Intestinal Epithelial Healing Research Articles

Single Cell Analysis of Human Colonoids Exposed to Uranium-Bearing Dust.

Uranium exposure remains an important environmental legacy and physiological health concern, with hundreds of abandoned uranium mines located in the Southwestern United States largely impacting underserved indigenous communities. The negative effects of heavy metals on barrier permeability and inhibition of intestinal epithelial healing have been described; however, transcriptomic changes within the intestinal epithelial cells and impacts on lineage differentiation are largely unknown. Herein, we sought to determine the molecular and cellular changes that occur in the colon in response to uranium bearing dust (UBD) exposure. Human colonoids from three biologically distinct donors were acutely exposed to UBD then digested for single cell RNA sequencing to define the molecular changes that occur to specific identities of colonic epithelial cells. Validation in colonoids was assessed using morphological and imaging techniques. Human colonoids acutely exposed to UBD exhibited disrupted proliferation and hyperplastic differentiation of the secretory lineage cell, enteroendocrine cells (EEC). Single-cell RNA sequencing also showed more EEC subtypes present in UBD-exposed colonoids. These findings highlight the significance of crypt-based proliferative cells and secretory cell differentiation using human colonoids to model major colonic responses to uranium-bearing particulate dust exposure. https://doi.org/10.1289/EHP13855.

1,25-Dihydroxy Vitamin D Enhances the Differentiation of Intestinal Stem Cells Ex Vivo and In Vivo

Abstract Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the intestines. Current therapies aim at suppressing inflammation to prevent further damage. However, healing already damaged intestinal epithelia is still an unmet medical need. In this regard, epithelial healing is achieved via intestinal stem cells (ISCs) that are located in the intestinal crypts. Considering the literature reports that the active vitamin D (1,25[OH]2D) is essential for the maintenance of Lgr5+ ISCs, the active proliferating ISCs, we recently proposed to deliver locally high 1,25(OH)2D concentrations to the proximity of intestinal crypts to promote intestinal stem cell functions. This local delivery was devised to avoid potential side effects associated with systemically high 1,25(OH)2D concentrations. Using 5-Bromo-20-deoxyuridine (BrdU) to label the actively proliferating ISCs, our previous data indicated that the locally delivered 1,25(OH)2D enhanced the migration and differentiation of Lgr5+ ISCs. However, although sparse in the crypts, other proliferating cells may be labeled with BrdU. This current study used a unique mouse strain to precisely trace Lgr5+ ISCs and consolidated our previous finding that the locally delivered 1,25(OH)2D augmented ISC differentiation. Besides, we generated pure Lgr5+ ISC lines that could be induced to differentiate on demand. Our data showed that, in vitro, 1,25(OH)2D enhanced the differentiation of Lgr5+ ISCs. Moreover, by knocking down the vitamin D receptor (VDR), we demonstrated that the 1,25(OH)2D functions in ISCs are mediated through VDR. Accordingly, our findings suggest that locally high 1,25(OH)2D concentrations are a promising strategy for intestinal epithelial healing. This study was partially supported by the National Institute of Health (NIH, USA) grant (1R21AI142170); the US Army Medical Research and Materiel Command grant (W81XWH-15-0240); and the GI Foundation of Loma Linda University Innovation grant (RIG-GI 681207-2967).

P074 SZN-1326 promotes colonic mucosal healing in an acute injury model of IBD by first accelerating epithelial regeneration and secondarily reducing inflammation

