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

Altered Chromosomal Positioning, Compaction, and Gene Expression with a Lamin A/C Gene Mutation

Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression.To investigate the hypothesis that mutant lamin A/C changes the lamina's ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction.These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered

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

In vitro processing and secretion of mutant insulin proteins that cause permanent neonatal diabetes

Permanent neonatal diabetes mellitus is a rare form of insulin-requiring diabetes presenting within the first few weeks or months of life. Mutations in the insulin gene are the second most common cause of this form of diabetes. These mutations are located in critical regions of preproinsulin and are likely to prevent normal processing or folding of the preproinsulin/proinsulin molecule. To characterize these mutations, we transiently expressed proinsulin-GFP fusion proteins in MIN6 mouse insulinoma cells. Our study revealed three groups of mutant proteins: 1) mutations that result in retention of proinsulin in the endoplasmic reticulum (ER) and attenuation of secretion of cotransfected wild-type insulin: C43G, F48C, and C96Y; 2) mutations with partial ER retention, partial recruitment to granules, and attenuation of secretion of wild-type insulin: G32R, G32S, G47V, G90C, and Y108C; and 3) similar to (2) but with no significant attenuation of wild-type insulin secretion: A24D and R89C. The mutant insulin proteins do not prevent targeting of wild-type insulin to secretory granules, but most appear to lead to decreased secretion of wild-type insulin. Each of the mutants triggers the expression of the proapoptotic gene Chop, indicating the presence of ER stress