PKR inhibition mediates endotoxin tolerance in macrophages through inactivation of PI3K/AKT signaling.

Following long‑term exposure to endotoxins, macrophages enter an immunosuppressive state that renders them unable respond to subsequent exposures to endotoxin, a phenomenon that is termed 'endotoxin tolerance'. Endotoxin tolerance increases the risks of secondary infection and mortality in patients with sepsis. In endotoxin‑tolerant macrophages, the mixed variation of gene transcription is referred to as macrophage reprogramming. The mechanisms underlying macrophage reprogramming remain unclear at present. Interferon‑induced double‑stranded RNA‑dependent protein kinase (PKR) is a widely expressed serine/threonine protein kinase. In addition to antiviral effects, PKR regulates the transcription of inflammatory cytokines by affecting transcription factors. However, the role of PKR in macrophage reprogramming remains to be elucidated. In the present study, the expression of inflammatory cytokines differed in lipopolysaccharide (LPS)‑tolerant RAW264.7 macrophages compared with LPS‑activated macrophages. Specifically, reverse transcription‑quantitative polymerase chain reaction results demonstrated that the mRNA levels of tumor necrosis factor‑α, interleukin‑1β (IL‑1β), C‑X‑C motif chemokine ligand 11, C‑C motif chemokine ligand (CCL17), CCL22 and suppressor of cytokine signaling 3 were decreased, and mRNAs levels of arginase‑1 (Arg1) and nitric oxide synthase 2 (iNOS) were increased, in LPS‑tolerant macrophages compared with LPS‑activated macrophages. Furthermore, western blot analysis demonstrated that the protein levels of phosphorylated (p)‑PKR were significantly decreased in the LPS‑tolerant cells. PKR activation with rotenone (10 µM) abrogated endotoxin tolerance by increasing the levels of the IL‑1β, CCL17 and CCL22 mRNAs and decreasing the levels of the Arg1 and iNOS mRNAs. Furthermore, western blotting demonstrated that AKT was markedly inactivated in endotoxin‑tolerant cells, as indicated by reduced p‑AKT levels. However, levels of p‑AKT were markedly increased following rotenone‑induced PKR activation in endotoxin‑tolerant cells. Ly294002 (10 µM), a phosphatidylinositol‑4,5‑bisphosphate 3‑kinase (PI3K)/AKT signaling inhibitor, partially reversed the rotenone‑induced alleviation of endotoxin tolerance. These results demonstrated that PKR inhibition mediated endotoxin tolerance in macrophages, and these effects were partially mediated by PI3K/AKT signaling. PKR may be a potential target for the treatment of endotoxin tolerance in patients with sepsis.