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This study reports a novel mechanism of NF-kappaB activation by the HIV-1 Tat transactivator. Based on the evidence that Tat enhanced the transcriptional activity of the p65 subunit of NF-kappaB (49,63,64), and physically interacted with the IkappaB-alpha repressor (50,51), we investigated the possibility that Tat could activate NF-kappaB via direct interaction with IkappaB-alpha and p65. To this end, the NF-kappaB activity was monitored in single round HIV-1 infection using RNA interference to silence the Tat expression. By this approach, we avoided the perpetuation of NF-kappaB activation signaling due to subsequent rounds of viral entry in cell culture propagation (30,31). Upon HIV-1 infection, the early NF-kappaB activation occurred concomitantly with IKK activation and IkappaB-alpha degradation in the absence of Tat. Soon after the shut off of IKK activity and new synthesis of IkappaB-alpha, the NF-kappaB activity was kept elevated in the presence of Tat, while it was down regulated upon silencing of the Tat gene. These findings indicate that in single round HIV-1 infection, Tat enhanced the NF-kappaB activity without affecting the IKK activity and the half-life of IkappaB-alpha. Similar kinetic of NF-kappaB activation occurred upon short-pulse of PMA in Tat-transfected HeLa cells, where Tat inhibited the post-activation turn off of NF-kappaB in presence of newly synthesized IkappaB-alpha.
The Tat-dependent activation of NF-kappaB correlated with the association of the viral protein with IkappaB-alpha, which interfered with the generation of the IkappaB-alpha/p65 complex. This evidence demonstrated that Tat competed for the binding of IkappaB-alpha to p65 and prevented the IkappaB-alpha repression of p65. As additional mechanism of NF-kappaB activation, Tat associated with p65 and increased the p65 binding affinity to the NF-kappaB enhancer; this evidence was obtained with recombinant proteins indicating that the stronger binding of p65 to DNA was a consequence of direct association with Tat, which likely caused a conformational change of p65. We also demonstrated that the Tat cysteine-rich sequence (C22,25,27) was involved in the binding to the RHD of p65 and NF-kappaB activation. Differently, the Tat arginine-rich sequence (R49,52,53,55,56,57) was required for the binding to the sixth ankyrin of IkappaB-alpha (50,51), and for releasing p65 from the IkappaB-alpha inhibition. Altogether these results demonstrated that Tat abolished the negative feedback regulation of NF-kappaB by hijacking the IkappaB-alpha repressor, shielding p65 from the IkappaB-alpha embrace, and enforcing the p65 binding to DNA.
Further evidence of Tat activation of NF-kappaB showed that wild-type Tat, and not the Tat mutants lacking the arginine- or cysteine-rich domains, up-regulated in vivo the expression of a number of NF-kappaB-responsive genes, including MIP-1alpha, CSF3, LTA, NFKBIA and TLR2. By ChIP analysis, we observed that Tat increased the recruitment of p65 to the NF-kappaB enhancers of the activated genes, and was recovered at the same sites. RNA interference of p65 abolished the Tat occupancy of the NF-kappaB enhancers indicating that the Tat binding to the NF-kappaB sites was mediated by p65. These results were consistent with the evidence that Tat displaced p65 from the binding to IkappaB-alpha (Figures 2D and 3C), and increased the p65 DNA binding affinity through association (Figure 4E and F). Thus, Tat likely increased the recruitment of p65 to the NF-kappaB-dependent promoters by associating with p65 and by interfering with the assembly of p65 with the repressor IkappaB-alpha.
Of interest, IkappaB-alpha was found associated to the NF-kappaB enhancers in absence of Tat, where it was displaced in presence of Tat. These results suggest a negative regulatory role of IkappaB-alpha in gene regulation through occupancy of specific promoters. Indeed, IkappaB-alpha was found associated with hystone deacetylases at the hes1 promoter, from where it was removed following TNF-alpha stimulation causing histone acetylation and hes1 transcriptional activation (65). A similar mechanism of gene regulation could apply to the NF-kappaB-dependent genes analysed in this study, where Tat could activate the gene expression by removing IkappaB-alpha and promoting the p65 loading (Figure 7).
The physiological relevance of Tat cross talk with NF-kappaB was demonstrated in the HIV-1 infection of human monocytes, where HIV-1-encoded Tat protein counteracted post-activation turn off of NF-kappaB, as shown by: (i) sustained NF-kappaB activity by Tat in presence of newly synthesized IkappaB-alpha; (ii) coimmunoprecipitation of Tat with IkappaB-alpha and p65; (iii) induction of MIP-1alpha expression dependent on Tat and p65; (iv) Tat occupancy of the MIP-1alpha NF-kappaB enhancer associated with increased recruitment of p65.
This study supports the pro-inflammatory action of Tat through physical interaction with p65 and IkappaB-alpha. Several inflammatory cytokines under the transcriptional control of NF-kappaB are hyper-expressed in HIV-1 infected individuals, including IL-6 (66,67), TNF-alpha (68), and MIP-1alpha (57-62,69-71). In the case of TNF-alpha and IL-6, Tat activated the cytokine expression by binding to the TAR-like stem loop of the 5' transcript, thus likely promoting the transcriptional elongation (33,37,47). Here, we have shown that the Tat-dependent transcriptional activation of a number of pro-inflammatory genes required the assembly of Tat with p65 at the NF-kappaB enhancer together with the displacement of IkappaB-alpha (Figure 5). Thus, in the set of distinct NF-kappaB-responsive genes, Tat acted as a component of the transcriptional initiation complex, which is consistent with previous reports on Tat promoting the transcription via DNA motifs (72,73).
Overproduction of pro-inflammatory cytokines is considered a major mechanism of immune deregulation (26) and neuron dysfunction in AIDS (74,75). In this scenario, inhibition of NF-kappaB activity could lead to a therapeutic strategy for counteracting the abnormal production of pro-inflammatory cytokines and chemokines in HIV-1 infection. NF-kappaB inhibitors, such as anti-oxidants, proteasome and IKK inhibitors, significantly reduced the HIV-1 replication and the associated inflammatory response (76,77); however, these NF-kappaB inhibitors acted indiscriminately in both HIV-1-infected and uninfected cells. The evidence that Tat activates NF-kappaB through direct interaction with IkappaB-alpha and p65 may lead to specific inhibitors to counteract the Tat pro-inflammatory action.

