Analysis of the mucosal chemokines CCL28, CXCL14, and CXCL17 in dry eye disease: An in vitro and clinical investigation

Experimental Eye Research 241 (2024) 109854 Available online 5 March 2024 0014-4835/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). Research article Analysis of the mucosal chemokines CCL28, CXCL14, and CXCL17 in dry eye disease: An in vitro and clinical investigation ☆ Alfredo Domínguez-L´ opez a , Marta Blanco-V´ azquez a , Andr´ es ´ Angel Calder´ on-García a , Carmen García-V´ azquez a , María J. Gonz´ alez-García a , c , Margarita Calonge a , b , c , Amalia Enríquez-de-Salamanca a , b , c , * a Institute of Applied Ophthalmobiology (IOBA), Universidad de Valladolid, Valladolid, Spain b OculoFacial Pain Unit, Institute of Applied Ophthalmobiology (IOBA), Universidad de Valladolid, Valladolid, Spain c Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Carlos III National Institute of Health, Spain ARTICLE INFO Keywords: Ocular surface Dry eye Mucosal chemokines Hyperosmolarity Inflammation CCL28 CXCL14 CXCL17 ABSTRACT Mucosal chemokines have antimicrobial properties and play an important role in mucosal immunity. However, little is known about their expression on the ocular surface. This study aimed to analyze the expression of the mucosal chemokines CCL28, CXCL14 and CXCL17 in corneal and conjunctival epithelial cells under in vitro dry eye (DE) conditions, and in conjunctival samples from healthy subjects and DE patients. Human corneal epithelial cells (HCE) and immortalized human conjunctival epithelial cells (IM-HConEpiC) were incubated under hyperosmolar (400–500 mOsM) or inflammatory (TNF- α 25 ng/mL) conditions for 6 h and 24 h to measure CCL28, CXCL14, and CXCL17 gene expression by RT-PCR and their secretion by immunobead-based analysis (CCL28, CXCL14) and ELISA (CXCL17). Additionally, twenty-seven DE patients and 13 healthy subjects were included in this study. DE-related questionnaires (OSDI, mSIDEQ and NRS) evaluated symptomatology. Ocular surface integrity was assessed using vital staining. Tactile sensitivity was measured with Cochet-Bonnet esthesiometer, and mechanic and thermal (heat and cold) sensitivity using Belmonte’s non-contact esthesiometer. Subbasal nerve plexus and dendritic cell density were analyzed by in vivo confocal microscopy. Conjunctival cells from participants were collected by impression cytology to measure mucosal chemokines gene expression by RTPCR. Our results showed that HCE and IM-HConEpiC cells increased CCL28, CXCL14, and CXCL17 secretion under hyperosmolar conditions. The gene expression of CCL28 was significantly upregulated in conjunctival samples from DE patients. CCL28 expression correlated positively with symptomatology, corneal staining, heat sensitivity threshold, and dendritic cell density. CXCL14 expression correlated positively with age, ocular pain, conjunctival staining, tactile sensitivity, and image reflectivity. CXCL17 expression correlated positively with corneal staining. These results suggest that corneal and conjunctival epithelial cells could be a source of CCL28, CXCL14, and CXCL17 on the ocular surface and that CCL28 might be involved in DE pathogenesis. 1. Introduction Chemokines are small cytokines that trigger the activation, recruitment, and migration of inflammatory cells. Their biological properties also include the development, proliferation, and differentiation of immune and non-immune cells. Furthermore, these molecules are secreted by a wide variety of cells, including leucocytes, fibroblasts, endothelial and epithelial cells, and neurons, among others (Chen et al., 2018; Griffith et al., 2014). Mucosal chemokines, such as CXC chemokine ligand 14 (CXCL14), and CXCL17, CC chemokine ligand 25 (CCL25), and CCL28, are constitutively expressed in mucosal tissues under homeostatic conditions (Hern´ andez-Ruiz and Zlotnik, 2017). CCL28 is known to induce the migration of IgA-secreting cells into the lamina propria of the intestine and into the mammary glands (Matsuo et al., 2018; Wilson and Butcher, 2004). In addition to their chemotactic activity (Meuter and Moser, 2008; Pisabarro et al., 2006), these chemokines exhibit also broad antimicrobial properties and play an important role in mucosal immunity (Berri et al., 2014; Hara and Tanegashima, 2012; Xiao et al., ☆ Partially presented at ARVO 2023. * Corresponding author. Institute of Applied Ophthalmobiology (IOBA) Universidad de Valladolid, Campus Miguel Delibes, Paseo de Bel´ en 17 Valladolid, Spain. E-mail address: [email protected] (A. Enríquez-de-Salamanca). Contents lists available at ScienceDirect Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer https://doi.org/10.1016/j.exer.2024.109854 Received 13 November 2023; Received in revised form 21 February 2024; Accepted 4 March 2024 Experimental Eye Research 241 (2024) 109854 2 2021). A large body of clinical studies has shown that the immune response plays a central role in the physiopathology of dry eye (DE). The correlation between proinflammatory mediators and clinical symptoms of DE has been widely reported (Lee et al., 2013; Li et al., 2020; Massingale et al., 2009; Mrugacz et al., 2017; Vandermeid et al., 2012). These include chemokines such as CCL2/MCP-1, CCL3/MIP-1 α , CCL4/MIP-1β, CCL5/RANTES, CCL15/MIP-5, CXCL5/ENA78, CXCL8/IL-8, CXCL9/Mig, CXCL10/IP-10, CXCL11/ITAC, and CX3C chemokine ligand 1 (CX3CL1/Fractalkine), which have been found to be increased in the tears of DE patients (Choi et al., 2012; Enríquez-de-Salamanca et al., 2010; Na et al., 2012; Yoon et al., 2010). CCL25 mucosal chemokine is mainly expressed in the small intestine and prostate in men but it has not been described to be expressed in the eye (Wang et al., 2019). Whereas, recent studies suggest the association of other mucosal chemokines with