Carfilzomib shows therapeutic potential for reduction of liver fibrosis by targeting hepatic stellate cell activation.

Because hepatic stellate cells (HSCs) play a major role in fibrosis, we focused on HSCs as a potential target for the treatment of liver fibrosis. In this study, we attempted to identify drug candidates to inactivate HSCs and found that several proteasome inhibitors (PIs) reduced HSC viability. Our data showed that a second-generation PI, carfilzomib (CZM), suppressed the expression of fibrotic markers in primary murine HSCs at low concentrations of 5 or 10 nM. Since CZM was not toxic to HSCs up to a concentration of 12.5 nM, we examined its antifibrotic effects further. CZM achieved a clear reduction in liver fibrosis in the carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis without worsening of liver injury. Mechanistically, RNA sequence analysis of primary HSCs revealed that CZM inhibits mitosis in HSCs. In the CCl4-injured liver, amphiregulin, which is known to activate mitogenic signaling pathways and fibrogenic activity and is upregulated in murine and human metabolic dysfunction-associated steatohepatitis (MASH), was downregulated by CZM administration, leading to inhibition of mitosis in HSCs. Thus, CZM and next-generation PIs in development could be potential therapeutic agents for the treatment of liver fibrosis via inactivation of HSCs without liver injury.

TLR9 activation in large wound induces tissue repair and hair follicle regeneration via γδT cells.

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.

Sustained amphiregulin expression in intermediate alveolar stem cells drives progressive fibrosis.

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.

Application of amphiregulin in IVM culture of immature human oocytes and pre-insemination culture for COCs in IVF cycles.

Background: 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). Methods: 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. Results: 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). Conclusion: 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.

Oxidized Phospholipids Regulate Tenocyte Function via Induction of Amphiregulin in Dendritic Cells.

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.

Stress-induced Rab11a-exosomes induce amphiregulin-mediated cetuximab resistance in colorectal cancer.

Exosomes are secreted vesicles made intracellularly in the endosomal system. We have previously shown that exosomes are not only made in late endosomes, but also in recycling endosomes marked by the monomeric G-protein Rab11a. These vesicles, termed Rab11a-exosomes, are preferentially secreted under nutrient stress from several cancer cell types, including HCT116 colorectal cancer (CRC) cells. HCT116 Rab11a-exosomes have particularly potent signalling activities, some mediated by the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). Mutant activating forms of KRAS, a downstream target of EGFR, are often found in advanced CRC. When absent, monoclonal antibodies, such as cetuximab, which target the EGFR and block the effects of EGFR ligands, such as AREG, can be administered. Patients, however, inevitably develop resistance to cetuximab, either by acquiring KRAS mutations or via non-genetic microenvironmental changes. Here we show that nutrient stress in several CRC cell lines causes the release of AREG-carrying Rab11a-exosomes. We demonstrate that while soluble AREG has no effect, much lower levels of AREG bound to Rab11a-exosomes from cetuximab-resistant KRAS-mutant HCT116 cells, can suppress the effects of cetuximab on KRAS-wild type Caco-2 CRC cells. Using neutralising anti-AREG antibodies and an intracellular EGFR kinase inhibitor, we show that this effect is mediated via AREG activation of EGFR, and not transfer of activated KRAS. Therefore, presentation of AREG on Rab11a-exosomes affects its ability to compete with cetuximab. We propose that this Rab11a-exosome-mediated mechanism contributes to the establishment of resistance in cetuximab-sensitive cells and may explain why in cetuximab-resistant tumours only some cells carry mutant KRAS.

Particulate matter facilitates amphiregulin-dependent lung cancer proliferation through glutamine metabolism.

