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

Abstract

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.

Fig. 1. Up-regulated GPR97 expression in neutrophils correlates with the disease status of inflammatory disorders.

a, b The immunohistochemical analyses of GPR97 expression in tissue sections of the early- a and late-stage b appendicitis. Reactivity of the anti-GPR97 mAb to GPR97 was shown as brownish staining. Scale bar, 100 μm. c The relative GPR97 staining intensities in tissue-infiltrating neutrophils of the early- (n = 11) and late-stage (n = 16) appendicitis tissues. Data are means ± SEM and p value was determined by one-sided unpaired student’s t-test. d Flow cytometry analyses of surface GPR97 levels of gated neutrophils of HC (n = 8) and bacterial sepsis patients (n = 5). Data are means ± SEM and p value was determined by two-sided unpaired student’s t-test. e, f Flow cytometry analyses of surface GPR97 levels of gated neutrophils of HC (n = 5 in e, 6 in f) and patients based on the disease category (GPA n = 8, MPA n = 6) e and status (remission n = 10, active n = 7) f. g The positive correlation of GPR97 levels with the ANCA titers of GPA (n = 8) and MPA (n = 6) patients. Data are means ± SEM and p value was determined by one-way ANOVA. HC healthy control, GPA granulomatosis with polyangiitis, MPA microscopic polyangiitis. Source data are provided in the Source Data file.

Fig. 2. Identification of a putative GPR97-ligand in a distinct human neutrophil subpopulation that is activated upon interaction with GPR97^E-mFc.

a Schematics of GPR97 receptor and GPR97^E-mFc probe. b List of cell samples screened for the expression of GPR97 and the putative GPR97-ligand. * indicates a subpopulation of primary human neutrophils. c A representative bimodal surface expression profile of the GPR97-ligand in human neutrophils detected by the GPR97^E-mFc probe. d Highly variable GPR97-ligand⁻ (blue) and GPR97-ligand⁺ (pink) neutrophil subsets in bloods of different donors (samples A to N). e, f Morphological changes of neutrophils induced by GPR97^E-mFc as observed by inverted light microscope e and Wright-Giemsa stain f. Scale bar, 20 μm. Experiments were repeated 3 times with similar results. g–j Phenotypic analyses of activated neutrophils induced by GPR97^E-mFc included up-regulated ROS production g, MPO activity (h, n = 6), expressional changes of specific CD markers (i, n = 7 for CD54 and n = 4 for others) and IL-8 production (j, n = 3) of independent samples. The mFc protein and fMLF were used as a negative and a positive control, respectively. Data are presented as means ± SEM and p value was determined by two-way ANOVA in h, j and one-way ANOVA in i. Source data are provided in the Source Data file.

Fig. 3. GPR97 is a binding partner and allosteric activator of mPR3.

a Far-western blot analyses of the putative GPR97-ligand(s)(black arrowheads). The EMR2^E-mFc protein was used as a negative control. Anti-CD11b and anti-GADPH Abs detected the corresponding protein markers of the membranous and cytoplasmic fractions, respectively. #, non-specific signals. Experiments were repeated 3 times with similar results. b FACS-based ligand-binding analysis of neutrophils pre-treated without or with phosphoinositide phospholipase C (PI-PLC). c Dot-plots of neutrophils double-stained with GPR97^E-mFc and anti-CD177 or anti-PR3 mAb. d Dot-plots (FSC vs. surface CD177 levels) of neutrophils incubated with GPR97^E-mFc. e IL-8 produced by unsorted and sorted neutrophils incubated with various reagents as indicated. n = 5 independent experiments. Cells treated with fMLF and LPS were included as positive controls. f Far-western blot analysis of specific GPR97-PR3 binding. Samples included total lysates of mock- and CD177-transfected HEK-293T cells as well as purified PR3. The arrowheads indicated the specific signals detected by the anti-PR3 mAb and the GPR97^E-mFc probe. Experiments were repeated 3 times with similar results. g, h The FACS-based PR3-binding assay of unsorted g and FACS-sorted h HEK-293T cells expressing CD177 or GPR97. The black arrows in g indicated the specific PR3-binding. FACS-sorted GPR97^high h and GPR97^low l HEK-293T cells were examined. Numbers on top of the bars indicated the percentage of PR3-binding cell populations. i ELISA-like PR3-binding analyses. CD177-mFc and GPR56-mFc proteins were included as the positive and negative controls, respectively. n = 6 biologically independent samples. j, k ELISA analysis of IL-8 secreted by neutrophils treated without or with indicated protease inhibitors. n = 5 j, 4 k independent experiments. Data are presented as means ± SEM and p value was determined by two-way ANOVA in e, i–k. ns non-significant. l, m The ex vivo mPR3 enzymatic activity of neutrophils incubated in the absence or presence of mFc or GPR97-mFc protein probe without or with protease inhibitors as indicated. Source data are provided in the Source Data file.

Fig. 4. Structural analysis of GPR97-ECR and mapping of the mPR3-binding regions.

a Cartoon representation of the GPR97-ECR structure obtained from X-ray crystallography experiments. The NTD is depicted in purple and the GAIN domain in green and beige (the 13^th β-strand). Cysteine residues involved in linking and stabilising the NTD are shown as yellow sticks. The red asterisk indicates the GPS cleavage site. b Close-up of the two disulphide bridges, C32-C50 and C54-C62, that are involved in the stabilisation of the NTD and GAIN domains. c Close-up on the extensive hydrophobic interactions stabilising the packing of the NTD against the GAIN domain. Residues from the NTD are depicted in purple and those from the GAIN domain are depicted in green. d, e Mapping of the mPR3-binding region(s) using the domain-swapped d and domain-truncated e GPR97-mFc probes as indicated. n = 5 d and 6 e independent experiments. f–h. Activated neutrophil phenotypes, namely morphological changes f, IL-8 production g, and the ex vivo mPR3 enzymatic activity h treated with various GPR97-mFc probes as indicated. n = 3 f, 4 g, and 6 h independent experiments. The bright and dim colors in f represent the percentage of irregular-shaped and round-shaped neutrophils, respectively. The results shown in h are relative Vmax of the ex vivo mPR3 enzymatic activity. Data are presented as means ± SEM and p value was determined by two-way ANOVA. Source data are provided in the Source Data file.

