Abstract
Bacterial sepsis involves a complex interaction between the host immune response and bacterial LPS. LPS binds Toll-like receptor (TLR) 4, which leads to the release of proinflammatory cytokines that are essential for a potent innate immune response against pathogens. The innate immune system is tightly regulated, as excessive inflammation can lead to organ failure and death. MicroRNAs have recently emerged as important regulators of the innate immune system. Here we determined the function of miR-718, which is conserved across mammals and overlaps with the 5' UTR of the interleukin 1 receptor-associated kinase (IRAK1) gene. As IRAK1 is a key component of innate immune signaling pathways that are downstream of most TLRs, we hypothesized that miR-718 helps regulate the innate immune response. Activation of TLR4, but not TLR3, induced the expression of miR-718 in macrophages. miR-718 expression was also induced in the spleens of mice upon LPS injection. miR-718 modulates PI3K/Akt signaling by directly down-regulating phosphatase and tensin homolog (PTEN), thereby promoting phosphorylation of Akt, which leads to a decrease in proinflammatory cytokine production. Phosphorylated Akt induces let-7e expression, which, in turn, down-regulates TLR4 and further diminishes TLR4-mediated proinflammatory signals. Decreased miR-718 expression is associated with bacterial burden during Neisseria gonorrhoeae infection and alters the infection dynamics of N. gonorrhoeae in vitro Furthermore, miR-718 regulates the induction of LPS tolerance in macrophages. We propose a role for miR-718 in controlling TLR4 signaling and inflammatory cytokine signaling through a negative feedback regulation loop involving down-regulation of TLR4, IRAK1, and NF-κB.
Highlights
Bacterial sepsis involves a complex interaction between the host immune response and bacterial LPS
Activation of Toll-like receptor 4 (TLR4), but not TLR3, induced the expression of miR-718-expressing cell line (miR-718) in macrophages. miR-718 expression was induced in the spleens of mice upon LPS injection. miR718 modulates PI3K/Akt signaling by directly down-regulating phosphatase and tensin homolog (PTEN), thereby promoting phosphorylation of Akt, which leads to a decrease in proinflammatory cytokine production
We prioritized miR-718 for further study, as it was within the 5Ј UTR of IRAK1, which encodes interleukin 1 receptor-associated kinase 1, a key component of Toll-like receptor (TLR) and IL-1R signaling pathways (Fig. 1, A and B)
Summary
Using our genome analysis pipeline, we identified several miRNAs that are hosted by innate immunity protein-coding genes (supplemental Table S1). The phosphorylated form of Akt was highly expressed in miR-718 macrophages (Fig. 5E, bottom panels), whereas it was downregulated in Anti-miR-718 macrophages (supplemental Fig. S4C, bottom panel) These data suggest that PTEN protein levels are decreased by overexpression of miR-718, which, in turn, enhances PI3K/Akt signaling. We confirmed these results by using two additional Akt inhibitors (API-1 and MK-2206) (supplemental Fig. S4, D and E) and Anti-miR-718 cells (supplemental Fig. S4F) Overall, these data indicated that, by targeting PTEN, miR-718 suppresses the production of proinflammatory cytokines through activation of Akt. LPS induces the release of proinflammatory cytokines through the innate immune receptor TLR4. TLR4 was already expressed at low levels in unstimulated miR-718 macrophages, and, LPS stimulation only caused a minor reduction in TLR4 levels To confirm these results, we used a strategy in which cell surface proteins were biotinylated and subsequently immunoprecipitated and purified using NeutrAvidin gel. These results suggest that miR-718 contributes to immune responses against N. gonorrhoeae infection
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