All Trans Retinoic Acid, Transforming Growth Factor β and Prostaglandin E2 in Mouse Plasma Synergize with Basophil-Secreted Interleukin-4 to M2 Polarize Murine Macrophages

Abstract

In previous studies we found that macrophages (MФs) from SH2-containing inositol-5'-phosphatase (SHIP) deficient mice are M2 polarized while their wild type (WT) counterparts are M1 polarized and that this difference in MФ phenotype can be recapitulated during in vitro derivation from bone marrow if mouse plasma (MP), but not fetal calf serum, is added to standard M-CSF-containing cultures. In the current study we investigated the mechanism by which MP skews SHIP-/- but not +/+ MФs to an M2 phenotype. Our results suggest that SHIP-/- basophils constitutively secrete higher levels of IL-4 than SHIP+/+ basophils and this higher level of IL-4 is sufficient to skew both SHIP+/+ and SHIP-/- MФs to an M2 phenotype, but only when MP is present to increase the sensitivity of the MФs to this level of IL-4. MP increases the IL-4 sensitivity of both SHIP+/+ and -/- MФs not by increasing cell surface IL-4 or CD36 receptor levels, but by triggering the activation of Erk and Akt and the production of ROS, all of which play a critical role in sensitizing MФs to IL-4-induced M2 skewing. Studies to identify the factor(s) in MP responsible for promoting IL-4-induced M2 skewing suggests that all-trans retinoic acid (ATRA), TGFβ and prostaglandin E2 (PGE2) all play a role. Taken together, these results indicate that basophil-secreted IL-4 plays an essential role in M2 skewing and that ATRA, TGFβ and PGE2 within MP collaborate to dramatically promote M2 skewing by acting directly on MФs to increase their sensitivity to IL-4.

Fig 1. MP-induced M2 skewing of SHIP-/- BM progenitors requires IL-4 and basophils but not IL-3.

A) Western blots of MФs derived from BM for 6 days with M-CSF ± IL-3 (10 ng/mL) or MP (5%). B) Arginase activity of MФs from SHIP-/- BM derived for 6 days with 10 ng/mL M-CSF ± IL-3 (10 ng/mL) or MP (5%) with 2.5 μg/mL of either an irrelevant or IL-4-neutralizing Ab. Data (mean ± SD) are representative of 3 independent experiments performed in triplicate. * p < 0.05 compared to all other conditions. C) Western blot of SHIP-/- MФs from (B) probed for Ym1, using SHC as a loading control. Dashed lines indicate where irrelevant lanes have been cropped out. All lanes are part of the same time-exposed film on the same gel. D) Western blot of SHIP-/- basophil-depleted (DX5^-) or basophils (DX5⁺) + DX5^- BM derived with M-CSF ± MP (5%). E) Arginase activity of SHIP-/- MФs derived from BM for 6 days with M-CSF ± MP (5%) or IL-3 (10 ng/mL) ± neutralizing Ab to IL-3 (2.5 μg/mL) or IL-4 (2.5 μg/mL). F) Western blot of cells corresponding to panel (E). *** p < 0.001 compared to relevant control.

Fig 2. MP increases the sensitivity of macrophages to IL-4-induced M2 skewing.

A) IL-4 levels secreted by basophils cultured at 5 x 10⁵ cells/mL for 24 h ± 5% MP or 10 ng/mL IL-3. Addition of DX5 Ab does not stimulate IL-4 production from SHIP-/- BM (data not shown). B) Arginase activity of mature MФs from the same BM used in panel (A) after 6 days of culture. All results (mean ± SD) are representative of 3 independent experiments. Black arrows indicate levels below detection. C) Western blots of mature MФs stimulated for 72 h with IL-4 (0.5 ng/mL) ± the indicated doses of MP. D) Western blots of mature MФs derived for 6 days from basophil-depleted BM with M-CSF + IL-4 (1 ng/mL) ± MP (5%). E) Western blot of MФs derived from BM for 6 days ± MP (5%) from SHIP+/+ and -/- basophil-depleted (DX5^-) BM in the bottom wells of a transwell system with basophils (DX5⁺ cells) of varying genotypes co-cultured in a contact-independent manner at a DX5^-: DX5⁺ ratio of 10:1. 0 = no added DX5⁺ cells. SHC was used as a loading control. All results are representative of 3 independent experiments. ** p < 0.01 compared to relevant control.***p < 0.001 compared to relevant control.

