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. Author manuscript; available in PMC: 2025 Sep 1.
Published in final edited form as: Mol Immunol. 2024 Jul 16;173:20–29. doi: 10.1016/j.molimm.2024.06.010

Endogenous IL-22 contributes to the pathogenesis of salivary gland dysfunction in the non-obese diabetic model of Sjögren’s syndrome

Fernanda Aragão Felix a,b,d, Jing Zhou a,d, Dongfang Li a, Shoko Onodera c, Qing Yu e
PMCID: PMC11343657  NIHMSID: NIHMS2010371  PMID: 39018744

Abstract

Sjögren’s syndrome is a systemic autoimmune disease primarily targeting the salivary and lacrimal glands. Our previous investigations have shown that administration of interleukin-22 (IL-22), an IL-10 family cytokine known for its complex and context-dependent effects on tissues, either protective- or detrimental, to salivary glands leads to hypofunction and pathological changes of salivary glands in C57BL/6 mice and in non-obese diabetic (NOD) mice, the latter being a commonly used model of Sjögren’s syndrome. This study aims to delineate the pathophysiological roles of endogenously produced IL-22 in the development of salivary gland pathologies and dysfunction associated with Sjögren’s disease in the NOD mouse model. Our results reveal that neutralizing IL-22 offered a protective effect on salivary gland function without significantly affecting the immune cell infiltration of salivary glands or the autoantibody production. Blockade of IL-22 reduced the levels of phosphorylated STAT3 in salivary gland tissues of NOD mice, while its administration to salivary glands had the opposite effect. Correspondingly, the detrimental impact of exogenously applied IL-22 on salivary glands was almost completely abrogated by a specific STAT3 inhibitor. Moreover, IL-22 blockade led to a downregulation of protein amounts of Ten-Eleven-Translocation 2, a methylcytosine dioxygenase critical for mediating interferon-induced responses, in salivary gland epithelial cells. IL-22 neutralization also exerted a protective effect on the salivary gland epithelial cells that express high levels of surface EpCAM and bear the stem cell potential, and IL-22 treatment in vitro hampered the survival/expansion of these salivary gland stem cells, indicating a direct negative impact of IL-22 on these cells. In summary, this study has uncovered a critical pathogenic role of the endogenous IL-22 in the pathogenesis of Sjögren’s disease-characteristic salivary gland dysfunction and provided initial evidence that this effect is dependent on STAT3 activation and potentially achieved through fostering Tet2-mediated interferon responses in salivary gland epithelial cells and negatively affecting the EpCAMhigh salivary gland stem cells.

Keywords: Sjögren’s disease, xerostomia, interferon, Ten-Eleven-Translocation 2, salivary gland stem cells

1. INTRODUCTION

Sjögren’s syndrome (SS) is a chronic systemic autoimmune disease predominantly affecting women and afflicts as many as 0.1% of the population, including 4 million individuals in the U.S. (Chiorini et al., 2009; Jin and Yu, 2013; Lee et al., 2009; Patel and Shahane, 2014; Voulgarelis and Tzioufas, 2010). The disease targets the salivary- and lacrimal glands, impairing their secretory function and causing the hallmark symptoms of dry mouth and dry eyes (Chiorini et al., 2009; Jin and Yu, 2013; Lee et al., 2009; Patel and Shahane, 2014; Voulgarelis and Tzioufas, 2010). Beyond these primary target tissues, SS also affects various other organs, causing a wide range of systemic symptoms (Chiorini et al., 2009; Jin and Yu, 2013; Lee et al., 2009; Patel and Shahane, 2014; Voulgarelis and Tzioufas, 2010). The pathogenic process of SS involves a complex collaboration and interplay of both innate and adaptive immune responses, with various major immune cell subsets and proflammatory mediators they produce, such as type I IFNs, IFNγ, TNFα, IL-4 and IL-17, playing key roles in the initiation, development and persistence of the disease (Baker et al., 2008; Cha et al., 2004; Frasca et al., 2012; Jin et al., 2013; Jin and Yu, 2013; Kang et al., 2011; Kulkarni et al., 2006; Lee et al., 2009; Mitsias et al., 2002; Nguyen and Peck, 2013; Szczerba et al., 2013; Tan et al., 2014; Voulgarelis and Tzioufas, 2010; Youinou and Pers, 2011; Zhou et al., 2017; Zhou and Yu, 2018). Despite significance advancements in the understanding of SS disease in recent years, there is still no effective treatment/biological therapies currently available (Seror et al., 2021).

