Background/Purpose: SLE is a leading cause of premature death, often from severe complications including lupus nephritis (LN), especially in disadvantaged groups. Despite advances in treatment, 10-30% of patients with LN progress to end-stage kidney disease. While disease-associated dysbiosis within gut microbiota has been reported, causative roles remain poorly understood. We found ~40% of LN flares are associated with gut blooms of Ruminococcus gnavus (RG) strains expressing a novel cell wall lipoglycan (LG)^1,2,3. We sought to define underlying in vivo pathogenic pathways.

Methods: Patients in three LN cohorts, NYU, and the validation cohorts: Bordeaux Hospital University and ITN_ACCESS_LN, were investigated with a quantitative bead-based assay for serum anti-LG IgG (Fig1A). Bulk whole-blood RNA-seq libraries were analyzed for gene and GSEA pathway representation. PF4/CXCL4 levels were assessed by ELISA and platelet-derived microparticles (MPs) by microfluorimetry. NETosis was measured via immunoassays for serum citrullinated histone-H3, DNA, and myeloperoxidase. To assess causation, naïve C57BL/6 female mice were gut-colonized with an LG-producing RG strain, and then whole-blood RNA-seq and flow cytometry were performed.

Results: In LN validation cohort patients with high anti-LG levels, whole-blood RNAseq revealed increased signatures for platelet activation and degranulation (qval< 4.4E-4), neutrophil degranulation (qval< 0.007), and myeloid leukocyte activation (qval< 0.002), independent of type I IFN signatures (Fig1B). Higher levels of PF4, a platelet granule protein, were associated with high anti-LG levels (p< 0.04)(Fig1C) and significantly higher NETosis fragments were also detectable in anti-LG positive LN patients (p< 0.02, Fig1D). Active-LN patients from the Bordeaux Hospital University cohort showed a positive trend between anti-LG levels and P-selectin+ platelet MPs. In these studies, anti-dsDNA levels did not correlate with platelet activation or NETosis. In mice colonized with LG-producing RG (Fig2A), blood-bulk RNAseq showed an increased platelet degranulation gene signature (p< 0.002, Fig2B). Notably, SPARC, PDGFA, and VTI1B were significantly increased in RG-colonized mice (p< 0.05), just as in LN patients. RG-colonization induced ex vivo platelet sensitization after thrombin challenge, detected as raised surface P-selectin levels (p<0.016)(Fig2C).

Conclusion: Our findings provide the first evidence that RG intestinal blooms trigger LN flares linked to platelet activation with MP formation and downstream NETosis (Fig3), in a type-I IFN-independent setting. Using the anti-LG antibody as a companion diagnostic, this set of active LN patients should be evaluated for therapeutic interventions of platelet activation and NETosis.

References:        
1. Azzouz, D et al. ARD 2019

2. Azzouz, D et al. ARD 2023

3. Silverman, GJ et al. NRR 2023

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-human-gut-pathobiont-drives-platelet-activation-with-microparticle-release-and-netosis-during-lupus-nephritis-flares/