Extended Data Fig. 2: Butyrate boosts efferocytosis via induction of efferocytotic transcriptional programs.

(a) Mature macrophages were conditioned with butyrate (1 mM) for 3d prior to efferocytosis assay. Conditioned macrophages were subsequently incubated with apoptotic mouse thymocytes co-labeled with CypHer5E and CTY at a 1:10 ratio for 1 h. Shown is summary data from flow cytometry analysis of efferocytosis efficiency (left, CypHer5E+ macrophages) and capacity (right, CTY MFI in macrophages). Data are from three independent experiments. (b) Representative flow cytometry plots of vehicle and butyrate (1 mM)-conditioned primary macrophages for the indicated time points from experiments shown in Fig. 2d. In some conditions, primary macrophages were conditioned for 3d followed by withdraw of butyrate for the indicated time point prior to efferocytosis assay. (c) Representative transluminescence microscopy image of vehicle and butyrate-treated primary macrophages from experiments performed in Fig. 2e. Scale bar is 50 µm. (d) Flow cytometry analysis and quantification of in vitro latex bead (n = 3) and fungal (zymosan particles) (n = 3) phagocytosis in primary macrophages conditioned with vehicle or butyrate (1 mM) for 3d. Data are from three independent experiments. (e) Efferocytosis-associated programs identified in butyrate-treated primary macrophages from experiments performed as in Fig. 2d. Data corresponds to analyses presented in Fig. 2i. (f) Putative efferocytosis-supporting programs identified in butyrate-treated primary macrophages from experiments performed as in Fig. 2d. Data corresponds to analyses presented in Fig. 2j. All bar graphs represent means + s.e.m. Statistics were performed by two-tailed t-test in a and d. *p < .05; ***p < .001. ns - not significant.

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