The mechanisms underlying tissue repair in response to damage have been one of main subjects of investigation. Here we leverage the wound-induced hair neogenesis (WIHN) models in adult mice to explore the correlation between degree of damage and the healing process and outcome. The multimodal analysis, in combination with single-cell RNA sequencing help to explore the difference in wounds of gentle and heavy damage degrees, identifying the potential role of toll-like receptor 9 (TLR9) in sensing the injury and regulating the immune reaction by promoting the migration of γδT cells. The TLR9 deficient mice or wounds injected with TLR9 antagonist have greatly impaired healing and lower WIHN levels. Inhibiting the migration of γδT cells or knockout of γδT cells also suppress the wound healing and regeneration, which can\'t be rescued by TLR9agonist. Finally, the amphiregulin (AREG) is shown as one of most important effectors secreted by γδT cells and keratinocytes both in silicon or in the laboratory, whose expression influences WIHN levels and the expression of stem cell markers. In total, our findings reveal a previously unrecognized role for TLR9 in sensing skin injury and influencing the tissue repair and regeneration by modulation of the migration of γδT cells, and identify the TLR9-γδT cells-areg axis as new potential targets for enhancing tissue regeneration.

Amphiregulin (AREG) stimulates human epithelial ovarian cancer (EOC) cell invasion by downregulating E-cadherin expression. YAP is a transcriptional cofactor that has been shown to regulate tumorigenesis. This study aimed to examine whether AREG activates YAP in EOC cells and explore the roles of YAP in AREG-induced downregulation of E-cadherin and cell invasion. Analysis of the Cancer Genome Atlas (TCGA) showed that upregulation of AREG and EGFR were associated with poor survival in human EOC. Treatment of SKOV3 human EOC cells with AREG induced the activation of YAP. In addition, AREG downregulated E-cadherin, upregulated Egr-1 and Slug, and stimulated cell invasion. Using gain- and loss-of-function approaches, we showed that YAP was required for the AREG-upregulated Egr-1 and Slug expression. Furthermore, YAP was also involved in AREG-induced downregulation of E-cadherin and cell invasion. This study provides evidence that AREG stimulates human EOC cell invasion by downregulating E-cadherin expression through the YAP/Egr-1/Slug signaling.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal fibrotic disease. Recent studies have highlighted the persistence of an intermediate state of alveolar stem cells in IPF lungs. In this study, we discovered a close correlation between the distribution pattern of intermediate alveolar stem cells and the progression of fibrotic changes. We showed that amphiregulin (AREG) expression is significantly elevated in intermediate alveolar stem cells of mouse fibrotic lungs and IPF patients. High levels of serum AREG correlate significantly with profound deteriorations in lung function in IPF patients. We demonstrated that AREG in alveolar stem cells is both required and sufficient for activating EGFR in fibroblasts, thereby driving lung fibrosis. Moreover, pharmacological inhibition of AREG using a neutralizing antibody effectively blocked the initiation and progression of lung fibrosis in mice. Our study underscores the therapeutic potential of anti-AREG antibodies in attenuating IPF progression, offering a promising strategy for treating fibrotic diseases.

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase that is frequently modified by glycosylation post-translationally. In cancer, EGFR amplifications and hotspot mutations such as L858R that promote proliferation have been detected in a significant fraction of non-small cell lung carcinomas and breast adenocarcinomas. Molecular dynamic simulations suggested that glycosylation at asparagine residue 361 (N361) promotes dimerization and ligand binding. We stably expressed glycosylation-deficient mutant EGFR N361A, with or without the oncogenic mutation L858R. Immunofluorescence and flow cytometry demonstrated that the mutants were each well expressed at the cell membrane. N361A decreased proliferation relative to wild-type EGFR as well as decreased sensitivity to ligands. Proximity ligation assays measuring co-localization of EGFR with its binding partner HER2 in cells revealed that N361A mutations increased co-localization. N361A, located near the binding interface for the EGFR inhibitor necitumumab, desensitized cells expressing the oncogenic EGFR L858R to antibody-based inhibition. These findings underline the critical relevance of post-translational modifications on oncogene function.

