Spatial Analysis of Neutrophil Subpopulations and Activity in Proliferative Lupus Nephritis Biopsies

Background: Neutrophils contribute to the glomerular inflammation and injury in lupus nephritis (LN). We recently reported the presence of neutrophils and granule constituents within glomeruli and tubules and that urine excretion of neutrophil granule proteins correlates with disease activity in human proliferative LN. Circulating neutrophils are not a homogeneous population in lupus patients, as both immature and mature low-density neutrophils (LDN) emerge in the literature. However, the role of different neutrophil subsets in LN remains to be established. The goal of this study was targeted spatial analysis of neutrophil subsets and granule marker expression in proliferative LN patient biopsies. Methods: Renal biopsy sections (FFPE) from 4 patients with class IV and IV/V LN and kidney wedge samples from 3 control donors (deceased donors unsuitable for transplantation) were used. A panel of 22 metal-tagged antibodies were used for imaging mass cytometry (IMC) analysis of samples. Antibodies to CD11b, CD15, CD16, MPO, CD66b, ARG1, MMP9 served as markers for neutrophils. Additional markers to glomeruli (Nephrin, WT1), tubule and epithelial cells (Na+/K+ ATPase, E-Cadherin, EpCAM), endothelial cells (CD31), macrophages (CD68, CD163) and general leukocytes (CD45) were used. Antibodies to alpha-smooth muscle, vimentin, and Ki67, P-ERK1/2 served to define degree of tissue injury and cell proliferation/activation. Serial sections of samples were PAS-stained for histopathology. Laser ablation of ROI of equal dimensions across all tissue samples carried out using the Hyperion Imaging system and MCD-files created by the CyTOF® Software were processed in MCD viewer to create pseudo-colored images for marker visualization. Results: Localization of neutrophil markers was increased in glomeruli of LN biopsies and correlated with glomerular damage shown by decreased abundance of nephrin and increased abundance of alpha-smooth muscle actin. CD68 localization to glomeruli increased in LN biopsies. Neutrophil markers CD66b, MPO, and CD11b distributed to both CD15 positive and negative cells. Conclusion: Co-localization of CD15 with other neutrophil markers suggests increased distribution of LDN in LN biopsies. High output imaging of LN patient biopsies allows differentiation of different neutrophil subsets and spatial distribution of immune cells in LN. Funding: NIDDK Support