Media from macrophages co-incubated with <i>Enterococcus faecalis</i> induces epithelial cell monolayer reassembly and altered cell morphology

Signal exchange between intestinal epithelial cells, microbes and local immune cells is an important mechanism of intestinal homeostasis. Given that intestinal macrophages are in close proximity to both the intestinal epithelium and the microbiota, their pathologic interactions may result in epithelial damage. The present study demonstrates that co-incubation of murine macrophages with E. faecalis strains producing gelatinase (GelE) and serine protease (SprE) leads to resultant condition media (CM) capable of inducing reassembly of primary colonic epithelial cell monolayers. Following the conditioned media (CM) exposure, some epithelial cells are shed whereas adherent cells are observed to undergo dissolution of cell-cell junctions and morphologic transformation with actin cytoskeleton reorganization resulting in flattened and elongated shapes. These cells exhibit marked filamentous filopodia and lamellipodia formation. Cellular reorganization is not observed when epithelial monolayers are exposed to: CM from macrophages co-incubated with E. faecalis GelE/SprE-deficient mutants, CM from macrophages alone, or E. faecalis (GelE/SprE) alone. Flow cytometry analysis reveals increased expression of CD24 and CD44 in cells treated with macrophage/E. faecalis CM. This finding in combination with the appearance colony formation in matrigel demonstrate that the cells treated with macrophage/E. faecalis CM contain a higher proportion progenitor cells compared to untreated control. Taken together, these findings provide evidence for a triangulated molecular dialogue between E. faecalis, macrophages and colonic epithelial cells, which may have important implications for conditions in the gut that involve inflammation, injury or tumorigenesis

Chmp 1A is a mediator of the anti-proliferative effects of All-trans Retinoic Acid in human pancreatic cancer cells

<p>Abstract</p> <p>Background</p> <p>We recently have shown that Charged multivesicular protein/Chromatin modifying protein1A (Chmp1A) functions as a tumor suppressor in human pancreatic tumor cells. Pancreatic cancer has the worst prognosis of all cancers with a dismal 5-year survival rate. Preclinical studies using ATRA for treating human pancreatic cancer suggest this compound might be useful for treatment of pancreatic cancer patients. However, the molecular mechanism by which ATRA inhibits growth of pancreatic cancer cells is not clear. The objective of our study was to investigate whether Chmp1A is involved in ATRA-mediated growth inhibition of human pancreatic tumor cells.</p> <p>Results</p> <p>We performed microarray studies using HEK 293T cells and discovered that Chmp1A positively regulated Cellular retinol-binding protein 1 (CRBP-1). CRBP-1 is a key regulator of All-trans retinoic acid (ATRA) through ATRA metabolism and nuclear localization. Since our microarray data indicates a potential involvement of Chmp1A in ATRA signaling, we tested this hypothesis by treating pancreatic tumor cells with ATRA <it>in vitro</it>. In the ATRA-responsive cell lines, ATRA significantly increased the protein expression of Chmp1A, CRBP-1, P53 and phospho-P53 at serine 15 and 37 position. We found that knockdown of Chmp1A via shRNA abolished the ATRA-mediated growth inhibition of PanC-1 cells. Also, Chmp1A silencing diminished the increase of Chmp1A, P53 and phospho-P53 protein expression induced by ATRA. In the ATRA non-responsive cells, ATRA did not have any effect on the protein level of Chmp1A and P53. Chmp1A over-expression, however, induced growth inhibition of ATRA non-responsive cells, which was accompanied by an increase of Chmp1A, P53 and phospho-P53. Interestingly, in ATRA responsive cells Chmp1A is localized to the nucleus, which became robust upon ATRA treatment. In the ATRA-non-responsive cells, Chmp1A was mainly translocated to the plasma membrane upon ATRA treatment.</p> <p>Conclusion</p> <p>Collectively our data provides evidence that Chmp1A mediates the growth inhibitory activity of ATRA in human pancreatic cancer cells via regulation of CRBP-1. Our results also suggest that nuclear localization of Chmp1A is important in mediating ATRA signaling.</p

CD44⁺CD24⁺ population enriched in epithelial cells treated with J774/ <i>E</i>. <i>faecalis</i> CMs independently on GelE/SprE.

<p><b>(A)</b> Representative results of flow cytometry analysis using CD44 and CD24 cell markers. <b>(B)</b> The percentage of CD44⁺CD24⁺ cells depending on epithelial monolayers applied CMs. Statistical analysis was performed using the one-way ANOVA with Bonferrony adjusted <i>p</i>-value for multiple comparisons where *<i>P</i> = 0.001, **<i>P</i><0.001 as compared to J774 CM, n = 3.</p

Media from macrophages co-incubated with <i>Enterococcus faecalis</i> induces epithelial cell monolayer reassembly and altered cell morphology

