Epithelial Repair Research Articles

Mesenchymal stem cell-based adjunctive therapy for Pseudomonas aeruginosa-induced keratitis: A proof-of-concept in-vitro study

PurposePseudomonas aeruginosa-induced keratitis is one of the most severe and challenging forms of corneal infection, owing to its associated intense inflammatory reactions leading to corneal necrosis and dense corneal scar with loss of vision. Since mesenchymal stem cells (MSCs) are reported to possess antimicrobial and immunomodulatory properties, they can be tested as an adjuvant treatment along with the antibiotics which are the current standard of care. This study aims to investigate the anti-bacterial and immunomodulatory roles of human bone marrow MSC-derived conditioned medium (MSC-CM) in P. aeruginosa-infected human corneal epithelial cells (HCECs) in vitro. MethodsThe effect of MSC-CM on the growth of clinical isolates of P. aeruginosa was evaluated by colony-forming unit assay. The expression of inflammatory cytokines (IL-6 and TNF-α) and an antimicrobial peptide (Lipocalin 2) in lipopolysaccharide-treated MSCs and HCECs was analyzed through ELISA. Corneal epithelial repair following infection with P. aeruginosa was studied through scratch assay. ResultsCompared to control (P. aeruginosa (5*105) incubated in DMEM (1 ml) at 37 °C for 16 h), MSC-CM significantly: i) inhibits the growth of P. aeruginosa (159*109 vs. 104*109 CFU/ml), ii) accelerates corneal epithelial repair following infection with P. aeruginosa (9% vs. 24% closure of the wounded area after 12 h of infection), and iii) downregulates the lipopolysaccharide-induced expression of IL-6, TNF-α and Lipocalin 2 in HCECs. A combination of MSC-CM with an antibiotic, Ciprofloxacin moderately regulated the expression of IL-6, TNF-α, and Lipocalin 2. ConclusionMSC-CM holds promise as an adjunctive therapeutic approach for P. aeruginosa-induced corneal epithelial damage.

Role of the GLP2–Wnt1 axis in silicon-rich alkaline mineral water maintaining intestinal epithelium regeneration in piglets under early-life stress

Stress-induced intestinal epithelial injury (IEI) and a delay in repair in infancy are predisposing factors for refractory gut diseases in adulthood, such as irritable bowel syndrome (IBS). Hence, it is necessary to develop appropriate mitigation methods for mammals when experiencing early-life stress (ELS). Weaning, as we all know, is a vital procedure that all mammalian newborns, including humans, must go through. Maternal separation (MS) stress in infancy (regarded as weaning stress in animal science) is a commonly used ELS paradigm. Drinking silicon-rich alkaline mineral water (AMW) has a therapeutic effect on enteric disease, but the specific mechanisms involved have not been reported. Herein, we discover the molecular mechanism by which silicon-rich AMW repairs ELS-induced IEI by maintaining intestinal stem cell (ISC) proliferation and differentiation through the glucagon-like peptide (GLP)2–Wnt1 axis. Mechanistic study showed that silicon-rich AMW activates GLP2-dependent Wnt1/β-catenin pathway, and drives ISC proliferation and differentiation by stimulating Lgr5+ ISC cell cycle passage through the G1–S-phase checkpoint, thereby maintaining intestinal epithelial regeneration and IEI repair. Using GLP2 antagonists (GLP23−33) and small interfering RNA (SiWnt1) in vitro, we found that the GLP2–Wnt1 axis is the target of silicon-rich AMW to promote intestinal epithelium regeneration. Therefore, silicon-rich AMW maintains intestinal epithelium regeneration through the GLP2–Wnt1 axis in piglets under ELS. Our research contributes to understanding the mechanism of silicon-rich AMW promoting gut epithelial regeneration and provides a new strategy for the alleviation of ELS-induced IEI.

Quercetin improves epithelial regeneration from airway basal cells of COPD patients.

