RBP-J is required for M2 macrophage polarization in response to chitin and mediates expression of a subset of M2 genes

Abstract

Development of alternatively activated (M2) macrophage phenotypes is a complex process that is coordinately regulated by a plethora of pathways and factors. Here, we report that RBP-J, a DNA-binding protein that integrates signals from multiple pathways including the Notch pathway, is critically involved in polarization of M2 macrophages. Mice deficient in RBP-J in the myeloid compartment exhibited impaired M2 phenotypes in vivo in a chitin-induced model of M2 polarization. Consistent with the in vivo findings, M2 polarization was partially compromised in vitro in Rbpj-deficient macrophages as demonstrated by reduced expression of a subset of M2 effector molecules including arginase 1. Functionally, myeloid Rbpj deficiency impaired M2 effector functions including recruitment of eosinophils and suppression of T cell proliferation. Collectively, we have identified RBP-J as an essential regulator of differentiation and function of alternatively activated macrophages.

Keywords: M2; RBP-J; arginase; chitin; macrophages.

Figure 1

A role for Rbpj in eosinophil recruitment in response to chitin administration. (A) Expression of CD11b and F4/80 in peritoneal exudates cells (PECs) harvested from WT and Rbpj cKO mice 1 d after peritoneal injection with chitin. Circles and numbers indicate percentage of macrophages (CD11b⁺F4/80⁺) in total PECs. Results from one representative experiment are shown. (B) Cumulative data showing percentage of macrophages as in (A) from three independent experiments. (C) Expression of CD45 and SiglecF in PECs harvested from WT and Rbpj cKO mice 1 d after peritoneal injection with chitin. Quadrants and numbers indicate percentage of eosinophils (CD45⁺SiglecF⁺) in total PECs. (D) Cumulative data showing percentage of eosinophils as in (C) using six pairs of littermate mice with desired genotypes. Errors bars indicate s.d. *P < 0.05 (two-tailed Student’s t-test)

Figure 2

Crucial role for Rbpj in regulating M2 macrophage polarization in response to chitin administration in vivo. Total mRNA was prepared from PECs isolated from WT and Rbpj cKO mice 24 h after administration of chitin, and mRNA expression of Arg1, MR, Ym1, Marco, Jmjd3, and IL-12p40 (relative to GAPDH mRNA) was measured using quantitative real-time PCR. ****P < 0.0001 (two-tailed Student’s t-test). *P < 0.05 (two-tailed Student’s t-test). Results are representative of four independent experiments, each with two-four pairs of littermate mice (analyzed individually)

Figure 3

Rbpj-deficient alternatively activated macrophages are defective in suppressing T cell proliferation. (A) PECs from control, PBS-injected WT mice (filled gray histogram), and day-1 chitin-elicited PECs from WT mice (black line) and Rbpj cKO (grey line) were co-cultured with CFSE-labeled LN cells from OT-II transgenic mice and cognate OVA peptide. Proliferation of CD4⁺ cells was examined at 96 h of co-culture. Results are representative of three independent experiments. (B) CFSE-labeled OT-II transgenic LN cells were co-cultured with control (filled gray histogram) or IL-4-treated (10 ng/mL, black line) BMDMs from WT or Rbpj cKO mice and cognate OVA peptide. Proliferation of CD4⁺ cells was examined at 96 h of co-culture. Results are representative of two independent experiments

Figure 4

A crucial role for Rbpj in regulating expression of a subset of M2 macrophage genes. BMDMs from WT and Rbpj cKO mice were stimulated with IL-4 (10 ng/mL) for the indicated times. (A) Immunoblot analysis of whole cell lysates using antibodies recognizing Arg1. Levels of SHP2 served as loading controls. Results are representative of three independent experiments. (B and C) mRNA expression (relative to GAPDH mRNA) was measured using quantitative real-time PCR. Cumulative results from three independent experiments are shown (errors bars indicate s.d.). P < 0.05, **P < 0.01 (two-tailed Student’s t-test)

Figure 5

RBP-J regulates Arg1 expression in macrophages independently of STAT6 and IRF8. (A) BMDMs from WT and Rbpj cKO mice were stimulated with 100 ng/mL of IL-4 for the indicated times. Whole cell lysates were analyzed with immunoblotting using antibodies recognizing STAT6 phosphorylated on Tyr641 (pSTAT6). Levels of SHP2 served as loading controls. Results are representative of three independent experiments. (B) BMDMs from WT and Rbpj cKO mice were stimulated with 10 ng/mL of IL-4 and 1 ng/mL of LPS for the indicated times. Whole cell lysates were analyzed with immunoblotting using antibody recognizing IRF8. Levels of p38 served as loading controls. Results are representative of three independent experiments. (C) mRNA expression of Arg1 (relative to GAPDH mRNA) was measured in BMDMs from WT and Irf8 ^−/− mice. Cumulative results from two independent experiments (errors bars indicate s.d.) are shown. (D) BMDMs from WT and Irf8 ^−/− mice were stimulated with 10 ng/mL of IL-4 for the indicated times. Whole cell lysates were analyzed with immunoblotting using antibodies against Arg1 and IRF8. Levels of SHP2 served as loading controls. Results are representative of three independent experiments