Macrophage-derived IL-1β enhances monosodium urate crystal-triggered NET formation

Abstract

Objective and design: Arthritic gout is caused by joint inflammation triggered by the damaging effects of monosodium uric acid (MSU) crystal accumulation in the synovial space. Neutrophils play a major role in mediating joint inflammation in gout. Along with neutrophils, other immune cells, such as macrophages, are present in inflamed joints and contribute to gout pathogenesis. Neutrophils form neutrophil extracellular traps (NETs) in response to MSU crystals. In the presence of MSU crystals, macrophages release IL-1β, a cytokine crucial to initiate gout pathogenesis and neutrophil recruitment. Our research investigated interactions between human macrophages and neutrophils in an in vitro model system and asked how macrophages affect NET formation stimulated by MSU crystals.

Materials or subjects: Human neutrophils and PBMCs were isolated from peripheral blood of healthy volunteers. PBMCs were differentiated into macrophages in vitro using human M-CSF.

Treatment: Human neutrophils were pretreated with macrophage-conditioned media, neutrophil-conditioned media, recombinant human IL-1β or anakinra prior to stimulation by MSU crystals.

Method: Interaction of neutrophils with MSU crystals was evaluated by live imaging using confocal microscopy. The presence of myeloperoxidase (MPO) and neutrophil elastase (NE) was measured by ELISA. NET formation was quantitated by Sytox Orange-based extracellular DNA release assay and NE-DNA ELISA. AggNET formation was assessed by macroscopic evaluation.

Results: We found that crystal- and cell-free supernatants of macrophages stimulated with MSU crystals promote MSU crystal-stimulated NET formation in human neutrophils. This observation was confirmed by additional assays measuring the release of MPO, NE, and the enzymatic activity of NE. MSU crystal-induced NET formation remained unchanged when neutrophil supernatants were tested. IL-1β is a crucial cytokine orchestrating the onset of inflammation in gout and is known to be released in large amounts from macrophages following MSU crystal stimulation. We found that recombinant IL-1β strongly promoted MSU crystal-induced NET formation in human neutrophils. Interestingly, IL-1β alone did not induce any NET release. We also found that clinical grade anakinra, an IL-1 receptor blocker, strongly reduced the NETosis-enhancing effect of macrophage supernatants indicating that IL-1β is mainly responsible for this effect.

Conclusions: Macrophage-derived IL-1β enhances MSU crystal-induced NET release in neutrophils. We identified a new mechanism by which macrophages and IL-1β affect neutrophil functions, and could contribute to the inflammatory conditions present in gout. Our results also revealed a new anti-inflammatory mechanism of anakinra.

Keywords: Gout; Interleukin-1 beta; Macrophage; Neutrophil extracellular traps (NETs).

Figure 1. Supernatants of macrophages, but not neutrophils, enhance extracellular DNA release from MSU crystal-stimulated human neutrophils

A) Dose-dependence of MSU crystal-stimulated NET release in human PMNs measured in the presence of the fluorescent, extracellular DNA-binding dye, Sytox Orange. Mean+/−S.E.M., n=3. B–D) Human macrophages differentiated with M-CSF for 5–7 days or neutrophils were exposed to 250 μg/ml MSU crystals for 5 hours. Collected supernatants were centrifuged to remove cells, crystals and debris and referred to as “PMN-conditioned medium” or “macrophage-conditioned medium”, respectively. The effects of conditioned media on NET formation were subsequently tested on human neutrophils stimulated in the absence (UT, untreated) or presence (MSU) of 250 μg/ml MSU crystals by Sytox Orange-based fluorescence. Fluorescence data were normalized on the 100 nM PMA-triggered signal as the positive control for NET formation. B) “PMN-conditioned media” of neither MSU crystal-stimulated (MSU PMN) nor untreated neutrophils (UT PMN) had any significant effect on NET release (No Sup). Mean+/−S.E.M., n=4, Dunnett’s test. C) “Macrophage-conditioned media” of MSU crystal-stimulated macrophages (MSU MAC) significantly increased NET formation in comparison to that seen in absence of conditioned media (No Sup). Conditioned media of macrophages without crystal stimulation (UT MAC, untreated) had no significant effect. Mean+/−S.E.M., n=11, Dunnett’s test. D) Representative confocal microscopy images of MSU crystal-stimulated human neutrophils show enhanced DNA release (Sytox Orange) by the MSU crystal-stimulated macrophage conditioned media compared (lower panels) to that without addition of conditioned media (upper panels) (n=3). 1) Merged images of transmitted light and Sytox Orange fluorescence and 2) three-dimensional Z-stack fluorescent images are shown for both experimental conditions. Black bars indicate 10 μm distance. RFU, relative fluorescence unit; PMA, phorbol myristate acetate; MAC, macrophage; PMN, neutrophil; MSU, monosodium urate; n.s., not significant; ****, p<0.0001.

