POS0152 PLATELET EXTRACELLULAR VESICLES MODULATE NEUTROPHIL STATE IN SYSTEMIC SCLEROSIS VIA HMGB1

Background

Platelet extracellular vesicles (Plt-EVs) expressing the damage-associated molecular pattern prototypic signal, high mobility group box 1 (HMGB1), accumulate in the blood of patients with systemic sclerosis (SSc) and per se promote autoimmunity, fibrosis and vascular inflammation [1, 2]. A poorly understood defect in critical players in sprouting angiogenesis, angiopoietins and the angiopoietin receptor, TIE2 is also an hallmark of SSc. Normal neutrophils respond to angiopoietin 1 and other stimuli by undergoing activation and upregulating the Tie2 expression [3].

Objectives

We reasoned that HMGB1 could be a putative candidate signal supporting neutrophils Tie2 overexpression, as it plays a critical role in neutrophil activation and is present as an EV-associated bioactive molecule in the blood of SSc patients[1, 2].

Methods

Neutrophil Tie2 expression was assessed in the blood of 39 patients with SSc and 43 healthy donors (HD) sex- aged matched, by flow cytometry and confirmed by immunogold at electron microscopy. Tie2 expression was determined in freshly purified normal neutrophils stimulated with Plt-EVs from SSc patients or from HD or stimulated with rHMGB1 in vitro. Moreover Plt-EVs purified from the plasma of SSc patients or from HD were injected in the tail vein of NSG mice (that are receptive to human tissues). 18 hours after injection the expression of Tie2 of neutrophils in blood and lung histology were analyzed. Box A and low molecular weight heparin (LMWH) have been used as HMGB1 antagonists in vitro and in vivo.

Results

Most SSc neutrophils express Tie2 (84.7±2.4% vs 8.3±1.1% in HD; p <0.0001) regardless of the extent of systemic inflammation. HMGB1 and Plt-EV expressing HMGB1 purified from the plasma of patients with SSc induced up-regulation of TIE2 in vitro. Tie2 induction was abrogated by Box A and LMWH. Plt-EVs from SSc patients (but not from the plasma of HD) injected in immune-deficient NSG mice dramatically upregulated Tie2 neutrophil expression. Neutrophil Tie2 upregulation abated in the presence of HMGB1 inhibitors, BoxA and LMWH. Immunohistochemitry revealed lung fibrosis associated with massive infiltration by Tie2+ neutrophils. Neutrophil ablation by liposome-encapsulated clodronate prevented SSc Plt-EV-induced lung fibrosis, thus causally linking neutrophil activation, Tie2 neutrophil-expressing pulmonary infiltration and tissue remodelling.

Conclusion

Our data highlight the role of the platelet HMGB1/neutrophil axis in SSc vascular inflammation and fibrosis.

References

[1]Manfredi AA; et al. Platelet Phagocytosis via P-selectin Glycoprotein Ligand 1 and Accumulation of Microparticles in Systemic Sclerosis. Arthritis Rheumatol. 2021. 10.1002/art.41926. [2]Maugeri N; et al. Platelet microparticles sustain autophagy-associated activation of neutrophils in systemic sclerosis. Sci Transl Med. 2018; 10. 10.1126/scitranslmed.aao3089. [3]Burnett A et al. Angiopoietin-1 enhances neutrophil chemotaxis in vitro and migration in vivo through interaction with CD18 and release of CCL4. Sci Rep. 2017; 7: 2332. 10.1038/s41598-017-02216-y.

Acknowledgements:

NIL.

Disclosure of Interests

None Declared.