Vasoactive intestinal peptide exerts therapeutic action by regulating PTEN in a model of Sjögren's disease

Abstract Introduction Sjögren's disease (SjD) is a chronic autoimmune disease characterized by the loss of the secretory function of the exocrine glands. At present, drugs that can both correct the immune imbalance and improve exocrine gland function are needed. Meanwhile, vasoactive intestinal peptide (VIP) has been reported as a candidate with anti‐inflammatory and immunoregulatory properties for treating autoimmune diseases. Methods Nonobese diabetic (NOD) mice and the primary splenic lymphocyte cells (SPLCs) were used to construct the SS model. The therapeutic effects of VIP for SjD by evaluating water consumption, histopathology, T cell subsets, and related cytokines. RT‐qPCR and Western blot analysis were used to identify the expression of the PTEN/PI3K/AKT pathway. Results We found that VIP therapy in NOD mice could increase the expression of PTEN and VIP/VPAC1 receptor, as well as decrease the PI3K/AKT pathway. In vitro, the results showed that the PTEN knockdown decreased the Treg/Th17 ratio and enhanced the phosphorylated PI3K/AKT pathway, which were reversed with VIP treatment. Conclusions VIP exerts potential therapeutic action in SjD by upregulating PTEN through the PI3K/AKT pathway and Treg/Th17 cell balance.


| INTRODUCTION
Sjögren's disease (SjD), also known as Sjögren's syndrome (SS), occurs in primary and secondary conditions defined by the impaired secretory function of the exocrine glands, especially the salivary and lacrimal glands. 1,2 According to epidemiological survey, the global prevalence of SS is 1 case per 1644 persons, and the female incidence rate is higher than the male. 3 The pathological feature of SS is the infiltration of lymphocytes in affected organs and tissue, as well as the increased levels of positive auto-antibodies. 4 Although the mechanism of this disease remains unclear, SS has been classically considered a disease of B cells on the basis of evidence that B cell activating factor (BAFF) is continuously detected in the damaged tissue. Then transition into the more recent research that has identified the participation of T-cell subsets in this process. 5 Several functional CD4+ T-cell subsets, including helper T 17 (Th17) cells and regulatory T (Treg) cells, modulate immunity activation by production of proinflammatory cytokines plus interaction with B cells. Treg cells are identified as a small subset of immune cells which can suppress the immune system and maintain immune homeostasis. 6,7 Genetic alteration resulting in a decrease in Treg cells can exacerbate sialadenitis. The study supports the idea that Treg cells play a prime representative in preventing autoimmunity of the salivary glands in healthy individuals. 8 Since the suppressive effects of Treg cells on some autoimmune diseases, Treg cells can confer tissue repair against the damaging effects during some stages of pSS. 9 Moreover, numerous studies have indicated that Th17 cells can contribute to the disease pathology of mouse models in the affected tissues. 10,11 As a prosecretory and vasodilating neuropeptide, the vasoactive intestinal peptide (VIP) has potent immunomodulatory effects on T cells. 12 Local gene therapy using an adenoviral construct encoding VIP could repair the salivary function in the model of SS. 13 Our former research found that treating with VIP in an SS model could reduce the immune injury of IL-17A to exocrine glands. 14 However, the molecular mechanism by which VIP signaling regulates the differentiation and function of T cells is still elucidated. 15 Phosphatase and Tensin homolog on chromosome 10 (PTEN), a tumor suppressor protein with dual specific protein and phospholipid phosphatase activity, can express ubiquitously and mediates cellular processes such as cell survival and apoptosis. 16 Mechanistically, PTEN maintains Treg cell stability and the metabolic balance between glycolysis and mitochondrial fitness. 17 Besides, PTEN acts as an antagonist of the phosphoinositide 3′ kinase (PI3K)/AKT pathway, thereby regulating the immune system. 18 The PI3K/AKT signaling pathway plays a pivotal role in balancing the Th17/Treg ratio and cell proliferation. 19 Both VIP and PTEN play an essential role in maintaining immune homeostasis. But whether there is a synergistic effect between the two is still unknown. In this study, we hypothesized that PTEN might involve in the underlining mechanisms of VIP in treating SS. The role of VIP in the regulation of the immune status and secretory function of salivary glands was examined in NOD mice and C57BL/6 mice. VIP and si-RNA targeting PTEN were used to intervene in splenic lymphocyte cells (SPLCs), and the intervention effect of VIP on the PTEN/PI3K/Akt pathway and the balance of Treg/Th17 was investigated.

