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Unique Toll-Like Receptor 4 Activation by NAMPT/PBEF Induces NFκB Signaling and Inflammatory Lung Injury.

Camp SM, Ceco E, Evenoski CL, Danilov SM, Zhou T, Chiang ET, Moreno-Vinasco L, Mapes B, Zhao J, Gursoy G, Brown ME, Adyshev DM, Siddiqui SS, Quijada H, Sammani S, Letsiou E, Saadat L, Yousef M, Wang T, Liang J, Garcia JG - Sci Rep (2015)

Bottom Line: Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown.Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS.The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Arizona Respiratory Center, The University of Arizona.

ABSTRACT
Ventilator-induced inflammatory lung injury (VILI) is mechanistically linked to increased NAMPT transcription and circulating levels of nicotinamide phosphoribosyl-transferase (NAMPT/PBEF). Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown. We now report that NAMPT/PBEF induces lung NFκB transcriptional activities and inflammatory injury via direct ligation of Toll-like receptor 4 (TLR4). Computational analysis demonstrated that NAMPT/PBEF and MD-2, a TLR4-binding protein essential for LPS-induced TLR4 activation, share ~30% sequence identity and exhibit striking structural similarity in loop regions critical for MD-2-TLR4 binding. Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS. The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.

No MeSH data available.


Related in: MedlinePlus

Extracellular NAMPT/PBEF- and LPS-induced NFκB pathway gene dysregulation is mediated by TLR4.(Panel A) Paraffin-embedded lung tissue was sectioned (10 μm) and immunostained with a p-NFκB antibody. Shown is a representative image demonstrating prominent NFκB activation and expression in capillary endothelium and alveolar epithelium from rPBEF-challenged wild type mice, whereas p-NFκB immunoreactivity was significantly reduced in rPBEF-challenged TLR4−/− mice. Scale bar = 100 μm. See Supplemental Figure 1 for IHC staining isotype controls. (Panel B) Heat maps reflecting the critical involvement of TLR4 in rPBEF- and LPS-mediated upregulation of NFκB pathway gene expression. Both rPBEF (40 μg/mouse, 4.5 hr) and LPS (2.5 mg/kg, 4 hr) mediate robust NFκB pathway increases in wild type mice whereas this expression was markedly reduced in TLR4−/− mice. Blue color indicates reduced gene expression, red color reflects increased gene expression. Bar graphs represent enriched pathways in mice challenged with rPBEF (40 μg/mouse, 4.5 hr) (Panel C) or LPS (2.5 mg/kg, 4 hr) (Panel D). The top ranking BIOCARTA pathways are listed for the genes differentially expressed between controls and wild-type mice challenged with rPBEF and LPS, respectively. The corresponding pathway patterns for the genes differentially expressed between the wild-type controls and TLR4−/− mice treated with rPBEF or LPS are also indicated. The genes dysregulated by LPS were identified using criteria of a false discovery rate (FDR) of <5% and a minimum of a 2-fold change. The genes dysregulated by rPBEF were identified by a cutoff of <10% FDR and >1.5-fold change. The gray dash line indicates the cutoff of significance (P-value < 0.05).
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f5: Extracellular NAMPT/PBEF- and LPS-induced NFκB pathway gene dysregulation is mediated by TLR4.(Panel A) Paraffin-embedded lung tissue was sectioned (10 μm) and immunostained with a p-NFκB antibody. Shown is a representative image demonstrating prominent NFκB activation and expression in capillary endothelium and alveolar epithelium from rPBEF-challenged wild type mice, whereas p-NFκB immunoreactivity was significantly reduced in rPBEF-challenged TLR4−/− mice. Scale bar = 100 μm. See Supplemental Figure 1 for IHC staining isotype controls. (Panel B) Heat maps reflecting the critical involvement of TLR4 in rPBEF- and LPS-mediated upregulation of NFκB pathway gene expression. Both rPBEF (40 μg/mouse, 4.5 hr) and LPS (2.5 mg/kg, 4 hr) mediate robust NFκB pathway increases in wild type mice whereas this expression was markedly reduced in TLR4−/− mice. Blue color indicates reduced gene expression, red color reflects increased gene expression. Bar graphs represent enriched pathways in mice challenged with rPBEF (40 μg/mouse, 4.5 hr) (Panel C) or LPS (2.5 mg/kg, 4 hr) (Panel D). The top ranking BIOCARTA pathways are listed for the genes differentially expressed between controls and wild-type mice challenged with rPBEF and LPS, respectively. The corresponding pathway patterns for the genes differentially expressed between the wild-type controls and TLR4−/− mice treated with rPBEF or LPS are also indicated. The genes dysregulated by LPS were identified using criteria of a false discovery rate (FDR) of <5% and a minimum of a 2-fold change. The genes dysregulated by rPBEF were identified by a cutoff of <10% FDR and >1.5-fold change. The gray dash line indicates the cutoff of significance (P-value < 0.05).

