IL-4 triggers salivary duct dilatation via SHH signaling and exacerbates Sjögren’s disease

SUMMARY

Primary Sjögren’s disease (pSjD) is a chronic autoimmune disease. Clinically, sialography and lip gland biopsy in patients with pSjD show characteristic ductal dilations. However, the roles of the immune responses in ductal dilation remain unknown. We show that Th2 cells and their core cytokine IL-4 promote salivary duct dilatation in human and experimental SjD. Specifically, striated duct dilation is accompanied by periductal lymphocyte infiltration, which is correlated with increased IL-4 levels. In vivo, IL-4 neutralization reduced ductal dilation. Mechanistically, IL-4 induces the formation of cyst-like structures in cultured embryonic submandibular glands of mice. At the molecular level, IL-4 activates SHH signaling pathway in striated duct epithelial cells, upregulating SNAI1 and suppressing Cadherin 1 expression. This process disrupts interepithelial adhesion, leading to ductal dilation. Thus, IL-4 drives salivary gland ductal dilation that interferes with salivary gland function in SjD. Our findings should have implications for a potential therapeutic target in clinical pSjD.

In brief

Zhang et al. demonstrate that type 2 immune response is a key driver of salivary duct dilatation, a hallmark of gland dysfunction in both experimental and clinical contexts of Sjögren’s disease.

Graphical abstract

INTRODUCTION

Sjögren’s disease (SjD), formerly known as Sjögren’s syndrome (SS) or Sicca syndrome, is a chronic autoimmune disease, with >90% of patients being women.¹ Primary SjD (pSjD) is characterized by destruction of exocrine glands, mainly salivary and lacrimal glands, with a high prevalence of oral and ocular dryness, affecting up to 90% of patients. Clinical symptoms include difficulty chewing or swallowing, pain, taste disturbances, and challenges in speaking for extended periods due to reduced saliva production. Additionally, 30%–50% of patients may experience intermittent inflammatory swelling of the major salivary and lacrimal glands, which significantly impacts their quality of life.^2,3 The impairment of salivary gland function in pSjD, resulting in decreased saliva secretion, is a multifactorial process involving genetic susceptibility, dysregulated immune responses, and glandular destruction and dysfunction.^4,5

A key diagnostic feature of pSjD is periductal focal lymphocytic sialadenitis (FLS) in the salivary glands, characterized by focal mononuclear cell infiltrates around ducts or acini.⁶ Additionally, lymphoepithelial lesions (LELs) are observed, where salivary gland ductal epithelial cells are surrounded by CD4⁺ T cells and B cells, with proliferating ductal cells present within the epithelial lining.⁶ However, the extent of lymphocytic infiltration in the salivary glands of patients with pSjD shows only a weak correlation with glandular dysfunction, suggesting that epithelial cell-specific defects, beyond lymphocyte infiltration, are crucial in salivary gland dysfunction. This indicates a cross-talk between epithelial cells and immune cells for the pathogenesis of pSjD.^7–9

Sialography is one of the auxiliary tools for diagnosing pSjD, categorizing disease severity based on the extent of ductal dilation, which ranges from punctate to globular dilation, cavitary formations, and glandular destruction.¹⁰ While salivary gland dysfunction is closely associated with ductal dilation, the mechanisms driving this dilation in pSjD remain unclear. Furthermore, the involvement of immune cells in ductal dilation and their interaction with ductal epithelial cells remain unknown.

It has been suggested that Th1/Th2 cell imbalance plays a role in the pathogenesis of SjD.^11,12 Previous studies have shown that pSjD is associated with abnormal Th1 activation and inflammatory cell infiltration.¹¹ In addition, Th2-related marker transcripts are present in the labial glands of patients with pSjD with severe B cell infiltration and germinal center (GC) formation, suggesting the involvement of both cell types in the disease.^11,13 Th2 cells primarily secrete cytokines IL-4, IL-5, and IL-13, with IL-4 levels being elevated in the salivary glands of patients with pSjD, especially those with marked B cell infiltration,^12,14–16 suggesting a potential role in the disease progression.

NOD.B10-H2^b(NOD.B10) mouse model is widely used for studying pSjD due to the morphological similarities between the submandibular glands (SMGs) of NOD mice and patients with pSjD.^17–19 Studies have shown that knockout of IL-4 in NOD.B10 mice can significantly restore salivary gland function, even in the presence of pSjD-like glandular lymphocytic infiltration.²⁰ The IL-4/STAT6 signaling pathway is also essential for the progression of pSjD-like disease, as Stat6-knockout mice do not develop glandular dysfunction.²¹ The presence of CD4⁺, CD8⁺ T cells, B cells, and the secretion of IL-4 were also detected in lymphocyte infiltration areas in the glandular tissue of other SjD models such as Id3^−/− mice.²² However, whether IL-4 is linked to ductal dilation remains unknown.

In this study, we show that IL-4 activates SHH signaling, which inhibits CDH1, resulting in ductal dilation in pSjD. Our findings could provide a theoretical basis for potential therapeutic approaches to alleviate salivary gland dysfunction in patients with pSjD.

