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. 2016 Sep 1;126(9):3296-312.
doi: 10.1172/JCI83585. Epub 2016 Aug 2.

EGFR regulates macrophage activation and function in bacterial infection

Dana M Hardbower, Kshipra Singh, Mohammad Asim, Thomas G Verriere, Danyvid Olivares-Villagómez, Daniel P Barry, Margaret M Allaman, M Kay Washington, Richard M Peek Jr, M Blanca Piazuelo, Keith T Wilson

EGFR regulates macrophage activation and function in bacterial infection

Dana M Hardbower et al. J Clin Invest. .

Abstract

EGFR signaling regulates macrophage function, but its role in bacterial infection has not been investigated. Here, we assessed the role of macrophage EGFR signaling during infection with Helicobacter pylori, a bacterial pathogen that causes persistent inflammation and gastric cancer. EGFR was phosphorylated in murine and human macrophages during H. pylori infection. In human gastric tissues, elevated levels of phosphorylated EGFR were observed throughout the histologic cascade from gastritis to carcinoma. Deleting Egfr in myeloid cells attenuated gastritis and increased H. pylori burden in infected mice. EGFR deficiency also led to a global defect in macrophage activation that was associated with decreased cytokine, chemokine, and NO production. We observed similar alterations in macrophage activation and disease phenotype in the Citrobacter rodentium model of murine infectious colitis. Mechanistically, EGFR signaling activated NF-κB and MAPK1/3 pathways to induce cytokine production and macrophage activation. Although deletion of Egfr had no effect on DC function, EGFR-deficient macrophages displayed impaired Th1 and Th17 adaptive immune responses to H. pylori, which contributed to decreased chronic inflammation in infected mice. Together, these results indicate that EGFR signaling is central to macrophage function in response to enteric bacterial pathogens and is a potential therapeutic target for infection-induced inflammation and associated carcinogenesis.