Sj¨ ogren’s syndrome; particularly, increased levels of CXCL17 in tears and saliva and reduced levels of CCL28 in saliva and serum have been found in patients with Sj¨ ogren’s syndrome (Hernandez-Molina et al., 2015; Hern´ andez-Ruiz et al., 2018; Yu et al., 2023). In addition, increased expression of the CXCL14 gene has been described in conjunctival samples from DE patients (Wei and Asbell, 2020). However, the expression levels of these mucosal chemokines on the ocular surface under homeostatic conditions and whether they are involved in the physiopathology of DE remain unknown. The aim of this study was to identify the presence of the mucosal chemokines CCL28, CXCL14, and CXCL17 in ocular surface epithelial cells and their potential involvement in DE. As inflammation and hyperosmolarity play an important etiological role in DE (Craig et al., 2017; Lee et al., 2014), we investigated the effect of hyperosmotic and inflammatory conditions on the in vitro production of the mucosal chemokines CCL28, CXCL14, and CXCL17 by corneal and conjunctival epithelial cells. In addition, we analyzed the gene expression of CCL28, CXCL14, and CXCL17 in conjunctival samples from healthy subjects and DE patients, and the correlation with their clinical parameters. 2. Materials and methods 2.1. Cell lines and culture conditions To perform the in vitro experiments, two ocular surface epithelial cell lines were used in this study: human corneal epithelial (HCE) cells (Araki-Sasaki et al., 1995) and immortalized human conjunctival epithelial (IM-HConEpiC) cells (García-Posadas et al., 2022). HCE cells were kindly provided by Dr. Arto Urti (University of Helsinki, Finland) and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12) +GlutaMax supplemented with fetal bovine serum (10%), human epithelial growth factor (10 ng/mL), insulin (5 g/mL), penicillin (100 U/mL) and streptomycin (0.1 mg/mL)) (Thermo Fisher Scientific, Rockford, IL, USA) as previously described (Katsinas et al., 2021). The passages used were from 30 to 35. IM-HConEpiC cells were purchased from Innoprot (P10870-IM Derio, Spain) and cultured in DMEM/F12 +GlutaMax supplemented with fetal bovine serum (10%), human epithelial growth factor (10 ng/mL), insulin (1 g/mL), penicillin (5000 U/mL) and streptomycin (5000 g/mL) (Katsinas et al., 2021). The passages used were from 10 to 15. Cell cultures were incubated at 37 ◦C under 5% CO 2 and 95% humidity. The medium was changed every second day, and daily observations were performed using a phase-contrast microscope. After the culture reached confluence, HCE and IM-HConEpiC cells were seeded in 24-well plates (3 ×10 4 cells/well). At 90% confluence, the cells were cultured with their respective medium without any additives for 24 h to regulate basal cytokine production. The cells were then incubated for 6 or 24 h under basal (300 mOsM) and hyperosmolar conditions (400, 450, or 500 mOsM) by adding NaCl (50, 75, or 100 mM) or inflammatory conditions with TNF- α (25 ng/mL) (Thermo Fisher Scientific) to mimic DE conditions. After 6 h of incubation, supernatants were discarded and adherent cells were lysed with RLT buffer (Qiagen, Hilden, Germany) containing 1% β-mercaptoethanol (Merck KGaA, Darmstadt, Germany) and stored at −80 ◦C for total RNA isolation. After 24 h of incubation, the supernatants were collected and stored at −80 ◦C for chemokine concentration measurement and plates with adherent cells were frozen at −80 ◦C until they were assayed for total protein content. Three independent experiments and two replicates for each treatment per experiment were performed. 2.2. Dry eye (DE) patients and healthy subjects The clinical study was approved by the University of Valladolid Ethics Committee (Ref: PI 15–301) and was conducted in accordance with the Declaration of Helsinki guidelines and Good Clinical Practices. Informed consent was obtained from all participants after explanation of the study protocol. Patients with a positive diagnosis of DE and healthy subjects were recruited in the present study. The criteria for defining DE condition were as we previously reported (Blanco-V´ azquez et al., 2022), having an ocular surface disease index (OSDI) score ≥13 and to present at least two of the following conditions in both eyes: fluorescein tear break-up time (TBUT) ≤7 s, corneal fluorescein staining ≥grade 1 (Oxford scale), conjunctival lissamine green staining ≥grade 1 (Oxford scale), and Schirmer test with topical anesthesia ≤5 mm in 5 min. Exclusion criteria were as previously described in (Blanco-V´ azquez et al., 2022), and included the following: any ocular surface disease other than DE in the last 3 months or any ocular surgery in the last 6 months; diagnosis of any systemic disease that could have an ocular component in the last 3 months; initiation of any systemic medication that could affect the ocular surface health in the previous 3 months; initiation of lacrimal punctum occlusion in the previous 3 months; contact lens wear in the 7 days before the study; on treatment with topical cyclosporine A or topical corticosteroids in the last month; or any topical medication, lubricants or blood derivatives in the 12 h before the study. A group of participants with the same exclusion criteria and negative diagnostic criteria for DE were selected as healthy subjects. 2.3. Clinical examination and sample collection All participants were evaluated after 30 min under normal controlled environment condition (23 ◦C temperature, 50% relative humidity and no localized air flow) in the Controlled Environment Laboratory (CELab) (www.visionrd.com/celab/) as we previously reported (Calonge et al., 2017; L´ opez-Miguel et al., 2014; Tes´ on et al., 2013), in order to avoid the influence of environmental conditions. Medical history and clinical questionnaires were completed during the 30 min of adaptation. After that, clinical test and sample collection were performed in the following sequence. 