Airway basal cells (BC) from patients with chronic obstructive pulmonary disease (COPD) regenerate abnormal airway epithelium and this was associated with reduced expression of several genes involved in epithelial repair. Quercetin reduces airway epithelial remodeling and inflammation in COPD models, therefore we examined whether quercetin promotes normal epithelial regeneration from COPD BC by altering gene expression. COPD BC treated with DMSO or 1 µM quercetin for three days were cultured at air/liquid interface (ALI) for up to 4 weeks. BC from healthy donors cultured at ALI were used as controls. Polarization of cells was determined at 8 days of ALI. The cell types and IL-8 expression in differentiated cell cultures were quantified by flow cytometry and ELISA respectively. Microarray analysis was conducted on DMSO or 1 µM quercetin-treated COPD BC for 3 days to identify differentially regulated genes (DEG). Bronchial brushings obtained from COPD patients with similar age and disease status treated with either placebo (4 subjects) or 2000mg/day quercetin (7 subjects) for 6 months were used to confirm the effects of quercetin on gene expression. Compared to placebo-, quercetin-treated COPD BC showed significantly increased transepithelial resistance, more ciliated cells, fewer goblet cells, and lower IL-8. Quercetin upregulated genes associated with tissue and epithelial development and differentiation in COPD BC. COPD patients treated with quercetin, but not placebo showed increased expression of two developmental genes HOXB2 and ELF3, which were also increased in quercetin-treated COPD BC with FDR < 0.001. Active smokers showed increased mRNA expression of TGF-β (0.067) and IL-8 (22.0), which was reduced by 3.6 and 4.14 fold respectively after quercetin treatment. These results indicate that quercetin may improve airway epithelial regeneration by increasing the expression of genes involved in epithelial development/differentiation in COPD. This study was registered at ClinicalTrials.gov on 6-18-2019. The study number is NCT03989271.

An inulin-type fructan CP-A from Codonopsis pilosula attenuates experimental colitis in mice by promoting autophagy-mediated inactivation of NLRP3 inflammasome

Inulin-type fructan CP-A, a predominant polysaccharide in Codonopsis pilosula, demonstrates regulatory effects on immune activity and anti-inflammation. The efficacy of CP-A in treating ulcerative colitis (UC) is, however, not well-established. This study employed an in vitro lipopolysaccharide (LPS)-induced colonic epithelial cell model (NCM460) and an in vivo dextran sulfate sodium (DSS)-induced colitis mouse model to explore CP-A's protective effects against experimental colitis and its underlying mechanisms. We monitored the clinical symptoms in mice using various parameters: body weight, disease activity index (DAI), colon length, spleen weight, and histopathological scores. Additionally, molecular markers were assessed through enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF), immunohistochemistry (IHC), and Western blotting assays. Results showed that CP-A significantly reduced reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), and interleukins (IL-6, IL-1β, IL-18) in LPS-induced cells while increasing IL-4 and IL-10 levels and enhancing the expression of Claudin-1, ZO-1, and occludin proteins in NCM460 cells. Correspondingly, in vivo findings revealed that CP-A administration markedly improved DAI, reduced colon shortening, and decreased the production of myeloperoxidase (MPO), malondialdehyde (MDA), ROS, IL-1β, IL-18, and NOD-like receptor protein 3 (NLRP3) inflammasome-associated genes/proteins in UC mice. CP-A treatment also elevated glutathione (GSH) and superoxide dismutase (SOD) levels, stimulated autophagy (LC3B, P62, Beclin-1, and ATG5), and reinforced Claudin-1 and ZO-1 expression, thereby aiding in intestinal epithelial barrier repair in colitis mice. Notably, the inhibition of autophagy via chloroquine (CQ) diminished CP-A's protective impact against colitis in vivo. These findings elucidate that CP-A's therapeutic effect on experimental colitis possibly involves mitigating intestinal inflammation through autophagy-mediated NLRP3 inflammasome inactivation. Consequently, inulin-type fructan CP-A emerges as a promising drug candidate for UC treatment.

Inflammation and epithelial repair predict mortality, hospital readmission, and growth recovery in complicated severe acute malnutrition.

Severe acute malnutrition (SAM) is the most high-risk form of undernutrition, particularly when children require hospitalization for complications. Complicated SAM is a multisystem disease with high inpatient and postdischarge mortality, especially in children with comorbidities such as HIV; however, the underlying pathogenesis of complicated SAM is poorly understood. Targeted multiplex biomarker analysis in children hospitalized with SAM (n = 264) was conducted on plasma samples, and inflammatory markers were assessed on stool samples taken at recruitment, discharge, and 12 to 24 and 48 weeks after discharge from three hospitals in Zimbabwe and Zambia. Compared with adequately nourished controls (n = 173), we found that at baseline, complicated SAM was characterized by systemic, endothelial, and intestinal inflammation, which was exacerbated by HIV infection. This persisted over 48 weeks despite nutritional recovery and was associated with children's outcomes. Baseline plasma concentrations of vascular endothelial growth factor, glucagon-like peptide-2, and intestinal fatty acid-binding protein were independently associated with lower mortality or hospital readmission over the following 48 weeks. Following principal components analysis of baseline biomarkers, higher scores of a component representing growth factors was associated with greater weight-for-height z score recovery and lower mortality or hospital readmission over the 48 weeks. Conversely, components representing higher gut and systemic inflammation were associated with higher mortality or hospital readmission. These findings highlight the interplay between inflammation, which damages tissues, and growth factors, which mediate endothelial and epithelial regeneration, and support further studies investigating interventions to reduce inflammation and promote epithelial repair as an approach to reducing mortality and improving nutritional recovery.