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Figures

Figure 1
Figure 1. Increased p-EGFR levels in human Gmacs in H. pylori–induced gastritis and precancerous intestinal metaplasia.
(A) Representative immunofluorescence images of human gastric biopsies with normal histology and active, nonatrophic gastritis. CD68 = red, EGFR p-Y1068 = green; merge = yellow; DAPI = blue. n = 4 normal samples and 5 gastritis samples. Scale bars: 50 μM. (B) Quantification of the percentage of CD68+p-EGFR+ macrophages among the total number of CD68+ cells in 10 fields per slide from images shown in A. The slides were analyzed in a blinded manner. n = 4 normal samples and 5 gastritis samples. ***P < 0.001, by 2-tailed Student’s t test. (C) Representative H&E-stained images and immunofluorescence images of gastric tissues from the Vanderbilt University TMA. CD68 = red; EGFR p-Y1068 = green; merge = yellow; DAPI = blue. n = 12 normal samples, 41 gastritis samples, and 9 intestinal metaplasia samples. Scale bars: 100 μM. (D) Quantification of the percentage of CD68+p-EGFR+ cells among the total number of cells in each individual core in the TMA, as determined by CellProfiler. n = 12 normal samples, 41 gastritis samples, and 9 intestinal metaplasia samples. *P < 0.05 and **P < 0.01, by 1-way ANOVA with the Kruskal-Wallis test, followed by the Mann-Whitney U test.
Figure 2
Figure 2. EGFR signaling in macrophages is induced by H. pylori infection in murine and human macrophages.
(A) Representative Western blot of EGFR p-Y1068 and p-S1046/7 in RAW 264.7 cells at various time points p.i. with H. pylori PMSS1 or stimulation with EGF (5 ng/ml). n = 3 biological replicates. (B) Densitometric analysis of the levels of p-Y1068 compared with levels of t-EGFR at 15 and 30 minutes p.i. n = 3 biological replicates. ***P < 0.001, by 1-way ANOVA with Newman-Keuls post test. (C) Densitometric analysis of p-S1046/7 levels compared with t-EGFR levels at 15 and 30 minutes p.i. n = 3 biological replicates. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Newman-Keuls post test. (D) Representative Western blot of EGFR at p-Y1068 and p-S1046/7 in THP-1 cells at 15 minutes p.i. with H. pylori PMSS1. Monocytes (– PMA) and macrophages (+ PMA) are represented in this blot. n = 3 biological replicates. (E) Representative Western blot of EGFR at p-Y1068 and p-S1046/7 in RAW 264.7 cells at 15 minutes p.i. with H. pylori PMSS1 ± 10 μM gefitinib. n = 3 biological replicates. (F) Representative immunofluorescence images of WT BMmacs infected with H. pylori PMSS1 ± 10 μM gefitinib at the indicated times. EGFR p-Y1068 = green; DAPI = blue. n = 5 biological replicates. Scale bars: 50 μM. (G) Representative immunofluorescence images of WT BMmacs infected with H. pylori PMSS1 ± 10 ng/ml anti–TNF-α or ± 25 ng/ml anti–HB-EGF at 30 minutes p.i. Green = EGFR p-Y1068; blue = DAPI. n = 3 biological replicates. Scale bars: 50 μM. Ctrl, control.
Figure 3
Figure 3. EgfrΔmye mice have significantly increased H. pylori burden but significantly decreased gastritis after chronic infection.
(A) Gastritis scores were assessed in a blinded manner by a gastrointestinal pathologist 4 months p.i. according to the updated Sydney System. *P < 0.05 and ***P < 0.001, by 1-way ANOVA with Newman-Keuls post test. (B) Representative H&E-stained images from infected mice in A. Scale bars: 100 μM. (C) Colonization of H. pylori SS1 was assessed by serial dilution and culture 4 months p.i. ***P < 0.01, by 1-way ANOVA with Newman-Keuls post test. n = 5–10 uninfected and 11–30 H. pylori SS1 mice per genotype (A and C). (D) Representative immunofluorescence images of p-EGFR from infected mice in A and B. EGFR p-Y068 = green; CD68 = red; merge = yellow; DAPI = blue. Solid arrowheads indicate CD68+p-EGFR+ macrophages; open arrowheads indicate CD68+p-EGFR– macrophages. Scale bars: 50 μM. (E) Immunofluorescence images of t-EGFR from infected mice in A and B. t-EGFR = green; CD68 = red; merge = yellow; DAPI = blue. Solid arrowheads indicate CD68+t-EGFR+ macrophages; open arrowheads indicate CD68+t-EGFR– macrophages. Scale bars: 50 μM. n ≥ 3 mice per genotype (D and E).
Figure 4
Figure 4. EgfrΔmye mice have significantly decreased chemokine production in gastric tissue.