Mentions: These in vitro results were extended to in vivo studies utilizing mice pretreated with the TLR4 inhibitor, RS-LPS, as well as TLR4−/− mice (Fig. 4). Consistent with our prior report12, rPBEF instillation produced marked increases in lung inflammatory indices in wild type mice (BAL protein levels, BAL PMNs, and BAL cell counts (Fig. 4A)) that were significantly attenuated by RS-LPS pretreatment (100 μg/mouse) (Fig. 4A) in wild type mice. Similar to NAMPT/PBEF challenge, RS-LPS pretreatment produced attenuation of VILI-induced pulmonary inflammation in wild-type mice (Fig. 4B). rPBEF-induced lung inflammation was also reduced in TLR4−/− mice compared to wild type mice (Fig. 4C). TLR4−/− mice demonstrated abolishment of the prominent rPBEF-induced NFκB phosphorylation in murine pulmonary EC (Fig. 5A) and reduced basal levels of NFκB signaling (Fig. 5B). Both rPBEF and LPS triggered similar, robust increases in expression of NFκB signaling genes in wild type animals that were markedly reduced in TLR4−/− mice (Fig. 5B). More importantly, the NFκB pathways gene ontology was significantly dysregulated by either rPBEF or LPS (as the top regulated pathways) with significant suppression of gene dysregulation in TLR4−/− mice (Fig. 5C,D). These results are consistent with the requirement for TLR4 participation in NAMPT/PBEF-induced pro-inflammatory activities and lung injury.


Unique Toll-Like Receptor 4 Activation by NAMPT/PBEF Induces NFκB Signaling and Inflammatory Lung Injury.

Camp SM, Ceco E, Evenoski CL, Danilov SM, Zhou T, Chiang ET, Moreno-Vinasco L, Mapes B, Zhao J, Gursoy G, Brown ME, Adyshev DM, Siddiqui SS, Quijada H, Sammani S, Letsiou E, Saadat L, Yousef M, Wang T, Liang J, Garcia JG - Sci Rep (2015)

