1763: Spatial Organization and Function of Disease-Associated Macrophages in Lupus Nephritis: Insights from Cross-Species Analyses

Background/Purpose: Myeloid cells are linked to kidney injury in lupus nephritis (LN) but lack targeted therapies, underscoring the need to better understand myeloid biology in LN. While LN mouse models provide biological systems to develop new therapies, identifying shared molecular features relevant to human disease is essential to fully optimize the effectiveness of these models. We recently compared kidney myeloid single-cell datasets from four LN mouse strains with different pathogenic backgrounds and 155 patients across various disease stages and histologic subtypes (Fig. 1). This revealed similar intrarenal myeloid subsets, including Disease-Associated Macrophages (D-Macs), which correlated with clinical disease in both species (Fig. 1C, F). In this follow-up study, we examined two aspects of myeloid biology in mouse and human LN: 1) the in situ distribution of D-Macs and myeloid subsets to evaluate whether analogous cells could interact similarly with their tissue microenvironment, and 2) functional characteristics of murine D-Macs relevant to LN pathogenesis.

Methods: To map myeloid localization, we used Xenium spatial transcriptomics on FFPE kidney sections from 2 mouse strains and human patients (n=6 LN pts. with proliferative or mixed histologic classes; mean ISN/RPS activity = 13.5; S.D = 5.8) and 2 controls, employing custom probes based on scRNA-seq and the multi-tissue panel. For functional assays, we performed ex vivo phagocytosis and metabolic tests on single-cell suspensions from myeloid cells isolated by flow from dissociated murine kidneys (pre- and post-nephritis), using fluorescent immune complexes (ICs) and ATP substrates to evaluate phagocytic capacity and metabolism—particularly of D-Macs—via flow cytometry.

Results: In situ mapping findings (Fig. 2): (1) In nephritic mice, D-Macs resided within glomeruli and periglomerular infiltrates, with strain-specific differences. In humans, D-Macs were most abundant within nephritic glomeruli. (2) Resident macrophages in both species localized mainly in the tubulointerstitium, with murine RMs forming rings around glomeruli. (3) Non-classical myeloid subsets were primarily restricted to glomeruli, with minimal presence in the interstitium. (4) Murine dendritic cells localized to parenchymal areas and aggregates, while human dendritic cells were mainly in the tubulointerstitium.

Murine D-Mac functions (Fig. 3): Compared to other monocyte subsets enriched in nephritic glomeruli (C1, NC1, NC2), D-Macs showed increased uptake of immune complexes and higher membrane potential and ATP levels, suggesting an alternatively activated state.

Conclusion: Across species, D-Macs, along with classical and non-classical monocytes, were positioned near injured glomeruli—within glomeruli in humans—while resident macrophages and dendritic cells mainly localize to the tubulointerstitium and periglomerular zones. Mouse studies further indicate D-Macs adopt an alternatively activated phenotype, potentially contributing to immune complex uptake that emerges in nephritic glomeruli that may modulate inflammation. These studies advance our understanding of D-Macs in LN and support the use of mouse models to study human-derived hypotheses.