mRNA and protein levels of the cytokines/chemokines (A) CXCL1, (B) CXCL10, (C) CXCL9, (D) CCL3, (E) CCL5, and (F) CCL4 were assessed by qRT-PCR and Luminex Multiplex Array, respectively, from gastric tissue 4 months p.i. with H. pylori SS1. *P < 0.05 and **P < 0.01, and ***P < 0.001. Statistical significance for A–F was calculated by 1-way ANOVA with the Kruskal-Wallis post test, followed by the Mann-Whitney U test. In all panels, n = 2–5 uninfected and 6–11 infected mice per genotype.
Figure 5
Figure 5. EgfrΔmye mice have significantly decreased M1 and Mreg cytokine production in gastric tissue and Gmacs.
(A) mRNA levels of the proinflammatory cytokines Nos2, Tnfa, and Il1b were assessed by qRT-PCR from gastric tissue 4 months p.i. with H. pylori SS1. *P < 0.05 and **P < 0.01. (B) mRNA levels of the antiinflammatory cytokines Il10, Tgfb, and Tnfsf14 (Light) were assessed by qRT-PCR from gastric tissue 4 months p.i. with H. pylori SS1. n = 3 uninfected mice and 5 infected mice per genotype (A and B). *P < 0.05 and **P < 0.01. Statistical significance in A and B was calculated by 1-way ANOVA with the Kruskal-Wallis post test, followed by the Mann-Whitney U test. (C) Measurement of IL-1β by ELISA in gastric tissues 4 months p.i. with H. pylori SS1. n = 4 uninfected and 10 infected mice per genotype. ***P < 0.001. (D) F4/80+ Gmacs were magnetically selected from the lamina propria at 48 hours p.i. with H. pylori SS1 and mRNA levels of Nos2 were assessed by qRT-PCR. n = 6 uninfected and 9–10 infected mice per genotype. *P < 0.05, **P < 0.01, and ***P < 0.001. Statistical significance in C and D was calculated by 1-way ANOVA with Newman-Keuls post test. (E) Protein levels of NOS2 were assessed in F4/80+ Gmacs 48 hours p.i. with H. pylori SS1. n = 4 uninfected and 6 infected mice per genotype. *P < 0.05 and **P < 0.01, by 1-way ANOVA with the Kruskal-Wallis test, followed by the Mann-Whitney U test.
Figure 6
Figure 6. EGFR signaling is critical for macrophage activation and function.
(A) mRNA levels of the M1 activation markers Nos2, Tnfa, and Il1b were assessed by qRT-PCR in WT BMmacs 24 hours p.i. with H. pylori PMSS1 ± 10 μM gefitinib. n = 3 biological replicates. ***P < 0.001. (B) mRNA levels of the M1 activation markers Nos2, Tnfa, and Il1b were assessed by qRT-PCR in Egfrfl/fl and EgfrΔmye BMmacs 24 hours p.i. with H. pylori PMSS1. n = 3 mice per genotype. ***P < 0.001. (C) Representative Western blot of NOS2 in RAW 264.7 cells 24 hours p.i. with H. pylori PMSS1 ± 150 nM AG1478 (AG) or 10 μM gefitinib (Gef). UT, untreated. n = 3 biological replicates. (D) Representative Western blot of NOS2 in WT BMmacs 24 hours p.i. with H. pylori PMSS1, SS1, or 7.13 ± 10 μM gefitinib. n = 3 biological replicates. (E) Measurement of NO2– from RAW 264.7 cell supernatants 24 hours p.i. with H. pylori PMSS1, SS1, or 7.13 ± 150 nM AG1478 or 300 nM PD153035. n = 5 biological replicates. †P < 0.05 versus PMSS1 alone; *P < 0.05 and **P < 0.01 versus SS1 alone; §P < 0.05 versus 7.13 alone. (F) Measurement of NO2– from Egfrfl/fl and EgfrΔmye BMmac supernatants 24 hours p.i. with H. pylori PMSS1. n = 3 mice per genotype. **P < 0.01. (G) Measurement of IL-1β in Egfrfl/fl and EgfrΔmye BMmac supernatants 24 hours p.i. with H. pylori PMSS1. n = 3 mice per genotype. **P < 0.01. Statistical significance in all panels was calculated by 1-way ANOVA with Newman-Keuls post test.
Figure 7
Figure 7. EGFR and NF-κB form a critical link in macrophages in response to H. pylori.
(A) Representative Western blot of MyD88 protein levels in Egfrfl/fl and EgfrΔmye BMmacs 30 minutes p.i. with H. pylori PMSS1. n = 3 biological replicates. (B) Representative Western blots of p-IKBK (also known as p-IKK) and p-NFKBIA (also known as p-IκBα) protein levels in Egfrfl/fl and EgfrΔmye BMmacs 30 and 45 minutes p.i. with H. pylori PMSS1. n = 3 biological replicates. (C) Representative Western blot of cytoplasmic and nuclear fractions of t-RELA (p65) in Egfrfl/fl and EgfrΔmye BMmacs 60 minutes p.i. with H. pylori PMSS1. n = 3 biological replicates. (D) Densitometric analysis of cytoplasmic and nuclear t-RELA. n = 3 biological replicates. **P < 0.01, by 2-tailed Student’s t test. (E) Western blot of cytoplasmic and nuclear fractions of p-RELA (p-p65) in Egfrfl/fl and EgfrΔmye BMmacs 60 minutes p.i. with H. pylori PMSS1. (F) Measurement of luminescence from an NF-κB luciferase reporter in immortalized BMmacs (NGL cells) at the indicated time points p.i. with H. pylori PMSS1 or SS1 ± 10 μM gefitinib. n = 4 biological replicates *P < 0.05 versus PMSS1 alone; §P < 0.05 versus SS1 alone, by 1-way ANOVA with Newman-Keuls post test.
