Evidence of Membranolytic Targeting and Intracellular Citrullinationin Neutrophils Isolated from Patients with Rheumatoid Arthritis

Neela Fatemeh Moadab¹, Tal Gazitt², Rayan Najjar¹, Ethan Le¹, J Lee Nelson³, Vijay Joshua⁴, Keith Elkon¹, Caroline Grönwall⁴ and Tomas Mustelin¹, ¹University of Washington, Seattle, WA, ²Carmel Hospital, Haifa, Israel, ³University of Washington and Fred Hutchinson Cancer Center, Seattle, WA, ⁴Karolinska Institutet, Stockholm, Sweden

Meeting: ACR Convergence 2024

Keywords: Anti-citrullinated Protein Autoantibodies (ACPAs), citrullination, neutrophils, rheumatoid arthritis

Session Information

Date: Saturday, November 16, 2024

Title: RA – Etiology & Pathogenesis Poster

Session Type: Poster Session A

Session Time: 10:30AM-12:30PM

Background/Purpose: Anti-citrullinated protein autoantibodies (ACPA) are diagnostic for rheumatoid arthritis (RA). The antigens recognized by these autoantibodies are produced by protein arginine deiminases (PADs), particularly PAD4. However, it remains unknown why and how PAD4 causes this aberrant citrullination in RA.

Methods: To be able to detect polymerized perforin pores on the surface of freshly isolated RA neutrophils, we developed a monoclonal antibody against the exposed N-terminal knob of perforin.

Results: Here, we report that poly-perforin pores are present on freshly isolated neutrophils from RA patients, but not on healthy donor neutrophils. Neutrophils with perforin pores also contained intracellular citrullinated proteins in the region adjacent to the pores. This response was replicated in vitro by treating neutrophils with purified perforin, which generated intense dots of anti-perforin immunofluorescence, calcium influx, and intracellular citrullination. Extensive neutrophil killing in Felty’s syndrome, an aggressive form of RA, correlated with particularly high ACPA, and PAD4 autoantibodies. In contrast, other forms of death, including NETosis, apoptosis, and pyroptosis,produced minimal citrullination.

Conclusion: We conclude that neutrophil targeting by perforin leading to intracellular citrullination takes place in patients with RA.

Generation and validation of the knob mAb (anti-perforinN). a, The 3-dimensional structure of the poly-perforin pore. b, Close up of the N-terminal knob highlighting the peptide His24-Glu46, which was used as immunogen conjugated to keyhole limpet hemocyanin. c, ELISA of anti-perforinN 4G10 using purified perforin. d, Coomassie Blue stain of 1µg and 0.3 µg of the purified perforin used in panel C. e, Immunofluorescence staining with anti-knob mAb 4G10 of neutrophils treated with 1 µg/ml of purified perforin for the indicated times. f, Quantitation of the response in panel E expressed as the number of brightly stained dots per neutrophil. g, Live cell imaging of two neutrophils loaded with the Fluo-5N calcium indicator and treated with 1 µg/ml of perforin at 37°C.

Poly-perforin pores on freshly isolated untreated RA neutrophils. a, Three representative neutrophils from an RA patient stained with the anti-knob mAb. b, Three representative neutrophils from a second RA patient. c, Three rep representative neutrophils from a third RA patient. d, Similar staining of three representative neutrophils from a healthy donor. e, Quantitation of the perforin pore-positive neutrophils in the same three patients, expressed as percent of all neutrophils, in four different fields containing approximately 100 cells. f, Quantitation of the number of bright perforin dots on each of the perforin-positive neutrophils from the same three RA patients. g, Close ups of two clusters of 3-4 bright dots

Citrullination in freshly isolated RA neutrophils with poly-perforin pores. a, A representative neutrophil from an RA patient stained with the anti-knob mAb and the ACPA mAb 10D07. b, Another neutrophil from a second RA patient. c, A third representative neutrophil from a third RA patient. d, Control staining of a neutrophil with secondary antibody alone.

Disclosures: N. Moadab: None; T. Gazitt: None; R. Najjar: None; E. Le: None; J. Nelson: Chimerocyte Inc, 8; V. Joshua: None; K. Elkon: None; C. Grönwall: None; T. Mustelin: Bristol-Myers Squibb(BMS), 2, Cugene, 2, Miro Bio, 2, QiLu Bopharma, 2, Rome therapeutics, 2.

To cite this abstract in AMA style:

Moadab N, Gazitt T, Najjar R, Le E, Nelson J, Joshua V, Elkon K, Grönwall C, Mustelin T. Evidence of Membranolytic Targeting and Intracellular Citrullinationin Neutrophils Isolated from Patients with Rheumatoid Arthritis [abstract]. Arthritis Rheumatol. 2024; 76 (suppl 9). https://acrabstracts.org/abstract/evidence-of-membranolytic-targeting-and-intracellular-citrullinationin-neutrophils-isolated-from-patients-with-rheumatoid-arthritis/. Accessed .

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