CXCL9, 10, 11/CXCR3 Axis Contributes to the Progress of Primary Sjogren’s Syndrome by Activating GRK2 to Promote T Lymphocyte Migration

Primary Sjogren’s syndrome (pSS) is a systemic autoimmune disease that causes dysfunction of secretory glands and the specific pathogenesis is still unknown. The CXCL9, 10, 11/CXCR3 axis and G protein-coupled receptor kinase 2 (GRK2) involved in many inflammation and immunity processes. We used NOD/Ltj mice, a spontaneous SS animal model, to elucidate the pathological mechanism of CXCL9, 10, 11/CXCR3 axis promoting T lymphocyte migration by activating GRK2 in pSS. We found that CD4 + GRK2, Th17 + CXCR3 was apparently increased and Treg + CXCR3 was significantly decreased in the spleen of 4W NOD mice without sicca symptom compared to ICR mice (control group). The protein levels of IFN-γ, CXCL9, 10, 11 increased in submandibular gland (SG) tissue accompanied by obvious lymphocytic infiltration and Th17 cells overwhelmingly infiltrated relative to Treg cells at the sicca symptom occurs, and we found that the proportion of Th17 cells was increased, whereas that of Treg cells was decreased in spleen. In vitro, we used IFN-γ to stimulate human salivary gland epithelial cells (HSGECs) co-cultured with Jurkat cells, and the results showed that CXCL9, 10, 11 was increased by IFN-γ activating JAK2/STAT1 signal pathway and Jurkat cell migration increased with the raised of cell membrane GRK2 expression. HSGECs with tofacitinib or Jurkat cells with GRK2 siRNA can reduce the migration of Jurkat cells. The results indicate that CXCL9, 10, 11 significantly increased in SG tissue through IFN-γ stimulating HSGECs, and the CXCL9, 10, 11/CXCR3 axis contributes to the progress of pSS by activating GRK2 to promote T lymphocyte migration.


INTRODUCTION
Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease that primarily affects the exocrine glands, with lymphocytes infiltrating, clinically characterized by eyes and mouth dryness [1]. Extraglandular manifestations also are prevalent in pSS, including cutaneous, pulmonary, renal, hematological, and neurological involvement [2,3]. At present, the specific pathogenesis of pSS is still unknown. The ductal cells of the salivary gland in pSS are characteristically surrounded by T cells and B cells infiltrate, T cells are overwhelming infiltrators in most phases of the disease, and the involvement of multiple T cell subsets suggests the extraordinary complexity of pSS pathogenesis [4,5].
The pathogenesis of pSS is intricate and it is mainly considered to be related to genetic, immune, and environmental factors at present [6]. The high expression of interferon-γ (IFN-γ) in submandibular gland (SG) tissue of pSS patients may be related to the pathogenesis of pSS and it plays an indispensable role in innate and adaptive immunity [7]. Chemokines are secreted proteins with low molecular weight (7-12 kDa), which play an important role in the inflammatory process through recruitment. IFN-γ induced chemokine subgroups include CXCL9/monokine induced by interferon (IFN)-γ (Mig), CXCL10/interferon-γ-induced protein-10 (IP-10), and CXCL11/interferon-inducible T cell α chemoattractant (I-TAC); CXCL9, 10, 11 are ligands of Chemokine receptor 3 (CXCR3) [8]. CXCL9, 10, 11 were significantly increased in exocrine glands tissue of pSS patients [9,10]. In SG tissues, recruited T lymphocytes may be responsible for enhanced IFN-γ production, which in turn stimulates CXCL9, CXCL10, and CXCL11 secretion from various cells, thus generating an amplification feedback loop and perpetuating the autoimmune process [11]. IFN-γ can promote the secretion of CXCL9, 10, 11 through activation of JAK2/ STAT1 signal pathway [12], and the specific mechanism of IFN-γ promoting CXCL9, 10, 11 secretion is still unclear in pSS. To clarify the role of the JAK2/ STAT1 pathway in the increase of CXCL9, 10, 11 in gland tissue of pSS patients, we used IFN-γ to stimulate Human submandibular gland epithelial cells (HSGECs) with tofacitinib (TOF), a pan-JAK inhibitor that blocks JAK1/2/3 kinase activity [13], in vitro.
CXCR3 is a 7-transmembrane domain G proteincoupled receptor, which is mainly expressed on immune cells, including T lymphocytes and natural killer cells [14,15]. CXCR3 plays an important role in T cell recruitment in various immune responses and autoimmune diseases [16,17]. The CXCL9, 10, 11/CXCR3 axis is mainly used for migration, differentiation and activation of immune cells [18]. The submandibular gland tissue of SS patients is infiltrated by a large number of T lymphocytes, and Th17 cells accounted for a large proportion, which may be related to the CXCL9, 10, 11/CXCR3 axis differential regulation of Th17 and Treg cells. G protein-coupled receptor kinase 2 (GRK2) are a family of seven soluble receptor modifying enzymes which are the basic regulators of GPCR activity [19]. GRK2 plays an irreplaceable role in CXCL9, 10, 11/CXCR3 axis for T lymphocytes migration, differentiation and activation [20].
To clarify the pathogenesis of CXCL9, 10, 11/ CXCR3 axis promotes T lymphocyte migration by activating GRK2 in pSS, we study pSS by following spontaneous disease development and progression in non-obese diabetic (NOD) mice. The expression of IFN-γ, CXCL9, 10, 11, Th17, and Treg cells was detected in submandibular gland tissues of NOD mice at different weeks of age, and the expression of CXCR3 and GRK2 on CD4 + T cells, Th17 and Treg cell membranes in spleen was detected. In vitro experiment, the effect of CXCL9, 10, 11/CXCR3 axis promotes Jurkat cell migration by activating GRK2 was studied by co-culture of HSGECs and Jurkat T cells.

