Introduction

Keratoconus is a progressive, corneal ectatic disorder characterized by thinning and anterior protrusion of the cornea, resulting in a conical shape [1]. This structural alteration leads to irregular astigmatism, myopia, and ultimately, decreased visual acuity [2]. The disease typically manifests during adolescence or early adulthood and may progress gradually over several years [3]. Keratoconus is widely regarded as a multifactorial condition involving genetic, biochemical, mechanical, and environmental factors that trigger an innate immune response associated with allergy and atopy [4]. The association of keratoconus with allergy, atopy, and chronic ocular surface irritation suggests that innate immune activation may contribute directly to the inflammatory microenvironment that promotes corneal stromal weakening and ectatic progression [5].

The disease primarily affects the corneal stroma, which constitutes most of the corneal thickness and is composed of an organized extracellular matrix (ECM) essential for corneal strength and transparency [6]. Studies have demonstrated that patients with keratoconus exhibit increased degradation of ECM components critical for maintaining corneal structure, leading to stromal thinning and ectasia [7]. Histopathological and biochemical analyses have identified several proteins involved in the innate immune response that contribute to ECM breakdown, altered collagen organization, and keratocyte apoptosis and necrosis within the stromal layer [8, 9].

Although keratoconus was classically defined as a non-inflammatory disorder, recent evidence indicates a strong inflammatory component [10, 11]. Elevated levels of cytokines such as interleukin-1α/β (IL-1α/β), IL-6, and tumor necrosis factor-alpha (TNF-α) have been detected in the tears, corneal tissue, and serum of keratoconus patients compared to healthy controls [12]. These proteins are key mediators of innate immune signaling and suggest sustained activation of inflammatory and oxidative pathways within the corneal microenvironment [13]. Importantly, many of these cytokines are downstream of pattern recognition receptor (PRR) engagement, positioning the innate response as a key driver of disease progression [14].

High mobility group box 1 (HMGB-1) is a nuclear chromatin-binding protein, in normal unstressed cells, it is predominantly localized within the nucleus, where it binds chromatin and contributes to DNA organization, transcriptional regulation, replication, repair, and maintenance of genomic stability [15]. HMGB-1 under conditions of cellular stress or injury is released into the extracellular environment where it functions as a damage-associated molecular pattern (DAMP) [16]. Extracellular HMGB-1 activates innate immunity signaling cascades by binding to PRRs such as the receptor for advanced glycation end products (RAGE) and toll-like receptors (TLRs: TLR2 and TLR4), leading to NF-κB activation, cytokine release, and recruitment of immune cells [17]. This positions HMGB-1 as a central mediator of inflammation, fibrosis, and tissue remodeling.

Recent studies have implicated HMGB-1 in several degenerative and fibrotic diseases, though its specific role in keratoconus remains incompletely understood [18]. Given that keratoconus is associated with ECM degradation, oxidative stress, and chronic inflammation, HMGB-1 expression may be associated with the inflammatory, oxidative, and ECM alterations observed in keratoconus [19]. Understanding the expression and localization of HMGB-1 in keratoconic corneas could therefore provide valuable insight into the mechanisms underlying disease progression.

Based on this background, we hypothesize that HMGB-1 expression is increased in keratoconic corneas and is associated with inflammatory and tissue-remodeling pathways. To test this hypothesis, this study investigates the expression patterns of HMGB-1 in corneal tissues from patients with keratoconus and Fuchs’ corneal dystrophy, compared with normal donor corneas, using immunofluorescence microscopy.

Materials and methods

Results

Quantitative immunofluorescence analysis revealed clear differences in HMGB-1 expression across normal, keratoconic, and Fuchs’ corneal dystrophy corneal tissues, highlighting disease-specific patterns of inflammatory and stress-associated signaling. Fluorescence intensity values were measured in a.u. and reflected HMGB-1 protein abundance within corneal tissue sections (Figure 1).

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Figure 1. Immunofluorescence analysis of high mobility group box 1 (HMGB-1) expression in human corneal tissue. Representative immunofluorescence (IF) images at 20× magnification of paraffin-embedded human corneal sections from normal, keratoconus, and Fuchs’ corneal dystrophy samples stained with anti-HMGB-1 antibody (red) and counterstained with 4',6-diamidino-2-phenylindole (DAPI) to label nuclei. Normal corneal tissue demonstrates low-level, diffuse HMGB-1 expression with preserved stromal architecture. In contrast, keratoconic corneas exhibit increased and irregular HMGB-1 signal with disrupted stromal organization, while Fuchs’ dystrophy samples show robust HMGB-1 immunoreactivity with widespread distribution throughout the corneal tissue. All images were acquired using identical imaging parameters, including magnification, exposure time, laser intensity, and gain settings, using a Nikon Eclipse Ti2 fluorescence microscope, to allow direct qualitative comparison across conditions.

