Miguel Quiros

Annexin A1-containing extracellular vesicles and polymeric nanoparticles promote epithelial wound repair.

Epithelial restitution is an essential process that is required to repair barrier function at mucosal surfaces following injury. Prolonged breaches in epithelial barrier function result in inflammation and further damage; therefore, a better understanding of the epithelial restitution process has potential for improving the development of therapeutics. In this work, we demonstrate that endogenous annexin A1 (ANXA1) is released as a component of extracellular vesicles (EVs) derived from intestinal epithelial cells, and these ANXA1-containing EVs activate wound repair circuits. Compared with healthy controls, patients with active inflammatory bowel disease had elevated levels of secreted ANXA1-containing EVs in sera, indicating that ANXA1-containing EVs are systemically distributed in response to the inflammatory process and could potentially serve as a biomarker of intestinal mucosal inflammation. Local intestinal delivery of an exogenous ANXA1 mimetic peptide (Ac2-26) encapsulated within targeted polymeric nanoparticles (Ac2-26 Col IV NPs) accelerated healing of murine colonic wounds after biopsy-induced injury. Moreover, one-time systemic administration of Ac2-26 Col IV NPs accelerated recovery following experimentally induced colitis. Together, our results suggest that local delivery of proresolving peptides encapsulated within nanoparticles may represent a potential therapeutic strategy for clinical situations characterized by chronic mucosal injury, such as is seen in patients with IBD.

The microenvironment of injured murine gut elicits a local pro-restitutive microbiota

The mammalian intestine houses a complex microbial community, which influences normal epithelial growth and development, and is integral to the repair of damaged intestinal mucosa1–3. Restitution of injured mucosa involves the recruitment of immune cells, epithelial migration and proliferation4,5. Although microenvironmental alterations have been described in wound healing6, a role for extrinsic influences, such as members of the microbiota, has not been reported. Here, we show that a distinct subpopulation of the normal mucosal-associated gut microbiota expands and preferentially colonizes sites of damaged murine mucosa in response to local environmental cues. Our results demonstrate that formyl peptide receptor 1 (FPR1) and neutrophilic NADPH oxidase (NOX2) are required for the rapid depletion of microenvironmental oxygen and compensatory responses, resulting in a dramatic enrichment of an anaerobic bacterial consortium. Furthermore, the dominant member of this wound-mucosa-associated microbiota, Akkermansia muciniphila (an anaerobic, mucinophilic gut symbiont7,8), stimulated proliferation and migration of enterocytes adjacent to the colonic wounds in a process involving FPR1 and intestinal epithelial-cell-specific NOX1-dependent redox signalling. These findings thus demonstrate how wound microenvironments induce the rapid emergence of ‘probiont’ species that contribute to enhanced repair of mucosal wounds. Such microorganisms could be exploited as potential therapeutics.

RhoGTPases, actomyosin signaling and regulation of the Epithelial Apical Junctional Complex

Epithelial cells form regulated and selective barriers between distinct tissue compartments. The Apical Junctional Complex (AJC) consisting of the tight junction (TJ) and adherens junction (AJ) control epithelial homeostasis, paracellular permeability and barrier properties. The AJC is composed of mutliprotein complexes consisting of transmembrane proteins that affiliate with an underlying perijunctional F-actin myosin ring through cytoplasmic scaffold proteins. AJC protein associations with the apical actin-myosin cytoskeleton are tightly controlled by a number of signaling proteins including the Rho family of GTPases that orchestrate junctional biology, epithelial homeostasis and barrier function. This review highlights the vital relationship of Rho GTPases and AJCs in controlling the epithelial barrier. The pathophysiologic relationship of Rho GTPases, AJC, apical actomyosin cytoskeleton and epithelial barrier function is discussed.