IL-22, an IL-10 family cytokine, is predominantly produced by immune cells and acts on non-immune cells, especially epithelial cells, keratinocytes and the epithelial stem/progenitor cells across various tissues (Bachmann et al., 2013; Muhl, 2013; Ouyang et al., 2011; Rutz et al., 2013; Rutz et al., 2014; Sabat et al., 2014; Sonnenberg et al., 2010). The actions of IL-22 are multifaceted and highly context-dependent, conferring both tissue-protective and tissue-damaging/proinflammatory outcomes (Bachmannn et al., 2013; Muhl, 2013; Sonnenberg et al., 2010). For instance, IL-22 attenuates ocular inflammatory pathologies in a murine model of dry eye disease (Ji et al., 2017), but promotes the development of a viral-infection-induced sialadenitis in mice by augmenting B cell responses (Barone et al., 2015). IL-22 and IL-22-producing cells are increased in salivary glands and blood of SS patients, positively correlating with the disease severity (Ciccia et al., 2012; Lavoie et al., 2016; Lavoie et al., 2011; Matsui and Sano, 2017; Schinocca et al., 2021; Tan et al., 2014). Moreover, IL-22 receptor is expressed at a higher level in the epithelial- and immune cells within the salivary glands of SS patients compared to the control subjects (Ciccia et al., 2012), suggesting a critical role for IL-22 in the pathogenesis of SS disease. Binding of IL-22 to its receptor, composed of IL-22Rα and IL-10Rβ (Ouyang and O'Garra, 2019; Sabat et al., 2014; Seror et al., 2021; Wolk et al., 2007; Xie et al., 2000), initiates the phosphorylation and activation of the JAK-STAT3 signaling pathway, and in some cases, the JAK-STAT1 pathway (Ouyang and O'Garra, 2019; Sabat et al., 2014; Seror et al., 2021; Wolk et al., 2007; Xie et al., 2000). The prominent tissue-protective and pro-regenerative actions of IL-22 are predominantly attributed to STAT3 activation, whereas its proinflammatory effects are mainly linked to STAT1 activation (Bachmann et al., 2013; Muhl, 2013; Ouyang and O'Garra, 2019; Saxton et al., 2021). Nevertheless, a recent study reveals that STAT3 can also enhance IFN-induced gene expression and augments inflammatory responses in salivary gland epithelial cells from SS patients (Charras et al., 2020), underscoring the complex and context-specific actions of these signaling pathways. The levels of both phosphorylated-STAT1 (pSTAT1) and pSTAT3 are elevated in the salivary glands of SS patients compared to the control subjects (Charras et al., 2020; Ciccia et al., 2015; Ciccia et al., 2012; Gandolfo and Ciccia, 2022; Pertovaara et al., 2016; Wakamatsu et al., 2006), correlating closely with the local IFNs and IL-22 levels, respectively (Ciccia et al., 2015; Ciccia et al., 2012; Gandolfo and Ciccia, 2022; Pertovaara et al., 2016; Wakamatsu et al., 2006). Moreover, IL-22Rα is also found to colocalize with pSTAT3 in salivary glands of SS patients (Ciccia et al., 2012).

Our recent study has revealed that administration of IL-22, which is elevated in SS patients and mouse models, exerts a detrimental impact on salivary gland function and integrity in both the non-obese diabetic (NOD) mouse model of SS disease and the wildtype C57BL/6 mice (Zhou et al., 2022a). We also identified a salivary gland-damaging effect of endogenously produced IL-22 in a model of salivary gland exocrinopathy induced by anti-CD3 antibodies in C57BL/6 mice (Zhou et al., 2022a). In the present study, we sought to further investigate the roles of endogenously derived IL-22 in the pathogenesis of SS-like salivary gland dysfunction and pathologies in the NOD mouse model.

2. MATERIAL and METHODS

2.1. Mice

The non-obese diabetic (NOD) mice (NOD/ShiLtJ strain) and C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed in the specific pathogen-free animal facility at the ADA Forsyth Institute. All the experimental protocols were approved by the Institutional Animal Care and Use Committee of the ADA Forsyth Institute. All the experimental protocols were approved by the Institutional Animal Care and Use Committee of the ADA Forsyth Institute and all the procedures were implemented in compliance with the “Guide for the Care and Use of Laboratory Animals” of the National Institutes of Health and the ARRIVE guidelines.

2.2. Cytokines and Antibodies

Recombinant murine IL-22 was purchased from Peprotech (Cranbury, NJ). Purified monoclonal anti-mouse IL-22 (8E11) and the isotype control, mouse IgG1, used for some of in vivo IL-22 blockade administration were kindly provided by Genentech Inc (South San Francisco, CA). In some experiments, rat anti-mouse IL-22 (IL22JOP, eBioscience) and its isotype control, rat IgG2a (BioXCell), were used. For immunohistochemistry, mouse anti-phosphorylated STAT3 antibody (clone 13A3-1, BioLegend), rabbit anti-Tet2 (212071, Proteintech), rabbit anti-P16 (PA5-119712, Invitrogen) were used. The respective secondary antibodies were purchased from Vector Laboratories. For flow cytometry, fluorescence conjugated antibodies against CD45 (clone 30-F11), CD4 (clone GK1.5), CD8α (clone 536-7), CD19 (clone 1D3), TCR-β (clone H57-597), IL-22 (clone Poly5164), CD16/32 (clone 93), EpCAM (G8.8), Ter119 (clone TER-119), Foxp3 (clone MF-14) were purchased from BioLegend (San Diego, CA), and anti-CD31 (clone 390) from eBioscience (San Diego, CA).

