Platelets Specifically Interact with Remigrating Neutrophils and Promote Immunogenic Cellular Death in Systemic Lupus

Marie nansion¹, Eloi Schmauch², Pierre Vacher³, Jean Sibilia⁴, Nathan Broussaudier¹, Adeline Bruchon¹, Pierre Grenot¹, Anne-Sophie Korganow⁵, Thierry Martin⁵, Sophie Caillard⁶, Anna Duval⁶, Jacques-Eric Gottenberg⁷, Raphaël Carapito¹, Séiamak Bahram¹, Patrick Blanco⁸ and Marc Scherlinger⁹, ¹UMR_INSERM 1109, Strasbourg, France, ²Broad Institute of MIT and Harvard, Cambridge, MA, USA., Boston, MA, ³Inserm U1045, Bordeaux, France, ⁴Strasbourg University Hospital, National reference center for autoimmune disease, Rheumatology, Strasbourg, France, ⁵Strasbourg University Hospital, National reference center for autoimmune disease, Clinical Immunology, Strasbourg, France, ⁶Nephrology department, HUS, Strasbourg, France, ⁷Rheumatology Department, Strasbourg University Hospital,, Strasbourg, France, ⁸UMR_CNRS 5164 Immunoconcept, BORDEAUX CEDEX, France, ⁹Strasbourg University Hospital; INSERM 1109, Strasbourg, France

Meeting: ACR Convergence 2024

Keywords: Adhesion, Adhesion molecules, Cell Death, Lupus nephritis, neutrophils, Systemic lupus erythematosus (SLE)

Session Information

Date: Monday, November 18, 2024

Title: Abstracts: SLE – Etiology & Pathogenesis

Session Type: Abstract Session

Session Time: 3:00PM-4:30PM

Background/Purpose: In SLE, platelet activation (express P-selectin+) follows the disease activity, which plays a role in thrombosis as well as in inflammation (1). Neutrophils express high levels of PSGL-1, the ligand for P-selectin, which predicts a physical interaction between neutrophils and activated platelets in SLE patients. Our objective is to explore the relationships between platelets and neutrophils in human SLE.

Methods: We prospectively included SLE patients satisfying the 2019 ACR-EULAR criteria. Clinical data including disease activity (SLEDAI-2K; active SLE if SLEDAI ≥6) and immunological parameters were retrieved. We conducted spectral cytometry (Cytek Aurora) on fresh whole blood using a 37-color panel including platelets, neutrophils, and other immune surface markers. Neutrophils were CD66b+ cells identified on the SSC/FSC gating. Platelet-neutrophil aggregates (PNA) were identified as neutrophils coexpressing CD41a ± CD62P (P-selectin) platelet-specific markers. Using FlowJo v10, we conducted supervised and unsupervised analysis of neutrophils based on their interaction with platelets to explore neutrophils heterogeneity. In vitro experiments were conducted to investigate the signaling induced by P-selectin engagement on neutrophils and the impact on their phenotype.

Results: We recruited 50 patients with repeat samples for 8 patients. Platelet-neutrophil aggregates are increased in patients with active SLE compared to inactive SLE and healthy donors (p = 0.005 and p < 0.001, respectively). PNA positively correlate with the SLEDAI (Spearman’s r = 0,49; p = 0.003) and follows disease activity in longitudinal analyses.
A non supervised analysis identified that platelet-neutrophils aggregates have an atypical phenotype (ICAM1^high, CXCR4^high, CXCR1^low; Fig. 1A), previously identified as activated neutrophils that remigrates from a peripheral tissue (ie, skin) to the kidney to induce tissue inflammation (2). We confirmed that these atypical neutrophils are increased in SLE patients (p < 0.05; Fig. 1B) and that these remigrant neutrophils preferentially aggregate with platelets (p < 0.0001; Fig. 1C). In vitro, P-selectin engagement on human neutrophils induces a PSGL-1/Syk/calcium/mitochondrialROS intracellular signalling which induces the upregulation of genes involved in NETosis (Fig 2A-C). Furthermore, P-selectin engagement promotes SLE-serum induced neutrophil death in vitro (p < 0.01; Fig 2D). Finally, in patients with lupus nephritis, scRNAseq whole-blood study successfully identified PNA and spatial transcriptomics (10X Visium) study of kidney biopsies is ongoing.