2.3.1. Clinical questionnaires The OSDI was measured with the following scores: 13–22 (mild), 23–32 (moderate), and 33–100 (severe) (Miller et al., 2010; Schiffman et al., 2000). The Modified Single-Item Dry Eye Questionnaire (mSIDEQ) was assessed on a scale from 0 to 4: 0 (absence of symptom), 1 (rarely felt), 2 (sometimes felt), 3 (always felt but without affecting daily activities), 4 (always felt with affected daily activities) (Tes´ on et al., 2013). The Numerical Rating Scale (NRS) was used to evaluate ocular pain intensity on a 0–10 scale: 0–1 (none), 2–4 (mild), 5–7 (moderate), 8–10 (severe) (Satitpitakul et al., 2017). The Hospital Anxiety and Depression Scale (HADS) was used to assess the level of anxiety and depression. The subscale cut-off points were 0–7 (normal, not presence of anxiety and/or depression), 8–10 (borderline, possible presence of anxiety and/or depression), and >10 (probable presence of anxiety and/or depression) (Snaith, 2003). A. Domínguez-L´ opez et al. Experimental Eye Research 241 (2024) 109854 3 2.3.2. Clinical tests Tear osmolarity was assessed in both eyes using a nanosmometer (TearLab Corporation, San Diego, CA, USA). Values >308 mOsm/L were considered abnormal (Wolffsohn et al., 2017). Tear stability was evaluated by measuring fluorescein tear break-up time (TBUT) with the SL-D7 slit lamp (Topcon Corporation, Tokyo, Japan). Sodium fluorescein strips (I-DEW FLO, Entod Research Cell UK Ltd, London, UK) were wetted with sodium chloride and applied to the inferior fornix. Of Three TBUT measurements were done in both eyes, and the mean was recorded. A TBUT below 7 s was considered to be abnormal (Craig et al., 2017). Corneal integrity was assessed in both eyes using fluorescein staining. According to the Oxford grading scale (range, 0–5) (Bron et al., 2003) and the Cornea and Contact Lens Research Unit (CCLRU) grading scale (range, 0–20) (Terry et al., 1993). Nasal and temporal bulbar conjunctiva integrity was assessed by staining the conjunctiva with lissamine green strips (I-DEW green, Entod Research Cell UK Ltd) according to the Oxford grading scale. For both scales, staining >1 was considered abnormal. Corneal tactile sensitivity of both central corneas was evaluated before and after the application of topical anesthesia using the Cochet–Bonnet esthesiometer (Luneau Ophthalmology, Chartres, France) as we previously reported (V´ azquez et al., 2022). Scores <10 mm suggested peripheral or nociceptive pain, and scores above than or equal to 10 mm suggested neuropathic or centralized pain (Crane et al., 2017; Dieckmann et al., 2017). Corneal sensitivity thresholds were measured using a Belmonte’s noncontact gas esthesiometer, as we previously described (Ca˜ nadas et al., 2021; L´ opez-De La Rosa et al., 2016). Briefly, the mechanical threshold was always determined first, and stimulation comprised a series of variable flows of medicinal air (0–200 mL/min). Thermal (heat and cold) thresholds were then evaluated using airflows at different temperatures (order randomized) and flows 10 mL/min below the mechanical threshold. Tear production was assessed using the Schirmer test with anesthesia in both eyes. A sterile Schirmer strip (I-DEW tear strips, Entod Research Cell UK Ltd) was placed after 5 min of instilling a drop of topical anesthetic (0.1% tetracaine and 0.4% oxibuprocaine, Alcon Cusí) in the external inferior fornix of both eyes. The wet length of the strips was recorded after 5 min with eyes closed, Values ≤5 mm were considered abnormal (Lemp et al., 2007). 2.3.3. Conjunctival impression cytology Conjunctival cells were collected by impression cytology (CIC) after topical anesthesia instillation, as previously described (L´ opez-de la Rosa et al., 2021). Briefly, one eye was selected randomly and a half of polyethersulfone membrane filter (Supor 200, pore size: 0.20 μ m, diameter: 13 mm; Gelman Laboratory, Ann Arbor, MI, USA) was gently applied on the upper-temporal bulbar conjunctiva for 10 s under moderate pressure. The filters were then submerged in 1 mL of RLT lysis buffer (Qiagen) containing 1% β-mercaptoethanol (Merck KGaA) and stored at −80 ◦C for subsequent isolation of total RNA. 2.3.4. In vivo confocal microscopy In vivo confocal microscopy (IVCM) was performed using the Rostock cornea module of Heidelberg Retina Tomograph 3 (Heidelberg Engineering GmbH, Heidelberg, Germany). The examination was performed as previously reported by our group (Ca˜ nadas et al., 2021) under topical anesthesia. At minimum of three good quality and non-overlapping images from the subbasal nerve plexus of the central cornea were obtained. Two masked evaluators analyzed the following image parameters: density of nerves, length of nerves, density of nerve branches, grade of nerve tortuosity, density of dendritic cells, presence of neuromas and image reflectivity. The mean value between the two observers for each parameter was computed for statistical analysis. The number of nerves (n/mm 2 ), nerve density ( μ m/mm 2 ) and length ( μ m/mm 2 ) were measured using the plugin Neuron J from the ImageJ software (https://i magej.nih.gov/ij/). Nerve branches, dendritic cells, and neuromas were manually counted using the multipoint tool of the ImageJ software, and the density was calculated (n/mm 2 ) (L´ opez-De La Rosa et al., 2018). The grade of nerve tortuosity was evaluated according to Oliveira-Soto and Efron with a (0–4) scale (Oliveira-Soto and Efron, 2001). 