2.3. In Vivo Administration of Recombinant IL-22 and Anti-IL-22 Antibody

To determine the roles of endogenously produced IL-22 in SS development, the neutralizing anti-IL-22 or the isotype control IgG was intraperitoneally injected into 6-weeks-old female NOD mice 3 times per week for 4 consecutive weeks. The tissues were harvested for analyses 2-3 days after the final injection, at 10 weeks of age. To assess the roles of STAT3 activity in IL-22-induction of salivary gland dysfunction, recombinant murine IL-22 (PeproTech) or the control PBS solution was directly injected into submandibular glands (SMGs) of 8 - 9 weeks old female NOD mice at 1 μg/lobe every other day for 3 days, along with the presence or absence of a specific, cell-permeable STAT3 inhibitor peptide (573096, Sigma-Aldrich).

2.4. Measurement of Salivary Flow Rate

Mice were weighed and intraperitoneally injected with 100 μL PBS-based secretagogue solution containing isoproterenol (1 mg/mL) and pilocarpine (2 mg/mL). One min after secretagogue injection, saliva was collected continuously for 5 min from the oral cavity of mice with a micropipette. The volume of saliva was measured and normalized to the body weight.

2.5. Immunohistochemistry and assessment of leukocyte infiltration of submandibular glands (SMGs)

Freshly collected SMG tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to 5 μm thickness. The sections were subsequently stained with hematoxylin and eosin (H&E) for the detection of leukocyte infiltration. The number of leucocyte foci (a leucocyte focus is defined as a cluster/aggregate of cells in the glands that contain at least 50 leukocytes), and leucocyte focus score (number of leukocyte foci withing a 4 mm2 tissue section) are assessed and calculated. The de-paraffinized and dehydrated SMG tissue sections were subjected to the antigen retrieval process and blocking procedure as needed, and stained with primary antibodies against pSTAT3, Tet-2 and P16, at 4°C overnight, followed by incubation with the appropriate secondary antibodies before the signal detection using VECTASTAIN Elite ABC Kits (Vector Laboratories) according to the manufacturer’s instruction. The stained tissues sections were imaged at 400× magnification under a light microscope, and the quantification was performed using the Image J 1.50i software. Briefly, for quantification of positively stained areas/cells, images were saved as splitted red, blue and green color Tiff files and appropriate segmentation of stained area was performed using color thresholding. The percentage of thresholded areas in each image was then measured, and that of 6-8 different images per sample was averaged and subjected to further calculation and statistical analysis. In addition, from three of the non-consecutive IHC stained SMG sections described above, the numbers of leukocytic foci (a leukocytic focus is defined as a cell aggregate containing at least 50 mononuclear cells) were also counted, and the highest number among the three was used for subsequent quantifications.

2.6. Antibody staining and flow cytometry

Single cell suspensions were prepared from freshly harvested SMGs or submandibular lymph nodes (smLNs), incubated with anti-CD16/32 antibody first before being stained with a combination of fluorescence-conjugated antibodies to surface markers CD45, CD4, CD8α, CD19, EpCAM, lineage markers Ter119 and CD31, at 4 °C for 30 min. Some of the cells were pre-treated with PMA plus ionomycin for 4 hours with GolgiStop (BioLegend) added in the last 2 hours, then fixed and permeabilized, and incubated with anti-IFN-γ, anti-IL-17A and anti-Foxp3 antibodies. The stained cells were subsequently washed and analyzed with a FACS Arial II flow cytometer (BD, Franklin Lakes, NJ) and the FlowJo V10 software (FlowJo, Ashland, OR).

2.7. Detection of anti-M3 muscarinic acetylcholine receptor (M3R)

The M3R peptide (AILFWQYFVGKRTVP), synthesized by Biomatik Corporation, was kindly provided by Dr. Toshihisa Kawai (Nova Southeastern University). A Nunc™ MaxiSorp™ flat-bottom 96-well plate (BioLegend) was coated with the M3R peptide solution (1 μg/mL) at 4 °C overnight and the non-specific binding sites on the plate were subsequently blocked with ELISA Assay Diluent A buffer (BioLegend). Serum samples, diluted at 1:80, were added to the plate and incubated at 4 °C overnight. The plates were incubated with biotinylated goat anti-mouse IgG antibody (Vector Laboratories, Newark, CA), Avidin-HRP solution and TMB substrate followed by reading as previously reported [28]. The absorbance was measured at 450 nm and 570 nm on a microplate reader (BioTek, Santa Clara, CA), and the absorbance at 450 nm subtracted by that at 570 nm was used as relative concentration of anti-M3R antibody in the serum.

