Resolution of sterile inflammation: role for vitamin C

Abstract

Introduction: Macrophage reprogramming is vital for resolution of acute inflammation. Parenteral vitamin C (VitC) attenuates proinflammatory states in murine and human sepsis. However information about the mechanism by which VitC regulates resolution of inflammation is limited.

Methods: To examine whether physiological levels of VitC modulate resolution of inflammation, we used transgenic mice lacking L-gulono-γ-lactone oxidase. VitC sufficient/deficient mice were subjected to a thioglycollate-elicited peritonitis model of sterile inflammation. Some VitC deficient mice received daily parenteral VitC (200 mg/kg) for 3 or 5 days following thioglycollate infusion. Peritoneal macrophages harvested on day 3 or day 5 were examined for intracellular VitC levels, pro- and anti-inflammatory protein and lipid mediators, mitochondrial function, and response to lipopolysaccharide (LPS). The THP-1 cell line was used to determine the modulatory activities of VitC in activated human macrophages.

Results: VitC deficiency significantly delayed resolution of inflammation and generated an exaggerated proinflammatory response to in vitro LPS stimulation. VitC sufficiency and in vivo VitC supplementation restored macrophage phenotype and function in VitC deficient mice. VitC loading of THP-1 macrophages attenuated LPS-induced proinflammatory responses.

Conclusion: VitC sufficiency favorably modulates macrophage function. In vivo or in vitro VitC supplementation restores macrophage phenotype and function leading to timely resolution of inflammation.

Figure 1

Vitamin C deficiency alters the progression of  TG-induced peritoneal inflammation. Plasma VitC levels were measured in VitC sufficient and deficient Gulo^−/− mice as well as in deficient mice treated daily with i.p. AscA for 3 days (N = 3–6 mice/group, ns = not significant).

Figure 2

Spatiotemporal profiling of inflammatory mediators following TG-induced peritoneal inflammation. Real time QPCR for IL-1β, TNFα, MCP-1, YM1, Arg1, and IL-10 mRNA from peritoneal macrophages elicited on day 3 (a) and day 5 (b) following TG-induced peritonitis from VitC sufficient and deficient Gulo^−/− mice. Following TG challenge, some VitC deficient mice were randomized to receive daily i.p. injection of VitC as AscA (200 mg/kg in saline) for a further 3 days (day 3, deficient + AscA) or 5 days (day 5, deficient + AscA) (N = 6 mice/group, ns = not significant).

Figure 3

LPS differentially activates proinflammatory gene expression in macrophages from vitamin C sufficient and deficient mice. Peritoneal macrophages elicited on day 3 following TG-induced peritonitis from VitC sufficient and deficient Gulo^−/− mice were exposed to LPS (50 ng/mL) for 4 hours. Macrophages from some VitC deficient mice were incubated with AscA (3 mM, 16 hours) prior to LPS exposure (deficient + AscA). Real time QPCR was performed for IL-1β (a) and TNFα (b) (N = 6/group). (c) Upper panel: representative western blot for expression of phospho-NFκB and NFκB from VitC deficient macrophages exposed to media alone (M), AscA (3 mM, 16 hours (AscA)), LPS (50 ng/mL) for 1 hour (LPS), or AscA for 16 hours followed by LPS for 1 hour (LPS + AscA). Lower panel: densitometry for normalized expression of phospho-NFκB from macrophages (N = 3/group). (d) Upper panel: representative western blot for expression of iNOS and actin from macrophages groups described in (c) and exposure to LPS (50 ng/mL) for 4 hour. Lower panel: densitometry for normalized expression of iNOS from macrophages (N = 3/group).

Figure 4

Vitamin C regulates macrophage function during the resolution of inflammation. (a) Macrophages were isolated on day 3 following TG-mediated peritonitis from VitC sufficient or deficient mice as well as in deficient mice treated daily  with i.p. AscA for 3 days and intracellular concentrations of VitC measured (N = 3–10 mice/group, ns = not significant). (b) Real time QPCR for Gal1 and 15-Lox from peritoneal macrophages elicited on day 3 following TG-induced peritonitis from VitC sufficient and deficient Gulo^−/− mice. Thirty minutes following TG challenge, some VitC deficient mice were randomized to receive i.p. injection of VitC as AscA (200 mg/kg in saline) for a further 3 days (deficient + AscA). (N = 6 mice/group, ns = not significant). (c) Real time QPCR for Gal1 and 15-Lox from peritoneal macrophages elicited on day 5 following TG-induced peritonitis from VitC sufficient and deficient Gulo^−/− mice. Thirty minutes following TG challenge, some VitC deficient mice were randomized to receive i.p. injection of VitC as AscA (200 mg/kg in saline) for a further 5 days (Deficient + AscA) (N = 6 mice/group, ns = not significant). (d) UPLC ESI-MS/MS quantification of resolvin D1 (RvD1) and E1 (RvE1) in peritoneal lavage on day 5 following TG-induced peritonitis from VitC sufficient and deficient Gulo^−/− mice (N = 3-4 mice/group, ns = not significant).

Figure 5

Vitamin C influences macrophage phenotype during resolution of inflammation. Flow cytometry for distribution of CD11b^high and CD11b^low population from macrophages isolated on day 3 and day 5 following TG-induced peritonitis in VitC sufficient or deficient mice (N = 5 mice/group, P < 0.05, CD11b^low day 3 versus day 5, ns = not significant).

Figure 6

Macrophages deficient in Vitamin C have reduced antioxidant capacity. Peritoneal macrophages elicited on day 3 following TG-induced peritonitis from VitC sufficient ((a)–(d)) and deficient Gulo^−/− ((e)–(h)) mice were exposed to 12.5, 25, and 50 μM H₂O₂ for 18 hours and probed with MitoTracker Red CMXRos. Macrophages from some VitC deficient mice were incubated with AscA (3 mM, 16 hours) prior to exposure to H₂O₂ followed by staining with MitoTracker Red CMXRos (Deficient + AscA, ((i)–(l))).

Figure 7

Vitamin C attenuates proinflammatory gene expression in human THP-1 monocyte/macrophages. THP-1 macrophages were exposed to media alone (Media), AscA (3 mM, 16 hours (AscA)), and LPS (50 ng/mL) for 4 hour (LPS) or AscA for 16 hours followed by LPS for 4 hour (LPS + AscA). (a) Real time QPCR for IL-6, IL-8, and TNFα was performed as described in Section 2 (N = 4/group; P < 0.05, Media versus LPS and LPS versus LPS + AscA). (b) Left panel: representative western blot for expression of phospho-NFκB and NFκB from THP-1 groups described above following exposure to LPS (50 ng/mL) for 0, 15, 30, and 60 minutes. Right panel: densitometry for normalized expression of phospho-NFκB from THP-1 (N = 4/group, P < 0.05, LPS versus LPS + AscA).