Tear Cytokine Profiling in Vernal Keratoconjunctivitis Identifies Immune Signatures Associated with Disease Severity

Tear Cytokine Profiling in Vernal Keratoconjunctivitis Identifies Immune Signatures Associated with Disease Severity | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 3 February 2026 V1 Latest version Share on Tear Cytokine Profiling in Vernal Keratoconjunctivitis Identifies Immune Signatures Associated with Disease Severity Authors : Kartik Goel , Prisha Warikoo , Abha Gour , Shailja Tibrewal , Virender Singh Sangwan , Mehak Sapra , and Anil Tiwari 0000-0003-0402-458X [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.177010931.16811284/v1 146 views 48 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: Vernal keratoconjunctivitis (VKC) is a chronic pediatric ocular allergic disease that can progress from seasonal to persistent inflammation with vision-threatening complications. While Th2 mechanisms are implicated, immune correlates of disease severity remain unclear. Methods: Tear samples from VKC patients (moderate intermittent and moderate persistent) and healthy controls were collected using Schirmer’s strips. Cytokine profiling was performed using the OLINK® Target 48 Cytokine Panel. Differential expression, correlation with clinical features, and pathway enrichment analyses were conducted. Results: IL-15, CXCL11, CXCL9, MMP12, and CCL13 were significantly elevated in VKC compared to controls, with higher levels in the persistent phenotype. These cytokines correlated with symptom duration, limbal involvement, and papillary hypertrophy. Pathway analysis revealed enrichment of IL-17, JAK–STAT, and chemokine signaling pathways. Conclusions: VKC severity is associated with distinct tear cytokine signatures, with the persistent phenotype showing enhanced chronic inflammatory signaling. Tear cytokines may serve as non-invasive biomarkers for disease monitoring and therapeutic stratification in paediatric VKC. Tear Cytokine Profiling in Vernal Keratoconjunctivitis Identifies Immune Signatures Associated with Disease Severity Kartik Goel 1 , Prisha Warikoo 1,3 , Abha Gour 1,2,4 , Shailja Tibrewal 5,6 , Virender Singh Sangwan 1, 4,6 , Mehak Sapra 1,2* , Anil Tiwari 1,2,6** 1. Eicher Shroff Centre for Stem Cell Research, Dr Shroff’s Charity Eye Hospital, New Delhi 2. Shroff-Pandorum Centre for Stem Cell Research, Pandorum Technologies Pvt. Ltd., New Delhi 3. College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, Arkansa 4. Department of Cornea and Anterior Segment Services, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India. 5. Department of Pediatric Ophthalmology, Strabismus and Neuro-ophthalmology, Dr Shroff’s Charity Eye, New Delhi, India 6. Dept of Ocular Genetics and Centre for Unknown and Rare Eye Diseases, Dr Shroff’s Charity Eye Hospital, New Delhi, India. Abstract Background: Vernal keratoconjunctivitis (VKC) is a chronic pediatric ocular allergic disease that can progress from seasonal to persistent inflammation with vision-threatening complications. While Th2 mechanisms are implicated, immune correlates of disease severity remain unclear. Methods: Tear samples from VKC patients (moderate intermittent and moderate persistent) and healthy controls were collected using Schirmer’s strips. Cytokine profiling was performed using the OLINK® Target 48 Cytokine Panel. Differential expression, correlation with clinical features, and pathway enrichment analyses were conducted. Results: IL-15, CXCL11, CXCL9, MMP12, and CCL13 were significantly elevated in VKC compared to controls, with higher levels in the persistent phenotype. These cytokines correlated with symptom duration, limbal involvement, and papillary hypertrophy. Pathway analysis revealed enrichment of IL-17, JAK–STAT, and chemokine signaling pathways. Conclusions: VKC severity is associated with distinct tear cytokine signatures, with the persistent phenotype showing enhanced chronic inflammatory signaling. Tear cytokines may serve as non-invasive biomarkers for disease monitoring and therapeutic stratification in paediatric VKC. Keywords: cytokines, ocular allergy, tear proteomics, vernal keratoconjunctivitis Key Message Vernal keratoconjunctivitis exhibits distinct tear cytokine signatures that correlate with disease severity and persistence. Elevated IL-15, CXCL9, CXCL11, MMP12, and CCL13 reflect chronic inflammatory activation in persistent VKC and associate with key clinical features. Tear-based cytokine profiling offers a non-invasive approach for disease monitoring and severity-guided therapeutic stratification in pediatric ocular allergy. INTRODUCITON Vernal keratoconjunctivitis (VKC) is a chronic, recurrent, and seasonally exacerbated allergic inflammation of the ocular surface that predominantly affects children and adolescents, particularly in warm, dry climates. The disease shows a higher prevalence in regions such as India, the Middle East, and Africa, with estimates suggesting it affects up to 3% of children in endemic zones. 1–3 . Clinically, VKC is characterized by intense ocular itching, photophobia, tearing, mucous discharge, and the development of giant papillae on the upper tarsal conjunctiva or limbal hypertrophy. While many cases follow a seasonal and self-limiting course, a substantial subset of patients—particularly in Indian and Asian populations—progresses to a moderate persistent or severe phenotype marked by perennial inflammation, conjunctival hypertrophy, limbal remodeling, and sight-threatening complications including punctate epithelial erosions, shield ulcers, and corneal scarring. This chronic evolution is frequently associated with prolonged steroid exposure and significant morbidity, underscoring the need for a deeper mechanistic understanding of disease persistence. 