Although many cohort studies have reported that long-term exposure to particulate matter (PM) causes lung cancer, the molecular mechanisms underlying the PM-induced increases in lung cancer progression remain unclear. We applied the lung cancer cell line A549 (Parental; A549.Par) to PM for an extended period to establish a mimic PM-exposed lung cancer cell line, A549.PM. Our results indicate that A549.PM exhibits higher cell growth and proliferation abilities compared to A549.Par cells in vitro and in vivo. The RNA sequencing analysis found amphiregulin (AREG) plays a critical role in PM-induced cell proliferation. We observed that PM increases AREG-dependent lung cancer proliferation through glutamine metabolism. In addition, the EGFR/PI3K/AKT/mTOR signaling pathway is involved in PM-induced solute carrier family A1 member 5 (SLC1A5) expression and glutamine metabolism. Our findings offer important insights into how lung cancer proliferation develops upon exposure to PM.

The growth factors amphiregulin and epiregulin are novel stimulators of feline ovarian granulosa cell functions.

This study aimed to investigate the impact of the epidermal growth factor receptor ligands amphiregulin (AREG) and epiregulin (EREG) on the fundamental functions of feline ovarian granulosa cells. Granulosa cells isolated from feline ovaries were incubated with AREG and EREG (0, 0.1, 1 or 10 ng/mL). The effects of these growth factors on cell viability, proliferation (assessed through BrdU incorporation), nuclear apoptosis (evaluated through nuclear DNA fragmentation) and the release of progesterone and estradiol were determined using Cell Counting Kit-8 assays, BrdU analysis, TUNEL assays and ELISAs, respectively. Both AREG and EREG increased cell viability, proliferation and steroid hormone release and reduced apoptosis. The present findings suggest that these epidermal growth factor receptor ligands may serve as physiological stimulators of feline ovarian cell functions.

CD133 Stimulates Cell Proliferation via the Upregulation of Amphiregulin in Melanoma.

CD133, a cancer stem cell (CSC) marker in tumors, including melanoma, is associated with tumor recurrence, chemoresistance, and metastasis. Patient-derived melanoma cell lines were transduced with a Tet-on vector expressing CD133, generating doxycycline (Dox)-inducible cell lines. Cells were exposed to Dox for 24 h to induce CD133 expression, followed by RNA-seq and bioinformatic analyses, revealing genes and pathways that are significantly up- or downregulated by CD133. The most significantly upregulated gene after CD133 was amphiregulin (AREG), validated by qRT-PCR and immunoblot analyses. Induced CD133 expression significantly increased cell growth, percentage of cells in S-phase, BrdU incorporation into nascent DNA, and PCNA levels, indicating that CD133 stimulates cell proliferation. CD133 induction also activated EGFR and the MAPK pathway. Potential mechanisms highlighting the role(s) of CD133 and AREG in melanoma CSC were further delineated using AREG/EGFR inhibitors or siRNA knockdown of AREG mRNA. Treatment with the EGFR inhibitor gefitinib blocked CD133-induced cell growth increase and MAPK pathway activation. Importantly, siRNA knockdown of AREG reversed the stimulatory effects of CD133 on cell growth, indicating that AREG mediates the effects of CD133 on cell proliferation, thus serving as an attractive target for novel combinatorial therapeutics in melanoma and cancers with overexpression of both CD133 and AREG.

Angiopoietin-like 4 is upregulated by amphiregulin and activates cell proliferation and migration through p38 kinase in head and neck squamous cell carcinoma.

