RGal Increases Intestinal Epithelial Barrier Function in a PKC‐Dependent Manner and Drives an Inflammatory Gene Expression Profile
Bibliographic record
Abstract
Background Loss of intestinal epithelial barrier function has been linked to inflammatory bowel disease (IBD). We previously showed that the dietary fibre, rhamnogalacturonan (RGal), reduces inflammation in a murine model of DSS colitis. Additionally, apical treatment of Caco2 human intestinal epithelial cells with RGal increases transepithelial electrical resistance (TER) and accelerates wound‐healing, but the underlying mechanisms remain unknown. Aims We aimed to determine the mechanisms of the RGal mediated increase in epithelial barrier function. (1) Given the ability of PKC to modulate barrier permeability we aimed to determine the role of PKC in the RGal‐mediated increase in epithelial barrier function. (2) We aimed to determine changes in gene expression following RGal treatment. Methods (1) To determine the role of PKC in the RGal‐induced increase in epithelial barrier function, Caco2 monolayers were mounted in Ussing Chambers, pre‐treated with pan protein kinase C (PKC), PKCz‐specific, or classical PKC inhibitors (GFX, PKCz pseudosubstrate or Go6976 respectively) and then treated with RGal. Change in TER and FITC‐dextran flux in response to RGal were assessed. (2) To determine the transcriptionally dependent effects of RGal on epithelial barrier function, Caco2 monolayers were treated apically for 6, 12 and 24h with RGal and RNAseq was performed. Genes with changes in expression more than 3‐fold were entered into Enrichr for pathway analysis. In order to confirm changes in gene expression from RNAseq, a multi‐array immunoassay was performed. Results (1) RGal (1 mg/mL) reduced FITC‐dextran flux by 52.0% (n=5, p<0.05) 30 min post‐treatment. The effect of RGal on macromolecular permeability was reduced with GFX pre‐treatment (500 nM) by 75.3% (n=5, p<0.01). However, the ability of GFX to block the RGal‐mediated increase in barrier function was not mimicked in monolayers pre‐treated with PKCz pseudosubstrate inhibitor (10 mM, n=6) or Go6976 (10 nM, n=5). (2) While RGal had no significant effect on tight junction gene expression 6h post‐treatment, it upregulated the expression of inflammatory response genes including neutrophil regulatory genes such as CXCL8. Consistent with gene expression data, multiplex protein assay revealed an upregulation in CXCL8 family chemokines and G‐CSF as early as 2h in cell lysates and 6h in the supernatant. Conclusions The RGal‐mediated increase in intestinal epithelial barrier function is dependent on PKC. Transcriptionally, RGal upregulates the expression of inflammatory mediators such as CXCL8. Understanding the mechanism of the RGal‐induced increase in intestinal epithelial barrier function may allow for leverage of these mechanisms to treat IBD. Support or Funding Information Project supported by the NSERC (Canada), J.S. supported by an UGREF from the American Physiological Society. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".