Sonic Hedgehog acts as a macrophage chemoattractant during regeneration of the gastric epithelium

Sonic Hedgehog (Shh), secreted from gastric parietal cells, contributes to the regeneration of the epithelium. The recruitment of macrophages plays a central role in the regenerative process. The mechanism that regulates macrophage recruitment in response to gastric injury is largely unknown. Here we tested the hypothesis that Shh stimulates macrophage chemotaxis to the injured epithelium and contributes to gastric regeneration. A mouse model expressing a myeloid cell-specific deletion of Smoothened (LysMcre/+;Smof/f) was generated using transgenic mice bearing loxP sites flanking the Smo gene (Smo loxP) and mice expressing a Cre recombinase transgene from the Lysozyme M locus (LysMCre). Acetic acid injury was induced in the stomachs of both control and LysMcre/+;Smof/f (SmoKO) mice and gastric epithelial regeneration and macrophage recruitment analyzed over a period of 7 days post-injury. Bone marrow-derived macrophages (BM-Mø) were collected from control and SmoKO mice. Human-derived gastric organoid/macrophage co-cultures were established, and macrophage chemotaxis measured. Compared to control mice, SmoKO animals exhibited inhibition of ulcer repair and normal epithelial regeneration, which correlated with decreased macrophage infiltration at the site of injury. Bone marrow chimera experiments using SmoKO donor cells showed that control chimera mice transplanted with SmoKO bone marrow donor cells exhibited a loss of ulcer repair, and transplantation of control bone marrow donor cells to SmoKO mice rescued epithelial cell regeneration. Histamine-stimulated Shh secretion in human organoid/macrophage co-cultures resulted in macrophage migration toward the gastric epithelium, a response that was blocked with Smo inhibitor Vismodegib. Shh-induced macrophage migration was mediated by AKT signaling. In conclusion, Shh signaling acts as a macrophage chemoattractant via a Smo-dependent mechanism during gastric epithelial regeneration in response to injury.

Sex Hormones, Growth Hormone, and the Cornea

The growth and maintenance of nearly every tissue in the body is influenced by systemic hormones during embryonic development through puberty and into adulthood. Of the ~130 different hormones expressed in the human body, steroid hormones and peptide hormones are highly abundant in circulation and are known to regulate anabolic processes and wound healing in a tissue-dependent manner. Of interest, differential levels of sex hormones have been associated with ocular pathologies, including dry eye disease and keratoconus. In this review, we discuss key studies that have revealed a role for androgens and estrogens in the cornea with focus on ocular surface homeostasis, wound healing, and stromal thickness. We also review studies of human growth hormone and insulin growth factor-1 in influencing ocular growth and epithelial regeneration. While it is unclear if endogenous hormones contribute to differential corneal wound healing in common animal models, the abundance of evidence suggests that systemic hormone levels, as a function of age, should be considered as an experimental variable in studies of corneal health and disease.

Colonic healing requires WNT produced by epithelium as well as Tagln+ and Acta2+ stromal cells

While Wnt signaling is clearly important for the intestinal epithelial homeostasis, the relevance of various sources of Wnt ligands themselves remains incompletely understood. Wnt blockage in distinct stromal cell types suggested obligatory functions of several stromal cell sources and yielded different observations. The physiological contribution of epithelial Wnt to tissue homeostasis remains unclear. We show here that blocking epithelial Wnts affected colonic Reg4+ epithelial cell differentiation, and impaired colonic epithelial regeneration after injury. Single cell RNA analysis of intestinal stroma showed that the majority of Wnt-producing cells were contained in transgelin (Tagln+) and smooth muscle actin alpha 2 (Acta2+) expressing populations. We genetically attenuated Wnt production from these stromal cells using Tagln-Cre and Acta2-CreER drivers, and found that Wnt blockage from either epithelium or Tagln+ and Acta2+ stromal cells impaired colonic epithelial healing after chemical-induced injury. Aggregated Wnt blockage from both epithelium and Tagln+ or Acta2+ stromal cells drastically diminished epithelial repair, increasing morbidity and mortality. These results from two uncharacterized stromal populations suggested that colonic recovery from colitis-like injury depends on multiple Wnt-producing sources.

Human microbiota. Friend? Enemy? Neighbors?

