Contributed equally
Interleukin (IL)-12 modulates the generation and function of various immune cells and plays a vital role in the pathogenesis of Sjögren's syndrome (SS). Myeloid-derived suppressor cells (MDSCs) are involved in autoimmune diseases by regulating various immune responses. However, it has not been confirmed whether inflammatory IL-12 participates in the progression of SS via regulating MSDCs. In the present study, the plasma levels of IL-12 were detected by ELISA in SS-like non-obese diabetic (NOD) mice. The mice were treated by intraperitoneal injection of IL-12 and anti-IL-12 antibody, respectively, and then the salivary flow rate was detected. The pathology of submandibular glands was evaluated in tissue sections stained with hematoxylin and eosin. The proportion of MDSCs was assessed by flow cytometry. The results showed that plasma IL-12 was significantly increased in the SS-like NOD mice comparing with that noted in the control mice. The exogenous IL-12 exacerbated SS-like symptoms of NOD mice and promoted the generation of both bone marrow (BM) and splenic MDSCs in the SS-like NOD mice. Of note, anti-IL-12 alleviated SS-like symptoms of NOD mice and inhibited the generation of BM and splenic MDSCs. Moreover, the generation of MDSCs was crippled in the IL-12-deficient C57BL/6 (
Sjögren's syndrome (SS), a common chronic systemic autoimmune disease with mononuclear infiltration in exocrine glands, is typically characterized by dry mouth and eyes (
Interleukin (IL)-12, consisting of two subunits, namely IL-12p40 and IL-12p35, has a powerful impact on the T cell responses in inflammation (
Myeloid-derived suppressor cells (MDSCs) were initially described as a heterogeneous population of immature myeloid cells with immune-regulating activity (
Previous studies have demonstrated that IL-12 plays a pro-inflammatory role in the pathogenesis of SS. IL-12 was reported to promote the recruitment of MDSCs and impair their suppressive function (
In the present study, increased plasma IL-12 was demonstrated in NOD mice which is one of the most accurate models in deciphering the pathologic mechanisms of SS (
In total, five female NOD mice (weight, 15–17 g; age, 3 weeks), 35 female NOD mice (weight, 19–22 g; age, 7 weeks), five female ICR mice (weight, 22–25 g; age, 3 weeks), five female C57BL/6 (B6) mice (weight, 22–25 g; age, 3 weeks) and 25 female
Non-obese diabetic (NOD) mouse as an appropriate model of Sjögren's syndrome (SS) has biochemical and immunological similarities with human SS (
The salivary flow rate of mice was measured as previously described (
For histological analysis, submandibular glands (SGs) were fixed with 4% paraformaldehyde at 4°C for 24 h, embedded in paraffin and cut into 3-µm-thick sections. Tissue sections were stained with hematoxylin and eosin (H&E). Each staining step was performed at room temperature. Images were captured using a light microscope (Olympus FSX100; Olympus Corporation; magnification, ×10).
Spleen single-cell suspensions were prepared after lysing red blood cells. The appropriate number of cells was pre-incubated with antibodies (eBioscience) in optimal concentration. For analysis of MDSCs, cells were pre-incubated with surface marker antibodies, including anti-mouse CD11b-APC and anti-mouse Gr1-PE and then analyzed on a FACS Calibur flow cytometer (BD Biosciences, Mountain View, CA, USA).
For analysis of the CD3+IFNγ+ T cells, the cells were incubated with 20 ng/ml phorbol myristate acetate (PMA) plus 1 µg/ml ionomycin with 5 µg/ml of brefeldin A (Enzo Life Sciences, Inc., East Farmingdale, NY, USA) at 37°C for 4 h. First, surface CD3 with anti-mouse CD3-APC was stained. Subsequently, cells were fixed with Cytofix/Cytoperm solution (BD Pharmingen), and then incubated with anti-mouse IFN-γ-PerCP-Cyanine 5.5 (eBioscience; clone no. XMG1.2; cat. no. 16-7311-81) and analyzed on a FACSCalibur flow cytometer (BD Biosciences).