| Animals
Eighteen female nonobese diabetic (NOD) mice and seven female C57BL/6 mice were accommodated in a specific pathogen-free facility. All mice were 8 weeks old and were raised under standard conditions (12 h light-dark cycle, 25-27°C, 40% humidity) with adequate water and food. At first, these samples acclimated for 1 week. A total of 21 mice with a group of seven mice were assigned. The NOD mice were divided randomly into a model group and a VIP therapy group, and the C57BL/6 mice were used as controls. The therapy group was given 1.5 nmol VIP (synthesized by China Peptides; purity ≥98%) via intraperitoneal (IP) injection every other day. The mice in the control and the model group were given pure water by IP injection. After VIP intervention for 8 weeks, mice were killed under an anesthetized state. The rest four NOD mice were used to isolate splenic lymphocyte cells (SPLCs) for preparation.

| Water intake, saliva flow rate, and histological analyses
The method of water intake and saliva flow rate were measured as published. 20 Saliva production was stimulated at a dose of 0.5 mg/kg pilocarpine (Yuanye Bio-Technology) through intraperitoneal injection. Water consumption was recorded weekly from Week 1 to Week 8, and the saliva flow rate was determined on Week 8. After removing and fixing with 4% paraformaldehyde in PBS, the submandibular glands (SMGs) were embedded in paraffin. Next, standard hematoxylin-eosin (H&E) staining was then directed on rehydrated paraffin segments. With the help of the BX43 microscope (Olympus), the Chisholm and Mason classification assessed the degree of lymphocytic infiltration of submandibular tissue. 21 PTEN, PI3K, and p-PI3K in SMGs were in immunohistochemical detection. Frozen tissue sections were embedded in optimum cutting temperature (OCT) compound and sectioned. After that, the tissues were incubated with anti-PTEN (ab267787; Abcam), anti-PI3 Kinase p85 alpha (ab225720; Abcam), anti-VIP (16233-1-AP; Proteintech), and after washes with goat anti-rabbit MaxVision Kit (MXB Biotechnologies), followed by DAB color reaction. Finally, the sections were counterstained with hematoxylin before dehydration and then cover-slipped. The brown staining represented a positive area. Using Image J software to define the pixels threshold to distinguish region of interest from background, the average optical density was measured by the percentage of the integrated optical density to the positive staining.
2.3 | Enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (qPCR) Spleen tissue was minced in PBS containing 1 mM PMSF and centrifuged at 12,000 g for 20 min at 4°C. The expression of IL-2 and IL-10 was measured using the Mouse IL-2 and Mouse IL-10 ELISA kit (both from MultiSciences) according to the manufacturer's instructions. Serum extraction method as published, 20 VIP levels in serum were measured using the mouse VIP ELISA kit (Elabscience). For qPCR analyses, total RNA from the spleen was individually isolated using Total RNA Extraction Reagent (Vazyme), and cDNAs were obtained using Hifair® II 1st-Strand cDNA Synthesis SuperMix (Yeason). Further quantitative analysis of genes was done by Hieff® qPCR SYBR Green Master Mix (Yeason) in a two-step reaction and performed using the QuantStudio 7 Flex (Thermo Fisher Scientific). Each sample was tested in triplicate, and the 2 −ΔΔ CT cycle threshold method was used for the analysis of relative gene expression. The sequences of qPCR primers are listed in Table 1 Primers sequences used for gene transcription levels determination.

| Statistical analysis
The Shapiro-Wilk normality and the KS normality test were used to check Gaussian-distribution of the data, all at T A B L E 1 Primers sequences used for gene transcription levels determination.

