneal opacity, neovascularization, HSV-1 DNA and production of TNF-α, IL-1ß, IFN-γ, and CCL2. A high number of corneal resident immune cells, including activated dendritic cells, was observed in mice exposed to polluted air; with a further significant influx of bone marrow-derived cells including GR1+ cells (neutrophils and inflammatory monocytes), CD11c+ cells (dendritic cells), and CD3+ (T cells) during acute corneal HSK. Moreover, mice exposed to polluted air showed a predominant Th1 type T cell response over Tregs in local lymph nodes during acute HSK with decreased corneal Tregs. These findings provide strong evidence that urban polluted air might trigger a local imbalance of innate and adaptive immune responses that exacerbate HSK severity. Taking this study into account, urban air pollution should be considered a key factor in developing ocular inflammatory diseases.

Herpes simplex virus-1 KOS-63 strain is virulent and causes titer-dependent corneal nerve damage and keratitis.

To investigate the acute clinical, immunological, and corneal nerve changes following corneal HSV-1 KOS-63 strain inoculation. Corneas of C57BL/6 mice were inoculated with either low dose (Ld) or high dose (Hd) HSV-1 KOS-63 or culture medium. Clinical evaluation was conducted up to 7 days post inoculation (dpi). Viral titers were assessed by standard plaque assay. Excised corneas were stained for CD45 and beta-III tubulin. Corneal flow cytometry was performed to assess changes in leukocyte subpopulations. Corneal sensation was measured using a Cochet-Bonnet esthesiometer. Naïve, sham-infected (post scarification), and McKrae-infected C57BL/6 corneas served as two negative and positive controls, respectively. Compared to Ld infected mice, Hd HSV-1 KOS-63 demonstrated higher incidence of corneal opacity (1.5 ×) and neovascularization (2.6 × ; p < 0.05). At 7 dpi Hd infected mice showed more severe corneal opacity (2.23 vs. 0.87; p = 0.0003), neovascularization (6.00 vs. 0.75; p < 0.0001), and blepharitis (3.11 vs. 2.06; p = 0.001) compared to the Ld group. At 3 dpi epitheliopathy was significantly larger in the Hd group (23.59% vs. 3.44%; p = 0.001). Similarly, corneal opacity was significantly higher in Hd McKrae-infected corneas as compared with Ld McKrae-infected corneas at 3 and 5 dpi. No significant corneal opacity, neovascularization, blepharitis, and epitheliopathy were observed in naïve or sham-infected mice. Higher viral titers were detected in corneas (1 and 3 dpi) and trigeminal ganglia (TG) (3 and 5 dpi) in Hd versus Ld KOS-63 groups (p < 0.05). Leukocyte density showed a gradual increase over time from 1 to 7 dpi in both KOS-63 and McKrae-infected corneas. Corneal flow cytometric analysis (3 dpi) demonstrated a higher percentage of Gr-1 + (71.6 vs. 26.3) and CD11b + (90.6 vs. 41.1) cells in Hd versus Ld KOS-63 groups. Corneal nerve density significantly decreased in both Hd KOS-63 and Hd McKrae infected corneas in comparison with naïve and sham-infected corneas. At 3 dpi corneal nerve density was lower in the Hd versus Ld KOS-63 groups (16.79 vs. 57.41 mm/mm2; p = 0.004). Corneal sensation decreased accordingly at 5 and 7 dpi in both Ld and Hd KOS-63-infected mice. Corneal inoculation with HSV-1 KOS-63 strain shows acute keratitis and nerve degeneration in a dose-dependent fashion, demonstrating virulence of this strain.

Dot1l Aggravates Keratitis Induced by Herpes Simplex Virus Type 1 in Mice via p38 MAPK-Mediated Oxidative Stress.

BACKGROUND: Disruptor of telomeric silencing 1-like (Dot1l) plays a vital role in biological processes as a well-known methyltransferase. However, its role in herpes simplex virus type 1- (HSV-1-) infected keratitis remains unclear. METHODS: In vitro and in vivo models were assessed to investigate the role of Dot1l in HSV-1 induced keratitis. C57BL/6 mice corneas were infected with HSV-1 for different days, with or without Dot1l inhibitor, to demonstrate the regulation of Dot1l in herpes simplex keratitis (HSK). Human corneal epithelial (HCE) cells were cultured and infected with HSV-1 to identify the molecular mechanisms involved. RESULTS: In this study, we found that Dot1l was positively related to HSK. Inhibition of Dot1l with EPZ004777 (EPZ) alleviated corneal injury, including oxidative stress and inflammation in vivo. Similarly, the inhibition of Dot1l with either EPZ or small interfering RNA (siRNA) showed an inhibitory effect on HSV-1-induced oxidative stress and inflammation in HCE cells. Moreover, our study revealed that the expression of p38 MAPK was elevated after HSV-1 infection in HCE cells, and the inhibition of Dot1l could reduce the increased expression of p38 MAPK induced by HSV-1 infection in vivo and in vitro. CONCLUSION: Our results demonstrated that the inhibition of Dot1l alleviated corneal oxidative stress and inflammation by inhibiting ROS production through the p38 MAPK pathway in HSK. These findings indicated that Dot1l might be a valuable therapeutic target for HSK.

Infection-Induced Porcine Ex Vivo Corneal Wound Model to Study the Efficacy of Herpes Simplex Virus-1 Entry and Replication Inhibitors.