Inflammation is a driving force of tendinopathy. The oxidation of phospholipids by free radicals is a consequence of inflammatory reactions and is an important indicator of tissue damage. Here, we have studied the impact of oxidized phospholipids (OxPAPC) on the function of human tenocytes. We observed that treatment with OxPAPC did not alter the morphology, growth and capacity to produce collagen in healthy or diseased tenocytes. However, since OxPAPC is a known modulator of the function of immune cells, we analyzed whether OxPAPC-treated immune cells might influence the fate of tenocytes. Co-culture of tenocytes with immature, monocyte-derived dendritic cells treated with OxPAPC (Ox-DCs) was found to enhance the proliferation of tenocytes, particularly those from diseased tendons. Using transcriptional profiling of Ox-DCs, we identified amphiregulin (AREG), a ligand for EGFR, as a possible mediator of this proliferation enhancing effect, which we could confirm using recombinant AREG. Of note, diseased tenocytes were found to express higher levels of EGFR compared to tenocytes isolated from healthy donors and show a stronger proliferative response upon co-culture with Ox-DCs, as well as AREG treatment. In summary, we identify an AREG-EGFR axis as a mediator of a DC-tenocyte crosstalk, leading to increased tenocyte proliferation and possibly tendon regeneration.

UNASSIGNED: Massive eosinophil infiltration into the esophagus is associated with subepithelial fibrosis and esophageal stricture in patients with eosinophilic esophagitis (EoE). However, the pathogenesis of esophageal fibrosis remains unclear.
UNASSIGNED: We sought to elucidate the cellular and molecular mechanisms underlying the induction of esophageal fibrosis.
UNASSIGNED: We established a murine model of EoE accompanied by fibrotic responses following long-term intranasal administration of house dust mite antigen. Using this murine model, we investigated the characteristics of immune cells infiltrating the fibrotic region of the inflamed esophagus using flow cytometry and histological analyses. We also analyzed the local inflammatory sites in the esophagus of patients with EoE using single-cell RNA sequencing, flow cytometry, and immunohistochemistry.
UNASSIGNED: Enhanced infiltration of both amphiregulin-producing and IL-5-producing TH2 cells was detected in the fibrotic area of the esophagus in mice subjected to repeated house dust mite exposure. Deletion of amphiregulin in CD4+ T cells ameliorates esophageal fibrosis. An analysis of human esophageal biopsy samples showed that the infiltration of amphiregulin-producing CD4+ T cells was higher in patients with EoE than in control patients. Furthermore, the number of infiltrated amphiregulin-producing CD4+ T cells was associated with the degree of esophageal fibrosis in patients with EoE.
UNASSIGNED: Amphiregulin, produced by TH2 cells, contributes to esophageal fibrosis in EoE and may be a therapeutic target.

OBJECTIVE: To analyze the biological effect and mechanism of areca nut extract (ANE) on human oral keratinocyte (HOK) cells.
METHODS: The effect of gradient concentration of ANE on the proliferation activity of HOK cells was analyzed by cell counting kit-8 (CCK-8) assays. The differentially expressed genes between the ANE group and control group HOK cells were analyzed by second-generation transcriptome sequencing. Real-time PCR and western blot were, respectively, used to analyze the expression of AREG gene and protein in HOK cells. After AREG gene overexpression or knockdown, the proliferation, migration, and expression of proteins related to epithelial-mesenchymal transformation (EMT), MAPK signal pathway in HOK cells were, respectively, detected by CCK-8, wound healing, transwell, and western blot assays.
RESULTS: ANE (500 μg/mL) promoted the proliferation and migration of HOK cells, ANE (2 mg/mL) promoted the EMT of HOK cells, and ANE (50 mg/mL) inhibited the proliferation of HOK cells. AREG knockdown inhibited ANE-induced proliferation and migration of HOK cells, while AREG overexpression promoted the proliferation and migration of HOK cells. Western blot assay showed that ANE activated MAPK signal pathway by upregulating AREG protein in HOK cells.
CONCLUSIONS: ANE promoted HOK cell proliferation, migration, and EMT by mediating AREG-MAPK signaling pathway.

