systematic-scoring-analysis-for-intestinal-inflammation-murine

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

University of Michigan

The intestinal epithelium forms a highly dynamic and selective barrier that controls absorption of fluid and solutes while restricting pathogen access to underlying tissues. Barrier properties are achieved by intercellular junctions that include an apical tight junction (TJ) and subjacent adherens junctions and desmosomes. The TJ tetraspan claudin proteins form pores between epithelial cells to control paracellular fluid and ion movement. In addition to regulation of barrier function, claudin family members control epithelial homeostasis and are expressed in a spatiotemporal manner in the intestinal crypt-luminal axis. This delicate balance of physiologic differential claudin protein expression is altered during mucosal inflammation. Inflammatory mediators influence transcriptional regulation, as well as endocytic trafficking, targeting, and retention of claudins in the TJ. Increased expression of intestinal epithelial claudin-1, -2, and -18 with downregulation of claudin-3, -4, -5, -7, -8, and -12 has been observed in intestinal inflammatory disorders. Such changes in claudin proteins modify the epithelial barrier function in addition to influencing epithelial and mucosal homeostasis. An improved understanding of the regulatory mechanisms that control epithelial claudin proteins will provide strategies to strengthen the epithelial barrier function and restore mucosal homeostasis in inflammatory disorders.

Intestinal epithelial claudins: expression and regulation in homeostasis and inflammation.

In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by dynamic crosstalk between immune cells and the epithelium; however, the mechanisms involved remain incompletely understood. Here, we report that IL-10 was rapidly induced following intestinal mucosal injury and was required for optimal intestinal mucosal wound closure. Conditional deletion of IL-10 specifically in CD11c-expressing cells in vivo implicated macrophages as a critical innate immune contributor to IL-10-induced wound closure. Consistent with these findings, wound closure in T cell- and B cell-deficient Rag1-/- mice was unimpaired, demonstrating that adaptive immune cells are not absolutely required for this process. Further, following mucosal injury, macrophage-derived IL-10 resulted in epithelial cAMP response element-binding protein (CREB) activation and subsequent synthesis and secretion of the pro-repair WNT1-inducible signaling protein 1 (WISP-1). WISP-1 induced epithelial cell proliferation and wound closure by activating epithelial pro-proliferative pathways. These findings define the involvement of macrophages in regulating an IL-10/CREB/WISP-1 signaling axis, with broad implications in linking innate immune activation to mucosal wound repair.

Macrophage-derived IL-10 mediates mucosal repair by epithelial WISP-1 signaling.

In vitro differentiation of human intestinal organoids (HIOs) from pluripotent stem cells is an unparalleled system for creating complex, multicellular three-dimensional structures capable of giving rise to tissue analogous to native human tissue. Current methods for generating HIOs rely on growth in an undefined tumour-derived extracellular matrix (ECM), which severely limits the use of organoid technologies for regenerative and translational medicine. Here, we developed a fully defined, synthetic hydrogel based on a four-armed, maleimide-terminated poly(ethylene glycol) macromer that supports robust and highly reproducible in vitro growth and expansion of HIOs, such that three-dimensional structures are never embedded in tumour-derived ECM. We also demonstrate that the hydrogel serves as an injection vehicle that can be delivered into injured intestinal mucosa resulting in HIO engraftment and improved colonic wound repair. Together, these studies show proof-of-concept that HIOs may be used therapeutically to treat intestinal injury.

Synthetic hydrogels for human intestinal organoid generation and colonic wound repair.

Pathobiology of several chronic inflammatory disorders, including ulcerative colitis and Crohn's disease is related to intermittent, spontaneous injury/ulceration of mucosal surfaces. Disease morbidity has been associated with pathologic release of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). In this report, we show that TNFα promotes intestinal mucosal repair through upregulation of the GPCR platelet activating factor receptor (PAFR) in the intestinal epithelium. Platelet activating factor (PAF) was increased in healing mucosal wounds and its engagement with epithelial PAFR leads to activation of epidermal growth factor receptor, Src and Rac1 signaling to promote wound closure. Consistent with these findings, delayed colonic mucosal repair was observed after administration of a neutralizing TNFα antibody and in mice lacking PAFR. These findings suggest that in the injured mucosa, the pro-inflammatory milieu containing TNFα and PAF sets the stage for reparative events mediated by PAFR signaling.

