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. Author manuscript; available in PMC: 2025 Oct 1.
Published in final edited form as: Biochim Biophys Acta Mol Cell Res. 2024 Aug 17;1871(7):119817. doi: 10.1016/j.bbamcr.2024.119817

The impact of alternate-day fasting on the salivary gland stem cell compartments in non-obese diabetic mice with newly established Sjögren's syndrome

Dongfang Li a, Shoko Onodera a,b, Qing Yu a, Jing Zhou a,*
PMCID: PMC11368138  NIHMSID: NIHMS2017858  PMID: 39159683

Abstract

Intermittent fasting exerts a profound beneficial influence on a spectrum of diseases through various mechanisms including regulation of immune responses, elimination of senescent- and pathogenic cells and improvement of stem cell-based tissue regeneration in a disease- and tissue-dependent manner. Our previous study demonstrated that alternate-day fasting (ADF) led to alleviation of xerostomia and sialadenitis in non-obese diabetic (NOD) mice, a well-defined model of Sjögren's syndrome (SS). This present study delved into the previously unexplored impacts of ADF in this disease setting and revealed that ADF increases the proportion of salivary gland stem cells (SGSCs), defined as the EpCAMhi cell population among the lineage marker negative submandibular gland (SMG) cells. Furthermore, ADF downregulated the expression of p16INK4a, a cellular senescence marker, which was concomitant with increased apoptosis and decreased expression and activity of NLRP3 inflammasomes in the SMGs, particularly in the SGSC-residing ductal compartments. RNA-sequencing analysis of purified SGSCs from NOD mice revealed that the significantly downregulated genes by ADF were mainly associated with sugar metabolism, amino acid biosynthetic process and MAPK signaling pathway, whereas the significantly upregulated genes related to fatty acid metabolic processes, among others. Collectively, these findings indicate that ADF increases the SGSC proportion, accompanied by a modulation of the SGSC property and a switch from sugar- to fatty acid-based metabolism. These findings lay the foundation for further investigation into the functionality of SGSCs influenced by ADF and shed light on the cellular and molecular mechanisms by which ADF exerts beneficial actions on salivary gland restoration in SS.

Keywords: Sjögren’s disease, xerostomia, intermittent fasting, cellular senescence

1. Introduction

Sjögren’s Syndrome (SS) is a chronic autoimmune disease primarily affecting salivary glands and lacrimal glands, leading to the hallmark symptoms of dry mouth and dry eyes [1-4]. Accumulating evidence showed that leukocyte infiltration of salivary and lacrimal glands and the production of autoantibodies critically contribute to glandular inflammation, destruction and subsequent secretory hypofunction [5-7]. Recent studies have revealed the presence of senescent cells (defined as p16INK4a-expressing cells) in the salivary glands, particularly within the salivary gland stem/progenitor cell (SGS/PC)-residing ductal compartments in the patients with SS [8]. Moreover, the proportion of senescent cells in the SGS/PC niche highly correlates with the severity of salivary gland inflammation and hyposalivation [8], suggesting a pivotal role for cellular senescence in SS. Therefore, elimination of senescent cells and rejuvenation of SGS/PC microenvironment in salivary glands could constitute critical cellular mechanisms for potential therapeutic interventions that can effectively mitigate SS-associated sialadenitis and xerostomia.

Intermittent fasting characterized by recurring and extended periods of food deprivation while maintaining unrestricted access to water has a demonstrated effectiveness in promoting longevity, extending health span and bolstering disease resistance in human and animal models [9-12]. During the process, fasting causes a metabolic switch from glucose to fatty acid utilization and elicits a series of highly orchestrated cellular responses and biological processes, such as immunomodulation and enhancement of stem cell-based tissue regeneration [12-17]. For instance, intermittent fasting mitigates tissue inflammation by reducing pro-inflammatory cytokine levels, diminishing the quantity and function of pathogenic immune cells and inhibiting NLRP3 inflammasome activity in multiple inflammatory and autoimmune contexts [13, 14, 16, 18]. Fasting also can augment the regenerative capacity of intestine-resident stem cells through Peroxisome Proliferator-Activated Receptor δ-driven fatty acid oxidation and promote dentate gyrus-resident neutral stem cell proliferation and neurogenesis in the central nervous system in mice [17, 19-21]. In addition, emerging evidence showed the potential of fasting or diets that mimic fasting to eliminate senescent and pathogenic cells via apoptosis or other forms of cell death, thereby contributing to the tissue homeostasis and disease amelioration [22-24].

