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Id Subject Object Predicate Lexical cue
T72 0-69 Sentence denotes Inflammasome-Mediated IL-1β Production in Humans with Cystic Fibrosis
T1 0-105 Sentence denotes Inflammasome-Mediated IL-1β Production in Humans with Cystic Fibrosis IL-1β Production in Cystic Fibrosis
T2 108-116 Sentence denotes Abstract
T73 117-127 Sentence denotes Background
T3 117-127 Sentence denotes Background
T74 128-287 Sentence denotes Inflammation and infection are major determinants of disease severity and consequently, the quality of life and outcome for patients with cystic fibrosis (CF).
T4 128-287 Sentence denotes Inflammation and infection are major determinants of disease severity and consequently, the quality of life and outcome for patients with cystic fibrosis (CF).
T75 288-346 Sentence denotes Interleukin-1 beta (IL-1β) is a key inflammatory mediator.
T5 288-346 Sentence denotes Interleukin-1 beta (IL-1β) is a key inflammatory mediator.
T76 347-428 Sentence denotes Secretion of biologically active IL-1β involves inflammasome-mediated processing.
T6 347-428 Sentence denotes Secretion of biologically active IL-1β involves inflammasome-mediated processing.
T77 429-526 Sentence denotes Little is known about the contribution of IL-1β and the inflammasomes in CF inflammatory disease.
T7 429-526 Sentence denotes Little is known about the contribution of IL-1β and the inflammasomes in CF inflammatory disease.
T78 527-651 Sentence denotes This study examines inflammasome-mediated IL-1β production in CF bronchial epithelial cell lines and human patients with CF.
T8 527-651 Sentence denotes This study examines inflammasome-mediated IL-1β production in CF bronchial epithelial cell lines and human patients with CF.
T79 653-660 Sentence denotes Results
T9 653-660 Sentence denotes Results
T80 661-886 Sentence denotes Bronchial epithelial cell lines were found to produce negligible amounts of basal or stimulated IL-1β compared to hematopoeitic cells and they did not significantly upregulate caspase-1 activity upon inflammasome stimulation.
T10 661-886 Sentence denotes Bronchial epithelial cell lines were found to produce negligible amounts of basal or stimulated IL-1β compared to hematopoeitic cells and they did not significantly upregulate caspase-1 activity upon inflammasome stimulation.
T81 887-1091 Sentence denotes In contrast, peripheral blood mononuclear cells (PBMCs) from both CF and healthy control subjects produced large amounts of IL-1β and strongly upregulated caspase-1 activity upon inflammasome stimulation.
T11 887-1091 Sentence denotes In contrast, peripheral blood mononuclear cells (PBMCs) from both CF and healthy control subjects produced large amounts of IL-1β and strongly upregulated caspase-1 activity upon inflammasome stimulation.
T82 1092-1243 Sentence denotes PBMCs from CF patients and controls displayed similar levels of caspase-1 activation and IL-1β production when stimulated with inflammasome activators.
T12 1092-1243 Sentence denotes PBMCs from CF patients and controls displayed similar levels of caspase-1 activation and IL-1β production when stimulated with inflammasome activators.
T83 1244-1340 Sentence denotes This IL-1β production was dependent on NF-κB activity and could be enhanced by priming with LPS.
T13 1244-1340 Sentence denotes This IL-1β production was dependent on NF-κB activity and could be enhanced by priming with LPS.
T84 1341-1502 Sentence denotes Finally, chemical inhibition of CFTR activity in control PBMCs and THP-1 cells did not significantly alter IL-1β or IL-8 production in response to P. aeruginosa.
T14 1341-1502 Sentence denotes Finally, chemical inhibition of CFTR activity in control PBMCs and THP-1 cells did not significantly alter IL-1β or IL-8 production in response to P. aeruginosa.
T85 1504-1514 Sentence denotes Conclusion
T15 1504-1514 Sentence denotes Conclusion
T86 1515-1624 Sentence denotes Hematopoeitic cells appear to be the predominant source of inflammasome-induced pro-inflammatory IL-1β in CF.
T16 1515-1624 Sentence denotes Hematopoeitic cells appear to be the predominant source of inflammasome-induced pro-inflammatory IL-1β in CF.
T87 1625-1778 Sentence denotes PBMCs derived from CF subjects display preserved inflammasome activation and IL-1β secretion in response to the major CF pathogen Pseudomonas aeruginosa.
T17 1625-1778 Sentence denotes PBMCs derived from CF subjects display preserved inflammasome activation and IL-1β secretion in response to the major CF pathogen Pseudomonas aeruginosa.
T88 1779-1954 Sentence denotes However, our data do not support the hypothesis that increased IL-1β production in CF subjects is due to an intrinsic increase in NF-κB activity through loss of CFTR function.
T18 1779-1954 Sentence denotes However, our data do not support the hypothesis that increased IL-1β production in CF subjects is due to an intrinsic increase in NF-κB activity through loss of CFTR function.
T19 1957-1969 Sentence denotes Introduction
T930 1970-2149 Sentence denotes Cystic fibrosis is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a chloride ion channel.
T20 1970-2149 Sentence denotes Cystic fibrosis is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a chloride ion channel.
T931 2150-2331 Sentence denotes CF remains one of the most common lethal genetic diseases in populations of European descent with the current average lifespan of CF patients approximately 40 years of age [1], [2].
T21 2150-2331 Sentence denotes CF remains one of the most common lethal genetic diseases in populations of European descent with the current average lifespan of CF patients approximately 40 years of age [1], [2].
T932 2332-2455 Sentence denotes Recurrent inflammatory pulmonary exacerbation is the primary cause of lung disease progression and ultimately, death in CF.
T22 2332-2455 Sentence denotes Recurrent inflammatory pulmonary exacerbation is the primary cause of lung disease progression and ultimately, death in CF.
T933 2456-2615 Sentence denotes Controlled inflammation is important for fighting infection, but in excess, it becomes destructive to host cells and to the architecture of the lungs [2], [3].
T23 2456-2615 Sentence denotes Controlled inflammation is important for fighting infection, but in excess, it becomes destructive to host cells and to the architecture of the lungs [2], [3].
T934 2616-2748 Sentence denotes In CF, airway epithelial cells have been shown to produce an exaggerated pro-inflammatory cytokine response to stimulation [4], [5].
T24 2616-2748 Sentence denotes In CF, airway epithelial cells have been shown to produce an exaggerated pro-inflammatory cytokine response to stimulation [4], [5].
T935 2749-2914 Sentence denotes It is unclear whether this heightened inflammatory response is intrinsic to cells lacking CFTR or whether it is a result of chronic polymicrobial infection [6], [7].
T25 2749-2914 Sentence denotes It is unclear whether this heightened inflammatory response is intrinsic to cells lacking CFTR or whether it is a result of chronic polymicrobial infection [6], [7].
T936 2915-3137 Sentence denotes Regardless of this controversy, identifying and targeting relevant inflammatory mediators is a critical step in developing more specific therapeutic approaches to control inflammation and improve health outcomes in CF [8].
T26 2915-3137 Sentence denotes Regardless of this controversy, identifying and targeting relevant inflammatory mediators is a critical step in developing more specific therapeutic approaches to control inflammation and improve health outcomes in CF [8].
T937 3138-3198 Sentence denotes Interleukin-1 beta (IL-1β) is a major inflammatory mediator.
T27 3138-3198 Sentence denotes Interleukin-1 beta (IL-1β) is a major inflammatory mediator.
T938 3199-3514 Sentence denotes Its physiological effects are diverse and potentially important to the pathogenesis of lung exacerbations in CF, including the generation of fever, the recruitment of inflammatory effector cells, the induction of other pro-inflammatory cytokines such as IL-6 and IL-8, and the shaping of T cell responses [9], [10].
T28 3199-3514 Sentence denotes Its physiological effects are diverse and potentially important to the pathogenesis of lung exacerbations in CF, including the generation of fever, the recruitment of inflammatory effector cells, the induction of other pro-inflammatory cytokines such as IL-6 and IL-8, and the shaping of T cell responses [9], [10].
T939 3515-3650 Sentence denotes Following initiation of the NF-κB signaling cascade, IL-1β is produced in the cytosol as a biologically inactive full-length pro-IL-1β.
T29 3515-3650 Sentence denotes Following initiation of the NF-κB signaling cascade, IL-1β is produced in the cytosol as a biologically inactive full-length pro-IL-1β.
T940 3651-3978 Sentence denotes Pro-IL-1β is subsequently converted into its active form by cytosolic protein complexes termed “inflammasomes.” Inflammasomes assemble in response to certain cellular danger signals and mediate the auto-activation of caspase-1 [9], [11], which cleaves pro-IL-1β and pro-IL-18 into their biologically active forms for secretion.
T30 3651-3978 Sentence denotes Pro-IL-1β is subsequently converted into its active form by cytosolic protein complexes termed “inflammasomes.” Inflammasomes assemble in response to certain cellular danger signals and mediate the auto-activation of caspase-1 [9], [11], which cleaves pro-IL-1β and pro-IL-18 into their biologically active forms for secretion.
T941 3979-4028 Sentence denotes Four distinct inflammasomes have been recognized.
T31 3979-4028 Sentence denotes Four distinct inflammasomes have been recognized.
T942 4029-4211 Sentence denotes These are the NLRP1 [12], NLRP3 [13], [14], NLRC4 [15], [16], and AIM2 inflammasomes [17], [18], which respond to a variety of different microbial signatures and danger signals [11].
T32 4029-4211 Sentence denotes These are the NLRP1 [12], NLRP3 [13], [14], NLRC4 [15], [16], and AIM2 inflammasomes [17], [18], which respond to a variety of different microbial signatures and danger signals [11].
T943 4212-4347 Sentence denotes P. aeruginosa, one of the most common and clinically relevant pathogens among CF patients, activates the NLRC4 inflammasome [19], [20].
T33 4212-4347 Sentence denotes P. aeruginosa, one of the most common and clinically relevant pathogens among CF patients, activates the NLRC4 inflammasome [19], [20].
T944 4348-4499 Sentence denotes Infection with P. aeruginosa triggers an increase in levels of IL-1β, IL-6, and IL-8 in bronchoalveolar lavage fluid (BALF) from patients with CF [21].
T34 4348-4499 Sentence denotes Infection with P. aeruginosa triggers an increase in levels of IL-1β, IL-6, and IL-8 in bronchoalveolar lavage fluid (BALF) from patients with CF [21].
T945 4500-4687 Sentence denotes Inflammasome responses depend on NF-κB signaling, where NF-κB is important in both the upregulation of specific inflammasome components [22], [23], as well as IL-1β expression [24], [25].
