Background
Calcium (Ca2+) is a known accelerator for gastric repair. However the mechanism by which Ca2+ mobilizes to promote repair remains unclear, and cannot be readily evaluated in vivo. Using ...gastric organoids derived from transgenic mice expressing a fluorescent Ca2+ reporter (yellow cameleon‐nano15; YC‐Nano), we previously observed intracellular Ca2+ increases in cells directly adjacent to a damaged cell. Using this Ca2+ sensor, we investigate the potential sources of intracellular Ca2+ essential for repair.
Methods
Gastroids generated from YC‐Nano mouse stomach corpus were cultured 4–5 days prior to experiments. Photodamage and resultant cell death was induced to 1–2 gastroid epithelial cells by ~3 sec high intensity 840 nm light. YC‐Nano reports Förster resonance energy transfer (FRET) from CFP to YFP in response to increased intracellular Ca2+. Change in intracellular Ca2+ was measured as FRET/CFP ratio, in cells adjacent to damaged cells. Inhibitors were used to test roles of Ca2+ channels (10 μM verapamil, 20 μM YM58483), intracellular Ca2+ (50 μM BAPTA/AM), Phospholipase C (10 μM U73122), and IP3R (50 μM 2‐APB).
Results
Unperturbed gastroid cells maintain a constant FRET ratio, indicating stable Ca2+ levels. In response to photodamage of the gastroid epithelium, increased levels of Ca2+ were observed specifically within the lateral membranes of cells neighboring the damaged cell. Chelation of intracellular Ca2+ by BAPTA/AM resulted in significant dampening of Ca2+ response, as well as blocking prompt repair. Inhibition of L‐type channels (verapamil) or store operated Ca2+ entry (YM58483) resulted in delaying repair and dampening intracellular Ca2+ response. Furthermore, inhibition of PLC (U73122) or IP3R (2‐APB) resulted in delayed repair and dampened Ca2+ response.
Conclusion
These results suggest both extracellular and intracellular Ca2+ sources are essential for supplying the Ca2+ that stimulates repair. The findings implicate an intracellular Ca2+ raise mediated via Ca2+ uptake via plasma membrane Ca2+ channels and intracellular Ca2+ release from the ER. Collectively this work indicates the usefulness of YC‐Nano to further assess intracellular Ca2+ dynamics and further investigate the signaling cascade behind Ca2+‐mediated repair.
Support or Funding Information
This work was supported by the National Institutes of Health (NIH) R01DK102551 (Montrose; Aihara) and F31DK115126 (Engevik). This project was also supported in part by the NIH P30 DK078392; Live Microscopy Core and DNA Sequencing and Genotyping Core of the Digestive Disease Research Core Center in Cincinnati.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Abstract only
Background
Calcium (Ca
2+
) is a known accelerator for gastric repair. However the mechanism by which Ca
2+
mobilizes to promote repair remains unclear, and cannot be readily evaluated
...in vivo
. Using gastric organoids derived from transgenic mice expressing a fluorescent Ca
2+
reporter (yellow cameleon‐nano15; YC‐Nano), we previously observed intracellular Ca
2+
increases in cells directly adjacent to a damaged cell. Using this Ca
2+
sensor, we investigate the potential sources of intracellular Ca
2+
essential for repair.
Methods
Gastroids generated from YC‐Nano mouse stomach corpus were cultured 4–5 days prior to experiments. Photodamage and resultant cell death was induced to 1–2 gastroid epithelial cells by ~3 sec high intensity 840 nm light. YC‐Nano reports Förster resonance energy transfer (FRET) from CFP to YFP in response to increased intracellular Ca
2+
. Change in intracellular Ca
2+
was measured as FRET/CFP ratio, in cells adjacent to damaged cells. Inhibitors were used to test roles of Ca
2+
channels (10 μM verapamil, 20 μM YM58483), intracellular Ca
2+
(50 μM BAPTA/AM), Phospholipase C (10 μM U73122), and IP3R (50 μM 2‐APB).
Results
Unperturbed gastroid cells maintain a constant FRET ratio, indicating stable Ca
2+
levels. In response to photodamage of the gastroid epithelium, increased levels of Ca
2+
were observed specifically within the lateral membranes of cells neighboring the damaged cell. Chelation of intracellular Ca
2+
by BAPTA/AM resulted in significant dampening of Ca
2+
response, as well as blocking prompt repair. Inhibition of L‐type channels (verapamil) or store operated Ca
2+
entry (YM58483) resulted in delaying repair and dampening intracellular Ca
2+
response. Furthermore, inhibition of PLC (U73122) or IP3R (2‐APB) resulted in delayed repair and dampened Ca
2+
response.
