1. Volume 93, Issue 3, Pages 615-625 (March 2018)
C5a receptor 1 promotes autoimmunity, neutrophil dysfunction and injury in experimental anti-myeloperoxidase glomerulonephritis 
Jonathan Dick, Poh-Yi Gan, Sharon L. Ford, Dragana Odobasic, Maliha A. Alikhan, Sven H. Loosen, Pam Hall, Clare L. Westhorpe, Anqi Li, Joshua D. Ooi, Trent M. Woodruff, Charles R. Mackay, A. Richard Kitching, Michael J. Hickey, Stephen R. Holdsworth 
Kidney International 
Volume 93, Issue 3, Pages (March 2018)
DOI: /j.kint
Copyright © 2017 International Society of Nephrology Terms and Conditions

2. Figure 1
Endogenous C5a promotes cellular and humoral immunity to myeloperoxidase (MPO). Cellular immune responses were measured 10 days after immunization of mice with native murine MPO (nmMPO) in Freund complete adjuvant. Compared with WT mice, C5aR1−/− mice showed (a) reduced proliferation of antigen restimulated cells from draining lymph nodes measured by 3H-thymidine, (b) reduced MPO-specific T helper (Th)1 response measured by interferon (IFN) gamma enzyme-linked ImmunoSpot. (c) Th17 response measured by interleukin (IL)-17A enzyme-linked ImmunoSpot was similar. (d) The proportion of CD4+ cells in the draining lymph nodes that were CD25+Foxp3+ T regulatory cells was increased in C5aR1−/− mice. (e) Representative flow cytometry plot gated on CD4+ cells showing CD25+Foxp3+ population. (f) Antibody responses were measured 28 days after nmMPO immunization. Compared with wild-type (WT) mice, C5aR1−/− mice exhibited reductions in total MPO-ANCA (anti-neutrophil cytoplasmic antibody) and IgG2b MPO-ANCA, titers of other subclasses were similar. (g) Total serum IgG 28 days after immunization was similar between groups. *P < 0.05, **P < 0.01, ***P < by Mann-Whitney U test. ns, not significant.
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

3. Figure 2
C5aR1 inhibition attenuates anti–myeloperoxidase (MPO) autoimmunity. Cellular immunity was measured 10 days after immunization with native murine MPO in Freund complete adjuvant in mice receiving the C5aR1 inhibitor PMX53 or vehicle delivered by osmotic infusion pump. C5aR1 inhibition resulted in (a) reduced proliferation of MPO restimulated cells (b) and reduced T helper (Th)1 response. (c) There was no difference in Th17 response or (d) the proportion of CD4+ cells that were CD25+Foxp3+. *P < 0.05, **P < 0.01 by Student t test. IL, interleukin; ns, not significant.
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

4. Figure 3
The C5aR1 on antigen-presenting cells modulates autoimmunity to myeloperoxidase. Bone marrow–derived dendritic cells (BMDCs) were grown from wild-type (WT) or C5aR1−/− mice. Day 8 BMDCs were stimulated for 24 hours with 25 nM rC5a and 1 μg/ml lipopolysaccharide before surface expression of costimulatory molecules and supernatant cytokines were analyzed. (a–d) WT BMDCs displayed higher expression of major histocompatibility complex (MHC)-II; other costimulatory molecules were not different. (e–g) C5aR1−/− BMDCs secrete less tumor necrosis factor (TNF) and interleukin (IL)-1β than WT DCs; levels of IL-12p70 were similar. (h) C5aR1−/− DCs secrete more IL-10 than WT DCs. (i) Day 8 BMDCs were incubated with 10 μg/ml DQ-OVA for 120 minutes before antigen uptake was analyzed by flow cytometry. Data expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < by Student t test (a–g) and Mann-Whitney U test (h and i). MFI, mean fluorescence intensity.
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

