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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 independently mediates rapid NFκB activation in human lung endothelium.(Panel A) Activation of NFκB signaling in human lung EC challenged with recombinant NAMPT/PBEF (rPBEF) (1 μg/ml, 5–60 min). Stimulated EC lysates were probed for p-NFκB (Ser536) or control β-actin. Heat-denatured rPBEF (HD-rPBEF) (1 μg/ml, 1 hr) served as a negative control confirming that rPBEF effects do not reflect endotoxin contamination. LPS (5 μg/ml, 1 hr) and TNF-α (100 ng/ml, 15 min) served as positive controls for NFκB signaling pathway activation. n = 3; representative blots shown. (Panel B) NAMPT/PBEF-mediated NFκB activation does not reflect endotoxin contamination. rPBEF or LPS were exposed to 100 °C for 5 min. Human lung ECs were treated with rPBEF (1 μg/ml), heat denatured (HD)-rPBEF (1 μg/ml), LPS (5 μg/ml), or HD-LPS (5 μg/ml) for 1 hr, EC lysates were then probed for p-NFκB (Ser536) or total NFκB. (Panel C) NAMPT/PBEF enzymatic inhibitor FK-866 fails to attenuate rPBEF-induced NFκB phosphorylation. Human lung ECs were treated with rPBEF (1 μg/ml, 1 hr) either without any pretreatment, with premixing with neutralizing NAMPT/PBEF pAb (100 μg/ml, 30 min), or pretreatment with FK-866 (0.1–10 μM, 1 hr). EC lysates were probed for p-NFκB (Ser536) and total NFκB. For (Panel B,C) bar graphs represent data as integrated density normalized to unstimulated control. n = 3–9 (B) or n = 4–6 (C); *p < 0.01 (B) or *p < 0.05 (C) vs rPBEF-stimulated control. (Panel D) Immunofluorescent monitoring of agonist-induced NFκB translocation to the nucleus. rPBEF (10 μg/ml) for 0–60 min induces NFκB translocation from the cytosol to the nuclei in human lung microvascular EC similar to EC challenge with TNF-α (100 ng/ml). Scale bar = 10 μm; n = 3; representative immunofluorescence images shown. (Panel E) rPBEF (10 ng/mL)-mediated increases in NFκB promoter-driven luciferase reporter activity in human lung EC assessed from 0–3 hr. NFκB promoter activity following rPBEF challenge was measured using a dual luciferase reporter assay and absorbance of the inducible NFκB promoter-responsive firefly luciferase construct was normalized to the absorbance of the constitutively active Renilla luciferase construct (internal control). Bar graphs represent data as percentage luciferase activity increase above control, n = 3; *p < 0.05 versus unstimulated controls.
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f1: Extracellular NAMPT/PBEF independently mediates rapid NFκB activation in human lung endothelium.(Panel A) Activation of NFκB signaling in human lung EC challenged with recombinant NAMPT/PBEF (rPBEF) (1 μg/ml, 5–60 min). Stimulated EC lysates were probed for p-NFκB (Ser536) or control β-actin. Heat-denatured rPBEF (HD-rPBEF) (1 μg/ml, 1 hr) served as a negative control confirming that rPBEF effects do not reflect endotoxin contamination. LPS (5 μg/ml, 1 hr) and TNF-α (100 ng/ml, 15 min) served as positive controls for NFκB signaling pathway activation. n = 3; representative blots shown. (Panel B) NAMPT/PBEF-mediated NFκB activation does not reflect endotoxin contamination. rPBEF or LPS were exposed to 100 °C for 5 min. Human lung ECs were treated with rPBEF (1 μg/ml), heat denatured (HD)-rPBEF (1 μg/ml), LPS (5 μg/ml), or HD-LPS (5 μg/ml) for 1 hr, EC lysates were then probed for p-NFκB (Ser536) or total NFκB. (Panel C) NAMPT/PBEF enzymatic inhibitor FK-866 fails to attenuate rPBEF-induced NFκB phosphorylation. Human lung ECs were treated with rPBEF (1 μg/ml, 1 hr) either without any pretreatment, with premixing with neutralizing NAMPT/PBEF pAb (100 μg/ml, 30 min), or pretreatment with FK-866 (0.1–10 μM, 1 hr). EC lysates were probed for p-NFκB (Ser536) and total NFκB. For (Panel B,C) bar graphs represent data as integrated density normalized to unstimulated control. n = 3–9 (B) or n = 4–6 (C); *p < 0.01 (B) or *p < 0.05 (C) vs rPBEF-stimulated control. (Panel D) Immunofluorescent monitoring of agonist-induced NFκB translocation to the nucleus. rPBEF (10 μg/ml) for 0–60 min induces NFκB translocation from the cytosol to the nuclei in human lung microvascular EC similar to EC challenge with TNF-α (100 ng/ml). Scale bar = 10 μm; n = 3; representative immunofluorescence images shown. (Panel E) rPBEF (10 ng/mL)-mediated increases in NFκB promoter-driven luciferase reporter activity in human lung EC assessed from 0–3 hr. NFκB promoter activity following rPBEF challenge was measured using a dual luciferase reporter assay and absorbance of the inducible NFκB promoter-responsive firefly luciferase construct was normalized to the absorbance of the constitutively active Renilla luciferase construct (internal control). Bar graphs represent data as percentage luciferase activity increase above control, n = 3; *p < 0.05 versus unstimulated controls.