<div><p>Signal exchange between intestinal epithelial cells, microbes and local immune cells is an important mechanism of intestinal homeostasis. Given that intestinal macrophages are in close proximity to both the intestinal epithelium and the microbiota, their pathologic interactions may result in epithelial damage. The present study demonstrates that co-incubation of murine macrophages with <i>E</i>. <i>faecalis</i> strains producing gelatinase (GelE) and serine protease (SprE) leads to resultant condition media (CM) capable of inducing reassembly of primary colonic epithelial cell monolayers. Following the conditioned media (CM) exposure, some epithelial cells are shed whereas adherent cells are observed to undergo dissolution of cell-cell junctions and morphologic transformation with actin cytoskeleton reorganization resulting in flattened and elongated shapes. These cells exhibit marked filamentous filopodia and lamellipodia formation. Cellular reorganization is not observed when epithelial monolayers are exposed to: CM from macrophages co-incubated with <i>E</i>. <i>faecalis</i> GelE/SprE-deficient mutants, CM from macrophages alone, or <i>E</i>. <i>faecalis</i> (GelE/SprE) alone. Flow cytometry analysis reveals increased expression of CD24 and CD44 in cells treated with macrophage/<i>E</i>. <i>faecalis</i> CM. This finding in combination with the appearance colony formation in matrigel demonstrate that the cells treated with macrophage/<i>E</i>. <i>faecalis</i> CM contain a higher proportion progenitor cells compared to untreated control. Taken together, these findings provide evidence for a triangulated molecular dialogue between <i>E</i>. <i>faecalis</i>, macrophages and colonic epithelial cells, which may have important implications for conditions in the gut that involve inflammation, injury or tumorigenesis.</p></div

Uniformity of morphological changes in both YAMC and C57BL/6 cells in response to conditioned media isolated after exposure of J774 macrophages to <i>E</i>. <i>faecalis</i> (Scale bars– 100 μm).

<p><b>(A)</b> Phase contrast microscopy images of primary mouse colon epithelial cells incubated with J774/<i>E</i>. <i>faecalis</i> CMs demonstrates reassembly of epithelial monolayer and morphological changes of adherent epithelial cells when J774/<i>E</i>. <i>faecalis</i> (GelE/SprE) CMs were used for co-incubation with J774; <b>(B)</b> The percentage of flattened spindle-shaped adherent epithelial cells. Four fields of phase contrast images from 3 independent experiments were taken for total cell count. Determination of morphological changes was based on cobblestone or spindle cell shape. The number of spindle-shaped epithelial cells in J774/WT CM and J774/ΔΔ/GS CM groups was significantly higher compared to J774/ΔΔ CM. Mean values are graphed and error bars represent standard error of the mean (SEM). Statistical analysis was performed using the one-way ANOVA with Bonferrony adjusted <i>p</i>-value for multiple comparisons where *<i>P</i> = 0.002, **<i>P</i> = 0.001 and ^#<i>P</i> = 0.001 as compared to J774/ΔΔ CM, n = 3.</p

Morphological and cytoskeletal changes in mouse primary epithelial cells (C57BL/6) induced by conditioned media from J774 macrophages co-incubated for 18h with <i>E</i>. <i>faecalis</i> GelE/SprE producing strains.

<p><b>(A)</b> Phase contrast microscopy images of primary mouse colon epithelial cells incubated with J774/<i>E</i>. <i>faecalis</i> CMs. Representative images of 10 experiments; <b>(B)</b> Immunostaining with rhodamine phalloidin for F-actin (red) and nucleus with DAPI (blue). Lamellipodia and filopodia formation are shown by white arrowheads and yellow arrows respectively; (<b>C)</b> Immunostaining for vimentin. Pink arrowheads indicate intermediate vimentin filaments. Scale bars– 20 μm.</p

Changes in expression and localization of epithelial cell-cell junction proteins E-cadherin and ZO-1 after 18 h of treatment of epithelial cell monolayers with macrophage/<i>E</i>. <i>faecalis</i> CM.

<p><b>(A)</b> Immunostaining for ZO-1 and E-cadherin. Scale bars– 20 μm; Area of ZO-1 and E-Cadherin fluorescence per cell in images in J774/WT CM and J774/ΔΔ/GS CM groups was significantly lower compared to J774 CM. Mean values are graphed and error bars represent standard error of the mean (SEM). Statistical analysis was performed using the one-way ANOVA with Bonferrony adjusted <i>p</i>-value for multiple comparisons where *<i>P</i> = 0.007, **<i>P</i> = 0.004 and ^#<i>P</i><0.001 as compared to J774 CM, n = 3. <b>(B)</b> Representative Western blot staining of E-cadherin in epithelial cells treated with CMs from macrophages co-incubated with <i>E</i>. <i>faecalis</i> strains. Results are typical of three independent experiments; <b>(C)</b> Expression of E-cadherin in epithelial cells treated with J774 CM and J774/<i>ΔΔ/GS</i> CM was significantly lower compared to J774 CM and consistent with fluorescent staining data for E-Cdherin. Statistical analysis was performed using the one-way ANOVA with Bonferrony adjusted <i>p</i>-value for multiple comparisons where *<i>P</i> = 0.005, **<i>P</i> = 0.002 as compared to J774 CM, n = 3. <b>(D)</b> Zymography of resulting epithelial cells CMs demonstrating the presence of active form of metalloproteinase MMP9 upon exposure to macrophage/E. <i>faecalis</i> CMs when GelE/SprE secreted strains (WT and ΔΔ/GS) were used.</p