RESULTS

IL-4 induces salivary gland ductal dilation in SjD

Histological examination of lip gland biopsy samples using hematoxylin and eosin (HE) staining from patients with advanced pSjD (Table S1) revealed FLS with notable lymphocytic infiltration. The luminal diameter of striated ducts was significantly enlarged in regions with lymphocytic infiltration compared to non-infiltrated areas, suggesting a correlation between lymphocytic infiltration and ductal dilation. Furthermore, the focus score showed a positive correlation with the diameter of the dilated ductus in this region (Figure 1A). Alcian blue-periodic acid-Schiff (AB-PAS) staining indicated that the secretory function of the duct was impaired in lymphocyte-infiltrated regions (Figure S1A).

Figure 1. IL-4 induces salivary gland ductal dilation in Sjögren’s disease.

(A) Representative H&E staining of labial gland slices from patients with pSjD. Arrowheads show the dilated striated duct. Scale bars, 100 μm (upper) and 50 μm (lower). Dashed boxes indicate magnified regions. Quantification of striated duct cross-sectional diameters and correlation analysis between focus score and ductal diameter in the corresponding region (right).

(B) Gene expression enrichment analysis of type II immune activation and IL-4 pathway activation in minor salivary glands from patients with pSjD compared to normal individuals.

(C) Representative immunofluorescence staining and fluorescence intensity measurement for KRT19 and IL-4 in labial glands slices from patients with pSjD. Dashed boxes indicate magnified regions. Scale bar, 50 μm.

(D) Representative H&E staining and luminal diameter measurements of striated ducts of submandibular gland (SMG) slices from mice in indicated groups. Arrowheads showing striated ducts. Dashed boxes indicate magnified regions. Scale bars, 200 μm (upper) and 50 μm (lower). Quantification of striated duct cross-sectional diameters and correlation analysis between focus score and ductal diameter in the corresponding region (right).

(E) Representative immunofluorescence staining and fluorescence intensity measurement for KRT19 and IL-4 in SMG from mice in indicated groups. Dashed boxes indicate magnified regions. Scale bar, 50 μm.

(F) RT-qPCR analysis of IL-4 pathway-related gene expression in indicated groups. The bars in graphs represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To investigate the role of lymphocytes in ductal dilation and identify the involvement of specific cell subsets, we conducted enrichment analysis on gene expression profiles of minor salivary glands from patients with pSjD and healthy individuals using the GEO dataset (GSE157159). The results indicated elevated type II immune signaling (Th2) and the activation of IL-4 pathway in labial glands in patients with pSjD (Figures 1B and S1B; Table S2). Subsequent immunofluorescence staining for IL-4 and KRT19 in labial glands in patients with pSjD showed significant IL-4 expression in the epithelium of dilated ducts within lymphocyte-infiltrated regions compared to non-lymphocyte-infiltrated areas (Figure 1C). This suggests that IL-4 plays a role in both lymphocyte infiltration and/or ductal dilation.

In 12-week-old NOD.B10 mice, we observed FLS and LELs in the SMGs. Striated ducts in lymphocyte-infiltrated regions exhibited dilation, whereas non-infiltrated areas did not (Figure 1D). The diameters of striated duct cross-sections were positively correlated with lymphocyte infiltration in the SMGs of NOD.B10 mice compared to C57BL/10 controls (Figure 1D). Treatment of NOD.B10 mice with the IL-4 neutralizing antibody resulted in reduced lymphocyte infiltration and ductal dilation, along with a significant improvement in salivary flow (Figures 1D and S1C). IL-4 neutralizing antibody treatment significantly reduced serum autoantibody levels in mice (Figure S1D).

We subsequently employed Masson’s trichrome staining to determine whether the observed ductal dilation was secondary to proximal fibrotic stenosis. Results showed no significant proximal fibrotic stenosis to the dilated lumens, indicating that the ductal dilation was not attributable to proximal fibrotic stenosis (Figure S1E). Immunofluorescence staining for KRT19 and IL-4 demonstrated elevated IL-4 expression in the dilated ductal epithelium of inflamed SMGs in 12-week-old NOD.B10 mice, and IL-4 was returned to control levels following the antibody treatment (Figure 1E). These findings suggest that IL-4 influences ductal morphology in pSjD. Immunofluorescence staining revealed that a subset of lymphocytes within the inflammatory foci in human labial gland and NOD.B10 mouse salivary gland samples co-expressed CD4 and IL-4. In contrast, IL-4 neutralizing antibody treatment led to a reduction in both the proportion of CD4⁺ cells and the number of CD4⁺ cells expressing IL-4 in the salivary glands of treated mice (Figure S1F). These findings suggested that CD4⁺ T cells were a potential source of IL-4 in pSjD, although we cannot fully exclude contributions from other cellular sources such as type 2 innate lymphoid cells (ILC2s) and macrophages.

Analysis of IL-4 receptor (IL-4R) with immunofluorescence staining revealed that it is located primarily in the ductal and some acinar epithelium in both human lip glands and in NOD.B10 SMGs (Figure S1G). We thus hypothesized that IL-4, which is produced by infiltrating T cells, signals through IL-4R in the ductal epithelium to influence ductal structure. Indeed, we determined an elevated expression of IL-4, IL-4R, and key transcriptional activators Stat6 and Jak1 in the lymphocyte-infiltrated ductal dilation areas compared to non-infiltration areas. Anti-IL-4 neutralization treatment significantly reduced the expression of aforementioned genes (Figure 1F). The results indicate that IL-4 signaling activation in the salivary glands in experimental and clinical pSjD.