T35 4500-4687 Sentence denotes Inflammasome responses depend on NF-κB signaling, where NF-κB is important in both the upregulation of specific inflammasome components [22], [23], as well as IL-1β expression [24], [25].
T946 4688-4782 Sentence denotes Previous studies support a role for IL-1β in the pathogenesis of CF inflammatory lung disease.
T36 4688-4782 Sentence denotes Previous studies support a role for IL-1β in the pathogenesis of CF inflammatory lung disease.
T947 4783-4968 Sentence denotes Levels of IL-1β are increased in BALF from CF patients with infection [21], [26], [27], [28] and this increase has been temporally associated with a clinical response to treatment [21].
T37 4783-4968 Sentence denotes Levels of IL-1β are increased in BALF from CF patients with infection [21], [26], [27], [28] and this increase has been temporally associated with a clinical response to treatment [21].
T948 4969-5087 Sentence denotes Polymorphisms in the IL1B gene have also been associated with varying degrees of disease severity in CF patients [29].
T38 4969-5087 Sentence denotes Polymorphisms in the IL1B gene have also been associated with varying degrees of disease severity in CF patients [29].
T949 5088-5355 Sentence denotes Murine models of CFTR dysfunction have exhibited significant increases in IL-1β expression or secretion in macrophages [30], [31], and support the hypothesis that the loss of CFTR increases NF-κB activation under basal and stimulatory conditions [4], [5], [32], [33].
T39 5088-5355 Sentence denotes Murine models of CFTR dysfunction have exhibited significant increases in IL-1β expression or secretion in macrophages [30], [31], and support the hypothesis that the loss of CFTR increases NF-κB activation under basal and stimulatory conditions [4], [5], [32], [33].
T950 5356-5619 Sentence denotes Finally, replacement of chloride ions with glutamate or gluconate in cell culture media increases secretion of IL-1β in response to NLRP3 stimulation by adenosine triphosphate (ATP) [34], implying an inhibitory role for extracellular chloride in NLRP3 activation.
T40 5356-5619 Sentence denotes Finally, replacement of chloride ions with glutamate or gluconate in cell culture media increases secretion of IL-1β in response to NLRP3 stimulation by adenosine triphosphate (ATP) [34], implying an inhibitory role for extracellular chloride in NLRP3 activation.
T951 5620-5746 Sentence denotes Taken together, these data implicate the involvement of IL-1β and consequently, the inflammasomes, in CF inflammatory disease.
T41 5620-5746 Sentence denotes Taken together, these data implicate the involvement of IL-1β and consequently, the inflammasomes, in CF inflammatory disease.
T42 5748-5755 Sentence denotes Results
T2604 5757-5864 Sentence denotes Airway epithelial cells do not produce significant amounts of IL-1β in response to inflammasome stimulation
T43 5757-5864 Sentence denotes Airway epithelial cells do not produce significant amounts of IL-1β in response to inflammasome stimulation
T2605 5865-5960 Sentence denotes The inflammasomes and their respective activators examined in this study are listed in Table 1.
T44 5865-5960 Sentence denotes The inflammasomes and their respective activators examined in this study are listed in Table 1.
T2606 5961-6027 Sentence denotes Cells were stimulated in accordance with the schedule in Figure 1.
T45 5961-6027 Sentence denotes Cells were stimulated in accordance with the schedule in Figure 1.
T2607 6028-6188 Sentence denotes In CF, airway epithelial cells have been shown to possess a hyper-inflammatory phenotype and produce an exaggerated pro-inflammatory cytokine response [4], [5].
T46 6028-6188 Sentence denotes In CF, airway epithelial cells have been shown to possess a hyper-inflammatory phenotype and produce an exaggerated pro-inflammatory cytokine response [4], [5].
T2608 6189-6446 Sentence denotes To determine if airway epithelial cells contribute to the increased IL-1β production in patients with CF, CF and control bronchial epithelial cell lines were stimulated with the inflammasome inducers P. aeruginosa strain PAO1 (PAO1) and LPS followed by ATP.
T47 6189-6446 Sentence denotes To determine if airway epithelial cells contribute to the increased IL-1β production in patients with CF, CF and control bronchial epithelial cell lines were stimulated with the inflammasome inducers P. aeruginosa strain PAO1 (PAO1) and LPS followed by ATP.
T2609 6447-6698 Sentence denotes IL-1β levels in cell culture supernatants were not greatly increased in either the CF or control cell lines (Fig. 2a–d), although a small increase in IL-1β production was detected in NuLi-1 and CuFi-1 cells, but not in S9 and IB3-1 cells, by 24 hours.
T48 6447-6698 Sentence denotes IL-1β levels in cell culture supernatants were not greatly increased in either the CF or control cell lines (Fig. 2a–d), although a small increase in IL-1β production was detected in NuLi-1 and CuFi-1 cells, but not in S9 and IB3-1 cells, by 24 hours.
T2610 6699-6867 Sentence denotes In contrast, these airway cells were highly responsive to other inflammatory stimuli, such as recombinant IL-1β, producing large quantities of IL-8 (Fig. 2a–d inserts).
T49 6699-6867 Sentence denotes In contrast, these airway cells were highly responsive to other inflammatory stimuli, such as recombinant IL-1β, producing large quantities of IL-8 (Fig. 2a–d inserts).
T50 6868-6952 Sentence denotes 10.1371/journal.pone.0037689.g001 Figure 1 Cell stimulation and inhibitor schedule.
T12680 6912-6952 Sentence denotes Cell stimulation and inhibitor schedule.
T12681 6953-7128 Sentence denotes Schedule outlines the timing of inhibitor addition and priming in relation to inflammasome stimulation (t = 0) for THP-1 reporter and PBMC cytokine quantification experiments.
T51 6953-7128 Sentence denotes Schedule outlines the timing of inhibitor addition and priming in relation to inflammasome stimulation (t = 0) for THP-1 reporter and PBMC cytokine quantification experiments.
T12682 7129-7234 Sentence denotes Inhibitor treatments and stimulations were carried out as described in the Materials and Methods section.
T52 7129-7234 Sentence denotes Inhibitor treatments and stimulations were carried out as described in the Materials and Methods section.
T53 7235-7391 Sentence denotes 10.1371/journal.pone.0037689.g002 Figure 2 Airway epithelial cells do not significantly contribute to IL-1β production in response to inflammasome stimuli.
T12809 7279-7391 Sentence denotes Airway epithelial cells do not significantly contribute to IL-1β production in response to inflammasome stimuli.
T12810 7392-7639 Sentence denotes Control cell lines ((A) S9, (C) NuLi-1) and their corresponding CF cell lines ((B) IB3-1, and (D) CuFi-1) cells were stimulated with P. aeruginosa (MOI = 10), ATP (5 mM), or IL-1β (10 ng/ml), for the indicated times (n = 3 individual experiments).
T54 7392-7639 Sentence denotes Control cell lines ((A) S9, (C) NuLi-1) and their corresponding CF cell lines ((B) IB3-1, and (D) CuFi-1) cells were stimulated with P. aeruginosa (MOI = 10), ATP (5 mM), or IL-1β (10 ng/ml), for the indicated times (n = 3 individual experiments).
T12811 7640-7709 Sentence denotes Cells were primed with LPS (100 ng/ml) for 4 hours where appropriate.
T55 7640-7709 Sentence denotes Cells were primed with LPS (100 ng/ml) for 4 hours where appropriate.
T12812 7710-7788 Sentence denotes Cell culture supernatants were assayed for IL-1β and IL-8 production by ELISA.
T56 7710-7788 Sentence denotes Cell culture supernatants were assayed for IL-1β and IL-8 production by ELISA.
T12813 7789-7866 Sentence denotes Insert shows IL-8 secretion in response to stimulation with IL-1β (10 ng/ml).
T57 7789-7866 Sentence denotes Insert shows IL-8 secretion in response to stimulation with IL-1β (10 ng/ml).
T58 7867-7934 Sentence denotes 10.1371/journal.pone.0037689.t001 Table 1 Inflammasome Activators.
T15013 7910-7934 Sentence denotes Inflammasome Activators.
T3090 7936-8050 Sentence denotes Airway epithelial cells do not significantly upregulate caspase-1 activity in response to inflammasome stimulation
T59 7936-8050 Sentence denotes Airway epithelial cells do not significantly upregulate caspase-1 activity in response to inflammasome stimulation
T3091 8051-8171 Sentence denotes To examine if inflammasome activation occurs in these airway cells, caspase-1 activity was quantified by flow cytometry.
T60 8051-8171 Sentence denotes To examine if inflammasome activation occurs in these airway cells, caspase-1 activity was quantified by flow cytometry.
T3092 8172-8283 Sentence denotes There was no significant increase upon stimulation with live PAO1 or LPS+ATP at the times examined (Fig. 3a–b).
T61 8172-8283 Sentence denotes There was no significant increase upon stimulation with live PAO1 or LPS+ATP at the times examined (Fig. 3a–b).
T3093 8284-8543 Sentence denotes Because previous studies have indicated a role for caspase-1 in the activation of NF-κB through Toll-like receptor (TLR) signaling [35], we examined whether chemical inhibition of caspase-1 altered NF-κB-dependent IL-6 production in response to P. aeruginosa.
T62 8284-8543 Sentence denotes Because previous studies have indicated a role for caspase-1 in the activation of NF-κB through Toll-like receptor (TLR) signaling [35], we examined whether chemical inhibition of caspase-1 altered NF-κB-dependent IL-6 production in response to P. aeruginosa.
T3094 8544-8679 Sentence denotes However, treatment with the caspase-1 inhibitor z-YVAD-fmk (YVAD) did not decrease IL-6 secretion by airway epithelial cells (Fig. 3c).
T63 8544-8679 Sentence denotes However, treatment with the caspase-1 inhibitor z-YVAD-fmk (YVAD) did not decrease IL-6 secretion by airway epithelial cells (Fig. 3c).
T64 8680-8819 Sentence denotes 10.1371/journal.pone.0037689.g003 Figure 3 Airway cells do not strongly upregulate caspase-1 activity in response to inflammasome stimuli.
T13116 8724-8819 Sentence denotes Airway cells do not strongly upregulate caspase-1 activity in response to inflammasome stimuli.
T13117 8820-8958 Sentence denotes S9 and IB3-1 cells were examined for caspase-1 activation following inflammasome stimulation with P. aeruginosa (MOI = 10) and ATP (5 mM).