2.4. Chemokine gene expression Total RNA from the HCE and IM-HConEpiC epithelial cells cultured in vitro and from the conjunctival cells collected in vivo by CIC was extracted using the commercial RNeasy Micro kit (Qiagen) according to the manufacturer’s protocol as previously described (L´ opez-de la Rosa et al., 2021). Briefly, RNA concentrations were measured using the Qubit RNA HS Assay kit (Life Technologies, Carlsbad, CA, USA) and the Qubit 4 fluorometer (Life Technologies). Reverse transcription was performed with 100 ng of RNA using the commercial iScript cDNA Synthesis kit (BioRad Laboratories Inc., Hercules, CA, USA) following the manufacturer’s instructions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed with 5 ng cDNA using the SsoAdvanced Universal SYBR Green Supermix (BioRad Laboratories Inc.) and primers in 20 μ l reaction volume. The primers used were CCL28 (qHsaCED0001386), CXCL14 (qHsaCID0008636), CXCL17 (qHsaCID0017116), and GAPDH (qHsaCED0038674) (BioRad Laboratories Inc.). The reaction was performed on a 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). For in vitro studies, specific mRNA levels were calculated after normalization of the results for each sample with those for GADPH mRNA (delta CT, ΔCT) and the 2 –ΔΔCt method (Schmittgen and Livak, 2008) was used to analyze the relative differences in specific mRNA levels between the groups. For conjunctival samples, the ΔCT values were compared by t-test between healthy controls and DE patients. 2.5. Chemokine concentration determination The concentrations of CCL28 and CXCL14 chemokines in the cell supernatants from the in vitro assays were measured simultaneously using X-MAP® technology with an immunobead-based assay (Milliplex HCYP4MAG-64 K Human Cytokine/Chemokine Magnetic Bead Panel IV, Merck Millipore, Burlington, MA, USA) in Magpix equipment (Luminex Corporation, Austin, TX, USA), according to the manufacturer’s instructions. Twenty-five microliters of undiluted sample per assay were used. Standard curves were used to convert fluorescence units to cytokine concentrations (pg/ml). Data were stored and analyzed using Belysa® software (Merck KGaA). The concentration of CXCL17 chemokine in the cell supernatants from the in vitro assays was measured using a commercial CXCL17 enzyme-linked immunosorbent assay (ELISA) kit (Sigma-Aldrich, Saint Louis, MO, USA) according to the manufacturer’s protocol. One hundred microliters of undiluted sample per assay were used. The optical density at 450 nm was measured using a SpectraMAx MS spectrophotometer (Molecular Devices, San Jose, CA, USA). Subsequently, absorbance values were used to calculate the concentrations (ng/mL) using the standard curve with SoftMax Pro software v4.8 (Molecular Devices). Chemokine concentration data were normalized to the corresponding protein content of each well, as determined by the bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific), according to the manufacturer’s instructions. 2.6. Statistical analysis Statistical analyses were performed using GraphPad Prism (version 9) (GraphPad Software, San Diego, California USA) and R software (version 4.2.2) (Foundation for Statistical Computing, Vienna, Austria). Quantitative data are expressed as mean ±standard deviation (SD), and qualitative variables are described in percentages. Normality A. Domínguez-L´ opez et al. Experimental Eye Research 241 (2024) 109854 4 assumptions were analyzed using the Shapiro–Wilk test. Student’s t-test was used to compare differences between two groups, one-way ANOVA to analyze multiple group comparisons, and chi-square test to analyze categorical variables. Spearman correlation coefficients were used to explore correlations between clinical parameters and gene expression levels using the inverse delta CT (-ΔCT). P-values <0.05 were considered statistically significant. 3. Results 3.1. Gene expression of CCL28, CXCL14, and CXCL17 by corneal and conjunctival epithelial cells under in vitro DE-simulated conditions HCE cells exposed to hyperosmolar conditions for 6 h increased gene expression of CCL28, CXCL14, and CXCL17 compared with basal condition (Fig. 1A); however, only CXCL14 gene expression at 450 mOsM was significantly increased (p <0.05). Instead, TNF- α (25 ng/mL) stimulation did not show significant effects on the gene expression levels of CCL28, CXCL14, or CXCL17 in HCE cells. In contrast, IM-HConEpiC cells incubated with hyperosmotic medium increased the gene expression of CXCL14 and CXCL17, whereas, TNF- α stimulation increased the gene expression of CCL28, CXCL14, and CXCL17 compared with basal condition. However, no significant differences in gene expression were observed in either condition (Fig. 1A). Individual CT values are shown in Supplementary Table 1. 3.2. CCL28, CXCL14, and CXCL17 protein secretion by corneal and conjunctival epithelial cells under in vitro DE-simulated conditions Chemokine concentrations were normalized to the corresponding protein content in each well. Total protein concentration was constant in each condition (50 ±8 μ g/mL) except for IM-HConEpiC cells at 500 mOsM, (35 ±7 μ g/mL) where a significant decrease (p <0.01) was observed. Chemokine concentrations without normalization are showed in Supplementary Table 2. HCE cells incubated for 24 h under hyperosmotic medium significantly increased the secretion of CXCL14 (p <0.05 at 450 and 500 mOsM) and CXCL17 (p <0.01 at 450 mOsM; p <0.05 at 500 mOsM) compared with unstimulated cells (Fig. 1B). In contrast, HCE cells stimulated with TNF- α did not significantly modify the secretion levels of CXCL14 and CXCL17. CCL28 secretion was not significantly affected either by hyperosmolar conditions or TNF- α stimulation. On the other hand, IM-HConEpiC cells exposed to hyperosmotic medium significantly increased the secretion of CCL28 (p <0.01 at 500 mOsM), CXCL14 (p <0.05 at 500 mOsM), and CXCL17 (p <0.05 at 450 mOsM). Contrarily, TNF- α stimulation did not affect the secretion of CCL28, CXCL14, and CXCL17 in IM-HConEpiC cells (Fig. 1B). 