Vitamin D impedes eosinophil chemotaxis via inhibiting glycolysis-induced CCL26 expression in eosinophilic chronic rhinosinusitis with nasal polyps

Abstract

Background

Chronic rhinosinusitis with nasal polyps (CRSwNP) is likely to relapse due to aberrant eosinophil infiltration. The deficiency of Vitamin D (VD) is associated with increased eosinophil infiltration in eosinophilic oesophagitis. However, the role of VD in eosinophilic CRSwNP (ECRSwNP) remains unclear. This study aims to explore the effects of VD on eosinophil chemotaxis in ECRSwNP and the underlying mechanisms.

Methods

Human nasal mucosal tissues were collected from the control group, patients with non-ECRSwNP and those with ECRSwNP. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of VD and CCL26 in the nasal mucosa, plasma, or human primary nasal epithelial cells (hNECs). hNECs and eosinophils from patients were cultured to investigate the effect of VD on eosinophil chemotaxis and CCL26 expression via eosinophil migration assay, Western blot, and ELISA. Transcriptome sequencing, pathway enrichment analysis, Western blot and immunohistochemical staining were used to determine the key signaling pathway involved in eosinophil chemotaxis.

Results

A significant decrease in VD levels was observed in the nasal mucosa of patients with ECRSwNP, which correlated with increased local eosinophil infiltration. Furthermore, pathway enrichment analysis suggested that glycolysis signaling was promoted in the ECRSwNP group, verified by enhanced expression of glycolytic key enzymes that were positively correlated with eosinophil infiltration in nasal mucosa from patients with ECRSwNP. VD suppressed eosinophil chemotaxis in vitro by inhibiting CCL26 expression. Glycolysis regulated CCL26 expression via the ERK pathway and lactate, which promoted the expression and stability of CCL26 protein. VD attenuated glycolysis, leading to decreased production of lactate and inactivation of the ERK pathway. The decrease in lactate production suppressed eosinophil chemotaxis. Moreover, the ERK pathway activator reversed the inhibitory effect of VD on eosinophil chemotaxis.

Conclusions

VD impedes eosinophil chemotaxis by inhibiting glycolysis - induced CCL26 expression via attenuating the activation of the ERK pathway and reducing lactate production. VD supplementation may be a novel strategy to treat ECRSwNP.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-025-02078-2.

Background

Chronic rhinosinusitis (CRS) is a chronic inflammatory disease of the nasal cavity and paranasal sinus mucosa with a global incidence of 5–15% [1]. CRS can be divided into CRS with nasal polyps (CRSwNP) and without nasal polyps [2]. CRSwNP is further classified as eosinophilic (ECRSwNP) or non-eosinophilic (non-ECRSwNP) based on the infiltration of local eosinophils [3]. ECRSwNP is characterized by severe symptoms and a high tendency of relapse after comprehensive treatment [4, 5]. Furthermore, the incidence of ECRSwNP has increased dramatically in recent decades [3, 6]. Therefore, understanding the pathogenesis of ECRSwNP and improving therapeutic efficacy are crucial.

Human nasal epithelial cells (hNECs) secrete various inflammatory factors under harmful stimuli and are involved in the occurrence and development of airway mucosal inflammation [7]. hNECs promote type 2 inflammation by producing thymic stromal lymphopoietin (TSLP) and interleukin (IL)-33, which induce eosinophil infiltration via signal transduction [8]. hNECs also produce eosinophil chemokines, including CCL11 (eotaxin-1), CCL24 (eotaxin-2), and CCL26 (eotaxin-3), which directly recruit eosinophils [8–11]. Activated eosinophils generate several type 2 cytokines, such as IL-4 and IL-13, which amplify local inflammation and stimulate hNECs to produce mucin [11]. Eosinophils also destroy the epithelial barrier through eosinophilic extracellular traps and participate in tissue remodeling by secreting transforming growth factor beta [11]. Therefore, interrupting nasal epithelial cell-related signal transduction and eosinophil migration toward the nasal mucosa is crucial for reducing inflammation in ECRSwNP.

Vitamin D (VD), a fat-soluble vitamin, regulates calcium and phosphorus metabolism [12]. VD is also involved in the innate and acquired immune responses. VD participates in anti-inflammatory and antioxidant reactions by regulating immune cell proliferation and differentiation and altering the secretion patterns of inflammatory mediators by inflammatory cells [13]. Moreover, VD inhibits the production of type 2 cytokines by CD4⁺ T cells to improve airway remodeling [14]. VD deficiency is associated with an abundance of eosinophils [15, 16], suggesting that VD is involved in regulating eosinophil infiltration. Serum VD levels in patients with CRSwNP are lower than those in controls [17, 18], and VD supplementation can alleviate the inflammatory response to CRSwNP [19]. However, the relationship between VD and eosinophil infiltration in patients with CRSwNP and whether VD inhibits eosinophil recruitment in CRSwNP remain unclear. Therefore, the present study investigated the effects of VD on eosinophil chemotaxis in ECRSwNP and the underlying mechanisms, thereby providing a theoretical basis for the role of VD in eosinophil infiltration in patients with ECRSwNP.