T65 8820-8958 Sentence denotes S9 and IB3-1 cells were examined for caspase-1 activation following inflammasome stimulation with P. aeruginosa (MOI = 10) and ATP (5 mM).
T13118 8959-9016 Sentence denotes Cells were primed with LPS for 5 hours where appropriate.
T66 8959-9016 Sentence denotes Cells were primed with LPS for 5 hours where appropriate.
T13119 9017-9329 Sentence denotes A representative histogram of % caspase-1-active cells is shown in (A) and the averaged values are shown in (B) (n = 3 separate experiments). (C) IB3-1 cells (5×104 cells/well) were pre-treated for 1 hour with increasing concentrations of z-YVAD-fmk (10–30 µM) prior to stimulation with P. aeruginosa (MOI = 50).
T67 9017-9329 Sentence denotes A representative histogram of % caspase-1-active cells is shown in (A) and the averaged values are shown in (B) (n = 3 separate experiments). (C) IB3-1 cells (5×104 cells/well) were pre-treated for 1 hour with increasing concentrations of z-YVAD-fmk (10–30 µM) prior to stimulation with P. aeruginosa (MOI = 50).
T13120 9330-9423 Sentence denotes Cell culture supernatants were collected after 6 hours and assayed for IL-6 by ELISA (n = 3).
T68 9330-9423 Sentence denotes Cell culture supernatants were collected after 6 hours and assayed for IL-6 by ELISA (n = 3).
T3401 9425-9553 Sentence denotes CD14 positive monocytes from CF patients and controls show similar increases in caspase-1 activity upon inflammasome stimulation
T69 9425-9553 Sentence denotes CD14 positive monocytes from CF patients and controls show similar increases in caspase-1 activity upon inflammasome stimulation
T3402 9554-9635 Sentence denotes Monocytes were identified in PBMC populations using CD14 as a phenotyping marker.
T70 9554-9635 Sentence denotes Monocytes were identified in PBMC populations using CD14 as a phenotyping marker.
T3403 9636-9899 Sentence denotes CD14 positive monocytes from CF patients and healthy controls showed a significant increase in caspase-1 activation upon stimulation with LPS+ATP, PAO1, and LPS+Poly(dA:dT) (Fig. 4a) but this activation was not different between CF and control subjects (Fig. 4b).
T71 9636-9899 Sentence denotes CD14 positive monocytes from CF patients and healthy controls showed a significant increase in caspase-1 activation upon stimulation with LPS+ATP, PAO1, and LPS+Poly(dA:dT) (Fig. 4a) but this activation was not different between CF and control subjects (Fig. 4b).
T72 9900-10054 Sentence denotes 10.1371/journal.pone.0037689.g004 Figure 4 PBMCs from CF patients and controls show similar increases in caspase-1 activity upon inflammasome activation.
T13406 9944-10054 Sentence denotes PBMCs from CF patients and controls show similar increases in caspase-1 activity upon inflammasome activation.
T13407 10055-10244 Sentence denotes PBMCs from CF patients (n = 6) and healthy controls (n = 6) were primed with LPS (10 ng/ml) for 5 hours prior to stimulation with ATP (5 mM) for 1 hour or Poly(dA:dT) (1 µg/ml) for 3 hours.
T73 10055-10244 Sentence denotes PBMCs from CF patients (n = 6) and healthy controls (n = 6) were primed with LPS (10 ng/ml) for 5 hours prior to stimulation with ATP (5 mM) for 1 hour or Poly(dA:dT) (1 µg/ml) for 3 hours.
T13408 10245-10310 Sentence denotes PBMCs were stimulated with P. aeruginosa strain PAO1 for 3 hours.
T74 10245-10310 Sentence denotes PBMCs were stimulated with P. aeruginosa strain PAO1 for 3 hours.
T13409 10311-10424 Sentence denotes A representative histogram of the % caspase-1-active cells is shown in (A) with the averaged values shown in (B).
T75 10311-10424 Sentence denotes A representative histogram of the % caspase-1-active cells is shown in (A) with the averaged values shown in (B).
T3629 10426-10520 Sentence denotes PBMCs from CF patients do not produce increased amounts of IL-1β upon inflammasome stimulation
T76 10426-10520 Sentence denotes PBMCs from CF patients do not produce increased amounts of IL-1β upon inflammasome stimulation
T3630 10521-10674 Sentence denotes Previous studies have shown that the loss of CFTR results in increased NF-κB activity and pro-inflammatory cytokine secretion [4], [5], [32], [36], [37].
T77 10521-10674 Sentence denotes Previous studies have shown that the loss of CFTR results in increased NF-κB activity and pro-inflammatory cytokine secretion [4], [5], [32], [36], [37].
T3631 10675-10887 Sentence denotes To further examine this relationship, PBMCs from CF patients and healthy adult controls were stimulated with PAO1, LPS+ATP, and LPS+Poly(dA:dT), to activate the NLRC4, NLRP3, and AIM2 inflammasomes, respectively.
T78 10675-10887 Sentence denotes To further examine this relationship, PBMCs from CF patients and healthy adult controls were stimulated with PAO1, LPS+ATP, and LPS+Poly(dA:dT), to activate the NLRC4, NLRP3, and AIM2 inflammasomes, respectively.
T3632 10888-11030 Sentence denotes By 24 hours of stimulation, CF PBMCs did not produce increased amounts of IL-1β (Fig. 5a) or IL-8 (Fig. 5b) when compared to healthy controls.
T79 10888-11030 Sentence denotes By 24 hours of stimulation, CF PBMCs did not produce increased amounts of IL-1β (Fig. 5a) or IL-8 (Fig. 5b) when compared to healthy controls.
T3633 11031-11180 Sentence denotes However, we did notice a transient decrease (P<0.001) in the amount of IL-1β produced by CF cells in response to LPS+ATP at 6 hours (data not shown).
T80 11031-11180 Sentence denotes However, we did notice a transient decrease (P<0.001) in the amount of IL-1β produced by CF cells in response to LPS+ATP at 6 hours (data not shown).
T3634 11181-11379 Sentence denotes Stimulation of PBMCs with P. aeruginosa that lacks exsA (PAO1ΔexsA), a key regulator of type III secretion, produced three-fold less IL-1β compared to the parental PAO1 strain by 24 hours (Fig. 5a).
T81 11181-11379 Sentence denotes Stimulation of PBMCs with P. aeruginosa that lacks exsA (PAO1ΔexsA), a key regulator of type III secretion, produced three-fold less IL-1β compared to the parental PAO1 strain by 24 hours (Fig. 5a).
T3635 11380-11490 Sentence denotes Inflammasome stimulation without priming did not result in any IL-1β production in either CF or control PBMCs.
T82 11380-11490 Sentence denotes Inflammasome stimulation without priming did not result in any IL-1β production in either CF or control PBMCs.
T3636 11491-11718 Sentence denotes Contrary to our hypothesis, these results indicate that PBMCs from CF patients do not display increased production of IL-1β or IL-8 with inflammasome activation nor do they suggest any increased basal or induced NF-κB activity.
T83 11491-11718 Sentence denotes Contrary to our hypothesis, these results indicate that PBMCs from CF patients do not display increased production of IL-1β or IL-8 with inflammasome activation nor do they suggest any increased basal or induced NF-κB activity.
T3637 11719-11848 Sentence denotes These results are consistent with our observation that caspase-1 activity is not different between CF and control PBMCs (Fig. 4).
T84 11719-11848 Sentence denotes These results are consistent with our observation that caspase-1 activity is not different between CF and control PBMCs (Fig. 4).
T85 11849-11958 Sentence denotes 10.1371/journal.pone.0037689.g005 Figure 5 PBMCs from CF patients do not produce increased amounts of IL-1β.
T13641 11893-11958 Sentence denotes PBMCs from CF patients do not produce increased amounts of IL-1β.
T13642 11959-12212 Sentence denotes PBMCs from CF patients (n = 17–20) and healthy controls (n = 15–19) were primed with LPS (10 ng/ml) overnight and stimulated with P. aeruginosa PAO1 (MOI = 1), P. aeruginosa PAO1 lacking exsA (MOI = 1), ATP (5 mM), or Poly(dA:dT) (1 µg/ml) for 24 hours.
T86 11959-12212 Sentence denotes PBMCs from CF patients (n = 17–20) and healthy controls (n = 15–19) were primed with LPS (10 ng/ml) overnight and stimulated with P. aeruginosa PAO1 (MOI = 1), P. aeruginosa PAO1 lacking exsA (MOI = 1), ATP (5 mM), or Poly(dA:dT) (1 µg/ml) for 24 hours.
T13643 12213-12324 Sentence denotes P. aeruginosa lacking exsA was used as a type III secretion control in comparison with wild-type P. aeruginosa.
T87 12213-12324 Sentence denotes P. aeruginosa lacking exsA was used as a type III secretion control in comparison with wild-type P. aeruginosa.
T13644 12325-12378 Sentence denotes Supernatants were assayed for (A) IL-1β and (B) IL-8.
T88 12325-12378 Sentence denotes Supernatants were assayed for (A) IL-1β and (B) IL-8.
T4383 12380-12454 Sentence denotes NF-κB activation is required for IL-1β and IL-8 responses to P. aeruginosa
T89 12380-12454 Sentence denotes NF-κB activation is required for IL-1β and IL-8 responses to P. aeruginosa
T4384 12456-12629 Sentence denotes We confirmed the dependence of PAO1-induced IL-1β and IL-8 production on NF-κB activation using THP-1 cells expressing a reporter driven by NF-κB and AP-1 response elements.
T90 12456-12629 Sentence denotes We confirmed the dependence of PAO1-induced IL-1β and IL-8 production on NF-κB activation using THP-1 cells expressing a reporter driven by NF-κB and AP-1 response elements.
T4385 12630-12878 Sentence denotes We found that stimulation of primed THP-1 reporter cells with heat-killed PAO1 produced the highest levels of NF-κB/AP-1 activation (Fig. 6a) and this correlated with IL-8 secretion (Fig. 6b) but negligible amounts of IL-1β were secreted (Fig. 6c).
T91 12630-12878 Sentence denotes We found that stimulation of primed THP-1 reporter cells with heat-killed PAO1 produced the highest levels of NF-κB/AP-1 activation (Fig. 6a) and this correlated with IL-8 secretion (Fig. 6b) but negligible amounts of IL-1β were secreted (Fig. 6c).
T4386 12879-13045 Sentence denotes Stimulation of primed THP-1 reporter cells with live PAO1 did not significantly increase NF-κB/AP-1 activity (Fig. 6a) or IL-8 secretion (Fig. 6b) over priming alone.
