Herpes simplex virus type 1 (HSV-1) triggers significant inflammation and immune dysregulation, particularly in immunocompromised hosts. MicroRNA-221 (miR-221) is implicated in viral pathogenesis and inflammatory responses, yet its role in HSV-1 infection remains undefined. This study demonstrates that miR-221 suppresses HSV-1 replication, with 100 nM miR-221 reducing plaque formation by approximately 70% and markedly decreasing infectious viral titers (TCID50) in vitro. Mechanistically, bioinformatic analyses, dual-luciferase reporting, and RNA immunoprecipitation (RIP) confirmed CAMK2A, a calcium-signaling kinase, as a direct target of miR-221. KEGG pathway mapping linked miR-221 to calcium signaling, complement activation, and extracellular matrix interactions. In vivo, miR-221 overexpression in HSV-1-infected mouse corneas reduced pro-inflammatory cytokines (TNF-α, IL-6) and elevated anti-inflammatory IL-10, while enhancing CD8+ T-cell and NK-cell activation. Critically, it attenuated inflammatory cell infiltration and tissue apoptosis. CAMK2A inhibition synergistically amplified these antiviral and anti-inflammatory effects, whereas CAMK2A overexpression reversed them. We conclude that miR-221 restricts HSV-1 replication and modulates host inflammation by recruiting RISC to silence CAMK2A, thereby disrupting calcium signaling and promoting immune activation. These findings identify the miR-221/CAMK2A axis as a promising therapeutic target for HSV-1-induced inflammation.

PurposeTo investigate corneal healing in a Pax6+/− mouse model of aniridia-associated keratopathy (AAK) when challenged with a mild epithelial wound.MethodsPax6+/− small-eye (Sey) mice (n = 15) and Pax6+/+ wild-type mice (n = 4) on the 129S1/SvImJ background underwent 1 mm central corneal mechanical epithelial debridement. Corneal status before and four weeks after wounding was evaluated by slit-lamp imaging. Longitudinal corneal thickness was measured using optical coherence tomography (OCT), and AAK was graded in Sey mice. Wounded corneas were additionally compared to unwounded contralateral corneas for AAK status. Immunofluorescence staining was used to assess inflammation, wound healing, and the presence of corneal blood and lymphatic vessels.ResultsIn wild-type mice, the central epithelial wound healed normally. In contrast, wounded corneas in Sey mice exhibited significantly accelerated AAK progression at 4 weeks compared to unwounded contralateral corneas (P = 0.011), with a corresponding increase in mean AAK grade relative to pre-wounding levels (P = 0.001). AAK grade correlated positively with increased central corneal thickness in wounded (P = 0.031) and unwounded (P = 0.0006) Sey mice. Wounded corneas in Sey mice exhibited strong hemangiogenesis and lymphangiogenesis and altered epithelial phenotype with decreased CK12 and elevated MUC5AC expression, along with an increase in F4/80, IL-6, and IL-1β inflammation markers. Aberrant epithelial differentiation and stem/progenitor maintenance (CK15, SOX9), and persistent tissue repair (Ki-67) were present and were consistent with disease progression.ConclusionsCentral epithelial wounding accelerates AAK progression in Sey mice, highlighting the epithelial fragility in Pax6-aniridia and providing an efficient and robust accelerated model of AAK progression for preclinical studies.

Single-cell RNA sequencing of mouse cornea during wound healing after infrared laser irradiation.

British anti-Lewisite (BAL) reduces the severity of systemic and local responses of the eye after exposure to the chemical warfare agent surrogate for Lewisite, phenylarsine oxide (PAO)

