Neutrophil-driven and interleukin-36γ-associated ocular surface inflammation in chronic Stevens-Johnson syndrome.

PURPOSE: This study aims to elucidate the tear proteome and understand the underlying molecular mechanisms involved in the ocular complications following Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). METHODS: Mass spectrometry (MS) was performed to quantify the tear fluid proteins from chronic SJS/TEN patients (n = 22 eyes) and age- and gender-matched controls (n = 22 eyes). The candidate proteins were validated using ELISA (n = 80 eyes) in tear samples and immunohistochemistry (IHC; n = 12) in eyelid margin specimens. These proteins were compared for significant differences based on age, gender, disease duration, and ocular severity. RESULTS: A total of 1692 tear fluid proteins were identified, of which 470 were significantly differentially regulated in chronic SJS/TEN. The top 10 significantly upregulated proteins were neutrophil secretions including neutrophil elastase (p < .0001), defensin (p < .0001), and matrix metalloproteinase 8 (p < .0001). The presence of neutrophils was confirmed by the upregulation of IL-8 (p < .001) in tears, a key cytokine known for recruiting neutrophils. Additionally, positive expression of myeloperoxidase was observed in the keratinized eyelid margins of SJS/TEN to validate the presence of neutrophils. Among 41 unique proteins identified by MS, IL-36γ (p < .01) was expressed in three SJS/TEN patients and was confirmed in SJS/TEN tears and eyelid margins by ELISA and IHC, respectively. IL-36γ was specifically expressed in the superficial layers of eyelid margin keratinized conjunctiva. The majority of the significantly downregulated proteins were lacrimal gland secretions such as lacritin (p < .0001) and opiorphin (p < .002). Neutrophil elastase (p < .02) was significantly elevated in patients with severe eyelid margin keratinization. CONCLUSION: Our observations indicate a clear correlation between eyelid margin keratinization and the expression of IL-36γ, potentially mediated by neutrophils recruited via IL-8. Future experimental studies are needed to test the role of therapies targeting IL-8 and/or IL-36γ in reducing eyelid margin keratinization and its associated ocular complications in SJS/TEN.

Development and validation of a reliable rabbit model of limbal stem cell deficiency by mechanical debridement using an ophthalmic burr.

A simple and reproducible method is necessary to generate reliable animal models of limbal stem cell deficiency (LSCD) for assessing the safety and efficacy of new therapeutic modalities. This study aimed to develop and validate a rabbit model of LSCD through mechanical injury. The corneal and limbal epithelium of New Zealand White rabbits (n = 18) were mechanically debrided using an ophthalmic burr (Algerbrush II) with a 1.0-mm rotating head after 360° conjunctival peritomy. The debrided eyes were serially evaluated for changes in corneal opacity, neo-vascularization, epithelial defect and corneal thickness using clinical photography, slit lamp imaging, fluorescein staining, and anterior segment optical coherence tomography scanning (AS-OCT). Following this, an assessment of histopathology and phenotypic marker expression of the excised corneas was conducted. The experimental eyes were grouped as mild (n = 4), moderate (n = 10), and severe (n = 4) based on the grade of LSCD. The moderate group exhibited abnormal epithelium, cellular infiltration in the stroma, and vascularization in the central, peripheral, and limbal regions of the cornea. The severe group demonstrated central epithelial edema, peripheral epithelial thinning with sparse goblet cell population, extensive cellular infiltration in the stroma, and dense vascularization in the limbal region of the cornea. A significant decrease in the expression of K12 and p63 (p < 0.0001) was observed, indicating the loss of corneal epithelium and limbal epithelial stem cells in the LSCD cornea. This study demonstrates that the Alger brush-induced mechanical debridement model provides a reliable model of LSCD with comprehensive clinic-pathological features and that is well suited for evaluating novel therapeutic and regenerative approaches.

Optimization and evaluation of tear protein elution from Schirmer's strips in dry eye disease.

PURPOSE: Extraction of tear protein from Schirmer's strip is a prerequisite for the proper identification and screening of biomarkers in dry eye disease. The study compares different methods of extraction of tear proteins from the Schirmer's strip. METHODS: Reflex tear was collected from healthy controls (HC; n = 12), Stevens-Johnson syndrome (SJS; n = 3) and dry eye disease (DED; n = 3) patients using capillary tube. This tear was used to measure the volume absorbed by Schirmer's strip per microliter. Different buffers (6) were used to compare the protein yield from the Schirmer's strip in four different conditions. The tear proteins extracted using the highest protein yield buffer were analyzed by mass spectrometry. RESULTS: A linear relationship between the tear volume and wetting length was observed (r = 0.0.997, n = 6). The highest yield was observed after incubation of the Schirmer's strip in 100 mM ammonium bicarbonate (ABC) with 0.25% Nonidet P-40(NP-40) at 4°C for an hour (P < 0.00005). The in-solution digestion of tear eluted in the above condition 100 Mm ABC + 0.25% NP-40 with one-hour incubation yielded a total of 2119 proteins in HC, SJS, and DED. The unique protein observed in SJS and DED was 0.6% and 17.9%, respectively. The significantly expressed proteins are associated with innate immune response, proteolysis, wound healing, and defense response. CONCLUSION: A method for extraction of protein from Schirmer's strip was optimized for increase in protein yield from the tear sample. SJS and DED tear samples have unique protein signature. The study will aid in better design of tear protein-based experimental study.

