BACKGROUND & AIMS: Intestinal fibrosis is one of the most threatening complications of Crohn's disease (CD). Intestinal fibrosis is usually the result of chronic inflammation, and T cell response is the main driver of intestinal inflammation. At present, there are few researches on the mechanism of Treg cells in intestinal fibrosis, and the role of Treg-derived AREG in intestinal fibrosis has not been studied.

METHODS: AREG and TGF-β expression were assessed in patients with CD with or without intestinal fibrosis by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). In this study, dextran sulfate sodium (DSS)-induced chronic colitis model in WT and Areg^-/-mice and T-cell transfer model with wild-type (WT) and Areg^-/- Treg cells were used. RNA-sequencing of human intestinal fibroblasts treated with or without AREG was performed. CD4⁺ T cell and human intestinal myofibroblasts expression of AREG were determined. The effect of AREG on proliferation/migration in human intestinal myofibroblasts was determined.

RESULTS: AREG and TGF-β expression were increased in fibrotic sites compared with nonfibrotic sites from patients with CD. Although DSS-induced more severe colitis in Areg^-/-mice, which developed less severe intestinal fibrosis compared with WT mice on DSS insults. Transfer of Areg^-/- Treg cells induced less severe fibrosis in Rag^-/- mice compared with WT Treg cells. TGF-β promoted AREG expression in Treg cells by activating Smad3. TGF-β also promoted the AREG expression in human intestinal myofibroblasts from CD patients with fibrosis. AREG promoted human intestinal fibroblast proliferation and motility by activating PI3K/AKT signaling. PI3K inhibitor suppressed AREG-induced fibroblast activation and proliferation, thus attenuated intestinal fibrosis.

CONCLUSION: These findings revealed that Treg and human intestinal myofibroblat-derived AREG induced by TGF-β promotes intestinal fibrotic responses in experimental colitis and human patients with CD. Thereby, AREG-PI3K/AKT might serve as a potential therapeutic target for fibrosis in CD.

<b>Figure 1. </b>The deficiency of AREG induces less severe intestinal fibrosis. <b>(A-G) </b>WT mice and <i>Areg<sup>-/- </sup></i>mice were administered with 3 cycles of DSS insults. In each cycle, mice were given 2.0% DSS (wt/vol) in drinking water for 7 days and control drinking water for 7 days. <b>(A-B) </b>Colon tissues were stained with masson trichrome. <b>(C–D) </b>α-SMA expression in colon tissues of mice was determined by WB. <b>(E-G) </b>Col1a1, Col6a1, and Col6a3 levels in colonic tissues were measured by qRT-PCR. <b>(H-L)</b> WT and <i>Areg<sup>-/- </sup></i>T cells were cultured under Treg conditions for 5 days and then transferred into <i>Rag<sup>-/-</sup> </i>mice. (<b>H-I</b>) Intestinal fibrosis was determined by masson trichrome. (<b>J-L</b>) Col1a1, Col6a1, and Col6a3 levels in colonic tissues were measured by qRT-PCR. <sup>*</sup><i>P</i> <0.05; <sup>**</sup><i>P</i> <0.01.

Figure 1. The deficiency of AREG induces less severe intestinal fibrosis. (A-G) WT mice and Areg^-/-mice were administered with 3 cycles of DSS insults. In each cycle, mice were given 2.0% DSS (wt/vol) in drinking water for 7 days and control drinking water for 7 days. (A-B) Colon tissues were stained with masson trichrome. (C–D) α-SMA expression in colon tissues of mice was determined by WB. (E-G) Col1a1, Col6a1, and Col6a3 levels in colonic tissues were measured by qRT-PCR. (H-L) WT and Areg^-/-T cells were cultured under Treg conditions for 5 days and then transferred into Rag^-/- mice. (H-I) Intestinal fibrosis was determined by masson trichrome. (J-L) Col1a1, Col6a1, and Col6a3 levels in colonic tissues were measured by qRT-PCR. ^*P <0.05; ^**P <0.01.

<b>Figure 2. </b>AREG promotes human intestinal myofibrosblasts (MFs) activation and proliferation through activation of PI3K/AKT pathway. Blockade of PI3K/AKT pathway suppresses intestinal fibrosis. <b>(A-D) </b>Human intestinal MFs were treated with or without AREG for 48h and then gene differences were analyzed by RNA-sequencing. <b>(E-F) </b>Human intestinal MFs were treated with or without AREG in the presence or absence of PI3K inhibitor, α-SMA were detected by IF <b>(E) </b>and WB <b>(F)</b>, and p-PI3K, p-AKT were determined by WB <b>(F)</b>. <b>(G) </b>Ki67 positive cells were analyzed by IF. <b>(H-I) </b>Col1a1 and Col6a1 expression were measured by qRT-PCR. (<b>J-M</b>) WT mice (n=6/group) were administered with 3 cycles of 1.5% DSS insults. Areg (550ng per mice) with or without LY294002 (0.5mg per mice) were injected intraperitoneally during the period of DSS administration. <b>(J-M)</b> Intestinal fibrosis was determined.

Figure 2. AREG promotes human intestinal myofibrosblasts (MFs) activation and proliferation through activation of PI3K/AKT pathway. Blockade of PI3K/AKT pathway suppresses intestinal fibrosis. (A-D) Human intestinal MFs were treated with or without AREG for 48h and then gene differences were analyzed by RNA-sequencing. (E-F) Human intestinal MFs were treated with or without AREG in the presence or absence of PI3K inhibitor, α-SMA were detected by IF (E) and WB (F), and p-PI3K, p-AKT were determined by WB (F). (G) Ki67 positive cells were analyzed by IF. (H-I) Col1a1 and Col6a1 expression were measured by qRT-PCR. (J-M) WT mice (n=6/group) were administered with 3 cycles of 1.5% DSS insults. Areg (550ng per mice) with or without LY294002 (0.5mg per mice) were injected intraperitoneally during the period of DSS administration. (J-M) Intestinal fibrosis was determined.

Hongjie Zhang

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