Fig 2: Tyrosine phosphorylation of TLR2 regulates NF-κB–mediated cytokine production.(A and B) Whole-cell lysates from primary murine macrophages derived from Tnks+/+ Tnks2fl/fl (WT) and Tnks–/– Tnks2fl/fl LysM-Cre (KO) mice, starved for 12 hours with 0.1% FBS and cultured in the presence of HKSA (107 cells/mL) for 0–30 minutes, were probed with the indicated antibodies for Western blot analysis. (C and G) Primary murine macrophages derived from Tnks+/+ Tnks2fl/fl and Tnks–/– Tnks2fl/fl LysM-Cre mice were starved for 12 hours with 0.1% FBS and cultured in the presence of HKSA (107 cells/mL) for 20 minutes, and SYK (C) or MyD88 (G) immune complexes were probed with the indicated antibodies for Western blot analysis. (D) Primary murine macrophages derived from Tnks+/+ Tnks2fl/fl and Tnks–/– Tnks2fl/fl LysM-Cre mice were starved for 12 hours with 0.1% FBS and cultured in the presence of HKSA (107 cells/mL) for 0–30 minutes, and pY immune complexes were probed with the indicated antibodies for Western blot analysis. (E) Whole-cell lysates from primary murine macrophages derived from Tnks+/+ Tnks2fl/fl and Tnks–/– Tnks2fl/fl LysM-Cre mice were probed with the indicated antibodies for Western blot analysis. (F) Nonradioactive pulse-chase assay to analyze turnover of endogenous TLR2 protein labeled with Click-IT Metabolic Labeling in primary murine macrophages derived from Tnks+/+ Tnks2fl/fl and Tnks–/– Tnks2fl/fl LysM-Cre mice. Biotinylated proteins were probed with the indicated antibodies, and percentages of TLR2 protein levels were plotted as a function of time. (H) qPCR analysis of Tnfa mRNA expression in primary murine macrophages derived from Tnks+/+ Tnks2fl/fl (WT) and Tnks–/– Tnks2fl/fl LysM-Cre (KO) mice and cultured in the presence or absence of SYK inhibitor (10–50 μM) and PP2 (10 μM); n = 3. P values were determined by unpaired t test (C, E, and G) or ANOVA with Tukey-Kramer post hoc test (A, B, D, and H). Data are presented as mean ± SEM. *P < 0.05.

Fig 3: Activation of the TLR2–NF-κB and mTOR signaling pathways by N. caninum infection impairs autophagy in mouse peritoneal macrophages (PMs). (A) Western blot analysis of TLR2, p62, LC3, p-IκBα, p-p65, and β-actin in macrophages from WT and TLR2−/− mice infected with N. caninum (MOI = 3) for 2, 12, and 24 h. Macrophages were treated with Pam3CSK4 (10 μg/ml) as a positive control. The rate of p62/β-actin was shown. (B) Confocal microscopy was used to detect the translocation of NF-κB from the cytoplasm to the nucleus in both WT and TLR2−/− mouse peritoneal macrophages. Scale bars: 10 μm. (C) Eight hours postinfection with N. caninum (MOI = 3), the accumulation of p62/SQSTM1 was examined by confocal microscopy in both the WT and TLR2−/− groups. Quantitative analysis of the number of p62 puncta. The graph represents the average data of at least 100 cells in each experimental group in three independent experiments. Scale bars: 10 μm. (D) WT and TLR2−/− mouse PMs were infected with N. caninum (MOI = 3) for 8 h and then immunoblotted for whole-cell lysis analysis of p-mTOR and p-AKT protein expression. The phosphorylated mTOR and AKT amounts were quantified by densitometric analysis. The data are expressed as the mean ± SEM from three separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns indicates not significant.

Fig 4: TLR2 tyrosine 647 is a phospho-switch that regulates TLR2 stabilization and activation.(A) Luciferase activity from an NF-κB reporter assay in HEK293T cells cotransfected with the indicated constructs; n = 3. (B) HEK293T cells cotransfected with the indicated constructs were treated with 10 μM MG132 for 8 hours before collection of cell lysates. pY immune complexes were probed with an anti-TLR2 antibody. (C, F, and G) HEK293T cells were cotransfected with the indicated constructs, and Myc-TLR2 (C), GST-MyD88 (F), or Flag-3BP2 (G) immune complexes were probed with the indicated antibodies for Western blot analysis. (D) HEK293T cells cotransfected with the indicated constructs were treated with 10 μM MG132 for 4 hours before collection of cell lysates. Myc-TLR2 immune complexes were probed with an anti-HA antibody. (E) Whole-cell lysates from HEK293T cells cotransfected with the indicated constructs were probed with the indicated antibodies for Western blot analysis. (H) Whole-cell lysates from primary murine macrophages derived from Sh3bp2+/+ and Sh3bp2–/– mice were probed with the indicated antibodies for Western blot analysis. (I) Primary murine macrophages derived from Sh3bp2+/+ and Sh3bp2–/– mice were lysed, and PPP1R11 immune complexes were probed with the indicated antibodies for Western blot analysis. P values were determined by unpaired t test (H and I) or ANOVA with Tukey-Kramer post hoc test (A–G). Data are presented as mean ± SEM. *P < 0.05.

Fig 5: Signaling events behind gut microbiota-derived small EVs in murine neutrophils. Murine bone marrow neutrophils were primed for 45 min by microbiota-derived small EVs (low concentration: 1 ng/mL; high concentration: 28.1 µg/mL) and later, on day 2, were re-challenged by 100 ng/mL LPS for 4 h as described above. Protein expression of TLR2 (A, n = 5), TLR4 (B, n = 5) and MyD88 (C, n = 5) were assayed by Western blotting and quantified (unprimed cells assigned as 1.0). Data are presented as scatter dot plots, mean + SEM, # p < 0.05, # versus unprimed condition (UP, gray bar).