The lupus mouse model was successfully established
To build a lupus mouse model, we used a previously validated method [13, 34, 35]. The levels of proteinuria (g/24 h) (1.69 ± 0.33), serum urea nitrogen (BUN) (32.7 ± 2.8), and creatinine (Cr) (1.45 ± 0.20) were higher in lupus mice than in control mice (0.55 ± 0.13, 11.33 ± 1.0, 0.33 ± 0.06, respectively, p < 0.05). Furthermore, the concentrations of ANA, anti-dsDNA, and anti-Sm autoantibodies (Fig. 1a) were significantly increased in the lupus mouse model (53.63 ± 2.28, 57.11 ± 1.42, 61.19 ± 4.77, respectively) (Fig. 1b), compared to those of the control mice (12.17 ± 0.81, 14.43 ± 0.71, 6.86 ± 0.53, respectively, p < 0.05) (Fig. 1a). Importantly, renal histopathology of lupus mouse tissues showed ischaemic and shrunk glomeruli to varying degrees, glomerular capillary stenosis and occlusion (1), increased mesangial matrix, proliferated mesangial cells, thickened basement membrane (2), degenerated vacuole granules of renal tubules (3), and infiltrated inflammatory cells in the renal interstitium (4), compared to those of control mice (Fig. 1c). The results suggested that the lupus mouse model was successfully established, and these mice were used in subsequent studies.
Depletion of Fractalkine and p38MAPK inhibitor, SB203580, ameliorated proteinuria and renal injury and reduced the serum concentration of autoantibodies in lupus mice
The results obtained for the seven groups are shown in Fig. 2 (a-c). The 24 h proteinuria and serum Cr and BUN levels of lupus mice significantly increased (1.81 ± 0.30, 1.44 ± 0.07 and 33.93 ± 3.17), compared to those of the control (0.52 ± 0.11, 0.35 ± 0.05 and 10.48 ± 1.71), Fractalkine-depleted (0.51 ± 0.13, 0.38 ± 0.11 and 9.82 ± 1.44), and SB203580 mice (0.64 ± 0.13, 0.34 ± 0.06 and 10.55 ± 2.17), respectively, p < 0.05. However, the 24 h proteinuria, serum Cr, and BUN levels decreased in the lupus mice when compared to lupus + Fractalkine-depleted (0.98 ± 0.10, 0.69 ± 0.09 and 21.81 ± 2.21) and lupus + SB203580 mice (0.97 ± 0.16, 0.67 ± 0.06 and 22.94 ± 2.05), respectively, p < 0.05. Serum Cr and BUN levels in lupus + Fractalkine-depleted + SB203580 mice (0.53 ± 0.09, 0.35 ± 0.05, and 10.7 ± 1.6), were much lower than those in lupus + Fractalkine-depleted and lupus + SB203580 mice (p < 0.05). The lupus mice also exhibited significantly higher levels of ANA (68.69 ± 4.64), anti-dsDNA (79.56 ± 7.50), and anti-Sm (54.69 ± 6.35) than those of the lupus + Fractalkine-depleted (50.25 ± 1.95, 53.25 ± 3.28, and 36.92 ± 4.25, respectively), and lupus + SB203580 mice (50.00 ± 2.69, 54.07 ± 3.14, and 34.16 ± 3.22, respectively, p < 0.05). In lupus + Fractalkine-depleted + SB203580 mice, the serum ANA, ds-DNA, and anti-Sm levels (25.52 ± 2.57, 25.07 ± 3.73, and 21.65 ± 3.20, respectively) was lower than those observed in lupus + Fractalkine-depleted and lupus + SB203580 mice (p < 0.05) (Fig. 2d). The PASM-stained renal tissues of control, Fractalkine-depleted, and SB203580 mice showed normal renal glomeruli and tubules, in contrast to the histopathological characteristics of lupus mouse model, which was described in the previous section. However, the symptoms of lupus + Fractalkine-depleted and lupus + SB203580 mice were significantly diminished than those of the lupus mice. Importantly, the lupus + Fractalkine-depleted + SB203580 mice showed significantly more reduced kidney injury, than both former groups (Fig. 2e).