5. Figure 4
C5aR1 on dendritic cells (DCs), but not T cells promotes anti-myeloperoxidase (MPO) autoimmunity. (a) Day 8 bone marrow–derived DCs (BMDCs) were incubated for 120 minutes with 10 μg/ml recombinant murine MPO before being matured for 18 hours with 1 μg/ml lipopolysaccharide. The cells were extensively washed before 1 × 106 cells were injected into wild-type (WT) mice. Cellular immunity was measured 10 days after immunization. (b) WT mice receiving C5aR1−/− MPO pulsed DCs had a reduced interferon (IFN) gamma producing lymphocytes, (c) similar interleukin (IL)-17A producing lymphocytes, and (d) an increased proportion of CD25+Foxp3+ T regulatory cells. (e) 5 × 106 CD4+Foxp3− cells from Foxp3GFP or C3aR−/−C5aR1−/−Foxp3GFP mice were injected into Rag1−/− recipients. These were immunized with native murine MPO in Freund complete adjuvant and cellular immunity was measured 10 days later. (f) There was no difference in the proportion of CD4+ IFN-γ+, IL-17A+, or Foxp3+ cells. *P < 0.05 by Student t test (b, c, and f) and Mann-Whitney U test (d). ns, not significant.
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

6. Figure 5
Autoimmunity transferred by C5aR1−/− dendritic cells (DCs) results in attenuated anti-myeloperoxidase (MPO) glomerulonephritis. (a) MPO autoimmunity was induced by immunization of WT mice with recombinant murine MPO pulsed wild-type (WT) or C5aR1−/− bone marrow–derived DCs (BMDCs). Glomerulonephritis was triggered by a sub-nephritogenic dose of anti–glomerular basement membrane (GBM) IgG. (b,c) Mice receiving C5aR1−/− BMDCs developed significantly less albuminuria and glomerular necrosis. (d) Segmental glomerular necrosis in mice receiving WT DCs. Sections stained with periodic acid–Schiff reagent, magnification ×400, bar = 30 μm. (e–g) The accumulation of CD4+ T cells, neutrophils, and macrophages in glomeruli was attenuated in mice receiving C5aR1−/− BMDCs. (h) MPO-ANCA (anti-neutrophil cytoplasmic antibody) titers were reduced in mice receiving C5aR1−/− DCs. *P < 0.05, **P < 0.01 by Student t test. GCS, glomerular cross-section. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

7. Figure 6
Myeloperoxidase (MPO)–anti-neutrophil cytoplasmic antibody (ANCA) induces neutrophil retention in the glomerular capillaries, which is attenuated by C5aR1 inhibition. Multiphoton microscopy was used to analyze neutrophil recruitment in wild-type mice treated with either MPO-ANCA or anti-OVA IgG (as control), following priming with systemic lipopolysaccharide. (a) Sequence of images showing an anti-Gr-1-PE stained neutrophil (red, outlined by fine dotted line) undergoing prolonged intravascular migration in the glomerulus following the administration of anti-MPO IgG. (Dotted line denotes migration path; time stamp shown below images.) Vasculature (blue) is labeled by Pacific Blue dextran. Bar = 10 μm (see also Supplementary Movies S1 and S2). (b) MPO-ANCA does not increase the number of adherent neutrophils in the glomerulus (glom). (c) MPO-ANCA causes an increase in glomerular dwell time due to prolonged retention of crawling, but not static neutrophils. (d) C5aR1 inhibition reduces glomerular crawling time compared with control. **P < 0.01 by Student t test. ns, not significant. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

8. Figure 7
Myeloperoxidase (MPO)–anti-neutrophil cytoplasmic antibody (ANCA)–induced glomerular neutrophil reactive oxygen species (ROS) production is abrogated by C5aR1 inhibition. (a) Multiphoton intravital microscopy image showing neutrophil ROS generation in a mouse following the administration of MPO-ANCA. Neutrophils were detected using anti-Gr-1 (green, arrowhead), whereas ROS generation was detected using the oxidant-sensitive fluorochrome dihydroethidium (DHE) (red, arrow). Bar = 10 μm. (b) MPO-ANCA induces ROS production by intraglomerular neutrophils as compared with similarly treated mice following anti-OVA administration. (c) Histogram showing time-course of ROS production in DHE-positive adherent neutrophils. (d) MPO-ANCA–induced ROS production in glomerular neutrophils is abrogated by C5aR1 inhibition. *P < 0.05, **P < 0.01 by Student t test (b) and Mann-Whitney U test (d). To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions