A22 USING INTESTINAL ORGANOIDS TO CHARACTERIZE THE NAIP-NLRC4 INFLAMMASOME RESPONSE IN THE INTESTINAL EPITHELIUM
Bibliographic record
Abstract
Abstract Background The NAIP-NLRC4 inflammasome is an important innate defence mechanism in intestinal epithelial cells (IECs) that protects the gut from invasive pathogens. NAIP-NLRC4 activation triggers pyroptotic cell death, releases active interleukin (IL)-18, and promotes the expulsion of infected enterocytes. Dysregulated inflammasomes result in exaggerated inflammation of the intestinal mucosa which is characterisitic of inflammatory bowel disease (IBD). There is increasing evidence that inflammasomes in IEC behave differently than in immune cells. However, a majority of inflammasome pathway characterization is studied immune cells. In addition, NAIP-NLRC4 activity in macrophages has been shown to be important for regulating the expression of nitric oxide (Nos2). The potential transcriptomic impact of NAIP-NLRC4 activity in IECs has yet to be explored. Purpose We will use intestinal orgnaoids to systematically characterize the NAIP-NLRC4 inflammasome pathway and identify the transcriptomic impact of NAIP-NLRC4 activity in IECs. To better define the role of NAIP-NLRC4 activity during a physiological infection, we have developed a novel ex vivo model of Shigella infection in 3D organoids. Method Organoids were derived from the ileal crypts of wild type (WT) and Nlrc4-/-, Casp1-/-, Pycard-/-, or Tlr5-/- mice and stimulated with Pam3CSk4, LPS, or flagellin. Inflammasome activation was assessed by Western blot (WB) and propidium iodide uptake. WT and Nlrc4-/- organoids were infected with WT or a non-invasive Shigella mutant and the inflammasome response was evaluated by WB and a colony forming unit assay. WT and Nlrc4-/- organoids were stimulated with flagellin and gene expression was assessed by RT-qPCR. Result(s) Basolateral organoid stimulation with bacterial ligands revealed a novel response of IECs to bacterial flagellin that results in pyroptosis and IL-18 processing. Basolateral internalization of flagellin occurred in a TLR5-independent manner. Inflammasome activation by flagellin was fully abrogated in Nlrc4-/- and Casp1-/- organoids while only IL-18 processing was affected in Pycard-/- organoids. Infection with only WT Shigella induced inflammasome activation in an NLRC4-dependent manner. Interestingly, flagellin stimulation of WT but not Nlrc4-/- organoids led to increased expression of Nos2. Furthermore, Nlrc4-/- organoids had significantly lower expression levels of cytokine genes Ccl20, Cxcl1, and Tnf following inflammasome activation. Conclusion(s) Our study demonstrates an integral role for epithelial NAIP-NLRC4 inflammasomes in the response to bacterial flagellin and Shigella infection. We have uncovered a novel response of IECs to basolateral flagellin stimulation and revealed that NAIP-NLRC4 is important for the transcriptional regulation of inflammatory genes. Further work will examine how NAIP-NLRC4 activation controls inflammation and epithelial integrity by analyzing the NLRC4-dependent transcriptome and the effects on cell proliferation, differentiation, and barrier integrity. Please acknowledge all funding agencies by checking the applicable boxes below CCC, CIHR Disclosure of Interest None Declared
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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) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| 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".