Zinc oxide nanoparticles exacerbate skin epithelial cell damage by upregulating pro-inflammatory cytokines and exosome secretion in M1 macrophages following UVB irradiation-induced skin injury

BackgroundZinc oxide nanoparticles (ZnONPs) are common materials used in skin-related cosmetics and sunscreen products due to their whitening and strong UV light absorption properties. Although the protective effects of ZnONPs against UV light in intact skin have been well demonstrated, the effects of using ZnONPs on damaged or sunburned skin are still unclear. In this study, we aimed to reveal the detailed underlying mechanisms related to keratinocytes and macrophages exposed to UVB and ZnONPs.ResultsWe demonstrated that ZnONPs exacerbated mouse skin damage after UVB exposure, followed by increased transepidermal water loss (TEWL) levels, cell death and epithelial thickness. In addition, ZnONPs could penetrate through the damaged epithelium, gain access to the dermis cells, and lead to severe inflammation by activation of M1 macrophage. Mechanistic studies indicated that co-exposure of keratinocytes to UVB and ZnONPs lysosomal impairment and autophagy dysfunction, which increased cell exosome release. However, these exosomes could be taken up by macrophages, which accelerated M1 macrophage polarization. Furthermore, ZnONPs also induced a lasting inflammatory response in M1 macrophages and affected epithelial cell repair by regulating the autophagy-mediated NLRP3 inflammasome and macrophage exosome secretion.ConclusionsOur findings propose a new concept for ZnONP-induced skin toxicity mechanisms and the safety issue of ZnONPs application on vulnerable skin. The process involved an interplay of lysosomal impairment, autophagy-mediated NLRP3 inflammasome and macrophage exosome secretion. The current finding is valuable for evaluating the effects of ZnONPs for cosmetics applications.

SOX9 switch links regeneration to fibrosis at the single-cell level in mammalian kidneys.

The steps governing healing with or without fibrosis within the same microenvironment are unclear. After acute kidney injury (AKI), injured proximal tubular epithelial cells activate SOX9 for self-restoration. Using a multimodal approach for a head-to-head comparison of injury-induced SOX9 lineages, we identified a dynamic SOX9 switch in repairing epithelia. Lineages that regenerated epithelia silenced SOX9 and healed without fibrosis (SOX9on-off). By contrast, lineages with unrestored apicobasal polarity maintained SOX9 activity in sustained efforts to regenerate, which were identified as a SOX9on-on Cadherin6pos cell state. These reprogrammed cells generated substantial single-cell WNT activity to provoke a fibroproliferative response in adjacent fibroblasts, driving AKI to chronic kidney disease. Transplanted human kidneys displayed similar SOX9/CDH6/WNT2B responses. Thus, we have uncovered a sensor of epithelial repair status, the activity of which determines regeneration with or without fibrosis.

Absence of the Klotho Function Causes Cornea Degeneration with Specific Features Resembling Fuchs Endothelial Corneal Dystrophy and Bullous Keratopathy.

The Klotho loss-of-function mutation is known to cause accelerated senescence in many organs, but its effects on the cornea have not been published. The present study aims to investigate the effects of the Klotho null mutation on cornea degeneration and to characterize the pathological features. Mouse corneas of Klotho homozygous, heterozygous, and wild-type mice at 8 weeks of age for both genders were subject to pathological and immunohistological examinations. The results show an irregular topography on the corneal surface with a Klotho null mutation. Histological examinations revealed a reduced corneal epithelial cell density, endothelial cell-shedding, and decreased cornea stromal layer thickness in the absence of the Klotho function. Furthermore, guttae formation and the desquamation of wing cells were significantly increased, which was comparable to the characteristics of Fuchs endothelial corneal dystrophy and bullous keratopathy. The mechanism analysis showed multi-fold abnormalities, including oxidative stress-induced cornea epithelium apoptosis and inflammation, extracellular matrix remodeling in the stroma, and a disruption of epithelial repair, presumably through the epithelial-mesenchymal transition. In conclusion, cornea degeneration was observed in the Klotho loss-of-function mutant mice. These pathological features support the use of Klotho mutant mice for investigating age-related cornea anomalies, including Fuchs endothelial corneal dystrophy, bullous keratopathy, and dry eye diseases.

Function of KvLQT1 potassium channels in a mouse model of bleomycin-induced acute lung injury.