2.8. Isolation, in vitro culture and IL-22 treatment of EpCAMhi salivary gland stem cells (SGSCs)

SMG tissues from 24-week-old female C57BL/6 mice were digested with 5 mL DMEM containing 10% FBS, 1% penicillin-streptomycin and 1.6 mg/ml collagenase type IV (C9891, Sigma-Aldrich). The cells were then filtered through a 100 μm nylon mesh, digested with 0.05% Trypsin-0.02% EDTA, and filtered again. The resulting single cells were incubated with antibodies against lineage markers (Ter119, CD31, and CD45) and EpCAM, before sorting for cells that were negative for propidium iodide and lineage markers and expressing high levels of EpCAM, which were enriched of SGSCs, on a BD FACSAria III cell sorter. The resulting Lineage−EpCAMhi SGSCs were cultured and expanded in the WRY medium, which was DMEM/F12 that contained Pen/Strep antibiotics, Glutamax (Invitrogen), N2 (Gibco), EGF (20 ng/ml; Sigma-Aldrich), FGF2 (20 ng/ml; PeproTech), insulin (10 μg/ml; Gibco), dexamethasone (1 μM; PeproTech), Y-27632 (10 μM; PeproTech) and recombinant murine Wnt3a (20 ng/ml, BioLegend). For the IL-22 treatment and proliferation assessment, the cells were seeded at a density of 2 × 105 cells per well in 96-well culture plates that had been pre-coated with 80 μg/ml Cultrex UltiMatrix RGF BME (R&D Systems), in the WRY medium that contained 10 ng/ml recombinant murine Wnt3a in the presence or absence of recombinant murine IL-22 (20 ng/ml, PeproTech) for 5 days.

2.9. MTT assay

The cultured cells were incubated in medium containing 1.09 mM MTT a final concentration of 1.09 mM at 37 °C. A medium alone control group with MTT incubation was included. After 2 h of incubation, all but 25 μL of medium was removed and 50 μL of DMSO was added to each well. After 10-min incubation at 37°C, the samples were read on a BioTek Synergy HT plate reader (Agilent Technologies) with the absorbance at 540 nm.

2.10. Statistical Analysis

Statistical significance was determined by One-way ANOVA, two-tailed Student’s t-test, or Mann-Whitney U test as appropriate. P values smaller than 0.05 were considered statistically significant.

3. Theory/calculation

We hypothesized that the intrinsically produced IL-22 plays a pivotal role in the development and onset of salivary gland dysfunction associated with SS in the NOD mice, acting in a STAT3-dependent manner. To test this hypothesis, we employed an approach involving the systemic delivery of a neutralizing antibody against IL-22, and examined the effects of IL-22 blockade on SS-characteristic salivary gland pathologies. In addition, we explored the requirement of the STAT3 signaling in IL-22-mediated effects by utilizing a specific STAT3 inhibitor peptide.

4. RESULTS

4.1. Neutralization of endogenous IL-22 impedes the development of salivary gland dysfunction in the NOD mice.

Female NOD mice that received repeated intraperitoneal injection of a neutralizing anti-IL-22 antibody between the ages of 6- and 10 weeks demonstrated a higher salivary flow rate compared to the control group that received the isotype IgG (Fig. 1A). IL-22 neutralization did not reduce leukocytic infiltration of the SMGs in a statistically significant manner, based on the leukocyte focus score (number of leukocytic foci within a 4 mm2 tissue area), even though there was a noticeable trend towards a decrease (Fig. 1B). In addition, the blockade of IL-22 blockade did not significantly affect the serum levels of anti-muscarinic receptor 3 autoantibodies (Fig. 1C).

Figure 1. Neutralization of IL-22 impedes the onset of hyposalivation in NOD mice without significantly affecting other SS parameters.

Figure 1.

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200 μg anti-IL-22 (8E11) or the isotype IgG was intraperitoneally injected to 6-week-old female NOD mice 3 times weekly for 4 weeks. Mice were analyzed 2 days after the last injection. (A) Salivary flow rate normalized to body weight. (B) Leukocytic focus score, defined as the number of leukocytic foci within a 4 mm2 tissue area. (C) ELISA of relative levels of anti-muscarinic acetylcholine receptor M3 (M3R) in 1:80 diluted sera.

4.2. IL-22 neutralization does not significantly affect local Th1/Th17 immune responses in NOD mice.

Flow cytometric analysis showed that IL-22 neutralization did not alter the proportion of total immune cells (CD45+), CD4- and CD8 T cells, and B cells in the SMGs compared to the isotype IgG-treated control group (Fig. 2A). Moreover, this intervention did not significantly affect the percentage of IFNγ- and IL-17-producing CD4- and CD8 T cells within the SMGs (Fig. 2A) or in the submandibular lymph nodes (smLNs, Fig. 2B).

Figure 2. Blockade of IL-22 does not significantly alter local T- and B cell responses.

Figure 2.

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200 μg anti-IL-22 (8E11) or the isotype control, mouse IgG1, was intraperitoneally injected to 6-week-old female NOD mice 3 times weekly for 4 weeks. Mice were analyzed 2 days after the last injection. (A) Percentage of total immune cells (CD45+) among live SMG cells and that of T- and B cells in the immune population analyzed by flow cytometry, and percentage of cytokine-producing cells among T cells in SMGs. (B) Percentage of cytokine-producing cells among T cells in the submandibular lymph nodes (smLNs).