Normal donor corneas demonstrated low and relatively uniform HMGB-1 fluorescence intensity, with a mean value of 338.6 ± 53.6 a.u. (Figure 2). Signal distribution within this group was narrow, consistent with minimal baseline inflammatory activity and homeostatic cellular turnover in healthy corneal tissue. HMGB-1 localization in normal samples appeared diffuse and low-level, supporting its expected role as a predominantly nuclear protein under non-pathologic conditions.

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Figure 2. Quantitative analysis of high mobility group box 1 (HMGB-1) immunofluorescence intensity in human corneal tissue. Scatter plot depicting HMGB-1 fluorescence intensity in arbitrary units (a.u.) measured from immunofluorescence images of normal, keratoconus, and Fuchs’ corneal dystrophy corneal samples. Each data point represents an individual region of interest (ROI) from the n = 3 tissues. Red horizontal lines denote group means and the bars represent standard deviation. Mean values were 338.6 a.u. for normal corneas, 486.2 a.u. for keratoconic corneas, and 783.5 a.u. for Fuchs’ endothelial dystrophy corneas. Welch’s one-way ANOVA demonstrated a significant overall group difference, F(2, 80.33) = 10.32, p = 0.00010. The two prespecified comparisons showed higher HMGB-1 intensity in keratoconus versus normal tissue [Welch t(61.17) = 2.87; Holm-adjusted p = 0.0057] and in Fuchs’ dystrophy versus normal tissue [Welch t(59.37) = 3.57; Holm-adjusted p = 0.0014].

In contrast, keratoconic corneas exhibited a significant increase in HMGB-1 expression, with a mean fluorescence intensity of 486.2 ± 395.2 a.u. (Figure 2). In the prespecified comparison with normal corneas, the mean difference was 147.7 a.u. (95% CI, 44.7–250.6), Welch t(61.17) = 2.87, Holm-adjusted p = 0.0057, with a Hedges’ g of 0.52. The broad distribution and high-intensity observations indicate substantial spatial variability in HMGB-1 expression within the keratoconus. This heterogeneity is consistent with the known patchy nature of stromal thinning, focal inflammation, and ECM disruption characteristic of keratoconus. These findings suggest increased cellular stress and inflammatory signaling within keratoconic corneal tissue.

Fuchs’ corneal dystrophy samples demonstrated the highest overall mean HMGB-1 fluorescence intensity 783.5 ± 964.2 a.u. (Figure 2). Examination of the fluorescence intensity distribution revealed pronounced variability within the Fuchs’ group. Three measurements reached the upper recorded intensity value of 4,095 a.u., contributing to the marked variance and right-skewed distribution. In the prespecified comparison with normal corneas, the mean difference was 444.9 a.u. (95% CI, 195.4–694.3), Welch t(59.37) = 3.57, Holm-adjusted p = 0.0014, with a Hedges’ g of 0.65. These findings indicate elevated but highly heterogeneous HMGB-1 immunoreactivity in the Fuchs’ dystrophy corneas. These high-intensity regions corresponded to localized areas of corneal stress, dense guttae formation, and focal inflammatory activation, rather than diffuse, uniform upregulation across the tissue. The presence of these localized HMGB-1 “hot spots” resulted in a substantially increased standard deviation within the Fuchs’ group. This finding highlights the importance of spatial heterogeneity in Fuchs’ corneal dystrophy and suggests that HMGB-1 expression in this condition may be driven by focal cellular injury and stress responses rather than a global increase throughout the cornea.

Welch’s one-way analysis of variance demonstrated a significant overall difference in maximum HMGB-1 fluorescence intensity among the three groups, Welch F(2, 80.33) = 10.32, p = 0.00010. The two prespecified disease-versus-normal comparisons remained statistically significant after Holm adjustment. The observed spatial heterogeneity, broad intensity distributions, and focal regions of high HMGB-1 expression in diseased tissues suggest that HMGB-1 may play an important role in corneal inflammatory signaling, cellular stress responses, and ECM remodeling. These findings support further investigation of HMGB-1 as a potential biomarker of corneal cellular stress and inflammation.

Discussion

This pilot study demonstrates increased expression of HMGB-1 in corneal tissues from patients with keratoconus and Fuchs’ corneal dystrophy, supporting a role for this protein in corneal inflammation and ECM remodeling. Quantitative immunofluorescence revealed a significant increase in HMGB-1 expression in keratoconic corneas compared with normal corneas, along with a variably elevated and spatially heterogeneous pattern in Fuchs’ corneal dystrophy. Together, these findings suggest that increased HMGB-1 expression may be associated with cellular stress, inflammation, and degenerative changes in corneal tissue.

