Dry eye disease (DED) is a multifactorial disease, and oxidative stress plays a crucial role in its pathogenesis. Recently, multiple studies have shown that upregulation of autophagy can protect the cornea from oxidative stress damage. The present study investigated the therapeutic effects of salidroside, the main component of Rhodiola crenulata, in both in vivo and in vitro dry eye models. The results showed that topical eye drop treatment with salidroside restored corneal epithelium damage, increased tear secretion, and reduced cornea inflammation in the DED mice. Salidroside activated autophagy through AMP-activated protein kinase (AMPK)-sirtuin-1 (Sirt1) signaling pathway, which promoted the nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2) and increased the expression of downstream antioxidant factors heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1). This process restored antioxidant enzyme activity, reduced reactive oxygen species (ROS) accumulation, and alleviated oxidative stress. The application of autophagy inhibitor chloroquine and AMPK inhibitor Compound C reversed the therapeutic efficacy of salidroside, validating the above findings. In conclusion, our data suggest that salidroside is a promising candidate for DED treatment.

We explored the therapeutic efficacy of Mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) and its inhibition of the functions of dendritic cells (DCs) in dry eye disease (DED). MSC-EVs were isolated from the culture supernatants of mesenchymal stromal cells (MSCs) and characterized. In vitro, human corneal epithelial cells (HCECs) were cultured in hyperosmotic medium to simulate the DED hyperosmotic environment and treated with MSC-EVs. Cell viability was assessed, and the expression of inflammatory cytokines was quantified. Next, we induced DED in female C57BL/6 mice and divided the mice into groups treated with either MSC-EVs or phosphate buffer solution (PBS) eye drops. Disease severity was assessed; mRNA expression of inflammatory cytokines was analyzed by RT-PCR; and Th17 cells were detected by flow cytometry. Lastly, we evaluated DCs by immunofluorescence and flow cytometric analysis to assess its amounts and maturation. MSC-EVs showed protective effects on HCECs under hyperosmotic stress in vitro, suppressing the expression of inflammatory cytokines. In vivo, mice topically treated with MSC-Evs presented reduced DED disease severity compared to PBS-treated mice. MSC-Evs downregulated the expression of inflammatory cytokines, including TNF-α, IL-6, and IL-1β, as well as the frequency of Th17 cells. Further investigation showed that MSC-EVs suppressed the increase of amounts and the maturation of DCs in DED. Changes of morphological characters of DCs were also inhibited by MSC-EVs. Our study revealed that MSC-EVs suppressed ocular surface inflammation by inhibiting DCs activation-mediated Th17 immune responses, explicating the therapeutic potential of MSC-EVs in DED and other ocular surface diseases.

Herpes simplex keratitis (HSK) is a blinding disease caused by herpes simplex virus type 1 (HSV-1) infection, and rapid eradication of the virus from the affected cornea is imperative. Nod-like receptors (NLRs) are intracellular innate immune sensors closely associated with cell death, inflammation and immune responses. In this study, we investigated the role of NLRP12 in the antiviral immunology in HSK and the underlying mechanisms. We found that NLRP12 expression was significantly decreased in HSV-1-infected human corneal epithelial cells (HCE-Ts) and HSK mouse corneas. Overexpression of NLRP12 significantly reduced viral replication in infected HCE-Ts and functioned through inflammasome-mediated pyroptosis and downstream IL-18-IFN-γ axis. In HSK mouse models, overexpression of NLRP12 reduced viral replication in the cornea and alleviated HSK symptoms. This resulted from enhanced antiviral immune responses including the activation of specific immune cells in both the cornea and the draining lymph nodes. Specifically, the NLRP12-IL-18-IFN-γ axis regulated the interaction between infected corneal epithelial cells and macrophages. In conclusion, our study identified a role of NLRP12 in mediating pyroptosis and regulating antiviral immune responses. This novel finding opens the possibilities of NLRP12 as a viable target in the therapeutic strategies for HSV-1 infection.