4–11 . The immunopathogenesis of VKC has classically been attributed to an IgE-mediated type I hypersensitivity reaction. Allergen exposure triggers mast cell degranulation with release of histamine, prostaglandins, and leukotrienes, leading to early-phase symptoms such as itching and conjunctival hyperaemia. 1 . Beyond this acute phase, VKC has been widely regarded as a T-helper 2 (Th2)–driven disorder, with elevated levels of IL-4, IL-5, and IL-13 detected in tears and conjunctival tissues. These cytokines promote IgE class switching, eosinophil recruitment and survival, mucus hypersecretion, and epithelial remodeling. 1,12,7,13–19 . A hallmark of VKC is the persistent infiltration of eosinophils into the conjunctiva, driven by chemokines such as eotaxin (CCL11) and RANTES (CCL5), with subsequent release of cytotoxic granule proteins including major basic protein and eosinophil cationic protein that disrupt epithelial integrity and contribute to corneal injury. 12,20 . However, accumulating evidence indicates that this Th2-centric paradigm may incompletely capture the immunological complexity of chronic VKC. Studies have demonstrated increased expression of Th1-associated cytokines, including IFN-γ and IL-12, as well as Th17-related mediators such as IL-17 and IL-6, particularly in more severe or long-standing disease 1,11,12,16,21–23 . In parallel, conjunctival fibroblasts and epithelial cells respond to inflammatory cues by producing matrix metalloproteinases, including MMP1, MMP9, and MMP12, thereby driving extracellular matrix degradation, tissue remodeling, and conjunctival fibrosis—features that distinguish persistent VKC from intermittent disease. Together, these findings suggest that VKC represents a complex, multicellular immune disorder involving overlapping Th2, Th1, and Th17 signaling networks rather than a purely allergic condition. Despite these advances, clinical grading of VKC continues to rely predominantly on symptom-based systems such as the Bonini classification and the 5-5-5 exacerbation grading scale, which do not incorporate molecular biomarkers. Consequently, the immunological correlates that distinguish intermittent from persistent VKC—and that may predict disease chronicity or progression—remain poorly defined. Most mechanistic studies to date have relied on conjunctival biopsies, serum measurements, or low-plex tear assays, approaches limited by invasiveness, low sensitivity, and restricted molecular coverage. This gap has likely reinforced an oversimplified Th2-dominant view of VKC pathogenesis, particularly at the ocular surface where disease activity is most clinically relevant 11,19,24,25 . Tear fluid provides a minimally invasive and highly informative window into the local ocular immune environment, offering clear advantages over tissue biopsies, especially in pediatric populations 2,26,27 Tears capture dynamic immune processes occurring at the ocular surface and are therefore well suited for biomarker discovery and disease stratification. Recent advances in high-sensitivity proteomic technologies, including the OLINK® proximity extension assay (PEA), enable multiplexed detection of inflammatory mediators from small tear volumes. While conventional multiplex assays have been applied to VKC, few studies have employed OLINK-based tear cytokine profiling, and even fewer have stratified patients according to clinical severity or disease persistence. 15,37,39 In this study, we applied OLINK® PEA-based tear proteomics to comprehensively profile inflammatory cytokines in VKC patients stratified as moderate intermittent or moderate persistent based on the Bonini grading system. We hypothesized that (i) persistent VKC is associated with a distinct cytokine signature that is not exclusively Th2-dominant, (ii) tear cytokine levels correlate with key clinical markers of disease severity and chronicity, and (iii) IL-4 is not a critical mediator in established VKC at the tear-film level. By integrating multiplex proteomics with clinical correlation and pathway analysis, this work aims to refine current immunological models of VKC progression and identify clinically actionable biomarkers to support improved disease stratification and management. METHODOLOGY 2.1 Study Design and Participants This was a cross-sectional observational study conducted at Dr Shroff’s Charity Eye Hospital (SCEH), New Delhi. The study was approved by the Intuitional review Board (IRB) of SCEH and a total of 15 patients were enrolled. VKC diagnosis was based on characteristic clinical features including giant papillae, limbal involvement, and corneal changes, assessed by slit-lamp microscopy. Classification into moderate or intermittent VKC was done according to symptom severity and frequency of exacerbations. Healthy controls were age- and sex-matched individuals without any history of ocular surface disease, allergy, or systemic inflammatory conditions. All participants provided written informed consent. 2.2 Clinical Data Collection Clinical parameters such as symptom severity, disease duration, presence and extent of papillary hypertrophy, limbal changes, and corneal manifestations (including shield ulcers and superficial punctate keratitis) were documented during ophthalmic evaluations. Patients were categorized based on symptoms into intermittent and persistent forms, aligning with standard classifications typically describing seasonal (intermittent) and perennial (persistent) clinical presentations. Disease severity grading followed the Bonini classification, a recognized system incorporating clinical parameters like conjunctival inflammation, papillary size, and corneal involvement to classify VKC severity into mild, moderate, and severe stages To minimize the potential influence of ongoing therapies, all subjects had discontinued any systemic and topical treatments, including corticosteroids, cyclosporine, tacrolimus, and antihistamines, at least two weeks before tear sample collection. 