Animals
The female NOD.ShiLtJ mice (commonly termed NOD) mice were used as the model of primary Sjögren's syndrome, NOD.ShiLtJ mice are an inbred strain developed by selection for diabetes from ICR mice, and ICR mice were used as controls [21]. All animals were purchased from Huafukang Biotechnology Co., Ltd., Beijing China. These mice were housed under specific pathogenfree conditions at the Institute of Clinical Pharmacology, Anhui Medical University. The room temperature was maintained at 25 °C with a 10:14-h light/dark cycle (lights on at 08:00 and off at 18:00). These mice had free access to standard mouse chow and water ad libitum. The experiments were approved by the Ethical Review Committee of Animal Experiments, Institute of Clinical Pharmacology, Anhui Medical University.

Measurement of salivary flow rate
The mice were anesthetized by intramuscular injection of 2.5 mg/ml tiletamine hydrochloride and zolazepam hydrochloride (0.06 ml/20 g) and injected intraperitoneally with pilocarpine (0.1 ml/20 g) to induce saliva secretion. Saliva was collected from the oral cavity 2 min later using preweighed sterile cotton balls for 10 min and then weighed. The amount of saliva was taken as the weight difference before and after soaking the cotton balls. Salivary flow rate is expressed as mg/10 min.

Salivary gland indexes and H&E staining analysis
The mice were weighed before sacrifice, and then submandibular glands (SG) were surgically removed and weighed under sterile conditions. The submandibular gland index (mg/g) = submandibular gland weight (mg)/ body weight (g). SG tissues from the mice were fixed in 4% paraformaldehyde, embedded in paraffin. The embedded tissues were sectioned at 5 μm thickness and mounted onto microscope slides. Slides were de-paraffinized by immersing in xylene then dehydrated in ethanol. Sections were stained with hematoxylin and eosin (H&E), and observed at 10 × and 40 × magnification. The grade of score was determined according to the severity of lymphocyte infiltration in SG tissues. The degrees of lymphocytic infiltration of the salivary gland were scored from 0 to 4 as described by Chisholm and Mason. Grade 0: absence of lymphocytes infiltration; grade 1: slight infiltrate of lymphocytes; grade 2: moderate infiltrate of lymphocytes or less than one focus per 4 mm 2 (focus was defined as an aggregate of 50 or more lymphocytes and histiocytes); grade 3: one focus per 4 mm 2 ; grade 4: more than one focus per 4 mm 2 [22].