Our previous study has demonstrated a beneficial effect of alternate-day fasting (ADF) on attenuating salivary gland inflammation and secretory hypofunction in non-obese diabetic (NOD) mice with newly established SS [25]. In the present study, we conducted an in-depth investigation to delineate the previously unexplored influence of ADF on salivary gland stem cell (SGSC) proportion, microenvironment and transcriptomic landscape, in the mouse model of this disorder. The results lay the groundwork for further exploration of SGSC activity and SGSC-mediated salivary gland regenerative process that are potentially influenced by ADF and offer insights into the cellular and molecular mechanisms by which ADF may counteract the pathological features of SS.

2. Materials and Methods

2.1. Mice and ADF intervention

Female NOD mice (NOD/ShiLtJ strain, Cat# 001976) were purchased from the Jackson Laboratory and maintained under specific pathogen-free condition at the ADA Forsyth Institute. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the ADA Forsyth Institute and performed in compliance with the National Institutes of Health guidelines for the care and use of laboratory animals.

For the fasting regimen, 10-week-old female NOD mice were fed every other day, and age- and sex-matched mice fed standard chow ad libitum served as controls. All mice had unrestricted access to water throughout the entire experiments.

2.2. Histologic Analysis

The procedures were performed as we previously described [26, 27]. Briefly, submandibular glands (SMGs) were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned to 5-micron thickness. For immunohistochemical staining, the sections were de-paraffinized, hydrated and then incubated with antibodies against p16INK4a (Invitrogen), NLRP3 (Thermo Scientific), IL-1β (BioLegend), IL-18 (Invitrogen), MCL-1 (Cell Signaling Technology), BCL-XL (Cell Signaling Technology) or BCL-2 (Invitrogen) overnight at 4°C using a VECTASTAIN Elite ABC Kit (Vector Laboratories) according to the manufacturer’s instructions.

2.3. In situ apoptosis detection

The paraffin embedded SMGs were de-paraffinized, hydrated and then subjected to apoptosis assay using in situ apoptosis detection kit (Abcam) following the manufacturer’s instructions. The stained sections were then imaged with a light microscope at 400× magnification. Quantification of positively stained aeras in the sections was performed using ImageJ 1.50i software.

2.4. Flow cytometry

SMGs were freshly harvested and then minced with sterilized surgical scissors on ice, followed by enzymatic digestion with a solution containing 0.63 mg/ml collagenase (Sigma-Aldrich), 0.5 mg/ml hyaluronidase (Sigma-Aldrich) and 6.25 mM CaCl2 (Fisher Scientific). The cell suspension was subsequently strained through 200 um- and then 50 um nylon meshes, and was further dissociated using 0.05 % trypsin-EDTA (Gibco). After being rinsed in cold PBS, the cells were stained with fluorescence-conjugated antibodies against CD45, CD31, Ter-119 and EpCAM antibodies for 30 min at 4°C followed by analyses or cell sorting with FACS Arial II flow cytometer (BD). The data analytical processing was conducted using FlowJo V10 software. All the antibodies utilized for flow cytometric staining were purchased from BioLegend.

