CSF2-dependent monocyte education in the pathogenesis of ANCA-induced glomerulonephritis

Abstract

Objectives: Myeloid cell activation by antineutrophil cytoplasmic antibody (ANCA) is pivotal for necrotising vasculitis, including necrotising crescentic glomerulonephritis (NCGN). In contrast to neutrophils, the contribution of classical monocyte (CM) and non-classical monocyte (NCM) remains poorly defined. We tested the hypothesis that CMs contribute to antineutrophil cytoplasmic antibody-associated vasculitis (AAV) and that colony-stimulating factor-2 (CSF2, granulocyte-macrophage colony-stimulating factor (GM-CSF)) is an important monocyte-directed disease modifier.

Methods: Myeloperoxidase (MPO)-immunised MPO^-/- mice were transplanted with haematopoietic cells from wild-type (WT) mice, C-C chemokine receptor 2 (CCR2)^-/- mice to abrogate CM, or transcription factor CCAAT-enhancer-binding protein beta (C/EBPβ)^-/- mice to reduce NCM, respectively. Monocytes were stimulated with CSF2, and CSF2 receptor subunit beta (CSF2rb)-deficient mice were used. Urinary monocytes and CSF2 were quantified and kidney Csf2 expression was analysed. CSF2-blocking antibody was used in the nephrotoxic nephritis (NTN) model.

Results: Compared with WT mice, CCR2^-/- chimeric mice showed reduced circulating CM and were protected from NCGN. C/EBPβ^-/- chimeric mice lacked NCM but developed NCGN similar to WT chimeric mice. Kidney and urinary CSF2 were upregulated in AAV mice. CSF2 increased the ability of ANCA-stimulated monocytes to generate interleukin-1β and to promote T_H17 effector cell polarisation. CSF2rb^-/- chimeric mice harboured reduced numbers of kidney T_H17 cells and were protected from NCGN. CSF2 neutralisation reduced renal damage in the NTN model. Finally, patients with active AAV displayed increased urinary CM numbers, CSF2 levels and expression of GM-CSF in infiltrating renal cells.

Conclusions: CMs but not NCMs are important for inducing kidney damage in AAV. CSF2 is a crucial pathological factor by modulating monocyte proinflammatory functions and thereby T_H17 cell polarisation.

Keywords: Autoantibodies; Autoimmune Diseases; Granulomatosis with polyangiitis; Inflammation; Systemic vasculitis.

Figure 1

CCR2^−/− chimeric mice are protected from anti-MPO-induced NCGN. (A) Experimental protocol describing the induction of NCGN in MPO^−/−. Chimeric mice were analysed 7–8 weeks following BM transplantation. (B) CCR2^−/− chimeric mice showed reduced renal damage compared with WT chimeric mice with significant reduction of crescentic and necrotic glomeruli. For each group, a representative image of a kidney section stained with PAS at high magnification (×40) is shown. (C) CCR2^−/− chimeric mice displayed reduced Ery by dipstick. Leu and Prot were similar in both groups. (D) Reduction in albuminuria by ELISA in CCR2^−/− chimeric mice. (E) Urinary NGAL levels by ELISA were similar in CCR2^−/− and WT chimeric mice. (F) Renal CD11b⁺Ly6G⁺ neutrophils, CD11b⁺Ly6G⁻Ly6C⁺ CMs and CD11b⁺Ly6G⁻Ly6C⁻MHC-II⁻CD11c^mid/+ NCMs were analysed by flow cytometry. Infiltration of CM was strongly reduced in CCR2^−/− chimeric mice, whereas infiltration of neutrophils and NCMs was similar in both groups. The number of immune cells is expressed per gram kidney and calculated using counting beads. (G) Representative flow cytometry plot showing that the renal infiltration of CM is strongly reduced in CCR2^−/− chimeric mice. *P<0.05, **P<0.01, ***P<0.001. CCR2, C–C chemokine receptor 2; AU, arbitrary unit; CM, classical monocyte; Ery, erythrocyturia; Leu, leukocyturia; mMPO, murine MPO; MPO, myeloperoxidase; NCGN, necrotising crescentic glomerulonephritis; NCM, non-classical monocyte; NGAL, neutrophil gelatinase-associated lipocalin; PAS, periodic acid-Schiff; Prot, proteinuria; WT, wild type.

