Nuclear retention of IL-1 alpha by necrotic cells: a mechanism to dampen sterile inflammation - PubMed (original) (raw)

Nuclear retention of IL-1 alpha by necrotic cells: a mechanism to dampen sterile inflammation

Nadia M Luheshi et al. Eur J Immunol. 2009 Nov.

Abstract

Sterile inflammation is a host response to tissue injury that is mediated by damage-associated molecular patterns released from dead cells. Sterile inflammation worsens damage in a number of injury paradigms. The pro-inflammatory cytokine IL-1 alpha is reported to be a damage-associated molecular pattern released from dead cells, and it is known to exacerbate brain injury caused by stroke. In the brain, IL-1 alpha is produced by microglia, the resident brain macrophages. We found that IL-1 alpha is actively trafficked to the nuclei of microglia, and hence tested the hypothesis that trafficking of IL-1 alpha to the nucleus would inhibit its release following necrotic cell death, limiting sterile inflammation. Microglia subjected to oxygen-glucose deprivation died via necrosis. Under these conditions, microglia expressing nuclear IL-1 alpha released significantly less IL-1 alpha than microglia with predominantly cytosolic IL-1 alpha. The remaining IL-1 alpha was immobilized in the nuclei of the dead cells. Thus, nuclear retention of IL-1 alpha may serve to limit inflammation following cell death.

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Figures

Figure 1

Figure 1. IL-1α nuclear localization in BV-2 microglia is inhibited by high local cell density

BV-2 microglia were left untreated (Ai) or LPS-treated (Aii, iii, B-E 1 μg/mL, 6 hours), IL-1α-immunostained (green) and co-stained with DAPI (blue). The % IL-1α-expressing cells containing nuclear IL-1α in individual fields of view was quantified by blind manual cell counting (B, C, E). IL-1α was intranuclear in some microglia (Aii, low local cell density) and cytosolic in others (Aiii, high local cell density) when microglia were cultured at 3.5 - 5 × 105 cells/mL. IL-1α localization was quantified in individual fields of view from microglia seeded at 0.25 - 5 × 105 cells/mL, and correlated with local cell density (B). Individual points represent single fields of view, linear regression line (solid line) R2 = 0.49 p<0.0001 vs. slope of zero. IL-1α localisation was also quantified in low density microglia (1 × 105 cells/mL) cultured with or without high density microglia (1 × 106 cells/mL) in transwell inserts (+TW or alone respectively, C). BV-2 microglia (1 × 105 cells/mL, isolectin B-4 stained, red, IB-4) were cultured alone (Di) or with HEK-293 cells (Dii). IL-1α localisation was quantified in microglia cultured with (HEK) or without (No HEK, E) HEK-293 cells. Data from n ≥ 3 independent experiments. Scale bars = 40 μm. ***p<0.0001, Student’s t-test.

Figure 2

Figure 2. Intranuclear IL-1α is retained by necrotic microglia

BV-2 microglia were seeded at low, medium or high density (1, 5 and 10 × 105 cells/mL respectively), LPS-treated (1μg/mL, 3 hours) and either kept in normoxic conditions or killed by 24 hours OGD. % total LDH (A) and IL-1α (B) release into media were quantified. Data are from n = 6 independent experiments. ***p<0.001 One-way ANOVA with post-hoc Bonferroni multiple comparison test. Remaining adherent cells in normoxia- (C) and OGD-treated (D) cultures of low density (Ci, Di) and high density BV-2 cells (Cii, Dii) were IL-1α-immunostained (green) with PI (red) and DAPI (blue) co-staining. Images are from one of n = 3 independent experiments. Scale bar = 40 μm.

Figure 3

Figure 3. IL-1α-GFP is retained by necrotic COS-7 cells

COS-7 cells transiently transfected with GFP or IL-1α-GFP were killed by ATP-depletion (30 minutes, 1 μM CCCP, 15mM 2-DOG) and OGD (24 hours). Control cells were kept under normoxic conditions and trypsinised. Necrotic, detached cells and live, trypsinised cells were collected and PI-stained. Cellular GFP and PI fluorescence was measured by FACS. Scatter plots (A) show PI and GFP fluorescence intensities of necrotic cells in one representative experiment. Dashed rectangle indicates PI+/GFP+ cells. Mean GFP fluorescence of GFP and IL-1α-GFP positive COS-7 cells was measured in live, trypsinised cells and in dead, PI positive cells (C). *p<0.05, **p<0.01, ***p<0.001, One-way ANOVA with post-hoc Bonferroni multiple comparison. The proportion of GFP fluorescence retained on cell death was calculated for GFP- and IL-1α-GFP-expressing cells (D). **p<0.01, Student’s t-test. Data from n = 3 independent experiments.

Figure 4

Figure 4. Intranuclear IL-1α is immobilised on depletion of cellular ATP

GFP or IL-1α-GFP-expressing COS-7 cells were ATP depleted (30 minutes, “+ inhibitors”, Aii, Bii) or maintained in imaging buffer with glucose “+ glucose” Ai, Bi). Confocal images (Ai, ii) show GFP and IL-1α-GFP intranuclear distribution in DAPI stained cells. Scale bar = 10 μm. Time-lapse confocal images (B) show fluorescence recovery after photobleaching of an intranuclear ROI (red square). Scale bar = 5 μm. Mean nucleoplasmic fluorescence recovery curves (C) and half times (t1/2, D) for fluorescence recovery are shown for GFP and IL-1α-GFP expressing cells with and without ATP depletion. Data from n = 15 individual cells per construct. ***p<0.001, *p<0.05, One-way ANOVA with post-hoc Bonferroni multiple comparison test.

Figure 5

Figure 5. Intranuclear IL-1α co-localizes with nuclear speckles and p300 on depletion of cellular ATP

IL-1α-GFP-expressing (green) COS-7 cells were ATP depleted (30 minutes), immunostained for p300 (a HAT), SC35 (nuclear speckle marker) or polII0 (active transcription site marker), and DAPI co-stained. Confocal images (A) are of representative cell nuclei from one of three independent experiments. Scale bar = 5 μm. The extent of co-localization of IL-1α-GFP with nuclear proteins and DNA (DAPI) was assessed using Pearson’s co-localization analysis (B). Data are from n ≥ 20 cells per co-stain.

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