Next, we additionally utilized an autoimmunization-induced experimental SjD model (ESS). The ESS model demonstrated a decreased salivary flow rate and dilation of the interlobular striated ducts (Figures S1H and S1I), accompanied by increased IL-4, IL-4R, and CD4 expression (Figures S1J and S1K).

IL-4 promotes salivary gland ductal dilation through the SHH signaling pathway

To understand the role and mechanisms of IL-4 in ductal dilation, we cultured SMGs from embryonic day 14.5 (E14.5) C57BL/6 mice, a critical stage for duct formation, with Th1 and Th2 cytokines, specifically interferon-gamma (IFN-γ) and IL-4, respectively. We found that significant ductal dilation occurred exclusively in the IL-4-treated group but not in the IFN-γ-treated or control groups (Figure 2A; S2A). Given that Sonic hedgehog (SHH) signaling regulates ductal dilation, we included Cyclopamine (Cyc), an inhibitor of the SHH pathway, into the cell culture and found that Cyc reversed IL-4-induced dilation; moreover, this effect occurred in a dose-dependent manner (Figure 2A; S2B).

Figure 2. IL-4 promotes salivary gland ductal dilation through the SHH signaling pathway.

(A) Representative morphogenesis and H&E staining of in vitro cultured E14.5 C57BL/6 mouse submandibular glands for 3 d in indicated groups (n = 5 per group). Dashed boxes indicate magnified regions, and arrowheads show salivary gland ducts. Scale bars, 200 μm (upper) and 50 μm (lower). Quantification of cross-sectional diameters of salivary gland ducts in embryonic organoid cultures (right).

(B) Representative immunofluorescence staining and fluorescence intensity measurements for SHH and GLI1 in embryonic salivary glands cultured in vitro. Dashed boxes indicate magnified regions. Scale bar, 50 μm.

(C) Representative immunofluorescence staining and fluorescence intensity measurements for SHH and GLI1 in labial glands from patients with pSjD and SMG from mice in indicated groups. Dashed boxes indicate magnified regions. Nuclear expression (white arrows). Scale bar, 50 μm.

(D) RT-qPCR analysis of SHH pathway gene expression (Shh, Smo, Ptch1, and Gli1) in the SMG from mice in indicated groups. The bars in graphs represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

In addition, immunofluorescence analysis demonstrated elevated expression of SHH and the transcription factor GLI1 in the IL-4-treated ducts, and their expression was reversed by Cyc treatment (Figures 2B and S2C). These findings indicate that IL-4 induces ductal dilation through SHH pathway.

We next determined that in the SMGs from NOD.B10 mice, the expression of SHH and GLI1 was substantially increased in the lymphocyte-infiltrated areas of the dilated ducts, and the expression was reduced back to normal levels following anti-IL-4 antibody treatment (Figures 2C and S2D). Importantly, the expression of SHH and GLI1 was also increased in the lip glands from patients with pSjD (Figure 2C). We further analyzed a publicly available RNA sequencing dataset of labial glands from patients with SjD (GSE157159) using GSEA; however, no significant upregulation of the SHH signaling pathway was detected in SjD. This finding may be attributed to the inherent limitation of bulk RNA sequencing, which cannot specifically resolve SHH signaling changes localized to the dilated ducts (Figure S2E). RT-qPCR analysis for SHH signaling genes in SMGs from 12-week-old NOD.B10 mice demonstrated upregulated expression of Shh, Smo, Gli1, while Ptch1 expression was downregulated in regions exhibiting lymphocyte infiltration and ductal dilation (Figure 2D).

Using the human salivary gland epithelial cell line A-253 derived from ductal epithelium, we confirmed the effects of IL-4 on the activation of the SHH pathway. We showed that IL-4 significantly enhanced the expression of SHH, resulting in elevated GLI1 expression, which was inhibited by Cyc treatment (Figures 3A and 3B). Comparative analysis revealed SHH signaling pathway activation was not detected in either IFN-γ-treated or control groups (Figure S3A). Consequently, IL-4 significantly increased the expression of Jak1, Jak2, and Stat6, as well as Shh pathway-related genes Shh, Smo, Gli1, and Gli2, and decreased the expression of Ptch1 (Figure 3C). Following Cyc treatment, the SHH signaling pathway was significantly suppressed in the IL-4-treated group, whereas no detectable inhibition was observed in either IFN-γ-treated or control groups (Figure 3C). Interestingly, other Th2 cytokines IL-5 and IL-13 did not affect the SHH pathway-related genes (Figure S3B), suggesting a specific function of IL-4 on duct dilation. The data collectively provide compelling evidence that IL-4 activation of the SHH pathway plays a key role in ductal dilation in SjD.

Figure 3. IL-4 activates SHH transcription factor GLI1 nuclear translocation in A-253 cells.

(A) Representative immunofluorescence staining and fluorescence intensity measurements for SHH and GLI1 of in vitro cultured A-253 cells in the indicated groups. Dashed boxes indicate magnified regions. Scale bar, 50 μm.

(B) Western blot analyses of SHH and GLI1 in A-253 cells.