Depletion of Fractalkine and p38MAPK inhibitor, SB203580, decreased the levels of renal Fractalkine, p-p38, IL-6, and IL-17, and increased those of renal Foxp3 in lupus mice
The concentrations of renal Fractalkine, p-p38, Foxp3, IL-6, and IL-17 proteins were compared between the different mouse groups by western blot and immunofluorescence. Western blot results showed a decreased expression of Foxp3 protein in lupus mice, when compared to that of the control mice, but an increased expression in lupus + Fractalkine-depleted and lupus + SB203580 mice, compared to lupus mice (p < 0.05) (Fig. 3). While the expression of Fractalkine, p-p38, IL-6, and IL-17 increased in lupus mice compared to control mice, that of lupus + Fractalkine-depleted and lupus + SB203580 mice decreased. Moreover, the Fractalkine, p-p38, IL-6, and IL-17 protein concentrations increased in Foxp3 but decreased in lupus + Fractalkine-depleted + SB203580 mice, when compared to those of lupus + Fractalkine-depleted and lupus + SB203580 mice (p < 0.05). The western blot results were further verified by immunofluorescence analysis. To this end, kidney sections of lupus mice were stained with antibodies against Fractalkine, p-p38, and Foxp3 proteins.
Furthermore, the average optical densities of FKN, p-p38, and Foxp3 are shown in Table 2. This semi-quantitative evaluation indicated that, compared to control mice, the expression of Foxp3 protein decreased in lupus mice, but compared to lupus mice, those levels increased in lupus + Fractalkine-depleted and lupus + SB203580 mice (p < 0.05). Fractalkine and p-p38 concentrations increased in lupus mice and decreased in lupus + Fractalkine-depleted and lupus + SB203580 mice. Moreover, the Foxp3 concentration increased and the Fractalkine and p-p38 expression decreased in lupus + Fractalkine-depleted + SB203580 mice, when compared to lupus + Fractalkine-depleted and lupus + SB203580 mice (p < 0.05) (Fig. 4).
Fractalkine levels were knocked down in Treg cells derived from the spleen of lupus mice
CD4+ CD25+ Treg cells were isolated and purified from the spleen of lupus mice by magnetic bead cell sorting. The purity of CD4+ CD25+ T cells was more than 95% (Fig. 5a). After optimising the purification method of CD4+CD25+T cells, we evaluated the effect of Fractalkine on the apoptosis of CD4+CD25+T cells after knocking down its expression. To this end, cells were infected with the lentiviral vector particle-Fractalkine or the hU6-MCS-Ubiquitin-EGFP-IRES-puromycin scramble negative control. After puromycin screening, the expression levels of Fractalkine were verified by western blot. As shown in Fig. 5b-c, the Fractalkine protein levels in the Fractalkine KD group (0.339 ± 0.018) were significantly lower than those in the control group (1.000 ± 0.000, p < 0.05); meanwhile, those in the negative control group did not change (0.999 ± 0.004, p > 0.05).
Knockdown of Fractalkine levels reduced cells apoptosis and suppressed the activation of p38MAPK signalling in Treg cells derived from lupus mice
Foxp3 mRNA and protein expression was significantly augmented in Treg cells from the Fractalkine KD lupus mouse after treatment with SB203580. On the other hand, the phosphorylation of p38MAPK was suppressed by Fractalkine KD and SB203580. U-46619 treatment produced the opposite effect than SB203580 in these analyses (Fig. 6a-c).
Annexin V-FITC/PI staining and FC analysis were performed to determine the degree of CD4+ CD25+ T cell apoptosis in all the groups after 48 h of culture. As shown in Fig. 7a-b, the apoptosis rate significantly decreased in the Fractalkine KD, SB203580, and Fractalkine KD + SB203580 groups, compared to that in the control group (p < 0.05), but significantly increased in the U-46619 group (p < 0.05). In addition, the apoptosis rates in the control, negative, and Fractalkine KD + U-46619 groups were not significantly different. Taken together, these data showed that SB203580 and Fractalkine KD inhibited the apoptosis of lupus mouse Treg cells, while U-46619 promoted it.
Protein and mRNA expression levels of several key apoptotic genes (Bax, Bcl-2, and Cyt-c) in mouse Treg cells from all groups were detected by western blot and RT-PCR, respectively. Bcl-2 mRNA and protein expression, as contrary to that of Bax and Cyt-c, was augmented in Fractalkine KD and/or SB203580-treated lupus mouse Treg cells. Moreover, treatment with U-46619 elicited the opposite effect (Fig. 7c-e). Taken together, these results suggested that Fractalkine regulated the activation of the p38MAPK signalling pathway to promote apoptosis of lupus mouse Treg cells.