Methods

Patients and samples

Nasal mucosal tissues were collected from controls and patients with non-ECRSwNP and ECRSwNP who underwent nasal endoscopic surgery between September 2020 and December 2022. CRSwNP was diagnosed in accordance with the European Position Paper on Rhinosinusitis and Nasal Polyps 2020 guidelines [1]. Patients who underwent endoscopic sinus surgery for simple nasal septum deviation (not caused by acute trauma and other factors), cerebrospinal fluid rhinorrhea, optic canal decompression, pituitary tumor surgery were regarded as control subjects. Patients with CRSwNP were divided into the non-ECRSwNP and ECRSwNP groups based on whether 10% eosinophils were present [20]. Patients with immunodeficiency, fungal sinusitis, coagulation disorder, cystic fibrosis or rickets were excluded in the study. None of the patients had received corticosteroids or antibiotics in the month prior to surgery. The patient characteristics are listed in Table S1.

Culture of primary hNECs

Fresh nasal mucosal tissue was cut up and soaked with Dispase enzyme II (Sigma-Aldrich, St Louis, MO, USA) at 4 °C overnight. The next day, tissues were digested with 0.25% trypsin (Thermo Scientific, New York, NY, USA) at 37 °C, and neutralized with Dulbecco’s Modified Eagle’s Medium (Thermo Scientific, New York, NY, USA), and thoroughly agitated, and filtered to obtain the cell suspension. After removal of fibroblasts, hNECs were obtained and cultured in 12-well plates with bronchial epithelial growth medium (Lonza, Basel, Switzerland) in an incubator under 5% CO₂ and 95% humidity at 37 °C.

VD (Calcitriol), 2-Deoxy-D-glucose (2-DG), Compound 3 K (C3K), PFK-015 (PFK15), and sodium lactate were purchased from Selleck Chemicals (Houston, TX, USA). PD98059 (PD) and tert-butylhydroquinone (tBHQ) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). These compounds were involved in the treatment of hNECs. Cycloheximide (GLPBIO, Montclair, CA, USA) was used in the protein stability assay. In this study, human homologous recombinant CCL26 protein (Shenzhou Yiqiao Biotechnology Co., Ltd., Beijing, China), TSLP protein (novoprotein, Suzhou, China), and human homologous recombinant IL-17 A protein (R&D System, Minnesota, MN, USA) were utilized.

Real-time quantitative polymerase chain reaction

RNA extraction and qRT-PCR were performed as described previously [21]. The relative expression levels of genes were calculated using the comparative method (2^−△△CT), with β-actin as an internal reference. The primer sequences are listed in Table S2.

Western blot

Western blot (WB) was performed according to the published methods [22]. The primary antibodies used were listed in Table S3. Each band was quantified with Image j software and normalized to the band of β-actin or GAPDH.

Immunohistochemical (IHC) staining

IHC staining was performed according to a previously published paper [21]. Primary antibodies used were listed in Table S3. The results of IHC staining were quantitatively analyzed using the average integrated optical density index with Image J software.

Immunofluorescence staining

Immunofluorescence staining was performed according to a previously published paper [23]. Cells of interest were incubated with primary antibodies (Table S3) at 4℃ overnight. On the following day, secondary antibodies conjugated with Alexa Fluor 594 (Thermo Scientific, New York, NY, USA) were incubated for 60 min. After staining with DAPI (Thermo Scientific, New York, NY, USA), the samples were observed.

Enzyme-linked immunosorbent assay

Tissue homogenate was obtained by grinding fresh nasal mucosa tissue with the specific buffers at a ratio of 1.0 mL buffer to every 0.1 g tissue after the removal of blood stains and mucus. VD was detected with a detection kit (Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). The supernatant from hNECs was collected and applied to CCL26 detection kits (Elabscience, Wuhan, China) and TSLP detection kits (Liankebio, Hangzhou, China), respectively.

Lactate detection

The supernatant from hNECs was collected and applied to lactate detection using a kit (Bioengineering Institute, Nanjing, China) based on the instructions of the reagents.

Eosinophil isolation and eosinophil migration assay

Peripheral blood from subjects was collected, and eosinophils were isolated according to the protocol of eosinophil extraction kit (Miltenyi Biotec, San Diego, Germany). The eosinophil migration assay was performed as described previously [24]. According to the published literature [25], eosinophils were stained with 5 µM CFDA-SE (GLBPIO, Montclair, CA, USA) for 8 min in advance and washed with phosphate-buffered saline. Subsequently, 300 µL of the supernatant taken from hNECs or fresh medium was added to the lower chamber. Eosinophils were resuspended in a medium to a volume of 150 µL, and added to the upper chamber at a density of 10 × 10⁴ cells per chamber. Eosinophils in the lower chamber were observed and counted using a fluorescence microscope after 2 h.