PurposeDiabetic keratopathy, a common ocular complication of diabetes, is characterized predominantly by corneal epithelial damage and peripheral nerve injury. This study examined the role of adiponectin (ADPN) in regulating the repair of the diabetic corneal epithelium and accompanying nerve injuries.MethodsRNA sequencing was performed on total RNA isolated from corneal epithelium of streptozotocin (STZ)-induced type 1 diabetic mice and type 2 diabetic BKS.Cg-Dock7m +/+ Leprdb/Nju (db/db) mice to identify differentially regulated pathways and interactions. ADPN receptor expression was assessed. Recombinant ADPN, ADPN receptor 1/2 siRNA, and a phosphorylated AKT (p-AKT) inhibitor were then utilized in diabetic mice and in human corneal epithelial cells (HCECs) cultured under high-glucose conditions to evaluate corneal wound healing responses.ResultsADPN receptor expression and p-AKT levels were downregulated in corneas of diabetic mice and in HCECs exposed to high glucose. Treatment with recombinant ADPN accelerated repair of corneal epithelial and nerve damage in both type 1 and type 2 diabetic mice, enhanced HCEC proliferation and migration under high-glucose conditions and activated AKT signaling. ADPN treatment also reduced neutrophil infiltration and inflammatory factor expression during wound repair. These beneficial effects were abolished by ADPN receptor 1 knockdown or AKT inhibition.ConclusionsOur results demonstrate that ADPN promotes the corneal epithelium and nerve regeneration in diabetic mice via activation of the AdipoR1/AKT signaling axis and suppression of inflammatory responses. These findings identify ADPN as a promising therapeutic candidate for promoting corneal epithelial wound healing in diabetic conditions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40662-025-00458-w.

Corneal fibrosis, a significant source of visual impairment, can result from keratocyte-to-myofibroblast transdifferentiation during wound healing. This study investigated the antifibrotic role of 1,25-dihydroxyvitamin D3 (1,25 Vit D) and the lesser-known vitamin D, 24,25-dihydroxyvitamin D3 (24,25 Vit D), in human and mouse corneal stromal cells (HSCs and MSCs) and in a Vit D receptor knockout (VDR KO) mouse model. Cells were treated with TGF-β1 ± Vit D metabolites and the expression of fibrotic and antifibrotic genes and proteins was evaluated. Both metabolites significantly reduced α-smooth muscle actin levels in HSCs, MSCs and organ-cultured mouse corneas (p < 0.05). They also upregulated the mRNA expression of BMP2, BMP6, BMPR2, and TGF-β3, as well as the protein expression of BMP6 and TGF-β3. VDR KO corneas subjected to alkali injury exhibited increased fibrotic responses and reduced CD45+ immune cell infiltration compared to wild-type controls. Notably, 24,25 Vit D exerted antifibrotic effects even in VDR KO cells, and the alternative 24,25 Vit D receptor FAM57B was expressed in all corneal cell layers. These results reveal consistent antifibrotic effects of both 1,25 and 24,25 Vit D across species, support the existence of VDR-independent mechanisms in the cornea, and offer new insights into potential therapeutic strategies for preventing corneal fibrosis.

Primary nociceptors are essentially characterized as afferent neurons carrying noxious sensory information from the periphery to the central nervous system. However, the information flow on primary nociceptors is bidirectional. Nociceptor peripheral terminals release a variety of mediators to the target organ near the injured area. These mediators promote sensitization of adjacent sensory neurons, vasodilation, and edema, affecting innate and adaptive immunity, leading to hyperalgesia and inflammation that often expands beyond the injured areas. While many studies associate these phenomena with the antidromic action-potential propagation along nociceptor terminals, direct evidence demonstrating that activation of a single nociceptor terminal can, through efferent signaling, recruit neighboring unstimulated terminals has been missing. In this study, using in vivo calcium imaging from the individual nociceptive terminals innervating the cornea of male mice, together with a computational approach, we demonstrated that brief activation of a single terminal in vivo by capsaicin was sufficient to evoke a response in a remote, unstimulated terminal, branching from the same nociceptor fiber. The activation of the remote unstimulated terminal was dependent on the activation of voltage-gated sodium and calcium channels. Moreover, we showed that in the model of acute hyperalgesia, following exposure to proinflammatory cytokines, the efferent signaling along nociceptive terminals increases, culminating in enhanced calcium signaling in the remote unstimulated terminals. This increase in intraterminal calcium could trigger an enhanced release of inflammatory mediators, affecting wider areas and terminals from adjacent unstimulated receptive fields, leading to the expansion of hyperalgesia and inflammation.

Impact of innate immune factors on the persistence of Staphylococcus aureus in the ocular environment.

In vivo removal of KS GAGs from murine cornea by intrastromal injection of keratanase II enzyme.