Immunohistochemistry
After deparaffinization, paraffin sections were dehydrated with ethanol. The tissue sections were incubated with IFN-γ, CXCL9, CXCL10, and CXCL11antibodies overnight at 4 °C, then stained with horseradish peroxidase (HRP) labeled goat anti mouse/rabbit IgG polymer. Antibodies used are listed in online supplementary table 1. The staining was visualized using DAB and counterstaining was performed with hematoxylin, and observed at 20 × magnification.

Immunofluorescence confocal microscopy
The paraffin section of SG tissue was incubated with CD4 (488 nm), CD25 (647 nm), FoxP3 (594 nm), IL-17 (647 nm), ROR-γ (594 nm) antibody, then stained with DAPI (401 nm), and then added Mounting Medium antipadding coverslipping. Antibodies used are listed in online supplementary table 1. SG tissue images were captured with a LEICA TCS SP8 confocal microscope (Leica, Germany) equipped with 63 × oil immersion objective. The average fluorescence intensity was used to analyze the positive staining via ImageJ 6.0 software.

Cell culture
HSGECs were purchased from the Zhejiang Mingzhou (Ningbo, China). Jurkat Clone E6-1 cells were obtained from Procell Life Science&Technology Co., Ltd. These cells were growing in the RPMI-1640 containing 10% FBS and penicillin/streptomycin (1%). The cells were maintained under a humidified atmosphere of 95% air and 5% CO2 at 37 °C. HSGECs were stimulated with IFN-γ (20 ng/ml) and cultured for 48 h with or without tofacitinib (TOF). Protein was extracted for Western blot analysis.

GRK2 siRNA transfection
According to the manufacturer's protocol, GenePharma siRNA Mate™ transfection reagent was dissolved in serum-free RPMI-1640, mixed with GRK2 siRNA after 5 min of stationary culture; the transfection effect of Jurkat cells was detected 48 h later.

Flow cytometry
Fresh spleens were collected from mice and processed in a plastic dish with sterile Mouse 1 × Lymphocyte Separation Medium to obtain a single-cell suspension. Cells were stained for surface antigens, fixed, permeabilised (fixation-permeabilisation buffer) and stained for intracellular cytokines. Antibodies used are listed in online supplementary table 1. CD4 + , Th17cells (CD4 + IL-17 + ROR-γ +), Treg cells (CD4 + CD25 + FoxP3 +), and GRK2 and CXCR3 on the cell surface were detected of spleen in Mice by Cytoflex ten color flow cytometer (BectmanCoulter, America). In vitro, the expression of GRK2 and CXCR3 on the surface of Jurkat cells in the upper chamber of Transwell was detected by Cytoflex ten color flow cytometer. Data of flow cytometry experiment were analyzed with cytExpert (BectmanCoulter, America) and FlowJo (Becton, Dickinson and Company, America) software.

Western blot analysis
SG tissues and cells were lysed in lysis buffer (RIPA: PMSF: Phosphatase inhibitors = 100: 1: 2). Lysates were centrifuged at 12,000 rpm for 15 min at 4℃ and the supernatant was collected. Then, the quantified protein was mixed with loading buffer (Beyotime, China) at a ratio of 4:1 and heated for 10 min. Samples were separated on a sodium dodecyl sulfate polyacrylamide gel and electrotransferred to PVDF nitrocellulose membranes, the membrane was blocked in 5% skim milk before being incubated with primary antibodies CXCL9, CXCL10, CXCL11, JAK2, p-JAK2, STAT1, p-STAT1 and β-actin (1:1000) at 4℃ overnight. Antibodies used are listed in online supplementary table 1. The membrane was washed and incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The membranes were finally imaged on an ImageQuant LAS 500 imager (GE Health care Systems, America) after applying ECL substrate, and the bands were semiquantified with ImageJ software.

