Anti-inflammatory effect of a selective IkappaB kinase-beta inhibitor in rat lung in response to LPS and cigarette smoke

Abstract

Rationale: IkappaB kinase (IKK) activates NF-kappaB which plays a pivotal role in pro-inflammatory response in the lung. NF-kappaB has been shown to be activated in alveolar macrophages and peripheral lungs of smokers and patients with chronic obstructive pulmonary disease. We investigated the anti-inflammatory effect of a highly selective and novel IKKbeta/IKK2 inhibitor, PHA-408 [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[gamma]indazole-3-carboxamide], in lungs of rat in vivo.

Methods: Adult Sprague-Dawley rats were administered orally with PHA-408 (15 and 45 mg/kg) daily for 3 days and exposed to LPS aerosol (once on day 3, 2 h post-last PHA-408 administration) or cigarette smoke (CS; 2h after PHA-408 administration for 3 days). Animals were sacrificed at 1, 4 and 24 h after the last exposure, and lung inflammatory response and NF-kappaB activation were measured.

Results: Oral administration of IKKbeta/IKK2 inhibitor PHA-408 significantly inhibited LPS- and CS-mediated neutrophil influx in bronchoalveolar lavage (BAL) fluid of rats. The levels of pro-inflammatory mediators in BAL fluid (CINC-1) and lungs (IL-6, TNF-alpha, IL-1beta and GM-CSF) were also reduced by PHA-408 administration in response to LPS or CS exposures. The reduced pro-inflammatory response in PHA-408-administered rats was associated with decreased nuclear translocation and DNA binding activity of NF-kappaB in response to LPS or CS.

Conclusion: These results suggest that IKKbeta/IKK2 inhibitor PHA-408 is a powerful anti-inflammatory agent against LPS- and CS-mediated lung inflammation.

Figure 1. IKK2 inhibitor (PHA-408) attenuated LPS- and CS-induced neutrophil influx in BAL fluid of rats

The neutrophil (A and B) and macrophage (C and D) numbers were counted in Diff-Quick stained cytospin slides, which were prepared using BAL cells collected by lung lavage in rats at 1, 4 and 24 h after LPS (A and C) or CS (B and D) exposures. IKKi (low) and IKKi (high) denote orally administered IKK2 inhibitor (PHA-408) at the doses of 15 and 45 mg/kg for 3 days, respectively at 2 h before LPS or CS exposure. PHA-408 was dissolved in the vehicle consisting of 0.5% carboxymethylcellulose and 0.025% Tween 20. Data are shown as mean ± SEM (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective saline- or air-exposed control group; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with respective LPS or CS exposed group. IKKi, IKK2 inhibitor.

Figure 1. IKK2 inhibitor (PHA-408) attenuated LPS- and CS-induced neutrophil influx in BAL fluid of rats

The neutrophil (A and B) and macrophage (C and D) numbers were counted in Diff-Quick stained cytospin slides, which were prepared using BAL cells collected by lung lavage in rats at 1, 4 and 24 h after LPS (A and C) or CS (B and D) exposures. IKKi (low) and IKKi (high) denote orally administered IKK2 inhibitor (PHA-408) at the doses of 15 and 45 mg/kg for 3 days, respectively at 2 h before LPS or CS exposure. PHA-408 was dissolved in the vehicle consisting of 0.5% carboxymethylcellulose and 0.025% Tween 20. Data are shown as mean ± SEM (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective saline- or air-exposed control group; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with respective LPS or CS exposed group. IKKi, IKK2 inhibitor.

Figure 2. IKK2 inhibitor (PHA-408) inhibited LPS- and CS-induced increase in pro-inflammatory cytokine levels in BAL fluid of rat lung

The level of CINC-1 was measured in BAL fluid collected by lung lavage in rat at 1, 4 and 24 h after LPS (A) or CS (B) exposures by ELISA. The levels of NF-κB-dependent pro-inflammatory cytokines, such as IL-6 (C), TNF-α (D), IL-1β (E), and GM-CSF (F) were measured in lungs obtained from rats at 1, 4 and 24 h post-LPS exposure. Two different doses of orally administered IKK2 inhibitor (PHA-408; at 2 h before LPS or CS exposures) were indicated as ‘low’ (15 mg/kg) and ‘high’ (45 mg/kg). Data are shown as mean ± SEM (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective saline- or air-exposed control group; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with respective LPS or CS exposed group. IKKi, IKK2 inhibitor; ND, Not detectable.

