Resolution of acute inflammation induced by monosodium urate crystals (MSU) through neutrophil extracellular trap-MSU
Cheng-Hsun Lu1,2, Chieh-Yu Shen1, Ko-Jen Li1
1Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, 10002, Taiwan.
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.
Insights
Neutrophil extracellular traps (NETs) aggregate with monosodium urate (MSU) crystals, resolving gouty arthritis inflammation. Large NET-MSU aggregates promote anti-inflammatory cytokine production and PMN polarization for resolution.
Area of Science:
- Immunology
- Cell Biology
- Inflammation Research
Background:
- Polymorphonuclear neutrophils (PMN) activation by monosodium urate (MSU) crystals drives acute gouty arthritis.
- Activated PMNs release neutrophil extracellular traps (NETs) that form NET-MSU aggregates, but their role in inflammation resolution is unclear.
Purpose of the Study:
- To investigate the molecular mechanisms by which NET-MSU aggregates contribute to the resolution of acute inflammation.
- To utilize a cell-based model to understand the dynamics of NET formation and its impact on inflammatory markers.
Main Methods:
- Differentiated HL-60 cells (dHL-60) were used as surrogate PMNs and activated with MSU crystals.
- NET formation, aggregate size, cytokine levels (IL-8, TNF-α, IL-1RA), cellular redox state, signaling regulators (ERK1/2, SHP-1, SHIP-1), and surface markers (N1, N2) were analyzed.
Main Results:
- MSU-activated dHL-60 cells formed NET-MSU aggregates, initially increasing inflammation (EIS 11.6).
- Prolonged incubation or addition of new cells/crystals led to inflammation resolution (EIS 1.53) with suppressed pro-inflammatory and enhanced anti-inflammatory cytokine production.
- Large NET-MSU aggregates promoted N1 to N2 PMN polarization, crucial for inflammation resolution.
Conclusions:
- Huge NET-MSU aggregates hinder the transformation of new PMNs to the N2 phenotype.
- This process enhances the production of the anti-inflammatory cytokine IL-1RA, contributing to the spontaneous resolution of MSU-induced acute inflammation.