Statistical analysis
The results are expressed as the mean ± SD. The data analysis used one-way ANOVA to compare the differences between groups; the pathological score of SG tissue was determined by Mann-Whitney U test. Statistical analyses were conducted. Spearman correlation analyses were used to assess for linear and nonlinear relationships and to ensure outliers would not obscure any existing correlations. p < 0.05 was regarded as statistically significance. The calculation was performed using IBM SPSS 26.0. The figures were constructed by GraphPad Prism 7.0.

NOD mice could be used as pSS animal models
Saliva secretions were stimulated by intra-peritoneal injections of a pilocarpine solution, we measured temporal changes in the saliva flow rates in NOD and ICR mice. We found the salivary flow rate no significant difference at 4 weeks of age in ICR and NOD mice. At 12 weeks of age, it showed a significantly decrease compared to salivary flow rates at 4 weeks of age, and we found that 12W and 16W NOD mice the salivary flow rate showed a significantly decrease compared with ICR mice of the same age ( Fig. 2A). NOD mice began to show tongue licking, scratching lips, and skin damage at the age of 12 weeks.
NOD mice were sacrificed at 4W, 8W, 12W, and 16W respectively. We measured the weight of the SG tissue to calculate the submandibular gland index, and then stained the SG tissue section with hematoxylin and eosin, and observed the pathology of the SG tissue under the microscope. We found that the submandibular gland index of 12W and 16W NOD mice increased significantly compared with ICR and 4W NOD mice (Fig. 2B), H&E staining pathological sections showed that there were severely lymphocytic infiltrates in SG tissues of 12W and 16W  , and ICR mice, the submandibular gland index (mg/g) = submandibular gland weight (mg)/body weight (g). C H&E staining of mice submandibular gland tissue, yellow arrow represents lymphocyte infiltration. Stained sections were observed at 10 × and 40 × magnification. D Pathological score of SG tissue and significance analysis of pathological score of mice with Mann-Whitney U test. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 vs ICR; #P < 0.05, ##P < 0.01 vs NOD 4W.   4 We analyzed correlation between the pathological score of SG tissue in mice and CD4 + GRK2, Th17 + CXCR3 in spleen by IBM SPSS 26.0. A Spearman correlation analysis between CD4 + GRK2 and pathological score. B Spearman correlation analysis between Th17 + CXCR3 and pathological score. C Linear regression analysis of CD4 + GRK2 and pathological score. D Linear regression analysis of Th17 + CXCR3 and pathological score. p < 0.05 was regarded as statistically significance. p values were denoted as follows: *P < 0.05, **P < 0.01. CXCL9, 10, 11/CXCR3 axis contributes to the progress of primary Sjogren's... NOD mice, and the infiltration mainly occurred around the ducts of SG tissues (Fig. 2C, yellow arrow), and this consistent with the features observed in salivary and lacrymal glands of human pSS. According to Chisholm and Mason evaluation criteria, we scored the pathology of SG tissues, and then used Mann-Whitney U test to analyze the statistically significance of pathological scores. We found that the pathological scores of 12W and 16W NOD mice were significantly higher than those of ICR and 4W NOD mice (Fig. 2D). To sum up, we believe that NOD mice have obvious sicca symptom in 12 weeks.

