Total Glucosides of Paeony Ameliorate Pristane-Induced Lupus Nephritis by Inducing PD-1 ligands+ Macrophages via Activating IL-4/STAT6/PD-L2 Signaling

Abstract

Macrophages, a major subset of innate immune cells, are main infiltrating cells in the kidney in lupus nephritis. Macrophages with different phenotypes exert diverse or even opposite effects on the development of lupus nephritis. Substantial evidence has shown that macrophage M2 polarization is beneficial to individuals with chronic kidney disease. Further, it has been reported that PD-1 ligands (PD-Ls) contribute to M2 polarization of macrophages and their immunosuppressive effects. Total glucosides of paeony (TGP), originally extracted from Radix Paeoniae Alba, has been approved in China to treat some autoimmune diseases. Here, we investigated the potentially therapeutic effects of TGP on lupus nephritis in a pristane-induced murine model and explored the molecular mechanisms regulating macrophage phenotypes. We found that TGP treatment significantly improved renal function by decreasing the urinary protein and serum creatinine, reducing serum anti-ds-DNA level and ameliorating renal immunopathology. TGP increased the frequency of splenic and peritoneal F4/80⁺CD11b⁺CD206⁺ M2-like macrophages with no any significant effect on F4/80⁺CD11b⁺CD86⁺ M1-like macrophages. Immunofluorescence double-stainings of the renal tissue showed that TGP treatment increased the frequency of F4/80⁺Arg1⁺ subset while decreasing the percentage of F4/80⁺iNOS⁺ subset. Importantly, TGP treatment increased the percentage of both F4/80⁺CD11b⁺PD-L1⁺ and F4/80⁺CD11b⁺PD-L2⁺ subsets in spleen and peritoneal lavage fluid as well as the kidney. Furthermore, TGP augmented the expressions of CD206, PD-L2 and phosphorylated STAT6 in IL-4-treated Raw264.7 macrophages in vitro while its effects on PD-L2 were abolished by pretreatment of the cells with an inhibitor of STAT6, AS1517499. However, TGP treatment did not affect the expressions of STAT1 and PD-L1 in Raw264.7 macrophages treated with LPS/IFN-γ in vitro, indicating a possibly indirect effect of TGP on PD-L1 expression on macrophages in vivo. Thus, for the first time, we demonstrated that TGP may be a potent drug to treat lupus nephritis by inducing F4/80⁺CD11b⁺CD206⁺ and F4/80⁺CD11b⁺PD-L2⁺ macrophages through IL-4/STAT6/PD-L2 signaling pathway.

Keywords: PD-1 ligand; PD-L1; PD-L2; lupus nephritis; macrophage polarization; total glueosides of paeony (TGP).

Figure 1

TGP treatment improves renal pathology and function in mice with pristane-induced lupus nephritis. (A) 24 weeks after pristane injection, 24-h urinary protein was determined every two weeks and mice with >0.4 mg/24 h urinary protein were also treated with total glucosides of paeony (TGP) in subsequent experiments starting at the same 24-week point. The effects of TGP on kinetics of 24-h urinary protein in pristane-induced lupus mice were observed. In addition, eight weeks after TGP treatment, the serum creatinine (Scr) (B) and serum anti-ds-DNA (C) were measured using sarcosine oxidase creatinine assay kits and murine anti-dsDNA standard enzyme-linked immunosorbent assay (ELISA) kits, respectively. Data are presented as means ± SD (n=5-6, ** p < 0.01 compared with control, and ^##p < 0.01 or ^#p < 0.05 compared with pristane-treated mice). (D) HE staining of the kidney in the pristane-induced lupus mice 8 weeks after TGP treatment (200×, with black arrows pointing to infiltrating immune cells).

Figure 2

TGP treatment promotes macrophage M2 polarization in pristane-induced lupus mice. Single-cell suspensions from spleens and peritoneal lavage fluids were collected 8 weeks after TGP treatment following pristane injection 24 weeks ago. The effects of TGP on the frequency of CD86⁺ (A) and CD206⁺ (B) subsets in the splenic and peritoneal F4/80⁺CD11b⁺ macrophages were analyzed via flow cytometry. (C, D) The percentages of CD86⁺ subset in splenic and peritoneal F4/80⁺CD11b⁺ macrophages are shown as means ± SD. (E, F) Shown also are the means ± SD of the percentages of CD206⁺ subset in splenic and peritoneal F4/80⁺CD11b⁺ macrophages. (n=6, ^*p < 0.05 compared with control, and ^#p<0.05 compared with pristane-treated mice).