2.5. Transcriptome analysis

Lin−EpCAMhi cell pellets were snap-frozen in liquid nitrogen and shipped to Genewiz, NJ with dry ice. After arrival, RNA exaction was performed using RNeasy Plus Universal Mini Kit (Qiagen). Following the validation of RNA quality and integrity, cDNA libraries were prepared and subjected to sequencing on the Illumina Hiseq platform, with a 2 × 150 bp paired-end configuration. The raw sequence data (.bcl files) generated by the sequencer were converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. The qualified sequencing reads were aligned to mouse genome reference sequence (UCSC mm10, NCBI) using STAR aligner v.2.5.2b. Unique gene hit counts were then determined via the Feature Counts tool of the Subread package v.1.5.2. Differential gene expression analysis between the fasting and control groups, incorporating biological replicates, was conducted using the DESeq2 R package. P-values were adjusted following the Benjamini-Hochberg correction method. Genes exhibiting adjusted p-values less than 0.05 and absolute log2 fold changes greater than 1 were identified as differentially expressed. Finally, enrichment analyses for the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms were carried out by analyzing the up- or downregulated DEGs using the Database for Annotation, Visualization, and Integrated Discover.

2.6. Statistical analysis

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

3. Results

3.1. ADF increases Lin−EpCAMhi cell proportion and decreases cellular senescence within the SMGs of NOD mice.

Patients with SS have been reported to exhibit a reduced number and diminished differentiation potential of SGSCs, which is accompanied by the presence of cellular senescence within the salivary glands, particularly in the SGS/PC-residing ductal compartments [8] [28]. Our previous study demonstrated a beneficial impact of ADF on mitigating tissue inflammation and secretory hypofunction of salivary glands in NOD mice with newly established SS [25]. Here, we delved deeper into the potential of ADF to affect the proportion of SGSCs and their senescent surroundings in the SMGs. 10-week-old NOD mice received ADF for 3 consecutive weeks or were fed standard chow ad libitum (AL), and were subsequently euthanized for various analyses at 13 weeks of age, when the mice have newly established SS as we previously described [25]. Flow cytometric analysis revealed a significant increase in the percentage of SGSCs, defined as cells expressing high level of EpCAM but negative for lineage markers [29], among the total SMG cell population and the total lineage marker-negative cell subset in the SMGs by ADF (Figure 1A). Immunohistochemical staining of SMG sections showed an elevated protein level of p16INK4a, a hallmark of cellular senescence, predominantly within the SGSC-residing ducts in NOD mice, compared to that of age- and gender-matched BALB/c mice (Figure 1B), which is consistent with the observations in SS patients [8]. Notably, the expression level of p16INK4a in the SMGs of NOD mice were significantly reduced by ADF (Figure 1C). Taken together, the protective effect of ADF from SS in NOD mice is accompanied by an increased frequency of SGSCs in the SMGs and a decline in cellular senescence in the SGSC compartments.

Figure 1. ADF increases Lin−EpCAMhi cell proportion and decreases cellular senescence within the SMGs of NOD mice.

Figure 1.

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10-week-old female NOD mice were subjected to alternate-day fasting (ADF) or fed with standard chow ad libitum (AL) for 3 weeks. (A) Representative FACS gating strategy for Lin−EpCAMhi cell population analyses (left panel). Mean percentage of Lin−EpCAMhi population among the total SMG cells or Lin− cells in the SMGs (n = 4 - 5) (right panel). (B) Immunohistochemical staining of SMG sections for p16INK4a from female BALB/c and NOD mice aged 13 weeks (Scale bar = 100 μm). Bar graph shows the average percentage of positively stained area (n = 4). (C) Immunohistochemical staining of SMG sections for p16INK4a from NOD mice in ADF or AL group (Scale bar = 100 μm). Bar graph shows the average percentage of positively stained area (n = 9).

3.2. ADF causes cell apoptosis in the SMGs of NOD mice.

Senescent cells have been reported to be resistant to apoptosis and can be eliminated through various strategies, including induction of apoptosis [30] [31]. We thus next examined whether ADF can induce apoptosis in the SMGs including ducts where p16INK4a is predominantly expressed. TUNEL-based in situ apoptosis detection assay on SMG sections showed that mice in the ADF group had a higher number of apoptotic cells in the SMGs, in particular within ducts, than the control subjects (Figure 2A). In line with this observation, the expression levels of anti-apoptotic molecules BCL-2, BCL-XL and MCL-1 in the SMGs were also decreased by ADF, as assessed by immunohistochemical staining (Figure 2B). Therefore, ADF led to cell apoptosis in the SMGs, including the SGSC compartments, in NOD mice, which is associated with local downregulation of BCL-2, BCL-XL and MCL-1.