4.3. The detrimental effects of IL-22 on the salivary glands in NOD mice require the activation of STAT3.

The primary signaling event triggered by IL-22 is the activation of JAK-STAT3 (Ouyang and O'Garra, 2019; Ouyang et al., 2011; Pickert et al., 2009). Even though the tissue-protective and pro-regenerative properties of IL-22 have been mostly ascribed to STAT3 activation (Bachmann et al., 2013; Muhl, 2013; Ouyang and O'Garra, 2019; Saxton et al., 2021), it has been shown that the STAT3 activity can also inflict damaging effects on certain tissues/cells in a context-dependent manner, including salivary gland epithelial cells of SS patients (Charras et al., 2020). Consequently, we examined the levels of pSTAT3 in the SMGs through immunohistochemical staining, which revealed that the pSTAT3 protein levels, quantified either in the total SMG areas that included the leukocyte foci or solely in the focus-free epithelial tissue areas, were significantly reduced following IL-22 neutralization (Fig. 3A). Conversely, pSTAT3 levels markedly increased upon intra-SMG administration of recombinant murine IL-22 (Fig. 3B). Consistent with these observations, the hyposalivation induced by IL-22 administration was almost completely abrogated by administering a specific cell-permeable STAT3 inhibitor in NOD mice (Fig. 3C), underscoring a functional importance of STAT3 in mediating the salivary gland-detrimental effects of IL-22.

Figure 3. Neutralizing IL-22 reduces phosphorylated-STAT3 and Tet2 levels in SMGs.

Figure 3.

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(A) 6-week-old female NOD mice were treated anti-IL-22 or the control IgG as described in Fig. 1. Representative images of immunohistochemical (IHC) staining for pSTAT3 in SMG sections (X400 Magnification; n=6-7). The graph on the left shows the percentage of total areas that are positively stained for pSTAT3, and the graph on the right shows the percentage of the focus-free areas that are positively stained for pSTAT3. (B) Recombinant murine IL-22 or the vehicle control solution was injected into SMGs of 8-week-old female NOD mice on day 0 and 2, and SMGs were harvested on day 3. IHC for pSTAT3 was performed and quantified as described above (n = 5-6). (C) PBS, or IL-22 in the presence or absence of a specific STAT3 inhibitor peptide was injected into SMGs of NOD mice as described in B, and the salivary flow rate relative to body weight is shown (n = 4-7).

4.4. Inhibition of IL-22 activity in NOD mice leads to a reduction in Tet2 protein levels in SMG epithelial tissues.

SS is characterized by a pronounced IFN molecular signature, with type I IFNs and IFNγ playing critical roles in its pathogenesis as evidence by multiple lines of studies including our own (Hall et al., 2015; Lessard et al., 2013; Nguyen and Peck, 2013; Peck and Nguyen, 2012; Voulgarelis and Tzioufas, 2010; Zhou and Yu, 2018; Zhou et al., 2022b). We consequently assessed the impact of IL-22 blockade on IFN responses, and found that it substantially reduced the levels of Tet2 protein (Fig. 4A), a methylcytosine dioxygenase that catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine and an integral molecular player in type I IFN- and IFNγ-induced inflammatory responses in plasmacytoid dendritic cells and islet β-cells, respectively (S. Ma et al., 2017; Rui et al., 2021; Stefan-Lifshitz et al., 2019). Furthermore, the Tet2 protein levels are significantly elevated in the salivary gland epithelial cells of SS patients, likely as a response to the increased amounts of proinflammatory cytokines, such as IFNγ and TNFα, in the salivary glands of SS patients (Lagos et al., 2018). Notably, Tet2 was detected in both immune cell foci and the epithelial cells of NOD mice, with a markedly greater expression in ductal epithelial cells compared to acinar cells (Fig. 4A).

Figure 4. IL-22 neutralization significantly reduces Tet2 protein levels in SMG epithelial cells in the NOD mice.

Figure 4.

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(A) Representative images of Tet2 IHC on SMGs of 10-week-old female NOD mice that had received intraperitoneal injection of anti-IL-22 antibody or the control IgG 3 times per week for 4 weeks (X400 Magnification; n=6-7). Graph shows the percentage of positively stained areas in Tet2 IHC sections. (B) Representative images of p16 IHC on SMGs of the same mice described in A (X400 Magnification; n=6-7). Graph shows the percentage of positively stained areas in the IHC sections. (C) Female NOD mice that received intra-SMG injection of IL-22 or the vehicle control solution twice, two days apart. Representative images of Tet2 IHC on SMGs (X400 Magnification) and the quantification of Tet2 IHC are shown.

