Fig 2: The disruption of the IRAK-M homeostatic circuit is due to miR-24-mediated suppression of Smad4.(a–h) BMMs from C57 BL/6 mice were cultured with M-CSF (10 ng ml-1) in the presence of LPS (0.1 ng ml-1) for 5 days and SP600125 or miR-24 antagomir was added to indicated cultures. Fresh LPS, SP600125 and miR-24 antagomir was added every 2 days. The mRNA and protein levels of SMAD4 and IRAK-M in the cell cultures were analysed by real-time reversre transcriptase–PCR and western blotting, respectively. (i) A schematic illustration of the SMAD4 3'-UTR and potential miR-24 binding sites. (j) Luciferase activity assays in 293T cells transfected with SMAD4 3'-UTR luciferase reporter plasmids in the presence of miR-24 mimic or the scramble control. (k) BMMs were cultured for 5 days and miR24 antagomir was transfected. On 7 days, the cells were stimulated by LPS (0.1 ng ml-1) for 4 h and then harvested for RNA co-immunoprecipitation (RIP). Quantified data are shown from cells with indicated treatment (n=3) and results are representative of three experiments. Error bars show means±s.e.m.; NS, not significant; *P<0.05, **P<0.01 and ***P<0.001; one-way analysis of variance.

Fig 3: Disruption of IRAK-M results in sustained pro-inflammatory programming of monocytes both in vivo and in vitro.(a) A schematic illustration of network dynamics underlying the establishment of non-resolving inflammation. (b–e) BMMs from WT C57 BL/6 mice or Irak-M-/- mice were cultured with M-CSF (10 ng ml-1) for 5 days. (b) JNK phosphorylation within CD11b+Ly6C++ monocytes. (c) The levels of miR-24-3p within CD11b+Ly6C++ monocytes . (d,e) The mRNA and protein levels of SMAD4. (f) The bifurcation diagram that illustrates a relatively tight suppression of MCP-1 expression in resting WT cells. Solid lines show two stable steady states (upper branch: MCP-1-positive state; lower branch: MCP-1-negative state). Dotted line is the unstable steady states. All cells reside at MCP-1-negative state because of the large energy barrier for a MCP-1-negative to MCP-1-positive state transition (red line). AU, conceptual arbitrary unit. (g) Deletion of IRAK-M allows leaky low-grade inflammation to occur as manifested in the leaky activation of MCP-1. Bifurcation diagram was plotted similar to (f). AU, arbitrary unit. (h) Flow cytometry analysis of MCP-1 levels within cultured CD11b+Ly6C++ monocytes from WT and Irak-M-/- mice(n=3). (i) Expression levels of SR-B1 within cultured CD11b+Ly6C++ monocytes from WT and Irak-M-/- mice (n=3). (j–l) ApoE-/- and ApoE-/-/Irak-M-/- mice were fed with HFD for 8 weeks. (j) Circulating CD11b+Ly6C++ and CD11b+Ly6C+ monocytes were examined by flow cytometry. Data are shown from ApoE-/- (n=5) and ApoE-/-/Irak-M-/- (n=6) mice. (k) The expression levels of miR-24 from splenocytes were analysed between ApoE-/- (n=4) and ApoE-/-/Irak-M-/- (n=4) mice. (l) The expression levels of SR-B1 within circulating monocytes were analyzed between ApoE-/- (n=5) and ApoE-/-/Irak-M-/- (n=6) mice. All data are representative of at least two similar experiments. Error bars show means±s.e.m.; NS, not significant; **P<0.01 and ***P<0.001; Student's t-test.