Figure 2. Macrophage supernatants increase MPO, HNE and NET release from human neutrophils following MSU crystal stimulation

Differentiated human macrophages were exposed to 250 μg/ml MSU crystals for 5 hours to collect and centrifuge their supernatants. This “macrophage-conditioned medium” (MSU MAC) was added to MSU crystal-stimulated human neutrophils and releases of A) MPO (n=9), B) HNE (n=9) and C) NETs (MPO-DNA, n=6) were quantitated by ELISA assays (mean+/−S.E.M., Dunnett’s test). As comparisons, PMNs were also treated with mock only (No MAC) or supernatants of macrophages without crystal stimulation (UT MAC, untreated). MPO, myeloperoxidase; HNE, human neutrophil elastase; MAC, macrophage. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 3. IL-1β promotes DNA release from human neutrophils stimulated with MSU crystals

A) MSU crystal-stimulation resulted in IL-1β release in human macrophages (MF) but not in human PMNs as measured by ELISA. Mean+/−S.E.M., n=4. Student’s t-test. B) Following treatment with increasing doses of recombinant human IL-1β, human neutrophils were left unstimulated or were stimulated with 100 or 250 μg/ml MSU crystals and DNA release was measured in presence of the membrane-impermeable DNA-binding dye, Sytox Orange. DNA release over 4 hours was presented as endpoint values (mean+/−S.E.M., n=3). C) Kinetics of the previous experiment (panel B) (one representative, n=4). Fluorescence data were normalized on the maximal signal obtained by stimulation with 100 nM PMA. RFU, relative fluorescence unit; MSU, monosodium urate; UT, untreated; PMA, phorbol myristate acetate; n.s., not significant; *, p<0.05; ***, p<0.001; ****, p<0.0001.

Figure 4. IL-1β enhances MSU crystal-stimulated releases of MPO, HNE and NETs from human neutrophils

Human neutrophils were treated with recombinant human IL-1β (0–5 ng/ml) prior to addition of 250 μg/ml MSU crystals. Neutrophils were also left unstimulated (UT, no crystals). Five hours later the following parameters were quantitated in the supernatants: A) MPO by commercial ELISA (n=5), B) HNE by ELISA (n=9), C) NETs (HNE-DNA ELISA, n=7) and D) HNE activity by commercial kit (n=7). NET release values were normalized on PMA (100%). Data are shown as mean+/−S.E.M. Significance was calculated using Dunnett’s test. *, p<0.05; **, p<0.01. MPO, myeloperoxidase; HNE, human neutrophil elastase; PMA, phorbol myristate acetate.

Figure 5. IL-1β increases MSU crystal-induced aggregated NET formation

250,000 human PMNs/well were incubated in wells of a 96-well microplate with IL-1β (10 ng/ml) and stimulated with 100 μg/ml MSU crystals or 100nM PMA for 15 hours in presence of the extracellular DNA-binding dye, Sytox Orange. Interactions of PMNs and crystals were followed using confocal microscopy every 15 minutes. A) Representative images taken at t=0, 2, 4, 6. hrs are shown (n=3). Black bars indicate 100 μm distances. Insets indicated by white frames are enlarged on the right side. B) Representative kinetics of Sytox Orange fluorescence curves followed for 8 hours (n=3, mean+/−S.D. at each time point). C) AggNET formation was evaluated by light microscopy after a 5-hour incubation of human PMNs with 1 mg/ml MSU crystal in presence or absence of 10 ng/ml human IL-1β (One representative image, n=3). MSU, monosodium urate; aggNET, aggregated NETs; RFU, relative fluorescence unit.

Figure 6. Anakinra inhibits the NET-enhancing effect of macrophage supernatants

A) Macrophages were isolated from nine independent donors (donors #4–12) and macrophage-conditioned media were collected when cells were left untreated (UT, no crystals) or stimulated with 250 mg/ml MSU crystals for 5 hours (MSU). Supernatants were cleaned, frozen and used in groups of three on PMNs isolated from three independent blood donors as indicated. PMNs were pretreated with 30 mg/ml anakinra for 1 hr before addition of 100 mg/ml MSU crystals and/or macrophage supernatants. EcDNA release was measured in PMNs for 6 hours following stimulation by the crystals using Sytox Orange dye. Results are expressed as percentage of maximal DNA release and shown as mean+/−S.D. of triplicates. B) Data shown in panel A) were further analyzed by calculating the MSU crystal-induced DNA release signal and its averages. Data are shown as mean+/−S.E.M. (n=12) and were analyzed by one-way ANOVA and Tukey’s post-hoc test. *, p<0.05; ***, p<0.001. MAC, macrophage; PMN, neutrophil; UT, untreated.