The reduction in Tet2 was more pronounced within the epithelial compartment compared to that in the immune cells, consistent with the notion that non-immune cells are the main targets of IL-22 (Ouyang et al., 2011; Rutz et al., 2013; Sabat et al., 2014). In contrast to Tet2, the levels of aquaporin 5 (AQP5) and claudin-1, a water channel protein and a tight junction protein respectively, that are critical for normal salivary production (Baker, 2016; T. Ma et al., 1999; Zhou et al., 2017; Zhou and Yu, 2018), were not discernably affected by IL-22 neutralization (data not shown). In addition, the level of p16, a major marker and promoter of cellular senescence (Liu et al., 2019; Zhang et al., 2022), was also unchanged following IL-22 blockade (Fig. 4B). Aligning with these findings, IL-22 administration to the SMGs led to a notable increase in Tet2 protein levels based on immunohistochemical staining and quantification (Fig. 4C). These findings collectively suggest that IL-22 may enhance and/or synergize with IFNs in SMG epithelial cells, including ductal/basal ductal cells and acinar cells, at least in part, through the upregulation of Tet2 expression.

4.5. IL-22 diminishes the size of the EpCAMhi SGSC compartment in NOD mice.

Given the function of IL-22 in promoting the expansion of various stem cell types, including skin- and intestinal stem cells, we investigated its impact on EpCAMhi SGSCs. Our study revealed that IL-22 neutralization in NOD mice led to a significant increase in the proportion of the Lineage−EpCAMhi population, which is enriched with SGSCs, within total SMG cells (Fig. 5A) (Maimets et al., 2016). Furthermore, treatment of purified Lineage−EpCAMhi cells/SGSCs from C57BL/6 mice with recombinant murine IL-22 markedly reduced the total metabolic activities (indicative of total cell viability/proliferation) after 5 days of culture as determined by the MTT assay (Fig. 5B). These data indicate that IL-22 can directly act on EpCAMhi SGSCs to limit their survival and/or proliferation.

Figure 5. Negative impact of IL-22 on the SGSC population.

Figure 5.

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(A) 6-week-old female NOD mice were treated with anti-IL-22 or the isotype IgG for 4 weeks as described in Fig. 3. The percentage of CD45− EpCAMhi cells, enriched of SGSCs, based on flow cytometric analysis, among total SMG cells is shown (n=6-7). (B) The Lineage−EpCAMhi cells, enriched with SGSCs, were electronically sorted from SMGs of 24-week-old female C57BL/6 mice and cultured in medium containing recombinant murine IL-22 (20 ng/ml) or control PBS solution for 5 days. Graph shows the value of MTT assays (absorbance 540 nm) indicative of the total metabolic activity (n=6).

5. DISCUSSION

In this study, we demonstrate that inhibiting the endogenous IL-22 activity in female NOD mice impedes the development and onset of salivary gland secretory dysfunction associated with the SS disease. Moreover, we reveal that the detrimental effects of IL-22 on salivary glands is dependent on STAT3 activation and coupled with a reduction in several molecular players in salivary gland epithelial cells that mediate the type I- and type 2 IFN responses. These results complement and extend our previous findings regarding a salivary gland-detrimental effect of IL-22, demonstrated through administration of exogenous IL-22 to NOD- and wildtype C57BL/6 mice and through blockade of endogenous IL-22 in an anti-CD3-induced exocrinopathy model in C57BL/6 mice.

IL-22 receptor signaling elicits the phosphorylation/activation of STAT3, and in some scenarios, that of STAT1 (Ouyang and O'Garra, 2019; Sabat et al., 2014; Seror et al., 2021; Wolk et al., 2007; Xie et al., 2000). The notable tissue-protective/pro-regenerative effects of IL-22 have been mostly attributed to STAT3 activation, whereas the proinflammatory actions of this cytokine are mainly ascribed to STAT1 activation (Bachmann et al., 2013; Muhl, 2013; Ouyang and O'Garra, 2019; Saxton et al., 2021). However, STAT3 activation in immune cells, such as T cells, often plays a proinflammatory and pathogenic role. Similarly, in certain non-immune cells, such as astrocytes, STAT3 activation contributes to tissue pathology and disease pathogenesis (Kim et al., 2023; Ni et al., 2023). Specifically pertinent to salivary tissues, a recent study has indicated that STAT3 enhances IFN-induced gene expression to amplify inflammatory responses in salivary gland epithelial cells patients with SS (Charras et al., 2020). These findings underscore the nuanced and context-dependent nature of the STAT3 function. Importantly, pSTAT3 levels are elevated in the salivary glands of SS patients, with pSTAT3 colocalizing with IL-22Rα (Charras et al., 2020; Ciccia et al., 2015; Ciccia et al., 2012; Gandolfo and Ciccia, 2022; Pertovaara et al., 2016; Wakamatsu et al., 2006). Our findings in this study reveal a pivotal role of the IL-22-STAT3 axis in the pathological impairment of salivary gland secretory function and the onset of clinical SS symptoms, at least in the context of the NOD mouse model.

