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Systemic inhibition of NF-kappaB activation protects from silicosis.

Di Giuseppe M, Gambelli F, Hoyle GW, Lungarella G, Studer SM, Richards T, Yousem S, McCurry K, Dauber J, Kaminski N, Leikauf G, Ortiz LA - PLoS ONE (2009)

Bottom Line: At the time of lung transplantation, we found the lungs of silica-exposed subjects to contain multiple foci of inflammatory cells and silicotic nodules with proximal TNFalpha expressing macrophage and NF-kappaB activation in epithelial cells.Using a mouse experimental model in which the endotracheal instillation of silica reproduces the silica-induced lung injury observed in humans we found that systemic inhibition of NF-kappaB activation with a pharmacologic inhibitor (BAY 11-7085) of IkappaB alpha phosphorylation decreased silica-induced inflammation and collagen deposition.In contrast, transgenic mice expressing a dominant negative IkappaB alpha mutant protein under the control of epithelial cell specific promoters demonstrate enhanced apoptosis and collagen deposition in their lungs in response to silica.

View Article: PubMed Central - PubMed

Affiliation: Division of Occupational and Environmental Medicine, Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT

Background: Silicosis is a complex lung disease for which no successful treatment is available and therefore lung transplantation is a potential alternative. Tumor necrosis factor alpha (TNFalpha) plays a central role in the pathogenesis of silicosis. TNFalpha signaling is mediated by the transcription factor, Nuclear Factor (NF)-kappaB, which regulates genes controlling several physiological processes including the innate immune responses, cell death, and inflammation. Therefore, inhibition of NF-kappaB activation represents a potential therapeutic strategy for silicosis.

Methods/findings: In the present work we evaluated the lung transplant database (May 1986-July 2007) at the University of Pittsburgh to study the efficacy of lung transplantation in patients with silicosis (n = 11). We contrasted the overall survival and rate of graft rejection in these patients to that of patients with idiopathic pulmonary fibrosis (IPF, n = 79) that was selected as a control group because survival benefit of lung transplantation has been identified for these patients. At the time of lung transplantation, we found the lungs of silica-exposed subjects to contain multiple foci of inflammatory cells and silicotic nodules with proximal TNFalpha expressing macrophage and NF-kappaB activation in epithelial cells. Patients with silicosis had poor survival (median survival 2.4 yr; confidence interval (CI): 0.16-7.88 yr) compared to IPF patients (5.3 yr; CI: 2.8-15 yr; p = 0.07), and experienced early rejection of their lung grafts (0.9 yr; CI: 0.22-0.9 yr) following lung transplantation (2.4 yr; CI:1.5-3.6 yr; p<0.05). Using a mouse experimental model in which the endotracheal instillation of silica reproduces the silica-induced lung injury observed in humans we found that systemic inhibition of NF-kappaB activation with a pharmacologic inhibitor (BAY 11-7085) of IkappaB alpha phosphorylation decreased silica-induced inflammation and collagen deposition. In contrast, transgenic mice expressing a dominant negative IkappaB alpha mutant protein under the control of epithelial cell specific promoters demonstrate enhanced apoptosis and collagen deposition in their lungs in response to silica.

Conclusions: Although limited by its size, our data support that patients with silicosis appear to have poor outcome following lung transplantation. Experimental data indicate that while the systemic inhibition of NF-kappaB protects from silica-induced lung injury, epithelial cell specific NF-kappaB inhibition appears to aggravate the outcome of experimental silicosis.

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TNFα receptors mediate NF-κB activation in experimental silicosis.Western blot (upper panel) representing expression of total and phosphorylated IκB (phospho IκB) in the lungs of silica-sensitive C57BL/6, or double (p55/p75−/−) TNFα receptor deficient mice following the intratracheal instillation of silica particles as described in the text. Lower panel illustrates the DNA binding activity of NF-κB in crude nuclear extracts from whole lung isolated from C57BL/6, and double (p55/p75−/−) TNFα receptor deficient mice after silica exposure. Silica induces two bands: a lower band representing the p50-p50 homo-dimer, present in both the C57BL/6 and the double (p55p75−/−) TNFα receptor deficient mice, and an upper band, only observed in the lungs of C57BL/6 mice, representing the p50-p60 heterodimer. Excess probe represents NF-κB binding in lung nuclear extract of a silica-treated mouse, assayed in the presence of excess unlabelled oligonucleotide as a competitor. Competition assays were performed using 400 times excess of unlabeled probe or NF-κB mutant oligonucleotide (Santa Cruz). Supershifts were performed by adding to the binding mixture antibodies to p50 (sc1190X) or to p65 (sc372X) or against C-Jun/AP-1 (sc44X) (Santa Cruz) before adding the labeled probe to the mixture.
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pone-0005689-g004: TNFα receptors mediate NF-κB activation in experimental silicosis.Western blot (upper panel) representing expression of total and phosphorylated IκB (phospho IκB) in the lungs of silica-sensitive C57BL/6, or double (p55/p75−/−) TNFα receptor deficient mice following the intratracheal instillation of silica particles as described in the text. Lower panel illustrates the DNA binding activity of NF-κB in crude nuclear extracts from whole lung isolated from C57BL/6, and double (p55/p75−/−) TNFα receptor deficient mice after silica exposure. Silica induces two bands: a lower band representing the p50-p50 homo-dimer, present in both the C57BL/6 and the double (p55p75−/−) TNFα receptor deficient mice, and an upper band, only observed in the lungs of C57BL/6 mice, representing the p50-p60 heterodimer. Excess probe represents NF-κB binding in lung nuclear extract of a silica-treated mouse, assayed in the presence of excess unlabelled oligonucleotide as a competitor. Competition assays were performed using 400 times excess of unlabeled probe or NF-κB mutant oligonucleotide (Santa Cruz). Supershifts were performed by adding to the binding mixture antibodies to p50 (sc1190X) or to p65 (sc372X) or against C-Jun/AP-1 (sc44X) (Santa Cruz) before adding the labeled probe to the mixture.