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Input: Tat counteracts the post-activation turn off of NF-kappaB in single round HIV-1-infection. Jurkat cells (5 x 107) were transfected with siRNA Tat or siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.Luc.R-E- virions (500 ng of p24) and harvested at the indicated time. (A) Tat expression was measured in total RNA by real-time PCR, while the luciferase activity was measured in whole cell extracts. (B) Nuclear extracts (10 microg) were analysed for the p65 and p50 binding to the NF-kappaB double-stranded oligonucleotide using the NF-kappaB Transcription Factor ELISA Assay kit (Cayman). (C) The expression of p65 and IkappaB-alpha was analysed by 12% SDS-PAGE and western blotting of nuclear or cytosolic extracts (20 microg) using anti-p65 and anti-IkappaB-alpha antibodies. Histone H1 and Hexokinase II were detected with specific antibodies as markers of nuclear and cytosolic extracts, respectively. Densitometry values (D) of the bands were expressed as fold increase above the control (mock). (D) IKK activity was measured in cytosolic extracts (100 microg) by using HTScan IKK Kinase Assay (Cell Signaling Technology). Values (mean +/- SE, n = 3) are shown.

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Input: Tat enhances the NF-kappaB activity following PMA stimulation by associating with IkappaB-alpha and counteracting the NF-kappaB repression. HeLa cells (5 x 106) were transfected with p3xFLAG-Tat or p3xFLAG empty vector (5 microg), and 48 h later were stimulated with PMA (5 min, 20 ng/ml) or left unstimulated, washed twice with DMEM and harvested at the indicated times. Nuclear and cytosolic extracts were prepared for further analysis. (A) Nuclear extracts were analysed for the p65, p50 and FLAG-Tat binding to the NF-kappaB double-stranded oligonucleotide using the NF-kappaB Transcription Factor ELISA assay kit (Cayman). (B) Nuclear and cytosolic extracts (20 microg) were separated by 12% SDS-PAGE and analysed by western blotting using the anti-p65, anti-FLAG, anti-Histone H1, anti-IkappaB-alpha and anti-Hexokinase-II antibodies. Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). (C) IKK activity was measured in cytosolic extracts (100 microg) by using HTScan IKK kinase assay (Cell Signaling Technology). (D) HeLa cells (5 x 106) were transfected with or without p3xFLAG-Tat (5 microg); 48 h later, cells were stimulated with PMA (5 min, 20 ng/ml) or left unstimulated, washed twice with DMEM, and harvested after 240 min. Whole cell extracts (1 mg) were immunoprecipitated with protein G-Sepharose-coupled anti-IkappaB-alpha antibody. Immunocomplexes were separated by 12% SDS-PAGE and analysed by western blotting with anti-p65, anti-FLAG and anti-IkappaB-alpha antibodies. Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). Values (mean +/- SE, n = 3) are shown.