Gene name
Sequence (5′-3′) alpha >0.05. Unpaired student t-test (two-tailed) was used to analyze the differences between the two experimental groups. One-way analysis of variance (ANOVA) with Tukey′s multiple-comparisons test was used for comparing means between groups considering only one independent factor. All analyses were acquired with GraphPad Prism 8.2.1 using p < 0.05 as statistically significant. The results are expressed as the means ± standard deviation (SD). Each experiment was repeated at least three times.

| Exogenous VIP relieved lymphocytic infiltration in the SMGs
Since the development and pathogenesis of NOD mice could be defined by the spontaneous infiltration of salivary gland lymphocytes under dry oral conditions. 22 We chose the NOD mice whose sialadenitis in the SMGs as the SS mice. C57BL/6 mice, known to be resistant to autoimmune diseases, were selected as the control group. 23 The average water consumption of the model and VIP groups was elevated starting around 5 weeks and continued till 8 weeks. But only 8 weeks after intervention, the water intake of the VIP therapy group was significantly lower compared with the SS model group (p < 0.05) ( Figure 1A). As shown in Figure 1B-G, the lymphocytic infiltration and formed lymphocytic foci were seen in the model of SS group. In contrast, the SMG from the control group was represented as a normal salivary gland. Lymphocytic infiltration was mild in the VIP group. The Chisholm-Mason grade was applied to assess the lymphocytic infiltration ( Figure 1H). Mice receiving VIP therapy, had a better overall histological condition than those from the model group (p < 0.05).
Consistently, compared to the mice from model group in Figure 1I, more salivary flow rate was seen than those in the VIP group (p < 0.05). These results indicated that exogenous VIP could effectively ameliorate sialadenitis with dry mouth in the SMGs of SS mice.

| Exogenous VIP increased PTEN and VIP expression in the SMGs of SS mice
As shown in Figure 2A,C, PTEN protein expression was detected in the ductal cells of and the serous acini of the control group and VIP group but not in the model of SS group ( Figure 2B). In contrast, detectable PI3K immunostaining was in the serous acini of the model of SS group and VIP group (Figures 2E,F) but not in the control mice ( Figure 2D). The decreased saliva secretion and atrophy of the salivary gland are associated with pathological changes of SS. This might explain why we do not observe the expression of PTEN in the serous acini of the SS group. Similarly, numerous VIP positive staining surrounding a ductal cell was seen in the control group and VIP therapy group ( Figures 2G,I) but not in the model of SS group ( Figure 2H). Overall, PTEN and VIP expression in the SMGs was significantly increased after VIP treatment ( Figure 2J).

| VIP regulated PTEN/PI3K/AKT pathway, VIP/VIP receptor signaling, and anti-inflammatory cytokines in the spleen of SS mice
The VIP and VIP receptors signaling, as an immunomodulatory factor, may play a protective role in the pathogenesis of autoimmune diseases. PTEN negatively regulates the PI3K/AKT pathway, and the mRNA of the PTEN/PI3K/Akt pathway was determined in the spleens from these groups.
In comparison to the model group, the expression of PTEN, VIP, and VPAC1 mRNA was increased in the VIP group ( Figure 3A,B) (p < 0.05). Conversely, the expression of PI3K and AKT mRNA from VIP and the control groups was significantly decreased than those in the SS mice ( Figure 3B) (p < 0.05). As shown in Figure 3C, the serum levels of VIP in SS mice were significantly lower than those in the control and VIP groups (p < 0.05). IL-10 is a wide spectrum of antiinflammatory activity, and IL-2 performs therapeutic ability in treating autoimmune diseases. The levels of IL-2 and IL-10 in the spleen tissue homogenates from SS mice were significantly lower than those in the control and VIP group ( Figure 3D-E) (p < 0.05). These results indicated that exogenous VIP regulated PTEN/PI3K/AKT pathway, VIP/ VIP receptor signaling, and related Treg cytokines in the spleen of SS Mice.

