Limits...
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 induces NFκB activation via TLR4 ligation in vitro.Panels A and B. Effects of TLR4 inhibitory strategies on rPBEF- and LPS-mediated NFκB phosphorylation in ECs. Cell lysates were immunoblotted for phospho-specific or total NFκB with experiments independently performed in triplicate (representative blots shown). TNF-α (100 ng/ml, 15 min) serves as a positive control for NFκB signaling activation. (Panel A) Inhibition of both rPBEF (1 μg/ml, 1 hr)- and LPS (5 μg/ml, 1 hr)-mediated NFκB phosphorylation (Ser536) in EC pretreated (1 hr) with neutralizing TLR4 polyclonal (pAb, 20 μg/ml) or TLR4 monoclonal antibodies (mAb, 10 μg/ml). Premixing of rPBEF, but not LPS, with neutralizing NAMPT/PBEF pAb (100 μg/ml, 30 min) similarly reduced NFκB phosphorylation. (Panel B) Inhibitory effects of 1 hr pretreatment with TLR4 pharmacologic inhibitors (RS-LPS [10 μg/ml], CLI-095 [5 μM] and OxPAPC [30 μg/ml]) on rPBEF- and LPS-mediated NFκB phosphorylation in ECs. (Panel C) Densitometric summary of the attenuation of rPBEF- and LPS-induced NFκB phosphorylation by TLR4 neutralizing antibodies and inhibitors. rPBEF-induced NFκB phosphorylation was significantly reduced to levels similar to those observed with pretreatment with anti-NAMPT/PBEF specific antibody. LPS-induced NFκB phosphorylation was unaffected by pretreatment with anti-NAMPT/PBEF specific antibody. Bar graphs represent data as integrated density normalized to rPBEF- or LPS-stimulated control. n = 3 independent experiments per condition; *p < 0.05 versus rPBEF-stimulated control, **p < 0.05 versus LPS-stimulated control. Pretreatment with inhibitors or neutralizing antibodies alone (without rPBEF or LPS stimulation) did not significantly differ from unstimulated controls (data not shown). (Panel D) Surface plasmon resonance (SPR) analysis (using Bio-Rad ProteOn XPR36 instrument and GLC sensor chips) demonstrates TLR4-NAMPT/PBEF binding interaction. Bethyl PBEF antibody was covalently bound to the chip surface, using standard direct immobilization, at a final immobilization level of 5000 RUs. rPBEF only (100 nM), rTLR4 only (1 μM), and rPBEF-rTLR4 pre-mixed (100 nM or 1 μM, respectively) analytes were then injected over the PBEF antibody coated surface. While rTLR4 did not bind to the PBEF antibody coated surface, pre-mixed rPBEF-rTLR4 resulted in increased binding response over rPBEF alone.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4536637&req=5

f3: Extracellular NAMPT/PBEF induces NFκB activation via TLR4 ligation in vitro.Panels A and B. Effects of TLR4 inhibitory strategies on rPBEF- and LPS-mediated NFκB phosphorylation in ECs. Cell lysates were immunoblotted for phospho-specific or total NFκB with experiments independently performed in triplicate (representative blots shown). TNF-α (100 ng/ml, 15 min) serves as a positive control for NFκB signaling activation. (Panel A) Inhibition of both rPBEF (1 μg/ml, 1 hr)- and LPS (5 μg/ml, 1 hr)-mediated NFκB phosphorylation (Ser536) in EC pretreated (1 hr) with neutralizing TLR4 polyclonal (pAb, 20 μg/ml) or TLR4 monoclonal antibodies (mAb, 10 μg/ml). Premixing of rPBEF, but not LPS, with neutralizing NAMPT/PBEF pAb (100 μg/ml, 30 min) similarly reduced NFκB phosphorylation. (Panel B) Inhibitory effects of 1 hr pretreatment with TLR4 pharmacologic inhibitors (RS-LPS [10 μg/ml], CLI-095 [5 μM] and OxPAPC [30 μg/ml]) on rPBEF- and LPS-mediated NFκB phosphorylation in ECs. (Panel C) Densitometric summary of the attenuation of rPBEF- and LPS-induced NFκB phosphorylation by TLR4 neutralizing antibodies and inhibitors. rPBEF-induced NFκB phosphorylation was significantly reduced to levels similar to those observed with pretreatment with anti-NAMPT/PBEF specific antibody. LPS-induced NFκB phosphorylation was unaffected by pretreatment with anti-NAMPT/PBEF specific antibody. Bar graphs represent data as integrated density normalized to rPBEF- or LPS-stimulated control. n = 3 independent experiments per condition; *p < 0.05 versus rPBEF-stimulated control, **p < 0.05 versus LPS-stimulated control. Pretreatment with inhibitors or neutralizing antibodies alone (without rPBEF or LPS stimulation) did not significantly differ from unstimulated controls (data not shown). (Panel D) Surface plasmon resonance (SPR) analysis (using Bio-Rad ProteOn XPR36 instrument and GLC sensor chips) demonstrates TLR4-NAMPT/PBEF binding interaction. Bethyl PBEF antibody was covalently bound to the chip surface, using standard direct immobilization, at a final immobilization level of 5000 RUs. rPBEF only (100 nM), rTLR4 only (1 μM), and rPBEF-rTLR4 pre-mixed (100 nM or 1 μM, respectively) analytes were then injected over the PBEF antibody coated surface. While rTLR4 did not bind to the PBEF antibody coated surface, pre-mixed rPBEF-rTLR4 resulted in increased binding response over rPBEF alone.