(C) RT-qPCR analysis of IL-4 receptors (Il-4ra and Il-13ra1), downstream transcription activators (Jak1, Jak2, and Stat6), and SHH pathway-related gene expression (Shh, Smo, Ptch1, Gli1, and Gli2) in A-253 cells in indicated groups. The bars in graphs represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

IL-4 downregulates CDH1 via Sonic Hedgehog to expand salivary duct

We next investigated the underlying mechanisms by which IL-4 regulates salivary duct expansion. The precise role of GLI1 in modulating epithelial cell alignment within salivary ducts, leading to abnormal cell arrangement and duct expansion, remains unclear. GLI1 is reported to negatively regulate CDH1, a key cell adhesion protein that, in coordination with F-actin, preserves ductal lumen integrity. We thus examined the expression of CDH1 and its upstream inhibitor SNAI1 and F-actin in the salivary glands of patients with pSjD. Immunofluorescence staining revealed an elevated SNAI1 but reduced CDH1 and F-actin expression in the expanded ductal epithelium within the lymphocyte infiltration centers in the lip glands of patients with pSjD, suggesting a loss of cell-cell adhesion. The expression of CDH1 and F-actin revealed disrupted cellular structure within these expanded ducts (Figures 4A and 4B). We also analyzed a publicly available RNA sequencing dataset of labial glands from patients with SjD (GSE157159). GSEA revealed significant downregulation of the epithelial cell adherens junction signaling pathway. The expression patterns of Snai1 and Cdh1 were consistent with our findings (Figure S2E). Similarly, in the SMGs of 12-week-old NOD.B10 mice, areas of lymphocyte infiltration displayed reduced CDH1 and F-actin expression compared to controls, while the ducts lacking lymphocyte infiltration showed no significant changes in SNAI1, CDH1, and F-actin expression (Figure S4A). Importantly, following IL-4 neutralization with its specific antibody, the expression of CDH1 and F-actin was restored in the ductal epithelium in the SMG of NOD.B10 mice, accompanied by reduced ductal expansion (Figures 4A and 4B). RT-qPCR analysis of cell-cell adhesion-related genes in SMGs in NOD.B10 mice revealed elevated expression of Snai1 but reduced expression of Cdh1 in the lymphocyte infiltration and ductal dilation areas at 12 weeks of age (Figure 4C). The neutralization of IL-4 reduced the aforementioned elevated genes in NOD.B10 mice. The data suggest that IL-4 suppresses Cdh1 expression, disrupting normal ductal cell adhesion in the SjD (Figure 4C).

Figure 4. IL-4 decreases CDH1 expression in dilated ductal epithelium in pSjD.

(A) Representative immunofluorescence staining and fluorescence intensity measurements for CDH1 and SNAI1 in labial glands of patients with pSjD and SMG from mice in indicated groups. Dashed boxes indicate magnified regions. Scale bar, 50 μm.

(B) Representative immunofluorescence staining and fluorescence intensity measurement for CDH1 and F-actin in labial glands of patients with pSjD and SMG from mice in indicated groups. Dashed boxes indicate magnified regions. Scale bar, 50 μm.

(C) RT-qPCR analysis of intercellular adhesion-related gene expression (Cdh1 and Snai1) in the SMG from mice in indicated groups. The bars in graphs represent mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001.

We next utilized a different approach to assess the expression of CDH1, SNAI1, and F-actin in embryonic salivary glands in culture. We observed that IL-4 treatment significantly increased SNAI1 expression in the abnormally expanded ducts, accompanied by a marked reduction in both CDH1 and F-actin expression compared to the untreated control groups (Figures 5A and 5B). Addition of Cyc, an SHH pathway inhibitor, to the IL-4-treated cells reversed the abnormal ductal expansion and restored CDH1 and F-actin expression (Figures 5A and 5B). Immunofluorescence quantification demonstrated no significant change in SNAI1, CDH1 expression levels, nor in F-actin cytoskeletal organization in either IFN-γ-treated or control groups following Cyc administration, highlighting Cyc’s selective inhibitory effects specific to IL-4-driven morphogenesis (Figure S4B). These findings indicate that IL-4 activates the SHH signaling pathway, leading to increased SNAI1 expression. SNAI1, in turn, downregulates CDH1, which disrupts cell adhesion within the ductal epithelium, resulting in ductal expansion.

Figure 5. IL-4 downregulates CDH1 via Sonic hedgehog to expand salivary duct and affect cell-cell adhesion junctions.

(A) Representative immunofluorescence staining and fluorescence intensity measurements for SNAI1 and CDH1 expression in embryonic salivary glands cultured in vitro under indicated conditions. Dashed boxes indicate magnified regions. Scale bar, 50 μm.

(B) Representative immunofluorescence staining and fluorescence intensity measurement for F-actin and CDH1 expression in embryonic salivary glands cultured in vitro under indicated conditions. Dashed boxes indicate magnified regions. Scale bar, 50 μm.

(C) Representative immunofluorescence staining and fluorescence intensity measurements for CDH1 expression of in vitro cultured A-253 cells under indicated conditions. Dashed boxes indicate magnified regions. Scale bar, 50 μm.

(D) Western blot analyses of SNAI1 and CDH1 in A-253 cells.

(E) RT-qPCR analysis of Cdh1 and Snai1 expression of in vitro cultured A-253 cells under indicated conditions. The bars in graphs represent mean ± SD. **p < 0.01, ****p < 0.0001.

