Schistosoma japonicum Cystatin Alleviates Sepsis Through Activating Regulatory Macrophages

Abstract

Multi-organ failure caused by the inflammatory cytokine storm induced by severe infection is the major cause of death for sepsis. Sj-Cys is a cysteine protease inhibitor secreted by Schistosoma japonicum with strong immunomodulatory functions on host immune system. Our previous studies have shown that treatment with Sj-Cys recombinant protein (rSj-Cys) attenuated inflammation caused by sepsis. However, the immunological mechanism underlying the immunomodulation of Sj-Cys for regulating inflammatory diseases is not yet known. In this study, we investigated the effect of Sj-Cys on the macrophage M2 polarization and subsequent therapeutic effect on sepsis. The rSj-Cys was expressed in yeast Pichia pastoris. Incubation of mouse bone marrow-derived macrophages (BMDMs) with yeast-expressed rSj-Cys significantly activated the polarization of macrophages to M2 subtype characterized by the expression of F4/80⁺ CD206⁺ with the elated secretion of IL-10 and TGF-β. Adoptive transfer of rSj-Cys treated BMDMs to mice with sepsis induced by cecal ligation and puncture (CLP) significantly improved their survival rates and the systemic clinical manifestations of sepsis compared with mice receiving non-treated normal BMDMs. The therapeutic effect of Sj-Cys-induced M2 macrophages on sepsis was also reflected by the reduced pathological damages in organs of heart, lung, liver and kidney and reduced serological levels of tissue damage-related ALT, AST, BUN and Cr, associated with downregulated pro-inflammatory cytokines (IFN-gamma and IL-6) and upregulated regulatory anti-inflammatory cytokines (IL-10 and TGF-β). Our results demonstrated that Sj-Cys is a strong immunomodulatory protein with anti-inflammatory features through activating M2 macrophage polarization. The findings of this study suggested that Sj-Cys itself or Sj-Cys-induced M2 macrophages could be used as therapeutic agents in the treatment of sepsis or other inflammatory diseases.

Keywords: Schistosoma japonicum; adoptive transfer; cysteine protease inhibitor; immunomodulation; macrophage; sepsis.

Figure 1

IMAC purification of recombinant Sj-Cys expressed in P. pastoris GS115. The rSj-Cys with His-tag at C-terminus was expressed as a soluble protein in the culture medium. After binding on the nickel column, rSj-Cys was eluted in a buffer containing imidazole. The purified protein was recognized by the anti-His antibody.

Figure 2

rSj-Cys induced M2 macrophage polarization and reduced LPS-induced M1 macrophages phenotype in vitro. (A) The flow cytometry experiments were repeated by adding live/dead cell staining, and re-gated to differentiate dead cells, adhere cells and to block Fc. (B) BMDMs were obtained by incubating mouse bone marrow cells (adherent) with M-CSF for 7 days. The mature BMDMs were defined as CD11b⁺F4/80⁺ subpopulations using FACS. (C–E) BMDMs were incubated with rSj-Cys (2 ug/ml), LPS (100 ng/ml), IL-4 (10 ng/ml) + IL-10 (10 ng/ml), rSj-Cys + LPS, rSj-Cys + IL-4 + IL-10, or PBS, respectively, for 24 h. The M1 (CD86) and M2 (CD206) markers were detected using FACS. n = 5. Data are expressed as mean ± SEM, ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

rSj-Cys induced BMDMs to secrete M2 macrophage-related cytokines IL-10 and TGF-β. Mature BMDMs were stimulated with PBS, rSj-Cys (2 ug/ml), LPS (100 ng/ml), IL-4 (10 ng/ml) + IL-10 (10 ng/ml), rSj-Cys + LPS, or rSj-Cys + IL-4 + IL-10 for 24 h. The levels of M2-related cytokines IL-10 (C), TGF-β (D), and M1-related cytokines IFN-gamma (A), IL-6 (B) were measured in the supernatant by ELISA. n = 5. The results are presented as mean ± SEM, ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

Adoptive transfer of rSj-Cys treated-BMDMs mitigated pathology caused by CLP-induced sepsis. (A) Mice adoptively transferred with rSj-Cys-treated BMDMs significantly increased their survival rate up to 72 h (80%) compared to mice receiving PBS (0%), LPS-treated BMDMs (0%), or untreated BMDMs (20%) during the same observation period (n = 10). (B, C) The results of histopathology of heart, lung, liver and kidney stained with H&E staining from mice 12 h after CLP and transferred with BMDMs treated with rSj-Cys, LPS or untreated BMDMs (n = 4). The pathological score comparison was shown on the right. The magnification ×400, scale bar = 100 µm. (D) The levels of ALT, AST, BUN, and Cr were measured in the sera from mice 12 h after receiving CLP and differently treated BMDMs (n = 4). The results are presented as mean ± SEM. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

rSj-Cys treated-BMDMs downregulated pro-inflammatory cytokines and upregulated regulatory cytokines in mice with sepsis. The levels of IFN-gamma (A), IL-6 (B), IL-10 (C), and TGF-β (D) were measured in the sera of mice in each group using ELISA. n = 4. The results are presented as mean ± SEM. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.