Valproic acid accelerates skin wound healing in mice via its anti-inflammatory and apoptotic cell clearance-promoting effects.

OBJECTIVE: To investigate the effects of valproic acid (VPA) on skin wound healing in mice. METHODS: Full-thickness wounds were created in mice, and then VPA was applied. The wound areas were quantified daily. In the wounds, granulation tissue growth, epithelialization, collagen deposition, and the mRNA levels of inflammatory cytokines were measured; furthermore, apoptotic cells were labeled. In vitro, VPA was added to RAW 264.7 cells (macrophages) stimulated with lipopolysaccharide, and apoptotic Jurkat cells were cocultured with the VPA-pretreated macrophages. Then, phagocytosis was analyzed, and the mRNA levels of phagocytosis-associated molecules and inflammatory cytokines were measured in the macrophages. RESULTS: VPA application significantly accelerated wound closure, granulation tissue growth, collagen deposition, and epithelialization. In wounds, the levels of tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß were decreased by VPA, whereas those of IL-10 and transforming growth factor-ß1 were increased. Additionally, VPA reduced the number of apoptotic cells. In vitro, VPA inhibited the inflammatory activation of macrophages and promoted the phagocytosis of apoptotic cells by macrophages. CONCLUSION: VPA accelerated skin wound healing, which could be partly attributable to its anti-inflammatory and apoptotic cell clearance-promoting effects, indicating that VPA could be a promising candidate for enhancing skin wound healing.

Erythropoietin mediates re-programming of endotoxin-tolerant macrophages through PI3K/AKT signaling and protects mice against secondary infection.

Initial lipopolysaccharide (LPS) exposure leads to a hypo-responsive state by macrophages to a secondary stimulation of LPS, known as endotoxin tolerance. However, recent findings show that functions of endotoxin-tolerant macrophages are not completely suppressed, whereas they undergo a functional re-programming process with upregulation of a panel of molecules leading to enhanced protective functions including antimicrobial and tissue-remodeling activities. However, the underlying molecular mechanisms are still elusive. Erythropoietin (EPO), a glycoprotein regulated by hypoxia-inducible factor 1α (HIF-1α), exerts anti-inflammatory and tissue-protective activities. Nevertheless, the potential effects of EPO on functional re-programming of endotoxin-tolerant macrophages have not been investigated yet. Here, we found that initial LPS exposure led to upregulation of HIF-1α/EPO in macrophages and that EPO enhanced tolerance in tolerized macrophages and mice as demonstrated by suppressed proinflammatory genes such as Il1b, Il6, and Tnfa after secondary LPS stimulation. Moreover, we showed that EPO improved host protective genes in endotoxin-tolerant macrophages and mice, such as the anti-bacterial genes coding for cathelicidin-related antimicrobial peptide (Cnlp) and macrophage receptor with collagenous structure (Marco), and the tissue-repairing gene vascular endothelial growth factor C (Vegfc). Therefore, our findings indicate that EPO mediates the functional re-programming of endotoxin-tolerant macrophages. Mechanistically, we found that PI3K/AKT signaling contributed to EPO-mediated re-programming through upregulation of Irak3 and Wdr5 expression. Specifically, IL-1 receptor-associated kinase 3 (IRAK3) was responsible for inhibiting proinflammatory genes Il1b, Il6, and Tnfa in tolerized macrophages after LPS rechallenge, whereas WDR5 contributed to the upregulation of host beneficial genes including Cnlp, Marco, and Vegfc. In a septic model of mice, EPO pretreatment significantly promoted endotoxin-tolerant re-programming, alleviated lung injury, enhanced bacterial clearance, and decreased mortality in LPS-tolerized mice after secondary infection of Escherichia coli. Collectively, our results reveal a novel role for EPO in mediating functional re-programming of endotoxin-tolerant macrophages; thus, targeting EPO appears to be a new therapeutic option in sepsis and other inflammatory disorders.

Erythropoietin Promotes Infection Resolution and Lowers Antibiotic Requirements in E. coli- and S. aureus-Initiated Infections.

Endogenous mechanisms underlying bacterial infection resolution are essential for the development of novel therapies for the treatment of inflammation caused by infection without unwanted side effects. Herein, we found that erythropoietin (EPO) promoted the resolution and enhanced antibiotic actions in Escherichia coli (E. coli)- and Staphylococcus aureus (S. aureus)-initiated infections. Levels of peritoneal EPO and macrophage erythropoietin receptor (EPOR) were elevated in self-limited E. coli-initiated peritonitis. Myeloid-specific EPOR-deficient mice exhibited an impaired inflammatory resolution and exogenous EPO enhanced this resolution in self-limited infections. Mechanistically, EPO increased macrophage clearance of bacteria via peroxisome proliferator-activated receptor γ (PPARγ)-induced CD36. Moreover, EPO ameliorated inflammation and increased the actions of ciprofloxacin and vancomycin in resolution-delayed E. coli- and S. aureus-initiated infections. Collectively, macrophage EPO signaling is temporally induced during infections. EPO is anti-phlogistic, increases engulfment, promotes infection resolution, and lowers antibiotic requirements.