Corneal infections by viruses and bacteria can result in ocular surface defects, ulcers, or wounds. Herpes simplex virus type-1 (HSV-1) is a human virus with global seroprevalence in the range of 60-90%. While the virus more commonly causes mucocutaneous lesions including ulcers on the face and mouth, it is also a leading cause of infection-associated blindness. In this chapter, we discuss an in-depth protocol required to evaluate corneal damage due to HSV-1 infection using porcine models of ex vivo infection. Our methods can be adapted to study similar infections caused by other viruses and bacteria.

Management of Stromal Herpes Simplex Virus Keratitis With Epithelial Ulceration Using Optical Coherence Tomography-Generated Corneal Thickness Maps.

PURPOSE: To report 2 cases of herpes simplex virus (HSV) stromal keratitis with epithelial ulceration that were managed using optical coherence tomography-generated pachymetric and corneal epithelial thickness maps. METHODS: Two patients with a history of HSV keratitis with nonhealing epithelial defects were referred to the Athens Vision Eye Institute. Anterior segment optical coherence tomography-generated pachymetric and corneal epithelial thickness maps showed subclinical stromal edema and irregular epithelium, thus indicating diagnoses of HSV stromal keratitis with epithelial ulceration. The patients were administered topical preservative-free dexamethasone and oral antiviral therapy. Steroid tapering was guided by pachymetric and corneal epithelial thickness maps at each follow-up visit. RESULTS: Both patients experienced initial healing of the epithelium and resolution of stromal inflammation. One patient had a recurrence of HSV stromal keratitis with epithelial defect 3 months after initial improvement, with pachymetric and corneal epithelial thickness maps indicating subclinical stromal edema. He was reintroduced to topical steroid therapy, and the stromal edema and epithelial defect subsequently resolved. Both patients have had no recurrences in the past year. CONCLUSIONS: Pachymetric and corneal epithelial thickness maps provide an objective assessment of stromal inflammation and the following 2 clinical advantages in the management of HSV stromal keratitis with epithelial ulceration: (1) they help differentiate it from HSV epithelial keratitis with geographic ulceration and neurotrophic keratopathy and (2) offer objective measurements to guide management with topical corticosteroids until resolution of stromal edema. Thus, treatment can be initiated in a timely manner, and the blinding complications of HSV stromal keratitis can be avoided.

Production of the Cytokine VEGF-A by CD4+ T and Myeloid Cells Disrupts the Corneal Nerve Landscape and Promotes Herpes Stromal Keratitis.

Herpes simplex virus type 1 (HSV-1)-infected corneas can develop a blinding immunoinflammatory condition called herpes stromal keratitis (HSK), which involves the loss of corneal sensitivity due to retraction of sensory nerves and subsequent hyperinnervation with sympathetic nerves. Increased concentrations of the cytokine VEGF-A in the cornea are associated with HSK severity. Here, we examined the impact of VEGF-A on neurologic changes that underly HSK using a mouse model of HSV-1 corneal infection. Both CD4+ T cells and myeloid cells produced pathogenic levels of VEGF-A within HSV-1-infected corneas, and CD4+ cell depletion promoted reinnervation of HSK corneas with sensory nerves. In vitro, VEGF-A from infected corneas repressed sensory nerve growth and promoted sympathetic nerve growth. Neutralizing VEGF-A in vivo using bevacizumab inhibited sympathetic innervation, promoted sensory nerve regeneration, and alleviated disease. Thus, VEGF-A can shape the sensory and sympathetic nerve landscape within the cornea, with implications for the treatment of blinding corneal disease.

Effect of Dry Eye Disease on the Kinetics of Lacrimal Gland Dendritic Cells as Visualized by Intravital Multi-Photon Microscopy.

The lacrimal gland (LG) is the main source of the tear film aqueous layer and its dysfunction results in dry eye disease (DED), a chronic immune-mediated disorder of the ocular surface. The desiccating stress (DS) murine model that mimics human DED, results in LG dysfunction, immune cell infiltration, and consequently insufficient tear production. To date, the immune cell kinetics in DED are poorly understood. The purpose of this study was to develop a murine model of intravital multi-photon microscopy (IV-MPM) for the LG, and to investigate the migratory kinetics and 3D morphological properties of conventional dendritic cells (cDCs), the professional antigen presenting cells of the ocular surface, in DED. Mice were placed in a controlled environmental chamber with low humidity and increased airflow rate for 2 and 4 weeks to induce DED, while control naïve transgenic mice were housed under standard conditions. DED mice had significantly decreased tear secretion and increased fluorescein staining (p < 0.01) compared to naïve controls. Histological analysis of the LG exhibited infiltrating mononuclear and polymorphonuclear cells (p < 0.05), as well as increased LG swelling (p < 0.001) in DED mice compared to controls. Immunofluorescence staining revealed increased density of cDCs in DED mice (p < 0.001). IV-MPM of the LG demonstrated increased density of cDCs in the LGs of DED mice, compared with controls (p < 0.001). cDCs were more spherical in DED at both time points compared to controls (p < 0.001); however, differences in surface area were found at 2 weeks in DED compared with naïve controls (p < 0.001). Similarly, 3D cell volume was significantly lower at 2 weeks in DED vs. the naïve controls (p < 0.001). 3D instantaneous velocity and mean track speed were significantly higher in DED compared to naïve mice (p < 0.001). Finally, the meandering index, an index for directionality, was significant increased at 4 weeks after DED compared with controls and 2 weeks of DED (p < 0.001). Our IV-MPM study sheds light into the 3D morphological alterations and cDC kinetics in the LG during DED. While in naïve LGs, cDCs exhibit a more dendritic morphology and are less motile, they became more spherical with enhanced motility during DED. This study shows that IV-MPM represents a robust tool to study immune cell trafficking and kinetics in the LG, which might elucidate cellular alterations in immunological diseases, such as DED.