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Input: Tat relieves p65 from the IkappaB-alpha inhibition. (A) Schematic representation of wild-type and mutant Tat proteins. (B) HA-IkappaB-alpha (5 microl) was incubated in presence or absence of FLAG-Tat, FLAG-Tat T,N(23,24)A, FLAG-Tat K(50,51)A, FLAG-Tat R(49-57)A or FLAG-Tat C(22,25,27)A (10 microl) using in vitro translated proteins. Wild-type and mutant Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS-PAGE and analysed by western blotting with anti-HA and anti-FLAG antibodies. (C) 35S-methionine-labeled p65 (5 microl) was incubated with in vitro translated HA-IkappaB-alpha (5 microl) in presence or absence of FLAG-Tat or FLAG-Tat R(49-57)A (5, 10 or 20 microl). HA-IkappaB-alpha was immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS-PAGE and analysed by western blotting with anti-HA and anti-FLAG antibodies, or autoradiography (35S-Met-p65). Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). (D) The p65 DNA binding activity was analysed by EMSA using in vitro translated proteins. 32P-labeled-NF-kappaB double-stranded oligonucleotide was incubated with p65 (0.5 microl) in presence or absence of HA-IkappaB-alpha (1 microl), FLAG-Tat or FLAG-Tat R(49-57)A (5 and 10 microl); competition of DNA binding was performed with 10 - up to 100-fold molar excess of unlabeled NF-kappaB double-stranded oligonucleotide. DNA/protein complexes were run on 6% PAGE-TBE and analysed by autoradiography. (E) p50-/-p65-/-MEFs (3 x 105 cells) were transfected with pkappaBLuc (0.5 microg) and pSV-beta-Gal (0.1 microg) with or without pRc/CMV-p65 (0.5 microg), pCMV4-HA-IkappaB-alpha (0.5 microg), p3xFLAG-Tat or p3xFLAG-Tat R(49-57)A (0.5, 1 and 2 microg). The luciferase activity was measured in cell extracts 48-h post-transfection and normalized to beta-galactosidase activity. Fold activation was calculated relative to transfection of the pkappaBLuc plasmid alone. Values (mean +/- SE, n = 3) are shown. As control of protein expression, aliquots of cell extracts (20 microg) were analysed by western blotting with anti-p65, anti-HA, anti-FLAG and anti-gamma-tubulin antibodies.

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Input: Tat associates with p65 increasing its DNA-affinity binding. (A) HeLa cells were transfected with pRc/CMV-3xHA-p65 (5 microg) in presence or absence of p3xFLAG-Tat, p3xFLAG-Tat T,N(23,24)A, p3xFLAG-Tat K(50,51)A, p3xFLAG-Tat R(49-57)A, or p3xFLAG-Tat C(22,25,27)A (5 microg). FLAG-Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS-PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (B) In vitro translated p65 (5 microl) was incubated with GST-Tat, GST-Tat T,N(23,24)A, GST-Tat K(50,51)A, GST-Tat R(49-57)A, GST-Tat C(22,25,27)A, or GST (5 microg) conjugated with Glutathione-Sepharose. Protein complexes were recovered by GST-pull down, separated by 12% SDS-PAGE, and analysed by western blotting with anti-p65 or anti-GST antibodies. (C) Schematic representation of wild-type and mutant p65 proteins. (D) HeLa cells were transfected with p3xFLAG-Tat (5 microg) in the presence or absence of pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65deltaC (1-318), or pRc/CMV-3xHA-p65deltaN (122-551) (5 microg). Wild-type and mutant HA-p65 proteins were immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS-PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (E) Recombinant p65 protein (100 ng; Active Motif Carlsbad, CA, USA) was 20 min incubated with 32P-labeled NF-kappaB double-stranded oligonucleotide in presence or absence of in vitro translated FLAG-Tat or FLAG-Tat C(22,25,27)A (5 microl); competitions were performed with 1.25- up to 40-fold molar excess of unlabeled oligonucleotide. DNA/protein complexes were separated by 6% PAGE in 0.5 x TBE buffer and analysed by autoradiography. (F) Densitometry of band-shifts shown in E.