The expression of GRK2 on CD4 + T cells was upregulated; CXCR3 expression on Th17 cells increased compared with Treg cells
We analyzed the populations of CD4 + GRK2 + , CD4 + CXCR3 + , Th17 cells (CD4 + IL-17 + ROR-γ +), Treg cells (CD4 + CD25 + FoxP3 +), Th17 + CXCR3 + , and Treg + CXCR3 in splenocytes of ICR mice and 4W, 8W, 12W, 16W NOD mice by flow cytometry. Compared with ICR mice, the expression of GRK2 on CD4 + T cells membrane was obviously increased in NOD mice without sicca symptoms (4W and 8W NOD mice), and the expression of GRK2 on CD4 + T cells membrane was significantly increased in NOD mice with sicca symptoms (12W and 16W NOD mice) compared to 4W NOD mice (Fig. 3A). However, the expression of CXCR3 on CD4 + T cells membrane has no significant difference in splenocytes of ICR and NOD mice (Fig. 3B). CXCR3 expression on Th17 membrane cells in spleen of NOD mice was significantly increased, and that on Treg cells membrane was significantly decreased compared to ICR mice (Fig. 3C, D), and CXCR3 expression on Th17 cells membrane of NOD mice was significantly higher than that on Treg cells membrane (Fig. 3G). Th17 cells in spleen of 16W NOD mice were significantly increase compared to ICR and 4W NOD mice (Fig. 3E), and Treg cells were significantly decrease (Fig. 3F). The proportion of Th17/Treg cells in 16W NOD mice was remarkably elevated compared to ICR and 4W NOD mice (Fig. 3H).

Correlation of the pathological score of SG tissue in mice with CD4 + GRK2, Th17 + CXCR3 in splenocytes
To further determine the relationship between CD4 + GRK2, Th17 + CXCR3 in splenocytes and the pathological score of SG tissue in mice, we used correlation analysis and linear regression to analyze quantitative and ranked data. We found that the pathological score is significantly correlated with CD4 + GRK2, Th17 + CXCR3 (Fig. 4A, B). With the increase of CD4 + GRK2 and Th17 + CXCR3 in mice spleen, there was a higher pathological score of SG tissue in mice (Fig. 4C, D). We can find the rising trend which shows the elevated frequency of CD4 + GRK2 and Th17 + CXCR3 could contribute to increased severity of lymphocytic infiltration in SG tissue, which is of great significance to the development of SS.
IFN-γ promotes HSGECs to secrete CXCL9, CXCL10, CXCL11 by activating JAK2/STAT1 pathway IFN-γ as a stimulator of CXCL9, CXCL10, and CXCL11 is highly expressed in salivary glands of pSS patients and animal models. Then, we used IFN-γ as a stimulus to study IFN-γ promote CXCL9, 10, 11 expressions by activating JAK2/STAT1 pathway in HSGECs. We found that after IFN-γ (20 ng/ml) stimulation of HSGECs for 24 h, compared with the control group without IFN-γ stimulation, the Phospho-JAK2, Phospho-STAT1, CXCL9, 10, 11 secreted by HSGECs was significantly increased, and after TOF administration, phosphorylation JAK2 and phosphorylation STAT1 were significantly inhibited, resulting in a significant reduction of CXCL9, 10 and 11 (Fig. 5A-D).  5 The changes of JAK2/STAT1 pathway protein and CXCL9, 10, 11 protein level of HSGECs stimulated by IFN-γ, and the changes of corresponding protein content in mice SG tissues were detected by Western blot and immunohistochemistry. A Western blot was used to detect the protein contents of p-JAK2, JAK2, p-STAT1, STAT1, CXCL9, CXCL10, and CXCL11 in HSGECs stimulated or not stimulated by IFN-γ and with or without tofacitinib. B-D Use ImageJ to quantitatively analyze HSGECs Western blot protein bands, and then use GraphPad Prism6.0 to statistical analysis and make statistical charts. E Western blot detection of CXCL9, CXCL10, CXCL11 protein content in SG tissues of ICR and 4W, 8W, 12W, 16WNOD mice. F Use ImageJ to quantitatively analyze Western blot protein bands in mouse SG tissues, and then use GraphPad Prism6.0 to make statistical analysis and make statistical charts. G Immunohistochemical detection of IFN-γ, CXCL9, CXCL10, CXCL11 protein levels in SG tissues of ICR and 4W, 8W, 12W, 16W NOD mice, the yellow in the tissue represents the corresponding protein antibody. Stained sections were observed at 20 × . Data are presented as mean ± SEM. B-D *P < 0.05, **P < 0.01 vs control, *P < 0.05, **P < 0.01 vs IFN-γ (20 ng/ml). F *P < 0.05, **P < 0.01 vs ICR; #P < 0.05, ##P < 0.01 vs NOD 4W. CXCL9, 10, 11/CXCR3 axis contributes to the progress of primary Sjogren's...
Western blot detected the protein levels of CXCL9, 10, 11 in SG tissues of ICR and 4W, 8W, 12W, 16W NOD mice (Fig. 5E). We found that the protein levels of CXCL9, 10, 11 in SG tissues of 12W and 16W NOD mice was significantly increased compare to ICR and 4W NOD mice (Fig. 5F). The levels of IFN-γ, CXCL9, 10, 11 proteins in SG tissue of mice detected by immunohistochemistry. We found that IFN-γ, CXCL9, 10, 11 proteins in SG tissue of 12W and 16W NOD mice were obviously upregulated compare with ICR, 4W and 8W NOD mice (Fig. 5G). Overall, these data confirm that IFN-γ significantly increased CXCL9, CXCL10 and CXCL11 in SG tissues of NOD sicca symptoms mice by activating JAK2/ STAT1 pathway.