Abstract Background Wnt signaling plays critical roles in regulating intestinal stem cell maintenance and differentiation. The inflammatory bowel disease, ulcerative colitis (UC), is characterized by epithelial lesions, and there is a need for therapies that promote mucosal healing in addition to limiting inflammation. The Surrozen proprietary Wnt mimetic, SZN-1326, is a FZD5,8 and LRP6-specific, full-length, effectorless, bi-specific IgG1 molecule that is well-suited for impacting the intestinal epithelium where FZD5 is highly expressed. Previously, we have shown that in an acute dextran sodium sulfate (DSS) epithelial injury model, where 4% DSS was provided in the drinking water for seven days followed by 1% DSS until termination at day 10, a single dose of SZN-1326 at 1 mg/kg or higher on Day 4 robustly induced intestinal epithelial healing and reduced inflammation, and we wanted to understand more thoroughly the mechanism underlying this activity. Methods Here, we report on additional studies in the same DSS model including scRNA-seq and tissue analysis timepoints at days 3, 4, 5, 6, 7, and 10 (6-days post treatment). Results DSS impacted all tissue layers by day 3. The direct effect of SZN-1326 on the epithelium occurred as early as 24-hours after treatment (day 5 of the experiment). SZN-1326 induced and/or increased Wnt target and cell cycle gene expression in the epithelium leading to progenitor cell expansion, and following this wave of enhanced progenitor proliferation, the cells transited toward colonocyte differentiation more quickly than the progenitor cells in control treated samples. Even in the presence of 1% DSS, relative to the anti-GFP treatment samples, the absorptive cell lineage in the SZN-1326-treated epithelium was more similar at a transcriptomic level to the uninjured condition. In addition, when comparing SZN-1326 treatment to anti-GFP at 24-hours, there were no differentially expressed genes in either the stroma or immune cells. However, analysis of the later timepoints revealed a robust reduction in cytokine/inflammatory signaling in the stromal and immune cells and a drastic reduction of infiltrating monocytes and neutrophils, which we validated in the tissue by immunohistochemistry. We detected this reduction by 72-hours after treatment, and it was more pronounced by day 10, 6-days after treatment, implicating a secondary effect. Conclusion In this acute damage context, SZN-1326 promoted mucosal healing by transiently inducing epithelial progenitor cell expansion and accelerating epithelial repair. Secondary to its initial impact on the epithelium, SZN-1326 reduced stromal and immune cytokine signaling and immune cell infiltration.

Pharmacological HIF-1 stabilization promotes intestinal epithelial healing through regulation of α-integrin expression and function.

Intestinal epithelia are critical for maintaining gastrointestinal homeostasis. Epithelial barrier injury, causing inflammation and vascular damage, results in inflammatory hypoxia, and thus, healing occurs in an oxygen-restricted environment. The transcription factor hypoxia-inducible factor (HIF)-1 regulates genes important for cell survival and repair, including the cell adhesion protein β1-integrin. Integrins function as αβ-dimers, and α-integrin-matrix binding is critical for cell migration. We hypothesized that HIF-1 stabilization accelerates epithelial migration through integrin-dependent pathways. We aimed to examine functional and posttranslational activity of α-integrins during HIF-1-mediated intestinal epithelial healing. Wound healing was assessed in T84 monolayers over 24 h with/without prolyl-hydroxylase inhibitor (PHDi) (GB-004), which stabilizes HIF-1. Gene and protein expression were measured by RT-PCR and immunoblot, and α-integrin localization was assessed by immunofluorescence. α-integrin function was assessed by antibody-mediated blockade, and integrin α6 regulation was determined by HIF-1α chromatin immunoprecipitation. Models of mucosal wounding and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis were used to examine integrin expression and localization in vivo. PHDi treatment accelerated wound closure and migration within 12 h, associated with increased integrin α2 and α6 protein, but not α3. Functional blockade of integrins α2 and α6 inhibited PHDi-mediated accelerated wound closure. HIF-1 bound directly to the integrin α6 promoter. PHDi treatment accelerated mucosal healing, which was associated with increased α6 immunohistochemical staining in wound-associated epithelium and wound-adjacent tissue. PHDi treatment increased α6 protein levels in colonocytes of TNBS mice and induced α6 staining in regenerating crypts and reepithelialized inflammatory lesions. Together, these data demonstrate a role for HIF-1 in regulating both integrin α2 and α6 responses during intestinal epithelial healing.NEW & NOTEWORTHY HIF-1 plays an important role in epithelial restitution, selectively inducing integrins α6 and α2 to promote migration and proliferation, respectively. HIF-stabilizing prolyl-hydroxylase inhibitors accelerate intestinal mucosal healing by inducing epithelial integrin expression.

Epithelial cell dysfunction in coeliac disease.

