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NF-κB inhibition after cecal ligation and puncture reduces sepsis-associated lung injury without altering bacterial host defense.

Li H, Han W, Polosukhin V, Yull FE, Segal BH, Xie CM, Blackwell TS - Mediators Inflamm. (2013)

Bottom Line: Treatment with the NF-κB inhibitor did not affect the ability of cultured macrophages to phagocytose bacteria and did not alter bacterial colony counts in blood, lung tissue, or peritoneal fluid at 24 hours after CLP.While BMS-345541 treatment did not alter mortality after CLP, our results showed a trend towards improved survival.Transiently blocking NF-κB activity after the onset of CLP-induced sepsis can effectively reduce acute lung injury in mice without compromising bacterial host defense or survival after CLP.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Medicine, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong Province 510080, China ; Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA ; Division of Respiratory Medicine, Nanjing Drum Tower Hospital & the Affiliated Hospital of Nanjing University, Nanjing, Jiangsu 210008, China.

ABSTRACT

Introduction: Since the NF-κB pathway regulates both inflammation and host defense, it is uncertain whether interventions targeting NF-κB would be beneficial in sepsis. Based on the kinetics of the innate immune response, we postulated that selective NF-κB inhibition during a defined time period after the onset of sepsis would reduce acute lung injury without compromising bacterial host defense.

Methods: Mice underwent cecal ligation and puncture (CLP). An NF-κB inhibitor, BMS-345541 (50 µg/g mice), was administered by peroral gavage beginning 2 hours after CLP and repeated at 6 hour intervals for 2 additional doses.

Results: Mice treated with BMS-345541 after CLP showed reduced neutrophilic alveolitis and lower levels of KC in bronchoalveolar lavage fluid compared to mice treated with CLP+vehicle. In addition, mice treated with CLP+BMS had minimal histological evidence of lung injury and normal wet-dry ratios, indicating protection from acute lung injury. Treatment with the NF-κB inhibitor did not affect the ability of cultured macrophages to phagocytose bacteria and did not alter bacterial colony counts in blood, lung tissue, or peritoneal fluid at 24 hours after CLP. While BMS-345541 treatment did not alter mortality after CLP, our results showed a trend towards improved survival.

Conclusion: Transiently blocking NF-κB activity after the onset of CLP-induced sepsis can effectively reduce acute lung injury in mice without compromising bacterial host defense or survival after CLP.

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Related in: MedlinePlus

Treatment with the IKK inhibitor (BMS-345541) inhibits NF-κB activation after CLP. Nuclear protein extracts from (a) spleen and (b) lung tissue were probed for p65 (RelA) by western blot. Densitometry shows p65 band density normalized by TATA binding protein (TBP). ANOVA test was used for comparison among multiple groups. Tukey post hoc tests were undertaken after ANOVA. There were significant differences for CLP versus sham or CLP+BMS mice (*P < 0.05 for CLP versus Ctrl, #P < 0.05 for CLP+BMS versus CLP), while there was no significant difference between sham and CLP+BMS mice. (c) Lung NF-κB DNA binding activity was also assessed in nuclear protein extracts by TransAM ELISA. ANOVA test was used for comparison among multiple groups. Tukey post hoc tests were undertaken after ANOVA. There were significant differences for CLP versus sham or CLP+BMS mice (*P < 0.05 for CLP versus Ctrl, #P < 0.05 for CLP+BMS versus CLP), while there was no significant difference between sham and CLP+BMS mice. Results are presented as mean ± SE, N = 3 per group. Each experiment was repeated three times.
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fig1: Treatment with the IKK inhibitor (BMS-345541) inhibits NF-κB activation after CLP. Nuclear protein extracts from (a) spleen and (b) lung tissue were probed for p65 (RelA) by western blot. Densitometry shows p65 band density normalized by TATA binding protein (TBP). ANOVA test was used for comparison among multiple groups. Tukey post hoc tests were undertaken after ANOVA. There were significant differences for CLP versus sham or CLP+BMS mice (*P < 0.05 for CLP versus Ctrl, #P < 0.05 for CLP+BMS versus CLP), while there was no significant difference between sham and CLP+BMS mice. (c) Lung NF-κB DNA binding activity was also assessed in nuclear protein extracts by TransAM ELISA. ANOVA test was used for comparison among multiple groups. Tukey post hoc tests were undertaken after ANOVA. There were significant differences for CLP versus sham or CLP+BMS mice (*P < 0.05 for CLP versus Ctrl, #P < 0.05 for CLP+BMS versus CLP), while there was no significant difference between sham and CLP+BMS mice. Results are presented as mean ± SE, N = 3 per group. Each experiment was repeated three times.

Mentions: Based on previous work using BMS-345541 to block NF-κB activation in vivo [6], we developed a protocol to target NF-κB activation by beginning BMS-345541 treatment after the initiation of systemic inflammation following CLP. Mice were treated with BMS-345541 or vehicle by peroral gavage beginning at 2 hours after CLP. BMS-345541 dosing was repeated at 6-hour intervals for a total of three doses. Multiple doses were used because of the short half-life of the compound in vivo [16]. To evaluate the efficacy of this intervention, we measured nuclear RelA (p65) levels by western blot analysis at 24 hours following CLP. As shown in Figures 1(a) and 1(b), BMS-345541 treatment blocked RelA (p65) nuclear translocation in spleens and lungs at this time point. In addition, NF-κB DNA-binding activity was reduced in the lungs of BMS-345541-treated mice compared to mice that received CLP and vehicle (Figure 1(c)). Together, these findings confirmed that BMS-345541 blocks CLP-induced NF-κB activity in our model.