Transcriptomic Profiling of Human Limbus-Derived Stromal/Mesenchymal Stem Cells-Novel Mechanistic Insights into the Pathways Involved in Corneal Wound Healing.

Limbus-derived stromal/mesenchymal stem cells (LMSCs) are vital for corneal homeostasis and wound healing. However, despite multiple pre-clinical and clinical studies reporting the potency of LMSCs in avoiding inflammation and scarring during corneal wound healing, the molecular basis for the ability of LMSCs remains unknown. This study aimed to uncover the factors and pathways involved in LMSC-mediated corneal wound healing by employing RNA-Sequencing (RNA-Seq) in human LMSCs for the first time. We characterized the cultured LMSCs at the stages of initiation (LMSC-P0) and pure population (LMSC-P3) and subjected them to RNA-Seq to identify the differentially expressed genes (DEGs) in comparison to native limbus and cornea, and scleral tissues. Of the 28,000 genes detected, 7800 DEGs were subjected to pathway-specific enrichment Gene Ontology (GO) analysis. These DEGs were involved in Wnt, TGF-ß signaling pathways, and 16 other biological processes, including apoptosis, cell motility, tissue remodeling, and stem cell maintenance, etc. Two hundred fifty-four genes were related to wound healing pathways. COL5A1 (11.81 ± 0.48) and TIMP1 (20.44 ± 0.94) genes were exclusively up-regulated in LMSC-P3. Our findings provide new insights involved in LMSC-mediated corneal wound healing.

Cytokeratin profile and keratinocyte gene expression in keratinized lid margins of patients with chronic Stevens-Johnson syndrome.

PURPOSE: To study the cytokeratin profile and keratinization-related gene expression in keratinized lid margins of chronic Stevens-Johnson syndrome (SJS) patients. METHODS: Posterior eyelid margins from 24 chronic SJS patients undergoing mucous membrane grafting and six healthy margins (orbital exenteration, fresh body donors) were studied using immunofluorescence staining (CK10, CK1, filaggrin, transglutaminase 1 (TGM1), (CK19, MUC5AC)) and quantitative PCR (keratinization-related genes-HBEGF, KGF, EGF, TGFα, TGFß, and TNFα). The staining and gene expression were studied separately in the lid margin epidermis (LME) and lid margin conjunctiva (LMC). RESULTS: The expression of CK 1/10, filaggrin, and TGM1 in the LMC was similar to the LME in SJS patients. CK19 was expressed only in the basal epithelial layer of the LMC with loss of MUC5AC expression. Increased expression of KGF (p ≤ 0.056), TNFα (p ≤ 0.02), and TGFα (p ≤ 0.01) was observed in the LME of SJS patients compared to normal LME. LMC of SJS patients showed an increased expression of HBEGF (p ≤ 0.002), EGF (p ≤ 0.0002), KGF (p ≤ 0.02), TNFα (p ≤ 0.04), TGFα (p ≤ 0.003), and TGFß (p ≤ 0.001) compared to normal LMC. Significant differences were observed in the expression of these genes between LME and LMC of SJS patients. These genes were validated using String analysis, which revealed the positive regulation of keratinization. CONCLUSION: In lid margins of SJS, there is an increased expression of keratinization-related genes compared to the normal lid margin. Keratinized LMC shares similar cytokeratin profile and keratinization gene expression as seen in cutaneous epithelium of SJS patients, indicating the possibility of the cutaneous epithelium as a source for keratinized LMC.

Differential expression of tear film cytokines in Stevens-Johnson syndrome patients and comparative review of literature.

To investigate the differential expression of tear cytokine levels among chronic Stevens-Johnson syndrome (SJS) patients to better understand the role of significantly altered cytokines in disease development. Tear samples were collected using Schirmer strips in 24 eyes of chronic SJS, 24 eyes of age and gender-matched controls, and 14 eyes of aqueous deficiency dry eye disease (DED) patients. The cytokine analysis was performed among 18 analytes which include pro-inflammatory, anti-inflammatory factors, and ELR-negative CXC chemokines. String analysis was performed for the significantly altered cytokines to understand their co-expression and role in the disease development. Additionally, a literature review was conducted to identify the signature cytokines present in chronic SJS tears. The differential expression of IL-6 (p ≤ 0.029), CXCL8/IL-8 (p ≤ 0.009), IL-1ß (p ≤ 0.041), IL-2 (p ≤ 0.025), IL-10 (p ≤ 0.053), and CXCL-10 (p ≤ 0.044) were observed in chronic SJS patients and healthy controls. Whereas, IL-6 (p ≤ 0.029), CXCL8/IL-8 (p ≤ 0.058), CCL4 (p ≤ 0.056), GM-CSF (p ≤ 0.0001) IL-10 (p ≤ 0.025), and CXCL-10 (p ≤ 0.010), were differentially expressed in SJS as compared to severe DED patients. String analysis of the significantly altered cytokines revealed the involvement of several biological processes including the chronic inflammatory response, nitric oxide synthesis, angiogenesis, and cellular response to drugs. Among all the cytokines evaluated, the expression of CXCL8/IL-8 and CXCL10 levels were consistently reported in the literature. There was a differential expression of tear cytokines in SJS when compared to DED and healthy controls. The differential expression of CXCL8/IL-8 and CXCL10 was in line with existing literature and their role in chronic SJS pathogenesis merits further evaluation.