P-205 IL-6-dependent STAT3(Ser727) Activation Regulates Excess TGF-β1 and Collagen Production in Patients with Fibrostenotic Crohn’s Disease and is Blocked by Static
Bibliographic record
Abstract
Our recent work showed that in patients with Montreal B2 Crohn’s disease TGF-β1 and TGF-β1-dependent collagen I are increased specifically in the intestinal smooth muscle of strictures compared to normal resection margin in the same patient. TGF-β1 and collagen I was not similarly increased in B1 or B3 phenotype Crohn’s. In muscle cells of normal intestine or normal resection margin in B2 patients STAT3(Y705) phosphorylation was high and STAT3(S727) phosphorylation was low. The opposite pattern was seen in muscle cells from strictures: phosphorylation of STAT3(Y705) decreased and STAT3(S727) increased. Since a consensus STAT3 binding element is present in the promoter of the TGFB gene, we hypothesized that IL-6-dependent STAT3 activity regulates increased TGF-β1 and Collagen I gene expression and could be blocked by the selective STAT3(S727) inhibitor, Stattic. Smooth muscle cells isolated from muscularis propria of patients undergoing ileal resection for Montreal B1, B2 and B3 Crohn’s disease were used to prepare RNA, cell lysates or primary cell culture. IL-6 was measured by ELISA. STAT3 phosphorylation was measured by immunoblot analysis and immunofluorescence. STAT3-TGFB DNA binding activity was measured by ChIP; transcriptional activity of TGFB gene was measured using a luciferase reporter assay. TGF-β1 and Collagen IαI RNA was measured by qRT-PCR. STAT3(Ser727) phosphorylation was selectively inhibited using 10µM Static. The participation of STAT3 in regulation of TGF-β1 and Collagen IαI expression was investigated in cells transfected with either a dominant negative STAT3(S727A), a constitutively active STAT3(S727E) or a dominant negative STAT3(Y705F) gene. IL-6 production by muscle cells from strictured intestine is significantly increased compared to normal resection margin in the same patient and significantly higher than that in affected regions of B1 or B3 patients or from non-Crohn’s disease patients (Fig. 1). Treatment of muscle cells with 10µg/mL IL-6 increased STAT3(S727) phosphorylation by 250% while basal STAT3(Y705) phosphorylation was unaffected. In cells treated with 10 µM Static, IL-6-induced STAT3(727) phosphorylation was inhibited 80% while STAT3(Y705) was unaffected. STAT3-TGFB DNA binding activity increased 5.5-fold in muscle cells in strictures compared to the normal resection margin and reflected increased STAT3(S727) and not STAT3(Y205) binding to TGFB. The same pattern of increased STAT3 and STAT3(S727) but not STAT3(Y705) binding activity to the SBE in the TGFB was seen in cells treated with 10 µg/mL IL-6. Treatment of cells with IL-6 increased TGFB transcriptional activity by 2.5-fold which was inhibited 60% by the STAT3(S727) inhibitor, Static. Transfect of cell with dnSTAT3(S727A) abolished and transfection of constitutively active STAT3(S727E) enhanced IL-6-induced STAT3(S727) phosphorylation TGFB transcriptional activity (Fig. 2) and TGF-β1 and collagen 1αI gene expression (Fig. 3). These findings indicate the STAT3(S727) phosphorylation mediates IL-6-induced upregulation of TGF-β1 and Collagen IαI in muscle cells of strictured intestine. The pathophysiology of IL-6-dependent STAT3(727) regulated TGF-β1 and collagen expression is unique in Crohn’s patients with a B2 phenotype compared to B1 and B3 disease. Increased STAT3(S727) activation, TGFB gene transcription and expression of TGF-β1 and Collagen IαI that result from smooth muscle cell IL-6 production can be inhibited using Static.
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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 | 0.000 |
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 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".