Our previous work indicated the presence of IL-22-producing B cells within salivary glands, noting an increase in these cells in SS-afflicted NOD mice compared to BALB/c mice. Moreover, in an adenovirus infection-induced SS model in C57BL/6 mice, both loss-of-function and gain-of-function studies demonstrate that IL-22 upregulates CXCL12 and −13 expression in SMG epithelial/stromal cells, which facilitates B cell accumulation and tertiary lymphoid organ (TLO) formation, thereby enhancing autoantibody production (Barone et al., 2015). In this study, however, IL-22 neutralization did not significantly affect the proportion of B cells in SMGs or the serum anti-M3R autoantibody levels. This discrepancy may stem from the differences in mouse models and methods used for IL-22 ablation. The prior study used a viral infection model combined with genetic IL-22 deletion (Barone et al., 2015), whereas our current study employed a spontaneous SS model along with antibody-mediated IL-22 neutralization. These differing results may shed light on the highly complex causes, pathogenic mechanisms and pathological presentations of this disease, as displayed in human patients and represented in various mouse models. Further investigations will be needed to fully elucidate the actions of IL-22 in B cell responses and B cell lymphomagenesis in this autoimmune condition.

SS is characterized by a prominent IFN signature within both salivary tissues and peripheral immune cells with the pivotal roles of type I IFNs and IFNγ in disease pathogenesis clearly demonstrated by multiple lines of studies using mouse models and/or human patient samples (Cha et al., 2004; Hall et al., 2015; Kulkarni et al., 2006; Lessard et al., 2013; Nguyen and Peck, 2013; Peck and Nguyen, 2012; Voulgarelis and Tzioufas, 2010; Zhou and Yu, 2018; Zhou et al., 2022b). Intriguingly, this study revealed that IL-22 blockade led to a downregulation in Tet2 protein levels in the SMGs. Tet2, a methylcytosine dioxygenase that catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine (Lagos et al., 2018; S. Ma et al., 2017; Rui et al., 2021; Stefan-Lifshitz et al., 2019), plays a crucial role in type I IFN production by plasmacytoid dendritic cells (S. Ma et al., 2017) and the inflammatory responses of islet β-cells triggered by both type I IFNs and IFNγ (Rui et al., 2021; Stefan-Lifshitz et al., 2019). Our finding indicates that Tet2 protein is expressed by both immune cells in the foci and epithelial cells in SMGs, with a particularly strong expression in the epithelial compartment, which aligns with the pertinent findings from SS patients (Lagos et al., 2018). That the reduction in Tet2 following IL-22 neutralization is more pronounced in epithelial cells is also consistent with the notion that non-immune cells are the major cellular targets of IL-22 (Ouyang et al., 2011; Rutz et al., 2013; Sabat et al., 2014). Hence, our findings present the first evidence that IL-22 may enhance, cooperate and/or synergize with IFNs in SMG epithelial cells, including ductal/basal ductal cells and acinar cells, potentially through the upregulation of Tet2 expression. Previous research by other groups have shown that Tet2 protein levels are elevated in salivary glands of SS patients and that proinflammatory cytokines TNFα and IFNγ can upregulate Tet2 expression in a human epithelial cell line with salivary gland epithelial cell features (Lagos et al., 2018). Additionally, another study demonstrated that in the same cell line, IFNα and IFNγ treatments, through activating STAT3, enhance the expression of Tet3, another member of the Tet protein family (Charras et al., 2020). Given that activation of STAT3 is a key signaling event induced by IL-22, IL-22 may upregulate Tet2 expression in an STAT3-dependent manner, a hypothesis that will be tested in future research. In addition, future investigations should also elucidate whether IL-22 can potentiate the production of type I IFNs in salivary tissues and their draining lymph nodes in addition to enhancing Tet2 expression. Another limitation of this study is that the impact of IL-22 blockade on the production of IFNγ and IL-17 in target tissues was only assessed by flow cytometry. Future work should also evaluate the secreted levels of these and other relevant proinflammatory cytokines.

Both type I IFN and IFNγ can directly act on salivary gland epithelial cells to disrupt their function via multiple mechanisms, including inducing apoptosis, reducing AQP5 levels, and interfering with normal expression and distribution of tight junction proteins (Baker et al., 2008; Jin et al., 2013; Kamachi et al., 2002; Kulkarni et al., 2006; Szczerba et al., 2013; Zhou and Yu, 2018; Zhou et al., 2022b). Importantly, type I IFN signaling has been shown to impair secretagogue-induced salivary secretion by acinar cells through interfering with Ca2+ mobilization without influencing cell survival/death (Nandula et al., 2013). In previous studies, we showed that IL-22 administration to C57BL/6 mice or IL-22 blockade in anti-CD3-treated C57BL/6 mice altered the levels of activated caspase-3 protein that were indicative of tissue apoptosis in the salivary glands (Zhou et al., 2022a). However, in the NOD mice, the level of activated caspase-3 and the degree of apoptosis are minimal throughout the course of SS development, suggesting that the apoptosis may not be a signification contributing factor to salivary gland pathology and hypofunction in this model. Moreover, our findings indicate that IL-22 blockade does not considerably affect the levels of AQP5 and claudin-1 proteins in the SMGs. In addition, despite the evidence that IL-22 induces senescence of hepatic stellate cells in a mouse model of liver fibrosis (Kong et al., 2012), blockade of IL-22 shows no impact on the expression levels of p16, a well-defined marker and mediator of cellular senescence, in SMGs in the NOD mice. Therefore, the beneficial impact of IL-22 blockade on salivary glands in the NOD mice is likely achieved, at least in part, by counteracting the direct impairing effects of type I IFNs on salivary secretion by acinar cells, independently of affecting cell death/survival, AQP5 and tight junction protein levels or cellular senescence in SMG tissues (Nandula et al., 2013).

