Ornithine decarboxylase regulates M1 macrophage activation and mucosal inflammation via histone modifications

Abstract

Macrophage activation is a critical step in host responses during bacterial infections. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine metabolism, has been well studied in epithelial cells and is known to have essential roles in many different cellular functions. However, its role in regulating macrophage function during bacterial infections is not well characterized. We demonstrate that macrophage-derived ODC is a critical regulator of M1 macrophage activation during both Helicobacter pylori and Citrobacter rodentium infection. Myeloid-specific Odc deletion significantly increased gastric and colonic inflammation, respectively, and enhanced M1 activation. Add-back of putrescine, the product of ODC, reversed the increased macrophage activation, indicating that ODC and putrescine are regulators of macrophage function. Odc-deficient macrophages had increased histone 3, lysine 4 (H3K4) monomethylation, and H3K9 acetylation, accompanied by decreased H3K9 di/trimethylation both in vivo and ex vivo in primary macrophages. These alterations in chromatin structure directly resulted in up-regulated gene transcription, especially M1 gene expression. Thus, ODC in macrophages tempers antimicrobial, M1 macrophage responses during bacterial infections through histone modifications and altered euchromatin formation, leading to the persistence and pathogenesis of these organisms.

Keywords: Helicobacter pylori; histone modifications; macrophage polarization; ornithine decarboxylase; polyamines.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Odc∆mye mice have significantly increased histologic gastritis, but significantly decreased H. pylori burden after chronic infection. (A) Representative immunoperoxidase staining for ODC in human gastric biopsies from patients with normal mucosa and with H. pylori+ gastritis. (Scale bars, 50 μm.) (B) Quantification of the percentage of ODC+ mononuclear cells in gastric biopsies from A. n = 4 normal biopsies and 20 H. pylori+ gastric biopsies with chronic active gastritis. *P < 0.05 by Mann–Whitney u test. H.P.F., high power field. (C) Representative immunofluorescence images of ODC from gastric biopsies in A and B. Green, ODC; red, CD68; yellow, merge; and blue, DAPI. Closed arrows indicate CD68+ODC+ macrophages. Boxed area indicates image shown in higher magnification. (Scale bars, 50 μm.) Note that different cases were used for representative images in A and C. (D) Quantification of the number of CD68+ODC+ cells in the cases from C. n = 5 normal biopsies and 10 H. pylori+ gastric biopsies with chronic active gastritis. **P < 0.05 by Student’s t test. (E) Histologic gastritis scores were assessed 4 mo p.i. by a gastrointestinal pathologist in a blinded manner according to the updated Sydney System. (F) Representative H&E images from infected mice in E. (Scale bars, 100 μm.) (G) Colonization of H. pylori SS1 was assessed by serial dilution and culture 4 mo p.i. In E and G, *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Newman–Keuls posttest. n = 8–10 uninfected and 15–19 H. pylori SS1-infected mice per genotype. (H) Correlation between histologic gastritis in E and H. pylori SS1 colonization levels in G. Correlation and significance determined by Pearson’s product-moment correlation test. (I) Representative immunofluorescence images of ODC from infected mice in E and G. Green, ODC; red, CD68; yellow, merge; and blue, DAPI. Closed arrows indicate CD68+ODC+ macrophages. Open arrows indicate CD68+ODC– macrophages. (Scale bars, 50 μm.) Data are displayed as mean ± SEM.

Fig. 2.

Cytokines and chemokines are significantly increased in Odc∆mye gastric tissues. Protein levels of the cytokines and chemokines CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CXCL1 (KC/GRO-α), CXCL2 (MIP-2), CXCL10 (IP-10), IL-17, and TNF-α were assessed by Luminex multiplex array in gastric tissues 4 mo p.i. with H. pylori SS1. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Kruskal–Wallis test, followed by Mann–Whitey u test. n = 4 uninfected and 8–9 H. pylori SS1-infected mice per genotype. Data are displayed as mean ± SEM.

Fig. 3.