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Input: Tat activates the p65-dependent expression of NF-kappaB-responsive genes by occupying the NF-kappaB enhancers and promoting the p65 recruitment with IkappaB-alpha displacement. HeLa cells (5 x 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49-57) or empty vector (10 microg); for p65 RNA interference, cells (5 x 106) were transfected with p3xFLAG-Tat (10 microg) and siRNA p65 or siRNA control (200 pmol). Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1alpha (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 x 106) were transfected with p3xFLAG-Tat, or empty vector (10 microg) and 48 h later ChIP was performed with anti-p65 antibody. Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 x 106) were transfected with empty vector, or p3xFLAG-Tat (10 microg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody. Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 x 106) were transfected with empty vector, p3xFLAG-Tat (10 microg), or empty vector plus siRNA IkappaB-alpha or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IkappaB-alpha antibody. Real-time PCR was performed with primers specific for the indicated promoters. Values (mean +/- SE, n = 3) are shown. The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P < 0.05).

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Input: In HIV-1 infection Tat sustains the NF-kappaB activity and enhances the MIP-1alpha expression via interaction with IkappaB-alpha and p65. U937 cells (5 x 107) were transfected with siRNA Tat, siRNA control (2 nmol), or left untransfected; 24 h later cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R-E- virions (500 ng of p24) and harvested at the indicated time. (A) Real-time PCR of total RNA measured the Tat expression; ELISA measured the amount of p24 in whole cell extracts. (B) The binding of p65 to the NF-kappaB double-stranded oligonucleotide was measured in nuclear extracts (10 microg) using the NF-kappaB Transcription Factor ELISA assay kit (Cayman). (C) The IkappaB-alpha content was analysed by 12% SDS-PAGE and western blotting of cytosolic extracts (20 microg) using anti-IkappaB-alpha antibody. Densitometry values (D) of the bands were expressed as fold increase above the control (mock). (D) IKK activity was measured in cytosolic cell extracts (100 microg) using HTScan IKK Kinase Assay (Cell Signaling Technology). (E) U937 cells (5 x 107) were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R-E- virions, or left uninfected. Twenty-four-hour post-infection, cell extracts (1 mg) were immunoprecipitated with protein G-Sepharose-coupled anti-FLAG antibody. Immunocomplexes were separated by 12% SDS-PAGE and analysed by western blotting with anti-p65, anti-FLAG and anti-IkappaB-alpha antibodies. (F) U937 cells (5 x 107) were transfected with siRNA Tat, siRNA p65, siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R-E- virions, or left uninfected. Twenty-four-hour post-infection, total RNA was analysed for MIP-1alpha expression by real-time PCR. (G and H) U937 cells (5 x 107) were transfected with siRNA Tat or siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R-E- virions, or left uninfected. Twenty-four-hour post-infection, ChIP was performed with anti-p65 (G) or anti-FLAG (H). Real-time PCR was performed with primers specific for MIP-1alpha and GAPDH promoters. Values (mean +/- SE, n = 3) are shown. The asterisks indicate statistically significant differences compared to the control (mock), according to the Student's t-test (P < 0.05).

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Input: Model of MIP-1alpha transcriptional activation by HIV-1 Tat. (A) In absence of Tat, IkappaB-alpha occupies the NF-kappaB enhancer of the MIP-1alpha promoter and likely interacts with transcriptional repressors, such as HDACs (65), to inhibit gene transcription. (B) Tat activates the MIP-1alpha expression by removing the IkappaB-alpha repressor from the NF-kappaB enhancer, and by increasing the binding of p65 NF-kappaB complex to the NF-kappaB enhancer.