The review is dedicated to the modern ideas about the composition of the human intestinal microbiome, factors that determine the bacterial «landscape» and affect its activity. The main functions of normal microflora have been described, including intestinal motility, protection of intestinal barrier against pathogenic bacteria, parasites, intestinal epithelial regeneration, metabolic and immunological functions, participation in digestive processes, synthesis of amino acids and proteins, antibiotics, vitamins, hormonally active substances, promoting the absorption of minerals and nutrients, preventing the development of pathological conditions. Determination of intestinal microbiota expression of the innate immune system explains the role of microflora in chronic inflammation and diseases such as liver fibrosis, metabolic syndrome, type 2 diabetes mellitus, atherosclerosis, cardiovascular, neurodegeneration and cancer. A promising way is the use of microorganisms with beneficial properties (probiotics), the necessary substrate for them (prebiotics), their metabolites (metabiotics) and complexes of pro‑ and prebiotics (synbiotics) not only to restore and regulate the intestinal microflora, but also as therapeutic agents in some diseases, in particular during eradication therapy of the bacterium Helicobacter pylori. The functions of some species and strains of beneficial bacteria have being discussed, in particular the strain Bacillus causii UBBC‑07, as well as the results of preclinical and clinical trials of its use at antibiotic‑associated diarrhea in the inhibition of Clostridium difficile. The issues of safety and facts of positive impact on human health of probiotic products of Probeez line (Organosyn Ltd.) are considered, which differ in the personification of appointments to certain segments of the population: Probeez®, Probeez® Femina, Probeez® Kids, Probeez® DUO and Probeez® Immuno contain successfully selected live probiotic bacteria. The available range of monocomponent (Probeez® with Kids, Probeez® DUO), multicomponent (Probeez®, Probeez® Femina) and synbiotic (Probeez® Immuno) products allows to choose the best option in specific conditions for their application not only in inflammatory diseases of the gastrointestinal tract, but also in chronic disorders of other systems and organs.

Death-associated protein kinase 3 (DAPK3) contributes to intestinal epithelial wound healing and the resolution of experimental colitis in mice

ABSTRACTVarious signaling molecules affecting epithelial restitution and wound healing are dysregulated in ulcerative colitis. Recent evidence demonstrates the necessity of Hippo-YAP/TAZ signaling, interceded by cytoskeletal remodeling, for intestinal regeneration. Death-associated protein kinase 3 (DAPK3) is a regulator of actin cytoskeleton reorganization that controls proliferation and apoptosis. Pharmacological inhibition of DAPK3 in Caco-2 human intestinal epithelial cells (IECs) with the HS38 compound augmented cell proliferation and enhanced wound closure. This phenotype corresponded with the increased colocalization of Yes-associated protein (YAP) with F-actin, which is indicative of YAP activation. The administration of HS38 impeded the resolution of intestinal injury and attenuated epithelial-specific proliferation after acute colitis induced by dextran-sodium-sulphate (DSS) in mice. During recovery from DSS-induced colitis, IEC proliferation was repressed, and mice exhibited increased disease severity when HS38 was applied to inhibit DAPK3. Moreover, HS38 treatment increased YAP nuclear localization in IECs, an indicator of signal activation. In summary, this study established DAPK3 as a key factor in intestinal epithelial regeneration and colitis progression by way of YAP signaling. Nevertheless, the role that DAPK3 play in different cell types will need further investigation to decipher the full consequence of DAPK3 inhibition on epithelial homeostasis.

Identification, Characterization, and Transcriptional Reprogramming of Epithelial Stem Cells and Intestinal Enteroids in Simian Immunodeficiency Virus Infected Rhesus Macaques

Epithelial cell injury and impaired epithelial regeneration are considered key features in HIV pathogenesis and contribute to HIV-induced generalized immune activation. Understanding the molecular mechanisms underlying the disrupted epithelial regeneration might provide an alternative approach for the treatment of HIV-mediated enteropathy and immune activation. We have observed a significant increased presence of α defensin5+ (HD5) Paneth cells and proliferating Ki67+ epithelial cells as well as decreased expression of E-cadherin expression in epithelial cells during SIV infection. SIV infection did not significantly influence the frequency of LGR5+ stem cells, but the frequency of HD5+ cells was significantly higher compared to uninfected controls in jejunum. Our global transcriptomics analysis of enteroids provided novel information about highly significant changes in several important pathways like metabolic, TCA cycle, and oxidative phosphorylation, where the majority of the differentially expressed genes were downregulated in enteroids grown from chronically SIV-infected macaques compared to the SIV-uninfected controls. Despite the lack of significant reduction in LGR5+ stem cell population, the dysregulation of several intestinal stem cell niche factors including Notch, mTOR, AMPK and Wnt pathways as well as persistence of inflammatory cytokines and chemokines and loss of epithelial barrier function in enteroids further supports that SIV infection impacts on epithelial cell proliferation and intestinal homeostasis.