Acute respiratory distress syndrome (ARDS) is characterized by an exacerbated inflammatory response, severe damage to the alveolar-capillary barrier and a secondary infiltration of protein-rich fluid into the airspaces, ultimately leading to respiratory failure. Resolution of ARDS depends on the ability of the alveolar epithelium to reabsorb lung fluid through active transepithelial ion transport, to control the inflammatory response, and to restore a cohesive and functional epithelium through effective repair processes. Interestingly, several lines of evidence have demonstrated the important role of potassium (K+) channels in the regulation of epithelial repair processes. Furthermore, these channels have previously been shown to be involved in sodium/fluid absorption across alveolar epithelial cells, and we have recently demonstrated the contribution of KvLQT1 channels to the resolution of thiourea-induced pulmonary edema in vivo. The aim of our study was to investigate the role of the KCNQ1 pore-forming subunit of KvLQT1 channels in the outcome of ARDS parameters in a model of acute lung injury (ALI). We used a molecular approach with KvLQT1-KO mice challenged with bleomycin, a well-established ALI model that mimics the key features of the exudative phase of ARDS on day 7. Our data showed that KvLQT1 deletion exacerbated the negative outcome of bleomycin on lung function (resistance, elastance and compliance). An alteration in the profile of infiltrating immune cells was also observed in KvLQT1-KO mice while histological analysis showed less interstitial and/or alveolar inflammatory response induced by bleomycin in KvLQT1-KO mice. Finally, a reduced repair rate of KvLQT1-KO alveolar cells after injury was observed. This work highlights the complex contribution of KvLQT1 in the development and resolution of ARDS parameters in a model of ALI.

Liquid plasma promotes angiogenesis through upregulation of endothelial nitric oxide synthase-induced extracellular matrix metabolism: potential applications of liquid plasma for vascular injuries

BackgroundApplications of nonthermal plasma have expanded beyond the biomedical field to include antibacterial, anti-inflammatory, wound healing, and tissue regeneration. Plasma enhances epithelial cell repair; however, the potential damage to deep tissues and vascular structures remains under investigation.ResultThis study assessed whether liquid plasma (LP) increased nitric oxide (NO) production in human umbilical vein endothelial cells by modulating endothelial NO synthase (eNOS) phosphorylation and potential signaling pathways. First, we developed a liquid plasma product and confirmed the angiogenic effect of LP using the Matrigel plug assay. We found that the NO content increased in plasma-treated water. NO in plasma-treated water promoted cell migration and angiogenesis in scratch and tube formation assays via vascular endothelial growth factor mRNA expression. In addition to endothelial cell proliferation and migration, LP influenced extracellular matrix metabolism and matrix metalloproteinase activity. These effects were abolished by treatment with NG-L-monomethyl arginine, a specific inhibitor of NO synthase. Furthermore, we investigated the signaling pathways mediating the phosphorylation and activation of eNOS in LP-treated cells and the role of LKB1-adenosine monophosphate-activated protein kinase in signaling. Downregulation of adenosine monophosphate-activated protein kinase by siRNA partially inhibited LP-induced eNOS phosphorylation, angiogenesis, and migration.ConclusionThe present study suggests that LP treatment may be a novel strategy for promoting angiogenesis in vascular damage.A1BBJsagnHJAo-Bf8DXnP6Video Graphical

Dual-Atom Nanozyme Eye Drops Attenuate Inflammation and Break the Vicious Cycle in Dry Eye Disease

HighlightsA dual-atom nanozyme (DAN) was successfully prepared based on Fe and Mn bimetallic single-atom embedded in N-doped carbon material and modified with hydrophilic polymer.The DAN possess excellent enzyme catalytic activity and attenuate dramatically inflammation by inhibiting the reactive oxygen species (ROS)/NLRP3 signal axis.The DAN break the vicious cycle in dry eye disease and is a potential strategy for treating dry eye disease.

Metformin alleviates benzo[a]pyrene-induced alveolar injury by inhibiting necroptosis and protecting AT2 cells

Exposure to benzo[a]pyrene (B[a]P) has been linked to lung injury and carcinogenesis. Airway epithelial cells express the B[a]P receptor AHR, so B[a]P is considered to mainly target airway epithelial cells, whereas its potential impact on alveolar cells remains inadequately explored. Metformin, a first-line drug for diabetes, has been shown to exert anti-inflammatory and tissue repair-promoting effects under various injurious conditions. Here, we explored the effect of chronic B[a]P exposure on alveolar cells and the impact of metformin on B[a]P-induced lung injury by examining the various parameters including lung histopathology, inflammation, fibrosis, and related signal pathway activation. MLKL knockout (Mlkl-/-) and AT2-lineage tracing mice (SftpcCre-ERT2;LSL-tdTomatoflox+/-) were used to delineate the role of necroptosis in B[a]P-induced alveolar epithelial injury and repair. Mice receiving weekly administration of B[a]P for 6 weeks developed a significant alveolar damaging phenotype associated with pulmonary inflammation, fibrosis, and activation of the necroptotic cell death pathway. These effects were significantly relieved in MLKL null mice. Furthermore, metformin treatment, which were found to promote AMPK phosphorylation and inhibit RIPK3, as well as MLKL phosphorylation, also significantly alleviated B[a]P-induced necroptosis and lung injury phenotype. However, the protective efficacy of metformin was rendered much less effective in Mlkl null mice or by blocking the necroptotic pathway with RIPK3 inhibitor. Our findings unravel a potential protective efficacy of metformin in mitigating the detrimental effects of B[a]P exposure on lung health by inhibiting necroptosis and protecting AT2 cells.