In a 2D A-253 cell culture system, we further confirmed the function of IL-4 in CDH1 expression through the SHH pathway. Immunofluorescence staining revealed reduced CDH1 expression in IL-4-treated cells, especially in the areas where the expression of CDH1 was absent, suggesting compromised cell adhesion. Cyc treatment restored CDH1 expression to levels comparable to the controls (Figure 5C). In addition, Western blot and RT-qPCR showed that IL-4 treatment significantly downregulated CDH1 expression while upregulating SNAI1 expression, and the addition of Cyc restored the expression levels of both. IFN-γ treatment had no significant effect on the expression of Cdh1 and Snai1 (Figures 5D and 5E).

Th2 cells promote ductal expansion in SMGs

To investigate the source of IL-4 in pSjD, we injected Th1 and Th2 cells into nude mice to simulate type I and type II immune environments (Figure 6A). Histological analysis of the SMGs from both control and Th1-injected groups showed no significant structural alterations. In contrast, SMGs from the Th2-treated group displayed lymphocytic infiltrates and ductal expansion, along with loss of acinar structures, resembling pSjD pathology (Figure 6B). Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining indicated a notable reduction in secretion within the Th2 group compared to controls (Figure 6C). Immunofluorescence staining further demonstrated that AQP5 expression was significantly reduced in the Th2-treated group compared with the other two groups, which closely resembled the pathological changes observed in pSjD (Figure S4C).

Figure 6. Th2 cells promote ductal expansion in SMGs through SHH activation and CDH1 reduction.

(A) Schematic of T cell injection workflow in nude mice with five experimental replicates per group.

(B) Representative H&E staining of SMG slices from nude mice in indicated groups. Scale bars, 100 μm (upper) and 50 μm (lower).

(C) Representative AB-PAS staining of SMG slices from nude mice in indicated groups. Dashed boxes indicate magnified regions. Scale bars, 100 μm (upper) and 50 μm (lower).

(D) Representative immunofluorescence staining and fluorescence intensity measurements for KRT19, IL-4, SHH, GLI1, SNAI1, CDH1, and F-actin in the SMG from nude mice in indicated groups. Dashed boxes indicate magnified regions. Scale bar = 50 μm.

(E) Western blot analyses of IL-4, SHH, GLI1, SNAI1, and CDH1 in the SMG of nude mice. The bars in graphs represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Immunofluorescence analysis showed significantly elevated IL-4 expression in the Th2 group epithelium, while no notable differences were observed in Th1 and control groups. Additionally, Th2 cell-treated SMGs demonstrated increased expression of SHH, GLI1, and SNAI1 and reduced expression of CDH1 and F-actin in the expanded ductal epithelium (Figure 6D), consistent with staining results in pSjD. Western blot analysis showed that the expressions of IL-4, SHH, GLI1, and SNAI1 in the Th2 treatment group were higher than those in the control group and the Th1 treatment group, while the expression of CDH1 was downregulated (Figure 6E). These data indicate that Th2 cells are an important source of IL-4 in SHH-mediated abnormal duct dilation in the salivary glands in pSjD.

DISCUSSION

Characteristic findings from labial and parotid gland biopsies in both experimental and clinical SjD reveal lymphocytic infiltrations surrounding striated ducts, consisting of both B and T lymphocytes.²³ Despite the distinctive morphological changes in salivary gland ducts of patients with SjD, the precise roles of immune cells remain poorly understood.^24–27 Here, we found that ductal dilation within the lymphoid invasion foci is linked to IL-4-producing Th2 cell immunity, which induces ductal dysfunction. RNA-seq analysis of salivary glands from patients with SjD reveals an enrichment of type II immune pathways, characterized by increased levels of Th2 cells, eosinophils, mast cells, basophils, and macrophages and elevated IL-4 levels compared to healthy individuals.^13,28,29 Notably, there is ongoing debate about whether the sialography features of SjD-specific terminal duct dilatation, including punctate dilation, globular dilation, cavitary formations, and glandular destruction, correlate with pathological striate duct dilatation.^30,31 However, previous studies have shown partial restoration of salivary gland function in IL-4 or STAT6 knockout NOD.B10 mice, and our findings provide insights into the potential mechanisms underlying this phenomenon.^20,21

This study uncovered that Th2 cells and their cytokine IL-4 activate the SHH/GLI1-SNAI1 axis, suppressing CDH1 expression and inducing salivary gland ductal dilation and functional impairment. Elevated levels of IL-4, SHH, and GLI1, along with reduced CDH1 expression around dilated ducts in NOD.B10 mice, and sequencing data from SjD labial glands further support this conclusion. However, the precise mechanisms by which the type II immune-activated SHH pathway induces SNAI1 and suppresses CDH1, as well as how reduced CDH1 leads to ductal dilation, remain to be elucidated.^32–36 Additionally, the relationship between abnormal ductal dilation and salivary gland dysfunction requires further investigation. Although we have clearly demonstrated here a critical role of Th2-producing IL-4 in the duct dilation in the SjD, the function and contribution of other IL-4-producing cells, such as innate lymphoid cell type 2 (ILC2) and other cells, and their crosstalk to Th2 cells remain to be investigated.³⁷

The impact of abnormal ductal dilatation on salivary gland function remains unclear. Under normal conditions, intercalated and striated ducts serve as key niches for salivary gland stem/progenitor cells (SGPCs), which proliferate and differentiate into new acinar and ductal cells, replacing damaged or senescent cells.³⁸ However, patients with SjD exhibit significantly fewer SGPCs with reduced differentiation capacity.³⁹ Consequently, it remains to be determined whether the abnormal ductal dilation is linked to the fate of ductal stem cells and whether epithelial-lymphocyte crosstalk influences SGPC apoptosis, thereby disrupting salivary gland cell proliferation and differentiation. These potential mechanisms and their effects on glandular function warrant further investigation.