Mentions: Leveraging our prior reports of NFκB transcriptome induction by recombinant NAMPT/PBEF (rPBEF)121718, complementary in vitro approaches were utilized to functionally examine the direct role of extracellular NAMPT/PBEF in NFκB pathway activation and innate immunity gene expression. Initial experiments assessed exogenous NAMPT/PBEF-mediated phosphorylation of NFκB (p-NFκB at Ser536) in human lung endothelial cells (EC) as an indication of NFκB activation. rPBEF, similar to LPS and TNF-α, increases phosphorylation of p-NFκB within 30 min with persistent elevation at 1 hr (Fig. 1A). Heat denaturing of rPBEF (HD-rPBEF) resulted in the elimination of NAMPT/PBEF’s capacity to induce NFκB activation, demonstrating that NAMPT/PBEF-mediated NFκB phosphorylation/activation does not reflect endotoxin contamination (Fig. 1A,B). rPBEF-induced NFκB activation was unaffected by inhibition of NAMPT enzymatic activity (FK-866), indicating that phosphoribosyl-transferase activity is not required (Fig. 1C).


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 independently mediates rapid NFκB activation in human lung endothelium.(Panel A) Activation of NFκB signaling in human lung EC challenged with recombinant NAMPT/PBEF (rPBEF) (1 μg/ml, 5–60 min). Stimulated EC lysates were probed for p-NFκB (Ser536) or control β-actin. Heat-denatured rPBEF (HD-rPBEF) (1 μg/ml, 1 hr) served as a negative control confirming that rPBEF effects do not reflect endotoxin contamination. LPS (5 μg/ml, 1 hr) and TNF-α (100 ng/ml, 15 min) served as positive controls for NFκB signaling pathway activation. n = 3; representative blots shown. (Panel B) NAMPT/PBEF-mediated NFκB activation does not reflect endotoxin contamination. rPBEF or LPS were exposed to 100 °C for 5 min. Human lung ECs were treated with rPBEF (1 μg/ml), heat denatured (HD)-rPBEF (1 μg/ml), LPS (5 μg/ml), or HD-LPS (5 μg/ml) for 1 hr, EC lysates were then probed for p-NFκB (Ser536) or total NFκB. (Panel C) NAMPT/PBEF enzymatic inhibitor FK-866 fails to attenuate rPBEF-induced NFκB phosphorylation. Human lung ECs were treated with rPBEF (1 μg/ml, 1 hr) either without any pretreatment, with premixing with neutralizing NAMPT/PBEF pAb (100 μg/ml, 30 min), or pretreatment with FK-866 (0.1–10 μM, 1 hr). EC lysates were probed for p-NFκB (Ser536) and total NFκB. For (Panel B,C) bar graphs represent data as integrated density normalized to unstimulated control. n = 3–9 (B) or n = 4–6 (C); *p < 0.01 (B) or *p < 0.05 (C) vs rPBEF-stimulated control. (Panel D) Immunofluorescent monitoring of agonist-induced NFκB translocation to the nucleus. rPBEF (10 μg/ml) for 0–60 min induces NFκB translocation from the cytosol to the nuclei in human lung microvascular EC similar to EC challenge with TNF-α (100 ng/ml). Scale bar = 10 μm; n = 3; representative immunofluorescence images shown. (Panel E) rPBEF (10 ng/mL)-mediated increases in NFκB promoter-driven luciferase reporter activity in human lung EC assessed from 0–3 hr. NFκB promoter activity following rPBEF challenge was measured using a dual luciferase reporter assay and absorbance of the inducible NFκB promoter-responsive firefly luciferase construct was normalized to the absorbance of the constitutively active Renilla luciferase construct (internal control). Bar graphs represent data as percentage luciferase activity increase above control, n = 3; *p < 0.05 versus unstimulated controls.