BACKGROUND: Angiopoietin-like 4 is a molecular hallmark that correlates with the growth and metastasis of head and neck squamous cell carcinoma, one of the most prevalent cancers worldwide. However, the molecular mechanisms by which angiopoietin-like 4 promotes head and neck squamous cell carcinoma tumorigenesis are unclear. METHODS: Using well-characterized cell lines of head and neck squamous cell carcinoma development, including human normal oral keratinocytes, dysplastic oral keratinocytes, oral leukoplakia-derived oral keratinocytes, and head and neck squamous cell carcinoma cell lines, HN13, HN6, HN4, HN12, and CAL27, we investigated the signaling pathways upstream and downstream of angiopoietin-like 4-induced head and neck squamous cell carcinoma tumorigenesis. RESULTS: We found that both epidermal growth factor receptor ligands, epithelial growth factor, and amphiregulin led to angiopoietin-like 4 upregulation in normal oral keratinocytes and dysplastic oral keratinocytes and cooperated with the activation of hypoxia-inducible factor-1 in this effect. Interestingly, amphiregulin and angiopoietin-like 4 were increased in dysplastic oral keratinocytes and head and neck squamous cell carcinoma cell lines, and amphiregulin-induced activation of cell proliferation was dependent on angiopoietin-like 4. Although both p38 mitogen-activated protein kinases (p38 MAPK) and protein kinase B (AKT) were activated by angiopoietin-like 4, only pharmacological inhibition of p38 MAPK was sufficient to prevent head and neck squamous cell carcinoma cell proliferation and migration. We further observed that angiopoietin-like 4 promoted the secretion of interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin-1 alpha (IL-1α), vascular endothelial growth factor, platelet-derived growth factor (PDGF), and tumour necrosis factor alpha (TNF-α), cytokines and chemokines previously implicated in head and neck squamous cell carcinoma pathogenesis. CONCLUSION: Our results demonstrate that angiopoietin-like 4 is a downstream effector of amphiregulin and promotes head and neck squamous cell carcinoma development both through direct activation of p38 kinase as well as paracrine mechanisms.

Amphiregulin secreted by umbilical cord multipotent stromal cells protects against ferroptosis of macrophages via the activating transcription factor 3-CD36 axis to alleviate endometrial fibrosis.

Endometrium fibrosis is the leading cause of uterine infertility. Macrophages participated in the occurrence and development of endometrial fibrosis. We previously reported that human umbilical cord multipotent stromal cells (hUC-MSCs) exerted their therapeutic effect in a macrophage-dependent manner in endometrial fibrosis. However precise mechanisms by which hUC-MSCs may influence macrophages in endometrial fibrosis remain largely unexplored. Here, we demonstrated that abnormal iron and lipid metabolism occurred in patients with intrauterine adhesions (IUA) and murine models. Ferroptosis has been proven to contribute to the progression of fibrotic diseases. Our results revealed that pharmacological activation of ferroptosis by Erastin aggravated endometrial fibrosis, while inhibition of ferroptosis by Ferrostatin-1 ameliorated endometrial fibrosis in vivo. Moreover, ferroptosis of macrophages was significantly upregulated in endometria of IUA murine models. Of note, transcriptome profiles revealed that CD36 gene expression was significantly increased in patients with IUA and immunofluorescence analysis showed CD36 protein was mainly located in macrophages. Silencing CD36 in macrophages could reverse cell ferroptosis. Dual luciferase reporter assay revealed that CD36 was the direct target of activation transcription factor 3 (ATF3). Furthermore, through establishing coculture system and IUA murine models, we found that hUC-MSCs had a protective role against macrophage ferroptosis and alleviated endometrial fibrosis related to decreased CD36 and ATF3. The effect of hUC-MSCs on macrophage ferroptosis was attributed to the upregulation of amphiregulin (AREG). Our data highlighted that macrophage ferroptosis occurred in endometrial fibrosis via the ATF3-CD36 pathway and hUC-MSCs protected against macrophage ferroptosis to alleviate endometrial fibrosis via secreting AREG. These findings provided a potential target for therapeutic implications of endometrial fibrosis.

Hepatocellular RECK as a Critical Regulator of Metabolic Dysfunction-associated Steatohepatitis Development.

BACKGROUND & AIMS: Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is an extracellular matrix regulator with anti-fibrotic effects. However, its expression and role in metabolic dysfunction-associated steatohepatitis (MASH) and hepatic fibrosis are poorly understood. METHODS: We generated a novel transgenic mouse model with RECK overexpression specifically in hepatocytes to investigate its role in Western diet (WD)-induced liver disease. Proteomic analysis and in vitro studies were performed to mechanistically link RECK to hepatic inflammation and fibrosis. RESULTS: Our results show that RECK expression is significantly decreased in liver biopsies from human patients diagnosed with MASH and correlated negatively with severity of metabolic dysfunction-associated steatotic liver disease (MASLD) and fibrosis. Similarly, RECK expression is downregulated in WD-induced MASH in wild-type mice. Hepatocyte-specific RECK overexpression significantly reduced hepatic pathology in WD-induced liver injury. Proteomic analysis highlighted changes in extracellular matrix and cell-signaling proteins. In vitro mechanistic studies linked RECK induction to reduced ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) and ADAM17 activity, amphiregulin release, epidermal growth factor receptor activation, and stellate cell activation. CONCLUSION: Our in vivo and mechanistic in vitro studies reveal that RECK is a novel upstream regulator of inflammation and fibrosis in the diseased liver, its induction is hepatoprotective, and thus highlights its potential as a novel therapeutic in MASH.

