IFN-γ Facilitates Corneal Epithelial Cell Pyroptosis Through the JAK2/STAT1 Pathway in Dry Eye

**Background:** Dry eye disease (DED) is a common multifactorial ocular surface disorder characterized by tear film instability, hyperosmolarity, and chronic inflammation. Pyroptosis, a lytic programmed cell death mediated by gasdermin D (GSDMD) and the NLRP3 inflammasome, has been implicated in DED pathogenesis. IFN-γ, a pleiotropic cytokine produced by NK and Th1 cells, is elevated in DED and promotes apoptosis, but its role in pyroptosis and the underlying molecular mechanisms remain unclear. The JAK/STAT pathway is a canonical downstream signaling cascade of IFN-γ, and its involvement in cell death and inflammation is well established. This study aimed to investigate whether IFN-γ facilitates corneal epithelial pyroptosis through the JAK2/STAT1 pathway in experimental dry eye.

**Methods:** Experimental dry eye was induced in adult wild-type (WT) C57BL/6 mice and Ifng-knockout (Ifng−/−) mice by subcutaneous injection of scopolamine (1.5 mg/0.3 mL, three times per day) and exposure to desiccating stress (30% humidity, air draft) for five days. Corneal epithelial defects were assessed by fluorescein sodium staining and quantified as percentage of defect area using ImageJ. Tear production was measured by phenol red thread test (15 seconds). Conjunctival goblet cells were evaluated by periodic acid-Schiff (PAS) staining. In vitro, an immortalized human corneal epithelial cell line (HCE-T) and primary human corneal epithelial cells (p-HCECs) were cultured in hyperosmolar medium (500 mOsM, achieved by adding 94 mM NaCl) with or without recombinant human IFN-γ (10 ng/mL). STAT1 was inhibited in vivo by subconjunctival injection of fludarabine (2.5 µM, 5 µL) or in vitro by siRNA targeting STAT1. ROS levels were measured using DCFH-DA reagent, and N-acetylcysteine (NAC) was used as a ROS scavenger. Gene expression was analyzed by qRT-PCR, and protein expression by Western blotting and immunofluorescence. Cell death was assessed by CCK-8 viability assay, LDH release assay, and TUNEL staining. RNA sequencing was performed on corneal tissues from control and DE mice.

**Key Results:** RNA sequencing revealed upregulation of IFNG, IFNGR1, IFNGR2, and interferon-inducible genes (OAS, GBP families) in corneas of DE mice. Ifng knockout significantly alleviated dry eye symptoms: corneal defect area was reduced (e.g., from ~40% in WT DE to ~15% in Ifng−/− DE on day 5, P<0.0001), tear secretion was less reduced (e.g., ~4 mm vs. ~2 mm wetting length, P<0.05), and goblet cell loss was partially reversed (e.g., ~80 vs. ~40 cells/field, P<0.01). Among JAK/STAT family genes, JAK2 and STAT1 showed the most pronounced increases in DE corneas. Phosphorylated JAK2 (p-JAK2) and p-STAT1 were significantly elevated in DE mice by Western blot. Subconjunctival injection of the STAT1 inhibitor fludarabine reduced corneal defects (e.g., ~20% vs. ~40% in DE, P<0.01) and preserved tear secretion (~3.5 mm vs. ~2 mm, P<0.01). Ifng knockout decreased TUNEL-positive corneal epithelial cells (e.g., ~10 vs. ~40 cells/section, P<0.0001) and reduced expression of pyroptosis markers NLRP3, ASC, C-CASP1, and N-GSDMD by Western blot and immunofluorescence. Similar results were obtained with neutralizing anti-IFN-γ antibody in WT DE mice. In vitro, IFN-γ (10 ng/mL) under hyperosmolar conditions (500 mOsM) for 24 hours significantly increased LDH release (e.g., ~40% vs. ~10% in control, P<0.0001), decreased cell viability (e.g., ~60% vs. ~100%, P<0.0001), and upregulated NLRP3, ASC, C-CASP1, and N-GSDMD protein levels. IFN-γ alone without hyperosmolarity had no effect. siRNA knockdown of STAT1 in HCE-T cells reduced pyroptosis-related gene and protein expression under hyperosmolar plus IFN-γ conditions. ROS levels were elevated in DE corneas and in HCE-T cells treated with IFN-γ plus hyperosmolarity. NAC treatment reduced ROS and suppressed NLRP3, ASC, C-CASP1, and N-GSDMD expression without affecting p-JAK2 or p-STAT1 levels, indicating that oxidative stress acts downstream of JAK2/STAT1.

**Clinical Implications:** This study identifies IFN-γ as a key driver of corneal epithelial pyroptosis in dry eye disease through the JAK2/STAT1 signaling pathway, with oxidative stress as a downstream mediator. Targeting the IFN-γ/JAK2/STAT1 axis, such as with STAT1 inhibitors or ROS scavengers, may represent a novel therapeutic strategy for DED. These findings provide mechanistic insight into the inflammatory pathogenesis of dry eye and support further investigation of JAK/STAT pathway inhibitors in clinical settings.