Current treatment options for SjD remain limited. While salivary gland lymphocyte infiltration is reversible, damage to the tissue structure is often irreversible, making SjD challenging to cure.^40,41 Given the early loss of salivary gland function in patients with pSjD and the late-stage development of LELs, the timing of therapeutic intervention is critical.⁸ Our study proposes a strategy targeting type II immunity as a potential treatment for SjD.

Limitations of the study

This study has several limitations. Firstly, technical constraints due to the scarcity of fresh human salivary gland tissue limited high-resolution, multi-modal validation. Consequently, we were unable to directly link epithelial state changes to local immune signaling at single-cell or spatial resolution. Secondly, although our findings indicate IL-4 signaling in promoting ductal dilatation and suggest Th2 cells as a potential in vivo source, contributions from other IL-4-producing populations cannot be excluded. Finally, our study did not address whether the observed ductal dilation is linked to alterations in ductal stem cell fate and how epithelial-lymphocyte crosstalk may influence salivary gland progenitor cell apoptosis and subsequent tissue repair.

RESOURCE AVAILABILITY

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Fu Wang (fuwang@dmu.edu.cn).

Materials availability

All materials, reagents, and transgenic lines generated in this article are available upon request to the lead contact.

Data and code availability

• Public RNA-seq data (GEO: GSE157159) were analyzed in this study. All data generated or analyzed are included in this article and its supplementary information files. The GEO accession number GSE157159 is provided in the key resources table.

• This study did not generate new code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

KEY RESOURCES TABLE.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse monoclonal anti-Cytokeratin 19	Proteintech	Cat# 60187-1-Ig; RRID:AB_10859834
Mouse monoclonal anti-IL-4	Proteintech	Cat# 66142-1-Ig; RRID:AB_2881539
Mouse monoclonal anti-GLI1	Proteintech	Cat# 66905-1-Ig; RRID:AB_2882232
Rabbit polyclonal anti-SHH	Proteintech	Cat# 20697-1-AP; RRID:AB_10694828
Rabbit polyclonal anti-IL-4R	Thermo Fisher Scientific	Cat# PA5-103142; RRID:AB_2852512
Mouse monoclonal anti-F-actin	Abcam	Cat# ab130935; RRID:AB_3696906
Rabbit polyclonal anti-AQP5	SangonBiotech	Cat# D260099-0010
Rabbit polyclonal anti-Cadherin-1	Abmart	Cat# TA0131F
Rabbit recombinant monoclonal anti-CD4	Abcam	Cat# ab288724; RRID:AB_2941893
Mouse monoclonal anti-SNAI1	Abcam	Cat# CL3700
IL-4 neutralizing antibody	BioXcell	Cat# BE0045; RRID:AB 1107707
Goat anti-rat IgG Alexa Fluor Plus 488	Abcam	Cat# ab150115; RRID: AB_2687948
Goat anti-rabbit IgG Alexa Fluor Plus 647	Abcam	Cat# ab150083; RRID: AB_2714032
Goat anti-rabbit IgG Alexa Fluor Plus 488	Abcam	Cat# ab150077; RRID: AB_2630356
Goat anti-mouse IgG Alexa Fluor Plus 488	Abcam	Cat# ab150113; RRID: AB_2576208
Biological samples
Human labial gland biopsy	This study	N/A
Mouse salivary gland tissue	This study	N/A
Chemicals, peptides, and recombinant proteins
Freund’s complete adjuvant	Sigma-Aldrich	Cat#F5881
Freund’s incomplete adjuvant	Sigma-Aldrich	Cat#F5506
Critical commercial assays
Mouse ANA ELISA Kit	ZCIBIO	Cat#ZC-38369
Deposited data
RNA-seq data	Oyelakin et al.29	GSE157159
Experimental models: Cell lines
Human: A-253	ATCC	CRL-7902
Experimental models: Organisms/strains
C57BL/6 mice	Liaoning Changsheng Biotechnology	N/A
C57BL/10 mice	Liaoning Changsheng Biotechnology	N/A
Balb/c mice	Liaoning Changsheng Biotechnology	N/A
Balb/c nude mice	Liaoning Changsheng Biotechnology	N/A
NOD.B10-H2b mice	Liaoning Changsheng Biotechnology	N/A
Oligonucleotides
Primers for RT-qPCR	This study	see Table S3
Software and algorithms
GraphPad Prism 9	GraphPad Software	https://www.graphpad.com/
ImageJ	NIH	https://imagej.net/

STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Mice

The C57BL/6, C57BL/10, Balb/c, Balb/c nude, and NOD.B10-H2b mice used in this study were provided and maintained by the Animal Experimental Center of Dalian Medical University. All mice were housed under specific-pathogen-free (SPF) conditions in individually ventilated cages. They were maintained on a 12-h light/12-h dark cycle. Standard rodent chow and water were provided ad libitum. All animal procedures were approved by the Animal Care and Use Committee of Dalian Medical University (Approval No.: AEE22006) and adhered to ARRIVE guidelines.