Re-entry into mitosis and regeneration of intestinal stem cells through enteroblast dedifferentiation in Drosophila midguts

Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis. In mammalian intestines, upon ablation of resident stem cells, the progenies of intestinal stem cells (ISCs) such as secretory cells and tuft cells can dedifferentiate to generate ISCs to drive epithelial regeneration, but whether and how the ISC progenies dedifferentiate to generate ISCs under physiological conditions remains unknown. Here we show that infection of pathogenic bacteria induces enteroblasts (EBs) as one type of ISC progenies to re-enter the mitotic cycle in the Drosophila intestine. The re-entry into mitosis is dependent on epithermal growth factor receptor (EGFR)-Ras signaling and ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive EBs cell-autonomously to re-enter into mitosis. In addition, we examined whether EBs gain ISC identity as a prerequisite to divide, but the immunostaining with stem cell marker Delta shows that these dividing EBs do not gain ISC identity. After employing lineage tracing experiments, we further demonstrate that EBs dedifferentiate to generate functional ISCs after symmetric divisions of EBs. Together, our study in Drosophila intestines uncovers a new role of EGFR-Ras signaling in regulating re-entry into mitosis and dedifferentiation during regeneration and reveals a novel mechanism by which ISC progenies undergo dedifferentiation through a mitotic division, which has important implication to mammalian tissue homeostasis and tumorigenesis.

Smooth muscle-specific MMP17 (MT4-MMP) regulates the intestinal stem cell niche and regeneration after damage

Smooth muscle is an essential component of the intestine, both to maintain its structure and produce peristaltic and segmentation movements. However, very little is known about other putative roles that smooth muscle cells may have. Here, we show that smooth muscle cells may be the dominant suppliers of BMP antagonists, which are niche factors essential for intestinal stem cell maintenance. Furthermore, muscle-derived factors render epithelium reparative and fetal-like, which includes heightened YAP activity. Mechanistically, we find that the membrane-bound matrix metalloproteinase MMP17, which is exclusively expressed by smooth muscle cells, is required for intestinal epithelial repair after inflammation- or irradiation-induced injury. Furthermore, we propose that MMP17 affects intestinal epithelial reprogramming after damage indirectly by cleaving diffusible factor(s) such as the matricellular protein PERIOSTIN. Together, we identify an important signaling axis that establishes a role for smooth muscle cells as modulators of intestinal epithelial regeneration and the intestinal stem cell niche.

Clinical and histological evaluation of a dual sequential application of fractional 10,600 nm and 1570 nm lasers, compared to single applications in a porcine model

The sequential application of fractional ablative/10,600 nm/CO2 followed by 1570 nm non-ablative laser treatment might produce better results than applying either laser treatment alone. However, histological data regarding the safety of this combination is lacking. This study aimed to assess and compare clinical effects, histological tissue damage, and wound healing after monochromatic and sequential fractional laser treatments. In this prospective porcine model study, three adult female pigs were each irradiated using three different wavelengths: (a) monochromatic fractional ablative CO2 laser; (b) monochromatic fractional non-ablative 1570 nm laser; (c) sequential fractional 10,600 nm/CO2 followed by 1570 nm laser treatment. There were six power levels in the monochromatic 1570 nm laser, five in the 10,600 nm/CO2, and five in the sequential treatment. The immediate skin reaction (ISR), crusting and adverse effects, was evaluated across different time points throughout the healing process. Wound biopsies were taken at immediately after (0) and at 3, 7, and 14 days after irradiation. Depth and width of craters, and width of coagulation zone were measured and compared. Similar ISR and crusting score values were obtained following the monochromatic and sequential irradiation in a similar dose–response manner. During 14 days of follow-up, the skin looked intact and non-infected with no signs of necrosis. The mean depth and width of craters were comparable only at the maximal energy level (240 mJ) of CO2 laser, with the coagulation size greater after the sequential treatment. In histology, a similar wound healing was evident. On day 3, crusts were observed above all lesions as was epithelial regeneration. The sequential irradiation with 10,600 nm/CO2 and 1570 nm lasers did not pose any additional risk compared to the risk of each laser alone.