HMGB-1 could be associated with this inflammatory cascade; however, the present study did not evaluate downstream signaling or establish whether HMGB-1 initiates or amplifies tissue injury. Upon cellular stress or injury, HMGB-1 translocates from the nucleus to the extracellular space, where it engages PRRs such as the RAGE receptor and TLR2 and TLR4 on corneal epithelial cells and keratocytes [19]. Activation of these receptors triggers NF-κB signaling and subsequent upregulation of proinflammatory cytokines including IL-1β, IL-6, and TNF-α [20]. These cytokines have been reported at elevated levels in keratoconic tears and corneal tissue, supporting the biological relevance of HMGB-1-mediated inflammatory signaling [21, 22]. The heterogeneous distribution of HMGB-1 observed in keratoconus aligns with the focal nature of stromal thinning and suggests localized regions of increased cellular stress and ECM degradation [23, 24]. These findings are consistent with prior studies demonstrating increased inflammatory cytokines, oxidative stress markers, and matrix metalloproteinase activity in keratoconic corneas, further supporting a model in which chronic low-grade inflammation contributes to progressive stromal weakening [25]. HMGB-1 may act upstream in this inflammatory cascade, amplifying tissue damage through sustained innate immune activation [26].

Fuchs’ endothelial dystrophy demonstrated the highest mean HMGB-1 fluorescence intensity and a significant ROI-level increase relative to normal corneal tissue. The distribution was markedly heterogeneous, with a broad variance, and three values at the upper recorded intensity of 4,095 a.u. The bimodal distribution of HMGB-1 expression observed in this group likely reflects regional differences in disease severity, with low-intensity regions resembling relatively preserved endothelium and high-intensity regions corresponding to areas of advanced corneal stress and guttae formation. Oxidative stress and mitochondrial dysfunction, both well-established features of Fuchs’ dystrophy, are known triggers of HMGB-1 release and may drive focal inflammatory amplification within affected corneal regions [27, 28]. The significant overall difference in HMGB-1 expression across all groups, as demonstrated by the ANOVA, supports disease-associated expression of HMGB-1 despite biological variability.

Fuchs’ endothelial corneal dystrophy was included as a disease comparison group, rather than as an age- or anatomically matched control. Fuchs’ dystrophy is characterized by endothelial cell loss, oxidative and mitochondrial stress, and degenerative changes involving the corneal endothelium and Descemet’s membrane. These cellular stressors may promote the release or increased expression of HMGB-1. Therefore, inclusion of the Fuchs’ group allowed us to assess whether elevated HMGB-1 expression was specific to keratoconus or was also observed in another corneal disorder associated with cellular injury and tissue degeneration. However, the two diseases affect different corneal compartments: keratoconus primarily involves the epithelium and stroma, whereas Fuchs’ dystrophy predominantly affects the endothelium and Descemet’s membrane. In addition, patients with Fuchs’ dystrophy are generally older than patients with keratoconus, and age-related changes, endothelial cell loss, and cumulative oxidative stress may independently influence HMGB-1 expression. Consequently, the Fuchs’ group should not be interpreted as a directly matched comparator for keratoconus. Rather, its elevated and heterogeneous HMGB-1 expression provides evidence that HMGB-1 may represent a broader marker of corneal cellular stress and injury. The keratoconus-versus-normal comparison therefore remains the primary analysis for evaluating the potential association between HMGB-1 and keratoconus, while the Fuchs’ group provides additional biological context regarding the specificity of HMGB-1 expression.

This study should be interpreted as a pilot study designed to better understand the HMGB-1 expression patterns in the pathogenesis of keratoconus. The small sample size, with three tissue specimens per group, represents a major limitation and restricts the statistical power of the study. Therefore, the findings should be considered exploratory rather than definitive. The small sample size reflects the limited availability of rare archived human corneal tissues, particularly keratoconic corneal specimens suitable for immunofluorescence analysis. Although this restricts statistical power, similarly small sample sizes have been reported in prior keratoconus immunohistochemistry studies, including “Transepithelial riboflavin/ultraviolet. A corneal cross linking in keratoconus: morphologic studies on human corneas.” Therefore, the present findings should be interpreted as preliminary and hypothesis-generating rather than definitive. Additional studies with larger sample sizes are needed to definitively determine whether HMGB-1 is significantly elevated in Keratoconus and Fuchs’ endothelial dystrophy and whether this expression correlates with disease severity or localized endothelial injury.

These findings identify increased HMGB-1 expression in keratoconus and Fuchs’ endothelial dystrophy as a potential marker of cellular stress, inflammation, and ECM remodeling in corneal pathology. The increased concentration in diseased tissues warrants further investigation. The present observational study does not establish that HMGB-1 has an active pathogenic role or that it represents a therapeutic target, further investigation is required.