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Input:
Inflammasome-Mediated IL-1beta Production in Humans with Cystic Fibrosis
Background
Inflammation and infection are major determinants of disease severity and consequently, the quality of life and outcome for patients with cystic fibrosis (CF). Interleukin-1 beta (IL-1beta) is a key inflammatory mediator. Secretion of biologically active IL-1beta involves inflammasome-mediated processing. Little is known about the contribution of IL-1beta and the inflammasomes in CF inflammatory disease. This study examines inflammasome-mediated IL-1beta production in CF bronchial epithelial cell lines and human patients with CF.
Results
Bronchial epithelial cell lines were found to produce negligible amounts of basal or stimulated IL-1beta compared to hematopoeitic cells and they did not significantly upregulate caspase-1 activity upon inflammasome stimulation. In contrast, peripheral blood mononuclear cells (PBMCs) from both CF and healthy control subjects produced large amounts of IL-1beta and strongly upregulated caspase-1 activity upon inflammasome stimulation. PBMCs from CF patients and controls displayed similar levels of caspase-1 activation and IL-1beta production when stimulated with inflammasome activators. This IL-1beta production was dependent on NF-kappaB activity and could be enhanced by priming with LPS. Finally, chemical inhibition of CFTR activity in control PBMCs and THP-1 cells did not significantly alter IL-1beta or IL-8 production in response to P. aeruginosa.
Conclusion
Hematopoeitic cells appear to be the predominant source of inflammasome-induced pro-inflammatory IL-1beta in CF. PBMCs derived from CF subjects display preserved inflammasome activation and IL-1beta secretion in response to the major CF pathogen Pseudomonas aeruginosa. However, our data do not support the hypothesis that increased IL-1beta production in CF subjects is due to an intrinsic increase in NF-kappaB activity through loss of CFTR function.

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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. 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]. Recurrent inflammatory pulmonary exacerbation is the primary cause of lung disease progression and ultimately, death in CF. 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]. In CF, airway epithelial cells have been shown to produce an exaggerated pro-inflammatory cytokine response to stimulation [4], [5]. 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]. 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].
Interleukin-1 beta (IL-1beta) is a major inflammatory mediator. 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]. Following initiation of the NF-kappaB signaling cascade, IL-1beta is produced in the cytosol as a biologically inactive full-length pro-IL-1beta. Pro-IL-1beta 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-1beta and pro-IL-18 into their biologically active forms for secretion. Four distinct inflammasomes have been recognized. 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].
P. aeruginosa, one of the most common and clinically relevant pathogens among CF patients, activates the NLRC4 inflammasome [19], [20]. Infection with P. aeruginosa triggers an increase in levels of IL-1beta, IL-6, and IL-8 in bronchoalveolar lavage fluid (BALF) from patients with CF [21]. Inflammasome responses depend on NF-kappaB signaling, where NF-kappaB is important in both the upregulation of specific inflammasome components [22], [23], as well as IL-1beta expression [24], [25].
Previous studies support a role for IL-1beta in the pathogenesis of CF inflammatory lung disease. Levels of IL-1beta 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]. Polymorphisms in the IL1B gene have also been associated with varying degrees of disease severity in CF patients [29]. Murine models of CFTR dysfunction have exhibited significant increases in IL-1beta expression or secretion in macrophages [30], [31], and support the hypothesis that the loss of CFTR increases NF-kappaB activation under basal and stimulatory conditions [4], [5], [32], [33]. Finally, replacement of chloride ions with glutamate or gluconate in cell culture media increases secretion of IL-1beta in response to NLRP3 stimulation by adenosine triphosphate (ATP) [34], implying an inhibitory role for extracellular chloride in NLRP3 activation. Taken together, these data implicate the involvement of IL-1beta and consequently, the inflammasomes, in CF inflammatory disease.

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Airway epithelial cells do not produce significant amounts of IL-1beta in response to inflammasome stimulation
The inflammasomes and their respective activators examined in this study are listed in Table 1. Cells were stimulated in accordance with the schedule in Figure 1. In CF, airway epithelial cells have been shown to possess a hyper-inflammatory phenotype and produce an exaggerated pro-inflammatory cytokine response [4], [5]. To determine if airway epithelial cells contribute to the increased IL-1beta 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. IL-1beta 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-1beta production was detected in NuLi-1 and CuFi-1 cells, but not in S9 and IB3-1 cells, by 24 hours. In contrast, these airway cells were highly responsive to other inflammatory stimuli, such as recombinant IL-1beta, producing large quantities of IL-8 (Fig. 2a-d inserts).