T92 12879-13045 Sentence denotes Stimulation of primed THP-1 reporter cells with live PAO1 did not significantly increase NF-κB/AP-1 activity (Fig. 6a) or IL-8 secretion (Fig. 6b) over priming alone.
T4387 13046-13199 Sentence denotes However, IL-1β production was greatly augmented over primed cells stimulated with heat-killed PAO1 or unprimed cells stimulated with live PAO1 (Fig. 6c).
T93 13046-13199 Sentence denotes However, IL-1β production was greatly augmented over primed cells stimulated with heat-killed PAO1 or unprimed cells stimulated with live PAO1 (Fig. 6c).
T4388 13200-13407 Sentence denotes This confirmed that NF-κB activation alone is not sufficient for maximal IL-1β secretion, but increased priming of NF-κB is capable of augmenting IL-1β production and secretion upon inflammasome stimulation.
T94 13200-13407 Sentence denotes This confirmed that NF-κB activation alone is not sufficient for maximal IL-1β secretion, but increased priming of NF-κB is capable of augmenting IL-1β production and secretion upon inflammasome stimulation.
T4389 13408-13757 Sentence denotes Dependency of these responses on NF-κB was confirmed by pharmacologic inhibition of NF-κB using the Bay11-7082 inhibitor of IκBα phosphorylation, which significantly reduced NF-κB/AP-1 activation (P<0.001) (Fig. 6d) and the subsequent production of IL-8 (P<0.01) (Fig. 6e) and IL-1β (P<0.001) (Fig. 6f) in response to both heat-killed and live PAO1.
T95 13408-13757 Sentence denotes Dependency of these responses on NF-κB was confirmed by pharmacologic inhibition of NF-κB using the Bay11-7082 inhibitor of IκBα phosphorylation, which significantly reduced NF-κB/AP-1 activation (P<0.001) (Fig. 6d) and the subsequent production of IL-8 (P<0.01) (Fig. 6e) and IL-1β (P<0.001) (Fig. 6f) in response to both heat-killed and live PAO1.
T4390 13758-13868 Sentence denotes These results were also verified in CF and control PBMCs for each inflammasome examined (P<0.001) (Fig. 6g–h).
T96 13758-13868 Sentence denotes These results were also verified in CF and control PBMCs for each inflammasome examined (P<0.001) (Fig. 6g–h).
T4391 13869-14046 Sentence denotes Overall these results confirm that NF-κB is an important modulator of IL-1β production and that increased activation of NF-κB augments inflammasome-mediated production of IL-1β.
T97 13869-14046 Sentence denotes Overall these results confirm that NF-κB is an important modulator of IL-1β production and that increased activation of NF-κB augments inflammasome-mediated production of IL-1β.
T98 14047-14194 Sentence denotes 10.1371/journal.pone.0037689.g006 Figure 6 NF-κB activation potentiates the degree of IL-1β production and secretion upon inflammasome activation.
T13935 14091-14194 Sentence denotes NF-κB activation potentiates the degree of IL-1β production and secretion upon inflammasome activation.
T13936 14195-14385 Sentence denotes THP-1 reporter cells were primed overnight with heat-killed P. aeruginosa and stimulated the next day with live P. aeruginosa or additional heat-killed P. aeruginosa for the times indicated.
T99 14195-14385 Sentence denotes THP-1 reporter cells were primed overnight with heat-killed P. aeruginosa and stimulated the next day with live P. aeruginosa or additional heat-killed P. aeruginosa for the times indicated.
T13937 14386-14510 Sentence denotes Cell culture supernatants were assayed for (A) NF-κB/AP-1 activity, (B) IL-8, and (C) IL-1β secretion (n = 3–6 experiments).
T100 14386-14510 Sentence denotes Cell culture supernatants were assayed for (A) NF-κB/AP-1 activity, (B) IL-8, and (C) IL-1β secretion (n = 3–6 experiments).
T13938 14511-14667 Sentence denotes Using the same stimulation method, THP-1 reporter cells were treated with Bay11-7082 (20 µM) for 1 hour prior to priming with heat-killed PAO1 or live PAO1.
T101 14511-14667 Sentence denotes Using the same stimulation method, THP-1 reporter cells were treated with Bay11-7082 (20 µM) for 1 hour prior to priming with heat-killed PAO1 or live PAO1.
T13939 14668-14779 Sentence denotes Supernatants were assayed at 24 hours for (D) NF-κB/AP-1 activity, (E) IL-8, and (F) IL-1β secretion (n = 3–5).
T102 14668-14779 Sentence denotes Supernatants were assayed at 24 hours for (D) NF-κB/AP-1 activity, (E) IL-8, and (F) IL-1β secretion (n = 3–5).
T13940 14780-15072 Sentence denotes PBMCs from CF patients (n = 11–15) and controls (n = 10–13) were treated with z-YVAD-fmk (20 µM) or Bay11-7082 (10 µM) and stimulated with live PAO1 (MOI = 1), ATP (5 mM), or Poly(dA:dT) (1 µg/ml) according to the schedule in Figure 1. (G) IL-1β and (H) IL-8 levels were measured at 24 hours.
T103 14780-15072 Sentence denotes PBMCs from CF patients (n = 11–15) and controls (n = 10–13) were treated with z-YVAD-fmk (20 µM) or Bay11-7082 (10 µM) and stimulated with live PAO1 (MOI = 1), ATP (5 mM), or Poly(dA:dT) (1 µg/ml) according to the schedule in Figure 1. (G) IL-1β and (H) IL-8 levels were measured at 24 hours.
T13941 15073-15228 Sentence denotes Statistical analysis was performed using two way ANOVA with Bonferroni correction for multiple comparisons. *, **, and *** signify P<0.05, 0.01, and 0.001.
T104 15073-15228 Sentence denotes Statistical analysis was performed using two way ANOVA with Bonferroni correction for multiple comparisons. *, **, and *** signify P<0.05, 0.01, and 0.001.
T5207 15230-15299 Sentence denotes Bay11-7082 inhibits pro-IL-1β production in response to P. aeruginosa
T105 15230-15299 Sentence denotes Bay11-7082 inhibits pro-IL-1β production in response to P. aeruginosa
T5208 15301-15411 Sentence denotes In addition to inhibition of NF-κB activity, Bay11-7082 can also directly inhibit the NLRP3 inflammasome [38].
T106 15301-15411 Sentence denotes In addition to inhibition of NF-κB activity, Bay11-7082 can also directly inhibit the NLRP3 inflammasome [38].
T5209 15412-15623 Sentence denotes To validate its use in this study as an NF-κB inhibitor, western blots for pro-IL-1β were performed alongside inhibition of CFTR activity by CFTRinh172 in response to PAO1 at 4 hours after stimulation (Fig. 7a).
T107 15412-15623 Sentence denotes To validate its use in this study as an NF-κB inhibitor, western blots for pro-IL-1β were performed alongside inhibition of CFTR activity by CFTRinh172 in response to PAO1 at 4 hours after stimulation (Fig. 7a).
T5210 15624-15740 Sentence denotes Our results indicate that Bay11-7082 prevents production of pro-IL-1β whereas CFTRinh172 does not seem to affect it.
T108 15624-15740 Sentence denotes Our results indicate that Bay11-7082 prevents production of pro-IL-1β whereas CFTRinh172 does not seem to affect it.
T5211 15741-15882 Sentence denotes This was further corroborated by the ability Bay11-7082 to inhibit IκBα degradation at 0.5, 1, and 1.5 hours post PAO1 stimulation (Fig. 7b).
T109 15741-15882 Sentence denotes This was further corroborated by the ability Bay11-7082 to inhibit IκBα degradation at 0.5, 1, and 1.5 hours post PAO1 stimulation (Fig. 7b).
T110 15883-15997 Sentence denotes 10.1371/journal.pone.0037689.g007 Figure 7 Bay11-7082 inhibits pro-IL-1β production in response to P. aeruginosa.
T14502 15927-15997 Sentence denotes Bay11-7082 inhibits pro-IL-1β production in response to P. aeruginosa.
T14503 15998-16183 Sentence denotes PMA-differentiated THP-1 cells were treated with 10 µM CFTRinh172 or 20 µM Bay11-7082 and harvested after (A) 4 hours (n = 3) or (B) 0.5, 1, and 1.5 hours (n = 3) stimulation with PAO1.
T111 15998-16183 Sentence denotes PMA-differentiated THP-1 cells were treated with 10 µM CFTRinh172 or 20 µM Bay11-7082 and harvested after (A) 4 hours (n = 3) or (B) 0.5, 1, and 1.5 hours (n = 3) stimulation with PAO1.
T14504 16184-16295 Sentence denotes One representative blot is shown with a graph of the averaged fluorescence intensity values over 3 experiments.
T112 16184-16295 Sentence denotes One representative blot is shown with a graph of the averaged fluorescence intensity values over 3 experiments.
T5497 16297-16384 Sentence denotes Disruption of CFTR activity does not increase IL-1β production in PBMCs and THP-1 cells
T113 16297-16384 Sentence denotes Disruption of CFTR activity does not increase IL-1β production in PBMCs and THP-1 cells
T5498 16385-16507 Sentence denotes A previous study has indicated a role for chloride ion concentration in suppression of NLRP3 inflammasome activation [34].
T114 16385-16507 Sentence denotes A previous study has indicated a role for chloride ion concentration in suppression of NLRP3 inflammasome activation [34].
T5499 16508-16725 Sentence denotes To determine whether CFTR dysfunction alters IL-1β production, THP-1 cells and PBMCs from CF patients and healthy controls were treated with the CFTR inhibitor, CFTRinh172, prior to simulation with live P. aeruginosa.
T115 16508-16725 Sentence denotes To determine whether CFTR dysfunction alters IL-1β production, THP-1 cells and PBMCs from CF patients and healthy controls were treated with the CFTR inhibitor, CFTRinh172, prior to simulation with live P. aeruginosa.
T5500 16726-16838 Sentence denotes Treatment with CFTRinh172 did not alter IL-1β or IL-8 production in control subjects or CF patients (Fig. 8a–b).
T116 16726-16838 Sentence denotes Treatment with CFTRinh172 did not alter IL-1β or IL-8 production in control subjects or CF patients (Fig. 8a–b).
T5501 16839-16977 Sentence denotes Similarly, IL-1β production was not different in monocyte-derived macrophages or THP-1 reporter cells treated with CFTRinh172 (Fig. 8c–d).
T117 16839-16977 Sentence denotes Similarly, IL-1β production was not different in monocyte-derived macrophages or THP-1 reporter cells treated with CFTRinh172 (Fig. 8c–d).