2.3 Tear Sample Collection and Storage Tear samples were collected from 15 patients, including 6 with moderate intermittent and 9 with moderate persistent VKC. Samples were collected from the singular, non-medicated eye of each patient using Schirmer’s strips placed in the lower conjunctival fornix for 5 minutes. Immediately after collection, the strips were transferred into sterile RNase/DNase-free microcentrifuge tubes and stored at –80°C until further processing. 2.4 Tear Protein Isolation and Quantification Stored Schirmer’s strips were thawed on ice and eluted in 60 μL of Phosphate - Buffered Saline (PBS), pH 7.4 (Himedia laboratories Pvt Ltd, Cat No. M1866) supplemented with cOmplete ™ Protease Inhibitor Cocktail (Hoffmann-La Roche AG, Catalogue No. 11697498001). The strips were vortexed and mechanically crushed on ice multiple times to enhance protein recovery. Eluates were then centrifuged, and total protein concentration was quantified using the Pierce™ BCA Protein Assay Kits (Thermo Scientific, Catalogue no. 23227) following standard protocols. 2.5 Cytokine Profiling Using OLINK Proteomics Eluted tear proteins were analysed using the OLINK® Target 48 Cytokine Panel 41 (OLINK Proteomics, Sweden), which quantifies 45 inflammation-related proteins using Proximity Extension Assay (PEA) technology. The resulting data included both Normalized Protein Expression (NPX) values, which are relative quantifications on a log2 scale, and absolute protein concentrations (expressed in pg/mL) for each cytokine. NPX values were used for statistical comparison and clustering analyses, while absolute values were used for fold change calculations and visualizations. All assays included internal controls, and inter-plate normalization was conducted as per the manufacturer’s standard data processing pipeline 37,42 . 2.6 Bioinformatic and Statistical Analysis All statistical and bioinformatic analyses were conducted using Jupyter Notebook with standard Python packages for data manipulation, statistical computation, and visualization. Outliers were identified and removed using the interquartile range (IQR) method. After normalization, fold changes were calculated for all cytokines by comparing VKC samples to healthy controls. Unpaired t-tests were used to assess statistical significance, with p < 0.05 considered significant. While statistical analyses were conducted in Python, GraphPad Prism 43 was used to generate bar graphs of absolute cytokine values for clearer visual presentation. To represent differential expression, volcano plots were generated based on calculated log2 fold changes and corresponding p -values. Heatmaps were created to compare cytokine expression profiles between VKC and healthy individuals, and further stratified to compare moderate intermittent and moderate persistent VKC subtypes, highlighting subgroup-specific differences. A Spearman correlation-based correlogram was constructed to explore associations between cytokine levels and clinical parameters such as symptom duration, papillae size, and clinical severity score. To assess biological relevance, significantly dysregulated cytokines were analyzed using STRING for pathway enrichment 44 , identifying associated KEGG pathways, Reactome pathways, and disease linkages. All visualization outputs—including volcano plots, heatmaps, PCA, and correlation matrices—were used to support interpretation of the cytokine profiling results. ChatGPT (OpenAI) was also used as a support tool to generate or troubleshoot specific Python code segments for data analysis and visualization workflows. RESULT Patient Demographics and Clinical Characteristics Fifteen patients with clinically confirmed vernal keratoconjunctivitis (VKC) and ten healthy controls were included. The VKC cohort was predominantly male (80%), with a median age of 13 years (range: 8–27 years). Based on clinical grading, patients were classified as moderate intermittent (n = 6) or moderate persistent VKC (n = 9). Disease duration ranged from acute onset (10 days) to over 10 years, with most persistent cases exhibiting symptoms for more than 3 years and frequent exacerbations. Seasonal triggers were reported in 67% of patients, while 33% showed a perennial course. The palpebral form was most common (47%), followed by mixed (33%) and limbal (13%) variants. Papillary hypertrophy was present in all patients; limbal papillae were observed in limbal and mixed subtypes. Horner–Trantas dots were noted in one patient, and superficial punctate keratitis in three. Ocular surface complications—including conjunctival scarring, corneal epithelial defects, and steroid-induced glaucoma—were more frequent in moderate persistent VKC (Supplementary Table 1). 3.2 Differential Expression of Tear Cytokines in VKC Tear cytokine profiling in VKC patients demonstrated a significant alteration in multiple inflammatory mediators compared to healthy controls (Supplementary Figure 1). In this study, 45 cytokines were analysed using the OLINK platform across 10 control and 15 VKC tear samples. Of these, 44 cytokines passed quality control—only Interleukin-4 (IL-4) failed detection and was excluded from analysis. Among the 44 analysable cytokines, 20 showed statistically significant differential expression (p < 0.05): 13 were upregulated and 7 were downregulated in VKC (Supplementary Figure 2). Among the upregulated cytokines, Chemokine (C-C motif) ligand 3 (CCL3) showed the highest fold change (log₂FC = 2.82, p = 0.0157), followed by CXCL11 (log₂FC = 2.18, p = 0.0116), CCL4 (log₂FC = 2.18, p = 0.0454), MMP1 (log₂FC = 1.74, p = 0.0079), and MMP12 (log₂FC = 1.57, p = 0.0239). Other significantly elevated cytokines included CXCL9, CCL13, CXCL8, TNFSF10, OLR1, TGF-α, and IL15. In contrast, CCL2 was the most significantly downregulated (log₂FC = –1.62, p = 0.0001), along with CSF3 (log₂FC = –1.37, p = 0.0456), IL18, CSF1, and IFNG. These findings highlight a distinct inflammatory signature in VKC tears involving immune cell recruitment and tissue remodelling. 