CXCL9, 10, 11/CXCR3 axis promotes Jurkat cell migration by activating GRK2
HSGECs and Jurkat cells were co-cultured to analyze the role of CXCL9, 10, 11/CXCR3 axis and GRK2 in T cell migration. We used GRK2 siRNA to successfully reduce the expression of GRK2 on the membrane of Jurkat cells for subsequent experiments (Fig. 6A). In the co-culture system of HSGECs and Jurkat cells, we found that the expression of GRK2 on Jurkat cell membrane in IFN-γ stimulated group was significantly increased, and GRK2 in TOF group was significantly reduced to the normal level (Fig. 6B), while the expression of CXCR3 on Jurkat cell membrane no significant difference in each group (Fig. 6C). The migration of Jurkat cells in IFN-γ stimulated group was significantly increased, while the migration of Jurkat cells in GRK2 siRNA group and TOF group was significantly decreased (Fig. 6D).
By detecting the expression of Th17 cells and Treg cells in the SG tissues of ICR and 4W, 8W, 12W, 16W NOD mice with immunofluorescence, we found that CD4 + T cells, Th17 cells and Treg cells in the SG tissue of 12W and 16W NOD mice were significantly increased (Fig. 6E, F). Using ImageJ software to analyze the average fluorescence intensity of SG tissue fluorescence charts, the results showed that compared with ICR and 4W NOD mice, Th17 cells and Treg cells in 12W and 16W NOD mice were significantly increased (Fig. 6G,  H), and Th17 cells were significantly higher than Treg cells (Fig. 6I).

