Inhibition of BRD4 Reduces Neutrophil Activation and Adhesion to the Vascular Endothelium Following Ischemia Reperfusion Injury

Abstract

Renal ischemia reperfusion injury (IRI) is associated with inflammation, including neutrophil infiltration that exacerbates the initial ischemic insult. The molecular pathways involved are poorly characterized and there is currently no treatment. We performed an in silico analysis demonstrating changes in NFκB-mediated gene expression in early renal IRI. We then evaluated NFκB-blockade with a BRD4 inhibitor on neutrophil adhesion to endothelial cells in vitro, and tested BRD4 inhibition in an in vivo IRI model. BRD4 inhibition attenuated neutrophil adhesion to activated endothelial cells. In vivo, IRI led to increased expression of cytokines and adhesion molecules at 6 h post-IRI with sustained up-regulated expression to 48 h post-IRI. These effects were attenuated, in part, with BRD4 inhibition. Absolute neutrophil counts increased significantly in the bone marrow, blood, and kidney 24 h post-IRI. Activated neutrophils increased in the blood and kidney at 6 h post-IRI and remained elevated in the kidney until 48 h post-IRI. BRD4 inhibition reduced both total and activated neutrophil counts in the kidney. IRI-induced tubular injury correlated with neutrophil accumulation and was reduced by BRD4 inhibition. In summary, BRD4 inhibition has important systemic and renal effects on neutrophils, and these effects are associated with reduced renal injury.

Keywords: BRD4; NFκB; inflammation; ischemia-reperfusion; neutrophils; tubule injury.

Figure 1

Early IRI is characterized by NFκB-mediated gene expression. (A) Venn diagram of genes identified following IRI and genes regulated by NFκB [19,20]. (B) Significantly enriched biological processes for the NFκB-mediated genes identified following IRI using Biological Networks Gene Ontology with Benjamini and Hochberg multiple testing correction (p < 0.05) and then run on Enrichment Map with Jaccard coefficient of 0.5 (p = 0.001; false discovery rate q-value = 0.05). Edge thickness represents the number of overlapping markers between two connecting nodes. (C) Heatmap representation of 34 NFκB-mediated genes following IRI at 4, 24 and 48 h with Euclidean hierarchical clustering. Log-transformed FPKM values for genes were converted into z-scores using mean values. Higher scores are indicated in red and lower scores in blue. (D) Flow diagram showing significant genes mediated by NFκB at 4, 24 and 48 h following IRI. (E–G) Volcano plots of NFκB-mediated genes following IRI at 4 (E), 24 (F), and 48 h (G). Independent Student t-tests (p < 0.05; red) with Benjamini and Hochberg multiple testing correction (q < 0.05; purple) was performed at each timepoint.

Figure 2

BRD4 inhibition attenuated NFκB, AP-1, and Smad-Binding Element (SBE) mediated luciferase activity. HK-2 cells were transfected with NFκB (A), AP-1 (B) or SBE (C) vectors and treated with 1 μM MS417 for 1 h followed by 10 ng/mL TNFa (NFκB), 50 ng/mL EGF (AP-1), or 5 ng/mL TGF-β1 (SBE) treatment. n = 3, *** p < 0.001, **** p < 0.0001.

Figure 3

MS417 treatment had no effect on cell viability up to 1 μM (A) BRD4 inhibition with MS417 had no effect on neutrophil cell viability at concentrations up to 1 μM MS417. MTT Cell Viability assay was performed following treatment of isolated human neutrophils with 1nM–1 mM MS417 for 1 h. (B) BRD4 inhibition improves cell viability of HUVEC following H/R. HUVEC were exposed to 1% O₂ for 2 h followed by treatment with 1 μM MS417 and 6, 12, and 24 h of re-oxygenation. Serum free media is in black, serum free media plus MS417 is in grey. Two-way ANOVA with Tukey was performed. n = 6, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

BRD4 inhibition attenuated neutrophil adhesion to the endothelium (A) BRD4 inhibition attenuated neutrophil adhesion to the endothelium with treatment to the neutrophils, endothelial cells, or combined. Isolated human neutrophils (10⁵ cells/well) or TNFα-stimulated HUVEC monolayers were treated with 1 μM MS417 for 1 h or left untreated and then co-incubated in various combinations as indicated. In some cases, MS417 was washed out as indicated. Cells were allowed to adhere for 30 min and any non-adherent cells were removed. Adhesion was measured using a fluorescent plate reader at excitation and emission wavelengths of 494 and 517 m, respectively. (N = neutrophils, H = HUVEC). (B) To better represent IRI, HUVEC were exposed to 1% O₂ for 2 h followed by 3 h of re-oxygenation. Neutrophil were treated with either 100 nm or 1 μM of MS417 and adhesion was measured as previously described. Two-way ANOVA with Tukey was performed. n = 6, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