Extracellular NAMPT/PBEF- and LPS-induced NFκB pathway gene dysregulation is mediated by TLR4.(Panel A) Paraffin-embedded lung tissue was sectioned (10 μm) and immunostained with a p-NFκB antibody. Shown is a representative image demonstrating prominent NFκB activation and expression in capillary endothelium and alveolar epithelium from rPBEF-challenged wild type mice, whereas p-NFκB immunoreactivity was significantly reduced in rPBEF-challenged TLR4−/− mice. Scale bar = 100 μm. See Supplemental Figure 1 for IHC staining isotype controls. (Panel B) Heat maps reflecting the critical involvement of TLR4 in rPBEF- and LPS-mediated upregulation of NFκB pathway gene expression. Both rPBEF (40 μg/mouse, 4.5 hr) and LPS (2.5 mg/kg, 4 hr) mediate robust NFκB pathway increases in wild type mice whereas this expression was markedly reduced in TLR4−/− mice. Blue color indicates reduced gene expression, red color reflects increased gene expression. Bar graphs represent enriched pathways in mice challenged with rPBEF (40 μg/mouse, 4.5 hr) (Panel C) or LPS (2.5 mg/kg, 4 hr) (Panel D). The top ranking BIOCARTA pathways are listed for the genes differentially expressed between controls and wild-type mice challenged with rPBEF and LPS, respectively. The corresponding pathway patterns for the genes differentially expressed between the wild-type controls and TLR4−/− mice treated with rPBEF or LPS are also indicated. The genes dysregulated by LPS were identified using criteria of a false discovery rate (FDR) of <5% and a minimum of a 2-fold change. The genes dysregulated by rPBEF were identified by a cutoff of <10% FDR and >1.5-fold change. The gray dash line indicates the cutoff of significance (P-value < 0.05).
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f5: Extracellular NAMPT/PBEF- and LPS-induced NFκB pathway gene dysregulation is mediated by TLR4.(Panel A) Paraffin-embedded lung tissue was sectioned (10 μm) and immunostained with a p-NFκB antibody. Shown is a representative image demonstrating prominent NFκB activation and expression in capillary endothelium and alveolar epithelium from rPBEF-challenged wild type mice, whereas p-NFκB immunoreactivity was significantly reduced in rPBEF-challenged TLR4−/− mice. Scale bar = 100 μm. See Supplemental Figure 1 for IHC staining isotype controls. (Panel B) Heat maps reflecting the critical involvement of TLR4 in rPBEF- and LPS-mediated upregulation of NFκB pathway gene expression. Both rPBEF (40 μg/mouse, 4.5 hr) and LPS (2.5 mg/kg, 4 hr) mediate robust NFκB pathway increases in wild type mice whereas this expression was markedly reduced in TLR4−/− mice. Blue color indicates reduced gene expression, red color reflects increased gene expression. Bar graphs represent enriched pathways in mice challenged with rPBEF (40 μg/mouse, 4.5 hr) (Panel C) or LPS (2.5 mg/kg, 4 hr) (Panel D). The top ranking BIOCARTA pathways are listed for the genes differentially expressed between controls and wild-type mice challenged with rPBEF and LPS, respectively. The corresponding pathway patterns for the genes differentially expressed between the wild-type controls and TLR4−/− mice treated with rPBEF or LPS are also indicated. The genes dysregulated by LPS were identified using criteria of a false discovery rate (FDR) of <5% and a minimum of a 2-fold change. The genes dysregulated by rPBEF were identified by a cutoff of <10% FDR and >1.5-fold change. The gray dash line indicates the cutoff of significance (P-value < 0.05).
Mentions: These in vitro results were extended to in vivo studies utilizing mice pretreated with the TLR4 inhibitor, RS-LPS, as well as TLR4−/− mice (Fig. 4). Consistent with our prior report12, rPBEF instillation produced marked increases in lung inflammatory indices in wild type mice (BAL protein levels, BAL PMNs, and BAL cell counts (Fig. 4A)) that were significantly attenuated by RS-LPS pretreatment (100 μg/mouse) (Fig. 4A) in wild type mice. Similar to NAMPT/PBEF challenge, RS-LPS pretreatment produced attenuation of VILI-induced pulmonary inflammation in wild-type mice (Fig. 4B). rPBEF-induced lung inflammation was also reduced in TLR4−/− mice compared to wild type mice (Fig. 4C). TLR4−/− mice demonstrated abolishment of the prominent rPBEF-induced NFκB phosphorylation in murine pulmonary EC (Fig. 5A) and reduced basal levels of NFκB signaling (Fig. 5B). Both rPBEF and LPS triggered similar, robust increases in expression of NFκB signaling genes in wild type animals that were markedly reduced in TLR4−/− mice (Fig. 5B). More importantly, the NFκB pathways gene ontology was significantly dysregulated by either rPBEF or LPS (as the top regulated pathways) with significant suppression of gene dysregulation in TLR4−/− mice (Fig. 5C,D). These results are consistent with the requirement for TLR4 participation in NAMPT/PBEF-induced pro-inflammatory activities and lung injury.

Bottom Line: Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown.Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS.The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Arizona Respiratory Center, The University of Arizona.

ABSTRACT
Ventilator-induced inflammatory lung injury (VILI) is mechanistically linked to increased NAMPT transcription and circulating levels of nicotinamide phosphoribosyl-transferase (NAMPT/PBEF). Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown. We now report that NAMPT/PBEF induces lung NFκB transcriptional activities and inflammatory injury via direct ligation of Toll-like receptor 4 (TLR4). Computational analysis demonstrated that NAMPT/PBEF and MD-2, a TLR4-binding protein essential for LPS-induced TLR4 activation, share ~30% sequence identity and exhibit striking structural similarity in loop regions critical for MD-2-TLR4 binding. Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS. The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.

No MeSH data available.


Related in: MedlinePlus