The intestinal epithelium limits host-luminal interactions and maintains gut homeostasis. Breakdown of the epithelial barrier and villous atrophy are hallmarks of coeliac disease. Besides the well characterized immune-mediated epithelial damage induced in coeliac mucosa, constitutional changes and early gluten direct effects disturb intestinal epithelial cells. The subsequent modifications in key epithelial signaling pathways leads to outnumbered immature epithelial cells that, in turn, facilitate epithelial dysfunction, promote crypt hyperplasia, and increase intestinal permeability. Consequently, underlying immune cells have a greater access to gluten, which boosts the proinflammatory immune response against gluten and positively feedback the epithelial damage loop. Gluten-free diet is an indispensable treatment for coeliac disease patients, but additional therapies are under development, including those that reinforce intestinal epithelial healing. In this chapter, we provide an overview of intestinal epithelial cell disturbances that develop during gluten intake in coeliac disease mucosa.

Physicochemical properties and potential beneficial effects of porphyran from Porphyra haitanensis on intestinal epithelial cells

This study examined the beneficial effects of porphyran from Porphyra haitanensis (PHP) on intestinal epithelial cells, in terms of cell proliferation and migration and elucidated the potential molecular mechanism of action of PHP. Purified PHP is a homogenous polysaccharide with a molecular weight of 2.01 × 105 Da, intrinsic viscosity [η] of 463.76 mL/g, and radius of gyration of 61.2 nm. When the intestinal epithelial wound healing activity of PHP was investigated in vitro using the IEC-6 cell line (intestinal epithelial cells-6), it was found that PHP could promote cell migration and proliferation. PHP enhanced the protein expression of cell division control protein 42, paxillin, and focal adhesion kinase, which suggest that PHP might modulate the expression of these proteins to improve intestinal epithelial healing. Thus, this study indicated that PHP could serve as a potential source of functional food constituents for intestinal epithelial protection and restoration.

Combined Restitutive Therapy for Treatment of Immunosuppressive Refractory Crohn Disease

Crohn’s Disease (CD) is believed to be the result of dys-regulated interactions between a genetically susceptible host and their commensal gut microbiota (1). CD affects over 600,000 Americans and treatment still faces many challenges (2). Pharmacotherapy centers on the use of anti-inflammatories and immunosuppressants (3). However, a sub-set of patients with CD are refractory to immunosuppressive therapy. Recent studies have implicated defects in autophagy and bacterial killing as a possible etiology leading to host-microbe dysregulation (4). This could explain the lack of response to immunosuppression seen in these CD patients. A recent study in adults with CD showed the potential for granulocyte-macrophage colony stimulating factor (GM-CSF) to enhance bacterial killing and restore intestinal homeostasis (5), but very little data exists in pediatric patients (6). Another study reports a role for low-dose naltrexone (LDN, a mu opioid antagonist) in helping restore the epithelial barrier in active CD (7). The mechanism of action of LDN is thought to be through activation of endogenous opioid signaling (8). Recent murine studies have shown that direct mu opioid receptor (MOR) activation can enhance intestinal epithelial healing and prevent epithelial apoptosis (9). Here, we report a combined intestinal-restitutive approach using GM-CSF and loperamide (a peripheral MOR agonist) to induce and maintain remission in a 17-year-old CD patient.

Hypoxia and Integrin-Mediated Epithelial Restitution during Mucosal Inflammation

Epithelial damage and loss of intestinal barrier function are hallmark pathologies of the mucosal inflammation associated with conditions such as inflammatory bowel disease. In order to resolve inflammation and restore intestinal integrity the mucosa must rapidly and effectively repair the epithelial barrier. Epithelial wound healing is a highly complex and co-ordinated process and the factors involved in initiating intestinal epithelial healing are poorly defined. In order for restitution to be successful there must be a balance between epithelial cell migration, proliferation, and differentiation within and adjacent to the inflamed area. Endogenous, compensatory epithelial signaling pathways are activated by the changes in oxygen tensions that accompany inflammation. These signaling pathways induce the activation of key transcription factors, governing anti-apoptotic, and proliferative processes resulting in epithelial cell survival, proliferation, and differentiation at the site of mucosal inflammation. In this review, we will discuss the primary processes involved in epithelial restitution with a focus on the role of hypoxia-inducible factor and epithelial integrins as mediators of epithelial repair following inflammatory injury at the mucosal surface.