A58 REGULATION OF INTESTINAL EPITHELIAL THYMIC STROMAL LYMPHOPOIETIN GENE EXPRESSION BY RETINOIC ACID RECEPTOR ALPHA
Bibliographic record
Abstract
Abstract Background We have targeted the retinoic acid receptor alpha (RARα) isoform in murine intestinal epithelial cells (IECs) to explore the role of epithelial intrinsic retinoid signaling in intestinal immune homeostasis. We noted a significant decrease in the CD11c+ and CD103+ dendritic cell (DC) subpopulations in the intestines of intestinal-epithelial specific RARα-deficient (RARα villin) mice. In the present study, we identified Thymic Stromal Lymphopoietin (TSLP) as a contributing factor that influences intrinsic RARα signaling within IECs. This cytokine preferentially stimulates CD103+ DCs to a more tolerogenic phenotype in the GI tract, induces Tregs and drives TH2 polarization. TSLP also influences immune homeostasis by modulating the activation of myeloid cells, inhibition of proinflamamtory DCs, and production of IL-12. Notably, TSLP levels are decreased in Crohn’s disease (CD) where inflammation is driven by IL-12. Aims We hypothesize that RA intrinsic signaling within IECs modulates TSLP expression via activation of RARα. We sought to generate a RARα -/- murine intestinal epithelial cell line utilizing the CRISPR/CAS9 system to examine the effects of RARa ablation. Second, we aim to establish RARα’s role in regulating intestinal epithelial TSLP expression in both the RARα villin mice and the CRISPR-generated RARα -/- cell line. Methods Exon 5 in the open reading frame of the RARα gene in Mode-K murine IECs was targeted using CRISPR/CAS9. CRISPR cleavage and knockdown of RARα activity was confirmed via surveyor/luciferase assays. The cell lines were phenotyped with regards to their morphology, proliferative ability, and viability. Next, we examined TSLP mRNA expression in the colons of RARα villin mice and the CRISPR RARα -/- knockout cell line via qRT-PCR under baseline and stimulated conditions. Wild-type (WT) and RARα-/- cells were stimulated with RARα-selective agonist, BMS753, and muramyl dipeptide (MDP), a NOD2-selective agonist. Results TSLP expression in surface colonic epithelium was elevated 2.3-fold in RARα villin mice compared to WT litter mates. TSLP expression was elevated 10-fold in the Mode-K RARα-/- cells versus parent cells at baseline. BMS753 stimulation resulted in a 15-fold increase in TSLP expression in the RARα-/- cells compared to baseline. When stimulated with MDP, a NOD2-selective agonist known to stimulate TSLP expression, TSLP expression was elevated 60-fold in the RARα-/- cells, but significantly impaired compared to the WT cells (400-fold). Conclusions TSLP expression is controlled by RARα in colonic IECs where it may act as a repressor of TSLP promoter transactivation under baseline conditions. This suggests an important role for RA on myeloid and T cell function via effects on intestinal epithelial TSLP expression. Funding Agencies CCCUniversity of Calgary, Department of Medicine
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| 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) | 0.002 | 0.001 |
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 source (direct Gemma or distilled Codex), 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".