GPR97 triggers inflammatory processes in human neutrophils via a macromolecular complex upstream of PAR2 activation

Neutrophils play essential anti-microbial and inflammatory roles in host defense, however, their activities require tight regulation as dysfunction often leads to detrimental inflammatory and autoimmune diseases. Here we show that the adhesion molecule GPR97 allosterically activates CD177-associated membrane proteinase 3 (mPR3), and in conjugation with several protein interaction partners leads to neutrophil activation in humans. Crystallographic and deletion analysis of the GPR97 extracellular region identified two independent mPR3-binding domains. Mechanistically, the efficient binding and activation of mPR3 by GPR97 requires the macromolecular CD177/GPR97/PAR2/CD16b complex and induces the activation of PAR2, a G protein-coupled receptor known for its function in inflammation. Triggering PAR2 by the upstream complex leads to strong inflammatory activation, prompting anti-microbial activities and endothelial dysfunction. The role of the complex in pathologic inflammation is underscored by the finding that both GPR97 and mPR3 are upregulated on the surface of disease-associated neutrophils. In summary, we identify a PAR2 activation mechanism that directs neutrophil activation, and thus inflammation. The PR3/CD177/GPR97/PAR2/CD16b protein complex, therefore, represents a potential therapeutic target for neutrophil-mediated inflammatory diseases.


Statistics
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Data
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Sample size
The minimal sample size needed to produce reliable measurements was determined for each type of analysis individually based on existing literature and our previous experience (e.g. PMID: 30559745, PMID: 28240246, or PMID: 31928845). Sample size was determined as being adequate by the magnitude and consistency of measurable differences between experimental groups. At least 3 independent experiments done in triplicate was considered significant for statistical analysis.
Data exclusions No outliers were excluded.

Replication
The reproducibility of experiments was performed using cells isolated from different donors to confirm the experimental findings. Number of each experiment is given in the manuscript. All experiments were performed in triplicates.
Randomization Sample randomization was not relevant to this study as each sample was divided into negative-, positive-control, and experimental groups and tested simultaneously in various experimental settings.

Blinding
Evaluation of the IHC results were done in blind by two different pathologists. Analysis of neutrophils from healthy control and patients was done in blind. Blinding samples of different cell types and primary cells were not relevant to this study as there is no prior knowledge of the expression characteristics of GPR97-ligand.

Gating strategy
The gating strategy of neutrophils from participants for studies in Figure 1 was described in the Supporting information file using FSC vs. SSC followed by CD16b staining.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.