Fig. 5. The CD177/GPR97/PAR2/CD16b receptor complex is required for efficient GPR97-mPR3 interaction that induces PAR2 activation.

a–c Flow cytometry analyses of GPR97-mPR3 interaction in HEK-293T cells expressing various receptors as indicated. a Surface levels of membrane-bound PR3 (green), GPR97^E-mFc (red), and mFc (blue) were detected using appropriate Abs. b, c The percentage of transfectants displaying the positive GPR97^E-mFc binding signal in the ligand-binding assay. n = 3 b, 4 c independent experiments. The underlined receptor in c indicates the substituted component of the core receptor complex. Data are means ± SEM and p value was determined by two-sided unpaired student’s t-test. d The PLA analyses showed the interaction signals of specific receptor pairs of the CD177-associated complex in resting neutrophils. Scale bar, 10 μm. Experiments were repeated 3 times with similar results. e, f Flow cytometry analyses of PAR2 proteolysis in neutrophils e and HEK-293T cells expressing the CD177-associated receptor complex f. Trypsin-treated neutrophils and mFc were included as a positive and a negative control, respectively. n = 5 independent experiments. Data are means ± SEM and p value was determined by one-way ANOVA. g ELISA analyses of IL-8 secreted by neutrophils incubated with the indicated reagents. n = 4 independent experiments. Data are means ± SEM and p value was determined by two-way ANOVA. ns, non-significant. h Schematic diagram of the PR3/CD177/GPR97/PAR2/CD16b complex in neutrophils. CD16, a green-colored receptor with two connected ovals; GPR97, a brown-colored 7TM receptor with two extracellular ovals; PAR2, a blue-colored 7TM receptor; PR3, a purple-colored soluble molecule; CD177, a red-colored receptor with two diamonds. Source data are provided in the Source Data file.

Fig. 6. Up-regulated GPR97 and PAR2 expression in activated neutrophils induces GPR97-PAR2 activation.

a, b Flow cytometry analyses of GPR97 and PAR2 expression in resting neutrophils treated without or with the azurophil granule degranulation stimulants (n = 4, except CD63 n = 3) a and aIgGs (GPR97 n = 3, PAR2 n = 4) b as indicated. a Samples include: 1, fresh neutrophils; 2, untreated neutrophils; 3, neutrophils treated with fMLF (1 μM) for 15 min; 4, neutrophils treated with cytochalasin B (cytoB, 5 μM) for 5 min, followed by fMLF (1 μM) for 10 min. CD63 expression was used as a degranulation marker of azurophil granule. c Relative PLA signals of indicated receptor pairs in resting (n = 3) and aIgG-treated (n = 3) neutrophils. d ELISA analyses of IL-8 secreted by neutrophils incubated for 3 h at 37 °C as indicated (n = 3). Data in a–d are presented as means ± SEM. P value was determined by two-way ANOVA in a, b, d and by one-sided unpaired student’s t-test in c. ns non-significant. e, f ELISA analyses of IL-8 secreted by neutrophils that were incubated with purified IgGs (0.5 mg/mL) from different PR3-ANCA and MPO-ANCA patients for 3 h at 37 °C as indicated. Data are presented as means ± SEM of one representative experiment done in triplicate. ENMD-1068: PAR2 antagonist; SAM11 and MAB3949: anti-PAR2 mAbs; G97-A and BGP: anti-GPR97 mAbs; 2A1: anti-EMR2 mAb. Source data are provided in the Source Data file.

Fig. 7. GPR97-mediated PAR2 activation enhances neutrophil-mediated bacterial phagocytosis and killing as well as endothelial cell activation and dysfunction.

a, b Phagocytosis a and killing b of live bacteria (E. coli n = 10, S. typhimurium n = 13 for phagocytosis assay n = 11 for killing assay, and S. aureus n = 8 for phagocytosis assay n = 11 for killing assay) by neutrophils incubated without or with GPR97^E-mFc. mFc was included as a negative control. Data are means ± SEM. P value was determined by one-way ANOVA. c The expressional analyses of cell activation markers (E-selectin, ICAM-1, VCAM-1 and eNOS) of HUVECs co-cultured with neutrophils in the absence or presence of GPR97^E-mFc. mFc was included as a negative control. HUVECs treated without or with LPS were used as a negative and a positive control, respectively. n = 3 independent experiments. Data are means ± SEM and p value was determined by two-sided unpaired student’s t-test. d, e Endothelial cell permeability assays of HUVECs co-cultured with neutrophils. d Assays were done in HUVEC-neutrophil co-culture in the absence or presence of GPR97^E-mFc for 20 h at 37 °C. mFc was included as a negative control. HUVECs alone treated without or with mFc were negative control groups, while those treated with thrombin were the positive control. n = 4 independent experiments. e The HUVEC-neutrophil co-culture was treated without or with aIgGs in the absence or presence of protease inhibitors/PAR2 antagonists/blocking Abs as indicated. HUVECs alone treated without or with aIgGs were included as controls. n = 3 independent experiments. Data are means ± SEM and p value was determined by two-way ANOVA. ns, non-significant. Source data are provided in the Source Data file.