Figure 1.
Identification of regulatory T cells and liver macrophages using flow cytometry.
A: liver macrophages were identified as CD11b/c positive cells after primary culture for 24 hours. B: The purity of Treg cells was examined after isolation using magnetic beads.
Figure 2.
Confirming induction of the severe acute pancreatitis model.
Assessment of pancreatic injury by HE staining. Pancreatitis was induced by pancreatic-ductal perfusion with 5% Sodium taurocholate. All rats were sacrificed at 24 h after disease model initiation. A: A typical photomicrographs of HE stained tissue obtained from control rat. B: A photomicrograph of pancreatic injury which includes pancreatic edema, neutrophil infiltration, necrosis and hemorrhage.
Figure 3.
Demonstration of M1 polarization of Liver macrophages isolated from the rats with acute pancreatitis.
Liver macrophages were obtained from CTRLthe rats post operation for 24 hours (all other groups). In vitro, macrophages from SAP rats treated with PBS for 4, 8 and 16 hours respectively (S+PBS4, S+PBS8, S+PBS16 group). and macrophages from CTRL rats undergone sham operation were treated with PBS for 4 h. The polarization of M1/M2 was assessed using real time PCR and immunofluorescence. A: mRNA level of M1 markers in the macrophages. B: mRNA level of M2 markers in the macrophages. C: Immuno-fluorescence examination of macrophage polarization. The significance of differing mRNA levels was analyzed while comparing with the CTRL group. Error bars indicate the mean ±S.E. *p<0.05, **p<0.01, ***p<0.001.
Figure 4.
Reverse the polarization of liver macrophages by IL-4 and Treg in vitro.
Liver macrophages were obtained from at 24 h after SAP model initiation. In vitro, macrophages were respectively treated with PBS, IL-4 (10 ng/ml) or Treg (macrophages: Treg ≈2∶1) for 8 hour (S+PBS, S+IL-4, S+Treg group). The M1/M2 polarization was assessed using real-time PCR and immunofluorescence. A: mRNA level of M1 markers in the macrophages. B: mRNA level of M2 markers in the macrophages. C: Immunofluorescence examination of the polarization of macrophages. The significance of differing mRNA levels was analyzed while comparing with the CTRL group. Error bars indicate the mean ±S.E. *p<0.05, **p<0.01, ***p<0.001.
Figure 5.
Treg cells reversed the polarization of Liver macrophages longer than IL-4 treatment.
In vitro, macrophages were treated with IL-4 (10 ng/ml) or Treg (macrophages: Treg ≈2∶1) for 4, 8 and 16 hour. The polarization of M1/M2 was assessed using real time PCR and immunofluorescence. A: mRNA level of M1 markers in the macrophages. B: mRNA level of M2 markers in the macrophages. The significance of differing mRNA levels was analyzed while comparing the two groups in the same time point using the indifferent T-test. Error bars indicate the mean ±S.E. *p<0.05.