Figure 2. ADF causes cell apoptosis in the SMGs of NOD mice.

Figure 2.

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10-week-old female NOD mice were subjected to ADF or fed with standard chow AL for 3 weeks. (A) TUNEL analysis of SMG sections (Scale bar = 50 μm). Bar graph shows the average percentage of positively stained area of TUNEL assay (n = 9). (B) Immunohistochemical staining of SMG sections for BCL-2, BCL-XL or MCL-1 (Scale bar = 100 μm). Bar graphs display the average percentage of positively stained areas (n = 9).

3.3. ADF reduces the expression levels of NLRP3 and its downstream products IL-1β and IL-18 in the SMGs.

Fasting has been demonstrated to effectively inhibit the activity of NLRP3 Inflammasomes [18], which are cytosolic multi-protein complexes and play a crucial role in various autoimmune and inflammatory diseases by facilitating the production of IL-1β and IL-18 [32-34]. NLRP3 inflammasomes can be activated by, and conversely, promote cellular senescence [35-38]. By immunohistochemical staining, we found that ADF significantly decreased NLRP3 expression level, along with a reduction of the downstream products of NLRP3 inflammasome, IL-β and IL-18, in the SMGs (Figure 3). These findings indicate the suppression of NLRP3 inflammasome pathway by ADF in the SMGs of NOD mice.

Figure 3. ADF reduces the expression levels of NLRP3 and their downstream products IL-1β and IL-18 in the SMGs.

Figure 3.

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10-week-old female NOD mice were subjected to ADF or fed with fed standard chow AL for 3 weeks. Immunohistochemical staining of SMG sections for NLRP3, IL-1β and IL-18 (Scale bar = 100 μm). Bar graphs display the average percentage of positively stained areas (n = 9).

3.4. Characterizing the transcriptomic profile of Lin−EpiCAMhi cells in the SMGs altered by ADF.

To comprehensively characterize the transcriptomic profile of SGSCs and the key pathways or biological processes that potentially contribute to the protective effects of ADF in SS, bulk RNA-sequencing was conducted on sorted Lin−EpCAMhi cells from the SMGs of NOD mice that received 3 weeks of ADF or were fed standard chow AL. Transcriptomic analysis identified a total of 506 differentially expressed genes (DEGs) between these two experimental groups, comprising 168 upregulated (adjusted p < 0.05 and Log2 fold change > 1) and 337 downregulated DEGs (adjusted p < 0.05 and Log2 fold change < −1) by ADF (Figure 4A). Hierarchical clustering analysis of these DEGs unveiled similar expression patterns within each group (Figure 4B).

Figure 4. ADF alters the transcriptomic profile of Lin−EpCAMhi cells in the SMGs.

Figure 4.

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Lin−EpCAMhi cells were electronically sorted from the SMGs of NOD mice that were subjected to ADF for a total of 3 weeks or fed standard chow AL. RNA-sequencing was performed, followed by bioinformatic analyses. (A) Volcano plot of the differentially expressed genes (DEGs). DEGs upregulated and downregulated by ADF are colored in red and blue, respectively (adjusted P value < 0.05 and absolute log2 fold change > 1). (B) Heatmap of hierarchical clustering analysis of the DEGs. Each row represents a single gene, and each column represents a sample. The colors indicate high (red), low (blue) and intermediate (white) expression values of the genes. (C) Significantly enriched KEGG pathways of the downregulated DEGs by fasting (FDR < 0.05). (D) Significantly enriched GO terms of the DEGs upregulated or downregulated by ADF (* FDR < 0.1; ** FDR < 0.05). (n = 2 - 3).