3.3 Functional Enrichment and Network Analysis of VKC-Associated Cytokines STRING network analysis (Figure 3A) revealed a dense protein–protein interaction network among the significantly dysregulated cytokines, highlighting key hubs such as CXCL8, CXCL9, IL15, TNFSF10, and MMP1. These genes are known to be involved in tightly connected inflammatory and chemokine signaling pathways. KEGG pathway enrichment (Figure 3B) demonstrated significant involvement of cytokine–cytokine receptor interaction, IL-17 signaling, JAK-STAT signaling, and Toll-like receptor pathways, all known contributors to allergic and chronic inflammatory responses. Reactome pathway enrichment (Figure 3C) supported these findings by indicating significant enrichment in interleukin signaling, chemokine receptor binding, and innate immune system activation. Disease-gene enrichment analysis (Figure 3D) further associated these cytokines with asthma, allergic diseases, bronchitis, and autoimmune conditions, reflecting the overlapping pathology of VKC. Tissue-specific enrichment (Figure 3E) revealed these cytokines to be predominantly expressed in inflammatory and myeloid-derived immune cells such as monocytes, macrophages, and T cells. GO Biological Process enrichment (Figure 3F) highlighted neutrophil chemotaxis, myeloid leukocyte activation, and inflammatory response as key biological events. Molecular Function analysis (Figure 3G) emphasized cytokine activity and chemokine receptor binding, specifically involving CCR1, CCR5, and CXCR3, known mediators of eosinophil and lymphocyte recruitment in allergic conjunctivitis. 3.4 Cytokine Shifts and Clinical Associations in VKC Subtypes Fold change analysis comparing VKC subtypes to healthy controls revealed that several cytokines were significantly upregulated in the persistent VKC group. Notably, CXCL11 exhibited the most prominent increase with a log₂ fold change of 2.72 ( p = 0.0186), followed by Oncostatin M (OSM) (log₂FC = 2.21, p = 0.0295), MMP1 (log₂FC = 1.72, p = 0.0320), CCL13 (log₂FC = 1.63, p = 0.0211), and CXCL9 (log₂FC = 1.42, p = 0.0006). These changes suggest enhanced chemotactic signaling and extracellular matrix remodeling in the persistent VKC subtype. While some of these cytokines also showed elevated expression in the intermittent group (e.g., OSM log₂FC = 3.09, MMP1 log₂FC = 1.77), their upregulation was more pronounced in the persistent form. The data highlight a distinct pattern of progressive immune activation and matrix degradation in VKC, with persistent cases exhibiting stronger cytokine shifts from baseline tear profiles observed in healthy individuals (Figure 4A). Spearman correlation analysis of the top 25 variables revealed that CXCL9, MMP12, CCL13, IL1B, and MMP1 showed strong positive correlations with papillae size. CXCL11, OSM, and IL6 were positively correlated with both symptom duration and papillae size, while CSF3, IL18, and EGF showed variable or negative correlations with these clinical parameters. Notably, CCL19, CCL4, and IL6 formed a cluster of inter-correlated cytokines. The pattern of correlations indicates distinct cytokine–clinical associations, particularly involving chemokines, matrix-modulating enzymes, and interleukins in VKC. DISCUSSION Vernal keratoconjunctivitis (VKC) is a chronic allergic disorder of the ocular surface that predominantly affects children and adolescents and poses a substantial clinical burden in tropical regions such as India. While many patients initially present with seasonal, self-limiting disease, a clinically important subset progresses to a moderate persistent phenotype characterized by chronic inflammation, reduced therapeutic responsiveness, and a heightened risk of conjunctival fibrosis and corneal complications. Understanding the immunological changes that accompany this transition is critical for improving disease stratification and guiding targeted interventions. In this study, we provide a comprehensive tear-based immune profile of VKC in an Indian cohort and demonstrate that disease persistence is associated with a cytokine landscape that extends beyond classical Th2-driven allergy. 1,2,5,45 . Tear cytokine profiling revealed a marked increase in inflammatory mediators in VKC patients compared to healthy controls, with significant upregulation of CCL3, CXCL9, CXCL11, MMP1, and MMP12. These molecules are centrally involved in leukocyte recruitment, chemotaxis, and extracellular matrix remodelling—processes that closely mirror the clinical features observed in VKC, including papillary hypertrophy, limbal thickening, and chronic conjunctival inflammation. Importantly, stratification of patients into moderate intermittent and moderate persistent subtypes uncovered distinct immunological differences associated with disease severity. Patients with moderate persistent VKC exhibited substantially higher levels of CXCL11, MMP1, and OSM, indicating a sustained, tissue-damaging inflammatory response rather than episodic allergic flares. A central and paradigm-shifting observation of this study is the complete absence of detectable IL-4 in VKC tear samples, despite the use of an ultrasensitive proximity extension assay. 1,2,5,20,39 . This finding contrasts with earlier reports emphasizing IL-4 as a key mediator of VKC pathogenesis, many of which relied on conjunctival tissue mRNA analysis or low-plex tear assays with limited sensitivity. Our data suggest that while IL-4 may contribute to early sensitization events or localized tissue responses, it is not a dominant effector cytokine at the tear-film level in established, moderate-to-severe VKC. This observation challenges the prevailing Th2-centric model and indicates that persistent VKC is maintained by alternative inflammatory mechanisms. 