Odc deletion in macrophages enhances M1 macrophage activation during H. pylori infection. (A) mRNA levels of proinflammatory cytokines Il1b, Il6, Il12a, Il12b, Tnfa, and the gene Nos2 were assessed by RT-PCR in gastric tissues 4 mo p.i. with H. pylori SS1. *P < 0.05, **P < 0.01. n = 3 uninfected and 5 H. pylori SS1-infected mice per genotype. (B) mRNA levels of proinflammatory cytokines Il1b, Il6, Il12a, Il12b, Tnfa, and Nos2 were assessed by RT-PCR in bone-marrow–derived macrophages (BMmacs) 24 h p.i. with H. pylori PMSS1. *P < 0.05, **P < 0.01. Statistical significance in A and B was calculated by one-way ANOVA with Kruskal–Wallis test, followed by Mann–Whitney u test. n = 6 mice per genotype. Note that the y axis values for Il1b, Il6, and Nos2 have been divided by a factor of 1,000. All samples were analyzed as fold change against Odcfl/fl uninfected control samples. No differences were observed in the fold changes for uninfected samples from all genotypes. (C) Secreted levels of IL-1β, IL-6, IL-12p70, and TNF-α were measured by ELISA from supernatants of BMmacs 24 h p.i. with H. pylori PMSS1. *P < 0.05, **P < 0.01 by one-way ANOVA with Newman–Keuls posttest. n = 3–6 mice per genotype. Note that none of the cytokines displayed were detected in supernatants from uninfected BMmacs. (D) Representative Western blot of NOS2 levels in BMmacs 24 h p.i. with H. pylori PMSS1. n = 3 biological replicates. (E) Measurement of NO2– from BMmac supernatants 24 h p.i. with H. pylori PMSS1. ***P < 0.001 by one-way ANOVA with Newman–Keuls posttest. n = 4 mice per genotype. Data are displayed as mean ± SEM.

Fig. 4.

The effects of Odc deletion in macrophages are due to putrescine depletion. (A) Measurement of polyamine levels in BMmacs 6 h and 24 h p.i. with H. pylori PMSS1 by mass spectrometry. ***P < 0.001 by one-way ANOVA with Newman–Keuls posttest. n = 3–4 mice per genotype. (B) mRNA levels of M1 cytokines Il1b, Tnfa, and Nos2 were assessed by RT-PCR in BMmacs 24 h p.i. with H. pylori PMSS1 ± 25 μM putrescine added 60 min before infection. (C) Measurement of NO2– from BMmac supernatants 24 h p.i. with H. pylori PMSS1 ± 25 μM putrescine added 60 min before infection. (D) mRNA levels of the M2 cytokine, Tgfb1, were assessed by RT-PCR in BMmacs 24 h p.i. with H. pylori PMSS1 ± 25 μM putrescine added 60 min before infection. In B–D, **P < 0.01, ***P < 0.001 vs. Odcfl/fl + PMSS1; @@P < 0.01, @@@P < 0.001 vs. Odcfl/fl + PMSS1 + putrescine; §§P < 0.01, §§§P < 0.001 vs. Odc∆mye + PMSS1 by one-way ANOVA with Newman–Keuls posttest. n = 4 biological replicates. Data are displayed as mean ± SEM.

Fig. 5.

Odc deletion in macrophages alters H3K4 and H3K9 modifications during H. pylori infection. (A) Representative Western blot of H3K4me1, H3K9ac, and H3K9me2/3 levels in BMmacs 24 h p.i. with H. pylori PMSS1. n = 3 biological replicates. (B) Densitometric analysis of H3K4me1, H3K9ac, and H3K9me2/3 levels in A. *P < 0.05, ***P < 0.001 by one-way ANOVA with Newman–Keuls posttest. n = 3 biological replicates. (C) Representative immunofluorescence images of H3K9ac in H. pylori-infected gastric tissues 4 mo. p.i. Green, H3K9ac; red, CD68; and blue, DAPI. (Scale bars, 50 μm.) Boxed area indicates image shown at higher magnification. (Scale bar, 10 μm.) n = 3 biological replicates per genotype. Note that a cell is considered double positive if the cell surface is red and the nucleus is green. (D) Expression of Gapdh and Iap was assessed by RT-PCR in BMmacs from Odcfl/fl and Odc∆mye mice 24 h p.i. with H. pylori PMSS1 with subsequent ChIP with the denoted antibodies. *P < 0.05. n = 3 biological replicates. (E) Expression of Il1b, Il6, Tnfa, and Nos2 promoter sequences was assessed by RT-PCR in BMmacs from Odcfl/fl and Odc∆mye mice 24 h p.i. with H. pylori PMSS1 with subsequent ChIP with the denoted antibodies. *P < 0.05. n = 3 biological replicates. In D and E, statistical significance was calculated by one-way ANOVA with Newman–Keuls posttest on square-root transformed data. Data are displayed as mean ± SEM.

Fig. 6.

Alterations in histone modifications and chromatin structure in ODC-deficient macrophages alters M1 macrophage activation during H. pylori infection. (A) mRNA levels of proinflammatory cytokines Il1b, Tnfa, and Nos2 were assessed by RT-PCR in BMmacs 24 h p.i. with H. pylori PMSS1 ± 5 μM BIX 01924 added 60 min before infection. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Kruskal–Wallis test, followed by Mann–Whitney u test. n = 5 mice per genotype. (B) mRNA levels of proinflammatory cytokines Il1b, Tnfa, and Nos2 were assessed by RT-PCR in BMmacs 24 h p.i. with H. pylori PMSS1 ± 10 μM anacardic acid added 60 min before infection. ***P < 0.001 by one-way ANOVA with Kruskal–Wallis test, followed by Mann–Whitney u test. n = 5 mice per genotype. Data are displayed as mean ± SEM.