Tissue transcriptome sequencing and bioinformatics analysis

Fresh tissue RNA sequencing was performed on a DNBSEQ platform, and 150-bp paired-end reads were generated by Beijing Genomics Institution (BGI, Shenzhen, China).

The GEO datasets were downloaded from the Gene Expression Omnibus website (https://www.ncbi.nlm.nih.gov/geo/). The SangerBox online analysis tool was used to evaluate eosinophil infiltration and perform enrichment analysis [26].

Kyoto Encyclopedia of Genes and Genomes (KEGG) database, with the website https://www.kegg.jp/kegg/, was used to acquire the biological processes related to VD in the compound module. And Cytoscape 3.0 software was further used to visualize the results.

Statistical analyses

Data were analyzed using SPSS software (version 20.0). Student’s t-tests or Mann–Whitney U tests were used for comparisons between two groups. Spearman’s test was used to identify bivariate correlations. P < 0.05 was considered significant.

Results

VD is deficient in patients with ECRSwNP and negatively correlates with eosinophil infiltration

The activated form of VD, 1,25-dihydroxyvitamin D (1,25(OH)2D) [27], was examined in the nasal mucosal tissue. Lower 1,25(OH)2D levels were observed in the ECRSwNP group than in the control (P < 0.01) and non-ECRSwNP groups (P < 0.05; Table S4; Fig. 1A). Consistently, serum 25-hydroxy VD (25(OH)D) expression in patients with non-ECRSwNP and ECRSwNP was lower than that in the control group (P < 0.01 and P < 0.001, respectively; Table S4; Fig. 1B). Additionally, 25(OH)D was negatively correlated with the visual analogue scale and Sino-Nasal Outcome Test-22 score of patients with CRSwNP, respectively (r = ˗0.49, P = 0.002, Fig. 1C; r = -0.45, P = 0.004, Fig. 1D). And a slight correction between 25(OH)D and Lund Mackay CT score without significance was observed (Figure S1A).

Fig. 1.

VD is deficient in patients with ECRSwNP and negatively correlates with eosinophil infiltration (A) Detection of nasal mucosal tissue 1,25(OH)2D in control patients (n = 6), patients with non-ECRSwNP (n = 16), and patients with ECRSwNP (n = 14). (B) Detection of serum 25(OH)D in control patients (n = 16), patients with non-ECRSwNP (n = 81), and patients with ECRSwNP (n = 55). (C, D) Serum 25(OH)D was negatively correlated with visual analogue scale and Sino-Nasal Outcome Test-22 score, respectively. (E) The quantitative analysis for ECP⁺ cells in nasal mucosal tissue (400× magnification). (F) 1,25(OH)2D negatively correlates with eosinophil infiltration in nasal mucosal tissue. (G) Representative IHC staining for ECP. (H) Representative nasal endoscopic examination of a patient with ECRSwNP before and after VD supplementation combined with glucocorticoid treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance; VAS, visual analogue scale; SNOT-22, Sino-Nasal Outcome Test-22; INCS: intranasal corticosteroids; OCS: oral corticosteroids

The number of eosinophil cationic protein (ECP) - positive cells in patients with ECRSwNP was higher than that in the non-ECRSwNP and control groups (P < 0.0001 for both; Fig. 1E, G). The number of ECP-positive cells was negatively correlated with 1,25(OH)2D levels (r = ˗0.51, P = 0.012; Fig. 1F). Similarly, peripheral eosinophils were significantly elevated in patients with ECRSwNP (Figure S1B, C), and the decreasing serum 25(OH)D levels were related to the increase in peripheral eosinophils (Figure S1D, E). Patients with ECRSwNP who showed poor efficacy after a single glucocorticoid treatment demonstrated greatly improved nasal symptoms after 3–4 weeks of glucocorticoid treatment combined with VD supplementation. Nasal endoscopy verified that nasal mucosal swelling was markedly alleviated (Fig. 1H).

VD inhibits CCL26-induced eosinophil chemotaxis

Eosinophil chemokines (IL-33, TSLP, CCL11, CCL24, and CCL26) were evaluated to investigate whether VD inhibited inflammation by suppressing eosinophil chemotaxis. The mRNA levels of TSLP, CCL11, CCL24, CCL26, and ECP in patients with ECRSwNP were evidently higher than those in the control and non-ECRSwNP groups (Fig. 2A–F). TSLP, CCL11, CCL24, and CCL26 expression was positively correlated with ECP (r = 0.60, P < 0.0001; r = 0.80, P < 0.0001; r = 0.90, P < 0.0001; and r = 0.93, P < 0.0001, respectively; Fig. 2G–J). Notably, the correlation between CCL26 and ECP was strongest. Expression profiles based on the external GSE36830 dataset (Fig. 2K) also showed that CCL26 and TSLP had the strongest correlation with ECP (Fig. 2L–O). Therefore, hNECs were treated with varying concentrations of VD for 24 h to clarify the effects of VD on TSLP and CCL26 expression. Both 10^˗6 and 10^˗8 M VD treatments decreased TSLP and CCL26 mRNA expression (Fig. 2P, Q), and 10^˗8 M VD was selected for further study. VD effectively reduced TSLP and CCL26 protein levels in hNECs supernatants (Fig. 2R, S).