EGF-like growth factors upregulate pentraxin 3 expression in human granulosa-lutein cells.

The epidermal growth factor (EGF)-like factors, comprising amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG), play a critical role in regulating the ovulatory process. Pentraxin 3 (PTX3), an essential ovulatory protein, is necessary for maintaining extracellular matrix (ECM) stability during cumulus expansion. The aim of this study was to investigate the impact of EGF-like factors, AREG, BTC, and EREG on the expression and production of PTX3 in human granulosa-lutein (hGL) cells and the molecular mechanisms involved. Our results demonstrated that AREG, BTC, and EREG could regulate follicular function by upregulating the expression and increasing the production of PTX3 in both primary (obtained from 20 consenting patients undergoing IVF treatment) and immortalized hGL cells. The upregulation of PTX3 expression was primarily facilitated by the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway, induced by these EGF-like factors. In addition, we found that the upregulation of PTX3 expression triggered by the EGF-like factors was completely reversed by either pretreatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, or knockdown of EGFR, suggesting that EGFR is crucial for activating the ERK1/2 signaling pathway in hGL cells. Overall, our findings indicate that AREG, BTC, and EREG may modulate human cumulus expansion during the periovulatory stage through the upregulation of PTX3.

Mucosal-associated invariant T cells promote ductular reaction through amphiregulin in biliary atresia.

BACKGROUND: Biliary atresia (BA) is a neonatal fibro-inflammatory cholangiopathy with ductular reaction as a key pathogenic feature predicting poor survival. Mucosal-associated invariant T (MAIT) cells are enriched in human liver and display multiple roles in liver diseases. We aimed to investigate the function of MAIT cells in BA. METHODS: First, we analyzed correlations between liver MAIT cell and clinical parameters (survival, alanine transaminase, bilirubin, histological inflammation and fibrosis) in two public cohorts of patients with BA (US and China). Kaplan-Meier survival analysis and spearman correlation analysis were employed for survival data and other clinical parameters, respectively. Next, we obtained liver samples or peripheral blood from BA and control patients for bulk RNA sequencing, flow cytometry analysis, immunostaning and functional experiments of MAIT cells. Finally, we established two in vitro co-culture systems, one is the rhesus rotavirus (RRV) infected co-culture system to model immune dysfunction of human BA which was validated by single cell RNA sequencing and the other is a multicellular system composed of biliary organoids, LX-2 and MAIT cells to evaluate the role of MAIT cells on ductular reaction. FINDINGS: Liver MAIT cells in BA were positively associated with low survival and ductular reaction. Moreover, liver MAIT cells were activated, exhibited a wound healing signature and highly expressed growth factor Amphiregulin (AREG) in a T cell receptor (TCR)-dependent manner. Antagonism of AREG abrogated the proliferative effect of BA MAIT cells on both cholangiocytes and biliary organoids. A RRV infected co-culture system, recapitulated immune dysfunction of human BA, disclosed that RRV-primed MAIT cells promoted cholangiocyte proliferation via AREG, and further induced inflammation and fibrosis in the multicellular system. INTERPRETATION: MAIT cells exhibit a wound healing signature depending on TCR signaling and promote ductular reaction via AREG, which is associated with advanced fibrosis and predictive of low survival in BA. FUNDING: This work was funded by National Natural Science Foundation of China grant (82001589 and 92168108), National Key R&D Program of China (2023YFA1801600) and by Basic and Applied Basic Research Foundation of Guangdong (2020A1515110921).

