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.

Fig. 1. Integrated landscape of myeloid cells in kidneys from mice and patients with lupus nephritis. (A) Kidney myeloid cells from 155 LN patients and 30 control biopsies were collected and processed for single-cell RNA seq with the AMP RA/SLE Network. (B) UMAP of human myeloid cells and cluster names. (C) Frequency of D-Mac (vs. all myeloid cells) is associated with activity index. A dot=1 pt. (D) Kidney myeloid cells from 4 lupus mouse strains were processed similarly and integrated with human data set. (E) UMAP of lupus murine myeloid cells and cluster names. (F) Frequency of D-Mac (vs. all myeloid cells) is associated with nephritis in all mouse models. A dot=1 mouse. Results in mice were confirmed by flow cytometry (not shown).

Fig. 2. In situ localization of analogous myeloid states in human and mouse kidney sections. (A-J) This series presents images of kidney sections and myeloid states within renal compartments from patients (A-D) and mice (G-J) with lupus nephritis. All panels are stained with hematoxylin and eosin (H&E), highlighting various subsets of monocytes and macrophages (D-Mac, C1, NC1, NC2, RM) as well as dendritic cells (Ccr7, DC1, DC2, pDC). The Activity Index (AI) and Chronicity Index (CI) are noted. Scale bars indicate measurements of 100 microns. (E-M) These panels quantify myeloid subsets by compartment in humans with lupus nephritis (E) and human control kidneys (F), as well as in lupus nephritis mouse models (K. NZB/W; L. Sle1.Yaa) and pre-nephritic mice (M. NZB/W). The left side of each panel displays the proportion of each indicated cell type within renal compartments across all samples. The right side presents the -log of the q-value, which indicates either depletion (blue) or enrichment (red) of the respective cell type for each renal compartment. Asterisks mark q-value < 0.05. (N) Areas of renal compartments that were analyzed in the spatial assessment. Abbreviations: TI = tubulointerstitial; Periglom. = Periglomerular. Definitions: Glomerulus, inside = area within annotated glomerular border. Glomerulus, border = area that includes -/+ 20 microns from annotated border; Periglomerular inflammation = area adjacent to at least 25% of annotated glomerular border containing >50% immune cells. Tubulointerstitial inflammatory aggregate = non-adjacent area outside of glomerular border with >10 contiguous immune cells.

Fig. 3. Selected functional assays of murine D-Macs and related subsets. A) Phagocytosis of APC-labeled ova-anti-ova immune complexes. * p < 0.05; ** p < 0.01 (non-parametric t-test for 2-group comparisons or Kruskal-Wallis ANOVA with Dunn’s correction for 3-group comparisons). B. Metabolic profile of murine renal myeloid subsets demonstrated by indicated macrophages or monocytes from pre-disease or nephritic NZB/W and Sle1.Yaa mice. Cells were stained with ATP biosensor (B-E) showing increased ATP in D-Macs and RMs that was not accounted for my differences in mitochondrial abundance or membrane potential (not shown). Pre-disease (black) and nephritis (red) samples are shown together as there was no difference between them.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/spatial-organization-and-function-of-disease-associated-macrophages-in-lupus-nephritis-insights-from-cross-species-analyses/