| VIP increased the expression of phosphorylated-PTEN and decreased the phosphorylated PI3K/AKT pathway
Primary SPLCs were cultured in a complete medium for 24 h. Then CCK-8 assay was used to measure the cell viability after the inducement of different concentrations of VIP inducement (1000, 750, 500, 250, 125, 62.5, and 0 ng/ μL) 12 h or 24 h ( Figure 4A). According to the results of CCK-8, the cell viability after treating 12 h was higher than those in treating 24 h for this screening. Additionally, a dose-dependent splenic lymphocyte proliferation caused by VIP was not seen. Then, at concentrations of 125 ng/μL and treating for 12 h were chosen in the following experiments. As shown in Figure 4B, mpten931 was successfully transfected into SPLCs. VIP time-dependently increased the expression of phosphorylated-PTEN after silencing PTEN with mpten931 ( Figure 4C). As shown in Figure 4D-G, the results showed that PTEN knockdown decreased phosphorylated-PTEN and increased p-PI3K/ total PI3K and p-AKT/total AKT ratio in SPLCs. However, decreased expressions of PI3K/AKT pathway by VIP were not observed in SPLC cells. Nevertheless, reduced phosphorylation of PTEN and increased p-PI3K/total PI3K, and p-AKT/total AKT ratio by mpten931 were abolished in the treatment with VIP.

| VIP regulated Treg/Th17 cell balance via PTEN pathway
The proportion of Treg and Th17 cells in the SPLCs was measured by flow cytometry (Figure 5A,B). The Treg cells were identified as CD4+ CD25+ Foxp3+, while Th17 cells were identified as CD4+ IL17A+. As shown in Figure 5C,D, the results showed an increase of the proportion of Th17 cells and a decrease of the proportion of Treg cells in the SPLCs with mpten931. However, treatment of VIP decreased the frequency of Th17 cells, and increased the frequency of Treg cells ( Figure 5E). Our results indicated that PTEN knockdown decreased the ratio of Treg/Th17 cells, which was reversed with VIP treatment.

