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Activated M1 macrophages suppress c-kit expression via TNF-α-mediated upregulation of miR-222 in Neonatal Necrotizing Enterocolitis

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Abstract

Background

Activation of intestinal macrophages is implicated in the pathogenesis of neonatal necrotizing enterocolitis (NEC), yet its precise mechanisms remain unclear.

Objective

The purpose of this study is to investigate the role of macrophages and TNF-α via an inflammatory MicroRNA in NEC.

Materials and methods

Immunofluorescence (IF) staining of CD68, iNOS, and Arg-1 was employed to identify phenotypes of macrophage in the intestines of NEC infants and NEC mice. Expression of TNF-α, c-kit, and miR-222 was evaluated by qRT-PCR, Western blot, and immunochemical staining from the tissue samples.

Results

Large number of M1 macrophage infiltration was found in the NEC intestines. Expression of CD68, iNOS, and TNF-α were significantly increased, while c-kit was decreased distinctly in the NEC group. In the early phase of NEC mouse model, inhibition of M1 macrophages reduced the incidence of NEC and intestinal inflammation. We found that TNF-α upregulated the expression of miRNA-222 and inhibited the expression of c-kit. Conversely, such decrease of c-kit expression could be reversed by miR-222 antagonists. Furtherly, dual-luciferase assay confirmed that c-kit can be inhibited by miR-222 directly.

Conclusion

Macrophages activation in NEC intestine results in an increased inflammatory response and TNF-α production, accompanied with miR-222 upregulation and c-kit suppression. Modulations of M1 macrophages, TNF-α or miR-222 may be potential therapeutic targets for NEC treatment.

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Abbreviations

Arg-1:

Arginase 1

CL:

Liposomal clodronate

DAPI:

4,6-Diamidino-2-phenylindole

ELAM-1:

Endothelial-leukocyte adhesion molecule 1

FBS:

Fetal bovine serum

HE:

Hematoxylin and eosin

ICCs:

Interstitial cells of Cajal

IF:

Immunofluorescence

IFN:

Interferon

iNOS:

Inducible NO synthase

IL:

Interleukin

LPS:

Lipopolysaccharide

NF-kB:

Nuclear factor kappa light chain enhancer of activated B cells

NEC:

Neonatal necrotizing enterocolitis

NS:

Normal saline

PAF:

Platelet activating factor

SCF:

Stem cell factor

SPF:

Specific pathogen free

TNF:

Tumor necrosis factor

TNFR1:

TNF receptor 1

VCAM-1:

Vascular cell adhesion molecule 1

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (2016YFE0203900), the National Natural Science Foundation of China (Nos. 81873541), and Sanming Project of Medicine in Shenzhen (SZSM201812055).

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Author notes

  1. Xue Xia and Di Wang authors have contributed equally to this work.

Authors and Affiliations

  1. Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China

    Xue Xia, Di Wang, Wenjing Wang, Chunlei Jiao, Xuyong Chen, Hongyi Zhang & Jiexiong Feng

  2. Department of Endocrinology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China

    Xue Xia

  3. Department of Neonatal Surgery, Wuhan Children’s Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China

    Lei Yu

  4. Department of General Surgery, Shenzhen Children’s Hospital, Shenzhen, People’s Republic of China

    Bin Wang

  5. Department of Pediatric Surgery, Fujian Medical University Union Hospital, Fuzhou, People’s Republic of China

    Feng Chen

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  1. Xue Xia
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Each named author has substantially contributed to conducting the research and drafting the manuscript.

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Correspondence to Jiexiong Feng.

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This study was approved by the Institutional Animal Care and Use of the Committee at Tongji Medical College (Wuhan, China) and was performed in accordance with the ethical guidelines.

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11_2021_1441_MOESM1_ESM.tif

Supplementary file1 Supplemental Fig. 1 Histologic scoring of intestines stained with HE in mouse NEC model. Representative examples of histologic scoring of intestine determined by HE staining as grade 0 (a, normal intestine), grade 1 (b, epithelial cell lifting or separation), grade 2 (c, necrosis to mid villus level), grade 3 (d, necrosis of entire villus), and grade 4 (e, transmural necrosis). Any injury of ≥grade 2 is considered to be consistent with NEC. Scale bar = 50 µm. Magnification: ×200. (TIF 3976 KB)

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Xia, X., Wang, D., Yu, L. et al. Activated M1 macrophages suppress c-kit expression via TNF-α-mediated upregulation of miR-222 in Neonatal Necrotizing Enterocolitis. Inflamm. Res. 70, 343–358 (2021). https://doi.org/10.1007/s00011-021-01441-6

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