3,46,47 Instead, our findings point toward a chemokine-driven and tissue-remodeling immune axis as a defining feature of chronic disease. The robust upregulation of CXCL9 and CXCL11 implicates CXCR3-mediated recruitment of activated T cells and innate immune populations, while elevated MMP1 and MMP12 reflect ongoing extracellular matrix degradation and structural remodeling of the ocular surface. 48 , 49 . Among these mediators, OSM emerged as a particularly strong marker of disease persistence. OSM is well recognized for its role in chronic inflammation, fibrosis, epithelial barrier dysfunction, and stromal activation, and its elevation in persistent VKC aligns closely with the clinical phenotype commonly observed in Indian patients—namely, perennial symptoms, corneal involvement, and conjunctival scarring. These findings suggest that OSM may serve both as a biomarker of disease chronicity and a potential therapeutic target. 42,48,49 . Although VKC has traditionally been described as a Th2-dominant disorder—particularly in European and Middle Eastern cohorts—our data highlight important population-specific immunological differences. In contrast to prior studies reporting elevated IL-4, IL-5, and IL-13, the Indian VKC cohort examined here displayed a cytokine profile dominated by chemokines, matrix remodeling enzymes, and inflammatory mediators associated with chronic immune activation. Dysregulated cytokines such as CXCL9, CXCL11, CCL3, MMP12, CCL8, CCL13, OLR1 (LOX-1), IL-15, and TGF-α are produced by a wide range of innate and stromal cell types, including monocytes, macrophages, endothelial cells, fibroblasts, epithelial cells, and multiple T-cell subsets. This broad cellular involvement underscores VKC as a complex immune-mediated disorder rather than a purely IgE-driven allergic condition and highlights the need for region-specific immune profiling. Pathway enrichment analyses further supported this interpretation, revealing significant involvement of IL-17 signaling, cytokine–cytokine receptor interaction, and JAK–STAT pathways—networks commonly associated with chronic inflammatory and autoimmune conditions. Correlation analyses demonstrated that CXCL9, CXCL11, MMP12, and OSM levels were significantly associated with clinical indicators such as disease duration and papillae size, strengthening their relevance as biomarkers of disease severity and progression. Although IL-17C was not among the most differentially expressed cytokines, its significant correlations with inflammatory mediators and clinical parameters suggest a contributory role in sustaining chronic inflammation. 28 A key strength of this study is the use of OLINK® proximity extension assay–based tear proteomics, which enabled highly sensitive, multiplex detection of low-abundance cytokines from minimal tear volumes. This platform overcomes many limitations of earlier VKC studies, including restricted analyte coverage and insufficient sensitivity, and allows unbiased pathway-level analysis of the ocular surface immune environment. The ability to capture complex immune signatures from non-invasive tear samples is particularly advantageous in paediatric populations and supports the translational potential of this approach. (Supplementary Material – Table 2). Several limitations should be acknowledged. The modest sample size and cross-sectional design limit causal inference and temporal interpretation. Longitudinal studies will be necessary to determine whether the identified cytokine shifts precede clinical progression or reflect established disease states. In addition, functional studies using conjunctival and limbal cell models will be critical to elucidate the mechanistic roles of OSM, CXCL11, and MMPs in VKC pathogenesis (Supplementary Table 3). In conclusion, this study provides a comprehensive tear-based immune profile of VKC in an Indian cohort and identifies a region-specific immunological signature distinct from classical Th2-dominant models. The absence of IL-4, coupled with increased chemokines, matrix-remodeling enzymes, and OSM, suggests that persistent VKC is driven by chronic, tissue-remodeling inflammation rather than ongoing allergic sensitization alone. These findings highlight the potential of tear-based molecular profiling to improve disease stratification, enable early identification of severe phenotypes, and guide the development of more effective, personalized therapeutic strategies. Limitations and Future Directions This study has some limitations. A larger number of samples is needed to improve the statistical significance of the results, and the inclusion of more VKC patient groups would make the findings more broadly applicable. Because samples were collected at only one time point, we were unable to track how cytokine levels change over the course of the disease or in response to treatment. For future research, it will be important to validate the identified molecular targets using advanced laboratory methods, expand the study to include a larger and more diverse patient population, and conduct in vitro experiments to better understand how these molecules contribute to VKC. These steps will help strengthen the findings and provide deeper insights into the disease mechanisms. Conclusion This study provides a comprehensive overview of tear cytokine dysregulation in VKC, identifying distinct molecular signatures that differentiate disease subtypes and correlate with clinical severity. In this Indian cohort, persistent VKC is characterized by a tear cytokine profile dominated by chemokines and