DISCUSSION
pSS is a prototypic autoimmune disorder, it is characterized by immune mediated injury of salivary and lacrimal glands, resulting in dry mouth and dry eyes, pSS mainly affects middle-aged women, and a 9:1 female to male sex ratio [23,24]. As a systemic disease, SS can affect almost any organ system, resulting in polymorphic clinical manifestations [25]. This disease has a great negative impact on the daily activities of patients. Similar to other autoimmune diseases, the etiology of pSS is still unclear. It is generally believed that it is mainly related to environmental factors, genetic, epigenetic factors, epithelial cells, innate immunity and adaptive immunity [26,27].
Congenital immune and adaptive immune disorders play a key role in the pathogenesis of SS through mechanisms involving interferon (IFN) pathway. Although type I IFNs are considered to be the main contributor to the pathogenesis of SS, recent data indicate that type II IFNs (IFN-γ) play a universal role in the pathogenesis of SS, subjects with IFN-γ activity have higher pathological scores, and the presence of inflammatory infiltration is closely related to IFN-γ activity [27][28][29][30]. In SS patients, IFN-γ can produce several cytokines and chemokines by acting on salivary gland epithelial cells (SGECs), leading to the recruitment and activation of T cells and B cells, thereby magnifying the inflammatory reaction and making the autoimmune process permanent [5,11].
Chemokines are small proteins that can induce chemotaxis and combine with chemokine receptors to promote the migration and differentiation of immune cells and cause tissue exosmosis [18]. In animal experiments, we found that IFN-γ, CXCL9, CXCL10, CXCL11 in SG tissues of NOD mice were significantly increased when sicca symptom occurred, accompanied by a large number of lymphocyte infiltration, and the salivary flow rate of mice was significantly reduced. It consistent with the features observed in clinical that infiltrated lymphocytes leading to xerostomia and keratoconjunctivitis sicca are the characteristics of human pSS [31]. In vitro experiment, we verified that IFN-γ promotes the generation of CXCL9, CXCL10, and CXCL11 through the JAK2/ STAT1 signaling pathway in HSGECs. CXCL9, 10, 11 have the same receptor CXCR3, CXCL9, 10, 11/CXCR3 axis mainly regulates the migration, differentiation and activation of immune cells [18].
GRK2 mainly acts as a phosphorylated and desensitized ligand activated G-protein coupled receptor. In addition, GRK2 responds to extracellular stress by promoting cell communication, thereby helping maintain internal balance [32,33]. In HSGECs and Jurkat cells were co-cultured experiments, we found that GRK2 played a significantly role in CXCL9, 10, 11/CXCR3 axis migration of Jurkat T cells. The increased of GRK2 on Jurkat T cell membrane contributes to cell migration. After we used GRK2 siRNA on Jurkat cells, Jurkat cell migration decreased significantly. GRK2 on CD4 + T cell membrane was obviously elevated, and CXCR3 on Th17 cell membrane was significantly higher than that on Treg cell membrane in NOD mice. This caused a great quantity of proinflammatory lymphocyte infiltration, when the SG tissue chemokines of NOD mice increased significantly, such as Th17 cells infiltrating into SG tissue, and the infiltrating pro-inflammatory lymphocytes will secrete pro-inflammatory cytokines to form a vicious circle, leading to the permanence of autoimmunity. Massive lymphocyte infiltration leads to decreased secretion of the affected gland [34,35]. We found that the increase of CD4 + GRK2, Th17 + CXCR3 in mice spleen promoted lymphocyte infiltration into SG tissue, and a large number of Th17 cells appear in SG tissues and peripheral tissues of mice, Treg cells are reduced, and the proportion of Th17/Treg cells is out of balance, it promotes the progression of pSS. We found that with the increase of CD4 + GRK2 and Th17 + CXCR3 in mice spleen, there was a higher pathological score of SG tissue in mice by correlation analysis. Then, we believe that IFN-γ accelerate CXCL9, 10, 11/CXCR3 axis to contribute to the development of pSS by activating GRK2 to promote T lymphocyte migration.
In conclusion, through the construction of an animal model of spontaneous Sjogren's syndrome in NOD mice and cell experiments, we proved that CXCL9, 10, 11/ CXCR3 axis participated in the regulation of T lymphocyte migration by activating GRK2 contribute to the pathogenesis of pSS. Inhibition of CXCL9, 10, 11/CXCR3 axis or GRK2 can reduce the migration of Jurkat T lymphocytes. This study clarified the mechanism of CXCL9, 10, 11/ CXCR3 axis promotes T lymphocyte migration by activating GRK2 under the action of IFN-γ, which contributes to the progress of pSS. It provided an experimental basis for the inhibition of CXCL9, 10, 11/CXCR3 axis or GRK2 as a potential therapeutic target of autoimmune disease pSS.