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Airway epithelial cells do not significantly upregulate caspase-1 activity in response to inflammasome stimulation
To examine if inflammasome activation occurs in these airway cells, caspase-1 activity was quantified by flow cytometry. There was no significant increase upon stimulation with live PAO1 or LPS+ATP at the times examined (Fig. 3a-b). Because previous studies have indicated a role for caspase-1 in the activation of NF-kappaB through Toll-like receptor (TLR) signaling [35], we examined whether chemical inhibition of caspase-1 altered NF-kappaB-dependent IL-6 production in response to P. aeruginosa. However, treatment with the caspase-1 inhibitor z-YVAD-fmk (YVAD) did not decrease IL-6 secretion by airway epithelial cells (Fig. 3c).

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CD14 positive monocytes from CF patients and controls show similar increases in caspase-1 activity upon inflammasome stimulation
Monocytes were identified in PBMC populations using CD14 as a phenotyping marker. 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).


Input:
PBMCs from CF patients do not produce increased amounts of IL-1beta upon inflammasome stimulation
Previous studies have shown that the loss of CFTR results in increased NF-kappaB activity and pro-inflammatory cytokine secretion [4], [5], [32], [36], [37]. 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. By 24 hours of stimulation, CF PBMCs did not produce increased amounts of IL-1beta (Fig. 5a) or IL-8 (Fig. 5b) when compared to healthy controls. However, we did notice a transient decrease (P<0.001) in the amount of IL-1beta produced by CF cells in response to LPS+ATP at 6 hours (data not shown). Stimulation of PBMCs with P. aeruginosa that lacks exsA (PAO1deltaexsA), a key regulator of type III secretion, produced three-fold less IL-1beta compared to the parental PAO1 strain by 24 hours (Fig. 5a). Inflammasome stimulation without priming did not result in any IL-1beta production in either CF or control PBMCs. Contrary to our hypothesis, these results indicate that PBMCs from CF patients do not display increased production of IL-1beta or IL-8 with inflammasome activation nor do they suggest any increased basal or induced NF-kappaB activity. These results are consistent with our observation that caspase-1 activity is not different between CF and control PBMCs (Fig. 4).

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NF-kappaB activation is required for IL-1beta and IL-8 responses to P. aeruginosa
We confirmed the dependence of PAO1-induced IL-1beta and IL-8 production on NF-kappaB activation using THP-1 cells expressing a reporter driven by NF-kappaB and AP-1 response elements. We found that stimulation of primed THP-1 reporter cells with heat-killed PAO1 produced the highest levels of NF-kappaB/AP-1 activation (Fig. 6a) and this correlated with IL-8 secretion (Fig. 6b) but negligible amounts of IL-1beta were secreted (Fig. 6c). Stimulation of primed THP-1 reporter cells with live PAO1 did not significantly increase NF-kappaB/AP-1 activity (Fig. 6a) or IL-8 secretion (Fig. 6b) over priming alone. However, IL-1beta production was greatly augmented over primed cells stimulated with heat-killed PAO1 or unprimed cells stimulated with live PAO1 (Fig. 6c). This confirmed that NF-kappaB activation alone is not sufficient for maximal IL-1beta secretion, but increased priming of NF-kappaB is capable of augmenting IL-1beta production and secretion upon inflammasome stimulation. Dependency of these responses on NF-kappaB was confirmed by pharmacologic inhibition of NF-kappaB using the Bay11-7082 inhibitor of IkappaBalpha phosphorylation, which significantly reduced NF-kappaB/AP-1 activation (P<0.001) (Fig. 6d) and the subsequent production of IL-8 (P<0.01) (Fig. 6e) and IL-1beta (P<0.001) (Fig. 6f) in response to both heat-killed and live PAO1. These results were also verified in CF and control PBMCs for each inflammasome examined (P<0.001) (Fig. 6g-h). Overall these results confirm that NF-kappaB is an important modulator of IL-1beta production and that increased activation of NF-kappaB augments inflammasome-mediated production of IL-1beta.

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Bay11-7082 inhibits pro-IL-1beta production in response to P. aeruginosa
In addition to inhibition of NF-kappaB activity, Bay11-7082 can also directly inhibit the NLRP3 inflammasome [38]. To validate its use in this study as an NF-kappaB inhibitor, western blots for pro-IL-1beta were performed alongside inhibition of CFTR activity by CFTRinh172 in response to PAO1 at 4 hours after stimulation (Fig. 7a). Our results indicate that Bay11-7082 prevents production of pro-IL-1beta whereas CFTRinh172 does not seem to affect it. This was further corroborated by the ability Bay11-7082 to inhibit IkappaBalpha degradation at 0.5, 1, and 1.5 hours post PAO1 stimulation (Fig. 7b).