3.3. Participants and clinical data Demographic characteristics and clinical findings are summarized in Table 1. A total of 27 DE patients (22 women and 5 men; mean age 64.96 ±9.74 years, range 43–73) and 13 healthy subjects (9 women and 4 men; mean age 59.38 ±9.35 years, range 48–89) were recruited in the study. No significant differences were found between the two groups in terms of sex (p =0.384) or age (p =0.135), indicating that the observed differences in chemokine gene expression levels and clinical parameters were related only to DE disease condition and not to demographic differences in age or sex between the groups. DE patients showed higher OSDI, mSIDEQ, and NRS scores than healthy subjects. TBUT was significantly decreased in the DE patients compared with the healthy subjects. In addition, DE patients showed an Fig. 1. Effect of hyperosmolarity and TNF- α on the gene expression and production of CCL28, CXCL14, and CXCL17 by HCE and IM-HConEpiC cells. A) CCL28, CXCL14, and CXCL17 gene expression levels after 6 h of hyperosmolar (400, 450 and 500 mOsm) or TNF- α (25 ng/mL) exposure. GAPDH expression was used as a housekeeping gene. Data are presented as gene relative expression ±standard deviation over basal condition (300 mOsm without TNF- α ). B) CCL28, CXCL14, and CXCL17 protein levels after 24 h of hyperosmolar (400, 450 and 500 mOsm) or TNF- α (25 ng/mL) exposure. Data are presented as fold change (pg/mg protein) ±standard deviation over basal condition. * Denotes p <0.05, ** denotes p <0.01. N =three independent experiments and two replicates for each treatment per experiment were performed. HCE: human corneal epithelial cells; IM-HConEpiC: immortalized human conjunctival epithelial cells. A. Domínguez-L´ opez et al. Experimental Eye Research 241 (2024) 109854 5 increase in the CCLRU and Oxford scales in corneal and conjunctival staining (Table 1). The information related to age, sex, OSDI, TBUT, Shirmer test and corneal and conjunctival staining for each DE patient and for the healthy controls separately is shown in Supplementary Table 3. Corneal tactile sensitivity measured by Cochet–Bonnet esthesiometry before topical anesthesia was significantly lower in DE patients than in healthy subjects. No significant differences were found between the two groups after topical anesthesia. In contrast, corneal mechanical sensitivity evaluated using the Belmonte’s non-contact gas esthesiometer was significantly higher in DE patients than in healthy subjects. However, no significant differences were found between both groups in thermal (heat and cold) thresholds (Table 1). Analysis of IVCM images revealed that DE patients exhibited a lower number and density of subbasal nerves than healthy subjects. The density of nerve branches was also significantly lower in the DE group. Moreover, DE patients showed a higher density of neuromas and dendritic cells than healthy subjects (Table 1). 3.4. Gene expression of CCL28, CXCL14, and CXCL17 in healthy subjects and DE patients and correlations with clinical data We examined the gene expression levels of CCL28, CXCL14, and CXCL17 in conjunctival cells collected by impression cytology from healthy subjects and DE patients. Gene expression of these chemokines was detected in all participants. The mean CT values for genes detected in CIC samples were 27.6 ±0.8 (CCL28), 23.51 ±2.5 (CXCL14), 19.28 ±0.9 (CXCL17), and 19.38 ±0.9 (GAPDH) for healthy subjects and 27.2 ±1.1 (CCL28), 23.87 ±3.1 (CXCL14), 19.51 ±1.1 (CXCL17), and 20.18 ±1.2 (GAPDH) for DE patients. Statistical analysis revealed that CCL28 was significantly increased in conjunctival samples from DE patients compared with that from healthy subjects (p <0.05) (Fig. 2). However, no significant differences were found for CXCL14 and CXCL17 between the two groups. We also investigated the correlations between gene expression levels of mucosal chemokines and the clinical parameters for the total sample (Fig. 3). The gene expression of CCL28 significantly correlated positively with the OSDI and mSIDEQ scores, with corneal staining (Oxford scale and CCLRU scale), with the heat sensitivity threshold, and with density of dendritic cells in the central cornea. CXCL14 expression levels correlated positively with age, NRS score, conjunctival staining (Oxford scale), corneal tactile sensitivity, and with image reflectivity. The gene expression of CXCL17 was positively correlated with corneal staining (Oxford scale) (Fig. 4). 4. Discussion Mucosal chemokines, including CCL28, CXCL14, and CXCL17, are constitutively expressed in mucosal tissues under homeostatic conditions. However, there is scarce information regarding their levels or expression in the ocular surface, either in healthy subjects or in DE patients. Additionally, ocular surface inflammation and tear hyperosmolarity are considered key processes in the pathogenesis of DE (Ganesalingam et al., 2019; Lee et al., 2014; Lemp et al., 2011; Mathews et al., 2017). In this study, we investigated the effects of hyperosmotic and inflammatory conditions on the in vitro gene expression and secretion of CCL28, CXCL14, and CXCL17 mucosal chemokines by corneal and conjunctival epithelial cells. In addition, we analyzed the gene expression of CCL28, CXCL14, and CXCL17 in conjunctival samples from healthy subjects and DE patients, and their correlations with clinical parameters. In vitro models of hyperosmolar stress using ocular surface epithelial cells have been widely used to simulate the hyperosmotic conditions of DE (Li et al., 2004; Ren et al., 2017; Versura et al., 2011; Zhang et al., 2022). Numerous studies have shown increased secretion of chemokines, inflammatory cytokines, and matrix metalloproteinases in corneal epithelial cells exposed to hyperosmotic media (Deng et al., 2014; Hua