Conclusion
These results suggest both extracellular and intracellular Ca
2+
sources are essential for supplying the Ca
2+
that stimulates repair. The findings implicate an intracellular Ca
2+
raise mediated via Ca
2+
uptake via plasma membrane Ca
2+
channels and intracellular Ca
2+
release from the ER. Collectively this work indicates the usefulness of YC‐Nano to further assess intracellular Ca
2+
dynamics and further investigate the signaling cascade behind Ca
2+
‐mediated repair.
Support or Funding Information
This work was supported by the National Institutes of Health (NIH) R01DK102551 (Montrose; Aihara) and F31DK115126 (Engevik). This project was also supported in part by the NIH P30 DK078392; Live Microscopy Core and DNA Sequencing and Genotyping Core of the Digestive Disease Research Core Center in Cincinnati.
This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in
The FASEB Journal
.
Background
Gastric epithelial repair, mediated by cell migration and dead cell exfoliation, is a rapid process. Actin dynamics, in collaboration with myosin, are essential for the cell migration ...during epithelial repair of damage. We previously demonstrated that actin dynamics play an important role in the repair of damage in vivo mouse stomach and in vitro gastric organoids (gastroids). To extend our current knowledge, we examined the repair of damage in gastroids generated from human actin‐GFP (HuGE) transgenic mouse stomach and investigated the involvement of actin polymerization and myosin in this process.
Methods
Gastroids were generated from gastric corpus of HuGE mice. Hoechst33342 (10 μg/ml) was added to image the nucleus. Selective high intensity illumination of single gastric epithelial cell nucleus (two‐photon; 730 nm light) caused a microscopic lesion via photodamage. Dynamic changes in actin‐GFP and damage size were quantified over time. In some cases, Lucifer yellow (20 μM) or Alexa647‐10K dextran (10 μM) was added to medium to determine cellular and/or epithelial leakage.
Results
In response to photodamage of gastroid epithelium, actin‐GFP expression increased within ~1 min in the lateral membranes neighboring the damaged cell, and stayed elevated in this position and in lamellipodia during the cellular migration inward that closes the gap at the basal pole of the dead cell. Synchronous dead cell exfoliation was observed. When no intentional damage was imposed, similar events occurred during natural cell shedding into the lumen of the gastroids. Assuming a bistable switch controlling actin polymerization, we constructed a mathematical model whose simulations recaptured the temporal order of the observed repair processes, and the model predicted actin‐mediated force led to the observed dead cell exfoliation. In undamaged gastroids, Cytochalasin D (inhibitor of actin assembly at F‐actin barded ends: 1 μM) did not alter total cellular actin‐GFP intensity, while Latrunculin A (inhibitor of G‐actin polymerization: 2 μM) reduced GFP intensity. Both inhibitors increased paracellular leakage in the gastroid over the time, and completely inhibited the damage‐induced increase of actin, as well as repair of damage. In contrast, in undamaged gastroids, Jasplakinolide (actin filament stabilizer: 1 μM) facilitated actin‐GFP accumulation in the membrane and decreased GFP in the cytosolic compartment in a time‐dependent manner. Short time exposures to Jasplakinolide or Latrunculin A were imposed such that there was not effect on basal actin‐GFP intensity. In this condition, Jasplakinolide did not affect repair of damage, but Latrunculin A completely inhibited repair. Blebbistatin (Myosin II inhibitor, 10 μM) significantly inhibited repair of damage and slowed dead cell ejection, with no effect on GFP‐actin accumulation.
Conclusion
New F‐actin polymerization or elongation is necessary to initiate gastric epithelial repair of damage. Furthermore, recruitment of myosin II supports generation of force to exfoliate the dead cell.
Support or Funding Information
NIH RO1 DK102551.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Abstract only
Background
Superficial injury to the gastrointestinal (GI) epithelium requires a rapid response to prevent further damage. In the stomach, initial gastric epithelial repair involves ...restitution, during which neighboring cells migrate to cover the damaged area within minutes. This process is regulated by several factors, including calcium and trefoil factor 2 (TFF2). In many cell types, changes in the actin cytoskeleton are necessary for cell migration, however its role within gastric epithelial repair of damage has yet to be elucidated. In this present study, we explore the role of actin in gastric repair of damage
in vivo
and
in vitro
gastric organoids (gastroids) using human actin‐GFP (HuGE) transgenic mouse stomach.
Methods
With an anesthetized mouse on the microscope stage, the surgically exposed stomach mucosa was continuously imaged by intravital confocal/two‐photon microscopy. Selective high intensity illumination of 3–5 gastric epithelial surface cells (730 nm light) caused a microscopic lesion in the epithelium via photodamage. Gastroids were created from gastric corpus tissue of HuGE mice. Hoechst33342 (10 μg/ml) was added to image the nucleus. Photodamage was induced in a single epithelial cell nucleus in the gastroid. Dynamic changes in actin‐GFP and damage size were quantified over time.