NF-κB inhibition after cecal ligation and puncture reduces sepsis-associated lung injury without altering bacterial host defense.

Li H, Han W, Polosukhin V, Yull FE, Segal BH, Xie CM, Blackwell TS - Mediators Inflamm. (2013)

Treatment with the IKK inhibitor (BMS-345541) inhibits NF-κB activation after CLP. Nuclear protein extracts from (a) spleen and (b) lung tissue were probed for p65 (RelA) by western blot. Densitometry shows p65 band density normalized by TATA binding protein (TBP). ANOVA test was used for comparison among multiple groups. Tukey post hoc tests were undertaken after ANOVA. There were significant differences for CLP versus sham or CLP+BMS mice (*P < 0.05 for CLP versus Ctrl, #P < 0.05 for CLP+BMS versus CLP), while there was no significant difference between sham and CLP+BMS mice. (c) Lung NF-κB DNA binding activity was also assessed in nuclear protein extracts by TransAM ELISA. ANOVA test was used for comparison among multiple groups. Tukey post hoc tests were undertaken after ANOVA. There were significant differences for CLP versus sham or CLP+BMS mice (*P < 0.05 for CLP versus Ctrl, #P < 0.05 for CLP+BMS versus CLP), while there was no significant difference between sham and CLP+BMS mice. Results are presented as mean ± SE, N = 3 per group. Each experiment was repeated three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3852536&req=5

fig1: Treatment with the IKK inhibitor (BMS-345541) inhibits NF-κB activation after CLP. Nuclear protein extracts from (a) spleen and (b) lung tissue were probed for p65 (RelA) by western blot. Densitometry shows p65 band density normalized by TATA binding protein (TBP). ANOVA test was used for comparison among multiple groups. Tukey post hoc tests were undertaken after ANOVA. There were significant differences for CLP versus sham or CLP+BMS mice (*P < 0.05 for CLP versus Ctrl, #P < 0.05 for CLP+BMS versus CLP), while there was no significant difference between sham and CLP+BMS mice. (c) Lung NF-κB DNA binding activity was also assessed in nuclear protein extracts by TransAM ELISA. ANOVA test was used for comparison among multiple groups. Tukey post hoc tests were undertaken after ANOVA. There were significant differences for CLP versus sham or CLP+BMS mice (*P < 0.05 for CLP versus Ctrl, #P < 0.05 for CLP+BMS versus CLP), while there was no significant difference between sham and CLP+BMS mice. Results are presented as mean ± SE, N = 3 per group. Each experiment was repeated three times.
Mentions: Based on previous work using BMS-345541 to block NF-κB activation in vivo [6], we developed a protocol to target NF-κB activation by beginning BMS-345541 treatment after the initiation of systemic inflammation following CLP. Mice were treated with BMS-345541 or vehicle by peroral gavage beginning at 2 hours after CLP. BMS-345541 dosing was repeated at 6-hour intervals for a total of three doses. Multiple doses were used because of the short half-life of the compound in vivo [16]. To evaluate the efficacy of this intervention, we measured nuclear RelA (p65) levels by western blot analysis at 24 hours following CLP. As shown in Figures 1(a) and 1(b), BMS-345541 treatment blocked RelA (p65) nuclear translocation in spleens and lungs at this time point. In addition, NF-κB DNA-binding activity was reduced in the lungs of BMS-345541-treated mice compared to mice that received CLP and vehicle (Figure 1(c)). Together, these findings confirmed that BMS-345541 blocks CLP-induced NF-κB activity in our model.

Bottom Line: Treatment with the NF-κB inhibitor did not affect the ability of cultured macrophages to phagocytose bacteria and did not alter bacterial colony counts in blood, lung tissue, or peritoneal fluid at 24 hours after CLP.While BMS-345541 treatment did not alter mortality after CLP, our results showed a trend towards improved survival.Transiently blocking NF-κB activity after the onset of CLP-induced sepsis can effectively reduce acute lung injury in mice without compromising bacterial host defense or survival after CLP.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Medicine, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong Province 510080, China ; Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA ; Division of Respiratory Medicine, Nanjing Drum Tower Hospital & the Affiliated Hospital of Nanjing University, Nanjing, Jiangsu 210008, China.

ABSTRACT

Introduction: Since the NF-κB pathway regulates both inflammation and host defense, it is uncertain whether interventions targeting NF-κB would be beneficial in sepsis. Based on the kinetics of the innate immune response, we postulated that selective NF-κB inhibition during a defined time period after the onset of sepsis would reduce acute lung injury without compromising bacterial host defense.

Methods: Mice underwent cecal ligation and puncture (CLP). An NF-κB inhibitor, BMS-345541 (50 µg/g mice), was administered by peroral gavage beginning 2 hours after CLP and repeated at 6 hour intervals for 2 additional doses.

Results: Mice treated with BMS-345541 after CLP showed reduced neutrophilic alveolitis and lower levels of KC in bronchoalveolar lavage fluid compared to mice treated with CLP+vehicle. In addition, mice treated with CLP+BMS had minimal histological evidence of lung injury and normal wet-dry ratios, indicating protection from acute lung injury. Treatment with the NF-κB inhibitor did not affect the ability of cultured macrophages to phagocytose bacteria and did not alter bacterial colony counts in blood, lung tissue, or peritoneal fluid at 24 hours after CLP. While BMS-345541 treatment did not alter mortality after CLP, our results showed a trend towards improved survival.

Conclusion: Transiently blocking NF-κB activity after the onset of CLP-induced sepsis can effectively reduce acute lung injury in mice without compromising bacterial host defense or survival after CLP.

Show MeSH
Related in: MedlinePlus