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Sci Rep. 2020;10(1):19867.

Toll-like receptor 4 regulates intestinal fibrosis via cytokine expression and epithelial-mesenchymal transition.

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제1저자 전유경 · 교신저자 고성준, 김지원

PMID 33199767 PMC7670421 DOI 10.1038/s41598-020-76880-y IF 4.9 소화기내과
소속 (7)
  1. Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea.
  2. Laboratory of Intestinal Mucosa and Skin Immunology, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea.
  3. Department of Dermatology, Boramae Medical Center, 20 Boramae-ro-5-gil, Dongjak-gu, Seoul, 07061, Republic of Korea.
  4. Division of Gastroenterology, Department of Internal Medicine, Boramae Medical Center, 20 Boramae-ro-5-gil, Dongjak-gu, Seoul, 07061, Republic of Korea. kjwjor@snu.ac.kr.
  5. Laboratory of Intestinal Mucosa and Skin Immunology, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea. kjwjor@snu.ac.kr.
  6. Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea. jel1206@snu.ac.kr.
  7. Laboratory of Intestinal Mucosa and Skin Immunology, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea. jel1206@snu.ac.kr.

Abstract

Intestinal fibrosis induced by chronic and recurrent colitis, which is exacerbated by bowel stenosis, stricture, and obstruction, is challenging to treat. Toll-like receptor 4 (TLR4) stimulates innate and acquired immunity in response to specific microbial components, but the role of TLR4 in intestinal fibrosis is largely unknown. We investigated its role in intestinal fibrosis using not only a murine fibrosis model but also human myofibroblasts and intestinal epithelial cells. Colon fibrosis was induced in TLR4-deficient (TLR4-/-) mice and its wild-type counterparts with 3% dextran sulfate sodium. Absence of TLR4 gene attenuated chronic inflammation and colonic macrophages infiltration; intestinal fibrosis and collagen deposition were suppressed. Also, the production of tumor necrosis factor-α, interleukin-12p40, and transforming growth factor-β was reduced in TLR4-deficient peritoneal macrophages. TLR4 was silenced in CCD-18Co cells by small interfering RNA (siRNA), and matrix metalloproteinase-1, tissue inhibitor of metalloproteinase, and collagen α1 expression was evaluated. Role of TLR4 in epithelial-mesenchymal transition (EMT) was evaluated in HCT116 cells. Suppression of TLR4 transcription by siRNAs affected myofibroblasts activity, collagen synthesis, and EMT in the human cancer cell line. Thus, we suggest that TLR4 can be an essential mediator in intestinal chronic inflammation and fibrosis, indicating that TLR4 signaling is a potential therapeutic target for intestinal fibrosis.

Figures

Figure 1
Figure 1 Comparison of clinical indices, gross appearance, and histologic grading in toll-like receptor 4-deficient (TLR4−/−) (n = 5) and wild-type mice (n = 5). (A) Wild-type mice experienced extensive weight loss compared to TLR4−/− mice during post dextran sulfate sodium treatment. (B) Disease activity index from day 5 to day 20 was higher in wild-type as compared to TLR4−/− mice. (C) Gross appearance of extracted colons showed bowel edema and fibrotic change in wild-type mice. However, in TLR4−/− mice, bowel edema was not marked. (D) Colon from TLR4−/− mice was longer than that from wild-type mice. Asterisks indicate significant differences (P-values < 0.05) between TLR4−/− and wild-type mice.
Figure 2
Figure 2 Representative hematoxylin–eosin (H&E) staining, Masson’s Trichrome (MT) staining, and immunohistochemistry. (A) H&E staining revealed prominent mucosal disruption and infiltration of inflammatory cells in wild-type colon. (magnification: × 100). Histological grading, determined by H&E staining, was higher in wild-type than TLR4−/− mice. (B) MT staining of colon tissue samples revealed more severe fibrosis in wild-type than in TLR4−/− mice (magnification: × 100). The proportion of MT-positive area, analyzed using ImageJ program, was significantly lower in TLR4−/− than in wild-type colon tissue. (C) Immunohistochemistry detected increased migration of F4/80-positive cells in wild-type, as compared to TLR4−/− mice (magnification: × 100 and × 400). The proportion of F4/80-positive region was markedly lower in TLR4−/− as compared with wild-type mice. (D) The proportion of α-smooth muscle actin (α-SMA)-positive cells was higher in distorted mucosal structure of wild-type than TLR4−/− mice (magnification: × 100 and × 400). Statistically significant results: *P-values < 0.05.
Figure 3
Figure 3 Comparison between the expression levels of inflammatory cytokines in peritoneal macrophages obtained from TLR4−/− mice (n = 3) and wild-type mice (n = 3) after lipopolysaccharides (LPS) stimulation (10 ng/mL for four hours). The transcription levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-12p40, and transforming growth factor-β (TGF-β) were measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). The results are representative data of three separate experiments. Asterisks indicate significant differences (P-values < 0.05) between two groups.
Figure 4
Figure 4 The effect of TLR4 silencing on gene expression in human colon myofibroblasts. Transfection with TLR4 small interfering RNA (siRNA) (n = 5) or control siRNA (n = 5) with or without LPS (10 ng/mL for four hours) stimulation was performed in CCD-18Co cells. The transcription levels of matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase (TIMP), and collagen α1 were evaluated by real-time RT-PCR. The expressions of TIMP and collagen α1 were decreased when TLR4 siRNA was treated regardless LPS stimulation. The expression of MMP-1 increased when TLR4 siRNA was treated without LPS, but it decreased when LPS was administrated. The results are representative data of three separate experiments. Statistically significant results: *P-values < 0.05.
Figure 5
Figure 5 The effect of TLR4 silencing on epithelial-mesenchymal transition in HCT116 cells. HCT116 cells were transfected with TLR4 siRNA (n = 5) or control siRNA (n = 5). After TLR4 silencing, LPS (10 ng/mL for four hours) was treated. The gene expression levels of vimentin were suppressed. The results are representative data of three separate experiments. Statistically significant results: *P-values < 0.05.

그림 출처: PubMed Central 공개 논문 (PMC7670421)

Conflict of interest statement

The authors declare no competing interests.

그 밖의 정보
출판 형태
Journal Article, Research Support, Non-U.S. Gov't
저널
Scientific reports · eISSN 2045-2322
전자 출판
2020-11-16
MeSH
Animals · Cell Line · Collagen · Colonic Diseases / chemically induced / genetics / immunology / pathology · Cytokines / metabolism · Dextran Sulfate / adverse effects · Disease Models, Animal · Epithelial-Mesenchymal Transition · Fibrosis · Gene Expression Regulation / drug effects · HCT116 Cells · Humans · Interleukin-12 Subunit p40 / metabolism · Macrophages, Peritoneal / metabolism · Mice · Signal Transduction · Toll-Like Receptor 4 / genetics · Transforming Growth Factor beta / metabolism · Tumor Necrosis Factor-alpha / metabolism
Substances
Cytokines · Interleukin-12 Subunit p40 · TLR4 protein, human · Tlr4 protein, mouse · Toll-Like Receptor 4 · Transforming Growth Factor beta · Tumor Necrosis Factor-alpha · Collagen · Dextran Sulfate
참고문헌
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