The experimental SjD model was established as previously described.^42,43 Briefly, the female C57BL/6 mice at 6 weeks old were immunized with submandibular gland autoantigen emulsified in complete Freund’s adjuvant (400 μg) on days 0 and were boosted with autoantigen emulsified in incomplete Freund’s adjuvant (200 μg) on day 14.

Culture of human salivary gland epithelial cell line A-253

A-253 line, a human salivary gland epithelial cell line obtained from the American Type Culture Collection (ATCC), was authenticated through short tandem repeat (STR) profiling and was negative for mycoplasma contamination prior to experiment. A-253 cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Treatments included 10 ng/mL IFN-γ and 20 ng/mL IL-4 as required. For SHH pathway inhibition, cells were pre-treated with 10 mM cyclopamine (Cyc) for 2 h before IFN-γ and IL-4 exposure. After 3 days of treatment, cells were fixed with 4% paraformaldehyde for 20 min.

METHOD DETAILS

Histological analysis of human lip gland tissue

All paraffin-embedded sections of human lip gland biopsies were obtained from the Stomatological Hospital at Dalian Medical University. The use of these samples was approved by the Ethics Committee of the Affiliated Stomatological Hospital at Dalian Medical University (Approval No.: 2024001). The informed consent was obtained from all subjects. All data were de-identified prior to analysis.

Isolation and sorting of Th cells

Eight-week-old female Balb/c mice were sacrificed to isolate the spleen and peripheral lymph nodes, which were immediately placed in PBS containing 2% antibiotics. After multiple washes with MACS buffer, the lymph nodes and spleens were separately processed into single-cell suspensions. Spleen suspensions were treated with 3 mL of red blood cell lysis buffer at 4°C for 15 min before combining with the lymph node suspensions. Naive CD4⁺ T cells were isolated using the CD4⁺ CD62L + T cell Isolation Kit (Miltenyi). The isolated cells were seeded on 96-well plates pre-coated with anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) antibodies and cultured in DMEM/Low Glucose (1 g/L) supplemented with 10% fetal bovine serum (FBS), 1% HEPES, 1% non-essential amino acids (NEAA), 1% sodium pyruvate, 1% penicillin-streptomycin, 1% L-glutamine, and 0.4% β-mercaptoethanol. Th1 conditions included IL-2 (4 ng/mL) and IL-12 (10 ng/mL), while Th2 conditions included IL-2 (4 ng/mL) and IL-4 (10 ng/mL). Cultures were incubated for three days at 37°C in a 5% CO₂ incubator.

Embryonic submandibular gland 3D culture

Submandibular glands from E14.5 C57BL/6 mouse embryos were isolated and cultured in collagen gel within the upper chamber of a 6-well Transwell plate. The lower chamber contained 900 μL of DMEM/F12 medium supplemented with 10% FBS, 10 μM Y-27632, 10 μg/mL EGF, 10 μg/mL FGF2, and 1% penicillin-streptomycin. The glands were maintained at the air-liquid interface at 37°C in a 5% CO₂ incubator, with the medium changed every two days. Morphological changes were observed and photographed on days 0, 1, 2, and 3. After three days, samples were fixed in 4% paraformaldehyde for 12 h.

IL-4 neutralizing antibody injection in NOD.B10 mice

Five 6-week-old female NOD.B10 mice received intraperitoneal injections of an IL-4 neutralizing antibody (25 μg per injection, BioXcell) every other day until they reached 12 weeks of age. At 12 weeks, the mice were sacrificed, and their submandibular glands were isolated and fixed in 4% paraformaldehyde at 4°C for 24 h for subsequent immunohistochemical analysis.

Measurement of salivary flow rate in mice

Saliva was collected at the same time weekly from 6-week-old C57BL/10 mice, untreated NOD.B10 mice, and NOD.B10 mice treated with an IL-4 neutralizing antibody. Mice were anesthetized with a ketamine (100 mg/mL) and xylazine (20 mg/mL) mixture, followed by an intraperitoneal injection of pilocarpine (5 mg/mL, 2 mL/kg) to stimulate salivation. Saliva was collected for 10 min using a capillary tube.

Intraperitoneal injection of T cells in mice

Fifteen 5-week-old female Balb/c nude immunodeficient mice were randomly assigned to control group, Th1 cell injection group, and Th2 cell injection group (5 mice per group). Th1 and Th2 cells were resuspended in PBS and administered via intraperitoneal injection at a dose of 1×10⁶ cells weekly for 4 weeks, while the control group received an equivalent volume of PBS. The salivary flow rate was monitored weekly.

HE and AB-PAS staining

For hematoxylin and eosin (HE) staining, tissue sections were deparaffinized, rehydrated, stained with hematoxylin and eosin, then dehydrated in graded ethanol, cleared in xylene, and mounted using neutral balsam. For Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining, sections were deparaffinized, rehydrated, and stained with Alcian blue in the dark for 30 min. After rinsing with distilled water, sections were treated with periodic acid solution for 5 min, followed by Schiff reagent for 10–20 min, and finally counterstained with hematoxylin. Sections were then dehydrated, cleared in xylene, and mounted with neutral balsam.

