Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis

Abstract

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology.

Figures

Figure 10. Apoptotic cells expressing phosphatidylserine-associated PR3 induce proinflammatory responses and favor Th9 polarization: in vivo switch to Th17 in the presence of PR3 ANCA.

Phagocytosis of apoptotic cells expressing phosphatidylserine-associated PR3 stimulates the production of inflammatory chemokines by macrophages (depicted in red) through the IL-1R1/MyD88 signaling pathway and NO production, thus promoting the recruitment of inflammatory cells expressing PR3, including neutrophils and monocytes. This process acts as an amplification loop and perpetuates inflammation. The presence of phosphatidylserine-associated PR3 on apoptotic cells generates a proinflammatory microenvironment, which facilitates the production of Th2/Th9- and Th1-activated CD4+ T cells through the interaction between pDCs and naive T cells. Importantly, the presence of anti-PR3 ANCA induces Th17 cell production. Increased G-CSF production by macrophages and by T cells may result in an increased PR3 biosynthesis and mobilization of neutrophils from BM, suggesting that PR3 participates in an autoamplification loop, which in turn sustains inflammation. Expression of phosphatidylserine-associated PR3 on apoptotic neutrophils, which prevents the resolution of inflammation, appears to be a key element in the pathogenesis of GPA. PMN, neutrophils.

Figure 9. Increased G-CSF secretion by macrophages and T cells was associated with increased expression of PR3 in mature neutrophils.

(A) G-CSF concentrations in supernatants from macrophages 24 hours after exposure to medium alone (white bars), apoptotic control cells (gray bars), and PR3-expressing (black bars) cells or (B) G-CSF concentrations in supernatants from pDCs preexposed to the above macrophage supernatants and cocultured with T cells (n = 4 mice per group, each concentration was determined in triplicate). (C) G-CSF concentrations in sera from healthy controls (n = 16) and GPA patients (n = 14). (D) Intracellular content of PR3 in neutrophils from healthy controls (n = 11) or GPA patients (n = 9) determined by flow cytometry analysis. (E) Western blot analysis of PR3 expression in neutrophils from healthy controls (n = 5) and GPA patients (n = 7) (upper panel). Results were quantified by densitometry and normalized to β-actin loading control. Values represent mean ± SEM. Similarly, Western blot analysis (lower panel) was also performed on neutrophils from healthy controls (n = 5) and healthy controls treated with recombinant G-CSF (n = 7). Results were quantified as above. (F) Western blot analysis of PR3 in supernatants of neutrophils treated with PMA, TNF-α, or f-MLF. A representative experiment is shown in the upper panel. The quantification of PR3 by densitometry of all the Western blots performed (healthy controls, n = 4; GPA patients, n = 5) is shown in the lower panel. Values represent mean ± SEM. Significant differences between groups were determined by ANOVA (A and F) or Mann-Whitney U test (C–E). *P < 0.05; **P < 0.01.

Figure 8. Neutrophils, pDCs, and macrophages were located in granulomatous inflammation from GPA patients.

(A) Double-positive CD123 (red staining) and IFN-α cells (brown staining) were located within the granulomatous lesion of lung tissue from GPA patients, and nuclei were counterstained in blue (black arrows). (B) IFN-α+ (brown staining) and CD123+ cells (red staining) were detected at the edge of a neutrophilic abscess in GPA (black arrow). (C–E) Serial sections of a neutrophilic microabscess showed PR3+ (C, brown staining) and LL37+ cells (C, red staining) surrounded by CD68+ macrophages (D, red staining) and IFN-α+ cells (E, brown staining). Lower panels depict higher magnification of the area inside the box in the upper panels. Data display typical images representative of 6 GPA patients. Scale bars: 50 µm (A, C–E, upper panels); 20 µm (B, C–E, lower panels).

Figure 7. CD4+ T cells from GPA patients display a skewed Th9 cell distribution.