TNFα promotes mucosal wound repair through enhanced platelet activating factor receptor signaling in the epithelium.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a tight barrier, separating luminal antigens and microbes from underlying tissue compartments. Mucosal damage results in a compromised epithelial barrier that can lead to excessive immune responses as observed in inflammatory bowel disease. Efficient wound repair is critical to reestablish the mucosal barrier and homeostasis. Intestinal epithelial cells (IEC) exclusively express the desmosomal cadherins, Desmoglein-2 and Desmocollin-2 (Dsc2) that contribute to mucosal homeostasis by strengthening intercellular adhesion between cells. Despite this important property, specific contributions of desmosomal cadherins to intestinal mucosal repair after injury remain poorly investigated in vivo. Here we show that mice with inducible conditional knockdown (KD) of Dsc2 in IEC (-Cre; 2 ) exhibited impaired mucosal repair after biopsy-induced colonic wounding and recovery from dextran sulfate sodium-induced colitis. In vitro analyses using human intestinal cell lines after KD of Dsc2 revealed delayed epithelial cell migration and repair after scratch-wound healing assay that was associated with reduced cell-matrix traction forces, decreased levels of integrin β1 and β4, and altered activity of the small GTPase Rap1. Taken together, these results demonstrate that epithelial Dsc2 is a key contributor to intestinal mucosal wound healing in vivo.

Desmocollin-2 promotes intestinal mucosal repair by controlling integrin-dependent cell adhesion and migration.

Food-triggered anaphylaxis can encompass a variety of systemic and intestinal symptoms. Murine-based and clinical studies have revealed a role for histamine and H1R and H2R-pathway in the systemic response; however, the molecular processes that regulate the gastrointestinal (GI) response are not as well defined. In the present study, by utilizing an IgE-mast cell (MC)-dependent experimental model of oral antigen-induced anaphylaxis, we define the intestinal epithelial response during a food-induced anaphylactic reaction. We show that oral allergen-challenge stimulates a rapid dysregulation of intestinal epithelial transcellular and paracellular transport that was associated with the development of secretory diarrhea. Allergen-challenge induced (1) a rapid intestinal epithelial Cftr-dependent Cl secretory response and (2) paracellular macromolecular leak that was associated with modification in epithelial intercellular junction proteins claudin-1, 2, 3 and 5, E-cadherin and desmosomal cadherins. OVA-induced Cftr-dependent Cl secretion and junctional protein degradation was rapid occurring and was sustained for 72 h following allergen-challenge. Blockade of both the proteolytic activity and Cl secretory response was required to alleviate intestinal symptoms of food-induced anaphylaxis. Collectively, these data suggest that the GI symptom of food-induced anaphylactic reaction, secretory diarrhea, is a consequence of CFTR-dependent Cl secretion and proteolytic activity.

Dysregulation of intestinal epithelial CFTR-dependent Cl ion transport and paracellular barrier function drives gastrointestinal symptoms of food-induced anaphylaxis in mice.

Article	Year
Intestinal epithelial claudins: expression and regulation in homeostasis and inflammation. Annals of the New York Academy of Sciences\| PubMed ID: 28493289
Macrophage-derived IL-10 mediates mucosal repair by epithelial WISP-1 signaling. The Journal of clinical investigation\| PubMed ID: 28783045
Synthetic hydrogels for human intestinal organoid generation and colonic wound repair. Nature cell biology\| PubMed ID: 29058719	2017
TNFα promotes mucosal wound repair through enhanced platelet activating factor receptor signaling in the epithelium. Mucosal immunology\| PubMed ID: 30971752
Desmocollin-2 promotes intestinal mucosal repair by controlling integrin-dependent cell adhesion and migration. Molecular biology of the cell\| PubMed ID: 31967937	2020
Dysregulation of intestinal epithelial CFTR-dependent Cl ion transport and paracellular barrier function drives gastrointestinal symptoms of food-induced anaphylaxis in mice. Mucosal immunology\| PubMed ID: 32576925	2021

Vicky Garcia-Hernandez

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