Figure 2. IKK2 inhibitor (PHA-408) inhibited LPS- and CS-induced increase in pro-inflammatory cytokine levels in BAL fluid of rat lung

The level of CINC-1 was measured in BAL fluid collected by lung lavage in rat at 1, 4 and 24 h after LPS (A) or CS (B) exposures by ELISA. The levels of NF-κB-dependent pro-inflammatory cytokines, such as IL-6 (C), TNF-α (D), IL-1β (E), and GM-CSF (F) were measured in lungs obtained from rats at 1, 4 and 24 h post-LPS exposure. Two different doses of orally administered IKK2 inhibitor (PHA-408; at 2 h before LPS or CS exposures) were indicated as ‘low’ (15 mg/kg) and ‘high’ (45 mg/kg). Data are shown as mean ± SEM (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective saline- or air-exposed control group; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with respective LPS or CS exposed group. IKKi, IKK2 inhibitor; ND, Not detectable.

Figure 2. IKK2 inhibitor (PHA-408) inhibited LPS- and CS-induced increase in pro-inflammatory cytokine levels in BAL fluid of rat lung

The level of CINC-1 was measured in BAL fluid collected by lung lavage in rat at 1, 4 and 24 h after LPS (A) or CS (B) exposures by ELISA. The levels of NF-κB-dependent pro-inflammatory cytokines, such as IL-6 (C), TNF-α (D), IL-1β (E), and GM-CSF (F) were measured in lungs obtained from rats at 1, 4 and 24 h post-LPS exposure. Two different doses of orally administered IKK2 inhibitor (PHA-408; at 2 h before LPS or CS exposures) were indicated as ‘low’ (15 mg/kg) and ‘high’ (45 mg/kg). Data are shown as mean ± SEM (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective saline- or air-exposed control group; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with respective LPS or CS exposed group. IKKi, IKK2 inhibitor; ND, Not detectable.

Figure 3. IKK2 inhibitor (PHA-408) attenuated LPS- and CS-induced IκBα degradation in rat lung

IκBα levels were measured by immunoblotting in cytosolic fractions of lungs collected at 1 h post-last exposure to LPS or CS. Gel pictures shown are representative of at least three separate experiments (A). The purity of cytosolic fraction was confirmed by the absence of nuclear protein, histone H3 (data not shown). GAPDH was used as a loading control. After densitometric analysis, the values were normalized against the loading controls (B). Data are shown as mean ± SEM (n = 3 per group). *P < 0.05, **P < 0.01, significant compared with respective saline- or air-exposed control group; ⁺P < 0.05, ⁺⁺P < 0.01, significant compared with respective LPS or CS exposed group. IKKi, IKK2 inhibitor; Sal, saline; Low, 15 mg/kg; High, 45 mg/kg.

Figure 4. IKK2 inhibitor (PHA-408) decreased LPS- and CS-induced nuclear translocation of NF-κB RelA/p65 in rat lung

Nuclear levels of RelA/p65 subunit of NF-κB were measured by immunoblotting at 1 h after LPS or CS exposure. The purity of nuclear fraction was shown by the presence of histone H3 (nuclear protein) and the absence of cytoskeleton protein, α-tubulin (data not shown). β-Actin was used as a loading control. After densitometric analysis, the values were normalized against the loading controls (B). Gel pictures shown are representative of at least three separate experiments. Data are shown as mean ± SEM (n = 3 per group). ***P < 0.001, significant compared with respective saline- or air-exposed control group; ⁺P < 0.05, ⁺⁺⁺P < 0.001, significant compared with respective LPS or CS exposed group. IKKi, IKK2 inhibitor; Sal, saline; Low, 15 mg/kg; High, 45 mg/kg.