Another notable insight generated by this study is the negative impact of IL-22 on the SGSCs that are negative for lineage markers and expressing high levels of surface EpCAM, as evidenced by both in vivo and in vitro investigations. This finding provides additional evidence for the highly context-dependent roles of IL-22, as it boosts stem cell activity and tissue regeneration in various contexts yet suppresses these processes in the salivary glands under the SS condition. The increase in the proportion of SGSCs within the SMGs of the NOD mice following IL-22 blockade, coupled with the decrease in the total metabolic activity of the cultured Lineage−EpCAMhi SGSCs in vitro in response to IL-22, suggests a negative regulation of IL-22 on SGSC survival and/or proliferation, which will merit additional investigations.

One of the limitations of this study, we did not assess the effects of IL-22 on the reported self-renewal and regenerative ability of differentiated acinar cells under certain conditions of tissue injury, stress and/or inflammation (Aure et al., 2015; Weng et al., 2019), a topic that merits further investigation in the future. Another limitation is that the cellular mechanisms underlying the negative impact of IL-22 on the EpCAMhi SGSC population has not been fully delineated. Future studies will aim to elucidate how IL-22 affects the survival/apoptosis and the proliferative status of SGSCs in vivo, using in situ and ex vivo assays to examine these cellular processes. Furthermore, while the effects of IL-22 on the protein levels of Tet2 and P16 have been determined, the corresponding changes in the mRNA levels of these molecules have not been assessed. This gap will be addressed in the future, along with a comprehensive transcriptomic analysis of SMG tissues following neutralization or provision of IL-22.

Finally, a recent study has shown that IL-22 can induce the endoplasmic reticulum stress response in intestinal epithelial cells, thereby exacerbating chronic colitis (Powell et al., 2020). In addition, it has been shown that salivary gland cells undergo atrophy in response to certain chemical compounds and drugs (Ungureanu et al., 2023), and a strong connection between endoplasmic reticulum stress-STAT3 axis and muscle atrophy has been reported (Zheng et al., 2023). Hence, exploring the potential of IL-22 to provoke endoplasmic reticulum stress responses and potentially induce atrophy in salivary gland epithelial tissues represents an interesting avenue for future investigations. These lines of research could generate novel insights into the mechanisms by which IL-22 influences salivary gland function and regeneration in the context of SS.

Conclusions:

The present study has identified a critical contribution of the endogenous IL-22 to the salivary gland dysfunction characteristics of Sjögren's disease, operating in a STAT3-dependent manner. It also offers preliminary evidence that the effect of IL-22 is achieved through enhancing Tet2-mediated interferon responses in salivary gland epithelial cells as well as negatively affecting the salivary gland stem cell compartment. These findings, along with additional future research on this topic, could facilitate the development of novel biological therapies for this challenging autoimmune disorder.

Highlights.

  • IL-22 neutralization protects salivary glands in a murine model of Sjögren's syndrome

  • STAT3 activity is required for the detrimental impact of IL-22 on salivary glands

  • IL-22 upregulates Ten-Eleven-Translocation 2 expression in salivary epithelial cells

  • IL-22 negatively impacts the size of the salivary gland stem cell compartment

ACKNOWLEDGMENTS

We thank Genentech Inc. for generously providing the neutralizing anti-IL-22 antibody and the isotype control IgG; Dr. Bo Ra You for the technical contribution to this study; Dr. Toshihisa Kawai for kindly providing the M3R peptides, and the staff at the ADA Forsyth Institute animal facility for their excellent animal care. FAF received a Ph.D. scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES –Process No. 88887.803395/2023-00), Brazil.

This study was supported by grants from NIH/NIAID (R03 AI142273, R56 AI181002), NIH/NIDCR (R56 DE023838, R21 DE031058) to QY, and NIH/NIDCR (R01 DE030646, R03 DE028033) to JZ.

Abbreviations:

SMG

Submandibular gland

SmLN

Submandibular lymph node

SGSC

Salivary gland stem cell

Footnotes

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Declaration of Generative AI and AI-assisted technologies in the writing process: During the preparation of this work the authors used ChatGPT Scholar AI tool solely to improve the readability and language. After using this tool, the authors reviewed the content carefully, edited it as needed, and take full responsibility for the content of the publication.

The authors declare that they have no competing interests.

All authors have no conflict of interests.

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