Research ArticleINFLAMMATION

Atypical complement receptor C5aR2 transports C5a to initiate neutrophil adhesion and inflammation

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Science Immunology  10 May 2019:
Vol. 4, Issue 35, eaav5951
DOI: 10.1126/sciimmunol.aav5951
  • Fig. 1 Role for C5aR2 on nonhematopoietic cells in IC-induced arthritis.

    (A) Schematic of C5aR2 BMC mice generation. WT or C5ar2-KO BM was transferred into lethally irradiated WT or C5ar2-KO mice. (B) Polymorphonuclear neutrophil (PMN) counts in the blood of BMC mice. (C to G) After reconstitutions, BMC mice were intraperitoneally treated with AST on days 0 and 2. n.s., not significant. (C) Clinical arthritis score. (D) Change in ankle thickness. (E) Number of neutrophils recovered from the SF on day 7 after AST. (F and G) Representative H&E staining and histopathological score on day 7 after AST. Arrowheads indicate inflammatory cell infiltration. Scale bars, 200 μm. Data indicate means ± SEM. n = 4 to 6 mice per group and P value calculated using ordinary one-way ANOVA with a post hoc Tukey’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 2 Role for ACKR1 on nonhematopoietic cells in IC-induced arthritis.

    (A) Schematic generation of ACKR1 BMC mice. WT or Ackr1-KO BM was transferred into lethally irradiated WT or Ackr1-KO mice. (B) Neutrophil counts in the blood of BMC mice. (C to G) BMC mice were intraperitoneally treated with AST on days 0 and 2. (B) Clinical arthritis score. (C) Change in ankle thickness. (D) Number of neutrophils in SF derived from each BMC mice on day 7 after AST. (E and F) Representative H&E staining and histopathological score of BMC mice on day 7 after AST. Arrowheads indicate inflammatory cell infiltration. Scale bars, 200 μm. Data indicate means ± SEM. n = 5 mice per group and P value calculated using ordinary one-way ANOVA with a post hoc Tukey’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 C5aR2 expression on joint endothelium.

    (A) C5ar2 mRNA levels in the ST of untreated WT mice and WT mice on day 7 after AST. Data indicate means ± SEM. n = 3 mice per condition and P value calculated using unpaired two-tailed Student’s t test. **P < 0.01. (B) Cell surface protein expression of C5aR2 on CD45CD31+ ECs, CD45CD31 cells, and CD45+ cells was determined by flow cytometry on live cells recovered from ST of untreated or day 7 AST-treated WT and C5ar2-KO mice. SSC-A, side scatter area; FSC-A, forward scatter area; FSC-H, forward scatter height. (C) Populations of C5aR2-expressing cells isolated from ST of untreated or day 7 AST-treated WT littermate controls and C5ar2-KO mice. n = 4 mice per group.

  • Fig. 4 Imaging joints of live WT, Ackr1-KO, and C5aR2-KO BMC mice on days 3 and 7 after AST.

    BMC (A) Schematic BMC mice generation. WT-LysM-GFP BM was transferred into lethally irradiated WT, Ackr1-KO, or C5ar2-KO mice. After reconstitution, joints were imaged on days 3 and 7 after AST. (B and C) In vivo joint imaging of WT, Ackr1-KO, or C5ar2-KO BMC mice for 35 min on day 3 (B) and day 7 (C) after AST. Scale bars, 50 μm. (D) Number of newly adherent neutrophils on joint endothelium of WT, Ackr1-KO, or C5ar2-KO BMC mice. (E) Number of newly extravasated neutrophils in joint tissue of WT, Ackr1-KO, or C5ar2-KO BMC mice. (F) Percentage of adherent neutrophils that extravasated within joints of WT, Ackr1-KO, or C5ar2-KO BMC mice. (G) Number of neutrophils observed in ST of WT, Ackr1-KO, or C5ar2-KO BMC. Data indicate means ± SEM. n = 3 to 4 mice per group and P value calculated using ordinary one-way ANOVA with a post hoc Tukey’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 5 In vivo superfusion assays.

    WT-LysM-GFP BM was transferred into lethally irradiated WT, Ackr1-KO, or C5ar2-KO mice as in Fig. 4. After reconstitution, the indicated chemoattractants (0.1 pmol) dissolved in sterile PBS (100 μl) were superfused onto surgically exposed joints, which were then imaged for 70 min. Cumulative graph of neutrophil adhesion (A, C, and E) and extravasation (B, D, and F) after (A and B) C5a application onto joints of WT and C5ar2-KO BMC mice, (C and D) CXCL1 application onto joints of WT and Ackr1-KO BMC, and (E and F) CXCL2 application onto joints of WT and Ackr1-KO BMC. Data indicate means ± SEM. n = 3 mice per group and P value calculated using ordinary one-way ANOVA with a post hoc Tukey’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 6 Sequential in vivo superfusion assays.

    WT-LysM-GFP BM was transferred into lethally irradiated WT, Ackr1-KO, or C5ar2-KO mice as in Fig. 4. After reconstitution, the indicated chemoattractants (0.1 pmol) dissolved in sterile PBS (100 μl) were superfused onto surgically exposed joints. (A and B) C5a application followed 35 min later by the application of other chemoattractants (CAs), including CXCL1, LTB4, or CXCL2 onto joints of C5ar2-KO BMC. (C and D) CXCL1 application followed 35 min later by the application of other CAs, inclduing C5a, LTB4, CXCL2, or PBS onto joints of WT and Ackr1-KO BMC. Data indicate means ± SEM. n = 3 mice per group and P value calculated using ordinary one-way ANOVA with a post hoc Tukey’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 7 Imaging C5ar2-dependent transport of C5a into the blood vessel lumen in vivo.