According to the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc.), cells from the spleen of 12-week old female NOD mice were labeled with CFSE. CFSE-labeled splenocytes were co-cultured without or with MDSCs at ratios of 2:1, 4:1 and 8:1 in 96-well round-bottom plates and were stimulated with the anti-CD3/CD28 antibody for 3 days. CFSE-labeled CD3+ T cells were analyzed using a FACSCalibur flow cytometer.
In the IL-12 treatment experiments, female 11-week-old NOD mice were injected with 40 µg/kg IL-12 (BioLegend) once a day for a total of 7 times or with the same volume of PBS.
In the anti-IL-12 treatment experiments, female 11-week-old NOD mice were injected with 4 mg/kg anti-IL-12 (BioLegend) once intraperitoneally (i.p.) and with commensurable Rat IgG2a isotype.
IL-12 consists of two subunits, IL-12p40 and IL-12p35, to the formation of the biologically active p70 compound. In our study, plasma IL-12p70 was assessed by a standard mouse sandwich ELISA kit (R&D Systems) according to the manufacturer's instructions.
The results of 3 independent experiments are presented as mean ± SEM. The two experimental groups were analyzed using Student's t-test. One-way ANOVA followed by the Least Significant Difference post hoc test were used to account for multiple comparisons. A P-value <0.05 was considered to indicate statistical significance.
To confirm the role of IL-12 in SS, plasma IL-12 was detected in the NOD mice and ICR control mice. The results showed that plasma IL-12 was significantly increased in the NOD mice with SS-like symptoms comparing with that noted in the ICR mice (
Previous studies have reported that MDSCs are significantly involved in autoimmune diseases by regulating various immune responses (
To verify the effects of IL-12 on the generation of MDSCs, NOD mice were treated i.p. with or without anti-IL-12 antibody, respectively, according to the treatment schedule (
To determine the regulatory effects of IL-12 on MDSCs, the percentages of MDSCs were detected in IL-12-deficient C57BL/6 (
The immunosuppressive function is an important characteristic of MDSCs. To confirm that CD11b+Gr-1+ cells that were determined are MDSCs in this study, T cells were co-cultured without or with MDSCs at the different ratios of 8:1, 4:1 and 2:1, respectively. The results showed that MDSCs had significant suppressive ability on T cell proliferation (
In the present study, it was demonstrated that IL-12 and MDSCs are involved in the disease progression of Sjögren's syndrome (SS). Plasma IL-12 was elevated in NOD mice, and IL-12 enhanced the expansion of MDSCs in the NOD mice. Anti-IL-12 decreased the percentages of MDSCs in the NOD mice, and IL-12 deficiency impaired the generation of MDSCs gradually with age. All of these results suggest a pathogenic role of IL-12 in SS by promoting the expansion of MDSCs.
IL-12 is known as an inflammatory cytokine involved in various autoimmune diseases (
MDSCs, possessing a prominent capacity to suppress T cell activation, have been regarded as a potent inducer for tumor immune escape. The immunosuppressive function is an important characteristic of MDSCs. T cells were co-cultured with MDSCs and the results showed that MDSCs had significant suppressive ability on T cell proliferation and decreased the percentages of CD3+IFNγ+ T cells. The expression of IFNγ indicates the activation level of T cells (
In the present study, our results demonstrated that IL-12 aggravated SS-like disease, while anti-IL-12 attenuated SS-like disease in NOD mice. Moreover, increased IL-12 and a higher percentage of MDSCs were associated with SS pathogenesis. Increased percentages of MDSCs in IL-12-treated NOD mice, decreased percentages of MDSCs in anti-IL-12-treated NOD mice and gradually reduced percentages of MDSCs in
MDSCs consist of two subsets: Granulocytic MDSCs (G-MDSCs) and monocytic MDSCs (M-MDSCs) (
The development and expansion of MDSCs are regulated by multiple factors which is a complex and gradual phenomenon. Among the related signaling groups, the inflammatory cytokines and damage-associated molecular patterns are important (
In conclusion, collectively, it was revealed that IL-12 deteriorates SS-like disease by enhancing MDSC expansion. These results provide new insight into the pathogenetic mechanisms and elucidate a potential novel therapy for SS.
Not applicable.
This research was supported by the National Natural Science Foundation of China (NSFC) (grant nos. 81571583 and 81770061 to GY; 31872732 to YH).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
YH and GY initiated, designed and supervised the project. JQ and DL carried out the experiments. XZ, YP and TW contributed to the animal experiments such as breeding procedures and tissue collection. GS and HD performed the flow cytometric analysis and conducted the data analyses. All authors were involved in writing the paper and approved the submitted version.
All animal experiments conformed to the Regulation of Animal Care Management of the Ministry of Public Health, P.R. China and were approved by the Ethics Committee of the Medical School of Nanjing University (Nanjing, China).
Not applicable.
The authors declare no competing interests.
Plasma IL-12 is increased in NOD mice with SS-like symptoms. (A) Plasma levels of IL-12 in 12-week-old NOD and ICR mice were detected by ELISA. (B) Plasma IL-12 and the saliva flow rate in 4- and 8-week-old NOD mice were detected. *P<0.05, n=5. Error bars indicate SEM. (C) H&E staining of SGs for histological analysis (magnification, ×100). Arrows indicate the locus of lymphocytic infiltration in SGs. IL-12, interleukin-12; SS, Sjögren's syndrome; SGs, submandibular glands; H&E, hematoxylin and eosin; W, week.
IL-12 promotes the development of MDSCs in bone marrow (BM) and spleen (SP) of NOD mice. Eleven-week-old NOD mice were intraperitoneally treated with PBS (Ctrl) or IL-12 (NOD+IL-12). (A) IL-12 treatment schedule. (B) Lymphocytic infiltration in SGs from mice. Arrows indicate the locus of lymphocytic infiltration in SGs. (C) Salivary flow rate of mice. (D) Representative flow cytometry graphs of BM and SP MDSCs (CD11b+Gr-1+). (E) Summary of flow cytometry results of BM and SP MDSCs. Error bars indicate SEM. *P<0.05, n=5. IL-12, interleukin-12; SS, Sjögren's syndrome; MDSCs, myeloid-derived suppressor cells; SGs, submandibular glands.
Anti-IL-12 alleviates SS and decreases the percentage of MDSCs in BM and SP of NOD mice. Eleven-week-old NOD mice were intraperitoneally treated with (NOD+Anti-IL-12) or without (Ctrl) anti-IL-12. (A) Anti-IL-12 treatment schedule. (B) Lymphocytic infiltration in SGs from mice. Arrows indicate the locus of lymphocytic infiltration in SGs. (C) Salivary flow rate of mice. (D) Representative flow cytometry graphs of BM and SP MDSCs. (E) Summary of flow cytometry results of BM and SP MDSCs. Error bars indicate SEM. *P<0.05, n=5. IL-12, interleukin-12; SS, Sjögren's syndrome; MDSCs, myeloid-derived suppressor cells; SGs, submandibular glands; BM, bone marrow; SP, spleen.
IL-12 deficiency impairs generation of MDSCs in C57BL/6 mice. (A) SP MDSCs were detected by flow cytometry in 8-week-old C57BL/6 (B6) and
MDSCs display T cell suppressive activity in SS mice. Cells from spleen of 12-week-old NOD mice were co-cultured without or with MDSCs in different ratios (MDSC:T) at 8:1, 4:1 and 2:1, respectively for 3 days. (A) Representative flow cytometry graphs (left) and the summary of flow cytometry results (right) of T cell proliferation. (B) Representative flow cytometry graphs (left) and the summary of flow cytometry results (right) of CD3+IFNγ+ T cells. Error bars indicate SEM. *P<0.05 and **P<0.01, n=5. SS, Sjögren's syndrome; MDSCs, myeloid-derived suppressor cells.