Neutrophil Heterogeneity Identifies an Association of LAMP1 With Proliferative Lupus Nephritis

Abstract

Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE) with limited biomarkers for early detection. While neutrophils contribute to SLE pathogenesis, their phenotypic heterogeneity in disease remains poorly characterized. Here, we used mass cytometry to profile blood neutrophils from patients with biopsy-confirmed proliferative LN and healthy controls. We identified a distinct population of activated neutrophils, marked by surface expression of lysosomal-associated membrane protein 1 (LAMP1/CD107a), that was virtually absent in healthy individuals. We demonstrate that LAMP1 resides intracellularly in resting neutrophils and translocates to the cell surface upon activation. Transcriptomic analysis revealed no difference in LAMP1 mRNA expression between patients with SLE and controls, confirming that surface LAMP1 reflects neutrophil activation rather than increased transcription. Soluble LAMP1 was significantly elevated in serum from patients with SLE compared with controls, with the highest levels in proliferative LN. In a large cohort of 225 patients with LN, urinary LAMP1 correlated with glomerular filtration rate, proteinuria, and histological activity indices. Together, our findings reveal LAMP1 as a marker of neutrophil activation in SLE and identify serum and urinary LAMP1 as potential noninvasive biomarkers for proliferative LN.

FIGURE 1

Mass cytometry analysis of blood leukocytes identifies increased expression of LAMP1 in patients with lupus nephritis compared with healthy donors. (A) Heatmap of the expression of 23 surface proteins on blood neutrophils from healthy donors (n = 10) and patients with LN (n = 21). (B) Volcano plot showing differential abundance of the examined surface markers in blood leukocytes in LN patients versus healthy donors. Positive difference in median abundance values indicates higher abundance in LN patients. (C) UMAP dimensionality reduction of the mass cytometry data of blood neutrophils from LN patients and healthy donors in 10 neutrophil meta‐clusters. (D) Marker protein expression and relative cell abundance of the neutrophil meta‐clusters. Clusters #8 and #10, which are increased in LN patients, are shown in the red boxes. (E, F) Abundance of LAMP1 (CD107a) (E) and CD18a (F) on neutrophils of patients with LN and healthy donors. Mann–Whitney U test: *p < 0.05; ***p < 0.001. UMAP: Uniform Manifold Approximation and Projection.

FIGURE 2

LAMP1 is stored intracellularly in secretory vesicles and mobilized upon neutrophil activation. (A) LAMP1 mRNA expression in peripheral blood from 58 healthy donors and 142 patients with SLE. (B) Surface and intracellular flow cytometry of LAMP1 in neutrophils from a healthy donor. Quantification of LAMP1 MFI on the neutrophil surface before and after permeabilization (healthy blood donors, n = 5). (C) Surface LAMP1 MFI of unpermeabilized neutrophils from healthy blood donors after incubation with fMLP and Cytochalasin B. (D) Confocal microscopy of healthy donor neutrophils, costained for LAMP1 and granule markers MPO (primary/azurophilic granules), LTF (secondary/specific granules), and CD35 (secretory granules). Scale bar: 5 µm. (E) Colocalisation of LAMP1 with granule markers calculated as Pearson correlation. Mann–Whitney U test: **p < 0.01. MFI, Median fluorescence intensity.

FIGURE 3

Soluble LAMP1 is increased in the serum of patients with SLE. (A) Soluble LAMP1 (sLAMP1) measured by ELISA in the serum of healthy donors (n = 11) and patients with SLE (n = 67). (B–D) Pearson correlation of serum LAMP1 with SLEDAI‐2K. (B) dsDNA autoantibodies (C), serum C3. (D) levels in patients with SLE. (E) Soluble LAMP1 is higher in patients with proliferative (class III/IV) LN compared with patients without proliferative LN. Mann–Whitney U test: *p < 0.05; ****p < 0.0001.

FIGURE 4

Urinary LAMP1 correlates with nephritis severity and histological activity in LN. (A) Urinary LAMP1 levels in healthy donors (n = 10) and LN patients (n = 225). (B–D) Correlation of (B) glomerular filtration rate (GFR), (C) proteinuria (g/g creatinine), and (D) Biopsy NIH activity index (D) with urinary LAMP1 (in pg/mg creatinine). (E) Correlation of LAMP1 with specific features of the NIH activity index of kidney biopsies.