    (A) Schematic of experimental design. WT, C5ar2-KO, and Ackr1-KO mice were intravenously injected with AF647-conjugated CD31 mAb (20 μg per mouse) and AF488-conjugated C5aR2 mAb (15 μg per mouse) 30 min before AF594-conjugated C5a was applied onto surgically exposed joints. Joints were then imaged by MP-IVM for 30 min in live mice. (B) Representative frames from in vivo imaging videos. Scale bars, 10 μm. J, joint tissue; B, blood vessel. (C) Mean fluorescence intensity of C5a deposition on joint endothelium. Data indicate means ± SEM. n = 4 mice per group and P value calculated using ordinary one-way ANOVA with a post hoc Tukey’s test for multiple comparisons. ****P < 0.0001.

  • Fig. 8 Sequential roles of atypical chemoattractant receptors C5aR2 and ACKR1 required for neutrophil arrest and subsequent TEM.

    The atypical chemoattractant receptors C5aR2 and ACKR1 are required for neutrophil entry into the joint and the development of inflammatory arthritis. In the KxB/N model of IC-induced inflammatory arthritis, the atypical C5a receptor, C5aR2, expressed on ECs transports C5a generated within the joint into the vessel lumen ①. C5a is then captured on the surface of ECs by heparan sulfate proteoglycans (HSPGs) where it is presented to circulating neutrophils and initiates C5aR1-mediated arrest ②. The atypical chemokine receptor, ACKR1, is also expressed on ECs where it transports CXCR2 ligands produced within the joint into the lumen of the overlying blood vessel ③ to initiate CXCR2-mediated neutrophil TEM and entry into the joint ④. Thus, atypical chemoattractant receptors collaborate with classical signaling chemoattractant receptors to control the recruitment of neutrophils into tissue sites of inflammation.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/35/eaav5951/DC1

    Fig. S1. C5aR2 contributes to the pathogenesis of IC-induced arthritis.

    Fig. S2. ACKR1 contributes to the pathogenesis of IC-induced arthritis.

    Fig. S3. C5aR2 expression on joint endothelium.

    Fig. S4. In vivo superfusion assays.

    Fig. S5. Immunofluorescence analysis of C5a expression on joint tissue harvested from WT and C5ar2-KO mice on day 7 after AST.

    Fig. S6. In vivo superfusion assay using AF594-C5a.

    Fig. S7. Analysis of CXCL1 transport into the lumen of joint blood vessels in WT, C5ar2-KO, and Ackr1-KO mice.

    Fig. S8. Representative gating strategy of Ly6G+ neutrophil.

    Movie S1. In vivo MP-IVM joint imaging of live WT, C5ar2-KO, and Ackr1-KO BMC mice on day 3 after AST.

    Movie S2. In vivo MP-IVM joint imaging of live WT, C5ar2-KO, and Ackr1-KO BMC mice on day 7 after AST.

    Movie S3. In vivo superfusion assay (application of C5a).

    Movie S4. In vivo superfusion assay (application of CXCL1).

    Movie S5. In vivo superfusion assay (application of CXCL2).

    Movie S6. Sequential in vivo superfusion assay (application of C5a followed by application of CXCL1, CXCL2, or LTB4).

    Movie S7. Sequential in vivo superfusion assay (application of CXCL1 followed by application of CXCL1, CXCL2, or LTB4).

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. C5aR2 contributes to the pathogenesis of IC-induced arthritis.
    • Fig. S2. ACKR1 contributes to the pathogenesis of IC-induced arthritis.
    • Fig. S3. C5aR2 expression on joint endothelium.
    • Fig. S4. In vivo superfusion assays.
    • Fig. S5. Immunofluorescence analysis of C5a expression on joint tissue harvested from WT and C5ar2-KO mice on day 7 after AST.
    • Fig. S6. In vivo superfusion assay using AF594-C5a.
    • Fig. S7. Analysis of CXCL1 transport into the lumen of joint blood vessels in WT, C5ar2-KO, and Ackr1-KO mice.
    • Fig. S8. Representative gating strategy of Ly6G+ neutrophil.
    • Legends for movies S1 to S7

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). In vivo MP-IVM joint imaging of live WT, C5ar2-KO, and Ackr1-KO BMC mice on day 3 after AST.
    • Movie S2 (.mp4 format). In vivo MP-IVM joint imaging of live WT, C5ar2-KO, and Ackr1-KO BMC mice on day 7 after AST.
    • Movie S3 (.mp4 format). In vivo superfusion assay (application of C5a).
    • Movie S4 (.mp4 format). In vivo superfusion assay (application of CXCL1).
    • Movie S5 (.mp4 format). In vivo superfusion assay (application of CXCL2).
    • Movie S6 (.mp4 format). Sequential in vivo superfusion assay (application of C5a followed by application of CXCL1, CXCL2, or LTB4).
    • Movie S7 (.mp4 format). Sequential in vivo superfusion assay (application of CXCL1 followed by application of CXCL1, CXCL2, or LTB4).

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