(A) Representative dot plots demonstrating the gating strategy used to identify Th1, Th2, Th17, and Th9 cells in human PBMCs. (B) The normalized ratio of Th2/Th1, Th17/Th1 and Th9/Th1 in circulating CD4+ T cells was determined in both healthy controls (white bars) and GPA patients (black bars). The percentages of Th9 cells were also calculated (n = 5 healthy controls and n = 6 GPA). (C and D) Sera from healthy controls (n = 16) and GPA patients (n = 12) were assessed for IL-4, IL-5, IL-17A, IL-9, and for IL-1β and IL-1RA using a Luminex assay. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Significant differences between groups were determined by multiple comparisons ANOVA (B–D).

Figure 6. pDCs exposed in vivo to a PR3-induced microenvironment triggered the production of Th9 cells, and addition of anti-PR3 ANCA facilitated Th17 cell polarization.

(A) pDCs isolated from mice following the i.v. injection of vehicle (white triangles), apoptotic controls (white circles), PR3- (black squares), or PR3/4H4A-expressing cells (black diamonds) were cultured with naive CD25–CD4+ T cells from OTII/Rag1–/– mice in the presence of OVA peptide. Flow cytometry analysis was performed 4 days later, and the percentages of Tregs (CD4+CD25+FOXP3+), Th9 (CD4+IL-9+), Th2 (CD4+IL-4+), Th1 (CD4+IFN-γ+), and Th17 (CD4+IL-17A+) cells determined. (B) The above experiments were also performed with the concomitant injection of anti-PR3 ANCA or control IgG in the presence of apoptotic control (white bars) or PR3-expressing cells (black bars). T cell polarization was analyzed using flow cytometry. Data are presented as mean ± SEM. n = 3 mice per group, each mice studied in triplicate. Significant differences between groups were determined by ANOVA with multicomparison analysis. *P < 0.05; **P < 0.01; ***P < 0.001. The experiment presented in A was reproduced twice with identical results.

Figure 5. pDCs exposed in vitro to a PR3-induced microenvironment triggered a skewed Th2/Th9 cell distribution.

(A) Representative dot plots demonstrating FOXP3 expression in CD4+CD25+ T cells and IFN-γ and IL-17A expression in CD4+ T cells. Flow cytometry analysis of naive CD4+CD25– OVA-specific OTII T cells cultured for 4 days with pDCs preexposed to macrophage supernatants generated from medium alone (white triangles), apoptotic controls (white circles), or apoptotic PR3-expressing cells (black squares). (B) Percentages of Tregs (CD4+CD25+FOXP3+), Th1 cells (CD4+IFN-γ+), and Th17 cells (CD4+IL-17A+) obtained following pDCs cocultured with naive T cells. (C) IL-4, IL-5, IL-9, IL-17, and IFN-γ secretion measured in media collected from the coculture experiments using a Luminex assay. Values are shown as mean ± SEM; n = 5 mice per group, each parameter determined in triplicates. *P < 0.05; **P < 0.01; ***P < 0.001. Significant differences between groups were determined by multicomparison ANOVA (B and C).

Figure 4. The proinflammatory response induced by PR3 on apoptotic cells was dependent on the IL-1R1/MyD88 signaling pathway and mediated by NO.

Thioglycolate-elicited macrophages from C57BL/6 (n = 11), Myd88–/– (n = 12), Tlr2–/– (n = 8), Tlr4–/– (n = 8), Il1r1–/– (n = 6), and Nos2–/– (n = 6) mice were cultured for 24 hours with apoptotic control or PR3-expressing cells and MCP-1 secretion was assessed in duplicate by ELISA. Values are shown as mean ± SEM. *P < 0.05; **P < 0.01. Significant differences between groups were determined by the Mann-Whitney U test.

Figure 3. Membrane expression of PR3 on apoptotic cells induced a proinflammatory response in macrophages and impaired their antiinflammatory reprogramming following efferocytosis in vitro.