Mentions: Given the prominent NFκB and TLR pathway gene signatures evoked by NAMPT/PBEF, we assessed TLR4 as a putative NAMPT/PBEF receptor. Human lung EC pretreated with either TLR4 antibodies (10–20 μg/ml, 1 hr) or TLR4 inhibitors (RS-LPS 10 μg/ml, CLI-095 5 μM, OxPAPC 30 μg/ml; 1 hr), were challenged with rPBEF or LPS and cell lysates immunoblotted for either phosphorylated or total NFκB. In each case, inhibition of TLR4 activity resulted in marked reductions in rPBEF-mediated Ser536 NFκB phosphorylation (Fig. 3A–C). The biologic activity of rPBEF was confirmed by NAMPT/PBEF-specific antibody inhibition (Fig. 3A,C). TLR4 inhibition also reduced LPS-mediated Ser536 NFκB phosphorylation. In contrast to rPBEF, however, LPS-mediated Ser536 NFκB phosphorylation was unaffected by a NAMPT/PBEF neutralizing antibody (Fig. 3A,C), strongly supporting TLR4 as a novel NAMPT/PBEF receptor. Surface plasmon resonance (SPR) analysis was next utilized to demonstrate direct molecular interaction of rTLR4 and rPBEF. Recombinant TLR4 failed to bind to the PBEF antibody coated surface whereas a previously mixed solution of rPBEF and rTLR4 resulted in strong increases in binding over rPBEF alone (Fig. 3D).


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 induces NFκB activation via TLR4 ligation in vitro.Panels A and B. Effects of TLR4 inhibitory strategies on rPBEF- and LPS-mediated NFκB phosphorylation in ECs. Cell lysates were immunoblotted for phospho-specific or total NFκB with experiments independently performed in triplicate (representative blots shown). TNF-α (100 ng/ml, 15 min) serves as a positive control for NFκB signaling activation. (Panel A) Inhibition of both rPBEF (1 μg/ml, 1 hr)- and LPS (5 μg/ml, 1 hr)-mediated NFκB phosphorylation (Ser536) in EC pretreated (1 hr) with neutralizing TLR4 polyclonal (pAb, 20 μg/ml) or TLR4 monoclonal antibodies (mAb, 10 μg/ml). Premixing of rPBEF, but not LPS, with neutralizing NAMPT/PBEF pAb (100 μg/ml, 30 min) similarly reduced NFκB phosphorylation. (Panel B) Inhibitory effects of 1 hr pretreatment with TLR4 pharmacologic inhibitors (RS-LPS [10 μg/ml], CLI-095 [5 μM] and OxPAPC [30 μg/ml]) on rPBEF- and LPS-mediated NFκB phosphorylation in ECs. (Panel C) Densitometric summary of the attenuation of rPBEF- and LPS-induced NFκB phosphorylation by TLR4 neutralizing antibodies and inhibitors. rPBEF-induced NFκB phosphorylation was significantly reduced to levels similar to those observed with pretreatment with anti-NAMPT/PBEF specific antibody. LPS-induced NFκB phosphorylation was unaffected by pretreatment with anti-NAMPT/PBEF specific antibody. Bar graphs represent data as integrated density normalized to rPBEF- or LPS-stimulated control. n = 3 independent experiments per condition; *p < 0.05 versus rPBEF-stimulated control, **p < 0.05 versus LPS-stimulated control. Pretreatment with inhibitors or neutralizing antibodies alone (without rPBEF or LPS stimulation) did not significantly differ from unstimulated controls (data not shown). (Panel D) Surface plasmon resonance (SPR) analysis (using Bio-Rad ProteOn XPR36 instrument and GLC sensor chips) demonstrates TLR4-NAMPT/PBEF binding interaction. Bethyl PBEF antibody was covalently bound to the chip surface, using standard direct immobilization, at a final immobilization level of 5000 RUs. rPBEF only (100 nM), rTLR4 only (1 μM), and rPBEF-rTLR4 pre-mixed (100 nM or 1 μM, respectively) analytes were then injected over the PBEF antibody coated surface. While rTLR4 did not bind to the PBEF antibody coated surface, pre-mixed rPBEF-rTLR4 resulted in increased binding response over rPBEF alone.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4536637&req=5

