Resolution of acute inflammation induced by monosodium urate crystals (MSU) through neutrophil extracellular trap-MSU aggregate-mediated negative signaling

Cheng-Hsun Lu; Chieh-Yu Shen; Ko-Jen Li; Cheng-Han Wu; Yu-Hsuan Chen; Yu-Min Kuo; Song-Chou Hsieh; Chia-Li Yu

doi:10.1186/s12950-024-00423-9

Resolution of acute inflammation induced by monosodium urate crystals (MSU) through neutrophil extracellular trap-MSU aggregate-mediated negative signaling

J Inflamm (Lond). 2024 Nov 27;21(1):50. doi: 10.1186/s12950-024-00423-9.

Authors

Cheng-Hsun Lu^{1

2}, Chieh-Yu Shen¹, Ko-Jen Li¹, Cheng-Han Wu³, Yu-Hsuan Chen¹, Yu-Min Kuo^{1

2}, Song-Chou Hsieh¹, Chia-Li Yu⁴

Affiliations

¹ Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, 10002, Taiwan.
² Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, No.1, Chang-Te Street, Taipei, 10048, Taiwan.
³ Department of Internal Medicine, National Taiwan University Hospital-Hsinchu Biomedical Park, No. 2, Sec. 1, Shengyi Road., Zhubei City, Hsinchu County, 302058, Taiwan.
⁴ Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, 10002, Taiwan. chialiyu@ntu.edu.tw.

Abstract

Background: Polymorphonuclear neutrophils (PMN) activation by monosodium urate crystals (MSU) is crucial to acute gouty arthritis and subsequent spontaneous remission within 7-10 days. Activated PMNs release neutrophil extracellular traps (NETs) that entrap MSU crystals, forming NET-MSU aggregates. Whether NET-MSU aggregates contribute to the resolution of acute inflammation remains to be elucidated. This study uses a cell-based approach to unveil their molecular bases.

Methods: All-trans retinoic acid-differentiated HL-60 cells (dHL-60) served as surrogate PMNs. NET release from MSU-activated dHL-60 was confirmed by detecting DNA, neutrophil elastase, and citrullinated histone 3, forming large NET-MSU aggregates. NET area was measured with Fiji software after SYTOX Green staining. Released pro-inflammatory cytokines IL-8 and TNF-α, and the anti-inflammatory cytokine IL-1RA in culture supernatants were quantified to calculate the estimate inflammation score (EIS). Cellular redox state was determined by a FRET-based sensor. Expression of intracellular positive (ERK1/2) and negative (SHP-1 and SHIP-1) cytokine signaling regulators was detected by western blot. qPCR detected mRNA expressions of CISH and SOCS1-SOCS7. Flow cytometry measured neutrophil N1 (CD54) and N2 (CD182) surface markers after staining with fluorescent-conjugated antibodies.

Results: Incubating dHL-60 with MSU for 4 h maximized NET-MSU aggregate formation and acute inflammation with an EIS of 11.6. Prolonging the incubation of dHL-60 + MSU to 22 h gradually raised the EIS to 19.40 without increasing NET area, due to reduced cellular redox capacity. Adding both new dHL-60 and new MSU crystals to the culture, mimicking the clinical scenario, increased NET area but conversely suppressed EIS to 1.53, indicating acute inflammation resolution. The resolution of acute inflammation following prolonged incubation was attributed to decreases in P-ERK and increases in P-SHP-1, SOCS2, SOCS3, and CISH gene expressions, which may suppress pro-inflammatory and enhance anti-inflammatory cytokine production. Moreover, the large NET-MSU aggregates facilitated N1 to N2 polarization, crucial for accelerating inflammation resolution.

Conclusion: We explored the potential molecular basis for the spontaneous resolution of MSU induced acute inflammation using a cell-based model in that huge NET-MSU aggregates frustrate the transformation of newly entering PMNs to the N2 phenotype, enhancing the production of the anti-inflammatory cytokine IL-1RA.

Keywords: Differentiated HL-60; Estimate inflammation score; Intracellular cytokine signaling regulator; Monosodium urate crystal (MSU); NET area; NET-MSU aggregate; Neutrophil extracellular trap (NET).