| DISCUSSION
In this study, we used NOD mice as the SS model and administered with VIP via intraperitoneal injection. The lymphocytic infiltration and an increased number of water consumption, as well as a decreased saliva rate, have been observed in the NOD mice. These results were basically consistent with the previous study about the SS model of mice. 24 VIP is considered an anti-inflammatory mediator that has multiple biological characteristics. It has been defined that VIP is involved in decelerating the progression of autoimmunity diseases, such as rheumatoid arthritis and osteoarthritis. 25 However, few studies investigate the therapeutic mechanism of VIP in treating SS. Here the alleviating effect of exogenous VIP was identified by clinical symptoms, histopathology, and antiinflammatory cytokines. IL-2 and IL-10 are related to cytokines in the anti-inflammation and functional Treg cells. Moreover, high-affinity IL-2 receptors are constitutively expressed on Treg cells, making these cell populations very sensitive. 26 Additionally, several studies have shown that VIP caused a progressive protection in the sialadenitis of NOD mice and upregulated the antiinflammatory factors. 27,28 Researchers have previously reported that VIP improved the secretory function and reduced the expression of IL-17A in a model of SS. 14 Thus, we did not specifically investigate the effects of VIP on the expression of IL-17A in NOD mice. Here, we revealed that exogenous VIP upregulated the levels of IL-2 and IL-10, as well as alleviated sialadenitis with dry mouth. These findings suggested that VIP may play the therapeutic role of SS. However, a broader panel of cytokines is still ongoing to investigate by which VIP altered in treating SS in the future work, such as interferon-γ and BAFF. The underlying mechanisms of VIP treatment in the disease are complex, and more evidence is needed.
SS is commonly known to be correlated with lymphomas, 29 which is the most severe complication associated with local chronic inflammatory stimulation of B cells. B cell lymphoma in SS frequently occurs in salivary glands, whereas PTEN suppresses tumor cell growth and survival. Soypacaci et al. 30 have reported that PTEN protein is expressed in 87.2% of SS outpatients in the salivary gland in a retrospective evaluation. However, the disadvantage of this research is the absence of a healthy control group. Here, our results indicated that PTEN has lower expression in salivary glands from the SS mice than those in the healthy group. Additionally, VIP therapy promoted the expression of PTEN in vivo and in vitro. Thus, the F I G U R E 3 Vasoactive intestinal peptide (VIP) upregulated PTEN, VIP receptor mRNA expression, and Treg-related cytokines in spleen. Q-PCR results for VIP mRNA, VIP receptors mRNA expression (A), and PTEN/PI3K/AKT pathway (B) in the spleen. The concentrations of VIP in the serum (C) were measured using ELISA kits (n = 7). The abundance of interleukin (IL)-10 (D) and IL-2 (E) in the splenic tissue homogenate supernatant was determined by ELISA as well (n = 7). Unpaired Student t-test was used in the analysis. *p < 0.05, **p < 0.01, ***p < 0.001. possibility of a synergistic effect exists between VIP and PTEN. Notably, PTEN antagonizes the PI3K-AKT pathway, which is a classic pro-inflammatory signaling pathway. The PI3K/AKT pathway enhances the levels of pro-inflammatory cytokines and chemokines, as well as regulates immune activities. In this study, inhibition of the PI3K/AKT pathway and increased VIP receptor VPAC1 by VIP intervention were observed in NOD mice. It agrees with a previous study that the activation VIP-receptor system decreases AKT activation. 31 Interestingly, accumulating data have demonstrated that activation of the PI3K/AKT pathway is involved in modulating the T cell subsets, including the balance of Treg/Th17 cells. 32,33 The function and differentiation of T cells are precisely, thus avoiding the impairment of normal organizations. In abnormal conditions, T-cell activation can induce inflammation and local damage. Therefore, the role of T cell suppression arouses widespread investigation for the treatment of autoimmune and chronic inflammation diseases. The imbalance of Treg/Th17 cells has been proven to be closely associated with SS, owing to the regulation of the immune system. Earlier studies have revealed an increase in the pathogenic Th17 cells in patients, which contribute to the pathogenesis of SS. 11 Since Treg cells are the main suppressors of the excessive activation of immune responses, the recent advances in the therapeutic strategy of SS are to target Treg homeostasis or its associated signaling pathway. 34 Here we found that PTEN knockdown decreased the Treg/Th17 ratio in SPLCs, and that VIP treatment increased the Treg/Th17 ratio and the expression of PTEN. The schematic figure incorporating these signaling pathways is shown in Figure 6. Our advancements might offer a theoretical foundation for the mechanistic understanding of VIP in treating SS. Additionally, the PI3K/AKT/mTOR pathway assumes a F I G U R E 5 The proportions of Treg and Th17 cells: The CD4+ events are shown on the plots. After splenic lymphocytes transfection, representative flow cytometry analysis of Foxp3+ CD25+ CD4+ Treg (A) and IL17A+ CD4+ Th17 (B) in ex vivo culture with 0 or 125 ng/μL vasoactive intestinal peptide (VIP) for 12 h. The frequency of Treg cells (C), Th17 (D), and the ratio of Treg/Th17 (E). *p < 0.05, **p < 0.01, ***p < 0.001.
F I G U R E 6 The schematic figure incorporating the signaling pathways. The red arrow represents promotion; the green arrow represents inhibition. vital role in modulating the autophagic reaction, and PTEN has pro-autophagic activities 35 Thus, whether VIP can protect the function of SMG cells by regulating the autophagy of the SMG epithelium is also worth paying attention to.
In conclusion, VIP exerts potential therapeutic action in SS by upregulating PTEN, acting partly through the PI3K/AKT pathway and the balance of Th17/Treg cells. Our findings may promote VIP as the potential novel SS therapeutics intervention.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
All mice care procedures and experiments were according to the ethical approval granted by the Animal Ethics Committee of Nanjing University of Chinese Medicine (No. 202101A006).