To develop and characterize a novel mouse model of granular corneal dystrophy type II (GCD2) using CRISPR/Cas9 technology and explore the underlying pathogenesis of transforming growth factor-beta-induced protein (TGFBIp) aggregation. CRISPR/Cas9 technology was employed to introduce the R124H mutation in the TGFBI gene of mice. Genomic sequencing and polymerase chain reaction confirmed the mutation. Phenotypic characteristics were evaluated through slit-lamp examination, optical coherence tomography, histological analysis, electron microscopy, and immunofluorescence, comparing wild-type (WT), heterozygous (HE), and homozygous (HO) mice. Transcriptome sequencing was conducted to identify the pathogenesis of GCD2. The findings were further validated through western blotting and transmission electron microscopy. The R124H mutation in TGFBI was successfully introduced, with breadcrumb-like deposits observed in the corneas of mutant mice, with HO mice displaying more severe phenotypes than HE mice. TGFBIp levels were elevated in HE and HO mice (both P < 0.001). Histological and electron microscopy analyses revealed abnormal collagen arrangement and TGFBIp deposits in the corneal stroma of the HE and HO mice. Transcriptome analysis indicated that the TGFBI-R124H mutation was associated with impaired autophagy, endocytosis, and extracellular matrix signaling. Additional experiments confirmed autophagy-related markers LC3 and SQSTM1 were upregulated in the corneas of mutant mice, accompanied by increased autophagosome formation in corneal keratocytes, indicating impaired autophagy flux in HE and HO mice. We established a GCD2 mouse model caused by the R124H mutation using CRISPR/Cas9, providing a reliable platform for understanding pathogenesis for GCD2.

PurposeHerpes simplex virus type 1 (HSV-1) infection of the human eye can lead to herpes simplex keratitis, which is the leading cause of infectious blindness worldwide. Inflammation invading the corneal stroma causes herpetic stromal keratitis (HSK). Interferon regulatory factor 6 (IRF6) is a member of the interferon regulatory factor family and is involved in the antiviral response against human papillomavirus 16. However, the mechanism related to the involvement of IRF6 in the host anti-HSV-1 response is unclear, and how latent infection and viral reactivation trigger corneal stromal inflammation remains unknown. Therefore, we investigated the role of IRF6 in HSV-1 infection.MethodsProteomic detection techniques indicated that IRF6 was expressed at low levels in the corneas of HSK model mice. The antiviral effects of IRF6 have been demonstrated in animal and cellular studies. HSV-1 replication was activated when IRF6 was silenced in human corneal epithelial cells (HCECs), and IRF6 overexpression inhibited viral replication. HSK was established after locally inoculating mouse corneas with lentivirus to overexpress IRF6.ResultsThe degree of inflammation was attenuated, and proinflammatory cytokine secretion was reduced in the mice overexpressing IRF6 compared with that in the lentivirus control mice, suggesting that IRF6 attenuates the severity of HSK. Silencing of protein kinase C delta and receptor-interacting serine/threonine kinase 4 reduced IRF6 phosphorylation and inhibited its degradation during HCEC infection with HSV-1.ConclusionThese findings indicate that IRF6 is involved in the immune response against HSV-1 virus, and IRF6 can be used as a therapeutic target for treating HSK.

TLR4 signal inhibition alleviates alkali-burn induced corneal neovascularization.

Blue light induces corneal epithelial damage by activating the NLRP3 inflammasome pathway.

Lacking TRPA1 Cation Channel Impairs Primary Closure of a Stromal Incision Injury in a Mouse Cornea.

Piezo2 is a mechanosensitive ion channel essential for touch and proprioception, yet the mechanisms that maintain this sensory modality in adult tissues are unknown. Using multiphoton imaging of the cornea in live mice, we discovered that the Cx3cr1 Cre locus targets not only macrophages, but also a distinct subset of nerves. Spatial-RNAseq resolved that Cx3cr1 Cre -driven labeling was uniquely enriched in Piezo2-expressing neurons, a result of temporal Cx3cr1 expression during development. Through lineage tracing, scRNAseq, and imaging, we identified a novel tripartite cellular niche at the epithelial basement membrane, comprised of monocyte-derived macrophages, nerves, and Schwann cells. Additional scRNAseq and genetic studies revealed that Schwann cell-derived IL34 maintained corneal macrophages. Through pharmacologic and genetic perturbations, we also demonstrate corneal macrophages selectively maintained the structure-function of Piezo2-enriched nerve endings, with disruption of this niche causing specific deficits in mechanosensation while preserving other sensory modalities. Altogether, we describe a novel tricellular niche in the cornea required for Piezo2-mediated touch sensation, suggesting new directions for investigating mechanosensory circuits including proprioception.