et al., 2015; Li et al., 2006; Zhang et al., 2022). Here we observed that HCE and IM-HConEpiC cells express mucosal chemokines CCL28, CXCL14 and CXCL17 under basal conditions and that hyperosmolar stress increases the secretion of CCL28, CXCL14 and CXCL17 in IM-HConEpiC cells and of CXCL14 and CXCL17 in HCE cells. This differential expression may be due to the fact that conjunctival epithelial cells are able to induce a more potent immune response than corneal epithelial cells due to their anatomical location. These results suggest that corneal and conjunctival epithelial cells could be an important source of mucosal chemokines on the ocular surface under homeostatic and hyperosmotic conditions as in DE. However, we found that TNF- α (25 ng/mL) stimulation for 6 and 24 h did not significantly alter the gene expression or secretion of CCL28, CXCL14, or CXCL17 in HCE and IM-HConEpiC cells. TNF- α stimulation has previously been shown to induce the upregulation of inflammatory cytokines and chemokines in corneal and conjunctival epithelial cells (Abeng´ ozar-Vela et al., 2015; Table 1 Demographic characteristics and clinical findings of DE patients and healthy subjects. Parameter Healthy subjects (n = 13) DE patients (n =27) p-value Age 59.38 ±9.35 64.96 ±9.74 0.135 Sex (female/male) 9/4 22/5 0.384 OSDI questionnaire (score: 0–100) 3.42 ±3.33 39.41 ± 21.11 <0.0001 mSIDEQ questionnaire (score: 0–28) 4.69 ±3.96 13.85 ±4.40 <0.0001 NRS ocular pain (score: 0–10) 0.23 ±0.59 4.33 ±3.06 <0.0001 HADS questionnaire - Anxiety (score: 0–21) 4.84 ±2.82 6.29 ±4.046 0.159 HADS questionnaire - Depression (score: 0–21) 1.69 ±1.60 3.51 ±3.60 0.106 Osmolarity (mOsm/L) 328.3 ±33.04 317.6 ± 25.15 0.178 TBUT (sec) 5.23 ±2.90 3.35 ±1.47 0.012 Corneal staining (Oxford scale: 0–5) 0.30 ±0.48 1.18 ±1.03 0.003 Corneal staining (CCLRU scale: 0–20) 1.00 ±1.22 3.55 ±2.60 0.0002 Conjunctival staining (Oxford scale: 0–5) 0.80 ±0.63 1.24 ±0.67 0.037 Schirmer test with anesthesia (mm/5 min; 0–35 mm) 8.69 ±6.11 10.70 ±8.79 0.357 Corneal tactile sensitivity without anesthesia (mm) 58.46 ±3.15 52.78 ± 11.55 0.021 Corneal tactile sensitivity with anesthesia (mm) 20.38 ±27.04 16.30 ± 23.39 0.418 Mechanical sensitivity threshold (ml/min) 141.5 ±54 101.9 ± 47.50 0.018 Cold sensitivity threshold (△◦C) −1.64 ±0.90 −1.46 ±2.45 0.358 Heat sensitivity threshold (△◦C) 1.09 ±0.89 5.13 ±17.56 0.137 Number of nerves (n/mm2) 65.34 ±20.51 43.40 ± 15.14 0.002 Density of nerves ( μ m/mm2) 15120 ±3754 9584 ±3464 0.0001 Length of nerves ( μ m/mm2) 1481 ±145 1407 ±228 0.132 Density of nerve branches (n/ mm2) 39.49 ±29.04 20.72 ± 14.91 0.025 Grade of nerve tortuosity (scale: 0–4) 2.59 ±0.45 2.833 ±0.55 0.106 Image reflectivity (gray units) 105.9 ±11.77 100.5 ±9.15 0.125 Number of neuromas (n/mm2) 0.09 ±0.30 1.46 ±3.37 0.006 Density of dendritic cells (n/ mm2) 7.57 ±7.28 89.51 ± 99.89 <0.0001 CCLRU: cornea and contact lens research unit grading scale; DE: dry eye; HADS: hospital anxiety and depression subscale; mSIDEQ: modified single item dry eye questionnaire; NRS: numerical rating scale; OSDI: ocular surface disease index; TBUT: tear break-up time. Data are presented as media ±standard deviation. Significant p values (<0.05) are denoted in bold. A. Domínguez-L´ opez et al. Experimental Eye Research 241 (2024) 109854 6 Enríquez-de-Salamanca et al., 2008; García-Posadas et al., 2022; Katsinas et al., 2021; McInnis et al., 2007). Other studies have analyzed the effect of TNF- α on the production of CCL28, CXCL14, and CXCL17 in other cell types. For instance, increased CCL28 gene expression was observed in epidermal keratinocytes stimulated with TNF- α (10 ng/mL) for 12 h (Shibata et al., 2010). In addition, airway epithelial cells stimulated with TNF- α (100 ng/mL) induced gene expression and secretion of CCL28 at 6 and 24 h, respectively (O’Gorman et al., 2005). Whereas, epidermal keratinocytes stimulated with TNF- α (10 ng/mL) did not induce CXCL14 and CXCL17 expression (Frederick et al., 2000; Oka et al., 2017). Moreover, other cytokines such as interferon-gamma (IFN-γ) and fibroblast growth factor 2 (FGF-β), have been shown to increase CXCL14 and CXCL17 expression in pericytes and epidermal keratinocytes (Oka et al., 2017; Wang et al., 2022). This discrepancy in results could be attributed to the TNF- α concentration and stimulation period used in these other studies. However, we have already shown an inflammatory effect of this same TNF- α concentration and time of stimulation in other studies by our group, that reported the secretion of inflammatory cytokines by IM-HConEpiC and HCE under the same TNF- α stimulation conditions (Abeng´ ozar-Vela et al., 2015; García-Po- sadas et al., 2022; Katsinas et al., 2021). Therefore, further studies are needed to clarify the effects of inflammatory cytokines on the expression of mucosal chemokines in corneal and conjunctival epithelial cells. To further corroborate our in vitro findings, we analyzed the gene expression of the mucosal chemokines in conjunctival cells obtained from DE patients and control healthy subjects. Chemokine expression on the ocular surface of DE patients has been extensively analyzed. Increased levels of CCL2, CCL3, CCL4, CCL5, CCL15, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, and CX3CL1 were detected in the tears of DE patients (Choi et al., 2012; Enríquez-de-Salamanca et al., 2010; Na et al., 2012; Yoon et al., 2010). In addition, increased gene expression of the chemokines CCL2, CXCL8, and CXCL12 and the chemokine receptors CCR2, CCR5, and CXCR4 was observed in conjunctival samples from DE patients (Gulati et al., 2006; Massingale et al., 2009; Nicolle et al., 2018). However, there is scarce information in the literature about mucosal chemokine expression in the ocular surface. Recently, Wei and Asbell