Biomimetic carbon monoxide delivery based on hemoglobin vesicles ameliorates acute pancreatitis in mice via the regulation of macrophage and neutrophil activity

Abstract

Macrophages play a central role in various inflammatory disorders and are broadly divided into two subpopulations, M1 and M2 macrophage. In the healing process in acute inflammatory disorders, shifting the production of M1 macrophages to M2 macrophages is desirable, because M1 macrophages secrete pro-inflammatory cytokines, whilst the M2 variety secrete anti-inflammatory cytokines. Previous findings indicate that when macrophages are treated with carbon monoxide (CO), the secretion of anti-inflammatory cytokine is increased and the expression of pro-inflammatory cytokines is inhibited, indicating that CO may have a potential to modulate the production of macrophages toward the M2-like phenotype. In this study, we examined the issue of whether CO targeting macrophages using a nanotechnology-based CO donor, namely CO-bound hemoglobin vesicles (CO-HbV), modulates their polarization and show therapeutic effects against inflammatory disorders. The results showed that the CO-HbV treatment polarized a macrophage cell line toward an M2-like phenotype. Furthermore, in an in vivo study using acute pancreatitis model mice as a model of an inflammatory disease, a CO-HbV treatment also tended to polarize macrophages toward an M2-like phenotype and inhibited neutrophil infiltration in the pancreas, resulting in a significant inflammation. In addition to the suppression of acute pancreatitis, CO-HbV diminished a subsequent pancreatitis-associated acute lung injury. This could be due to the inhibition of the systemic inflammation, neutrophil infiltration in the lungs and the production of HMGB-1. These findings suggest that CO-HbV exerts superior anti-inflammatory effects against inflammatory disorders via the regulation of macrophage and neutrophil activity.

Keywords: Macrophage; acute pancreatitis; carbon monoxide; liposome; polarization.

Figure 1.

(A) CO release from HbV (open circle) or CO-HbV (closed circle) in the presence of 100% FBS. Either HbV or CO-HbV was mixed with 100% FBS in a glass vial. At stipulated time after mixing, the CO concentration in the head space of the glass vial was determined using gas chromatography with a CO-analyzer. Data are mean ± SD (n = 3 per group). (B) CO concentration in blood after CO-HbV administrating to healthy mice. Mice were single injected CO-HbV, and CO concentrations in blood were measured using CO analyzer. Data are mean ± S.D. (n = 4).

Figure 2.

The changes of mRNA expression of (A) TNF-α, (B) NOS2, (C) CD163 and (D) MRC1 after incubation with either M1-trophic cytokines (LPS and IFN-γ), M2-trophic cytokines (IL-13 and IL-4), CO-HbV or HbV in RAW 264.7 cells. TNF-α and NOS2: M1 macrophage marker, CD163 and MRC1: M2 macrophage marker. Error bars indicate the S.E. of six separate experiments. *p < .05,**p < .01 versus non-treatment.

Figure 3.

The plasma levels of (A) amylase, (B) lipase and (C) pancreatic weight after CO-HbV administrating to acute pancreatitis model mice. Data are mean ± S.D. (n = 6 per group). **p < .01 versus control. (D) Micrographs of pancreas slides stained with HE. (E) Immunological staining of pancreatic slices for MPO (red, upper) and NO₂-Tyr (red, lower). Blue staining represents the nuclei immunostained with DAPI (counterstain). Scale bars: 100 μm.

Figure 4.

M1 and M2 polarization of macrophages in the pancreas of acute pancreatitis model mice. The mRNA expressions of (A) NOS2, (B) TNF-α, (C) IL-10, and (D) MRC1 were determined using pancreas tissue collected at 12 h after the start of cerulein administration. Data are mean ± S.E. (n = 6 per group). **p < .01 versus control.

Figure 5.

The levels of (A) TNF-α, (Β) IL-6, (C) IL-1β, and (D) IL-10 in the pancreas of acute pancreatitis model mice. Pancreas tissue collected at 12 h after the start of cerulein administration. Data are mean ± S.D. (n = 6 per group). **p < .01 versus control.

Figure 6.

Evaluation of distant lung injury after CO-HbV treatment in acute pancreatitis model mice. (A) Lung wet/dry ratio were determined using one lobe of the left lung collected at 12 h after the start of cerulein administration. Data are mean ± S.D. (n = 6 per group). **p < .01 versus control. (B) Micrographs of lung section stained with HE and (C) immunological staining of lung slices for MPO (red, upper) and NO₂-Tyr (red, lower). Blue staining represents the nuclei immunostained with DAPI (counterstain). Scale bars: 100 μm.

Figure 7.

Pancreatic and plasma HMGB1 levels in acute pancreatitis model mice. HMGB1 level in (A) the pancreas and (B) plasma were determined using pancreas and plasma collected at 12 h after the start of cerulein administration. Data are mean ± S.D. (n = 6 per group). **p < .01 versus control.