T5502 16978-17085 Sentence denotes IL-8 (Fig. 8e) and NF-κB activity (Fig. 8f) were also unchanged in CFTRinh172-treated THP-1 reporter cells.
T118 16978-17085 Sentence denotes IL-8 (Fig. 8e) and NF-κB activity (Fig. 8f) were also unchanged in CFTRinh172-treated THP-1 reporter cells.
T119 17086-17217 Sentence denotes 10.1371/journal.pone.0037689.g008 Figure 8 Disruption of CFTR activity does not increase IL-1β production in PBMCs or macrophages.
T14670 17130-17217 Sentence denotes Disruption of CFTR activity does not increase IL-1β production in PBMCs or macrophages.
T14671 17218-17502 Sentence denotes PBMCs from CF patients (n = 15) and controls (n = 13) were treated with CFTRinh172 (10 µM) for 18 hours prior to stimulation with live PAO1 (MOI = 1). (A) IL-1β and (B) IL-8 production was measured at 24 hours. (C) Monocytes from controls (n = 3) were differentiated into macrophages.
T120 17218-17502 Sentence denotes PBMCs from CF patients (n = 15) and controls (n = 13) were treated with CFTRinh172 (10 µM) for 18 hours prior to stimulation with live PAO1 (MOI = 1). (A) IL-1β and (B) IL-8 production was measured at 24 hours. (C) Monocytes from controls (n = 3) were differentiated into macrophages.
T14672 17503-17620 Sentence denotes Macrophages were treated with CFTRinh172, stimulated as per monocytes, and measured for IL-1β production at 24 hours.
T121 17503-17620 Sentence denotes Macrophages were treated with CFTRinh172, stimulated as per monocytes, and measured for IL-1β production at 24 hours.
T14673 17621-17807 Sentence denotes THP-1 reporter cells were treated with CFTRinh172 24 hours prior to stimulation with PAO1 and measured for (D) IL-1β secretion, (E) IL-8, and (F) NF-κB/AP-1 activity at 24 hours (n = 4).
T122 17621-17807 Sentence denotes THP-1 reporter cells were treated with CFTRinh172 24 hours prior to stimulation with PAO1 and measured for (D) IL-1β secretion, (E) IL-8, and (F) NF-κB/AP-1 activity at 24 hours (n = 4).
T123 17809-17819 Sentence denotes Discussion
T6037 17820-18006 Sentence denotes Levels of IL-1β are increased in the BALF of CF patients but the cellular source of this cytokine and its production in the context of targeted inflammasome activation are still unclear.
T124 17820-18006 Sentence denotes Levels of IL-1β are increased in the BALF of CF patients but the cellular source of this cytokine and its production in the context of targeted inflammasome activation are still unclear.
T6038 18007-18176 Sentence denotes We first studied airway epithelial cells due to their role in barrier function, proximity to infection, and ability to produce high levels of pro-inflammatory cytokines.
T125 18007-18176 Sentence denotes We first studied airway epithelial cells due to their role in barrier function, proximity to infection, and ability to produce high levels of pro-inflammatory cytokines.
T6039 18177-18416 Sentence denotes However, we found that bronchial epithelial cells do not produce significant amounts of IL-1β and do not show a significant increase in caspase-1 activation in response to PAO1 and LPS+ATP, in comparison to hematopoeitic mononuclear cells.
T126 18177-18416 Sentence denotes However, we found that bronchial epithelial cells do not produce significant amounts of IL-1β and do not show a significant increase in caspase-1 activation in response to PAO1 and LPS+ATP, in comparison to hematopoeitic mononuclear cells.
T6040 18417-18526 Sentence denotes Hematopoeitically-derived cells, such as monocytes and macrophages, appear to be a principal source of IL-1β.
T127 18417-18526 Sentence denotes Hematopoeitically-derived cells, such as monocytes and macrophages, appear to be a principal source of IL-1β.
T6041 18527-18726 Sentence denotes CFTR is expressed in alveolar macrophages [32], [39] as well as in PBMCs at both the mRNA and protein level [40], [41], and its loss is frequently associated with an augmented inflammatory phenotype.
T128 18527-18726 Sentence denotes CFTR is expressed in alveolar macrophages [32], [39] as well as in PBMCs at both the mRNA and protein level [40], [41], and its loss is frequently associated with an augmented inflammatory phenotype.
T6042 18727-19110 Sentence denotes Despite our findings indicating that bronchial epithelial cells when grown in vitro are unlikely to be significantly involved in the direct production of IL-1β (Fig. 2a–d), others have shown their capacity to respond to alveolar macrophage-derived IL-1β and to amplify the inflammatory response through the induction of chemokines and recruitment of inflammatory effector cells [42].
T129 18727-19110 Sentence denotes Despite our findings indicating that bronchial epithelial cells when grown in vitro are unlikely to be significantly involved in the direct production of IL-1β (Fig. 2a–d), others have shown their capacity to respond to alveolar macrophage-derived IL-1β and to amplify the inflammatory response through the induction of chemokines and recruitment of inflammatory effector cells [42].
T6043 19111-19338 Sentence denotes This interaction may constitute a critical component to effective host defense and diminishing the capacity of host cells to respond to IL-1β may leave the host susceptible to infections by pathogens such as P. aeruginosa [43].
T130 19111-19338 Sentence denotes This interaction may constitute a critical component to effective host defense and diminishing the capacity of host cells to respond to IL-1β may leave the host susceptible to infections by pathogens such as P. aeruginosa [43].
T6044 19339-19485 Sentence denotes Conversely, overproduction of IL-1β can also play a key role in chronic inflammatory responses and cause damage to the lung parenchyma [44], [45].
T131 19339-19485 Sentence denotes Conversely, overproduction of IL-1β can also play a key role in chronic inflammatory responses and cause damage to the lung parenchyma [44], [45].
T6045 19486-19694 Sentence denotes Although we hypothesized that CF cells would secrete increased amounts of IL-1β, we found that IL-1β production in CF PBMCs was not increased upon inflammasome stimulation as compared to controls (Fig. 5a–b).
T132 19486-19694 Sentence denotes Although we hypothesized that CF cells would secrete increased amounts of IL-1β, we found that IL-1β production in CF PBMCs was not increased upon inflammasome stimulation as compared to controls (Fig. 5a–b).
T6046 19695-19924 Sentence denotes This was in contrast to a previous study from our group, which showed increased IL-1β production by CF PBMCs in response to LPS [46], although this difference may be accounted for by technical issues in stimulation time and dose.
T133 19695-19924 Sentence denotes This was in contrast to a previous study from our group, which showed increased IL-1β production by CF PBMCs in response to LPS [46], although this difference may be accounted for by technical issues in stimulation time and dose.
T6047 19925-20089 Sentence denotes Moreover, IL-1β production was not increased in CF PBMCs with inflammasome stimulation alone as would be anticipated if there were basal levels of NF-κB activation.
T134 19925-20089 Sentence denotes Moreover, IL-1β production was not increased in CF PBMCs with inflammasome stimulation alone as would be anticipated if there were basal levels of NF-κB activation.
T6048 20090-20319 Sentence denotes Cells deficient in CFTR are thought to exhibit an increased basal level of NF-κB activity, which leads to increased pro-inflammatory cytokine production including an increased availability of pro-IL-1β for cleavage and secretion.
T135 20090-20319 Sentence denotes Cells deficient in CFTR are thought to exhibit an increased basal level of NF-κB activity, which leads to increased pro-inflammatory cytokine production including an increased availability of pro-IL-1β for cleavage and secretion.
T6049 20320-20488 Sentence denotes This amplification of IL-1β secretion was shown by priming THP-1 monocytes and PBMCs with heat-killed P. aeruginosa or LPS prior to stimulation with live P. aeruginosa.
T136 20320-20488 Sentence denotes This amplification of IL-1β secretion was shown by priming THP-1 monocytes and PBMCs with heat-killed P. aeruginosa or LPS prior to stimulation with live P. aeruginosa.
T6050 20489-20600 Sentence denotes This dramatically increased IL-1β secretion over stimulation with live P. aeruginosa without priming (Fig. 6c).
T137 20489-20600 Sentence denotes This dramatically increased IL-1β secretion over stimulation with live P. aeruginosa without priming (Fig. 6c).
T6051 20601-20742 Sentence denotes Similarly, if CF PBMCs expressed increased basal NF-κB activity, there would be an increase in IL-1β secretion in the absence of LPS priming.
T138 20601-20742 Sentence denotes Similarly, if CF PBMCs expressed increased basal NF-κB activity, there would be an increase in IL-1β secretion in the absence of LPS priming.
T6052 20743-20819 Sentence denotes However, no increase in IL-1β was observed under basal or primed conditions.
T139 20743-20819 Sentence denotes However, no increase in IL-1β was observed under basal or primed conditions.
T6053 20820-20898 Sentence denotes Studies investigating the production of IL-1β have been somewhat inconsistent.
T140 20820-20898 Sentence denotes Studies investigating the production of IL-1β have been somewhat inconsistent.
T6054 20899-21160 Sentence denotes A study by Reininger et al. [43] provided evidence that human bronchial epithelial cells possessing the ΔF508 CFTR mutation had a slightly reduced capacity to produce IL-1β and lacked the ability to induce an early NF-κB activation in response to P. aeruginosa.
T141 20899-21160 Sentence denotes A study by Reininger et al. [43] provided evidence that human bronchial epithelial cells possessing the ΔF508 CFTR mutation had a slightly reduced capacity to produce IL-1β and lacked the ability to induce an early NF-κB activation in response to P. aeruginosa.
T6055 21161-21411 Sentence denotes Conversely, a study by Kotrange et al. [31] found that murine bone marrow-derived macrophages expressing ΔF508-CFTR produced increased amounts of IL-1β when compared to macrophages expressing normal CFTR in response to Burkholderia cenocepacia K56-2.
T142 21161-21411 Sentence denotes Conversely, a study by Kotrange et al. [31] found that murine bone marrow-derived macrophages expressing ΔF508-CFTR produced increased amounts of IL-1β when compared to macrophages expressing normal CFTR in response to Burkholderia cenocepacia K56-2.
T143 21412-21537 Sentence denotes The differentiation of monocytes into macrophages may partly account for the differences observed with the study by Kotrange.
T6056 21412-21726 Sentence denotes The differentiation of monocytes into macrophages may partly account for the differences observed with the study by Kotrange. Inflammasome-mediated IL-1β production by monocytes and PBMCs does differ from macrophages [47], [48], [49] and macrophages are found to have higher expression of CFTR over monocytes [50].
