Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: “Defining the role of myeloid populations in lupus nephritis: a spatially resolved multi-omics approach”

ABSTRACT Background and aims Accelerating Medicines Partnership (AMP), discovered that urinary PR3, a neutrophil degranulation product, is associated with histological activity, indicating the involvement of neutrophil degranulation in proliferative lupus nephritis (LN), the most aggressive type of LN. Although mature neutrophils with classical polylobate nuclei are rare in LN kidney biopsies, recent evidence suggests that immature myeloid cells undergoing degranulation play a role in the pathogenesis of LN. We aimed to investigate the presence of PR3+ cells in LN kidney, their association with histopathological features and to define their immunophenotype. Methods We performed multiplexed histology using serial immunohistochemistry (sIHC) on LN kidney biopsies to quantify the expression of PR3 and multiple cell lineage markers (20-plex). Image analysis including deconvolution, cell segmentation, glomerular annotation, and quantitative histology was performed using Indica HALO. The analysis was limited to the renal cortex. Results A total of 33 patients with LN who underwent a clinically indicated kidney biopsy were enrolled: 14 (42.4%) with pure proliferative LN (ISN/RPS class III or IV), 14 (42.4%) with pure membranous LN (ISN/RPS class V) and 5 (15.2 %) with a mixed phenotype (ISN/RPS class III or IV + V). PR3+ cells were identified in all biopsies and most of them did not show a polylobate nucleus. The majority of PR3+ cells were in the tubulointersitium. While the tubulointerstitium contained the majority of PR3+ cells, it is noteworthy that the density of these cells was higher in the glomeruli due to their smaller area. PR3+ cell abundance was higher in proliferative LN, especially in the glomeruli. Glomerular PR3+ cell density very strongly correlated with histological activity measured by the NIH Activity Index. PR3+ cells displayed a unilobate nucleus. Most PR3+ cells coexpressed MPO. We identified 3 subsets of PR3+ cells based the coexpression of other lineage markers. One group coexpressed CD66b, CD11b, CD15, and (variably) CD10 indicating a degranulating phenotype. The other group expressed CD14, CD163 indicating a phagocytic phenotype. Conclusions PR3+ cells are abundant in LN, increased in proliferative LN, and are strongly associated with histological activity thereby characterizing a more aggressive phenotype. This population densely infiltrated the glomeruli emphasizing a potential role in the endothelial pathogenic process. There were two main subsets of kidney-infiltrating PR3+ cells characterized by a phagocytic or a degranulating phenotype; both were mononucleated suggesting a monocyte or nonsegmented neutrophil lineage. It was previously showed the association between urinary PR3 and histological activity suggesting that intrarenal PR3+ cells are actively degranulating and therefore likely inducing kidney damage. These findings nominate PR3+ cells as a potential therapeutic target.