Figure 2

Absence of NCMs in C/EBPβ^−/− chimeric mice does not affect anti-MPO-induced NCGN. (A) Experimental scheme describing the induction of NCGN in MPO^−/− mice. (B) No difference in histological renal damage between C/EBPβ^−/− and WT chimeric mice. For each group, a representative image of a kidney section stained with PAS at high magnification (×40) is shown. (C) C/EBPβ^−/− chimeric mice showed no difference in Leu, Prot and Ery by dipstick compared with C/EBPβ^+/+ chimeric mice. (D) Albuminuria by ELISA was similar in both groups. (E) Urinary NGAL levels by ELISA were similar in both groups. (F) Renal CD11b⁺Ly6G⁺ neutrophils, CD11b⁺Ly6G⁻Ly6C⁺ CMs and CD11b⁺Ly6G⁻Ly6C⁻MHC-II⁻CD11c^mid/+ NCMs were analysed by flow cytometry. Infiltration of NCM was strongly reduced in C/EBPβ^−/− chimeric mice, whereas infiltration of neutrophils and CM was similar in both groups. The number of immune cells is expressed per gram kidney and calculated using counting beads. (G) Representative flow cytometry plot showing that the renal infiltration of NCM is strongly reduced in C/EBPβ^−/− chimeric mice. **P<0.01. AU, arbitrary unit; C/EPBβ, CCAAT–enhancer-binding protein beta; CM, classical monocyte; Ery, erythrocyturia; Leu, leukocyturia; MPO, myeloperoxidase; mMPO, murine MPO; NCGN, necrotising crescentic glomerulonephritis; NCM, non-classical monocyte; NGAL, neutrophil gelatinase-associated lipocalin; PAS, periodic acid-Schiff; Prot, proteinuria; WT, wild type.

Figure 3

CSF2 expression is increased in kidneys from mice with anti-MPO-induced NCGN. (A) Immunofluorescence images show strong CSF2 expression in kidney sections from mice with AAV compared with Ctrl mice. A representative image at magnification ×40 is shown for each group. 4',6-Diamidin-2-phenylindol (DAPI) was used to stain nuclei (blue). (B) CSF2 level in urine by ELISA is increased in AAV mice compared with Ctrl mice. (C) CSF2 level by ELISA in renal lysates is increased in AAV mice compared with Ctrl mice. (D) Csf2 mRNA by RT-PCR expression in kidney lysates is increased in AAV mice compared with Ctrl mice. (E) Correlation between the percentage of crescents and the amount of renal CSF2. **P<0.01, ***P<0.001. AAV, antineutrophil cytoplasmic antibody-associated vasculitis; AU, arbitrary unit; CSF2, colony-stimulating factor-2; Ctrl, control; MPO, myeloperoxidase; NCGN, necrotising crescentic glomerulonephritis.

Figure 4

Csf2rb^−/− chimeric mice are protected from anti-MPO-induced NCGN. (A) Experimental settings describing the induction of NCGN. (B) Csf2rb^−/− chimeric mice developed less renal damage compared with WT chimeric mice with a reduction of crescentic and necrotic glomeruli. A representative image of a kidney section stained with PAS at high magnification (×40) is shown for each group. (C) Leu, Prot and Ery by urine dipstick are similar in both groups. (D) Albuminuria and (E) NGAL urine levels by ELISA were similar in both groups. (F) Renal infiltration of immune cells was analysed by flow cytometry. Infiltration of CD11b⁺Ly6G⁺ neutrophils and CD11b⁺Ly6G⁻Ly6C⁺ CMs was similar in both groups, whereas CD11b⁺Ly6G^-Ly6C⁻MHC-II⁻CD11c^mid/+ NCM infiltration was slightly increased in Csf2rb^−/− chimeric mice compared with WT chimeric mice. The number of immune cells is expressed per gram kidney and calculated using counting beads. *P<0.05. AU, arbitrary unit; CM, classical monocyte; Csf2rb, CSF2 receptor subunit beta; Ery, erythrocyturia; Leu, leukocyturia; MPO, myeloperoxidase; NCGN, necrotising crescentic glomerulonephritis; NCM, non-classical monocyte; NGAL, neutrophil gelatinase-associated lipocalin; PAS, periodic acid-Schiff; Prot, proteinuria; WT, wild type.