T144 21538-21726 Sentence denotes Inflammasome-mediated IL-1β production by monocytes and PBMCs does differ from macrophages [47], [48], [49] and macrophages are found to have higher expression of CFTR over monocytes [50].
T6057 21727-21910 Sentence denotes However, as monocytes and other PBMCs express CFTR [50], [51] and produce large amounts of IL-1β, they are adequate models to examine the effects of CFTR function on IL-1β production.
T145 21727-21910 Sentence denotes However, as monocytes and other PBMCs express CFTR [50], [51] and produce large amounts of IL-1β, they are adequate models to examine the effects of CFTR function on IL-1β production.
T6058 21911-21977 Sentence denotes Other hematopoeitic cells may also contribute to IL-1β production.
T146 21911-21977 Sentence denotes Other hematopoeitic cells may also contribute to IL-1β production.
T6059 21978-22160 Sentence denotes For example, neutrophil counts can be significantly increased in the lungs of CF patients [52], [53], [54] and may produce mature IL-1β through caspase-1 independent mechanisms [55].
T147 21978-22160 Sentence denotes For example, neutrophil counts can be significantly increased in the lungs of CF patients [52], [53], [54] and may produce mature IL-1β through caspase-1 independent mechanisms [55].
T6060 22161-22260 Sentence denotes The role of NF-κB activation in inflammasome activation and IL-1β secretion is not straightforward.
T148 22161-22260 Sentence denotes The role of NF-κB activation in inflammasome activation and IL-1β secretion is not straightforward.
T6061 22261-22406 Sentence denotes Studies have revealed an essential role for NF-κB activation in the production of pro-IL-1β and inflammasome components such as NLRP3 [22], [23].
T149 22261-22406 Sentence denotes Studies have revealed an essential role for NF-κB activation in the production of pro-IL-1β and inflammasome components such as NLRP3 [22], [23].
T6062 22407-22599 Sentence denotes In contrast, deletion of IKKβ, a kinase essential in NF-κB activation, increases IL-1β secretion in murine macrophages [56], [57] and demonstrates a dual role for NF-κB in regulation of IL-1β.
T150 22407-22599 Sentence denotes In contrast, deletion of IKKβ, a kinase essential in NF-κB activation, increases IL-1β secretion in murine macrophages [56], [57] and demonstrates a dual role for NF-κB in regulation of IL-1β.
T6063 22600-22791 Sentence denotes To address this uncertainty in our experiments we also quantified IL-8, an important CF cytokine and marker of NF-κB activation [58], and found no differences between CF and control subjects.
T151 22600-22791 Sentence denotes To address this uncertainty in our experiments we also quantified IL-8, an important CF cytokine and marker of NF-κB activation [58], and found no differences between CF and control subjects.
T6064 22792-22932 Sentence denotes Similarly, levels of intracellular pro-IL-1β in THP-1 cells were dependent on NF-κB activity and did not increase with CFTRinh172 treatment.
T152 22792-22932 Sentence denotes Similarly, levels of intracellular pro-IL-1β in THP-1 cells were dependent on NF-κB activity and did not increase with CFTRinh172 treatment.
T6065 22933-23081 Sentence denotes Subsequent treatment of THP-1 cells and PBMCs with the NF-κB inhibitor Bay11-7082 significantly inhibited both IL-1β and IL-8 secretion (Fig. 6d–h).
T153 22933-23081 Sentence denotes Subsequent treatment of THP-1 cells and PBMCs with the NF-κB inhibitor Bay11-7082 significantly inhibited both IL-1β and IL-8 secretion (Fig. 6d–h).
T6066 23082-23173 Sentence denotes Therefore, the IL-1β and IL-8 responses observed were both dependent upon NF-κB activation.
T154 23082-23173 Sentence denotes Therefore, the IL-1β and IL-8 responses observed were both dependent upon NF-κB activation.
T6067 23174-23454 Sentence denotes Priming with heat-killed P. aeruginosa, like LPS, is unable to induce a strong IL-1β response as compared to live P. aeruginosa (Fig. 6c), but generated greater NF-κB/AP-1 activation (Fig. 6a) and IL-8 secretion (Fig. 6b) than live bacteria despite stimulation at equivalent MOIs.
T155 23174-23454 Sentence denotes Priming with heat-killed P. aeruginosa, like LPS, is unable to induce a strong IL-1β response as compared to live P. aeruginosa (Fig. 6c), but generated greater NF-κB/AP-1 activation (Fig. 6a) and IL-8 secretion (Fig. 6b) than live bacteria despite stimulation at equivalent MOIs.
T6068 23455-23594 Sentence denotes This may be indicative of the different degree and quality of the inflammatory response generated by live as opposed to dead bacteria [59].
T156 23455-23594 Sentence denotes This may be indicative of the different degree and quality of the inflammatory response generated by live as opposed to dead bacteria [59].
T6069 23595-23784 Sentence denotes Potential shortcomings of these experiments include its translatability to lung disease and issues related to the hypermutability of P. aeruginosa during the evolution of chronic infection.
T157 23595-23784 Sentence denotes Potential shortcomings of these experiments include its translatability to lung disease and issues related to the hypermutability of P. aeruginosa during the evolution of chronic infection.
T6070 23785-24274 Sentence denotes Although the responses measured in peripheral blood cells may not completely reflect the responses occurring in the CF lung, PBMCs have a number of useful advantages: (i) PBMCs are not subject to alterations that may emerge from long-term cell culture, cloning and immortalization, and (ii) PBMCs express a large repertoire of innate immune receptors and secrete a broad array of cytokines and chemokines allowing comprehensive analysis of the modulation of inflammatory responses by CFTR.
T158 23785-24274 Sentence denotes Although the responses measured in peripheral blood cells may not completely reflect the responses occurring in the CF lung, PBMCs have a number of useful advantages: (i) PBMCs are not subject to alterations that may emerge from long-term cell culture, cloning and immortalization, and (ii) PBMCs express a large repertoire of innate immune receptors and secrete a broad array of cytokines and chemokines allowing comprehensive analysis of the modulation of inflammatory responses by CFTR.
T6071 24275-24412 Sentence denotes Consideration must also be given to the nature of P. aeruginosa infection and genotypic changes in P. aeruginosa as infection progresses.
T159 24275-24412 Sentence denotes Consideration must also be given to the nature of P. aeruginosa infection and genotypic changes in P. aeruginosa as infection progresses.
T6072 24413-24543 Sentence denotes P. aeruginosa mediates inflammasome activation through its type III secretion system (T3SS) and the NLRC4 inflammasome [19], [20].
T160 24413-24543 Sentence denotes P. aeruginosa mediates inflammasome activation through its type III secretion system (T3SS) and the NLRC4 inflammasome [19], [20].
T6073 24544-24678 Sentence denotes However, clones of P. aeruginosa established during chronic infection may accumulate mutations in virulence factors such as exsA [60].
T161 24544-24678 Sentence denotes However, clones of P. aeruginosa established during chronic infection may accumulate mutations in virulence factors such as exsA [60].
T6074 24679-24961 Sentence denotes By employing a deletion mutant in exsA, the key regulator in T3SS transcription, we confirmed that the T3SS is important for IL-1β secretion (Fig. 5a–b), and that depending on the adaptation in type III secretion, the host IL-1β response may be up or downregulated [19], [60], [61].
T162 24679-24961 Sentence denotes By employing a deletion mutant in exsA, the key regulator in T3SS transcription, we confirmed that the T3SS is important for IL-1β secretion (Fig. 5a–b), and that depending on the adaptation in type III secretion, the host IL-1β response may be up or downregulated [19], [60], [61].
T6075 24962-25179 Sentence denotes In conclusion, our data are consistent with a role for hematopoietic cells, not airway epithelial cells, as the major source of inflammasome-mediated IL-1β production in the lungs in response to ATP and P. aeruginosa.
T163 24962-25179 Sentence denotes In conclusion, our data are consistent with a role for hematopoietic cells, not airway epithelial cells, as the major source of inflammasome-mediated IL-1β production in the lungs in response to ATP and P. aeruginosa.
T6076 25180-25318 Sentence denotes Furthermore, we find little evidence to support an increased IL-1β inflammatory response to NF-κB/Inflammasome stimulation in CF patients.
T164 25180-25318 Sentence denotes Furthermore, we find little evidence to support an increased IL-1β inflammatory response to NF-κB/Inflammasome stimulation in CF patients.
T6077 25319-25533 Sentence denotes Further studies are warranted to determine if adaptations of P. aeruginosa during the course of chronic lung infection alters inflammasome activation, and whether this can be correlated with disease severity in CF.
T165 25319-25533 Sentence denotes Further studies are warranted to determine if adaptations of P. aeruginosa during the course of chronic lung infection alters inflammasome activation, and whether this can be correlated with disease severity in CF.
T166 25535-25556 Sentence denotes Materials and Methods
T9309 25558-25574 Sentence denotes Ethics Statement
T167 25558-25574 Sentence denotes Ethics Statement
T168 25575-25701 Sentence denotes Blood samples were obtained with informed written consent from control subjects and CF patients at the BC Children's Hospital.
T9310 25575-25770 Sentence denotes Blood samples were obtained with informed written consent from control subjects and CF patients at the BC Children's Hospital. Consent was obtained for children by their parent or legal guardian.
T169 25702-25770 Sentence denotes Consent was obtained for children by their parent or legal guardian.
T9311 25771-25854 Sentence denotes Subjects 7 years of age and older were required to provide informed assent as well.
T170 25771-25854 Sentence denotes Subjects 7 years of age and older were required to provide informed assent as well.
T9312 25855-25929 Sentence denotes Protocols were approved by the Clinical Research Ethics Board (H09-01192).
T171 25855-25929 Sentence denotes Protocols were approved by the Clinical Research Ethics Board (H09-01192).
T9454 25931-25943 Sentence denotes Cell Culture
T172 25931-25943 Sentence denotes Cell Culture
T173 25944-26056 Sentence denotes CF (IB3-1 and CuFi-1) and control (S9 and NuLi-1) cells were obtained from the American Type Culture Collection.
T9455 25944-26195 Sentence denotes CF (IB3-1 and CuFi-1) and control (S9 and NuLi-1) cells were obtained from the American Type Culture Collection. IB3-1 cells were derived from a patient expressing the ΔF508 and W1282X mutations and CuFi-1 were derived from a ΔF508 homozygous patient.
T174 26057-26195 Sentence denotes IB3-1 cells were derived from a patient expressing the ΔF508 and W1282X mutations and CuFi-1 were derived from a ΔF508 homozygous patient.
T9456 26196-26379 Sentence denotes S9 cells are IB3-1 cells that have been transfected with CFTR using an adeno-associated viral vector and NuLi-1 cells were derived from a patient possessing a wild-type CFTR genotype.
T175 26196-26379 Sentence denotes S9 cells are IB3-1 cells that have been transfected with CFTR using an adeno-associated viral vector and NuLi-1 cells were derived from a patient possessing a wild-type CFTR genotype.
T9457 26380-26510 Sentence denotes THP1-XBlue cells stably express a secreted embryonic alkaline phosphatase (SEAP) reporter inducible by NF-κB and AP-1 (Invivogen).
T176 26380-26510 Sentence denotes THP1-XBlue cells stably express a secreted embryonic alkaline phosphatase (SEAP) reporter inducible by NF-κB and AP-1 (Invivogen).
T9458 26511-26601 Sentence denotes Cells were cultured as recommended by their respective suppliers using standard protocols.
T177 26511-26601 Sentence denotes Cells were cultured as recommended by their respective suppliers using standard protocols.
T9459 26602-26760 Sentence denotes S9 and IB3-1 cells were cultured in basal LHC-8 (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, and 1 mM sodium pyruvate.
T178 26602-26760 Sentence denotes S9 and IB3-1 cells were cultured in basal LHC-8 (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, and 1 mM sodium pyruvate.
T9460 26761-27023 Sentence denotes NuLi-1/CuFi-1 cells were cultured in BEBM serum-free medium (Lonza) with supplement bullet kit (EGF, hydrocortisone, bovine pituitary extract, transferrin, bovine insulin, triiodothyronine, epinephrine, retinoic acid), 2 mM L-glutamine, and 1 mM sodium pyruvate.
T179 26761-27023 Sentence denotes NuLi-1/CuFi-1 cells were cultured in BEBM serum-free medium (Lonza) with supplement bullet kit (EGF, hydrocortisone, bovine pituitary extract, transferrin, bovine insulin, triiodothyronine, epinephrine, retinoic acid), 2 mM L-glutamine, and 1 mM sodium pyruvate.
T9461 27024-27183 Sentence denotes PBMCs from CF patients and controls were cultured in RPMI-1640 (Hyclone) supplemented with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate (complete RPMI).
T180 27024-27183 Sentence denotes PBMCs from CF patients and controls were cultured in RPMI-1640 (Hyclone) supplemented with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate (complete RPMI).
T9462 27184-27332 Sentence denotes THP1-XBlue cells were cultured in complete RPMI with the addition of zeocin (100 µg/ml) to select for cells expressing the SEAP NF-κB/AP-1 reporter.
T181 27184-27332 Sentence denotes THP1-XBlue cells were cultured in complete RPMI with the addition of zeocin (100 µg/ml) to select for cells expressing the SEAP NF-κB/AP-1 reporter.
T9463 27333-27516 Sentence denotes Prior to stimulation, bronchial epithelial cell lines were plated in coated [62] 96-well plates (BD Biosciences) at 3×104 cells/well unless indicated, and allowed to adhere overnight.
T182 27333-27516 Sentence denotes Prior to stimulation, bronchial epithelial cell lines were plated in coated [62] 96-well plates (BD Biosciences) at 3×104 cells/well unless indicated, and allowed to adhere overnight.
T9464 27517-27694 Sentence denotes Plates for S9 and IB3-1 stimulations were coated in a mixture of bovine serum albumin (100 µg/ml), fibronectin (10 µg/ml), and bovine collage type I (30 µg/ml) (BD Biosciences).
T183 27517-27694 Sentence denotes Plates for S9 and IB3-1 stimulations were coated in a mixture of bovine serum albumin (100 µg/ml), fibronectin (10 µg/ml), and bovine collage type I (30 µg/ml) (BD Biosciences).
T9465 27695-27785 Sentence denotes Plates for NuLi-1 and CuFi-1 were coated with collagen type IV (60 µg/ml) (Sigma Aldrich).
T184 27695-27785 Sentence denotes Plates for NuLi-1 and CuFi-1 were coated with collagen type IV (60 µg/ml) (Sigma Aldrich).
T9466 27786-27886 Sentence denotes PBMCs were plated in 96-well plates at a density of 1.5×105 cells/well in 200 µl (7.5×105 cells/mL).
T185 27786-27886 Sentence denotes PBMCs were plated in 96-well plates at a density of 1.5×105 cells/well in 200 µl (7.5×105 cells/mL).
T9467 27887-28106 Sentence denotes THP-1 reporter cells were differentiated into a macrophage-like phenotype using 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich) for 24 hours at a density of 1×105 cells/well in 200 µl (5×105 cells/ml).
T186 27887-28106 Sentence denotes THP-1 reporter cells were differentiated into a macrophage-like phenotype using 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich) for 24 hours at a density of 1×105 cells/well in 200 µl (5×105 cells/ml).
T9468 28107-28194 Sentence denotes Cells were washed with PBS and allowed to rest a further 42 hours prior to stimulation.
T187 28107-28194 Sentence denotes Cells were washed with PBS and allowed to rest a further 42 hours prior to stimulation.
T10414 28196-28224 Sentence denotes CF and control subject PBMCs
T188 28196-28224 Sentence denotes CF and control subject PBMCs
T10415 28225-28384 Sentence denotes The diagnosis of CF was established by typical clinical features, increased sweat chloride concentrations (>60 mmol/l), and detection of CF-inducing mutations.
T189 28225-28384 Sentence denotes The diagnosis of CF was established by typical clinical features, increased sweat chloride concentrations (>60 mmol/l), and detection of CF-inducing mutations.
T10416 28385-28576 Sentence denotes All patients with CF were clinically stable at the time of blood donation, and we excluded any subjects who were receiving systemic corticosteroids due to potential immunomodulatory activity.
T190 28385-28576 Sentence denotes All patients with CF were clinically stable at the time of blood donation, and we excluded any subjects who were receiving systemic corticosteroids due to potential immunomodulatory activity.
T10417 28577-28635 Sentence denotes Control samples were provided by healthy adult volunteers.
T191 28577-28635 Sentence denotes Control samples were provided by healthy adult volunteers.
T10418 28636-28846 Sentence denotes In previously published work we have demonstrated that TLR-mediated inflammatory responses are stable in humans from birth to 60 years old [63], therefore did not age-match the CF patients and control subjects.
T192 28636-28846 Sentence denotes In previously published work we have demonstrated that TLR-mediated inflammatory responses are stable in humans from birth to 60 years old [63], therefore did not age-match the CF patients and control subjects.
T10419 28847-29019 Sentence denotes Peripheral blood was collected in sodium heparin tubes (BD Biosciences) and PBMCs were isolated using density gradient centrifugation on Ficoll-Paque™ Plus (GE Healthcare).
T193 28847-29019 Sentence denotes Peripheral blood was collected in sodium heparin tubes (BD Biosciences) and PBMCs were isolated using density gradient centrifugation on Ficoll-Paque™ Plus (GE Healthcare).
T10420 29020-29114 Sentence denotes The layer containing PBMCs was isolated, washed twice in PBS and resuspended in complete RPMI.
T194 29020-29114 Sentence denotes The layer containing PBMCs was isolated, washed twice in PBS and resuspended in complete RPMI.
T10421 29115-29217 Sentence denotes Cells were enumerated by trypan blue exclusion using the Countess automated cell counter (Invitrogen).
T195 29115-29217 Sentence denotes Cells were enumerated by trypan blue exclusion using the Countess automated cell counter (Invitrogen).
T10422 29218-29377 Sentence denotes For derivation of macrophages from monocytes, monocytes were allowed to adhere to plastic for 2 hours in RPMI 1640 after which non-adherent cells were removed.
T196 29218-29377 Sentence denotes For derivation of macrophages from monocytes, monocytes were allowed to adhere to plastic for 2 hours in RPMI 1640 after which non-adherent cells were removed.
T10423 29378-29480 Sentence denotes Monocytes were allowed to differentiate in RPMI 1640 supplemented with 10% human AB serum for 10 days.
T197 29378-29480 Sentence denotes Monocytes were allowed to differentiate in RPMI 1640 supplemented with 10% human AB serum for 10 days.
T10861 29482-29526 Sentence denotes Cell stimulation and cytokine quantification
T198 29482-29526 Sentence denotes Cell stimulation and cytokine quantification
T10862 29527-29619 Sentence denotes Bronchial epithelial cells were plated and allowed to adhere overnight prior to stimulation.
T199 29527-29619 Sentence denotes Bronchial epithelial cells were plated and allowed to adhere overnight prior to stimulation.
T10863 29620-29765 Sentence denotes Bronchial epithelial cells were rested or primed with LPS for 5 hours and stimulated with live P. aeruginosa PAO1 or ATP for the times indicated.
T200 29620-29765 Sentence denotes Bronchial epithelial cells were rested or primed with LPS for 5 hours and stimulated with live P. aeruginosa PAO1 or ATP for the times indicated.
T10864 29766-29888 Sentence denotes PBMCs and THP-1 reporter cells were either rested or primed with LPS (Invivogen) or heat-killed PAO1 overnight (16 hours).
T201 29766-29888 Sentence denotes PBMCs and THP-1 reporter cells were either rested or primed with LPS (Invivogen) or heat-killed PAO1 overnight (16 hours).
T10865 29889-30040 Sentence denotes The next day the cells were challenged with live PAO1, PAO1ΔexsA, ATP (Invivogen), or Poly(dA:dT) (Sigma Aldrich) for the times indicated (see Fig. 1).
T202 29889-30040 Sentence denotes The next day the cells were challenged with live PAO1, PAO1ΔexsA, ATP (Invivogen), or Poly(dA:dT) (Sigma Aldrich) for the times indicated (see Fig. 1).
T10866 30041-30206 Sentence denotes For stimulations with Poly(dA:dT), lipofectamine LTX was used at a 1∶1 (w∶v) ratio of µg of DNA to µl of lipofectamine and was mixed 30 minutes prior to stimulation.
T203 30041-30206 Sentence denotes For stimulations with Poly(dA:dT), lipofectamine LTX was used at a 1∶1 (w∶v) ratio of µg of DNA to µl of lipofectamine and was mixed 30 minutes prior to stimulation.
T10867 30207-30296 Sentence denotes The NF-κB inhibitor Bay11-7082 (Invivogen) was added to cultures 1 hour prior to priming.
T204 30207-30296 Sentence denotes The NF-κB inhibitor Bay11-7082 (Invivogen) was added to cultures 1 hour prior to priming.