Fig. 2.

VD inhibits CCL26 and TSLP expression in hNECs (A–F) The expression of IL-33, TSLP, CCL11, CCL24, CCL26, and ECP in the nasal mucosa was detected by qRT-PCR. (G–J) Correlations between ECP and TSLP (G), CCL11 (H), CCL24 (I), and CCL26 (J) mRNA levels. (K) Heat map of eosinophil infiltration evaluation and TSLP, CCL11, CCL24, and CCL26 mRNA expression based on the GSE36830 dataset. (L–O) Correlations between eosinophil infiltration and CCL11 (L), CCL24 (M), TSLP (N), and CCL26 (O) mRNA levels based on the GSE36830 dataset. (P, Q) TSLP (P) and CCL26 (Q) in hNECs were detected by qRT-PCR. (R, S) ELISA detection of TSLP (R) and CCL26 (S) proteins in the supernatant of hNECs from the control and VD-treated groups (10^− 8 M). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance

The identification of eosinophils isolated from the peripheral blood of patients with CRSwNP was confirmed through immunofluorescence staining (Figure S2A). Next, the migration assay was carried out as described (Figure S2B), indicating that CCL26, rather than TSLP, significantly promoted the chemotaxis of eosinophils (Figure S2C-F). Considering that CCL26 would directly drive the eosinophil migration [28], while TSLP is mainly reported to promote eosinophil chemotaxis through activating stromal immune cells to secret some cytokines, especially Th2 cytokines [29], we further investigated whether VD could inhibit CCL-26 induced eosinophil migration. The eosinophil migration assay (Fig. 3A) demonstrated that 10^˗8 M VD effectively inhibited eosinophil chemotaxis because the eosinophil count decreased after VD treatment (Fig. 3B, C). We previously reported that IL-17 A facilitated eosinophil inflammation [30]. In addition, IL-17 A was found to be overexpressed in the nasal mucosae of patients with ECRSwNP (Figure S3 A), with a positive correlation with ECP expression (Figure S3 B). To investigate whether IL-17 A regulated eosinophil chemotaxis, hNECs was treated with IL-17 A subsequently. Elevated CCL26 expression was observed in hNECs after IL-17 A (300ng/mL) stimulation (Figure S3C), thereby facilitating eosinophil chemotaxis by the supernatant from hNECs treated with IL-17 A (Figure S3D, E). Further investigation suggested that VD not only inhibited the expression of CCL26 induced by IL-17 A (Fig. 3D), but also effectively blocked eosinophil chemotaxis (Fig. 3E, F).

Fig. 3.

VD inhibits eosinophil chemotaxis (A) Schematic diagram of eosinophil migration assay. (B, C) Representative results of migration assays and quantitative analysis of eosinophils treated with supernatants of hNECs from the control and VD-treated (10^− 8 M) groups (40× magnification). (D) ELISA detection of CCL26 proteins in the supernatant of hNECs from the control, IL-17 A-treated, IL-17 A + VD-treated groups. (E, F) Representative results of migration assays and quantitative analysis of eosinophils treated with supernatant of hNECs from the control, IL-17 A-treated, IL-17 A + VD-treated groups (40× magnification). *, P < 0.05; EOS, eosinophils; hNECs, human nasal epithelial cells

Glycolysis is enhanced in ECRSwNP and positively correlates with eosinophil infiltration

We used the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to determine the biological activities regulated by VD and to investigate the potential mechanisms through which VD inhibits eosinophil chemotaxis and decreases CCL26 expression. Notably, VD was involved in regulating metabolic activity (Fig. 4A, S4A). Nasal mucosal tissues from patients with CRSwNP and controls were subjected to transcriptome sequencing. Glycolysis was the only metabolic signaling pathway among the top 10 differentially regulated pathways in patients with CRSwNP compared to the controls (Fig. 4B). Based on the GSE72713 dataset, glycolysis-related gene expression (Figure S4B) and glycolysis signals were increased in patients with ECRSwNP compared to those in the non-ECRSwNP and control groups (Figure S4C, D). Lactate dehydrogenase A (LDHA) and key glycolytic enzymes, including hexokinase-2 (HK2), phosphofructokinase, liver type (PFKL), and pyruvate kinase M2 (PKM2), were upregulated in patients with ECRSwNP compared to those in the non-ECRSwNP and control groups (Fig. 4C–F). Moreover, HK2, PFKL, and PKM2 expressions were enhanced in the nasal mucosal epithelium of patients with ECRSwNP (Fig. 4G, H). Lactate levels in patients with ECRSwNP were also markedly higher than those in the control and non-ECRSwNP groups (Fig. 4I).

Fig. 4.

Glycolysis enhances in ECRSwNP and correlates with eosinophil infiltration (A) Activities regulated by VD were obtained using the KEGG database. (B) The top 10 differential signaling pathways upregulated in the CRSwNP group compared with the control group were acquired based on nasal mucosal tissue transcriptome sequencing. (C-F) HK2, PFKL, PKM2, and LDHA mRNA in nasal mucosa from the control (n = 15), non-ECRSwNP (n = 20), and ECRSwNP groups (n = 19) were detected by qRT-PCR. (G-H) Representative IHC staining and quantitative analysis for HK2, PFKL, and PKM2 in the nasal mucosa of control, non-ECRSwNP and ECRSwNP patients (400× magnification). (I) Detection of lactate in nasal mucosa tissue of control patients (n = 7), patients with non-ECRSwNP (n = 16), and patients with ECRSwNP (n = 13). (J-M) Correlations between ECP and HK2 (J), PFKL (K), PKM2 (L), LDHA (M) at mRNA level. (N–P) Correlations between tissue eosinophil infiltration and HK2 (N), PFKL (O), and PKM2 (P) expression levels in mucosal epithelium. *, P < 0.05; **, P < 0.01; ****; P < 0.0001; ns, no significance

We also analyzed the relationship between glycolysis and eosinophil infiltration. HK2, PFKL, PKM2, and LDHA mRNA levels were positively correlated with those of ECP (Fig. 4J–M) (r = 0.84, P < 0.0001; r = 0.79, P < 0.0001; r = 0.78, P < 0.0001; and r = 0.62, P < 0.0001, respectively). Additionally, HK2, PFKL, and PKM2 expression in epithelial cells was positively associated with the eosinophil count in the nasal mucosa (r = 0.32, P = 0.036; r = 0.55, P < 0.001; and r = 0.39, P = 0.010, respectively; Fig. 4N–P). This finding was also verified using GSE72713 dataset analysis. The increase in eosinophil infiltration in the nasal tissue of patients with ECRSwNP was accompanied by intense glycolysis signal (Figure S4E), and HK2 and PFKL mRNA levels were positively correlated with eosinophil infiltration (Figure S4F).

Glycolysis regulates eosinophil chemotaxis by promoting CCL26 expression via lactate in hNECs

Quantitative HK2, PFKL, PKM2, and LDHA mRNA expression levels in the nasal mucosa were positively correlated with those of CCL26 (r = 0.87, P < 0.0001; r = 0.86, P < 0.0001; r = 0.81, P < 0.0001; and r = 0.70, P < 0.0001, respectively; Fig. 5A–D). Thus, we speculated that epithelial cell glycolysis drives eosinophil chemotaxis by promoting CCL26 expression. According to the published literatures [30, 31], we enhanced the glycolytic phenotype of hNECs via IL-17 A stimulation. IL-17 A upregulated the HK2, PFKL, and PKM2 protein levels in hNECs (Fig. 5E). Inhibiting glycolysis with 2-DG (a HK2 inhibitor) and C3K (a PKM2 inhibitor) effectively decreased CCL26 expression (Fig. 5F, S5A); however, the inhibition of glycolysis with PFK15 (a PFKL inhibitor) did not induce CCL26 downregulation (Figure S5B). Treatment with 2-DG (Fig. 5G, H) and C3K (Figure S5C, D) also reversed the IL-17 A-induced upregulation of CCL26 protein levels in hNECs and its secretion from hNECs. Enhanced glycolysis increased eosinophil chemotaxis, whereas 2-DG treatment effectively disrupted IL-17 A-induced eosinophil chemotaxis (Fig. 5I, J). IL-17 A also induced lactate production (Fig. 5K). Further investigation suggested that sodium lactate not only promoted CCL26 expression but enhanced its stability (Fig. 5L, M).

Fig. 5.

Glycolysis regulates CCL26-induced eosinophil chemotaxis (A–D) Correlations between CCL26 and HK2 (A), PFKL (B), PKM2 (C), and LDHA (D) at mRNA levels in the nasal mucosa. (E) Representative WB images and quantitative analysis of HK2, PFKL, PKM2 in hNECs treated with IL-17 A. (F) Representative WB images and quantitative analysis of CCL26 in whole cell lysates of hNECs treated with 2-DG. (G) Representative WB images and quantitative analysis of CCL26 in whole cell lysates of hNECs pretreated with IL-17 A (300 ng/mL) for 1 h and then stimulated with or without 2-DG for 24 h. (H) ELISA detection of CCL26 in hNECs supernatant from the control, IL-17 A, or IL-17 A + 2-DG (5mM)-treated groups. (I, J) Representative results of migration assays and quantitative analysis of eosinophils treated with hNECs supernatant from the control, IL-17 A (300 ng/mL) or IL-17 A + 2-DG (5mM)-treated groups (40× magnification). (K) Detection of lactate in supernatant of hNECs. (L, M) Detection of CCL26 in whole cell lysates of hNECs treated with sodium lactate (40mM) or combined with cycloheximide (CHX). *, P < 0.05; **, P < 0.01; ns, no significance

Glycolysis regulates eosinophil chemotaxis via extracellular signal-regulated kinase (ERK) pathway

Gene set enrichment analysis of the GSE72713 dataset showed that the ERK pathway was upregulated in the ECRSwNP group compared to the control and non-ECRSwNP groups (Fig. 6A, B). We further tested ERK activation in the nasal tissues. Phosphorylated ERK (p-ERK) was predominantly expressed in the nucleus of the nasal mucosal epithelium. Expression of p-ERK in the nasal mucosal epithelium of patients with ECRSwNP was higher than that in the non-ECRSwNP and control groups (Fig. 6C, D). IL-17 A treatment promoted p-ERK expression in hNECs (Figure S6A, B), and an ERK pathway inhibitor interfered with p-ERK upregulation (Fig. 6E, F). ERK pathway inhibition remarkably reduced CCL26 expression in hNECs and their supernatants (Fig. 6G, H) and decreased eosinophil chemotaxis (Fig. 6I, J). Activating the ERK pathway with tBHQ promoted p-ERK protein expression (Figure S6C), and dose-dependently increased eosinophil migration (Figure S6D, E). Furthermore, inhibition of glycolysis impeded activation of ERK pathway (Fig. 6K).

Fig. 6.

Glycolysis regulates eosinophil chemotaxis via ERK pathway (A, B) GSEA of the ERK pathway based on the GSE72713 dataset. (C, D) Representative IHC staining and quantitative analysis of p-ERK in tissue epithelium of control patients (n = 9), patients with non-ECRSwNP (n = 23), and patients with ECRSwNP (n = 19). (E–J) hNECs pretreated with PD for 1 h and then stimulated with or without IL-17 A (300 ng/mL) for 24 h. (E) Representative IF staining of p-ERK in hNECs. (F–G) Representative WB images and quantitative analysis of p-ERK (F) and CCL26 (G) in lysates of hNECs. (H) ELISA detection of CCL26 in hNECs supernatant. (I, J) Representative results of migration assays and quantitative analysis of eosinophils treated with hNECs supernatant from the control, IL-17 A, and IL-17 A + PD groups (40× magnification). (K) Representative WB and quantitative analysis of p-ERK in whole cell lysates of hNECs after treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001

VD inhibits eosinophil chemotaxis by interrupting glycolysis

Next, we speculated whether VD inhibited eosinophil chemotaxis via attenuating glycolysis to disrupt ERK pathway activation or lactate production. As expected, VD disrupted p-ERK expression in hNECs (Fig. 7A). Similarly, VD suppressed HK2, PFKL, and PKM2 expression in hNECs (Fig. 7B). Moreover, VD and lactate levels were observed negatively correlated in the nasal mucosa from patients with CRSwNP (Fig. 7C). And lactate production also decreased in the supernatant of hNECs after VD treatment (Fig. 7D). Furthermore, VD disrupted eosinophil chemotaxis induced by IL-17 A-mediated glycolysis, which was partially reversed by activating the ERK pathway with tBHQ. The addition of glycolysis inhibitor to inhibit lactate production effectively prevented the reversal of ERK activation on eosinophil chemotaxis. CCL26 was added to the lower chamber in eosinophil migration assay next. Therefore, human homologous recombinant CCL26, combined with tBHQ and 2-DG treatment, restored eosinophil chemotaxis, which VD suppressed (Fig. 7E, F) (Fig. 8).

Fig. 7.

VD inhibits eosinophil chemotaxis by blocking glycolysis (A–B) hNECs were pretreated with IL-17 A (300 ng/mL) for 1 h and then stimulated with or without VD (10^− 8 M) for 24 h. (A) Representative WB and quantitative analysis of ERK pathway proteins in whole cell lysates of hNECs. (B) Representative WB images and quantitative analysis of HK2, PFKL and PKM2 in whole cell lysates of hNECs. (C) Correlation between lactate and 1,25(OH)2D in the nasal mucosa. (D) Detection of lactate in the hNECs supernatant. (E, F) hNECs pretreated with IL-17 A (300 ng/mL) for 1 h and then stimulated with VD (10^− 8 M), followed by tBHQ (20 µM) or 2-DG (5 mM) for 24 h with or without human homologous recombinant CCL26 protein (0.1µM) for 1 h. Representative results of migration assays and quantitative analysis of eosinophils treated with the hNECs supernatant are shown (40× magnification); VD was dissolved in DMSO for use; * P < 0.05; ** P < 0.01

Fig. 8.