A61 ELUCIDATING INTESTINAL EPITHELIAL INFLAMMASOME MECHANISMS THAT REPAIR THE MUCOSAL BARRIER DURING ENTERIC INFECTION

Abstract Background To protect the gastrointestinal tract from infection, intestinal epithelial cells (IECs) lining the mucosa possess cytosolic complexes called inflammasomes which detect pathogenic insult and rapidly initiate inflammation to stop infectious spread. Active inflammasomes mediate crucial effector functions such as epithelial cell extrusion, pyroptotic cell death, and the release of interleukin (IL)-18 and prostaglandin E2 (PGE2). Recent literature has shown that IL-18 and PGE2 mediate intestinal repair following epithelial damage from DSS-induced colitis. Exogenous administration of IL-18 in inflammasome deficient mice improves survival and reduces pathology. PGE2 activates intestinal cellular reprogramming to drive intestinal wound healing. Whether inflammasome derived IL-18 and PGE2 mediate epithelial repair during enteric infection is unclear. Aims Using murine small intestinal organoids (mSIOs), we will investigate how IL-18 and PGE2 production by intestinal inflammasomes directly impacts IECs at both transcriptional and physiological levels. We hypothesize that inflammasome derived IL-18 and PGE2 will act on the intestinal epithelium resulting in transcriptional reprogramming that promotes tissue regeneration and barrier reinforcement. Methods WT and Nlrc4-/- mSIOs were stimulated with flagellin or FlaTox to activate the NAIP-NLRC4 inflammasome. The transcriptomic landscape was assessed with bulk-RNA sequencing and inflammasome activation was assessed by Western blot. IL-18 and PGE2 dependent gene targets were identified using Il-18-/- and indomethacin treated mSIOs by RT-qPCR. Results Bulk-RNA sequencing revealed an inflammasome activation dependent transcriptional signature in murine IECs upon NAIP-NLRC4 inflammasome activation. GO term analysis showed these genes to be involved in actin cytoskeleton rearrangement, epidermal growth factor signaling, and regulation of cell-cell adhesion. The expression of PGE2 gene targets were significantly reduced by indomethacin treatment while IL-18 gene targets remained unaffected in Il-18-/- IECs. FlaTox induced the same transcriptional changes but substantially amplified their expression. FlaTox but not flagellin led to a significant drop in Lgr5 (stem cells) and Lyz1 (Paneth cell) expression and an increase in Ly6a, a marker of fetal-like stem cells. Conclusions This data demonstrates that inflammasome signaling causes transcriptional reprogramming of IECs that activates pathways involved in maintaining epithelial barrier integrity. While IL-18 had no effects on gene expression at 4 hours, PGE2 paracrine signaling contributed to the expression of wound healing genes. Stronger inflammasome mediated damage with FlaTox amplified the repair response at the transcript level and we observed transient reprogramming of IECs into a fetal-like state suggesting active tissue regeneration. Funding Agencies CCC, CIHREPIC: Emerging &amp; Pandemic Infections Consortium

Injectable and Thermosensitive Hydrogel with Platelet-Rich Plasma for Enhanced Biotherapy of Skin Wound Healing.

The rapid and effective healing of skin wounds resulted from severe injuries and full-layer skin defects remains a pressing clinical challenge in contemporary medical practice. The reduction of wound infection and rapid healing is helpful to rebuild and repair skin tissue. Here, a thermosensitive chitosan-based wound dressing hydrogel incorporating β-glycerophosphate (GP), hydroxy propyl cellulose (HPC), graphene oxide (GO), and platelet-rich plasma (PRP) is developed, which exhibits the dual functions of antibacterial properties and repair promotion. GP and HPC enhance the mechanical properties through forming hydrogen bonding connection, while GO produces local heat under near-infrared light, leading to improved blood circulation and skin recovery. Notably, antibacterial properties against Pseudomonas aeruginosa, and control-release of growth factors from PRP are also achieved based on the system. In vitro experiments reveal its biocompatibility, and ability to promote cell proliferation and migration. Animal experiments demonstrate that the epithelial repair and collagen deposition can be promoted during skin wound healing in Sprague Dawley rats. Moreover, a reduction in wound inflammation levels and the improvement of wound microenvironment are observed, collectively fostering effective wound healing. Therefore, the composite hydrogel system incorporated with GO and PRP can be a promising dressing for the treatment of skin wounds.

