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

Abstract

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 <i>Il1b</i>, <i>Il6</i>, and <i>Tnfa</i> 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 (<i>Cnlp</i>) and macrophage receptor with collagenous structure (<i>Marco</i>), and the tissue-repairing gene vascular endothelial growth factor C (<i>Vegfc</i>). 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 <i>Irak3</i> and <i>Wdr5</i> expression. Specifically, IL-1 receptor-associated kinase 3 (IRAK3) was responsible for inhibiting proinflammatory genes <i>Il1b</i>, <i>Il6</i>, and <i>Tnfa</i> in tolerized macrophages after LPS rechallenge, whereas WDR5 contributed to the upregulation of host beneficial genes including <i>Cnlp</i>, <i>Marco</i>, and <i>Vegfc</i>. 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 <i>Escherichia coli</i>. 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.