Microglial cell secretion of serpin‐like trypsin‐inhibitory activity: potential regulation of proteinase‐activated receptor‐2 (PAR2)‐mediated inflammatory signalling
Bibliographic record
Abstract
Microglial cells function as a relay station from the periphery to the central nervous system (CNS). In part, microglial cells signal to their microenvironment via cytokines and other agonists. We hypothesized that microglial cells can also secrete proteinases, known to signal via proteinase‐activated receptors (PARs) in the CNS. PARs, which are members of the G protein‐coupled receptor superfamily, are activated upon cleavage of their extracellular N‐terminus to unmask a tethered receptor‐activating sequence (PMID: 22453980). However, the role(s) of PARs and their activating proteinases in a microglial cell environment remains to be elucidated. Using an EOC 13.31 (ATCC® CRL‐2468) mouse neonatal‐derived microglial cell culture model (EOC cells), we evaluated the presence of functional PARs (RT‐PCR and calcium signaling assays), and their secretion of proteinases, proteinase‐inhibitors, or both. Cell‐secreted proteinase activity was assessed 1. using a microtiter plate chromogenic substrate cleavage assay and 2. a PAR cleavage assay whereby proteolysis releases an N‐terminal‐luciferase signal from an N‐Luc‐tagged PAR (PMID: 26957205). Proteinase‐inhibitory activity was monitored using the same assays by observing the ability of the EOC cell supernatants to block substrate cleavage caused by thrombin and trypsin in the microtiter plate or N‐Luc release assay. We found that the EOC cells express mRNA for PARs 1 and 2, which are known to cause inflammatory responses. However, there was no calcium response upon stimulation of either PAR by their selective peptide agonists, TFLLR‐amide and 2‐furoyl‐LIGRLO‐amide for PARs 1 and 2, respectively. PAR‐cleaving proteinase activity in the EOC cell supernatants was not detected by neither the microtiter nor the PAR N‐Luc release assay. In contrast, the EOC cell supernatants contained a serpin‐like trypsin inhibitor that blocked substrate cleavage by trypsin (but not thrombin) in both the microtiter plate and PAR2 N‐Luc release assay. Further, the EOC supernatants blocked the ability of trypsin to cleave/activate PAR2 in a target HEK cell PAR2 calcium signaling assay. We conclude that in addition to secreting cytokines and other agonists, microglial cells produce a serpin‐like trypsin inhibitor that may modulate the inflammatory activity of proteinases in the CNS microenvironment. We aim to identify the secreted trypsin inhibitor using a proteomic and activity‐based probe labelling approach.
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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) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 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".