Mechanisms driving fasting-induced protection from genotoxic injury in the small intestine.

Genotoxic agents such as doxorubicin (DXR) can cause damage to the intestines that can be ameliorated by fasting. How fasting is protective and the optimal timing of fasting and refeeding remain unclear. Here, our analysis of fasting/refeeding-induced global intestinal transcriptional changes revealed metabolic shifts and implicated the cellular energetic hub mechanistic target of rapamycin complex 1 (mTORC1) in protecting from DXR-induced DNA damage. Our analysis of specific transcripts and proteins in intestinal tissue and tissue extracts showed that fasting followed by refeeding at the time of DXR administration reduced damage and caused a spike in mTORC1 activity. However, continued fasting after DXR prevented the mTORC1 spike and damage reduction. Surprisingly, the mTORC1 inhibitor, rapamycin, did not block fasting/refeeding-induced reduction in DNA damage, suggesting that increased mTORC1 is dispensable for protection against the initial DNA damage response. In Ddit4-/- mice [DDIT4 (DNA-damage-inducible transcript 4) functions to regulate mTORC1 activity], fasting reduced DNA damage and increased intestinal crypt viability vs. ad libitum-fed Ddit4-/- mice. Fasted/refed Ddit4-/- mice maintained body weight, with increased crypt proliferation by 5 days post-DXR, whereas ad libitum-fed Ddit4-/- mice continued to lose weight and displayed limited crypt proliferation. Genes encoding epithelial stem cell and DNA repair proteins were elevated in DXR-injured, fasted vs. ad libitum Ddit4-/- intestines. Thus, fasting strongly reduced intestinal damage when normal dynamic regulation of mTORC1 was lost. Overall, the results confirm that fasting protects the intestines against DXR and suggests that fasting works by pleiotropic - including both mTORC1-dependent and independent - mechanisms across the temporally dynamic injury response.NEW & NOTEWORTHY New findings are 1) DNA damage reduction following a 24-h fast depends on the timing of postfast refeeding in relation to chemotherapy initiation; 2) fasting/refeeding-induced upregulation of mTORC1 activity is not required for early (6 h) protection against DXR-induced DNA damage; and 3) fasting increases expression of intestinal stem cell and DNA damage repair genes, even when mTORC1 is dysregulated, highlighting fasting's crucial role in regulating mTORC1-dependent and independent mechanisms in the dynamic recovery process.

WSB1, a Hypoxia-Inducible E3 Ligase, Promotes Myofibroblast Accumulation and Attenuates Alveolar Epithelial Regeneration in Mouse Lung Fibrosis

Idiopathic pulmonary fibrosis is a progressive interstitial lung disease for which there is no curative therapy available. Repetitive alveolar epithelial injury repair, myofibroblast accumulation, and excessive collagen deposition are key pathologic features of idiopathic pulmonary fibrosis, eventually leading to cellular hypoxia and respiratory failure. The precise mechanism driving this complex maladaptive process remains inadequately understood. WD repeat and suppressor of cytokine signaling box containing 1 (WSB1) is an E3 ubiquitin ligase, the expression of which is associated strongly with hypoxia, and forms a positive feedback loop with hypoxia-inducible factor 1α (HIF-1α) under anoxic condition. This study explored the expression, cellular distribution, and function of WSB1 in bleomycin (BLM)-induced mouse lung injury and fibrosis. WSB1 expression was highly induced by BLM injury and correlated with the progression of lung fibrosis. Significantly, conditional deletion of Wsb1 in adult mice ameliorated BLM-induced pulmonary fibrosis. Phenotypically, Wsb1-deficient mice showed reduced lipofibroblast to myofibroblast transition, but enhanced alveolar type 2 proliferation and differentiation into alveolar type 1 after BLM injury. Proteomic analysis of mouse lung tissues identified caveolin 2 as a potential downstream target of WSB1, contributing to BLM-induced epithelial injury repair and fibrosis. These findings unravel a vital role for WSB1 induction in lung injury repair, thus highlighting it as a potential therapeutic target for pulmonary fibrosis.

Dopamine Receptor 1 Treatment Promotes Epithelial Repair of Corneal Injury by Inhibiting NOD-Like Receptor Protein 3-Associated Inflammation.