To further elucidate the biological pathways and processes affected by ADF, these up- and downregulated DEGs were analyzed separately utilizing the Database for Annotation, Visualization and Integrated Discovery. KEGG pathway analysis revealed that the downregulated DEGs were significantly enriched in six pathways, which were related to cell metabolism, mucin type O-glycan band amino acid biosynthesis, the MAPK signaling, nucleotide sugar biosynthesis and amino- and nucleotide sugar metabolism (FDR < 0.05), whereas no pathways exhibited significant enrichment in the upregulated DEGs (FDR > 0.1) (Figure 4C). GO function and enrichment analyses showed that the most downregulated DEGs were related to GO terms membrane (Cellular Component), among others such as lipid metabolic process (Biological Process), Golgi membrane (Cellular Component), cell surface (Cellular Component), neuronal cell body (Cellular Component) and polypeptide N-acetylgalactosaminyl transferase activity (Molecular Function) (FDR < 0.05 or 0.1) (Figure 4D, right panel). The most upregulated DEGs were associated with fatty acid metabolic process (Biological Process), N-acetylglucosamine metabolic process (Biological Process) and extracellular matrix (Cellular Component) (FDR < 0.05 or 0.1) (Figure 4D, left panel). In addition, the GO terms extracellular region (Cellular Component) and extracellular space (Cellular Component) (FDR < 0.05 or 0.1) were enriched in both the upregulated and the downregulated DEGs (Figure 4D), indicating significant and complex changes in the processes related to the extracellular environment as a result of ADF.

4. Discussion

Our previous study elucidated the beneficial impact of ADF on mitigating salivary gland inflammation and hypofunction in NOD mice with newly established SS [25]. Extending these findings, the current study further assessed the influence of ADF on the SGSC proportion, property and transcriptomic landscape using the same mouse model of SS. The findings provide crucial insights into the cellular and molecular mechanisms that underpin the protective action of ADF and warrant future investigations on SGSC functionality affected by this regimen in SS.

Intermittent fasting has garnered a great amount of attention for its potential in reducing aging-related complications and promoting longevity and health span [12, 16, 39, 40]. While SS is not specifically classified as an aging-related ailment, there is an increased prevalence of senescent cells presents in the salivary glands, particularly in the ductal compartments where salivary SGS/PCs reside, in patients with this disorder [8, 29]. In this study, we validated the increased protein level of a senescent marker p16INK4a in the SMGs, which is most abundantly expressed in the ductal compartments in NOD mice with newly established SS. Senescent cells accumulating in tissues can disturb the microenvironment in the stem cell niches by secreting pro-inflammatory cytokines, chemokines and other signaling molecules, impeding stem cell function and eventually contributing to tissue degeneration and hypofunction [41, 42]. Our findings showed that ADF not only increased the frequency of Lin−EpCAMhi cell subset among the total Lin− cell population and the overall SMG cells, but also reduced p16INK4a expression in salivary gland ducts, suggesting a favorable impact on SGSC expansion. Nevertheless, the specific impact of ADF on SGSC proliferation and differentiation requires further elaboration through in vivo and in vitro studies. Additionally, future research with SGSC/P cells identified by other specific markers such as Lin−CD24+c-Kit+Sca1+ or CD24hiCD29hi is necessary to validate and extend our findings, which will offer more comprehensive insights into how SGS/PCs respond to this diet regimen in SS. In addition to SGSC cells, the salivary gland ductal compartments also harbor lineage-restricted salivary gland progenitor cells, such as KRT5+ or Axin 2+ cells, that are capable of duplication to replenish ductal cells in steady state and exhibit plasticity deviating from their original lineage to regenerate acinar cells post severe damage [43]. The influence of ADF on these progenitor cells and the specific cell types prone to senescence in the salivary gland ducts await further delineation.

A characteristic of senescent cells is their resistance to apoptosis, and strategies disrupting anti-apoptotic pathways have shown promise in effectively eliminating cellular senescence in various tissues including the SGS/PC compartments in mouse models with radiation-induced hyposalivation [31, 44]. Several lines of evidence showed that fasting regimens can trigger apoptosis in pathogenic or cancer cells, thereby promoting tissue homeostasis and disease amelioration [22-24]. Consistent with this, our study here demonstrated that accompanying reduction of senescent cells, ADF promoted apoptosis and suppressed the expression of anti-apoptotic proteins, including BCL-2, BCL-XL and MCL-1, in the SMGs, predominantly within the ducts. Further investigations are required to ascertain whether ADF selectively induces apoptosis in senescent cells within the salivary glands.