VD inhibits eosinophil chemotaxis in chronic rhinosinusitis with nasal polyps. VD, vitamin D; EOS, eosinophil

Discussion

VD deficiency is common in patients with chronic inflammatory diseases [32, 33]. We compared serum 25(OH)D levels between patients with CRSwNP and controls. The serum 25(OH)D levels in patients with ECRSwNP and non-ECRSwNP were remarkably lower than those in the control group. Serum 25(OH)D levels were negatively correlated with the visual analogue scale score and Sino-Nasal Outcome Test-22 score, suggesting their potential as a marker of CRSwNP severity. In this study, 1,25(OH)2D expression was lower in the nasal mucosa of patients with ECRSwNP than in those without ECRSwNP. VD deficiency promotes eosinophil infiltration and aggravates oesophageal inflammation [15]. We found that 1,25(OH)2D levels were negatively correlated with eosinophil infiltration in patients with CRSwNP, and VD supplementation alleviated the inflammatory reactions within the nasal mucosa of patients with ECRSwNP, suggesting that VD could inhibit inflammatory reactions related to eosinophils. Moreover, VD inhibited eosinophil chemotaxis in vitro. Therefore, VD may alleviate eosinophilic infiltration by inhibiting eosinophil chemotaxis; however, the specific mechanism remains unclear.

hNECs are physical barriers in the airway, and disrupting the epithelial barrier is vital for ECRSwNP pathogenesis [7]. hNECs secrete several eosinophil chemokines and type 2-related cytokines to recruit eosinophils [8]. In the present study, CCL26 and TSLP mRNA levels in patients with ECRSwNP dramatically increased compared to those in patients without ECRSwNP and were positively correlated with ECP expression. Moreover, compared to CCL24 and CCL11, the increase in CCL26 mRNA levels was higher in patients with ECRSwNP compared with those of the non-ECRSwNP group [33]. Notably, these three chemokines directly recruit eosinophils. CCL26 possesses a higher efficiency for eosinophil chemotaxis than CCL11 and CCL24 in patients with asthma and can persistently induce eosinophil migration [28, 35]. Furthermore, CCL26 directly recruits eosinophils in vitro, whereas TSLP regulates eosinophil recruitment indirectly via cytokines secreted by interstitial immune cells [35]. In this study, CCL26 directly drove eosinophil chemotaxis while TSLP failed to do so. And VD effectively reduced CCL26 secretion by hNECs and inhibited eosinophil recruitment in vitro. Therefore, VD disrupts eosinophil chemotaxis by suppressing CCL26 production in hNECs.

Abnormal glucose metabolism is associated with various airway inflammatory responses [36–38]. Glucose concentration in the airway surface fluid dramatically increases in patients with CRS. Additionally, enhanced glucose concentration in nasal secretions facilitates glucose uptake and glycolysis, thereby upregulating IL-1α, and IL-1β production in hNECs [39]. Blocking glycolysis in hNECs inhibits the production of these cytokines. Furthermore, enhanced glycolysis promotes the release of inflammatory factors from airway epithelial cells, whereas inflammation is alleviated after blocking glycolysis [40]. However, the role of glycolysis in driving eosinophilic inflammation remains to be investigated. In this study, glycolysis was upregulated in ECRSwNP tissues. Based on the KEGG analysis, glycolysis is a potential target of VD. Furthermore, the levels of key glycolytic enzymes and lactate products in the nasal tissues were higher in the ECRSwNP group than in the non-ECRSwNP group. The expression of key glycolytic enzymes was positively correlated with tissue eosinophil infiltration. Glycolytic blockade downregulated CCL26 protein levels in hNECs, implying that glycolysis may be involved in eosinophil infiltration into the nasal mucosa of patients with CRSwNP through CCL26 regulation.