Lycium barbarum glycopeptide reduces inflammation and fibrosis in corneal injury by modulating the NF-κB/NLRP3/IL-1 β signaling pathway and microRNA-21a-5p/SMAD7.

Small Extracellular Vesicle–Mediated Peptide Delivery to the Mouse Corneal Endothelium In Vivo

Diabetes mellitus (DM) patients are at higher risk for infections, which are often more severe. This study investigated the role of aconitate decarboxylase 1 (Acod1) and its product, itaconate, in innate defense against bacterial keratitis and its impairment in type 1 DM mice. Wild-type or normal (NL), streptozotocin-induced DM, and Acod1-/- mice were inoculated with Pseudomonas aeruginosa (Pa) with or without 4-octyl itaconate (4-OI), a cell-permeable derivative of itaconate. Keratitis severity was determined by photography, clinical scores, Pa burden (cfu), and myeloperoxidase (MPO) activity. Gene expression was determined by quantitative PCR. Immune and Acod1-positive cells were determined by immunohistochemistry. DM mice expressed lower levels of Acod1 in B6 mouse corneas, and Pa infection triggered its upregulation, mostly in infiltrated cells. Acod1 deficiency increased the severity of Pa keratitis and significantly augmented the expression of Il-1β, Il-1ra, and Ccl3, but not Ccl2, at 1day post-infection (dpi). Acod1-/- increased neutrophil but decreased macrophage infiltration. 4-OI prevented Pa infection in NL corneas (P = 6.7E-05) and alleviated Pa keratitis in DM corneas (P = 0.000204) at 3 dpi. Hyperglycemia augmented Pa infection-induced Il-1β, Il-1ra, and particularly Ccl3, but not Ccl2. In DM corneas, 4-OI greatly dampened the expression of CCL3 but not CCL2, compared to DM corneas without the treatment. The presence of 4-OI significantly reduced the severity of Pa keratitis in Acod1-deficient mice. Acod1/itaconate is crucial for mediating protective immune responses against Pa infection in both NL and DM corneas. Acod1 activation and/or itaconate-based therapies may offer promising adjunctive treatments for microbial keratitis in patients with diabetes.

We previously showed that selective suppression of CD44 in the corneal epithelium leads to structural abnormalities in the mouse cornea. Our comparative studies of young and aged ocular biopsies revealed that CD44 expression is downregulated in aged corneas, while leucine-rich repeats and immunoglobulin-like domain 1 (Lrig1+) stem cells remain preserved in the peripheral limbus. These findings suggest an age-related shift in the corneal stem cell compartmentalization, characterized by impaired CD44 expression in the central cornea and preservation of Lrig1+ stem cells in the limbus, which become the main stem cells in the senescent cornea. To investigate this further, we performed topical tamoxifen-inducible, diphtheria toxin-mediated ablation of Lrig1+ stem cells in mouse corneas. We then assessed both activated and non-activated beta-catenin expression in wild-type (WT) and CD44 knockout (CD44KO) mice, given that CD44 modulates the Wingless-related integration site (Wnt) pathway. Our results indicate that two distinct stem cell populations operate in the mouse cornea: Lrig1-derived stem cells and Wnt-activity/CD44-dependent stem cells. The Lrig1-derived cells act as a reservoir of quiescent stem cells that regenerate the cornea upon injury, whereas under homeostatic conditions, the Wnt-activity/CD44-dependent stem cells are primarily responsible for corneal renewal. In the aged cornea, the loss of CD44 expression leads to reduced Wnt signaling, making the tissue increasingly dependent on Lrig1+ stem cells for regeneration. In mice, Lrig1+ stem cells are capable of sustaining permanent corneal renewal, even in the absence of CD44.

Essential roles of sensory nerve in maintenance of cornea-phenotype in mice.