The ST2+ Treg/amphiregulin axis protects from immune-mediated hepatitis.

Introduction: The alarmin IL-33 has been implicated in the pathology of immune-mediated liver diseases. IL-33 activates regulatory T cells (Tregs) and type 2 innate lymphoid cells (ILC2s) expressing the IL-33 receptor ST2. We have previously shown that endogenous IL-33/ST2 signaling activates ILC2s that aggravate liver injury in murine immune-mediated hepatitis. However, treatment of mice with exogenous IL-33 before induction of hepatitis ameliorated disease severity. Since IL-33 induces expression of amphiregulin (AREG) crucial for Treg function, we investigated the immunoregulatory role of the ST2+ Treg/AREG axis in immune-mediated hepatitis. Methods: C57BL/6, ST2-deficient (Il1rl1-/-) and Areg-/- mice received concanavalin A to induce immune-mediated hepatitis. Foxp3Cre+ x ST2fl/fl mice were pre-treated with IL-33 before induction of immune-mediated hepatitis. Treg function was assessed by adoptive transfer experiments and suppression assays. The effects of AREG and IL-33 on ST2+ Tregs and ILC2s were investigated in vitro. Immune cell phenotype was analyzed by flow cytometry. Results and discussion: We identified IL-33-responsive ST2+ Tregs as an effector Treg subset in the murine liver, which was highly activated in immune-mediated hepatitis. Lack of endogenous IL-33 signaling in Il1rl1-/- mice aggravated disease pathology. This was associated with reduced Treg activation. Adoptive transfer of exogenous IL-33-activated ST2+ Tregs before induction of hepatitis suppressed inflammatory T-cell responses and ameliorated disease pathology. We further showed increased expression of AREG by hepatic ST2+ Tregs and ILC2s in immune-mediated hepatitis. Areg-/- mice developed more severe liver injury, which was associated with enhanced ILC2 activation and less ST2+ Tregs in the inflamed liver. Exogenous AREG suppressed ILC2 cytokine expression and enhanced ST2+ Treg activation in vitro. In addition, Tregs from Areg-/- mice were impaired in their capacity to suppress CD4+ T-cell activation in vitro. Moreover, application of exogenous IL-33 before disease induction did not protect Foxp3Cre+ x ST2fl/fl mice lacking ST2+ Tregs from immune-mediated hepatitis. In summary, we describe an immunoregulatory role of the ST2+ Treg/AREG axis in immune-mediated hepatitis, in which AREG suppresses the activation of hepatic ILC2s while maintaining ST2+ Tregs and reinforcing their immunosuppressive capacity in liver inflammation.

A distinct "repair" role of regulatory T cells in fracture healing.

Regulatory T cells (Tregs) suppress immune responses and inflammation. Here, we described the distinct nonimmunological role of Tregs in fracture healing. The recruitment from the circulation pool, peripheral induction, and local expansion rapidly enriched Tregs in the injured bone. The Tregs in the injured bone displayed superiority in direct osteogenesis over Tregs from lymphoid organs. Punctual depletion of Tregs compromised the fracture healing process, which leads to increased bone nonunion. In addition, bone callus Tregs showed unique T-cell receptor repertoires. Amphiregulin was the most overexpressed protein in bone callus Tregs, and it can directly facilitate the proliferation and differentiation of osteogenic precursor cells by activation of phosphatidylinositol 3-kinase/protein kinase B signaling pathways. The results of loss- and gain-function studies further evidenced that amphiregulin can reverse the compromised healing caused by Treg dysfunction. Tregs also enriched in patient bone callus and amphiregulin can promote the osteogenesis of human pre-osteoblastic cells. Our findings indicate the distinct and nonredundant role of Tregs in fracture healing, which will provide a new therapeutic target and strategy in the clinical treatment of fractures.

Amphiregulin Switches Progenitor Cell Fate for Lineage Commitment During Gastric Mucosal Regeneration.