tissue-remodeling mediators, including CXCL11, MMP1, and OSM, reflecting a shift toward chronic, self-sustaining inflammation and increased risk of irreversible tissue damage. Notably, the absence of detectable IL-4 challenges the classical Th2-centric paradigm of VKC pathogenesis and suggests that alternative immune pathways drive established disease. These findings underscore important population-specific differences from Western cohorts and highlight the need to tailor therapeutic strategies to the underlying immunological landscape of Indian patients. By redefining disease mechanisms and identifying biomarkers of chronicity, OLINK-based tear proteomics emerges as a powerful, non-invasive approach to advance precision diagnosis, guide targeted therapy, and inform future translational research in ocular allergy.Top of Form Bottom of Form Conflict of Interest Statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript. Funding Information This research was supported by the Department of Biotechnology (DBT) Ramalingaswami Re-entry Fellowship (BTHRD/35/02/2006), Centre for Rare and Unknown Eye Disorders (CURED – Proj. No. 1973) and institutional funding from the Eicher Shroff Centre for Stem Cell Research (ESCSCR). Acknowledgement The authors sincerely acknowledge the support and cooperation of the members of the Eicher Shroff Centre for Stem Cell Research (ESCSCR) for their technical assistance and valuable discussions throughout the study. We are grateful to the fellows and clinicians at Dr. Shroff’s Charity Eye Hospital for their support in patient recruitment, clinical evaluation, and sample collection. We also thank the doctors and staff of Ram Manohar Lohia (RML) Hospital for their collaboration and assistance in clinical coordination. The authors appreciate the contributions of all study participants, without whom this research would not have been possible. References 1. Sacchetti M, Plateroti R, Bruscolini A, Giustolisi R, Marenco M. Understanding Vernal Keratoconjunctivitis: Beyond Allergic Mechanisms. Life 2021, Vol 11, Page 1012 . 2021;11(10):1012. doi:10.3390/LIFE11101012 2. Bonini S, Coassin M, Aronni S, Lambiase A. Vernal keratoconjunctivitis. Eye . 2004;18(4):345-351. doi:10.1038/SJ.EYE.6700675;KWRD=MEDICINE 3. Uchio E, Kimura R, Migita H, Kozawa M, Kadonosono K. Demographic aspects of allergic ocular diseases and evaluation of new criteria for clinical assessment of ocular allergy. Graefe’s Archive for Clinical and Experimental Ophthalmology . 2008;246(2):291-296. doi:10.1007/S00417-007-0697-Z 4. Shoji J, Inada N, Sawa M. Evaluation of Novel Scoring System Named 5-5-5 Exacerbation Grading Scale for Allergic Conjunctivitis Disease. Allergology International . 2009;58(4):591-597. doi:10.2332/ALLERGOLINT.09-OA-0100 5. Bonini S, Sacchetti M, Mantelli F, Lambiase A. Clinical grading of vernal keratoconjunctivitis. Curr Opin Allergy Clin Immunol . 2007;7(5):436-441. doi:10.1097/ACI.0B013E3282EFB726 6. Oray M, Toker E. Tear cytokine levels in vernal keratoconjunctivitis: The effect of topical 0.05% cyclosporine a therapy. Cornea . 2013;32(8):1149-1154. doi:10.1097/ICO.0B013E31828FFDF8 7. Bilotta MT, Vacca P, Esposito M, et al. Immunophenotyping in Vernal Keratoconjunctivitis: Schirmer Test for Therapy Response Prediction. Allergy: European Journal of Allergy and Clinical Immunology . Published online 2025. doi:10.1111/ALL.16525, 8. Shoji J, Inada N, Sawa M. Evaluation of eotaxin-1, -2, and -3 protein production and messenger RNA expression in patients with vernal keratoconjunctivitis. Jpn J Ophthalmol . 2009;53(2):92-99. doi:10.1007/s10384-008-0628-5 9. Chigbu DGI, Karbach NJ, Abu SL, Hehar NK. Cytokines in Allergic Conjunctivitis: Unraveling Their Pathophysiological Roles. Life 2024, Vol 14, Page 350 . 2024;14(3):350. doi:10.3390/LIFE14030350 10. Zicari AM, Capata G, Nebbioso M, et al. Vernal Keratoconjunctivitis: An update focused on clinical grading system. Ital J Pediatr . 2019;45(1). doi:10.1186/S13052-019-0656-4 11. Pattnaik L, Acharya L. A comprehensive review on vernal keratoconjunctivitis with emphasis on proteomics. Life Sci . 2015;128:47-54. doi:10.1016/J.LFS.2015.01.040 12. Leonardi A, Curnow SJ, Zhan H, Calder VL. Multiple cytokines in human tear specimens in seasonal and chronic allergic eye disease and in conjunctival fibroblast cultures. Clinical and Experimental Allergy . 2006;36(6):777-784. doi:10.1111/J.1365-2222.2006.02499.X;PAGE:STRING:ARTICLE/CHAPTER 13. Artesani MC, Esposito M, Buzzonetti L, Di Nardo G, Fiocchi AG, Mennini M. Vernal keratoconjunctivitis: The burden of being allergic. Journal of Allergy and Clinical Immunology: Global . 2025;4(2). doi:10.1016/J.JACIG.2025.100421, 14. KANEHARA S, YAMAGUCHI M, HIRATSUKA Y, AMANO S, EBIHARA N. Effects of Allergic Predisposition on Corneal Biomechanical Properties in Normal Corneas: A Retrospective Study. Juntendo medical journal . 2025;71(1):JMJ24-0028-OA. doi:10.14789/EJMJ.JMJ24-0028-OA 15. Demirtas N, Senol O, Cinici E, et al. Nontargeted metabolomics analysis in allergic conjunctivitis. Eur J Ophthalmol . Published online 2025. doi:10.1177/11206721251338495, 16. Vichyanond P, Pacharn P, Pleyer U, Leonardi A. Vernal keratoconjunctivitis: A severe allergic eye disease with remodeling changes. Pediatric Allergy and Immunology . 2014;25(4):314-322. doi:10.1111/pai.12197 17. Okano M, Hirahara K, Kiuchi M, et al. Interleukin-33-activated neuropeptide CGRP-producing memory Th2 cells cooperate with somatosensory neurons to induce conjunctival itch. Immunity . 2022;55(12):2352-2368.e7. doi:10.1016/j.immuni.2022.09.016 18. Yang X, Liu P, Zhao X, et al. Sulforaphane inhibits cytokine-stimulated chemokine and adhesion molecule expressions in human corneal fibroblasts: Involvement of the MAPK, STAT, and NF-κB signaling pathways. Exp Eye Res . 