Figure 5

CSF2 increases the capacity of anti-MPO stimulated monocytes to release IL-1β and polarise T_H17 CD4⁺ T cells. (A) TNFα-primed human or murine neutrophils were isolated and stimulated with either isotype IgG or anti-MPO IgG. Unstimulated cells were used as negative controls. Addition of recombinant murine or human CSF2 (10 ng/mL) increases IL-1β generation by TNFα-primed-monocytes stimulated with anti-MPO as measured in culture medium by ELISA. (B, C) CD4⁺ T cells were sorted from murine spleen and cultured in medium containing anti-IFN-γ, anti-IL-4, anti-IL-2 antibodies, together with supernatants from TN-α-primed monocytes stimulated with either isotype IgG or anti-MPO IgG. Supernatants from unstimulated monocytes were used as negative control. Addition of recombinant murine CSF2 (10 ng/mL) increases the capacity of TNFα-primed anti-MPO stimulated monocytes to induce T_H17 polarisation. A representative flow cytometry plot is shown. (D) Renal T_H17 CD4⁺ T cells were analysed by flow cytometry in WT and Csf2rb^−/− chimeric mice. **P<0.01, ***P<0.001. CSF2, colony-stimulating factor-2; Csf2rb, CSF2 receptor subunit beta; Ctrl, control; IFN-γ, interferon gamma; IL, interleukin; MPO, myeloperoxidase; TNFα, tumour necrosis factor alpha; WT, wild type.

Figure 6

CSF2 and monocyte subsets in patients with AAV and HCs. (A) Human kidney biopsies from patients with AAV were analysed for in situ expression of Csf2 usingRNAscope Probe-Hs-CSF2 mRNA and appropriate positive and negative controls. Counterstaining was done with haematoxylin. A representative picture at ×40 magnification is shown for both groups (left: negative control, right: RNAscope for Csf2). (B) CSF2 protein in human urines was measured by ELISA. CSF2 was increased in urine from patients with active AAV compared with HCs. (C, D) Myeloid cells in urines from patients with active AAV with kidney involvement (kAAV), patients in Rem and HCs were analysed by flow cytometry. Neutrophils, monocytes and the three different monocyte subsets were quantified and expressed as cell number per 100 mL urine. Total neutrophil and monocyte numbers are significantly higher in patients with kAVV compared with HCs. CD14⁺CD16⁻ CMs, CD14⁺CD16⁺ IMs and CD14⁻CD16⁺ NCMs are significantly increased in urine from patients with kAAV compared with HC. In addition, CMs are increased in urine from patients with kAAV compared with patients with AAV in Rem. (E) Dot plots representing the distribution of the different monocyte subsets in urine from patients with kAAV and Rem. *P<0.05, **P<0.01, ***P<0.001. AAV, antineutrophil cytoplasmic antibody-associated vasculitis; CM, classical monocyte; CSF2, colony-stimulating factor-2; HC, healthy control; IM, intermediary monocyte; kAAV, antineutrophil cytoplasmic antibody-associated vasculitis kidney manifestation; NCM, non-classical monocyte; ns, not significant; Rem, remission.

Figure 7

CSF2 neutralisation attenuates crescentic glomerulonephritis. (A) Experimental protocol describing the induction of cGN in C57Bl/6J mice. Animals were sacrificed 6 days after the induction of cGN with NTS. Anti-CSF2 antibodies were injected daily. Control mice received the same amount of isotype. (B) Albuminuria and urine NGAL were determined by ELISA. (C) Mice treated with anti-CSF2 antibodies showed reduced renal damage compared with control mice with significant reduction of crescentic and necrotic glomeruli. For each group, a representative image of a kidney section stained with PAS at high magnification (×40) is shown. **P<0.01, ***P<0.001. cGN, crescentic glomerulonephritis; CSF2, colony-stimulating factor-2; NGAL, neutrophil gelatinase-associated lipocalin; NTS, nephrotoxic serum; PAS, periodic acid-Schiff.