Carboxyamidotriazole Regulates the Function of Salivary Gland Epithelial Cells and B Cells to Alleviate Experimental Sjögren's Disease in Mice

Abstract

Sjögren's disease (SjD), a systemic autoimmune disease, suffers from restricted treatment choices. The activation of salivary gland epithelial cells and abnormal auto-reactive B cells, triggering cytokine and autoantibody generation, is key to its immunopathogenesis. Carboxyamidotriazole (CAI) was reported to have anti-inflammatory properties by reducing cytokines, yet its role in SjD was unknown. In this research, we targeted to probe CAI's potential treatment effect on SjD-like NOD/Ltj mice and its mechanism. Utilizing the salivary glands of these mice, we employed HE staining, ELISA, immunohistochemistry and flow cytometry. Findings revealed that CAI augmented salivary secretion, decreased water intake and serum autoantibody levels, suppressed histological alterations and lymphocyte foci, and diminished inflammatory factors such as IL-1β and IL-6. It also blocked IκBα degradation and p65 nuclear translocation. In vitro, CAI restrained IL-6 secretion from stimulated SGECs and halted Raji B cells' proliferation at G0/G1 stage. Overall, CAI shows an anti-SjD effect in NOD/Ltj mice, probably by regulating relevant cells and deactivating the NF-κB pathway.

Keywords: B cells; NOD/Ltj mice; Sjögren′s disease; carboxyamidotriazole; cytokines; salivary gland epithelial cells.

Figure 1

CAI alleviated SjD-like manifestation in NOD/Ltj model mice. A The average water intake of mice was measured and calculated during the experiment. B The salivary flow of the mice was assessed at the age of 12 weeks and 20 weeks. C, D SG index (C) and spleen index (D) of mice were evaluated at the age of 20 weeks. ^#P < 0.05, ^##P < 0.01 compared with the control group; ^*P < 0.05, ^**P < 0.01 compared with PEG 400 group. ^△P < 0.05, ^△△P < 0.01 compared with the age of 12 weeks, n = 8-12.

Figure 2

CAI attenuated lymphocytes infiltration in NOD/Ltj mice. A Representative HE staining images of SGs from each group. White arrows represent lymphocytic infiltration. Original magnification × 200, bar=100 μm. B The severity of gland tissue lesions was assessed for histological scores as defined in Materials and Methods. ^##P < 0.01 compared with the control group; ^**P < 0.01 compared with PEG 400 group, n = 10-14.

Figure 3

CAI decreased the levels of SS-related autoantibodies. Mice were sacrificed at the age of 20 weeks, and serum was collected from the peripheral blood. A The level of anti-SSA/Ro level was detected by ELISA kit. B The level of anti-SSB/La was detected by ELISA kit. C The level of IgG was detected by ELISA kit. ^##P < 0.01 compared with the control group; ^*P < 0.05, ^**P < 0.01 compared with PEG 400 group, n = 6-8.

Figure 4

CAI reduced the concentrations of immune-inflammatory cytokine in NOD/Ltj mice. Mice were sacrificed at the age of 20 weeks, SGs were extracted and homogenized. The levels of IL-1β (A), IL-6 (B), TNF-α (C), CXCL1/GRO-α (D) in homogenates were analyzed by ECL kit. ^##P < 0.01 compared with the control group; ^*P < 0.05, ^**P < 0.01 compared with the PEG 400 group, n = 5-6.

Figure 5

CAI inhibited NF-κB activation in NOD/Ltj mice. A Representative IHC staining of the NF-κB p65 subunit in SGs of the indicated groups. B Representative IHC staining of IκB in SGs of the indicated groups. Original magnification×200, bar=100 μm. C, D Semiquantitative analysis of the immunohistochemical results as defined in Materials and Methods. ^##P < 0.01 compared with the control group; ^*P < 0.05, ^**P < 0.01 compared with PEG 400 group, n = 7-9.

Figure 6

CAI downregulated stimulus-induced IL-6 secretion in SGECs. A SGECs were incubated with vehicle (0.1% DMSO) or CAI (20 and 40 μmol/L) together with LPS (1 µg/mL) for 24 h. IL-6 level in the supernants were detected by ELISA kit. B, C SGECs were incubated with vehicle (0.1% DMSO) or CAI (20 and 40 μmol/L) together with TNF-α (100 ng/mL) for 24 h (B) and 48 h (C), respectively. IL-6 level in the supernants was detected by ELISA kit. D Effect of CAI on SGECs viability was evaluated using a CCK-8 assay. ^##P < 0.01 compared with the control group; ^*P < 0.05, ^**P < 0.01 compared with stimulus-alone group, n = 4-6.

Figure 7

CAI inhibited B cells proliferation via G0/G1 cell cycle arrest. A, B Raji cells were incubated with vehicle (0.1% DMSO) or CAI (5, 10, 20 and 40 μmol/L) for 24 h (A) and 48 h (B). The effect of CAI on cells proliferation was measured using a CCK-8 assay. C Raji cells were incubated with vehicle (0.1% DMSO) or low concentration of CAI (5 and 10 μmol/L) for 24 h. The effect of CAI on cell cycle progression was tested by flow cytometry-based PI/RNase staining. The representative images from each group were shown. D Statistics analysis of flow cytometry. ^*P < 0.05, ^**P < 0.01 compared with DMSO group, n = 3.

Figure 8

CAI did not induce apoptosis in Raji cells. A Raji cells were incubated with vehicle (0.1% DMSO) or low concentration of CAI (5 and 10 μmol/L) for 24 h. The effect of CAI on cell cycle progression was tested by flow cytometry-based Annexin V/PI staining. The representative images from each group were shown. B Statistics analysis of flow cytometry, n = 4-5.