CBP Bromodomain Inhibition Rescues Mice From Lethal Sepsis Through Blocking HMGB1-Mediated Inflammatory Responses

Abstract

CREB binding protein (CBP), a transcriptional coactivator and acetyltransferase, is involved in the pathogenesis of inflammation-related diseases. High mobility group box-1 protein (HMGB1) is a critical mediator of lethal sepsis, which has prompted investigation for the development of new treatment for inflammation. Here, we report that the potent and selective inhibition of CBP bromodomain by SGC-CBP30 blocks HMGB1-mediated inflammatory responses in vitro and in vivo. Our data suggest that CBP bromodomain inhibition suppresses LPS-induced expression and release of HMGB1, when the inhibitor was given 8 h post LPS stimulation; moreover, CBP bromodomain inhibition attenuated pro-inflammatory activity of HMGB1. Furthermore, our findings provide evidence that SGC-CBP30 down-regulated rhHMGB1-induced activation of MAPKs and NF-κB signaling by triggering the reactivation of protein phosphatase 2A (PP2A) and the stabilization of MAPK phosphatase 1 (MKP-1). Collectively, these results suggest that CBP bromodomain could serve as a candidate therapeutic target for the treatment of lethal sepsis via inhibiting LPS-induced expression and release of HMGB1 and suppressing the pro-inflammatory activity of HMGB1.

Keywords: CREB binding protein; MAPK phosphatase 1; high mobility group box-1 protein; protein phosphatase 2A; sepsis.

Figure 1

SGC-CBP30 treatment prevented LPS-induced lethal endotoxemia and CLP-induced sepsis. (A) Male BALB/c mice received SGC-CBP30 (19.3 mg/kg; intraperitoneal injection) 30 min or 8 h after a lethal dose of LPS (10 mg/kg; intraperitoneal injection). Similar treatments with DEX (1.3 mg/kg; intraperitoneal injection) or saline were taken as control. Mouse survival rate was monitored continuously for 72 h after LPS treatment. Blood was collected after 18 h, and serum HMGB1 (B) and TNF-α (C) were determined by ELISA. (D) Male BALB/c mice were subjected to CLP and SGC-CBP30 (19.3 mg/kg; intraperitoneal injection) was administrated to the mice 30 min or 8 h after surgical procedure. Survival of mice was monitored for 72 h. Serum levels of HMGB1 (E) and TNF-α (F) 18 h after CLP were measured using ELISA. In (A, D), Kaplan-Meier analysis was used to analyze the survival rate of septic mice. n=10 mice/group. In (B, C, E, F), graphs show mean ± SD. n=6 mice/group. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

Effect of SGC-CBP30 on tissue damage in sepsis mice. (A: LPS-, B: CLP-) Lung tissues, colon tissues, liver tissues, and kidney tissues were collected and subjected to H&E staining 18 h after the models were established, and examined by light microscopy (× 400). Scale bar: 100 μm. Histological scoring of tissue injury were evaluated as described in the Materials and Methods section. For lung tissues, the following parameters were evaluated: (a) alveolar septal thickness, (b) interstitial edema, (c) infiltration of inflammatory cells, (d) alveolar congestion/collapse; colon tissues: (a) bleeding ulcers in the intestinal mucosa, (b) interstitial edema, (c) infiltration of inflammatory cells, (d) disorganized architecture with intestinal gland; liver tissues: (a) centrilobular necrosis, (b) hepatocyte edema, (c) infiltration of inflammatory cells, (d) central venous congestion; kidney tissues: (a) epithelial cell brush-border loss, (b) interstitial edema, (c) infiltration of inflammatory cells, (d) glomerular shrinkage. Graphs show mean ± SD. n=5 mice/group. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

SGC-CBP30 plus ciprofloxacin combination therapy protected mice against lethal sepsis. (A) Male BALB/c mice were administered ciprofloxacin (7.5 mg/kg) intravenously at 1 h after CLP, and thereafter administered SGC-CBP30 (19.3 mg/kg) at 8 h after CLP. Survival of mice was monitored for up to 72 h. At 18 h post the onset of sepsis, serum levels of HMGB1 (B) and TNF-α (C) were determined by ELISA. (D) Mice were euthanized 18 h after surgeries and selected organs were collected. Lung, colon, liver, kidney of septic mice was stained with hematoxylin and eosin, and examined by light microscopy (×400). Scale bar: 100 μm. Histological scoring of tissue injury was evaluated as described in the Materials and Methods section. For lung tissues, the following parameters were evaluated: (a) alveolar septal thickness, (b) interstitial edema, (c) infiltration of inflammatory cells, (d) alveolar congestion/collapse; colon tissues: (a) bleeding ulcers in the intestinal mucosa, (b) interstitial edema, (c) infiltration of inflammatory cells, (d) disorganized architecture with intestinal gland; liver tissues: (a) centrilobular necrosis, (b) hepatocyte edema, (c) infiltration of inflammatory cells, (d) central venous congestion; kidney tissues: (a) epithelial cell brush-border loss, (b) interstitial edema, (c) infiltration of inflammatory cells, (d) glomerular shrinkage. In (a), Kaplan-Meier analysis was used to analyze the survival rates of septic mice. n=10 mice/group. In (B–D), graphs show mean ± SD. n=5 or 6 mice/group. ***p < 0.001.