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f1: Extracellular NAMPT/PBEF independently mediates rapid NFκB activation in human lung endothelium.(Panel A) Activation of NFκB signaling in human lung EC challenged with recombinant NAMPT/PBEF (rPBEF) (1 μg/ml, 5–60 min). Stimulated EC lysates were probed for p-NFκB (Ser536) or control β-actin. Heat-denatured rPBEF (HD-rPBEF) (1 μg/ml, 1 hr) served as a negative control confirming that rPBEF effects do not reflect endotoxin contamination. LPS (5 μg/ml, 1 hr) and TNF-α (100 ng/ml, 15 min) served as positive controls for NFκB signaling pathway activation. n = 3; representative blots shown. (Panel B) NAMPT/PBEF-mediated NFκB activation does not reflect endotoxin contamination. rPBEF or LPS were exposed to 100 °C for 5 min. Human lung ECs were treated with rPBEF (1 μg/ml), heat denatured (HD)-rPBEF (1 μg/ml), LPS (5 μg/ml), or HD-LPS (5 μg/ml) for 1 hr, EC lysates were then probed for p-NFκB (Ser536) or total NFκB. (Panel C) NAMPT/PBEF enzymatic inhibitor FK-866 fails to attenuate rPBEF-induced NFκB phosphorylation. Human lung ECs were treated with rPBEF (1 μg/ml, 1 hr) either without any pretreatment, with premixing with neutralizing NAMPT/PBEF pAb (100 μg/ml, 30 min), or pretreatment with FK-866 (0.1–10 μM, 1 hr). EC lysates were probed for p-NFκB (Ser536) and total NFκB. For (Panel B,C) bar graphs represent data as integrated density normalized to unstimulated control. n = 3–9 (B) or n = 4–6 (C); *p < 0.01 (B) or *p < 0.05 (C) vs rPBEF-stimulated control. (Panel D) Immunofluorescent monitoring of agonist-induced NFκB translocation to the nucleus. rPBEF (10 μg/ml) for 0–60 min induces NFκB translocation from the cytosol to the nuclei in human lung microvascular EC similar to EC challenge with TNF-α (100 ng/ml). Scale bar = 10 μm; n = 3; representative immunofluorescence images shown. (Panel E) rPBEF (10 ng/mL)-mediated increases in NFκB promoter-driven luciferase reporter activity in human lung EC assessed from 0–3 hr. NFκB promoter activity following rPBEF challenge was measured using a dual luciferase reporter assay and absorbance of the inducible NFκB promoter-responsive firefly luciferase construct was normalized to the absorbance of the constitutively active Renilla luciferase construct (internal control). Bar graphs represent data as percentage luciferase activity increase above control, n = 3; *p < 0.05 versus unstimulated controls.
Mentions: Leveraging our prior reports of NFκB transcriptome induction by recombinant NAMPT/PBEF (rPBEF)121718, complementary in vitro approaches were utilized to functionally examine the direct role of extracellular NAMPT/PBEF in NFκB pathway activation and innate immunity gene expression. Initial experiments assessed exogenous NAMPT/PBEF-mediated phosphorylation of NFκB (p-NFκB at Ser536) in human lung endothelial cells (EC) as an indication of NFκB activation. rPBEF, similar to LPS and TNF-α, increases phosphorylation of p-NFκB within 30 min with persistent elevation at 1 hr (Fig. 1A). Heat denaturing of rPBEF (HD-rPBEF) resulted in the elimination of NAMPT/PBEF’s capacity to induce NFκB activation, demonstrating that NAMPT/PBEF-mediated NFκB phosphorylation/activation does not reflect endotoxin contamination (Fig. 1A,B). rPBEF-induced NFκB activation was unaffected by inhibition of NAMPT enzymatic activity (FK-866), indicating that phosphoribosyl-transferase activity is not required (Fig. 1C).

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