BRD4 attenuated the gene expression of NFκB-mediated genes. (A–C) Exposure time was optimized by treating PTECs with H₂O₂ for 0, 1, 6, and 24 h before cells were collected to qPCR analysis of IL6 (A), CXCL2 (B) and CCL2 (C). (D–F) In a subsequent set of studies, cells were treated with 1 μM MS417 for 2 h followed by H₂O₂ for 6 h followed by qPCR. n = 3, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 6

BRD4 inhibition attenuated gene expression in vivo following IRI. (A) A schematic representing the in vivo experimental design. C57BL/6 mice were treated with 1 μM MS417 daily for 7 days by oral gavage before unilateral IRI. Mice were sacrificed at 6 (B), 24 (C), and 48 (D) hours post-IRI and kidney tissue was collected for qPCR analysis of pro-inflammatory cytokines (CCL2 and TNFα) and adhesion molecules (ICAM1 and VCAM1). n = 7–11, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

BRD4 inhibition attenuated absolute neutrophil counts in the bone marrow, circulation and kidney tissue following IRI (A) Flow cytometry gating strategy in kidney tissue. Mouse PMNs were gated based on Ly6G^+ve/F4/80^−ve. Doublets were excluded using SSC-W x SSC-H. (B–D) C57BL6 mice were treated with 1 μM MS417 daily for 7 days by oral gavage before unilateral IRI. Bone marrow (B), blood (C) and kidney tissue (D) was collected at 6, 24, and 48 h following IRI and absolute neutrophil counts were quantified by flow cytometry. ANOVA with Fischers LSD test was performed. n = 7–11, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 8

BRD4 inhibition attenuated neutrophil infiltration in kidney tissue following IRI (A–C) Semi-quantitative scoring of neutrophil infiltration on Ly6B.2 clone 7/4 stained kidney sections from 6 h (A), 24 h (B), and 48 h (C) performed in a blinded manner. (D) Representative images of neutrophil infiltration on Ly6B.2 clone 7/4 stained kidney sections performed at 24 h in Sham, IRI and IRI + MS417 mice. Images are at 1.6× magnification with high magnification inserts at 20×. n = 7–9, * p < 0.05, ** p < 0.01.

Figure 9

BRD4 inhibition attenuated CD66a up-regulation on kidney neutrophils following IRI but had no effect on CD11b expression. C57BL/6 mice were treated with 1 μM MS417 daily for 7 days by oral gavage before unilateral IRI. (A,B) Representative histograms of CD66a and CD11b expression for kidney tissue neutrophils (CD16^high/side scatter area (SSC-A)^high) in mice comparing Sham and IRI plus/minus MS417 treatment at 24 h post-IRI. Bone marrow (C,F), blood (D,G) and kidney tissue (E,H) was collected at 6, 24, and 48 h following IRI and CD66a+ and CD11b+ neutrophils were quantified by flow cytometry. MFI = mean fluorescence intensity. ANOVA with Fischers LSD test was performed. n = 7–11, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 10

BRD4 inhibition attenuated tubular injury following IRI. (A–C) Semi-quantitative scoring of tubular injury on PAS sections performed in a blinded manner at 6 h (A), 24 h (B), and 48 h (C). (D) Representative images of PAS staining performed at 24 h in Sham, IRI and IRI + MS417 mice. Images are at 1.6× magnification with high magnification inserts at 20×. n = 7–9, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 11

BRD4 inhibition attenuated expression of injury markers following IRI. (A–C) Mice were sacrificed at 6 (A), 24 (B), and 48 h (C) and kidney tissue was collected for qPCR analysis of injury markers (HAVCR1 and LCN2). n = 7–11, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 12

Neutrophil infiltration is related to tubular injury following IRI. Correlation plot of semi-quantitative scoring performed in a blinded manner of tubular injury and neutrophil infiltration at 24 h-post IR (R2 = 0.7663, p = 0.004). n = 8 (two sets of mice had the same scores so are overlapped).