Abnormal IL-17 A response was implicated in the pathogenesis of many disorders, making it the most studied member in IL-17 family [41]. According to our previous research, CD8 + cytotoxic T lymphocytes were major IL-17 A producers in nasal tissues of CRSwNP [42]. In addition, we also found that IL-17 A was also expressed in nasal mucosal epithelial cells [23]. For CRS, IL-17 A levels in the type 2 inflammatory-dominant cluster module are markedly increased in the nasal mucosa of patients with CRS [43, 44]. Previously, we observed that IL-17 A was highly expressed in epithelial cells and participated in the inflammatory response of sinusitis through the ERK-pyroptosis axis [23]. And IL-17 A facilitated the type 2 immune response in a CRS mouse model to increase eosinophil infiltration in the nasal mucosa [30]. Furthermore, IL-17 A promotes epithelial damage repair by regulating glycolysis [31]. Nevertheless, these studies failed to elucidate the precise function of IL-17 A in eosinophilic inflammatory responses. In this study, we uncovered the function of IL-17 A in promoting eosinophil migration, which might be responsible for the intensified eosinophil inflammatory responses following IL-17 A treatment [30]. In this study, IL-17 A stimulation of hNECs promoted key glycolytic enzyme expression, lactate production, and CCL26 expression. The supernatant from hNECs treated with IL-17 A contributed to eosinophil migration. However, glycolysis inhibitors counteracted the effects of IL-17 A on CCL26 expression and eosinophil chemotaxis. VD also inhibited IL-17 A-induced glycolysis, CCL26 expression, and eosinophil chemotaxis. Recent studies demonstrated that lactate engaged in disease development by regulating essential transcriptional activity, modulating protein function and protein stability [45, 46]. Our research suggested that lactate enhanced CCL26 stability. Moreover, VD reduced lactate production, contributing to the decline of CCL26 expression in hNECs.

The ERK pathway is vital for transmitting extracellular signals to the nucleus [47], which is associated with eosinophilic inflammation and eosinophil migration [48]. Blocking the ERK pathway reduces eosinophil infiltration into the mouse oesophageal mucosa [49]. It’s reported that 15-Lipoxygenase 1 regulated CCL26 expression through ERK pathway [50]. In this study, p-ERK expression was upregulated in the nasal epithelium of patients with ECRSwNP compared to that in patients without ECRSwNP. ERK pathway activation could promote eosinophil chemotaxis by upregulating CCL26 expression in hNECs. Further investigation showed that inhibition of glycolysis inactivated ERK pathway, suggesting that glycolysis regulated eosinophilic chemotaxis was partially attributable to ERK pathway.

However, some limitations remain to be tackled. First, the limited number of clinical cases involving steroid therapy combined with VD makes it challenging to determine whether VD reduces inflammation via glucocorticoid sensitization or the function of VD to inhibit eosinophil chemotaxis on its own. Also, VDR is expressed in diverse cells including stromal immune cells, making it possible that VD might attenuate inflammation by targeting these inflammatory cells directly. Second, TSLP could exacerbate eosinophil inflammation via stromal immune cells. Whether VD diminishes inflammation via disturbing TSLP signal transduction deserves to be studied. Moreover, lactate enables the transcriptional expression of target genes through lactylation [51, 52], which needs to be investigated further. Additionally, employing an air-liquid interface culture is needed to enhance the persuasive power of relative findings in the future.

Conclusions

VD inhibits eosinophil chemotaxis by downregulating glycolysis and ERK pathway, suggesting the potential inhibitory effect of VD on eosinophil inflammation. This study also uncovers the crucial role of IL-17 A in exacerbating eosinophil inflammatory responses However, further comprehensive in vitro experiments and clinical data are needed to provide reliable evidence for VD-assisted clinical treatment of ECRSwNP to enhance curative effects in the future.

Electronic supplementary material

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Abbreviations

CRS

Chronic rhinosinusitis

CRSwNP

Chronic rhinosinusitis with nasal polyps

ECRSwNP

Eosinophilic chronic rhinosinusitis with nasal polyps

hNECs

Human nasal epithelial cells

TSLP

Thymic stromal lymphopoietin

VD

Vitamin D

1,25(OH)2D

1,25-dihydroxyvitamin D

25(OH)D

25-hydroxyvitamin D

ECP

Eosinophil cationic protein

EOS

Eosinophil

GSEA

Gene Set Enrichment Analysis

HK2

Hexokinase-2

PFKL

Phosphofructokinase, liver type

PKM2

Pyruvate kinase M2

ERK

Extracellular signal-regulated kinase

INCS

Intranasal corticosteroids

OCS

Oral corticosteroids

VAS

Visual analog scale

SNOT-22

Sino-Nasal Outcome Test-22

2-DG

2-deoxy-D-glucose

tBHQ

Tert-butylhydroquinone

KEGG

Kyoto Encyclopedia of Genes and Genomes

Author contributions

Weiqiang Huang and Yue Li performed the most of experiments, and Weiqiang Huang drafted the article. Xia Li, Zizhen Huang, Xifu Wu and Xiaoping Lai collected human nasal mucosa samples. Junming Ma, Yanjie jiang, Jianqi Wang, Xiaohong Chen, Haotian Wu and Donglin Li helped culture human nasal epithelial cells. Yana Zhang, Lihong Chang and Gehua Zhang contributed to manuscript revision. Gehua Zhang provided major funding support. All authors approved the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (82271145, 82071020, 82101194, 82371121), the Natural Science Foundation of Guangdong Province (2023A1515012430), the Science and Technology Planning Project of Guangzhou (202002030034, 202102010167, 2023A03J0208).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. Informed consent was obtained from all patients.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.