2022;216. doi:10.1016/j.exer.2022.108946 19. Bortolotti M, Palmigiano A, Messina A, et al. Identification of Tear Fluid Biomarkers in Vernal Keratoconjunctivitis Using iTRAQ Quantitative Proteomics. Invest Ophthalmol Vis Sci . 2010;51(13):1931-1931. 20. Leonardi A, Sathe S, Bortolotti M, Beaton A, Sack R. Cytokines, matrix metalloproteases, angiogenic and growth factors in tears of normal subjects and vernal keratoconjunctivitis patients. Allergy: European Journal of Allergy and Clinical Immunology . 2009;64(5):710-717. doi:10.1111/J.1398-9995.2008.01858.X;PAGE:STRING:ARTICLE/CHAPTER 21. Leonardi A, Daull P, Rosani U, et al. Evidence of epithelial remodelling but not epithelial–mesenchymal transition by transcriptome profiling in vernal keratoconjunctivitis. Allergy: European Journal of Allergy and Clinical Immunology . 2022;77(11):3460-3462. doi:10.1111/ALL.15450;PAGE:STRING:ARTICLE/CHAPTER 22. Micera A, Di Zazzo A, De Piano M, et al. Tissue remodeling in adult vernal keratoconjunctivitis. Exp Eye Res . 2022;225. doi:10.1016/j.exer.2022.109301 23. Leonardi A, Borghesan F, Depaoli M, Plebani M, Secchi AG. Procollagens and inflammatory cytokine concentrations in tarsal and limbal vernal keratoconjunctivitis. Exp Eye Res . 1998;67(1):105-112. doi:10.1006/exer.1998.0499 24. Di Zazzo A, Zhu AY, Nischal K, Fung SSM. Vernal keratoconjunctivitis in adults: a narrative review of prevalence, pathogenesis, and management. Frontiers in Ophthalmology . 2024;4:1328953. doi:10.3389/FOPHT.2024.1328953/BIBTEX 25. Pong JCF, Chu CY, Li WY, et al. Association of Hemopexin in Tear Film and Conjunctival Macrophages With Vernal Keratoconjunctivitis. Archives of Ophthalmology . 2011;129(4):453-461. doi:10.1001/ARCHOPHTHALMOL.2011.41 26. Leonardi A, Daull P, Garrigue JS, et al. Conjunctival transcriptome analysis reveals the overexpression of multiple pattern recognition receptors in vernal keratoconjunctivitis. Ocular Surface . 2021;19:241-248. doi:10.1016/j.jtos.2020.09.009 27. Rastegar Rad N, Rastgarrad N. Topical tacrolimus versus dexamethasone in managing shield ulcer of vernal keratoconjunctivitis. Medical Hypothesis, Discovery, and Innovation in Ophthalmology . 2024;13(4):160-168. doi:10.51329/MEHDIOPHTHAL1507, 28. Leonardi A, De Dominicis C, Motterle L. Immunopathogenesis of ocular allergy: A schematic approach to different clinical entities. Curr Opin Allergy Clin Immunol . 2007;7(5):429-435. doi:10.1097/ACI.0B013E3282EF8674, 29. Hehar NK, Chigbu DGI. Vernal Keratoconjunctivitis: Immunopathological Insights and Therapeutic Applications of Immunomodulators. Life 2024, Vol 14, Page 361 . 2024;14(3):361. doi:10.3390/LIFE14030361 30. Jiang J, Yang X, Du F, Zheng W, Wang J. The therapeutic effect of IPL on children with refractory seasonal allergic conjunctivitis. BMC Ophthalmol . 2025;25(1). doi:10.1186/S12886-025-04058-Z, 31. Leonardi A, Donato A, Rosani U, Di Stefano A, Cavarzeran F, Brun P. Endoplasmic Reticulum Stress and Unfolded Protein Response in Vernal Keratoconjunctivitis. Invest Ophthalmol Vis Sci . 2024;65(4):23-23. doi:10.1167/IOVS.65.4.23 32. Mehta JS, Chen WL, Cheng ACK, et al. Diagnosis, Management, and Treatment of Vernal Keratoconjunctivitis in Asia: Recommendations From the Management of Vernal Keratoconjunctivitis in Asia Expert Working Group. Front Med (Lausanne) . 2022;9:882240. doi:10.3389/FMED.2022.882240/BIBTEX 33. Montan P. Immunological and inflammatory mechanisms in ocular allergy with special reference to vernal keratoconjunctivitis. Clinical and experimental studies. Acta Ophthalmol Scand . 2001;79(2):212-212. doi:10.1034/J.1600-0420.2001.079002212.X 34. Nebbioso M, Zicari AM, Celani C, Lollobrigida V, Grenga R, Duse M. Pathogenesis of Vernal Keratoconjunctivitis and Associated Factors. Semin Ophthalmol . 2015;30(5-6):340-344. doi:10.3109/08820538.2013.874483;SUBPAGE:STRING:ACCESS 35. Irkec M, Bozkurt B, Kerimoglu H, Orhan M, Us D. Tear Cytokines in Patients with Vernal Keratoconjunctivitis (VKC) Responding Clinically to Topical Olopatadine or Additional Rimexolone. Invest Ophthalmol Vis Sci . 2002;43(13):106-106. 36. Zhou L, Beuerman RW. Tear analysis in ocular surface diseases. Prog Retin Eye Res . 2012;31(6):527-550. doi:10.1016/J.PRETEYERES.2012.06.002, 37. Gijs M, Adelaar TI, Vergouwen DPC, et al. Tear Fluid Inflammatory Proteome Analysis Highlights Similarities Between Keratoconus and Allergic Conjunctivitis. Invest Ophthalmol Vis Sci . 2023;64(15). doi:10.1167/IOVS.64.15.9, 38. Ito Y, Usui-Ouchi A, Ebihara N. Galectin-3, a damage-associated molecular pattern, in tears of patients with vernal keratoconjunctivitis. Jpn J Ophthalmol . 2023;67(4):431-439. doi:10.1007/S10384-023-00994-9, 39. Leonardi AA, Calder V, Curnow JS, Sathe S, Sack R. Metalloproteases, Cytokines and Growth Factors Detection in Tears of Vernal Keratoconjunctivitis (VKC) Patients by Antibody Array Analysis. Invest Ophthalmol Vis Sci . 2006;47(13):5871-5871. 40. Assarsson E, Lundberg M, Holmquist G, et al. Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability. PLoS One . 2014;9(4). doi:10.1371/JOURNAL.PONE.0095192, 41. Olink Target 48 — Olink®. Accessed May 15, 2025. https://olink.com/products/olink-target-48 42. Gurumurthy S, Bhambhani V, Agarwal S, Srinivasan B, Iyer G. Tear cytokines and their relevance as biomarkers in ocular surface inflammatory diseases. Journal of Cornea and Ocular Surface . 2023;1(2):120-129. doi:10.4103/JCOS.JCOS_23_23 43. Home - GraphPad. Accessed May 15, 2025. https://www.graphpad.com/ 44. Szklarczyk D, Kirsch R, Koutrouli M, et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res . 2023;51(1 D):D638-D646. doi:10.1093/NAR/GKAC1000 45. Vernal Keratoconjunctivitis - EyeWiki. Accessed May 11, 2025. https://eyewiki.org/Vernal_Keratoconjunctivitis 46. Seo DH, Corr M, Patel S, et al. Chemokine CXCL9, a marker of inflammaging, is associated with changes of muscle strength and mortality in older men. Osteoporosis International . 