Figure 5. IKK2 inhibitor (PHA-408) decreased LPS- or CS-induced NF-κB-DNA binding in rat lung

NF-κB-DNA binding was measured by EMSA in nuclear proteins form LPS (A) or CS (B) exposed rat lung using γ-³²P-labeled NF-κB oligonucleotide. The specificity of NF-κB signals was determined by specific and nonspecific competitor reactions by adding non-labeled NF-κB (specific) and AP-1 (non-specific) oligonucleotides to HeLa nuclear extracts (before the addition of γ-³²P labeled NF-κB oligonucleotides). Positive and negative control reactions were also performed using HeLa nuclear extracts and water, respectively. Quantification of band intensities from three independent experimental results was determined by a densitometry (C and D). Gel pictures shown are representative of at least three separate experiments. NF-κB DNA-binding activity in response to LPS (E) or CS (F) exposure in the lung nuclear protein was determined using Trans AM transcription factor p65 ELISA kit. *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective saline- or air-exposed control groups; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with respective LPS or CS exposed group. Positive, Positive control; Negative, negative control; SC, Specific competitor; NSC, Nonspecific competitor; IKKi, IKK2 inhibitor; Low, 15 mg/kg; High, 45 mg/kg.

Figure 5. IKK2 inhibitor (PHA-408) decreased LPS- or CS-induced NF-κB-DNA binding in rat lung

NF-κB-DNA binding was measured by EMSA in nuclear proteins form LPS (A) or CS (B) exposed rat lung using γ-³²P-labeled NF-κB oligonucleotide. The specificity of NF-κB signals was determined by specific and nonspecific competitor reactions by adding non-labeled NF-κB (specific) and AP-1 (non-specific) oligonucleotides to HeLa nuclear extracts (before the addition of γ-³²P labeled NF-κB oligonucleotides). Positive and negative control reactions were also performed using HeLa nuclear extracts and water, respectively. Quantification of band intensities from three independent experimental results was determined by a densitometry (C and D). Gel pictures shown are representative of at least three separate experiments. NF-κB DNA-binding activity in response to LPS (E) or CS (F) exposure in the lung nuclear protein was determined using Trans AM transcription factor p65 ELISA kit. *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective saline- or air-exposed control groups; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with respective LPS or CS exposed group. Positive, Positive control; Negative, negative control; SC, Specific competitor; NSC, Nonspecific competitor; IKKi, IKK2 inhibitor; Low, 15 mg/kg; High, 45 mg/kg.

Figure 5. IKK2 inhibitor (PHA-408) decreased LPS- or CS-induced NF-κB-DNA binding in rat lung

NF-κB-DNA binding was measured by EMSA in nuclear proteins form LPS (A) or CS (B) exposed rat lung using γ-³²P-labeled NF-κB oligonucleotide. The specificity of NF-κB signals was determined by specific and nonspecific competitor reactions by adding non-labeled NF-κB (specific) and AP-1 (non-specific) oligonucleotides to HeLa nuclear extracts (before the addition of γ-³²P labeled NF-κB oligonucleotides). Positive and negative control reactions were also performed using HeLa nuclear extracts and water, respectively. Quantification of band intensities from three independent experimental results was determined by a densitometry (C and D). Gel pictures shown are representative of at least three separate experiments. NF-κB DNA-binding activity in response to LPS (E) or CS (F) exposure in the lung nuclear protein was determined using Trans AM transcription factor p65 ELISA kit. *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective saline- or air-exposed control groups; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with respective LPS or CS exposed group. Positive, Positive control; Negative, negative control; SC, Specific competitor; NSC, Nonspecific competitor; IKKi, IKK2 inhibitor; Low, 15 mg/kg; High, 45 mg/kg.

Figure 5. IKK2 inhibitor (PHA-408) decreased LPS- or CS-induced NF-κB-DNA binding in rat lung

NF-κB-DNA binding was measured by EMSA in nuclear proteins form LPS (A) or CS (B) exposed rat lung using γ-³²P-labeled NF-κB oligonucleotide. The specificity of NF-κB signals was determined by specific and nonspecific competitor reactions by adding non-labeled NF-κB (specific) and AP-1 (non-specific) oligonucleotides to HeLa nuclear extracts (before the addition of γ-³²P labeled NF-κB oligonucleotides). Positive and negative control reactions were also performed using HeLa nuclear extracts and water, respectively. Quantification of band intensities from three independent experimental results was determined by a densitometry (C and D). Gel pictures shown are representative of at least three separate experiments. NF-κB DNA-binding activity in response to LPS (E) or CS (F) exposure in the lung nuclear protein was determined using Trans AM transcription factor p65 ELISA kit. *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective saline- or air-exposed control groups; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with respective LPS or CS exposed group. Positive, Positive control; Negative, negative control; SC, Specific competitor; NSC, Nonspecific competitor; IKKi, IKK2 inhibitor; Low, 15 mg/kg; High, 45 mg/kg.