Patients with refractory dry eye disease (DED) often face the threat of diminished visual quality and have limited responses to existing treatments. Ocular injection of Mesenchymal stem cells (MSCs) has recently emerged as a promising new therapeutic strategy for DED. Topical eye drops are the clinical favorable choice for drug administration in DED. To date, the clinical use of MSC eye drops has not been reported in settings. This clinical trial represents a groundbreaking exploration into the preliminary therapeutic potential and safety of umbilical cord MSC eye drops for patients with refractory DED, including both non-Sjögren's dry eye (NSDE) and Sjögren's syndrome dry eye (SSDE). The study also aimed to investigate the possible underlying mechanisms. In this open-label, prospective, single-arm, self-controlled trial, 11 NSDE and 5 SSDE patients received twice-daily MSC eye drops for two weeks, subsequent follow-up visits were scheduled at 4 weeks and 12 months after treatment. The primary efficacy was evaluated using the ocular surface disease index (OSDI) score, tear meniscus height (TMH), non-invasive break-up time (NIBUT), Schirmer I test (SIT), and corneal fluorescein staining (CFS) score. Secondary assessments focused on the evaluation of lipid layer, meibomian gland function, and bulbar conjunctival redness. Safety was monitored by recording adverse events (AEs) throughout the study. Changes in tear levels of interleukin-6 (IL-6), IL-17A, Mucin 5AC (MUC5AC), C–C chemokine ligand 20 (CCL20) and IL-23, along with proteomic alterations, were compared between baseline and T-week2. Significant clinical improvements were observed in most symptoms and signs following MSC eye drops treatment in both NSDE and SSDE patients, particularly in tear production as measured by SIT and TMH, and the alleviation of meibomian gland blockage. The therapeutic effect on OSDI, NIBUT, and the lipid layer was more pronounced in NSDE patients compared to SSDE. No serious AEs were reported during the treatment and follow-up period. Post-treatment reductions in tear levels of IL-6 and IL-17A, along with an increase in MUC5AC, further confirmed the efficacy. Tear proteomic analysis indicated that the efficacy of MSC eye drops is associated with the inhibition of inflammation caused by T helper 17 (Th17) cells in both NSDE and SSDE groups. In this prospective exploratory clinical study, we have demonstrated that MSC eye drops might offer clinical efficacy and manageable safety in treating refractory DED for the first time, potentially bringing a new perspective on the treatment of such patients. Our research represents a preliminary exploratory endeavor, paving the way for future large-scale randomized positive-controlled trials. Trial registration: ClinicalTrials.gov, NCT05784519. Registered 28 February 2023, https://clinicaltrials.gov/study/NCT05784519 .

Herpes simplex keratitis (HSK) is a severe, infectious corneal disease caused by herpes simplex virus type 1 (HSV-1) infection. The increasing prevalence of acyclovir resistance, the side effects of hormonal drugs, and the ease of recurrence after surgery have made it crucial to develop new methods of treating HSK. HSV-1 evades the host immune response through various mechanisms. Therefore, we explored the role of the immunogenic cell death inducer PKHB1 peptide in HSK. After subconjunctival injection of PKHB1 peptide, we observed the ocular surface lesions and survival of HSK mice and detected the virus levels in tear fluid, corneas, and trigeminal ganglions. We found that PKHB1 peptide reduced HSV-1 levels in the eye and alleviated the severity of HSK. Moreover, it increased the number of corneal infiltrating antigen-presenting cells (APCs), such as macrophages and dendritic cells, and CD8⁺ T cells in ocular draining lymph nodes. We further observed that PKHB1 peptide promoted the exposure of calreticulin, as well as the release of ATP and high-mobility group box 1 in HSV-1-infected cells in vitro. Our findings suggested that PKHB1 peptide promoted the recruitment and maturation of APCs by inducing the release of large amounts of damage-associated molecular patterns from infected cells. APCs then phagocytized antigenic materials and translocated to the lymph nodes, triggering a cytotoxic T lymphocyte-dependent immune response that ultimately alleviated HSK.