Mentions: We have previously reported that TNFα receptors play a fundamental role in experimental silicosis and TNFα receptor deficient mice are protected from silica-induced lung fibrosis [12], [20], [21]. Here we show that TNFα-receptor mediated signaling is an important determinant of the NF-κB activation in the mouse lung in response to silica. Instillation of silica particles increased phophorylated IκB moiety (Figure 4). This increase was followed by IκB rapid degradation and eventual disappearance of (total IκB) in the lungs of silica-sensitive C57BL/6 mice. In contrast to C57BL/6 mice, these changes in IκB did not take place in the lungs of double (p55p75) TNFα receptor deficient mice developed in the same silica-sensitive C57BL/6 genetic background. Silica-induced degradation of phospho IκB is associated with NF-κB activation in the lungs of C57BL/6 (Figure 4). Analysis of NF-κB gel shift in lung nuclear extracts show that silica induces two bands: a lower band representing the p50-p50 (NFKB1-NFKB1) homodimer, present in both the C57BL/6 and the double (p55p75−/−) TNFα receptor deficient mice, and an upper band, only observed in the lungs of C57BL/6 mice, representing the p50-p65 (NFKB1:RELA) heterodimer (Figure 4). Supershift assays confirmed that the lower band could be competed by excess cold, but not by mutant, probe while the upper band could be displaced by incubation of the nuclear extract with an antibody against the p50 or the p65 complex (Figure 4). These data indicate that silica induces a canonical, TNFα receptor-mediated, activation of NF-κB in the mouse lung.


Systemic inhibition of NF-kappaB activation protects from silicosis.

Di Giuseppe M, Gambelli F, Hoyle GW, Lungarella G, Studer SM, Richards T, Yousem S, McCurry K, Dauber J, Kaminski N, Leikauf G, Ortiz LA - PLoS ONE (2009)

TNFα receptors mediate NF-κB activation in experimental silicosis.Western blot (upper panel) representing expression of total and phosphorylated IκB (phospho IκB) in the lungs of silica-sensitive C57BL/6, or double (p55/p75−/−) TNFα receptor deficient mice following the intratracheal instillation of silica particles as described in the text. Lower panel illustrates the DNA binding activity of NF-κB in crude nuclear extracts from whole lung isolated from C57BL/6, and double (p55/p75−/−) TNFα receptor deficient mice after silica exposure. Silica induces two bands: a lower band representing the p50-p50 homo-dimer, present in both the C57BL/6 and the double (p55p75−/−) TNFα receptor deficient mice, and an upper band, only observed in the lungs of C57BL/6 mice, representing the p50-p60 heterodimer. Excess probe represents NF-κB binding in lung nuclear extract of a silica-treated mouse, assayed in the presence of excess unlabelled oligonucleotide as a competitor. Competition assays were performed using 400 times excess of unlabeled probe or NF-κB mutant oligonucleotide (Santa Cruz). Supershifts were performed by adding to the binding mixture antibodies to p50 (sc1190X) or to p65 (sc372X) or against C-Jun/AP-1 (sc44X) (Santa Cruz) before adding the labeled probe to the mixture.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2682759&req=5