revealed the upregulation of CXCL14 gene expression in conjunctival samples from DE patients (Wei and Asbell, 2020). In this study, we found no differences in CXCL14 gene expression between healthy subjects and DE patients, but a positive correlation with age was observed. Therefore, the discrepancy in CXCL14 expression might be attributed to the age difference between controls and DE patients in the study by Wei and Asbell. In addition, we demonstrated the upregulation of CCL28 gene expression in conjunctival samples from DE patients. It is important to note that the conjunctival cells collected by impression cytology although mainly epithelial, also include immune cells (particularly in DE patients). Therefore, the upregulation of CCL28 gene expression may be related to these cell populations. Other studies have described increased CCL28 levels in inflammatory conditions such as inflamed colon, gingivitis, chronic periodontitis, generalized aggressive periodontitis, rheumatoid arthritis, and celiac disease. Furthermore, it has been observed that CCL28 expression is induced by proinflammatory cytokines, and that it correlates with disease severity (Chen et al., 2015; Ertugrul et al., 2013; Ogawa et al., 2004; Rashidiani et al., 2017). Several studies have investigated the correlation between chemokine Fig. 2. Conjunctival gene expression of CCL28, CXCL14, and CXCL17 in healthy subjects and DE patients. Data represent the inverse delta CT (-ΔCT) ± standard deviation. GAPDH expression was used as a housekeeping gene. *P <0.05. DE: Dry eye. Fig. 3. Correlations between chemokine gene expression levels in conjunctiva and clinical parameters in the total sample (healthy subjects and DE patients). Data represent the rho correlation coefficients colored according to their values: blue and red for positive and negative values, respectively. Color intensity and bubble size correspond to correlation strength; the darker the color and bigger the bubble, the higher the correlation. Statistically significant correlation coefficients are shown in bold. CCLRU: cornea and contact lens research unit grading scale; mSIDEQ: modified single item dry eye questionnaire; NRS: numerical rating scale; OSDI: ocular surface disease index; TBUT: tear break-up time. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) A. Domínguez-L´ opez et al. Experimental Eye Research 241 (2024) 109854 7 levels and clinical signs of DE patients. We previously reported that the concentrations of CXCL8 and CX3CL1 in the tears of DE patients correlate with age (Enríquez-de-Salamanca et al., 2010). In addition, the levels of CCL2, CCL15, CXCL5, CXCL8, and CX3CL1 were reported to correlate significantly with the severity of DE (Massingale et al., 2009; Na et al., 2012). The tear concentrations of CCL5 and CXCL11 were significantly correlated with keratoepitheliopathy in DE patients (Choi et al., 2012; Yoon et al., 2010). In this study, we extended these studies by analyzing the correlation between conjunctival gene expression of CCL28, CXCL14, and CXCL17 and clinical severity and ocular damage parameters in DE patients. Our results showed that the gene expression levels of CCL28 were significantly and positively correlated with the severity of DE-related symptoms analyzed by OSDI and mSIDEQ questionnaires. In addition, CCL28, CXCL14, and CXCL17 gene levels Fig. 4. Significant correlations between A) CCL28, B) CXCL14, and C) CXCL17 conjunctival gene expression levels and clinical parameters in the total subject sample (healthy þDE patients). The open circles (⚬) represent healthy subjects and solid circles (●) represent DE patients. The solid blue line represents the best-fit line, and the gray area indicates the 95% confidence interval. Data represent the inverse delta CT (-ΔCT). CCLRU: cornea and contact lens research unit grading scale; mSIDEQ: modified single item dry eye questionnaire; NRS: numerical rating scale; OSDI: ocular surface disease index. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) A. Domínguez-L´ opez et al. Experimental Eye Research 241 (2024) 109854 8 correlated significantly and positively with epithelial damage (analyzed by vital staining) in the cornea (CCL28 and CXCL17) and conjunctiva (CXCL14). The relationship between chemokine levels and corneal sensitivity and pain in DE patients has also been studied previously (Choi et al., 2012). In a previous study by our group, we found a significant correlation between CXCL8 tear levels and ocular pain in DE patients (Enríquez-de-Salamanca et al., 2010). Herein, we showed that the gene expression of CCL28 was significantly and positively correlated with the heat sensitivity threshold and that the gene expression of CXCL14 was correlated with corneal tactile sensitivity and ocular pain intensity. Recent studies suggest that corneal sensitivity abnormalities may be partially caused by alterations in subbasal nerves and immune cells in the cornea of DE patients. Reduced subbasal nerve plexus and increased inflammatory cells in the cornea have been reported to correlate with disease severity (Aggarwal et al., 2021; Labb´ e et al., 2013; Tepelus et al., 2017). According to these studies, our results revealed a reduction in nerve density in the subbasal nerve plexus and an increase in dendritic cells in the cornea of DE patients. We also found that the gene expression of CCL28 and CXCL14 correlated significantly and positively with the density of dendritic cells and image reflectivity, respectively. In recent years, some chemokines and their receptors have been reported to directly modulate neuronal activity and trigger neuroinflammatory processes and pain (Gonçalves dos Santos et al., 2020). It has been reported that CXCL14 is involved in the development of paclitaxel-induced neuropathic pain and that knockdown