UNASSIGNED: To examine the effect of intraocularly applied amphiregulin antibody on physiological axial elongation in young nonhuman primates.
UNASSIGNED: The experimental study included six male 12-months-old macaque nonhuman primates (body weight:2.46 ± 0.25kg;range:2.20-2.90kg). In the experimental group (n=3 animals), three intravitreal injections of amphiregulin antibody (100μg/50μl) were applied to the left eyes at intervals of 4-6 weeks, and injections of phosphate buffered solution (50μl) were applied to the right eyes. Three other animals were assigned to a blank control group.
UNASSIGNED: During the study period of 23.6 weeks, axial length in the experimental group did not change in the left eyes (18.91 ± 0.37mm to 18.94 ± 0.67mm;P=0.90), while it linearly increased in the right eyes (18.87 ± 0.38mm to 19.24 ± 0.53mm;P=0.056) and in the control group (left eyes:19.15 ± 0.22mm to 19.48 ± 0.22mm;P=0.009; right eyes:19.17 ± 0.15 mm to 19.46 ± 0.23 mm;P=0.024). The interocular difference in axial elongation increased in the experimental group from -0.11 ± 0.12mm at 4 weeks after baseline to -0.34 ± 0.15mm at the study end, while in the control group, the interocular side difference did not change significantly (from 0.01 ± 0.10 mm to 0.03 ± 0.08 mm;P=0.38). The difference in the interocular difference in axial elongation between the two groups was significant at 8 weeks (P=0.01), 15 weeks (P=0.007), and at study end (P=0.02). The interocular difference in axial length correlated with the interocular difference in vitreous cavity length (standardized regression coefficient beta:0.85;P<0.001). The interocular axial length difference was inversely associated with the interocular refractive error difference (beta:-0.49;P<0.001).
UNASSIGNED: Intraocularly applied amphiregulin antibody (100μg) reduced the physiological ocular axial elongation in juvenile nonhuman primates.

Accumulating evidence shows that inflammation is essential for embryo implantation and decidualization. Histamine, a proinflammatory factor that is present in almost all mammalian tissues, is synthesized through decarboxylating histidine by histidine decarboxylase (HDC). Although histamine is known to be essential for decidualization, the underlying mechanism remains undefined. In the present study, histamine had no obvious direct effects on in vitro decidualization in mice. However, the obvious differences in HDC protein levels between day 4 of pregnancy and day 4 of pseudopregnancy, as well as between delayed and activated implantation, suggested that the blastocyst may be involved in regulating HDC expression. Furthermore, blastocyst-derived tumor necrosis factor α (TNFα) significantly increased HDC levels in the luminal epithelium. Histamine increased the levels of amphiregulin (AREG) and disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) proteins, which was abrogated by treatment with famotidine, a specific histamine type 2 receptor (H2R) inhibitor, or by TPAI-1 (a specific inhibitor of ADAM17). Intraluminal injection of urocanic acid (HDC inhibitor) on day 4 of pregnancy significantly reduced the number of implantation sites on day 5 of pregnancy. TNFα-stimulated increases in HDC, AREG and ADAM17 protein levels was abrogated by urocanic acid, a specific inhibitor of HDC. Additionally, AREG treatment significantly promoted in vitro decidualization. Collectively, our data suggests that blastocyst-derived TNFα induces luminal epithelial histamine secretion, and histamine increases mouse decidualization through ADAM17-mediated AREG release.

UNASSIGNED: Amphiregulin (AR) is a growth factor that resembles the epidermal growth factor (EGF) and serves various functions in different cells. However, no systematic studies or reports on the role of AR in human oocytes have currently been performed or reported. This study aimed to explore the role of AR in human immature oocytes during in vitro maturation (IVM) and in vitro fertilization (IVF) in achieving better embryonic development and to provide a basis for the development of a pre-insemination culture medium specific for cumulus oocyte complexes (COCs).
UNASSIGNED: First, we examined the concentration of AR in the follicular fluid (FF) of patients who underwent routine IVF and explored the correlation between AR levels and oocyte maturation and subsequent embryonic development. Second, AR was added to the IVM medium to culture immature oocytes and investigate whether AR could improve the effects of IVM. Finally, we pioneered the use of a fertilization medium supplemented with AR for the pre-insemination culture of COCs to explore whether the involvement of AR can promote the maturation and fertilization of IVF oocytes, as well as subsequent embryonic development.
UNASSIGNED: A total of 609 FF samples were examined, and a positive correlation between AR levels and blastocyst formation was observed. In our IVM study, the development potential and IVM rate of immature oocytes, as well as the fertilization rate of IVM oocytes in the AR-added groups, were ameliorated significantly compared to the control group (All P < 0.05). Only the IVM-50 group had a significantly higher blastocyst formation rate than the control group (P < 0.05). In the final IVF study, the maturation, fertilization, high-quality embryo, blastocyst formation, and high-quality blastocyst rates of the AR-added group were significantly higher than those of the control group (All P < 0.05).
UNASSIGNED: AR levels in the FF positively correlated with blastocyst formation, and AR involvement in pre-insemination cultures of COCs can effectively improve laboratory outcomes in IVF. Furthermore, AR can directly promote the in vitro maturation and developmental potential of human immature oocytes at an optimal concentration of 50 ng/ml.