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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

TLR4 is the novel receptor for extracellular NAMPT/PBEF- and VILI-induced NFκB activation and inflammatory lung injury in vivo.Panel A. Compared to saline-challenged controls (VEH), intratracheal instillation of rPBEF (40 μg/mouse) in wild type mice produces robust increases in BAL protein levels, in the percentage of BAL PMNs, and in BAL total cell counts, findings consistent with prior reports12. These rPBEF–mediated inflammatory lung indices were significantly reduced both in mice pretreated with the TLR4 inhibitor RS-LPS (100 μg/mouse, i.p.) (Panel A) and in TLR4−/− mice (Panel C) indicating that TLR4 is required for NAMPT/PBEF-induced pro-inflammatory activities and lung injury. Results are expressed as mean ± SEM; n = 3–6 per condition; *p < 0.05 for VEH/WT vs VEH/WT + rPBEF and **p < 0.05 VEH/WT + rPBEF vs RS/TLR4−/− + rPBEF, using Anova non-parametric Newman-Keuls Multiple Comparison Test. (Panel B) Exposure to VILI (40 ml/kg, 4 hr) in wild type mice produces significant increases in BAL protein levels, in the percentage of BAL PMNs, and in BAL cell counts. These rPBEF–mediated inflammatory indices were significantly reduced in mice pretreated with the TLR4 inhibitor RS-LPS (100 μg/mouse), indicating that TLR4 is required for NAMPT/PBEF-induced pro-inflammatory activities and lung injury. Results are expressed as mean ± SEM; n = 3–6 per condition; *p < 0.05 for VEH vs VEH + VILI and **p < 0.05 VEH + VILI vs RS + VILI.
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f4: TLR4 is the novel receptor for extracellular NAMPT/PBEF- and VILI-induced NFκB activation and inflammatory lung injury in vivo.Panel A. Compared to saline-challenged controls (VEH), intratracheal instillation of rPBEF (40 μg/mouse) in wild type mice produces robust increases in BAL protein levels, in the percentage of BAL PMNs, and in BAL total cell counts, findings consistent with prior reports12. These rPBEF–mediated inflammatory lung indices were significantly reduced both in mice pretreated with the TLR4 inhibitor RS-LPS (100 μg/mouse, i.p.) (Panel A) and in TLR4−/− mice (Panel C) indicating that TLR4 is required for NAMPT/PBEF-induced pro-inflammatory activities and lung injury. Results are expressed as mean ± SEM; n = 3–6 per condition; *p < 0.05 for VEH/WT vs VEH/WT + rPBEF and **p < 0.05 VEH/WT + rPBEF vs RS/TLR4−/− + rPBEF, using Anova non-parametric Newman-Keuls Multiple Comparison Test. (Panel B) Exposure to VILI (40 ml/kg, 4 hr) in wild type mice produces significant increases in BAL protein levels, in the percentage of BAL PMNs, and in BAL cell counts. These rPBEF–mediated inflammatory indices were significantly reduced in mice pretreated with the TLR4 inhibitor RS-LPS (100 μg/mouse), indicating that TLR4 is required for NAMPT/PBEF-induced pro-inflammatory activities and lung injury. Results are expressed as mean ± SEM; n = 3–6 per condition; *p < 0.05 for VEH vs VEH + VILI and **p < 0.05 VEH + VILI vs RS + VILI.

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)

TLR4 is the novel receptor for extracellular NAMPT/PBEF- and VILI-induced NFκB activation and inflammatory lung injury in vivo.Panel A. Compared to saline-challenged controls (VEH), intratracheal instillation of rPBEF (40 μg/mouse) in wild type mice produces robust increases in BAL protein levels, in the percentage of BAL PMNs, and in BAL total cell counts, findings consistent with prior reports12. These rPBEF–mediated inflammatory lung indices were significantly reduced both in mice pretreated with the TLR4 inhibitor RS-LPS (100 μg/mouse, i.p.) (Panel A) and in TLR4−/− mice (Panel C) indicating that TLR4 is required for NAMPT/PBEF-induced pro-inflammatory activities and lung injury. Results are expressed as mean ± SEM; n = 3–6 per condition; *p < 0.05 for VEH/WT vs VEH/WT + rPBEF and **p < 0.05 VEH/WT + rPBEF vs RS/TLR4−/− + rPBEF, using Anova non-parametric Newman-Keuls Multiple Comparison Test. (Panel B) Exposure to VILI (40 ml/kg, 4 hr) in wild type mice produces significant increases in BAL protein levels, in the percentage of BAL PMNs, and in BAL cell counts. These rPBEF–mediated inflammatory indices were significantly reduced in mice pretreated with the TLR4 inhibitor RS-LPS (100 μg/mouse), indicating that TLR4 is required for NAMPT/PBEF-induced pro-inflammatory activities and lung injury. Results are expressed as mean ± SEM; n = 3–6 per condition; *p < 0.05 for VEH vs VEH + VILI and **p < 0.05 VEH + VILI vs RS + VILI.
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f4: TLR4 is the novel receptor for extracellular NAMPT/PBEF- and VILI-induced NFκB activation and inflammatory lung injury in vivo.Panel A. Compared to saline-challenged controls (VEH), intratracheal instillation of rPBEF (40 μg/mouse) in wild type mice produces robust increases in BAL protein levels, in the percentage of BAL PMNs, and in BAL total cell counts, findings consistent with prior reports12. These rPBEF–mediated inflammatory lung indices were significantly reduced both in mice pretreated with the TLR4 inhibitor RS-LPS (100 μg/mouse, i.p.) (Panel A) and in TLR4−/− mice (Panel C) indicating that TLR4 is required for NAMPT/PBEF-induced pro-inflammatory activities and lung injury. Results are expressed as mean ± SEM; n = 3–6 per condition; *p < 0.05 for VEH/WT vs VEH/WT + rPBEF and **p < 0.05 VEH/WT + rPBEF vs RS/TLR4−/− + rPBEF, using Anova non-parametric Newman-Keuls Multiple Comparison Test. (Panel B) Exposure to VILI (40 ml/kg, 4 hr) in wild type mice produces significant increases in BAL protein levels, in the percentage of BAL PMNs, and in BAL cell counts. These rPBEF–mediated inflammatory indices were significantly reduced in mice pretreated with the TLR4 inhibitor RS-LPS (100 μg/mouse), indicating that TLR4 is required for NAMPT/PBEF-induced pro-inflammatory activities and lung injury. Results are expressed as mean ± SEM; n = 3–6 per condition; *p < 0.05 for VEH vs VEH + VILI and **p < 0.05 VEH + VILI vs RS + VILI.
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