p47(phox) directs murine macrophage cell fate decisions

Abstract

Macrophage differentiation and function are pivotal for cell survival from infection and involve the processing of microenvironmental signals that determine macrophage cell fate decisions to establish appropriate inflammatory balance. NADPH oxidase 2 (Nox2)-deficient chronic granulomatous disease (CGD) mice that lack the gp91(phox) (gp91(phox-/-)) catalytic subunit show high mortality rates compared with wild-type mice when challenged by infection with Listeria monocytogenes (Lm), whereas p47(phox)-deficient (p47(phox-/-)) CGD mice show survival rates that are similar to those of wild-type mice. We demonstrate that such survival results from a skewed macrophage differentiation program in p47(phox-/-) mice that favors the production of higher levels of alternatively activated macrophages (AAMacs) compared with levels of either wild-type or gp91(phox-/-) mice. Furthermore, the adoptive transfer of AAMacs from p47(phox-/-) mice can rescue gp91(phox-/-) mice during primary Lm infection. Key features of the protective function provided by p47(phox-/-) AAMacs against Lm infection are enhanced production of IL-1α and killing of Lm. Molecular analysis of this process indicates that p47(phox-/-) macrophages are hyperresponsive to IL-4 and show higher Stat6 phosphorylation levels and signaling coupled to downstream activation of AAMac transcripts in response to IL-4 stimulation. Notably, restoring p47(phox) protein expression levels reverts the p47(phox)-dependent AAMac phenotype. Our results indicate that p47(phox) is a previously unrecognized regulator for IL-4 signaling pathways that are important for macrophage cell fate choice.

Figure 1

p47^phox−/− macrophages discriminate activation signal in vitro. Bone marrow–derived macrophages were stimulated overnight with 1000 units/mL IL-4, heat-killed Lm (HKL/γ or LPS/γ). A: Real-time PCR quantification of Ym1 and iNos. Values are relative to expression of the gene encoding RPS29. Data represent three independent experiments including three to four of each genotype per experiment. *P < 0.05. B: Total cell lysates from BMMacs stimulated overnight with IL-4 or LPS/γ were blotted with anti-Ym1 or anti–β-actin. Results are representative of two independent experiments including three of each genotype per experiment.

Figure 2

p47^phox protein attenuates IL-4 signaling in macrophages independent of the gp91^phox catalytic subunit. A: Immunoblot analysis of STAT6 phosphorylation in inflammatory macrophages from each genotype stimulated with IL-4 for 20 minutes as indicated. Results of one representative experiment of three independent experiments are shown. B: Real-time PCR analysis of Arg1, Ym, and FIZZ1 mRNA from inflammatory macrophages from each genotype stimulated with IL-4 for 18 hours as indicated. Values are relative to expression of the gene encoding RPS29. Data represent three independent experiments including three of each genotype per experiment. *P < 0.05.

Figure 3

Restoring p47^phox protein attenuates signaling downstream of the IL-4R. Immunoblot analysis of p47^phox−/− bone marrow–derived macrophages (BMMacs) expressing human p47^phox (hNCF1). BMMacs were transduced by viral infection with hNCF1 or control vector (rGFP). A: Immunoblot analysis of IL-4–stimulated STAT6 phosphorylation. Quantification relative to total STAT6 is indicated. B: Immunoblot analysis of IL-4 stimulated Ym1 and ArgI expression. Results are representative of three independent experiments including three of each genotype per experiment.

Figure 4

Enhanced Lm killing by p47^phox−/− AAMacs. Mice were infected with 5 × 10⁴ CFU of Lm by i.p. injection. Tissues were harvested 3 days postinfection. A:Lm peritoneal bacterial burdens 3 days postinfection. Data are mean (± SEM) for six individual mice from two separate experiments with three of each genotype. B:In vitro Lm killing by WT and p47^phox−/− BMMacs primed overnight as indicated, before infection with 5 × 10⁵ CFU Lm. Data are mean (± SEM) of seven independent experiments with pooled cells from three of each genotype/experiment. **P < 0.001. C:In vitro Lm killing by p47^phox−/− BMMacs primed overnight as indicated, before infection with 5 × 10⁵ CFU Lm. Data are mean (± SEM) of three independent experiments with pooled cells from three p47^phox−/− mice/experiment. **P < 0.001.

Figure 5

H&E-stained tissue sections from gp91^phox−/− (A–C), p47^phox−/− (D–F), and WT (G–I) mice showing differences in severity of pathology in multiple tissue types. Changes in the liver and spleen of gp91^phox−/− mice at 3 days post-Lm infection were more severe than in those of p47^phox−/− and WT mice and show evidence of tissue necrosis (original magnification, ×200). Lungs of gp91^phox−/− and p47^phox−/− contained infiltrates of macrophages and multinucleated giant cells and crystalline pneumonia (original magnification, ×400). Results are representative of five each genotype examined.

Figure 6

Immunofluorescence for YM1/YM2 (A) and MMR protein (B) in the lung of a p47^phox−/− mouse 3 days post Lm infection. YM1/YM2–positive crystals are present within the cytoplasm of macrophages and multinucleated cells (original magnification, ×400). Results are representative of five each genotype examined. Splenocyte cultures from Lm-infected mice were incubated for 5 hours with 1 μmol/L OVA_323–339 and/or OVA_257–264 peptides for CD4 and CD8 T lymphocyte stimulation, respectively. Monensin was added for the final 2 hours of culture, and the percentage of CD4⁺ or CD8⁺ lymphocytes expressing IL-4 was determined by flow cytometry (C). Data are mean (± SEM) of six individual mice from two separate experiments with 3 of each genotype/experiment. *P = 0.043.

Figure 7

Enhanced survival of gp91^phox−/− mouse recipients of p47^phox−/− AAMacs. A: gp91^phox−/− Mouse survival after adoptive transfer of 10 × 10⁶Lm or thioglycolate (TGCL)) elicited p47^phox−/− inflammatory macrophages before Lm infection. PBS indicates survival of gp91^phox−/− control recipients that received PBS by i.v. injection. n = 10 per group. P = 0.048. B: Tissue severity scores for gp91^phox−/− recipients of Lm or TGCL elicited p47^phox−/− inflammatory macrophages. n = 5 per group. *P < 0.05.