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. 2018 Oct 15;201(8):2492-2501.
doi: 10.4049/jimmunol.1800003. Epub 2018 Aug 31.

Neutrophils Promote Amphiregulin Production in Intestinal Epithelial Cells through TGF-β and Contribute to Intestinal Homeostasis

Feidi Chen  1 , Wenjing Yang  2   3 , Xiangsheng Huang  2 , Anthony T Cao  2 , Anthony J Bilotta  2   4 , Yi Xiao  2 , Mingming Sun  2   3 , Liang Chen  2   3 , Chunyan Ma  2 , Xiuping Liu  5 , Chang-Gong Liu  5 , Suxia Yao  2 , Sara M Dann  2   4 , Zhanju Liu  3 , Yingzi Cong  6   2
Affiliations

Neutrophils Promote Amphiregulin Production in Intestinal Epithelial Cells through TGF-β and Contribute to Intestinal Homeostasis

Feidi Chen et al. J Immunol. .

Abstract

Neutrophils are the first responders to sites of inflammation when the intestinal epithelial barrier is breached and the gut microbiota invade. Despite current efforts in understanding the role of neutrophils in intestinal homeostasis, the complex interactions between neutrophils and intestinal epithelial cells (IECs) is still not well characterized. In this study, we demonstrated that neutrophils enhanced production of amphiregulin (AREG), a member of the EGFR ligand family, by IECs, which promoted IEC barrier function and tissue repair. Depletion of neutrophils resulted in more severe colitis in mice because of decreased AREG production by IECs upon dextran sodium sulfate (DSS) insult. Administration of AREG restored epithelial barrier function and ameliorated colitis. Furthermore, neutrophil-derived TGF-β promoted AREG production by IECs. Mechanistically, TGF-β activated MEK1/2 signaling, and inhibition of MEK1/2 abrogated TGF-β-induced AREG production by IECs. Collectively, these findings reveal that neutrophils play an important role in the maintenance of IEC barrier function and homeostasis.

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Conflict of interest statement

Disclosures: The authors report no financial conflict of interests.

Figures

Figure 1.
Figure 1.. Neutrophil conditioned media induces AREG expression by IECs.
Intestinal enteroids were cultured with or without neutrophil conditioned media for 6 h followed by microarray analysis. (A) Hierarchical clustering of genes that were differentially (|Fold change| > 2) expressed between the two groups (n = 2). Heat map of some differentially expressed probes between the two groups (n = 2). (B) Scatterplot displaying the log2 fold change in expression between the two groups (n = 2). (C) mRNA expression levels of AREG in the enteroids were measured by qRT-PCR and normalized to GAPDH. (D) Primary IECs from either small intestines (SB) or large intestines (LB) and treated with or without neutrophil conditioned media for 6 h, and mRNA expression level of AREG measured by qRT-PCR and normalized to GAPDH. (E) mRNA expression level of AREG in the MSIE cells treated with neutrophil conditioned media (CM) for 6 h. Data are presented as mean ± SEM of three independent experiments. CM, neutrophil conditioned media; None, media alone. *P < 0.05, **P < 0.01 Student’s t test.
Figure 2.
Figure 2.. AREG protects the intestinal epithelium from DSS-induced injury.
WT mice were administered 2% DSS in drinking water for 7 days, followed by 3 days of water. One group of mice received 15μg of AREG every 2 days. (A) Relative body weight change. (B) Blinded histopathological scoring. (C) Representative images of H&E-stained colon sections. Scale bar, 100 μm. N = 4 per group per experiment. Data are representative of 3 independent experiments; *p<0.05 Student’s t test.
Figure 3.
Figure 3.. Neutrophils confer protection against colitis through induction of AREG by IECs.
WT mice were administered 2% DSS in drinking water for 7 days, followed by 3 days of water. The mice were given Ly6G-depleting antibody i.p. every 3 days. One group of mice received AREG every 2 days and the other group received PBS. One group of mice treated with DSS were administrated with IgG to serve as control. (A) Relative change in body weight. (B) Representative images of H&E-stained colon sections. Scale bar, 100 μm. (C) Blinded histopathological scoring. (D) Protein level of inflammatory cytokines in colonic culture supernatant. (E) mRNA expression level of HNF4α, TJP1 in isolated IECs. (F) FITC-dextran level in plasma was determined. n = 4 per group. Data are representative of 3 independent experiments; ND, not detectable; *P < 0.05, **P < 0.01, ***P < 0.001 unpaired Student’s t test, one-way ANOVA, nonparametric Mann–Whitney U test.
Figure 4.
Figure 4.. TGFβ induces AREG production in IEC.
(A) MSIE cells were treated with different cytokines/factors for 6 h, and mRNA expression level of AREG was measured by qRT-PCR and normalized against GAPDH. (B) Level of TGFβ was determined in neutrophil conditioned media versus media control. (C) mRNA expression level of AREG in the MSIE cells treated with 20 μg/ml TGFβ over time, determined by qRT-PCR and normalized against GAPDH. (D and E) Neutrophil conditioned media was pre-treated with 10 μg/ml anti-TGFβ antibody and then added into MSIE cell cultures. (D) mRNA expression level (24 h) and (E) protein level of AREG (24 h) were measured by qRT-PCR and Western blot, respectively. (F) mRNA expression level of AREG in primary IECs isolated from either SB or LB. Data are presented as mean ± SEM of three independent experiments. CM, neutrophil conditioned media; None, media alone. *P < 0.05, **P < 0.01, ***P < 0.001 Student’s t test, one-way ANOVA.
Figure 5.
Figure 5.. Neutrophils produce TGFβ which regulates IEC production of AREG in the setting of inflammation.
(A) mRNA expression level of TGFβ in bone marrow, peritoneal cavity, spleen neutrophils, with Treg as positive control. (B) Protein levels of TGFβ in the colonic tissue culture were analyzed by ELISA. (C) Soluble AREG was measured in colonic culture supernatant via ELISA. (D and E) IECs were isolated from the colon of mice, mRNA expression level (G) and protein level (E) of AREG were measured in IECs. (F) mRNA expression level of ADAM17 in the colonic IECs of mice. (G) Intestinal enteroids were cultured with or without neutrophil conditioned media for 6 h, mRNA expression levels of ADAM17 in the enteroids were measured by qRT-PCR and normalized to GAPDH. Data are representative of 3 independent experiments; PMN, neutrophil; *P < 0.05, **P < 0.01 unpaired Student’s t test, one-way ANOVA, nonparametric Mann–Whitney U test.
Figure 6.
Figure 6.. TGFβ induction of AREG in IECs is MEK1/2-dependent.
MSIE cells were treated with or without TGFβ. (A) The phosphorylated ERK1/2 (pERK1/2) was determined by Western blot (1 h). (B and C) MEK1/2 was inhibited or knockdown in MSIE cells in the presence of TGFβ. mRNA expression level of AREG was measured by qRT-PCR in the MSIE cells treated with (B) specific inhibitors or (C) siRNA. Data are presented as mean ± SEM of three independent experiments; NT, no treatment; NT siRNA, non-targeting siRNA. *P < 0.05, **P < 0.01, ***P < 0.001 one-way ANOVA.
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