Background: The complement system is important for neutrophil priming and activation in autoimmune disease. Avacopan, a complement 5a receptor (C5AR1) antagonist, was approved in October 2021 as add-on therapy for anti-neutrophil cytoplasmic antibody (ANCA)–associated vasculitis. The majority of ANCA autoantibodies target the neutrophil granule proteins proteinase 3 (PR3) or myeloperoxidase (MPO) to form the two main disease subtypes. The kidney is a major target organ in both subtypes of ANCA vasculitis, with the prognostic classification based on the proportions of normal, crescentic, and sclerotic glomeruli on biopsy. Here, we investigated baseline renal biopsy and longitudinal blood single cell expression patterns of C5AR1 in an ANCA vasculitis cohort.

Objectives: To characterize C5AR1 expression across disease-relevant tissues in the two main subtypes of ANCA vasculitis.

Methods: Forty-one patients with ANCA vasculitis were enrolled in an observational study. Twenty-four diagnostic biopsy sections were processed for spatial transcriptomics (at 55 µm resolution) following annotation of the different glomerular lesions by a board-certified pathologist. Using a combination of gene expression, microenvironment prediction and histological feature locations, we identified molecular niches associated with the histopathology annotations. Additionally, 80 longitudinal whole-blood samples from the 41 patients were analyzed using single-cell RNA sequencing. The single-cell data was also used to infer monocyte-infiltration in the kidney spatial transcriptomics data with cell-type deconvolution. Statistical significance was calculated with a pairwise Wilcoxon test with Bonferroni correction. Here we report mean values ± standard deviation.

Results: Biopsy samples were taken at diagnosis, before patients were assigned to treatment. Among samples with a biopsy available, 42% were anti-PR3 autoantibody-positive and 58% were anti-MPO autoantibody-positive. Spatial transcriptomics showed overall higher glomerular C5AR1 expression in the PR3-ANCA group, as this group had more crescentic glomeruli. Analyses of the glomeruli by lesion type across patients showed that the average expression of C5AR1 was elevated in crescentic compared with normal glomeruli (0.37±0.28 vs 0.18±0.29 log-norm counts per million, p = 0.00032). The crescentic glomeruli also had higher monocyte/macrophages infiltration compared to normal glomeruli (3.76±1.61% vs 1.06±0.83% of the niche area, p < 0.0001), which could contribute to the higher C5AR1 expression observed in this glomerular lesion type. Single-cell blood analyses showed high C5AR1 expression in basophils (0.97±0.26 log-normalized counts per million), monocytes (1.21±0.25), and neutrophils (1.97±0.36), with the highest levels in neutrophils (p. adj. <0.0001). Longitudinal analysis revealed lower neutrophil C5AR1 expression during peak disease activity at diagnosis (PR3: 1.79±0.22, MPO: 1.84± 0.40, PR3: vs MPO p. adj = 0.622), followed by gradual increases over time during recovery (6m PR3: 2.15±0.15, MPO: 2.27± 0.36, p. adj PR3 vs MPO = 0.093, p. adj BL vs 6m PR3: 0.00066, p. adj BL vs 6m MPO 0.004).

Conclusions: Using spatial and single-cell omics, we identified dynamic C5AR1 expression patterns in ANCA vasculitis – and among ANCA-subtypes. C5AR1 expression was the highest in crescentic glomeruli, which also showed immune cell infiltration. In peripheral blood, neutrophils were the primary C5AR1-expressing cell type, starting with lower baseline expression levels in both ANCA subtypes during highly active disease followed by gradual increases over time during the recovery period. Future studies should assess how temporal changes in C5AR1 expression influences Avacopan efficacy in ANCA vasculitis.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: Melinda Barkhuizen Employee of Evotec International, Kay Schreiter Employee of Evotec International, Hanna Schebet Employee of Evotec International, Björn Tampe Research compensation from Evotec International and CSL Vifor, and honoraria/ travel support from CSL Vifor, Michael Zeisberg Received research compensation from Evotec International, Ramon Oliveira Vidal Employee of Evotec International, Menglin Zheng Employee of Evotec International, Ilya Komarov Employee of Evotec International, Britta Seip Employee of Evotec International, Janne Schoening Employee of Evotec International, Varsha Poondi Krishnan Employee of Evotec International, Helge Stark Employee of Evotec International, Kulwadee Thanamit Employee of Evotec International, Philipp Skroblin Employee of Evotec International, Uwe Andag Employee of Evotec International.