Fig 2: A model of MyD88-dependent TLR signaling events in the presence and absence of IRAK4 kinase activity. A, activation of TLR dimers at membranes leads to rapid recruitment of the TIR adaptor MyD88 and the ordered assembly of an oligomeric signaling platform termed the myddosome, which comprises four layers: 2× molecules of MyD88, 4× molecules of MyD88, 4× molecules of IRAK4, and 4× molecules of IRAK1/2. Myddosome formation facilitates IRAK4 autophosphorylation and subsequent phosphorylation of IRAK1. The E3 Ub ligase, TRAF6, is then thought to simultaneously activate signaling pathways leading to the transcriptional activity of NF-κB, AP-1 and cAMP-response element–binding protein (via the MAPK pathway), and IRF5, which act in concert for the production of inflammatory cytokines. B, in the absence of IRAK4 kinase activity, the myddosome assembles into a more stable complex following TLR activation. The activation of NF-κB and MAPK signaling is only moderately affected by IRAK4 inhibition.

Fig 3: IRAK4 kinase inhibition stabilizes TLR-induced myddosome interactions. A, BMDMs were pretreated with either DMSO or 20 μm I4 inhb for 30 min. Cells were then left untreated or stimulated with 1 μg/ml LPS for a further 30 min. WCLs were then subjected to MyD88 IP followed by immunoblotting for IRAK4, IRAK1, P-IRAK4, and MyD88 or directly subjected to immunoblotting with the same antibodies. MyD88 immunoblotting also acted as a loading control. The data presented are representative of three independent experiments. B, WT iBMDMs were treated as in A, before WCLs were subjected to MyD88 IP followed by immunoblotting for IRAK4, IRAK1, and MyD88 or directly subjected to immunoblotting with the same antibodies. The data presented are representative of three independent experiments. C, BMDMs were pretreated with either DMSO or 20 μm I4 inhb for 30 min. Cells were then left untreated or stimulated with either 500 ng/ml P3C or 0.5 μm CpG DNA 1826 for a further 30 min. WCLs were then subjected to MyD88 IP followed by immunoblotting for IRAK4, IRAK1, P-IRAK4, and MyD88 or directly subjected to immunoblotting with the same antibodies. MyD88 immunoblotting also acted as a loading control. The data presented are representative of three independent experiments. D, Irak4−/− iBMDMs expressing IRAK4-mCitrine were treated as in A before WCLs were subjected to GFP IP, followed by immunoblotting for MyD88 and GFP, or directly subjected to immunoblotting for P-IRAK4, IRAK1, GFP, and MyD88. The data presented are representative of three independent experiments. E, Irak4−/− iBMDMs expressing either mCitrine alone, IRAK4-mCitrine, or IRAK4 KD-mCitrine were stimulated with 1 μg/ml LPS for 30 min before WCLs were subjected to GFP IP followed by immunoblotting for MyD88 and GFP or directly subjected to immunoblotting with the same antibodies. The data presented are representative of four independent experiments. F, BMDMs were pretreated with either DMSO or 20 μm I4 inhb for 30 min. Cells were then left untreated or stimulated with 1 μg/ml LPS for a further 2 or 4 h. WCLs were then subjected to MyD88 IP, followed by immunoblotting for IRAK4, IRAK1, P-IRAK4, and MyD88, or directly subjected to immunoblotting with the same antibodies. MyD88 immunoblotting also acted as a loading control. The data presented are representative of three independent experiments.

Fig 4: IRAK4 kinase activity is critical for MyD88-dependent cytokine production. A, BMDMs were pretreated with either DMSO or a 20 µm concentration of a selective IRAK4 inhibitor (I4 inhb) for 30 min. Cells were then left untreated (UT) or stimulated with either 1 µg/ml LPS, 500 ng/ml P3C, or 0.5 µm CpG DNA 1826 for a further 4 h. Cells were then lysed for RNA isolation and mRNA analysis by qPCR. Data are presented as the mean ± S.E. (error bars) (DMSO versus I4 inhb: *, p < 0.05; **, p < 0.01) combined from three independent experiments. B, BMDMs were pretreated with either DMSO or 0.2–20 µm I4 inhb for 30 min. Cells were then left untreated (UT) or stimulated with either 1 µg/ml LPS, 500 ng/ml P3C, or 0.5 µm CpG DNA 1826 for a further 4 h. Supernatants were then collected and subjected to ELISA to determine the levels of secreted TNF. Data are presented as the mean ± S.E. (DMSO versus I4 inhb: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001) combined from four individual experiments. C, Irak4-/- iBMDMs expressing either mCitrine alone, IRAK4-mCitrine, or IRAK4 KD-mCitrine were stimulated with 1 µg/ml LPS, 500 ng/ml P3C, 0.5 µm CpG DNA 1826, or 500 µg/ml CMA (a murine STING ligand) for 4 h. Supernatants were then collected and subjected to ELISA to determine the levels of secreted TNF. Data are presented as the mean ± S.E. (IRAK4-mCit versus IRAK4 KD-mCit: ns, not significant; *, p < 0.05; ***, p < 0.001) combined from three individual experiments.

Fig 5: IRAK4 kinase activity is dispensable for NF-?B and MAPK activation downstream of TLRs. A–C, BMDMs were pretreated with either DMSO or a 20 µm concentration of a selective I4 inhb for 30 min. Cells were then left untreated or stimulated with 1 µg/ml LPS (A) or 500 ng/ml P3C (B) for an additional 30, 60, or 90 min. WCLs were then subjected to immunoblotting for IRAK1, P-IRAK4, NF-?B P-p65, P-p38 MAPK, and actin as a loading control. Data presented are representative of at least three independent experiments. C, cells were then left untreated (UT) or stimulated with 500 ng/ml P3C for an additional 4 h before fixation and staining with an NF-?B P-p65 antibody, followed by a specific secondary antibody conjugated to an Alexa-488 fluorophore. Samples were then examined by confocal microscopy. Representative images are presented from three biological replicates. Scale bar, 10 µm. D, NF-?B P-p65 nuclear translocation was then quantified by examining the mean fluorescence intensity of the Alexa-488 signal in cell nuclei. Nuclear intensity was made relative to DMSO-untreated samples and presented as the mean ± S.E. (error bars) (DMSO versus IRAK4 inhb: *, p < 0.05; **, p < 0.01) combined from BMDMs derived from three individual mice (biological replicates).