(A) Thioglycolate-elicited macrophages were cultured for 24 hours in vitro with medium alone, with control apoptotic cells or with apoptotic cells expressing either PR3 or PR3/4H4A. Cytokine (IL-6, IL-1β, TNF-α, IL-12p70) and chemokine (RANTES, KC, MIP-1α, and MCP-1) production were assessed using a Luminex assay (n = 4 mice per group, each cytokine measured in triplicate; mean ± SEM). (B) iNOS expression in macrophages was evaluated using Western blot analysis (135 kDa), and β-actin was used as a loading control. Results represent 3 independent experiments. (C) Macrophages previously exposed to medium alone, apoptotic control cells, or PR3 or PR3/4H4A cells were stimulated with LPS (10 ng/ml) for 24 hours. Culture media was assessed for IL-6, GM-CSF, IL-12p70, and MIP-1α using a Luminex assay. Values are represented as mean ± SEM, n = 4 mice per group, each cytokine measured in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001. Significant differences between groups were determined by multicomparison ANOVA (A and C).

Figure 2. Membrane expression of PR3 on apoptotic cells triggered a proinflammatory response in vivo that required serine protease activity.

(A) Apoptotic control (CT) (white circles ) or PR3-expressing (black squares) cells were injected i.p. and peritoneal lavage fluid collected after 2 hours. IL-6, TNF-α, KC, G-CSF, IL-12p70, MCP-1, IL-2, and IL-1β were assessed using the proinflammatory mouse cytokine assay (n = 5 mice per group, each cytokine measured in triplicate). (B) Using flow cytometry, polarization of F4/80+ macrophages was examined by assessing expression of various cell-surface markers, including CD11b, TLR4, MHCII, CD11c, CD206, CD23, and CD16/CD32 (n = 4 mice per group, each marker measured in duplicate). (C) Apoptotic control (n = 8), PR3- (n = 6), or PR3/S203A-expressing cells (n = 8, white diamonds) were injected i.p. in mice for 2 hours as in A and MCP-1 measured in duplicates in the peritoneal lavage by ELISA. (D) PBS (white triangles), apoptotic control, or PR3-expressing cells were injected i.p. 72 hours after peritonitis was induced with thioglycolate. The peritoneal lavage fluid was collected after a further 24 hours and concentrations of MIP-1α, MIP-1β, MCP-1, and RANTES determined (n = 5 mice per group, each cytokine measured in triplicate). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Significant differences between groups were determined by multicomparison ANOVA (A–C) or Mann-Whitney U test (D).

Figure 1. PR3 membrane expression on apoptotic neutrophils was increased in GPA.

(A and B) Membrane expression of PR3 and CD11b on apoptotic neutrophils (CD15+Annexin V+7AAD–) from healthy controls (HC) (n = 8) and GPA patients (n = 10) was assessed in whole blood maintained at 37°C for 20 hours. Values are presented as mean ± SEM. **P < 0.01. (C) PR3 expression on unstimulated neutrophils (basal) correlated with PR3 expression on TNF-α–stimulated neutrophils. (D) No correlation in membrane expression of PR3 (mean fluorescence intensity [MFI]) on basal and apoptotic neutrophils was observed. Data are presented for n = 19 healthy controls (white circles) and n = 15 GPA (black squares) with a mean age of 47.6 ± 3.2 versus 51.7 ± 4.4 years, respectively, and with a similar sex ratio, provided in Supplemental Table 1. (E) Within granulomatous inflammation in GPA, PR3+ (green, upper right panel) and cleaved caspase 3+ (red, lower left panel) cells detected by immunofluorescence analyses using confocal microscopy colocalized within the same cell (merge, lower right panel). Nuclei visualized using DAPI are depicted in gray (upper left panel). Scale bars: 5 μm. Significant differences between groups were determined by Mann-Whitney U test (A and B), and correlations were assessed using Pearson’s tests (C and D).