T10868 30297-30386 Sentence denotes If no priming was involved, inhibitor was added 1 hour prior to inflammasome stimulation.
T205 30297-30386 Sentence denotes If no priming was involved, inhibitor was added 1 hour prior to inflammasome stimulation.
T10869 30387-30498 Sentence denotes The CFTR inhibitor CFTRinh172 (Sigma Aldrich) was added to cultures 18 hours prior to inflammasome stimulation.
T206 30387-30498 Sentence denotes The CFTR inhibitor CFTRinh172 (Sigma Aldrich) was added to cultures 18 hours prior to inflammasome stimulation.
T10870 30499-30609 Sentence denotes The caspase-1 inhibitor z-YVAD-fmk (Biovision) was added to cultures 1 hour prior to inflammasome stimulation.
T207 30499-30609 Sentence denotes The caspase-1 inhibitor z-YVAD-fmk (Biovision) was added to cultures 1 hour prior to inflammasome stimulation.
T208 30610-30658 Sentence denotes Supernatants were collected and stored at −20°C.
T10871 30610-30798 Sentence denotes Supernatants were collected and stored at −20°C. Cytokines released into supernatants from PBMCs stimulated with inflammasome activators were quantified using sandwich ELISA (eBioscience).
T209 30659-30798 Sentence denotes Cytokines released into supernatants from PBMCs stimulated with inflammasome activators were quantified using sandwich ELISA (eBioscience).
T11380 30800-30814 Sentence denotes Immunoblotting
T210 30800-30814 Sentence denotes Immunoblotting
T11381 30815-30995 Sentence denotes 1×106 cells were seeded in 12-well plates, stimulated as indicated, and lysed in RIPA buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific).
T211 30815-30995 Sentence denotes 1×106 cells were seeded in 12-well plates, stimulated as indicated, and lysed in RIPA buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific).
T11382 30996-31073 Sentence denotes Protein concentrations were determined by Bradford assay (Thermo Scientific).
T212 30996-31073 Sentence denotes Protein concentrations were determined by Bradford assay (Thermo Scientific).
T11383 31074-31194 Sentence denotes Lysates were resolved by electrophoreses on 10% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore).
T213 31074-31194 Sentence denotes Lysates were resolved by electrophoreses on 10% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore).
T11384 31195-31352 Sentence denotes Blots were blocked for 1 hour at room temperature and probed overnight at 4°C for pro-IL-1β (Santa Cruz), IκBα (Cell Signaling), or β-actin (Cell Signaling).
T214 31195-31352 Sentence denotes Blots were blocked for 1 hour at room temperature and probed overnight at 4°C for pro-IL-1β (Santa Cruz), IκBα (Cell Signaling), or β-actin (Cell Signaling).
T11385 31353-31485 Sentence denotes Blots were subsequently probed with fluorescently-labeled secondary antibodies, IRDye® 680 or 800CW (LI-COR Biosciences) for 1 hour.
T215 31353-31485 Sentence denotes Blots were subsequently probed with fluorescently-labeled secondary antibodies, IRDye® 680 or 800CW (LI-COR Biosciences) for 1 hour.
T11386 31486-31641 Sentence denotes Both blocking and probing steps were carried out in tris-buffered saline (G Biosciences) containing 5% bovine serum albumin and 0.1% TWEEN 20 (Calbiochem).
T216 31486-31641 Sentence denotes Both blocking and probing steps were carried out in tris-buffered saline (G Biosciences) containing 5% bovine serum albumin and 0.1% TWEEN 20 (Calbiochem).
T11387 31642-31777 Sentence denotes Blots were imaged on a LI-COR Odyssey infrared imaging system (LI-COR Biosciences) and quantified using the included analysis software.
T217 31642-31777 Sentence denotes Blots were imaged on a LI-COR Odyssey infrared imaging system (LI-COR Biosciences) and quantified using the included analysis software.
T11755 31779-31815 Sentence denotes Quantification of caspase-1 activity
T218 31779-31815 Sentence denotes Quantification of caspase-1 activity
T11756 31816-31902 Sentence denotes Bronchial epithelial cells were plated in 6-well plates at 5×105 cells/well overnight.
T219 31816-31902 Sentence denotes Bronchial epithelial cells were plated in 6-well plates at 5×105 cells/well overnight.
T11757 31903-32018 Sentence denotes Cells were primed with LPS for 5 hours and stimulated with ATP for 1 hour or stimulated with live PAO1 for 3 hours.
T220 31903-32018 Sentence denotes Cells were primed with LPS for 5 hours and stimulated with ATP for 1 hour or stimulated with live PAO1 for 3 hours.
T11758 32019-32072 Sentence denotes PBMCs were stimulated the same day as blood donation.
T221 32019-32072 Sentence denotes PBMCs were stimulated the same day as blood donation.
T11759 32073-32301 Sentence denotes PBMCs were seeded in a 96-well plate at a density of 4.5×105 cells/well (2.5×106 cells/ml), primed with LPS for 5 hours and then stimulated with ATP for 1 hour or Poly(dA:dT) for 3 hours or stimulated with live PAO1 for 3 hours.
T222 32073-32301 Sentence denotes PBMCs were seeded in a 96-well plate at a density of 4.5×105 cells/well (2.5×106 cells/ml), primed with LPS for 5 hours and then stimulated with ATP for 1 hour or Poly(dA:dT) for 3 hours or stimulated with live PAO1 for 3 hours.
T11760 32302-32456 Sentence denotes Caspase-1 activity was measured using FLICA (Immunochemistry Technologies), a cell-permeable fluorescent probe (FAM-YVAD-fmk) that binds active caspase-1.
T223 32302-32456 Sentence denotes Caspase-1 activity was measured using FLICA (Immunochemistry Technologies), a cell-permeable fluorescent probe (FAM-YVAD-fmk) that binds active caspase-1.
T11761 32457-32596 Sentence denotes Cells were incubated 1 hour with FLICA at 37°C and stained with PE-Cy7-conjugated anti-CD14 antibodies (eBioscience) to identify monocytes.
T224 32457-32596 Sentence denotes Cells were incubated 1 hour with FLICA at 37°C and stained with PE-Cy7-conjugated anti-CD14 antibodies (eBioscience) to identify monocytes.
T11762 32597-32750 Sentence denotes The gating strategy consisted of including live cells that were CD14 positive which were subsequently analyzed for the frequency of FLICA positive cells.
T225 32597-32750 Sentence denotes The gating strategy consisted of including live cells that were CD14 positive which were subsequently analyzed for the frequency of FLICA positive cells.
T12140 32752-32777 Sentence denotes NF-κB/AP-1 Activity Assay
T226 32752-32777 Sentence denotes NF-κB/AP-1 Activity Assay
T12141 32778-32918 Sentence denotes Supernatants from THP-1 reporter cells were incubated with Quanti-Blue substrate (Invivogen) at 37°C and allowed to develop for 16–18 hours.
T227 32778-32918 Sentence denotes Supernatants from THP-1 reporter cells were incubated with Quanti-Blue substrate (Invivogen) at 37°C and allowed to develop for 16–18 hours.
T12142 32919-33166 Sentence denotes Quanti-Blue contains a substrate for alkaline phosphatase and changes in the amount of NF-κB/AP-1 activity were quantified by optical density (λ = 655) measured using a SpectraMax 384 Plus plate reader and SoftMax Pro software (Molecular Devices).
T228 32919-33166 Sentence denotes Quanti-Blue contains a substrate for alkaline phosphatase and changes in the amount of NF-κB/AP-1 activity were quantified by optical density (λ = 655) measured using a SpectraMax 384 Plus plate reader and SoftMax Pro software (Molecular Devices).
T12298 33168-33185 Sentence denotes Bacterial strains
T229 33168-33185 Sentence denotes Bacterial strains
T230 33186-33287 Sentence denotes P. aeruginosa laboratory strains PAO1 and the PAO1ΔexsA mutant were obtained from Dr. Robert Hancock.
T12299 33186-33452 Sentence denotes P. aeruginosa laboratory strains PAO1 and the PAO1ΔexsA mutant were obtained from Dr. Robert Hancock. P. aeruginosa strains PAO1 and PAO1ΔexsA were grown from overnight cultures in Luria Bertani (LB) broth and LB+streptomycin (150 µg/ml) until mid-logarithmic phase.
T231 33288-33452 Sentence denotes P. aeruginosa strains PAO1 and PAO1ΔexsA were grown from overnight cultures in Luria Bertani (LB) broth and LB+streptomycin (150 µg/ml) until mid-logarithmic phase.
T12300 33453-33548 Sentence denotes Cells were washed once in PBS and resuspended in PBS to an optical density of 0.5 (λ = 600 nm).
T232 33453-33548 Sentence denotes Cells were washed once in PBS and resuspended in PBS to an optical density of 0.5 (λ = 600 nm).
T12301 33549-33674 Sentence denotes To prepare heat-killed bacteria, live PAO1 was resuspended in PBS to an optical density of 0.5 and heated at 60°C for 1 hour.
T233 33549-33674 Sentence denotes To prepare heat-killed bacteria, live PAO1 was resuspended in PBS to an optical density of 0.5 and heated at 60°C for 1 hour.
T12302 33675-33863 Sentence denotes For stimulations, live PAO1 was resuspended to an optical density of 0.5 in PBS and further diluted in culture medium prior to stimulation to achieve the desired multiplicity of infection.
T234 33675-33863 Sentence denotes For stimulations, live PAO1 was resuspended to an optical density of 0.5 in PBS and further diluted in culture medium prior to stimulation to achieve the desired multiplicity of infection.
T12303 33864-33964 Sentence denotes Heat-killed PAO1 was added in a volume equivalent to that used to achieve an MOI of 1 for live PAO1.
T235 33864-33964 Sentence denotes Heat-killed PAO1 was added in a volume equivalent to that used to achieve an MOI of 1 for live PAO1.
T12606 33966-33976 Sentence denotes Statistics
T236 33966-33976 Sentence denotes Statistics
T12607 33977-34054 Sentence denotes All graphs display the mean ± SEM and were generated with Prism 5 (Graphpad).
T237 33977-34054 Sentence denotes All graphs display the mean ± SEM and were generated with Prism 5 (Graphpad).
T12608 34055-34176 Sentence denotes Statistical significance was determined by performing one or two-way ANOVA and the Bonferroni post-test where applicable.
T238 34055-34176 Sentence denotes Statistical significance was determined by performing one or two-way ANOVA and the Bonferroni post-test where applicable.