Inflammation-induced desmoglein-2 ectodomain shedding compromises the mucosal barrier

Proinflammatory cytokines promote desmoglein-2 (Dsg2) ectodomain shedding in intestinal epithelial cells. Epithelial exposure to Dsg2 ectodomains compromises intercellular adhesion while also promoting proliferation. These findings identify mechanisms by which mucosal inflammation–induced cleavage of Dsg2 influences intestinal epithelial homeostasis.

Intestinal epithelial claudins: expression and regulation in homeostasis and inflammation

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.

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

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.

IFNγ-induced suppression of β-catenin signaling: evidence for roles of Akt and 14.3.3ζ

Nuclear Akt1 phosphorylates 14.3.3ζ at serine 58 to inhibit β-catenin transactivation. The results outline a dual function of Akt1, which suppresses intestinal epithelial cell proliferation during intestinal inflammation.

Intracellular Desmoglein-2 cleavage sensitizes epithelial cells to apoptosis in response to pro-inflammatory cytokines

Desmosomal cadherins mediate intercellular adhesion and have also been shown to regulate homeostatic signaling in epithelial cells. We have previously reported that select pro-inflammatory cytokines induce Dsg2 ectodomain cleavage and shedding from intestinal epithelial cells (IECs). Dsg2 extracellular cleaved fragments (Dsg2 ECF) function to induce paracrine pro-proliferative signaling in epithelial cells. In this study, we show that exposure of IECs to pro-inflammatory cytokines interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) resulted in Dsg2 intracellular cleavage and generation of a ~55 kDa fragment (Dsg2 ICF). Dsg2 intracellular cleavage is mediated by caspase-8 and occurs prior to Dsg2 extracellular cleavage and the execution of apoptosis. Expression of exogenous Dsg2 ICF in model IECs resulted in increased sensitivity to apoptotic stimuli and apoptosis execution. Additionally, expression of the Dsg2 ICF repressed the anti-apoptotic Bcl-2 family member proteins Bcl-XL and Mcl1. Taken together, our findings identify a novel mechanism by which pro-inflammatory mediators induce modification of Dsg2 to activate apoptosis and eliminate damaged cells, while also promoting release of Dsg2 ECF that promotes proliferation of neighboring cells and epithelial barrier recovery.

PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery

In vitro differentiation of human pluripotent stem cell (hPSC)-derived organoids (HOs) facilitates the production of multicellular three-dimensional structures analogous to native human tissues. Most current methods for the generation of HOs rely on Matrigel, a poorly defined basement membrane derivative secreted by Engelbreth–Holm–Swarm mouse sarcoma cells, limiting the potential use of HOs for regenerative medicine applications. Here, we describe a protocol for the synthesis of a fully defined, synthetic hydrogel that supports the generation and culture of HOs. Modular, cell-encapsulating hydrogels are formed from a four-armed poly(ethylene glycol) macromer that has maleimide groups at each terminus (PEG-4MAL) and is conjugated to cysteine-containing adhesive peptides and cross-linked via protease-degradable peptides. The protocol also includes guidelines for the localized in vivo delivery of PEG-4MAL hydrogel–encapsulated HOs to injured mouse colon. The PEG-4MAL hydrogel supports the engraftment of the HOs and accelerates colonic wound repair. This culture and delivery strategy can thus be used to develop HO-based therapies to treat injury and disease. Hydrogel and tissue preparation and subsequent encapsulation can be performed within 2.5–3.5 h. Once HOs have been cultured in synthetic hydrogels for at least 14 d, they can be prepared and delivered to the mouse colon in under 5 h.This protocol describes how to use a fully defined, synthetic hydrogel to support the in vitro generation and culture of human organoids derived from pluripotent stem cells and the in vivo delivery of hydrogel-encapsulated organoids into mouse colon.

Publisher Correction: PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery

In the version of this protocol originally published, the caption for Fig. 3 was erroneously placed with Fig. 4, and that for Fig. 4 was placed with Fig. 3. This error has been corrected in the HTML and PDF versions of the paper.