BACKGROUND & AIMS: Isthmic progenitors, tissue-specific stem cells in the stomach corpus, maintain mucosal homeostasis by balancing between proliferation and differentiation to gastric epithelial lineages. The progenitor cells rapidly adopt an active state in response to mucosal injury. However, it remains unclear how the isthmic progenitor cell niche is controlled during the regeneration of damaged epithelium. METHODS: We recapitulated tissue recovery process after acute mucosal injury in the mouse stomach. Bromodeoxyuridine incorporation was used to trace newly generated cells during the injury and recovery phases. To define the epithelial lineage commitment process during recovery, we performed single-cell RNA-sequencing on epithelial cells from the mouse stomachs. We validated the effects of amphiregulin (AREG) on mucosal recovery, using recombinant AREG treatment or AREG-deficient mice. RESULTS: We determined that an epidermal growth factor receptor ligand, AREG, can control progenitor cell lineage commitment. Based on the identification of lineage-committed subpopulations in the corpus epithelium through single-cell RNA-sequencing and bromodeoxyuridine incorporation, we showed that isthmic progenitors mainly transition into short-lived surface cell lineages but are less frequently committed to long-lived parietal cell lineages in homeostasis. However, mucosal regeneration after damage directs the lineage commitment of isthmic progenitors towards parietal cell lineages. During recovery, AREG treatment promoted repopulation with parietal cells, while suppressing surface cell commitment of progenitors. In contrast, transforming growth factor-α did not alter parietal cell regeneration, but did induce expansion of surface cell populations. AREG deficiency impairs parietal cell regeneration but increases surface cell commitment. CONCLUSIONS: These data demonstrate that different epidermal growth factor receptor ligands can distinctly regulate isthmic progenitor-driven mucosal regeneration and lineage commitment.

Investigating the impact of antibiotic-induced dysbiosis on protection from Clostridium difficile colitis by mouse colonic innate lymphoid cells.

Innate lymphoid cells (ILCs) play a critical role in maintaining intestinal health in homeostatic and diseased conditions. During Clostridium difficile infection (CDI), IL-33 activates ILC2 to protect from colonic damage and mortality. The function of IL-33 and ILC is tightly regulated by the intestinal microbiota. We set out to determine the impact of antibiotic-induced disruption of the microbiome on ILC function. Our goal was to understand antibiotic-induced changes in ILC function on susceptibility to C. difficile colitis in a mouse model. We utilized high-throughput single-cell RNAseq to investigate the phenotypic features of colonic ILC at baseline, after antibiotic administration with or without IL-33 treatment. We identified a heterogeneous landscape of colonic ILCs with gene signatures of inflammatory, anti-inflammatory, migratory, progenitor, plastic, and antigen-presenting ILCs. Antibiotic treatment decreased ILC2 while coordinately increasing ILC1 and ILC3 phenotypes. Notably, Ifng+, Ccl5+, and Il23r+ ILC increased after antibiotics. IL-33 treatment counteracted the antibiotic effect by downregulating ILC1 and ILC3 and activating ILC2. In addition, IL-33 treatment markedly induced the expression of type 2 genes, including Areg and Il5. Finally, we identified amphiregulin, produced by ILC2, as protective during C. difficile infection. Together, our data expand our understanding of how antibiotics induce susceptibility to C. difficile colitis through their impact on ILC subsets and function.IMPORTANCEClostridium difficile infection (CDI) accounts for around 500,000 symptomatic cases and over 20,000 deaths annually in the United States alone. A major risk factor of CDI is antibiotic-induced dysbiosis of the gut. Microbiota-regulated IL-33 and innate lymphoid cells (ILCs) are important in determining the outcomes of C. difficile infection. Understanding how antibiotic and IL-33 treatment alter the phenotype of colon ILCs is important to identify potential therapeutics. Here, we performed single-cell RNAseq of mouse colon ILCs collected at baseline, after antibiotic treatment, and after IL-33 treatment. We identified heterogeneous subpopulations of all three ILC subtypes in the mouse colon. Our analysis revealed several potential pathways of antibiotic-mediated increased susceptibility to intestinal infection. Our discovery that Areg is abundantly expressed by ILCs, and the protection of mice from CDI by amphiregulin treatment, suggests that the amphiregulin-epidermal growth factor receptor pathway is a potential therapeutic target for treating intestinal colitis.