To elucidate the influence of dopamine receptor 1 (DRD1) on the proliferation of mouse corneal epithelial cells (MCECs) under inflammatory conditions. In vitro, immortalized MCECs (iMCECs) were treated with IL-1β, with and without pcDNA3.1_DRD1. Primary MCECs (pMCECs) were exposed to IL-1β, with and without DRD1 agonist (A68930). Cell proliferation was quantified using the Cell Counting Kit-8 (CCK-8) assay and immunofluorescence staining for Ki-67 and p63. Expression levels of NOD-like receptor protein 3 (NLRP3), IL-1β, and IL-6 were assessed. To establish a corneal injury model in mice, a 2-mm superficial keratectomy was performed. Either 0.1% A68930 or PBS was topically administered three times daily to the injured eyes for up to 5days post-injury. Immunofluorescence analysis was employed to evaluate the expression of Ki-67, p63, and CD45 in mouse corneas. Western blotting and real-time quantitative PCR were utilized for quantitative analysis of DRD1, NLRP3, IL-1β, and IL-6 in mouse corneas. Corneal epithelial regeneration was monitored through fluorescein sodium staining for a duration of up to 5days following the injury. Overexpression of DRD1 and A68930 promoted MCEC proliferation and suppressed the expression of NLRP3, IL-1β, and IL-6 in vitro. Topical application of the 0.1% A68930 following mechanical corneal injury in mice led to increased Ki-67 and p63 expression compared to PBS treatment. Furthermore, topical administration of the 0.1% A68930 reduced the expression of CD45, NLRP3, IL-1β, and IL-6. Analysis with fluorescein sodium indicated accelerated corneal epithelial regeneration in the 0.1% A68930 treatment group. DRD1 treatment counteracts NLRP3-associated inflammation and facilitates epithelial repair of corneal injury.

P2Y1 RECEPTOR SIGNALING INFLUENCES EPITHELIAL RESPONSE TO INTESTINAL INFLAMMATION

Abstract Purines are among the most influential and ancient biochemical molecules in evolutionary history. In recent years, several studies have identified that nucleotides are released from cells during infection and tissue injury and serve as paracrine signaling molecules that help coordinate host defense and repair responses. These important extracellular signaling molecules activate purinergic receptors, of which there are 19 different purinergic receptor subtypes, which are classified into P1 and P2 receptors. Several studies have revealed crucial roles for P2 purinergic receptors during inflammatory and infectious diseases; however, these studies have largely examined purinergic signaling in immune cells and the enteric nervous system. Yet, while epithelial cells serve as the first barrier against infection and inflammation, the role of purinergic signaling within the gastrointestinal tract remains largely unknown. Our group recently discovered a novel and prominent role for P2Y1 in mediating host responses during rotavirus infection. To extend these studies, we sought to elucidate the P2 purinergic receptor repertoire expressed within gut epithelium and assess their functional role during insult. Using the Human Protein Atlas, we queried single-cell RNA sequencing data for P2 purinergic receptor expression in small intestine, colon, and rectum. In silico analysis revealed high expression of P2Y1, P2Y2, P2Y11 and P2X4 among the purinergic receptors throughout the gastrointestinal tract. Interestingly, P2Y1 showed highest expression in intestinal enterocytes and goblet cells. Further, we compared P2 receptor expression between commonly used, cancer-derived intestinal cell lines (T84, HT29 and Caco2) and crypt-derived human intestinal organoids (HIOs). Both HIOs and T84 and Caco2 cell lines had similar mRNA expressions of P2Y1, P2Y2, P2Y11 and P2X4. However, live calcium imaging studies showed robust responses to P2Y receptor-specific agonists in HIOs, particularly P2Y1 and P2Y2 agonists, but the cancer-derived cell lines failed to respond to these agonists, suggesting these cell lines have defects in P2Y signaling. Functionally, blocking P2Y1, but not P2Y2, significantly delayed wound healing in epithelial scratch assays, indicating a potential role in epithelial injury repair. To delineate the role of epithelial P2Y1 in vivo, we generated VillinCre P2Y1flex/flox mice. At baseline, the knockout animals did not exhibit any overt intestinal defects; however, in the DSS-induced colitis model, the epithelial P2Y1 KO mice had significantly greater weight loss and epithelial damage than their wildtype littermates. These findings confirm that the gastrointestinal tract express P2Y1, P2Y2, P2Y11 and P2X4 purinergic receptors and P2Y1 signaling plays important functional roles in both gastrointestinal epithelial homeostasis and repair of injury.