Results
The unperturbed actin‐GFP intracellular distribution was similar in gastric epithelium between
in vivo
and
in vitro
gastroids generated from the HuGE mice. In response to photodamage of either
in vivo
gastric epithelium or
in vitro
gastroid epithelium, actin‐GFP fluorescence decreased in the damaged cell. Subsequently, actin‐GFP expression increased in the lateral membranes neighboring the damaged cell, followed by migration inward to close the gap at the basal pole of the dead cell, in parallel with dead cell exfoliation into the lumen. This repair of damage and focal increase of actin‐GFP was significantly blocked by the calcium chelator EDTA (10 mM in the lumen
in vivo
, 1 mM in the medium
in vitro
) or the inhibition of actin polymerization (1 μM Cytochalasin D in the medium
in vitro
). Phalloidin (actin) staining in the gastroids, at 5 and 10 min after photodamage, showed that endogenous actin selectively increased at the leading edge of cell migration/restitution that restored epithelial continuity after cell exfoliation. Conversely, immunofluorescence of E‐cadherin increased in the trailing edge of the same restituting cells. AMD3100 (CXCR4/TFF2 inhibitor, 1 μM) and FAK inhibitor (1 μM) significantly slowed repair of damage but did not impact actin‐GFP dynamics.
Conclusion
Actin dynamics regulated by extracellular calcium but not CXCR4 are a component of gastric restitution.
Support or Funding Information
NIH RO1 DK102551 (M.H. Montrose).
Abstract only
Background
Trefoil factor 2 (TFF2) plays an important role in promoting gastric epithelial repair of damage. We previously demonstrated that the action of TFF2 requires the ...sodium‐hydrogen exchanger isoform 2 (NHE2) for rapid gastric epithelial repair of damage. Dissecting molecular pathways is challenging
in vivo
, so the mechanism of interaction between TFF2 and NHE2 remains unknown. Recently, 3D primary culture of gastric epithelial cells known as gastric organoids has been established. Gastric organoids contain differentiated gastric epithelial cell types and to some degree mimic
in vivo
gastric epithelium. We tested the hypothesis that gastric organoids can be a useful
in vitro
model to elucidate the mechanisms of gastric epithelial repair of damage, by investigating if epithelial restitution in mouse gastric organoids was TFF2‐and/or NHE‐dependent.
Methods
Gastric organoids were generated from isolated fundic tissue of wild‐type (WT), TFF2 knockout (TFF2
−/−
), or NHE2 knockout (NHE2
−/−
) mice. Gastric organoids were cultured for 4–5 days prior to experiments. Damage was induced in gastric organoids by 5s exposure of 3 epithelial cells to high intensity two‐photon 720 nm light (photodamage), resulting in damage to targeted cells and subsequent cell death. Progression of damage and repair was evaluated using confocal/2‐photon microscopy, imaging cell nuclei (10 μg/ml Hoechst33342) and paracellular permeability between the basolateral and luminal compartments of organoids (20 μM Lucifer yellow LY added to culture medium).
Results
Gastric organoids derived from TFF2
−/−
mice (311 ± 49 μm diameter, n=13) were significantly (p< 0.01) smaller than those derived from WT mice (514 ± 43 μm diameter, n=34) 5 days after passaging. In contrast, organoids derived from NHE2
−/−
(604 ± 45 μm diameter, n=35) were larger than those derived from WT. Consistent with
in vivo
findings, Western blot showed TFF2 protein was upregulated in NHE2
−/−
organoids, while NHE2 was downregulated in TFF2
−/−
organoids. In unperturbed organoids, LY did not leak into the luminal space of the gastric organoids over 90 min, confirming integrity of the epithelial barrier. Photodamage increased LY leakage into the WT organoid. Subsequently, dead cell exfoliation occurred coincident with migration of neighboring cells to restore a continuous epithelium in the damaged area within 8 ± 1 min (n=14). In preliminary experiments, photodamage in TFF2
−/−
and NHE2
−/−
gastric organoids resulted in a similar intensity of LY fluorescence in the organoid, however there was a 2‐and 3‐fold delay of recovery from LY leakage in the repair of damage in TFF2
−/−
and NHE2
−/−
, respectively, accompanied by delay of damaged cell exfoliation. Basolateral application of EIPA (pan NHE inhibitor, 100 μM) in WT organoids also caused delay in the recovery of LY leakage and damaged cell exfoliation, while Hoe 694 (NHE 1 inhibitor, 100 μM) did not affect gastric restitution.