Elastin microfibril interface-located protein 1 in fibroblasts is regulated by amphiregulin and interleukin-1α produced by keratinocytes.

OBJECTIVE: The structure of elastic fibres changes with ageing. Elastin microfibril interface-located protein 1 (EMILIN-1) is known to contribute to structural changes in elastic fibres. EMILIN-1 is one of the components of elastic fibres and also colocalizes with oxytalan fibres near the epidermis. Therefore, EMILIN-1 may be affected by epidermal-dermal interactions. The purpose of this study is to identify the key factors involved in epidermal-dermal interactions during the structural degeneration of elastic fibres. METHODS: Keratinocytes and fibroblasts were co-cultured, and changes in elastic fibre-related proteins were evaluated. Additionally, cytokine arrays were used to identify the factors involved in epidermal-dermal interactions. RESULTS: EMILIN-1 expression in fibroblasts was increased in the presence of keratinocytes, and its expression decreased when keratinocytes were stressed. Amphiregulin (AREG) and interleukin-1α (IL-1α) were identified as the keratinocyte-derived cytokines that influence the production of EMILIN-1, which is secreted by the fibroblasts. EMILIN-1 expression was promoted by AREG and decreased by IL-1α via an increase in cathepsin K (a catabolic enzyme). AREG and IL-1α were associated with changes in EMILIN-1 levels in fibroblasts. CONCLUSION: The findings suggest that the suppression of IL-1α expression and promotion of AREG expression in the epidermis could be a new approach that prevents the wrinkles and sagging caused by the structural changes in elastic fibres.

OBJECTIF: La structure des fibres élastiques change avec le vieillissement. La protéine située à l'interface des microfibrilles d'élastine 1 (EMILIN­1) est connue pour sa contribution aux changements structurels des fibres élastiques. EMILIN­1 est l'un des composants des fibres élastiques et colocalise également avec les fibres oxytalanes à proximité de l'épiderme. Par conséquent, EMILIN­1 peut être affectée par des interactions épidermiques­dermiques. Cette étude a objectif d'identifier les facteurs clés impliqués dans les interactions épidermiques­dermiques au cours de la dégénérescence structurelle des fibres élastiques. MÉTHODES: Co­cultiver les kératinocytes et les fibroblastes, et évaluer les changements dans les protéines liées aux fibres élastiques. De plus, identifier les facteurs impliqués dans les interactions épidermique­dermique des puces à l'aide des cytokines. RÉSULTATS: L'expression de l'EMILIN­1 dans les fibroblastes était augmentée en présence des kératinocytes, et son expression a diminué lorsque les keratinocytes ont été stressés. L'amphiréguline (AREG) et l'interleukine­1α (IL­1α) étaient identifiées comme les cytokines dérivées des kératinocytes qui influencent la production d'EMILIN­1, qui est sécrétée par les fibroblastes. L'expression d'EMILIN­1 était favorisée par l'AREG et diminuée par l'IL­1α via une augmentation de la cathepsine K (une enzyme catabolique). L'AREG et l'IL­1α étaient liés aux changements du niveau d'EMILIN­1 dans les fibroblastes. CONCLUSION: Les résultats montrent que la suppression de l'expression de l'IL­1α et la promotion de l'expression de l'AREG dans l'épiderme pourraient être une nouvelle approche qui prévient les rides et l'affaissement dus aux changements structurels des fibres élastiques.

Amphiregulin in lung diseases: A review.

Amphiregulin is a member of the EGFR family, which is involved in many physiological and pathological processes through its binding with EGFR. Studies have found that amphiregulin plays an important role in the occurrence and development of lung diseases. This paper mainly reviews the structure and function of amphiregulin and focuses on the important role of amphiregulin in lung diseases.