Conclusion: Platelet-neutrophil aggregates are increased in SLE and are enriched in an atypical subpopulation of neutrophils believed to remigrate to SLE target organs. Through P-selectin signalling, platelet promotes neutrophil immunogenic death in SLE and represents a novel therapeutic target.

References:

1) Scherlinger et al. Nat Rev Immunol 2023 Aug;23(8):495-510

2) Skopelja-Gardner et al. Proc. Natl. Acad. Sci. 2021 Jan 19;118(3):e2019097118.

ICAM1high CXCR4high CXCR1low neutrophils preferentially bind with platelets in SLE.
(A) UMAP of neutrophils from 20 active SLE patients showing expression patterns of platelet markers (CD41a & CD62p), adhesion marker/chemokine receptors (ICAM1, CXCR4, CXCR1) and receptors involved in neutrophil cell death (CD40, FAS). (B) Gating strategy and cumulative results of ICAM1high CXCR4high CXCR1low (remigrating) neutrophils in SLE patients. (C) Platelet neutrophil aggregates in total and remigrating neutrophils in SLE patients. *, p < 0.05 using Mann-Whitney test; ****, p < 0.0001 using Wilcoxon's test.

P-selectin engagement on neutrophils primes neutrophil immunogenic death. (A) 10^7 human neutrophils were cultured 8 hours with or without P-selectin 200ng/mL. After RNA extraction, RNAseq was conducted. Results showing the pathway enrichment analysis of the P-selectin culture condition. P-values are corrected for multiple analysis. (B) Human neutrophils were incubated with the Cali-520 calcium-sensitive fluorophore, and single-cell fluorescence was acquired real time using an epifluorescence microscope (Olympus, IX70) after P-selectin addition to the medium (200ng/mL). Neutrophils were preincubated with an anti-PSGL_1 antibody (red curve; clone KPL_1, 1/100) or a Syk inhibitor (blue curve; 1µM). (C) The same experience was conducted using the MitoSox mitochondrial reactive oxygen species (ROS) marker. (D) Human neutrophils were freshly sorted using MACSXpress and cultured overnight with autologous or SLE patient serum 20% ± P-selectin 20%. 7-AAD/Annexin staining shows the percentage of living cells at the end of the experiments. *, p < 0.05; **, p < 0.01 using paired ANOVA with correction for multiple analysis.

Disclosures: M. nansion: None; E. Schmauch: None; P. Vacher: None; J. Sibilia: None; N. Broussaudier: None; A. Bruchon: None; P. Grenot: None; A. Korganow: None; T. Martin: None; S. Caillard: None; A. Duval: None; J. Gottenberg: AbbVie, 2, BMS, 2, 5, Galapagos, 2, Gilead, 2, Lilly, 2, MSD, 2, Novartis, 2, Pfizer, 2, 5; R. Carapito: None; S. Bahram: None; P. Blanco: None; M. Scherlinger: Abbvie, 2, Amgen, 2, AstraZeneca, 2, Bristol-Myers Squibb(BMS), 2, Fresenius Kabi, 2, GlaxoSmithKlein(GSK), 2, Innate pharma, 2, Nordic Pharma, 2, Novartis, 2, Roche, 2, Sandoz, 2.

To cite this abstract in AMA style:

nansion M, Schmauch E, Vacher P, Sibilia J, Broussaudier N, Bruchon A, Grenot P, Korganow A, Martin T, Caillard S, Duval A, Gottenberg J, Carapito R, Bahram S, Blanco P, Scherlinger M. Platelets Specifically Interact with Remigrating Neutrophils and Promote Immunogenic Cellular Death in Systemic Lupus [abstract]. Arthritis Rheumatol. 2024; 76 (suppl 9). https://acrabstracts.org/abstract/platelets-specifically-interact-with-remigrating-neutrophils-and-promote-immunogenic-cellular-death-in-systemic-lupus/. Accessed .

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