Figure 4

Inhibitory effect of SGC-CBP30 on HMGB1 release and expression in LPS-stimulated cells. THP-1 cells (A) and primary MPM cells (B) were treated with SGC-CBP30 (4 μM) at 8 h after stimulation with LPS (500 ng/ml). After LPS stimulation for the indicated time, HMGB1 release was measured by ELISA. THP-1 cells were treated with LPS (500 ng/ml) for 8 h and then incubated with SGC-CBP30 (4 μM). After the indicated time of LPS stimulation, nuclear and cytoplasmic fractions were analyzed by Western blot using anti-HMGB1 antibody (C). Cells were incubated with mouse anti-HMGB1 antibody and then incubated with Alexa Flour 555-conjugated anti-mouse (red) secondary antibody. The nuclei were counterstained with DAPI (blue). The location of HMGB1 was observed under a confocal laser microscope. Scale bar: 10 μm (D). The cell lysates were immunoprecipitated with anti-HMGB1 antibody, followed by immunoblotting with anti-acetyl lysine and anti-HMGB1 antibodies (E). Quantification of HMGB1 transcripts by real-time PCR with GAPDH as the internal control (F). Whole cell lysates were subjected to immunoblotting with anti-HMGB1 and anti-β-actin antibodies (G). Data shown were representative of three independent experiments. Error bars indicate mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Effects of CBP suppression on HMGB1-mediated inflammatory response. THP-1 cells (A) and primary MPM cells (B) were pretreated with SGC-CBP30 (4 μM) for 2 h and then stimulated with rhHMGB1 (500 ng/ml) for 12 h; the production of TNF-α, IL-1β, and IL-6 were measured by ELISA. (C) THP-1 cells and primary MPM cells were pretreated with or without SGC-CBP30 (4 μM) for 2 h followed by stimulation with rhHMGB1 (100 ng/ml) for 1 h. Cell lysates were prepared and the levels of phospho-MAPKs, phospho-IKKα/β and phospho-IκBα were determined by Western blot analysis. (D) THP-1 cells were transfected with CBP shRNA or negative control shRNA, and after 72 h, stimulated with rhHMGB1 (100 ng/ml) for 1 h. The cell lysates were subjected to immunoblotting analysis using indicated antibodies. Data shown were representative of three independent experiments. Error bars indicate mean ± SD. **p < 0.01; ***p < 0.001.

Figure 6

CBP suppression prevented rhHMGB1 from reducing PP2A activity and facilitating MKP-1 degradation. (A) THP-1 cells and primary MPM cells were pretreated with SGC-CBP30 (4 μM) for 2 h, followed by rhHMGB1 (100 ng/ml) treatment for 1 h. Whole cell lysates were immunoblotted using anti-CBP, anti-p-PP2A(Y307), anti-methyl-PP2A-Cα/β, anti-PP2A-Cα/β, anti–p-MKP-1(S359) or anti–MKP-1 antibodies, respectively. (B) THP-1 cells were transfected with CBP shRNA or negative control shRNA, and 72 h after transfection, cells were stimulated with or without rhHMGB1 (100 ng/ml) for 1 h. Cell lysates were subjected to immunoblotting with indicated antibodies. THP-1 cells were treated the same as in (A), and cell lysates were immunoprecipitated with anti-PP2A-Cα/β then immunoblotted with anti–PME-1 and anti-PTPA antibodies (C). The mRNA expression levels of MKP-1 were detected by RT-PCR (D). THP-1 cells were pretreated with SGC-CBP30 (4 μM) for 2 h, followed by rhHMGB1 (100 ng/ml) for 1 h in the presence or absence of MG132 (20 μM). Cell lysates were immunoprecipitated using anti–MKP-1 antibody, followed by immunoblotting using antibody against ubiquitin (Ub) (E). Cells were transfected with CBP shRNA, and 72 h after transfection, then treated with or without rhHMGB1 (100 ng/ml) for 1 h in the presence or absence of MG132 (5, 10, or 20 μM). Western blot analysis was performed using anti-p-MKP-1 (S359) or anti-MKP-1 antibodies (F). Data shown were representative of three independent experiments. Error bars indicate mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., no significance.

Figure 7

CBP bromodomain inhibitor SGC-CBP30 prevented rhHMGB1-induced inflammatory response by rescuing PP2A/MKP-1. THP-1 cells were pretreated with SGC-CBP30 (4 μM), and then with okadaic acid (100 mM) (A) or RO 31-8220 (5 μM) (B) for 1 h, followed by treatment with rhHMGB1 (100 ng/ml) for 1 h. Whole cell lysates were subjected to Western blotting using indicated antibodies. Cells were transfected with Flag-PP2A (C) or Flag-MKP-1 (D) and pretreated with SGC-CBP30 (4 μM) for 2 h, followed by treatment with rhHMGB1 (100 ng/ml) for 1 h. Whole cell lysates were subjected to Western blotting using indicated antibodies. Cells were transfected with PP2A shRNA (E) or MKP-1 shRNA (F), followed by pretreatment with SGC-CBP30 (4 μM) for 2 h and then stimulation with rhHMGB1 (100 ng/ml) for 1 h. Whole cell lysates were subjected to Western blotting using indicated antibodies. Data shown were representative of three independent experiments. Error bars indicate mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 8

Schematic illustration of CBP bromodomain inhibitor prevents sepsis-related HMGB1 secretion and blocks HMGB1 pro-inflammatory activity. The potent and selective CBP inhibitor, SGC-CBP30, significantly rescues mice from LPS-/CLP-induced sepsis model. In addition, SGC-CBP30 suppresses LPS-induced HMGB1 expression and cytoplasmic translocation. Furthermore, by triggering the reactivation of PP2A and the stabilization of MKP-1, CBP bromodomain inhibition prevents rhHMGB1-stimulated activation of MAPKs and NF-κB pathways and production of proinflammatory cytokines.