Dynamic ocular surface secretome analysis in keratoconus patients under scleral lens intervention

To characterize the temporal dynamics of the ocular surface secretome in response to scleral lens (SL) wear in keratoconus and to explore potential molecular mechanisms. Ocular surface secretions were collected from four patients with keratoconus at baseline, 1 hour, and 1 month after initiating SL wear. Proteins were extracted, digested to peptides, and profiled by label-free data-independent acquisition (DIA) mass spectrometry. Temporal differences were assessed by principal component analysis, differential protein identification, and Mfuzz time-series clustering. Functional enrichment analyses were performed for biological processes and signaling pathways. Across all samples, 16,119 peptides corresponding to 2,617 proteins were identified, substantially expanding ocular surface secretome coverage and encompassing proteins involved in immune defense, metabolic regulation, and epithelial homeostasis. Within 1 hour of SL wear, there was rapid and significant upregulation of immunoglobulins; stress-inducible molecules such as MICB and IRF8; and antioxidant enzymes including GPX3 and SOD1, indicating acute immune activation and oxidative stress responses. At 1 month, LTF, SLPI, and BPI—together with proteins linked to epithelial remodeling, remained elevated, consistent with reinforcement of ocular surface barrier function and innate immunity. Mfuzz clustering resolved three dominant temporal patterns: (i) a “rise-then-fall” trajectory reflecting acute immune and stress responses; (ii) a general monotonic increase consistent with enhanced humoral immunity; and (iii) a continuous decrease suggestive of adaptive changes in carbohydrate and energy metabolism. Enrichment analyses highlighted processes related to innate and humoral immune responses, epithelial maintenance, oxidative stress pathways, and metabolic regulation. SL wear induces both immediate and short-term adaptations on the ocular surface in keratoconus. Proteomic changes reveal rapid activation of local immune and antioxidant defenses, followed by sustained enhancement of humoral immunity and coordinated metabolic reprogramming. • First construction of a temporal atlas of the ocular surface secretome in keratoconus patients under scleral lens wear • In-depth proteome analysis identifying 2,617 proteins involved in immune defense, metabolism, and epithelial homeostasis • Revealed three distinct temporal patterns indicating acute immune activation and subsequent metabolic adaptation