Immunofluorescence staining of A-253 cells

A-253 cell slides were washed in PBS, fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 15 min, and blocked with goat serum for 30 min. Slides were then incubated with primary antibodies overnight at 4°C. Following PBS washes, slides were incubated with fluorescent secondary antibodies for 1 h in the dark and mounted with an antifade medium containing DAPI. The secondary antibodies used were as follows: Goat Anti-mouse IgG H&L conjugated with Alexa Fluor 488 (abcam ab150113), diluted at 1:500; Goat Anti-rat IgG H&L conjugated with Alexa Fluor 488 (abcam ab150115), diluted at 1:500; Goat Anti-rabbit IgG H&L conjugated with Alexa Fluor 488 (abcam ab150077), diluted at 1:500; and Goat Anti-Rabbit IgG H&L (Alexa Fluor 647) preadsorbed(abcam ab150083), diluted at 1:500.

Immunofluorescence staining of paraffin-embedded tissue

Sections were deparaffinized, rehydrated, and subjected to antigen retrieval under pressure. After permeabilization, sections were blocked with goat serum for 1 h, incubated with primary antibodies overnight at 4°C, washed, and incubated with fluorescent secondary antibodies at 37°C for 30 min. Sections were mounted with DAPI-containing antifade mounting medium.

RT-qPCR analysis

Total RNA was extracted using the RNeasy Mini Kit (Qiagen), and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). RT-qPCR was performed using the Bio-Rad Real-Time PCR System, and data were analyzed using the 2 −ΔΔCt method. Primer sequences are listed in Table S3.

Protein extraction and Western Blotting analysis

Total protein lysates were obtained by homogenizing tissues or cells in Laemmli sample buffer. Protein samples were separated by SDS-PAGE using a 12% resolving gel and a 5% stacking gel. A total of 20μg of protein was loaded into each lane. Electrophoresis was performed at a constant voltage of 80 V for the stacking gel and 120 V for the resolving gel until the dye front reached the bottom of the gel. After electrophoresis, the proteins were transferred onto a PVDF membrane for subsequent immunoblot analysis. For immunoblotting, membranes were probed with specific primary antibodies followed by appropriate secondary detection reagents.

ELISA measurement of mouse antinuclear antibody (ANA)

Autoantibodies were detected using the Mouse ANA ELISA Kit (ZC-38369) following the manufacturer’s instructions. Briefly, the target antigen was pre-coated onto the 96-well microplate to form the solid phase. Standards or mouse serum samples were added to the wells, allowing the target autoantibodies to bind to the immobilized antigen. This was followed by the addition of a horseradish peroxidase (HRP)-conjugated detection antibody. After incubation, unbound components were removed by thorough washing. Tetramethylbenzidine (TMB) substrate was then added for color development. The absorbance (O.D.) at 450 nm was measured using a microplate reader, and autoantibody concentrations were calculated accordingly.

Ducts measurement

The luminal diameter of striated ducts was measured using ImageJ software. Measurements were performed on five histological sections, each obtained from a different patient or mouse. For each section, three representative striated ducts were randomly selected in both lymphocytic infiltrated and non-infiltrated regions, resulting in a total of 15 ducts per condition (infiltrated vs. non-infiltrated). Only ducts with a clear and complete transverse profile were included. The average luminal diameter of each duct was calculated from the maximal cross-sectional span.

Public RNA sequencing data analysis

Public RNA-seq data (GSE157159) were downloaded from the Gene Expression Omnibus (GEO). Because GEO provided a curated TPM expression matrix, raw FASTQ files were not reprocessed; instead, the TPM matrix was used directly for all downstream analyses. Low-abundance genes were filtered out, duplicated gene symbols were collapsed by averaging, and expression values were log2-transformed as log2(TPM +1). Differential expression between Sjögren’s syndrome and control samples was assessed using the limma package with Benjamini–Hochberg multiple-testing correction, and genes with FDR <0.05 and |log2FC| > 1 were considered differentially expressed. Pathway activity at the individual-sample level was quantified using single-sample gene set enrichment analysis (ssGSEA) implemented in the GSVA package (method = “ssgsea”), applied to the normalized log2(TPM +1) expression matrix to generate enrichment scores for each gene set in each sample. Differences in ssGSEA scores between groups were evaluated using limma with BH correction. Selected gene expression patterns and ssGSEA enrichment scores were visualized as Z-score–scaled heatmaps generated with the pheatmap package. All analyses were conducted in R (v4.1.1) using in-house scripts for data processing and visualization, without custom algorithms.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis

Data are presented as mean ± SEM. The statistical significance between two groups was calculated by the Two-tailed t test. For multiple groups, significance was evaluated by One-way ANOVA with Bonferroni multiple comparisons test in GraphPad Prism 9.5.0 (CA, USA), with p < 0.05 considered statistically significant.

Supplementary Material

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2026.117132.

Highlights.

• IL-4 induces salivary gland ductal dilation in Sjögren’s disease (SjD)

• IL-4 promotes salivary gland ductal dilation through the SHH signaling pathway

• Activated SHH pathway leads to ductal dilation by upregulating SNAI1 to repress CDH1

• IL-4 derived from Th2 cells plays a key role in SHH-mediated duct dilation in pSjD