Fig 3. MP enhances IL-4-induced M2 skewing via ROS and activation of Erk and Akt in MФs.

A) Surface IL-4R expression on WT MФs cultured for 48 h ± 10 ng/mL IL-4, 5% MP, or 5% MP + 0.5 ng/mL IL-4, as assessed by FACS using PE-conjugated anti-IL-4Rα. B) Western blot of WT MФs exposed to 5% MP ± etomoxir (100 μM) ± 0.5 ng/mL IL-4 for 72 h and probed for Arg1. Grb2 was used as a loading control. C) Western blot of WT MФs treated or not with 10% FCS, 5% MP ± 0.5 ng/mL or 10 ng/mL IL-4 for 5, 15, 30 or 60 min. The blot was probed for pSTAT6, pAKT, pERK1/2 and ERK1/2. Grb2 was used as a loading control. D) Western blot of WT MФs exposed for 72 h to the indicated combinations of low dose IL-4 (0.5ng/mL) and 5% MP ± the MEK inhibitor U0126 (5 μM) or the PI3K inhibitor LY294002 (5 μM). E) Western blot of WT MФs exposed for 72 h to the indicated combinations of low dose IL-4 (0.5ng/mL) and 5% MP ± N-acetylcysteine (NAC, 2 mM) or rotenone (0.25 μM). F) Western blot of WT MФs exposed for 72 h to the indicated combinations of low dose IL-4 (0.5ng/mL) and 5% MP ± low and high doses of N-acetylcysteine (NAC), L-buthionine-S,R-sulfoximine (BSO), or dithiothreitol (DTT). All results are representative of 2 or 3 independent experiments.

Fig 4. Identification of the factors in MP responsible for enhancing IL-4-induced M2 skewing of MФs.

A) Western blot of mature SHIP+/+ and -/- MФs stimulated for 72 h with 5% MP + 0.5 ng/mL IL-4 ± nothing (C), vehicle control (DMSO) (V) or 1 μM of the TGFβ inhibitor, SB505124. B) Western blot showing the effect of charcoal stripping (CS) on the ability of MP to induce M2 skewing WT MФs in the presence of 0.5 ng/mL IL-4 compared to intact MP. C) Western blot showing the effects of MP delipidation (DL MP) and the recovered lipid fraction (Lip Fx) on the ability to M2 skew WT MФs in the presence of 0.5 ng/mL IL-4. D) Arginase activity of SHIP-/- BM MФs derived with increasing doses of TGFβ and PGE₂. All data points are the mean ± SEM of experimental triplicates of 2 independent experiments. E) Western blot showing the effect of an ATRA dose response ± 0.5 ng/mL IL-4 on M2 skewing of mature WT MФs after culture for 48 h. F) Western blot showing the effect of the combination of 5 μM BMS, 1 μM SB505124 and 20 μM SC28125 as well as 3 mM NAC, 5 μM UO126, and 5 μM LY294002 on ATRA (4 nM) + TGFβ (2 ng/mL) + PGE₂ (3 ng/mL) induced M2 skewing of WT MФs ± 0.5 ng/mL IL-4 after 48 h in culture. G) Western blot showing the effect of BMS SB505124 and SC58125 at the same concentrations as in (F) and the combination of all three on MP-induced M2 skewing of WT MФs ± 0.5 ng/ml IL-4 after 48 hr in culture. For panels C, F and G, densitometry was performed and expression levels of Arg1, relative to Grb2 levels, are indicated. All results are representative of 2 or 3 independent experiments.

Fig 5. Proposed model of M2-skewing of SHIP+/+ and -/- MФs.

SHIP-/-, but not +/+, basophils constitutively secrete sufficient IL-4 to M2 skew SHIP+/+ or -/- progenitors or mature MФs but only in the presence of ATRA, TGFβ and PGE₂ in MP. These MP factors synergize with low levels of IL-4 to M2 skew the MФs under non-inflamed conditions.