f3: Extracellular NAMPT/PBEF induces NFκB activation via TLR4 ligation in vitro.Panels A and B. Effects of TLR4 inhibitory strategies on rPBEF- and LPS-mediated NFκB phosphorylation in ECs. Cell lysates were immunoblotted for phospho-specific or total NFκB with experiments independently performed in triplicate (representative blots shown). TNF-α (100 ng/ml, 15 min) serves as a positive control for NFκB signaling activation. (Panel A) Inhibition of both rPBEF (1 μg/ml, 1 hr)- and LPS (5 μg/ml, 1 hr)-mediated NFκB phosphorylation (Ser536) in EC pretreated (1 hr) with neutralizing TLR4 polyclonal (pAb, 20 μg/ml) or TLR4 monoclonal antibodies (mAb, 10 μg/ml). Premixing of rPBEF, but not LPS, with neutralizing NAMPT/PBEF pAb (100 μg/ml, 30 min) similarly reduced NFκB phosphorylation. (Panel B) Inhibitory effects of 1 hr pretreatment with TLR4 pharmacologic inhibitors (RS-LPS [10 μg/ml], CLI-095 [5 μM] and OxPAPC [30 μg/ml]) on rPBEF- and LPS-mediated NFκB phosphorylation in ECs. (Panel C) Densitometric summary of the attenuation of rPBEF- and LPS-induced NFκB phosphorylation by TLR4 neutralizing antibodies and inhibitors. rPBEF-induced NFκB phosphorylation was significantly reduced to levels similar to those observed with pretreatment with anti-NAMPT/PBEF specific antibody. LPS-induced NFκB phosphorylation was unaffected by pretreatment with anti-NAMPT/PBEF specific antibody. Bar graphs represent data as integrated density normalized to rPBEF- or LPS-stimulated control. n = 3 independent experiments per condition; *p < 0.05 versus rPBEF-stimulated control, **p < 0.05 versus LPS-stimulated control. Pretreatment with inhibitors or neutralizing antibodies alone (without rPBEF or LPS stimulation) did not significantly differ from unstimulated controls (data not shown). (Panel D) Surface plasmon resonance (SPR) analysis (using Bio-Rad ProteOn XPR36 instrument and GLC sensor chips) demonstrates TLR4-NAMPT/PBEF binding interaction. Bethyl PBEF antibody was covalently bound to the chip surface, using standard direct immobilization, at a final immobilization level of 5000 RUs. rPBEF only (100 nM), rTLR4 only (1 μM), and rPBEF-rTLR4 pre-mixed (100 nM or 1 μM, respectively) analytes were then injected over the PBEF antibody coated surface. While rTLR4 did not bind to the PBEF antibody coated surface, pre-mixed rPBEF-rTLR4 resulted in increased binding response over rPBEF alone.
Mentions: Given the prominent NFκB and TLR pathway gene signatures evoked by NAMPT/PBEF, we assessed TLR4 as a putative NAMPT/PBEF receptor. Human lung EC pretreated with either TLR4 antibodies (10–20 μg/ml, 1 hr) or TLR4 inhibitors (RS-LPS 10 μg/ml, CLI-095 5 μM, OxPAPC 30 μg/ml; 1 hr), were challenged with rPBEF or LPS and cell lysates immunoblotted for either phosphorylated or total NFκB. In each case, inhibition of TLR4 activity resulted in marked reductions in rPBEF-mediated Ser536 NFκB phosphorylation (Fig. 3A–C). The biologic activity of rPBEF was confirmed by NAMPT/PBEF-specific antibody inhibition (Fig. 3A,C). TLR4 inhibition also reduced LPS-mediated Ser536 NFκB phosphorylation. In contrast to rPBEF, however, LPS-mediated Ser536 NFκB phosphorylation was unaffected by a NAMPT/PBEF neutralizing antibody (Fig. 3A,C), strongly supporting TLR4 as a novel NAMPT/PBEF receptor. Surface plasmon resonance (SPR) analysis was next utilized to demonstrate direct molecular interaction of rTLR4 and rPBEF. Recombinant TLR4 failed to bind to the PBEF antibody coated surface whereas a previously mixed solution of rPBEF and rTLR4 resulted in strong increases in binding over rPBEF alone (Fig. 3D).

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