Figure 3. Lamtor1 is required for IL-4−elicited activation of mTORC1, which is necessary for M2 polarization.
(a) Western blots for previously known essential M2−polarizing factors in IL-4−stimulated BMDMs. See Supplementary Fig. 8a for confirmation of the fractionation. (b) Immunofluorescence confocal microscopy for phospho-S6 ribosomal protein indicated decreased mTORC1 activity in Lamtor1-deficient BMDM. Scale bars indicate 20 μm. (c) Flow cytometry showed that Lamtor1-deficient BMDMs were smaller in cell sizes than wild-type BMDMs. (d) Time course for acute activation of mTORC1, mTORC2 and JAK-STAT6 pathway during the stimulation by IL-4 (that is, M2-polarizing condition). (e) Time course for acute activation of mTORC1 and mTORC2 in BMDMs stimulated by 100 ng ml−1 of LPS. (f–h) Inhibition of mTORC1 by an mTOR inhibitor selectively abolished M2 polarization, but did not block M1 polarization. (f) Real-time PCR for M2 signature genes. M2 macrophages were polarized with IL-4 stimulation and concomitant mTOR inhibition. See Supplementary Fig. 8b for exclusion of unintended suppressive effect of the vehicle. (g) Real-time PCR for an M1 signature gene in wild-type BMDMs. M1 macrophages were polarized with IFNγ and LPS stimulation and concomitant mTOR inhibition. (h) Real-time PCR showed that M2 polarization of wild-type BMDMs was blocked even at 8 h of IL-4 stimulation and concomitant inhibition of mTOR. (i,j) Engulfed apoptotic cells activated mTORC1, and decreased IL-6 production from LPS-stimulated BMDMs. (i) BMDMs that had phagocytosed apoptotic thymocytes showed active mTORC1, even in the absence of amino acids in culture medium. Western blots for phosphorylation of S6K are shown as mTORC1 activity. (j) BMDMs that had phagocytosed apoptotic thymocytes showed decreased production of IL-6 after stimulation by LPS (100 ng ml−1) in amino acids-free medium. IL-6 concentration measured by ELISA (left panel), and calculated percentage of the decrease (right panel) are shown. In left panel, BMDMs that did not engulf apoptotic thymocytes are shown as white bars, and ones that engulfed apoptotic thymocytes are shown as black bars. ΦWT and ΦKO are defined as in Fig. 1. The representative results of three independent experiments are shown (a–g). *P<0.05, **P<0.01, ***P<0.001. Error bars show s.d.