pone-0005689-g004: TNFα receptors mediate NF-κB activation in experimental silicosis.Western blot (upper panel) representing expression of total and phosphorylated IκB (phospho IκB) in the lungs of silica-sensitive C57BL/6, or double (p55/p75−/−) TNFα receptor deficient mice following the intratracheal instillation of silica particles as described in the text. Lower panel illustrates the DNA binding activity of NF-κB in crude nuclear extracts from whole lung isolated from C57BL/6, and double (p55/p75−/−) TNFα receptor deficient mice after silica exposure. Silica induces two bands: a lower band representing the p50-p50 homo-dimer, present in both the C57BL/6 and the double (p55p75−/−) TNFα receptor deficient mice, and an upper band, only observed in the lungs of C57BL/6 mice, representing the p50-p60 heterodimer. Excess probe represents NF-κB binding in lung nuclear extract of a silica-treated mouse, assayed in the presence of excess unlabelled oligonucleotide as a competitor. Competition assays were performed using 400 times excess of unlabeled probe or NF-κB mutant oligonucleotide (Santa Cruz). Supershifts were performed by adding to the binding mixture antibodies to p50 (sc1190X) or to p65 (sc372X) or against C-Jun/AP-1 (sc44X) (Santa Cruz) before adding the labeled probe to the mixture.
Mentions: We have previously reported that TNFα receptors play a fundamental role in experimental silicosis and TNFα receptor deficient mice are protected from silica-induced lung fibrosis [12], [20], [21]. Here we show that TNFα-receptor mediated signaling is an important determinant of the NF-κB activation in the mouse lung in response to silica. Instillation of silica particles increased phophorylated IκB moiety (Figure 4). This increase was followed by IκB rapid degradation and eventual disappearance of (total IκB) in the lungs of silica-sensitive C57BL/6 mice. In contrast to C57BL/6 mice, these changes in IκB did not take place in the lungs of double (p55p75) TNFα receptor deficient mice developed in the same silica-sensitive C57BL/6 genetic background. Silica-induced degradation of phospho IκB is associated with NF-κB activation in the lungs of C57BL/6 (Figure 4). Analysis of NF-κB gel shift in lung nuclear extracts show that silica induces two bands: a lower band representing the p50-p50 (NFKB1-NFKB1) homodimer, present in both the C57BL/6 and the double (p55p75−/−) TNFα receptor deficient mice, and an upper band, only observed in the lungs of C57BL/6 mice, representing the p50-p65 (NFKB1:RELA) heterodimer (Figure 4). Supershift assays confirmed that the lower band could be competed by excess cold, but not by mutant, probe while the upper band could be displaced by incubation of the nuclear extract with an antibody against the p50 or the p65 complex (Figure 4). These data indicate that silica induces a canonical, TNFα receptor-mediated, activation of NF-κB in the mouse lung.

Bottom Line: At the time of lung transplantation, we found the lungs of silica-exposed subjects to contain multiple foci of inflammatory cells and silicotic nodules with proximal TNFalpha expressing macrophage and NF-kappaB activation in epithelial cells.Using a mouse experimental model in which the endotracheal instillation of silica reproduces the silica-induced lung injury observed in humans we found that systemic inhibition of NF-kappaB activation with a pharmacologic inhibitor (BAY 11-7085) of IkappaB alpha phosphorylation decreased silica-induced inflammation and collagen deposition.In contrast, transgenic mice expressing a dominant negative IkappaB alpha mutant protein under the control of epithelial cell specific promoters demonstrate enhanced apoptosis and collagen deposition in their lungs in response to silica.

View Article: PubMed Central - PubMed

Affiliation: Division of Occupational and Environmental Medicine, Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT

Background: Silicosis is a complex lung disease for which no successful treatment is available and therefore lung transplantation is a potential alternative. Tumor necrosis factor alpha (TNFalpha) plays a central role in the pathogenesis of silicosis. TNFalpha signaling is mediated by the transcription factor, Nuclear Factor (NF)-kappaB, which regulates genes controlling several physiological processes including the innate immune responses, cell death, and inflammation. Therefore, inhibition of NF-kappaB activation represents a potential therapeutic strategy for silicosis.

Methods/findings: In the present work we evaluated the lung transplant database (May 1986-July 2007) at the University of Pittsburgh to study the efficacy of lung transplantation in patients with silicosis (n = 11). We contrasted the overall survival and rate of graft rejection in these patients to that of patients with idiopathic pulmonary fibrosis (IPF, n = 79) that was selected as a control group because survival benefit of lung transplantation has been identified for these patients. At the time of lung transplantation, we found the lungs of silica-exposed subjects to contain multiple foci of inflammatory cells and silicotic nodules with proximal TNFalpha expressing macrophage and NF-kappaB activation in epithelial cells. Patients with silicosis had poor survival (median survival 2.4 yr; confidence interval (CI): 0.16-7.88 yr) compared to IPF patients (5.3 yr; CI: 2.8-15 yr; p = 0.07), and experienced early rejection of their lung grafts (0.9 yr; CI: 0.22-0.9 yr) following lung transplantation (2.4 yr; CI:1.5-3.6 yr; p<0.05). Using a mouse experimental model in which the endotracheal instillation of silica reproduces the silica-induced lung injury observed in humans we found that systemic inhibition of NF-kappaB activation with a pharmacologic inhibitor (BAY 11-7085) of IkappaB alpha phosphorylation decreased silica-induced inflammation and collagen deposition. In contrast, transgenic mice expressing a dominant negative IkappaB alpha mutant protein under the control of epithelial cell specific promoters demonstrate enhanced apoptosis and collagen deposition in their lungs in response to silica.

Conclusions: Although limited by its size, our data support that patients with silicosis appear to have poor outcome following lung transplantation. Experimental data indicate that while the systemic inhibition of NF-kappaB protects from silica-induced lung injury, epithelial cell specific NF-kappaB inhibition appears to aggravate the outcome of experimental silicosis.

Show MeSH
Related in: MedlinePlus