NANOPARTICLE ENCAPSULATION OF THE SPECIALIZED PRO-RESOLVING MEDIATOR MARESIN-2: A NOVEL APPROACH FOR PROMOTING MUCOSAL REPAIR IN THE INTESTINE

Abstract Epithelial cells form protective barriers at mucosal surfaces and play a critical role in maintaining tissue homeostasis. Active inflammation in inflammatory bowel disease (IBD) is associated with epithelial barrier disruption and mucosal erosions/wounds that contribute to the inflammatory response and disease symptoms. Therefore, efficient repair of epithelial erosions/wounds is crucial for restoring intestinal barrier function and resolving mucosal inflammation. In response to injury, epithelial cells proliferate and migrate to cover denuded surfaces and restore critical barrier properties. Resident and recruited immune cells at sites of injury release factors that play an important role in orchestrating mucosal repair. Bioactive lipid mediators released by leukocytes, including leukotrienes and specialized pro-resolving mediators (SPMs), regulate epithelial signaling pathways to influence mucosal repair processes. Liquid chromatography-mass spectrometry (LCMS) analyses of healing colonic mucosal wounds performed to detect SPMs identified high levels of MaR2 in healing colonic wounds. In vitro studies using primary colonic epithelial cells revealed MaR2-driven increases in epithelial wound repair that were potentiated by the pro-inflammatory cytokines TNFα and IFNγ. While MaR2 increased epithelial migration by activating cell matrix adhesion proteins, including Src-paxillin and focal adhesion kinase, we did not observe any influence of MaR2 on the activation of cyclin D1/D2 or on epithelial proliferation. Importantly, in vivo studies demonstrated enhanced colonic mucosal wound repair and recovery from dextran sulfate-induced colitis in mice administered MaR2. Given the thermolabile nature of MaR2 (and associated ultracold storage requirements), thermostable polylactic acid (PLA) nanoparticles (NP) containing MaR2 (PLA MaR2) were generated. PLA MaR2 nanoparticles retained bioactivity following extended storage at room temperature and exhibited potent effects on in vivo colonic biopsy induced wound repair. Taken together these data reveal that thermostable MaR2 containing nanoparticles represent a promising new therapeutic approach for regenerating epithelial barrier function and restoring mucosal homeostasis in individuals with IBD.

BIOACTIVE INTERLEUKIN-1 DETECTED IN IBD PATIENT INTESTINAL BIOPSIES IS A HALLMARK OF ULCERS AND CORRELATES WITH TRANSCRIPTOMIC ASSESSMENTS, INCLUDING AN ULCER-ASSOCIATED GENE MODULE

Abstract BACKGROUND Interleukin (IL)-1 and its post-translationally modified and released forms, IL-1α and IL-1β, have been identified as key molecules to maintain mucosal homeostasis and to drive inflammatory bowel disease (IBD). Deep cellular phenotypic, transcriptional, and in vitro data highlight the importance of IL-1-associated cytokine networks and IL-1 producing cells in severe and anti-TNF non-responsive IBD. Nevertheless, little is known about the presence of bioactive IL-1 proteins within the cell-free gastrointestinal (GI) mucosal environment and associated function in severe IBD and ulceration. METHODS We established a highly sensitive functional assay (~500-fold greater than ELISA) to assess bioactive extracellular IL-1 protein from previously cryopreserved GI biopsies. We performed an ex vivo study in a cohort of Crohn’s disease (CD, n=23), ulcerative colitis (UC, n=18) and non-IBD (n=17) patients, with biopsies from paired inflamed and uninflamed intestinal regions per patient (primarily colon but also ileal). We assessed differential IL-1 bioactivity, including IL-1α vs IL-1β contributions, and performed RNA-seq of the matched cellular compartments of the samples. An ulcer-associated gene signature derived from RNA-seq analysis was evaluated in a single-cell RNA-seq cohort (n=42) of very early onset IBD including monogenic disorders. RESULTS The extent of bioactive intestinal mucosal IL-1α and IL-1β corresponded with disease and ulcer severity in both CD and UC. The most extreme signals were seen in select CD patients with deep ulceration. Bioactive IL-1α was the predominant contributor to total IL-1 signal in most patients although several with ulcers displayed an IL-1β-predominant signal. Matched RNA-seq analysis demonstrated enhanced IL-1 transcripts, correlating with IL-1 bioactivity, and a weighted gene co-expression network analysis (WGCNA) revealed a compelling ulcer-specific module enriched in IL-1 signaling genes and wound repair. We validated our findings in several published (e.g., RISK cohort of ileal CD) as well as unpublished independent adult and pediatric datasets from our group. Deconvolution of the ulcer-specific gene module and projection onto an unpublished single-cell RNA-seq cohort of very early onset IBD patients (including IBD-causative mutations such as IL-10R deficiency characterized by deep ulceration) implicated specific stromal and myeloid populations in ulcer biology. CONCLUSION Mucosal ulceration in IBD is associated with bioactive IL-1α and IL-1β proteins, and transcriptomic evaluation of the same correlates with bioactive signal. An ulcer-associated gene module, validated in other datasets, sheds light on IL-1 biology in intestinal epithelial repair. Results strongly suggest the IL-1 signaling pathway being an attractive and precision-based therapeutic target in subsets of IBD patients.