Extended Data Fig. 7: Related to Figs. 6 and 7: Additional in-vivo and in-vitro data on the role of AhR in efferocytosis-induced resolution.
From: Efferocytosis drives a tryptophan metabolism pathway in macrophages to promote tissue resolution
a, BMDMs were incubated for 1 h with 100 μM Kyn alone or with control (non-PS) or PS beads ± Kyn and then assayed for Cyp1a1 and Ido1 after a 3-h chase (n = 4 samples/group). b, BMDMs incubated with ACs, PS-beads (PS), Kyn, or PS-beads + Kyn were assayed for Tgfb1 after a 6-h chase (n = 4 samples/group). c, BMDMs incubated with 50 μM Trp, PS-beads (PS), Trp and PS-beads, or 100 μM Kyn and PS-beads for 1 h were assayed for Cyp1a1 and Il10 after a 3-h or 6-h chase, respectively (n = 3 biological replicates/group). d, BMDMs pre-treated ± U0126 were incubated with ACs for 1 h and then immunoblotted for AhR and ARNT after a 3-h chase (n = 3 samples/group). e, BMDMs treated with PS-beads (PS) and Kyn ± U0126 for 1 h were assayed for Tgfb1 or Ido1 after a 3-h chase or 6-h chase, respectively (n = 3 biological replicates/group). f, BMDMs incubated ± ACs ± CH223191 for 1 h were assayed Hsp90 or Xap2 after a 3-h chase (n = 3 biological replicates/group). g, Scr-, siHsp90-, or siXap2-transfected BMDMs were incubated with PKH26-labelled ACs and quantified for percent PKH26+ macrophages (arrows). Scale bar, 50 μm (n = 3 biological replicates/group). h, Proposed pathway (created using BioRender.com): Trp form an efferocytosed AC (AC1) is transported into the macrophage by SLC36A4 and then converted to Kyn by IDO1. Kyn and activated ERK induce Hsp90 and Xap2, leading to AhR-ARNT-mediated transcription of Tgfb1, Il10, and Ido1 and Rac1-mediated AC2 internalization (continuing efferocytosis). i-k, The thymi of the mice from Fig. 7 were immunostained for AhR (red) and Mac2 (green) and quantified for AhR MFI in Mac2+ areas (arrows). Also shown are thymus weight and F4/80+ macrophages/thymus (n = 8 mice/group). All mRNA data are expressed relative to the indicated control groups. Data are mean ± SEM, and significance was determined by two-tailed Student’s t-test or one-way ANOVA with Fisher’s LSD post-hoc analysis.