Figure 3

TGP treatment upregulates macrophage expression of PD-Ls in pristane-induced lupus mice. Single-cell suspensions from spleens and peritoneal lavage fluids were collected 8 weeks after TGP treatment following pristane injection 24 weeks ago. The effects of TGP on the frequency of PD-L1⁺ (A) or PD-L2⁺ (B) subsets in splenic and peritoneal F4/80⁺CD11b⁺ macrophages were determined via FACS. (C, D) The percentages of PD-L1⁺ subset in splenic and peritoneal F4/80⁺CD11b⁺ macrophages are shown as means ± SD. (E, F) Shown are the means ± SD of the percentages of PD-L2⁺ subset in splenic and peritoneal F4/80⁺CD11b⁺ macrophages. (n=5-6, *p < 0.05 compared with control, and ^##p < 0.01 or ^#p < 0.05 compared with pristane treated mice).

Figure 4

TGP treatment increases renal PD-Ls⁺ macrophage infiltration in pristane-induced lupus mice. (A) Immunofluorescence double-stainings for F4/80 and iNOS or (B) for F4/80 and Arg1 in the kidney of pristane-induced LN mice 8 weeks after TGP treatment (400×). (C) Kidney infiltrating cells were isolated 8 weeks after TGP treatment following pristane injection 24 weeks ago and the total infiltrating macrophages were gated on CD45⁺F4/80⁺CD11b⁺ population. The percentages of kidney infiltrating CD86⁺ (D), CD206⁺ (E), PD-L1⁺ (F) or PD-L2⁺ (G) macrophages were determined via flow cytometer, as shown by the bar graphs, in which data are presented as means ± SD (n=5-6, ^**p < 0.01 compared with control, and ^#p < 0.05 compared with pristane-treated mice).

Figure 5

TGP promotes PD-Ls-expressing macrophage polarization in vitro. (A) Representative FACS plots show the expression of PD-L1 in CD86^- and CD86⁺ Raw264.7 macrophages stimulated with LPS/IFN-γ. (B) Representative FACS plots display the expression of PD-L2 in CD206^- and CD206⁺ Raw264.7 macrophages stimulated with IL-4. (C) The expression of CD86 or PD-L1 in LPS/IFN-γ-treated Raw264.7 cells was determined via FACS. (D) The expression of CD206 or PD-L2 in IL-4-treated Raw264.7 cells was determined via FACS. (E–H) Data are shown as the mean percentages of CD86 (E) and PD-L1 (F) expression in LPS/IFN-γ-treated Raw264.7 cells or CD206 (G) and PD-L2 (H) expression in IL-4-treated Raw264.7 cells. Data are presented as means ± SD (n=3-4, ^**p<0.01 or ^*p < 0.05 compared with control, and ^##p < 0.01 or ^#p < 0.05 compared with LPS/IF-γ or IL-4 group without TGP).

Figure 6

TGP augments PD-L2 expression on macrophages via phosphorylating STAT6. (A) Raw264.7 cells were pretreated with LPS/IFN-γ for 12 h and treated with TGP for another 48 h, then protein expressions of p-STAT1, STAT1 and PD-L1 were detected via western blot analyses. (B) Raw264.7 cells were pretreated with IL-4 for 12 h and treated with TGP for another 48 h, the protein expressions of p-STAT6, STAT6 and PD-L2 were then detected via western blot analyses. (C) To determine if TGP regulates PD-L2 expression via STAT6 pathway, Raw264.7 cells were treated with a STAT6 inhibitor, AS1517499, and detected for the expressions of p-STAT6 and PD-L2. Overall, GAPDH was used as internal references. The expressions of p-STAT1, STAT1, PD-L1, p-STAT6, STAT6 and PD-L2 were calculated relatively to GAPDH. Data are presented as means ± SD (n=3, ^**p<0.01 or ^*p<0.05, as indicated for direct comparisons, ns, not significant).