Figure 8
Figure 8. EGFR signaling and MAPK1/3 signaling are linked in regulating macrophage activation in response to H. pylori.
(A) Representative Western blot of p-MAPK1/3 levels in WT BMmacs ± 10 μM gefitinib or ± 50 μM ERKi at 15 and 30 minutes p.i. with H. pylori PMSS1. n = 3 biological replicates. (B) Representative Western blot of p-MAPK1/3 levels in Egfrfl/fl and EgfrΔmye BMmacs ± 50 μM ERKi at 15 and 30 minutes p.i. with H. pylori PMSS1. n = 3 biological replicates. (C) mRNA levels of the M1 activation markers Nos2 and Il1b were assessed by qRT-PCR in WT BMmacs ± 10 μM gefitinib and/or ± 50 μM ERKi 24 p.i. with H. pylori PMSS1. n = 5 mice. ***P < 0.001. (D) mRNA levels of the Mreg activation markers Il10 and Tgfb were assessed by qRT-PCR in WT BMmacs ± 10 μM gefitinib and/or ± 50 μM ERKi 24 p.i. with H. pylori PMSS1. n = 5 mice. ***P < 0.001. (E) mRNA levels of the M1 activation markers Nos2 and Il1b were assessed by qRT-PCR in Egfrfl/fl and EgfrΔmye BMmacs ± 50 μM ERKi 24 p.i. with H. pylori PMSS1. n = 3 mice per genotype. **P < 0.01 and ***P < 0.001. (F) mRNA levels of the Mreg activation markers Il10 and Tgfb were assessed by qRT-PCR in Egfrfl/fl and EgfrΔmye BMmacs ± 50 μM ERKi 24 p.i. with H. pylori PMSS1. n = 3 mice per genotype. ***P < 0.001. Statistical significance in C–F was calculated by 1-way ANOVA with Newman-Keuls post test.
Figure 9
Figure 9. EGFR deficiency in macrophages leads to a diminished Th17 response to H. pylori.
(A) mRNA levels of Il17a were assessed by qRT-PCR in gastric tissues from Egfrfl/fl, EgfrΔmye, and LysMCre/Cre mice 4 months p.i. with H. pylori SS1. n = 3 uninfected and 6 infected mice per genotype. *P < 0.05 and **P < 0.01. (B) IL-17 protein levels were assessed by ELISA in gastric tissues from Egfrfl/fl and EgfrΔmye mice 4 months p.i. with H. pylori SS1. n = 4 uninfected and 10 infected mice per genotype. **P < 0.01 and ***P < 0.001. (C) Flow cytometric assessment of CD4+IL-17+ T cells from GLNs of Egfrfl/fl and EgfrΔmye mice 4 months p.i. with H. pylori SS1. Isolated T cells were cultured in 96-well plates containing 5 μg/ml anti-CD3 and 1 μg/ml anti-CD28. Cells were then stimulated with 20 ng/ml PMA and 1 μg/ml ionomycin for 4 hours. n = 3 uninfected and 7 infected mice per genotype. *P < 0.05 and **P < 0.01. (D) mRNA levels of the Th17 markers Rorc and Il17 were assessed by qRT-PCR from magnetically selected CD4+ T cells from the gastric lamina propria of Egfrfl/fl and EgfrΔmye mice 4 months p.i. with H. pylori SS1. n = 3 uninfected and 6–7 infected mice per genotype. **P < 0.01 and ***P < 0.001. Statistical significance in all panels was calculated by 1-way ANOVA with Newman-Keuls post test.
Figure 10
Figure 10. EGFR deficiency in macrophages leads to an enhanced Treg response during H. pylori infection.
(A) mRNA levels of Foxp3 were assessed by qRT-PCR in gastric tissues from Egfrfl/fl and EgfrΔmye mice 4 months p.i. with H. pylori SS1. **P < 0.01. n = 3 uninfected and 6 infected mice per genotype. (B) Assessment of CD4+CD25+FOXP3+ T cells from GLNs of Egfrfl/fl and EgfrΔmye mice 4 months p.i. with H. pylori SS1 by flow cytometry. Isolated T cells were not cultured or were stimulated as in Figure 9C. n = 3 uninfected and 6–7 infected mice per genotype. *P < 0.05 and **P < 0.01. (C) mRNA levels of the Treg markers Foxp3, Il10, and Tgfb were assessed by qRT-PCR from magnetically selected CD4+ T cells from the gastric lamina propria of Egfrfl/fl and EgfrΔmye mice 4 months p.i. with H. pylori SS1. n = 3 uninfected and 6–7 infected mice per genotype. *P < 0.05 and **P < 0.01. Statistical significance in all panels was calculated by 1-way ANOVA with Newman-Keuls post test.
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References

    1. Benoit M, Desnues B, Mege JL. Macrophage polarization in bacterial infections. J Immunol. 2008;181(6):3733–3739. doi: 10.4049/jimmunol.181.6.3733. - DOI - PubMed
    1. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 2008;8(12):958–969. doi: 10.1038/nri2448. - DOI - PMC - PubMed
    1. Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol. 2011;11(11):723–737. doi: 10.1038/nri3073. - DOI - PMC - PubMed
    1. Mosser DM. The many faces of macrophage activation. J Leukoc Biol. 2003;73(2):209–212. doi: 10.1189/jlb.0602325. - DOI - PubMed
    1. Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep. 2014;6: - PMC - PubMed

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