Conclusion
Gastric organoids have a TFF2‐ and NHE‐dependent epithelial repair of damage, paralleling previous
in vivo
studies. Therefore, gastric organoids can be a useful tool to elucidate mechanisms involved in epithelial repair of damage.
Support or Funding Information
Supported by NIH R01 DK102551
ABSTRACT Multiple studies have implicated microbes in the development of inflammation, but the mechanisms remain unknown. Bacteria in the genus Fusobacterium have been identified in the intestinal ...mucosa of patients with digestive diseases; thus, we hypothesized that Fusobacterium nucleatum promotes intestinal inflammation. The addition of >50 kDa F. nucleatum conditioned media, which contain outer membrane vesicles (OMVs), to colonic epithelial cells stimulated secretion of the proinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor (TNF). In addition, purified F. nucleatum OMVs, but not compounds <50 kDa, stimulated IL-8 and TNF production; which was decreased by pharmacological inhibition of Toll-like receptor 4 (TLR4). These effects were linked to downstream effectors p-ERK, p-CREB, and NF-κB. F. nucleatum >50-kDa compounds also stimulated TNF secretion, p-ERK, p-CREB, and NF-κB activation in human colonoid monolayers. In mice harboring a human microbiota, pretreatment with antibiotics and a single oral gavage of F. nucleatum resulted in inflammation. Compared to mice receiving vehicle control, mice treated with F. nucleatum showed disruption of the colonic architecture, with increased immune cell infiltration and depleted mucus layers. Analysis of mucosal gene expression revealed increased levels of proinflammatory cytokines (KC, TNF, IL-6, IFN-γ, and MCP-1) at day 3 and day 5 in F. nucleatum-treated mice compared to controls. These proinflammatory effects were absent in mice who received F. nucleatum without pretreatment with antibiotics, suggesting that an intact microbiome is protective against F. nucleatum-mediated immune responses. These data provide evidence that F. nucleatum promotes proinflammatory signaling cascades in the context of a depleted intestinal microbiome. IMPORTANCE Several studies have identified an increased abundance of Fusobacterium in the intestinal tracts of patients with colon cancer, liver cirrhosis, primary sclerosing cholangitis, gastroesophageal reflux disease, HIV infection, and alcoholism. However, the direct mechanism(s) of action of Fusobacterium on pathophysiological within the gastrointestinal tract is unclear. These studies have identified that F. nucleatum subsp. polymorphum releases outer membrane vesicles which activate TLR4 and NF-κB to stimulate proinflammatory signals in vitro. Using mice harboring a human microbiome, we demonstrate that F. nucleatum can promote inflammation, an effect which required antibiotic-mediated alterations in the gut microbiome. Collectively, these results suggest a mechanism by which F. nucleatum may contribute to intestinal inflammation.
Microbial metabolites, including B complex vitamins contribute to diverse aspects of human health. Folate, or vitamin B
, refers to a broad category of biomolecules that include pterin, ...para-aminobenzoic acid (pABA), and glutamate subunits. Folates are required for DNA synthesis and epigenetic regulation. In addition to dietary nutrients, the gut microbiota has been recognized as a source of B complex vitamins, including folate. This study evaluated the predicted folate synthesis capabilities in the genomes of human commensal microbes identified in the Human Microbiome Project and folate production by representative strains of six human intestinal bacterial phyla. Bacterial folate synthesis genes were ubiquitous across 512 gastrointestinal reference genomes with 13% of the genomes containing all genes required for complete
folate synthesis. An additional 39% of the genomes had the genetic capacity to synthesize folates in the presence of pABA, an upstream intermediate that can be obtained through diet or from other intestinal microbes. Bacterial folate synthesis was assessed during exponential and stationary phase growth through the evaluation of expression of select folate synthesis genes, quantification of total folate production, and analysis of folate polyglutamylation. Increased expression of key folate synthesis genes was apparent in exponential phase, and increased folate polyglutamylation occurred during late stationary phase. Of the folate producers, we focused on the commensal
to examine host-microbe interactions in relation to folate and examined folate receptors in the physiologically relevant human enteroid model. RNAseq data revealed segment-specific folate receptor distribution. Treatment of human colonoid monolayers with conditioned media (CM) from wild-type
did not influence the expression of key folate transporters proton-coupled folate transporter (PCFT) or reduced folate carrier (RFC). However, CM from
containing a site-specific inactivation of the
gene, which prevents the bacteria from synthesizing a polyglutamate tail on folate, significantly upregulated RFC expression. No effects were observed using
with a site inactivation of
, which results in no folate production. This work sheds light on the contributions of microbial folate to overall folate status and mammalian host metabolism.
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