NLRP3 Inflammasome is a cytosolic multi-protein complex that assembles in response to pathogens and tissue damage and causes the maturation of IL-1β and IL-18, thereby contributing to tissue inflammation [45]. The effectiveness of intermittent fasting in reducing the NLRP3 protein level and attenuating the NLRP3 inflammasome activity has been reported in both preclinical and clinical studies on various diseases including inflammatory and autoimmune disorders [18, 46, 47]. In SS patients, the elevated expression of NLRP3 inflammasome components and functional products, such as NLRP3, IL-1β and IL-18, was observed in the salivary gland tissues, saliva and peripheral blood mononuclear cells, and positively correlated with the disease severity [48-51]. In mouse models, activation of P2X7R, an upstream activator of NLRP3 inflammasome, promoted mature IL-1β production and exacerbated salivary gland inflammation [52], and antagonism of P2X7R ameliorated sialadenitis and improved salivary secretion [49], indicating a disease-promoting role of NLRP3 inflammasome pathway in SS. Nevertheless, our previously study demonstrated that inhibition of NLRP3 inflammasome formation with MCC950 exacerbated SS pathologies and hyposalivation in NOD mice [53]. Thus, the relationship between NLRP3 inflammasome and SS pathogenesis is more complex than previously understood. In the present study, ADF not only suppressed the activation of NLRP3 inflammasome but also reduced NLRP3 protein level in the SMGs of in NOD mice. There is a critical need to further investigate the complex functionality of NLRP3 inflammasome altered by ADF in the SS-disease setting.

As mentioned above, while the impact of ADF on the protein levels of P16, BCL-2, BCL-XL ,MCL-1, NLRP3, IL-1β and IL-18 has been determined, the corresponding changes in their mRNA levels have not been examined. This gap will be addressed by quantitative RT-PCR in future studies.

Another well-documented consequence of intermittent fasting is the metabolic switch from glucose utilization to fatty acid oxidation for energy production [12]. In the Lin−EpCAMhi population of NOD mice, ADF markedly downregulated genes involved in nucleotide sugar biosynthesis while upregulating those associated with fatty acid metabolism, suggesting a decreased demand for the nucleotide sugars and a heightened reliance on fatty acids as an energy substrate during the fasting period. ADF also led to the downregulation of lipid metabolism-related genes, which may reflect the reduced lipid accumulation. Additionally, ADF attenuated the transcription of genes involved in MAPK signaling pathways, which are integral to various critical cellular processes such as proliferation, differentiation, stress responses and apoptosis [54, 55]. This modulation highlights the complexity of the cellular responses to intermittent energy deprivation, paving the way for further exploration of how the MAPK signaling affected by ADF is involved in SGSC functionality in SS. Despite the insightful revelations from our RNA sequencing analysis, these findings should be interpreted with caution due to limited sample size. Further studies with a larger sample size are imperative to solidify these observations and potentially reveal additional DEGs and pathways in SGSCs that are significantly influenced by ADF in the SS-disease setting.

Highlights.

  • Fasting increases Lin−EpCAMhi cells and reduces senescence in the salivary glands

  • Fasting induces apoptosis in salivary gland tissues

  • Fasting lowers NLRP3, IL-β and IL-18 protein levels in the salivary glands

  • Fasting regulates genes involved in sugar and fat metabolism in Lin−EpCAMhi cells

Acknowledgement

We thank the staff of the animal facility at the ADA Forsyth Institute for their work. This study was supported by grants from NIH/NIDCR (R01 DE030646, R03 DE028033) to JZ, and NIH/NIDCR (R56 DE023838, R21 DE031058) and NIH/NIAID (R03 AI142273) to QY.

Footnotes

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Disclosure

The authors have no competing financial interests.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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