2024;35(10):1789. doi:10.1007/S00198-024-07160-Y 47. Hardison JL, Wrightsman RA, Carpenter PM, Lane TE, Manning JE. The chemokines CXCL9 and CXCL10 promote a protective immune response but do not contribute to cardiac inflammation following infection with Trypanosoma cruzi. Infect Immun . 2006;74(1):125-134. doi:10.1128/IAI.74.1.125-134.2006, 48. Wolf CL, Pruett C, Lighter D, Jorcyk CL. The clinical relevance of OSM in inflammatory diseases: a comprehensive review. Front Immunol . 2023;14:1239732. doi:10.3389/FIMMU.2023.1239732 49. Patel P, Rai V, Agrawal DK. Role of oncostatin-M in ECM remodeling and plaque vulnerability. Mol Cell Biochem . 2023;478(11):2451-2460. doi:10.1007/S11010-023-04673-8, Figure Legends Figure 1. Representative clinical images demonstrating key ocular features in Moderate Intermittent and Moderate Persistent VKC. Moderate Intermittent VKC : Giant papillae are visible on the upper tarsal conjunctiva, accompanied by mild limbal papillae, localized corneal haze under fluorescein staining, and mild conjunctival hyperemia . Moderate Persistent VKC : More pronounced tarsal papillary hypertrophy is observed, along with prominent limbal thickening, extensive corneal epithelial damage and fluorescein uptake (third row), and diffuse conjunctival hyperemia. Figure 2. Tear cytokine profile in VKC compared to healthy controls. (a) Heatmap representing the fold change of significantly differentially expressed cytokines, showing marked upregulation of CCL3, CXCL11, MMP1, MMP12, OLR1, CCL13, IL15, CXCL8, TNFSF10, CXCL9, and others in VKC tears. (b) Volcano plot illustrating the distribution of cytokines based on fold change and statistical significance, highlighting both upregulated and downregulated targets. (c) Bar graph depicting the expression levels and statistical significance of highly upregulated cytokines, with CXCL9 ( p = 0.0007), CCL13 ( p = 0.0006), and CXCL8 ( p = 0.0007) among the most significant. (d) Bar graph showing significantly downregulated cytokines, including CCL2 ( p = 0.0003) and CSF3 ( p = 0.0047), along with their expression range and significance. Figure 3. Network interaction and enrichment analysis of differentially expressed tear cytokines in VKC. (A) STRING network visualization of significantly dysregulated tear cytokines in VKC patients, showing protein–protein interactions with high confidence scores. Prominent nodes include CXCL8, TNFSF10, CCL3, CXCL11, CXCL9, IL15, MMP1, and MMP12. (B) KEGG pathway enrichment analysis highlighting cytokine–cytokine receptor interaction, chemokine signaling, IL-17 signaling, and JAK-STAT signaling pathways. (C) Reactome pathway enrichment indicating significant involvement in interleukin signaling, chemokine receptor activity, and immune system modulation. (D) Disease-gene association analysis showing enrichment in autoimmune and respiratory conditions such as asthma, bronchitis, and allergic diseases. (E) Tissue-specific enrichment demonstrating association with immune cells including inflammatory cells, monocytes, macrophages, and T lymphocytes. (F) Gene Ontology (GO) enrichment for Biological Processes revealing pathways like neutrophil chemotaxis, inflammatory response, and myeloid leukocyte activation. (G) GO enrichment for Molecular Functions emphasizing cytokine activity, receptor binding, and chemokine receptor interaction, including specific binding to CCR1, CCR5, and CXCR3. Figure 4. Comparative analysis of significant tear cytokines in VKC subgroups and their correlation with clinical features. (A)Heatmap displaying fold change values of the same cytokines in moderate intermittent and moderate persistent VKC groups relative to healthy controls, highlighting elevated fold changes particularly for OSM, CCL3, and CXCL11 in moderate persistent cases. (B) Correlogram illustrating Spearman’s correlation between cytokine levels and clinical parameters including symptom duration, limbal reaction, SPK score, and papillae size. Circle size and color intensity indicate the strength and direction of correlation Figure 5: (A) Venn diagram representing the differences and similarities in cytokine between Italian and Indian patients with Vernal Keratoconjunctivitis (VKC). (B) Schematic representation of cytokine profiles in VKC subgroups and highlights their possible links to clinical signs like papillae size, limbal reaction, and how long symptoms last. It also shows which pathways are involved, such as IL-17 signalling and JAK-STAT signalling, and points out certain cytokines that may be linked to ongoing or worsening inflammation. Supplementary Material File (images.pptx) Download 3.87 MB Information & Authors Information Version history V1 Version 1 03 February 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Kartik Goel Dr Shroff's Charity Eye Hospital Delhi View all articles by this author Prisha Warikoo Dr Shroff's Charity Eye Hospital Delhi View all articles by this author Abha Gour Dr Shroff's Charity Eye Hospital Delhi View all articles by this author Shailja Tibrewal Dr Shroff's Charity Eye Hospital Delhi View all articles by this author Virender Singh Sangwan Dr Shroff's Charity Eye Hospital Delhi View all articles by this author Mehak Sapra Dr Shroff's Charity Eye Hospital Delhi View all articles by this author Anil Tiwari 0000-0003-0402-458X [email protected] Dr Shroff's Charity Eye Hospital Delhi View all articles by this author Metrics & Citations Metrics Article Usage 146 views 48 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Kartik Goel, Prisha Warikoo, Abha Gour, et al. Tear Cytokine Profiling in Vernal Keratoconjunctivitis Identifies Immune Signatures Associated with Disease Severity. Authorea . 03 February 2026. 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