of CXCL14 in the dorsal horn significantly attenuates mechanical allodynia (Liu et al., 2020). Moreover, the upregulation of CXCL14 was observed in a chronic pain model induced by local inflammation of the dorsal root ganglion in rats (Strong et al., 2012). CXCL17 administration has been shown to induce strong pain-related behaviors in naïve mice and to increase hypersensitivity to mechanical or thermal stimuli after chronic constriction injury. In addition, the study showed that blockade of the receptor for CXCL17 prevented the hypersensitivity (Rojewska et al., 2019). Although our results show a correlation between these mucosal chemokines and corneal sensitivity and corneal nerve damage, further studies are needed to determine whether they play a role in the development of ocular pain associated with DE. Given their multiple functions in mucosal tissues, it is important to know whether CCL28, CXCL14, and CXCL17 are involved in antimicrobial activity and/or immune cell recruitment on the ocular surface of DE patients. The imbalance in ocular surface microbiota composition has recently been analyzed in DE patients with and without Sj¨ ogren’s syndrome-associated DE (Andersson et al., 2021; Kim et al., 2022; Qi et al., 2021; Song et al., 2022). In addition, significant correlations were found between the composition of the microbiome and the inflammatory response on the ocular surface of those patients and healthy subjects (Cano-Ortiz et al., 2020). However, we do not know whether it is the imbalance in the homeostasis of the microbiome that alters mucosal chemokine expression levels or vice versa. Therefore, future studies are required to clarify the role of mucosal chemokines in ocular surface antimicrobial activity and immune cell recruitment. This study has some limitations. First, the in vitro experiments were performed using two immortalized human epithelial cell lines, which may have some alterations in metabolic activity and immune response. Therefore, future experiments using primary cell cultures are needed to identify mucosal chemokine production by corneal and conjunctival cells under hyperosmolar and inflammatory conditions. Second, the relatively small sample of participants included in the study, and that the DE patients were not grouped according to the severity of their disease. It would be interesting to analyze larger cohorts of patients to evaluate the presence of CCL28, CXCL14, and CXCL17 and also to be able to compare its expression in DE patients with different levels of disease severity (normal, mild to moderate, and severe). Another limitation may be that our sample study size did not allow us to analyze separately the mucosal chemokine gene expression depending on the sex of our participants. Although no significant differences were found in sex distribution between healthy subjects and DE patients, it might be interesting to analyze whether chemokine expression would change between both sexes. It could also be relevant the fact that HCE cells derive from a human cornea obtained from a female donor (Araki-Sasaki et al., 1995), whereas no information was available regarding the mal- e/female origin of the IM-HConEpiC cells. Finally, this study quantified the gene expression of mucosal chemokines in conjunctival samples but mRNA expression of a gene does not necessarily correlate with protein concentration. Therefore, future experiments quantifying the actual protein levels of CCL28, CXCL14, and CXCL17 in tear samples should be performed to support our findings. In conclusion, our study showed that gene expression and secretion of CCL28, CXCL14, and CXCL17 mucosal chemokines are induced in HCE and IM-HConEpiC cells under hyperosmolar conditions, a fact that is etiologically related with DE. We also described the gene expression of these chemokines in conjunctival samples from healthy subjects, and the upregulation of CCL28 gene expression in DE patients. In addition, significant positive correlations were found between the gene expression of CCL28, CXCL14, and CXCL17 and the clinical severity of DE. All these results suggest that mucosal chemokines may be involved in the pathogenesis of DE and open the possibility that they might be considered as biomarkers for this disease and even potential therapeutic targets. Future studies analyzing protein levels of CCL28, CXCL14, and CXCL17 in tears of healthy subjects and DE patients should be performed to further increase the knowledge of the role of these chemokines on the ocular surface under homeostatic conditions and DE pathogenesis. Funding This work was supported by the Ministry of Education, Science, Technology and Innovation of Mexico City (SECTEI) [SECTEI/160/ 2021, ADL]; the Ministry of Science, Innovation, and Universities, Spain [Grant SAF-2016-77080-P, MICIU/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe. PI: AES and MJG]; and [Grant PID 2022-142578OB-I00, MICIU/AEI/10.13039/501100011033 and by “ERDF /EU. PI: AES]. Declaration of competing interest The authors have declared that no conflict of interest exists. CRediT authorship contribution statement Alfredo Domínguez-L´ opez: Writing – review & editing, Writing – original draft, Methodology, Investigation, Conceptualization. Marta Blanco-V´ azquez: Writing – review & editing, Methodology, Investigation, Data curation. Andr´ es ´ Angel Calder´ on-García: Methodology, Investigation. Carmen García-V´ azquez: Methodology. María J. Gonz´ alez-García: Writing – review & editing, Validation, Supervision, Resources, Project administration. Margarita Calonge: Writing – review & editing, Resources. Amalia Enríquez-de-Salamanca: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Data availability Data will be made available on request. Appendix A. 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