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Disruption of CFTR activity does not increase IL-1beta production in PBMCs and THP-1 cells
A previous study has indicated a role for chloride ion concentration in suppression of NLRP3 inflammasome activation [34]. To determine whether CFTR dysfunction alters IL-1beta 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. Treatment with CFTRinh172 did not alter IL-1beta or IL-8 production in control subjects or CF patients (Fig. 8a-b). Similarly, IL-1beta production was not different in monocyte-derived macrophages or THP-1 reporter cells treated with CFTRinh172 (Fig. 8c-d). IL-8 (Fig. 8e) and NF-kappaB activity (Fig. 8f) were also unchanged in CFTRinh172-treated THP-1 reporter cells.

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Levels of IL-1beta 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. 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. However, we found that bronchial epithelial cells do not produce significant amounts of IL-1beta and do not show a significant increase in caspase-1 activation in response to PAO1 and LPS+ATP, in comparison to hematopoeitic mononuclear cells. Hematopoeitically-derived cells, such as monocytes and macrophages, appear to be a principal source of IL-1beta. 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.
Despite our findings indicating that bronchial epithelial cells when grown in vitro are unlikely to be significantly involved in the direct production of IL-1beta (Fig. 2a-d), others have shown their capacity to respond to alveolar macrophage-derived IL-1beta and to amplify the inflammatory response through the induction of chemokines and recruitment of inflammatory effector cells [42]. This interaction may constitute a critical component to effective host defense and diminishing the capacity of host cells to respond to IL-1beta may leave the host susceptible to infections by pathogens such as P. aeruginosa [43]. Conversely, overproduction of IL-1beta can also play a key role in chronic inflammatory responses and cause damage to the lung parenchyma [44], [45].
Although we hypothesized that CF cells would secrete increased amounts of IL-1beta, we found that IL-1beta production in CF PBMCs was not increased upon inflammasome stimulation as compared to controls (Fig. 5a-b). This was in contrast to a previous study from our group, which showed increased IL-1beta production by CF PBMCs in response to LPS [46], although this difference may be accounted for by technical issues in stimulation time and dose. Moreover, IL-1beta production was not increased in CF PBMCs with inflammasome stimulation alone as would be anticipated if there were basal levels of NF-kappaB activation. Cells deficient in CFTR are thought to exhibit an increased basal level of NF-kappaB activity, which leads to increased pro-inflammatory cytokine production including an increased availability of pro-IL-1beta for cleavage and secretion. This amplification of IL-1beta secretion was shown by priming THP-1 monocytes and PBMCs with heat-killed P. aeruginosa or LPS prior to stimulation with live P. aeruginosa. This dramatically increased IL-1beta secretion over stimulation with live P. aeruginosa without priming (Fig. 6c). Similarly, if CF PBMCs expressed increased basal NF-kappaB activity, there would be an increase in IL-1beta secretion in the absence of LPS priming. However, no increase in IL-1beta was observed under basal or primed conditions. Studies investigating the production of IL-1beta have been somewhat inconsistent. A study by Reininger et al. [43] provided evidence that human bronchial epithelial cells possessing the deltaF508 CFTR mutation had a slightly reduced capacity to produce IL-1beta and lacked the ability to induce an early NF-kappaB activation in response to P. aeruginosa. Conversely, a study by Kotrange et al. [31] found that murine bone marrow-derived macrophages expressing deltaF508-CFTR produced increased amounts of IL-1beta when compared to macrophages expressing normal CFTR in response to Burkholderia cenocepacia K56-2. The differentiation of monocytes into macrophages may partly account for the differences observed with the study by Kotrange. Inflammasome-mediated IL-1beta 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]. However, as monocytes and other PBMCs express CFTR [50], [51] and produce large amounts of IL-1beta, they are adequate models to examine the effects of CFTR function on IL-1beta production. Other hematopoeitic cells may also contribute to IL-1beta production. For example, neutrophil counts can be significantly increased in the lungs of CF patients [52], [53], [54] and may produce mature IL-1beta through caspase-1 independent mechanisms [55].
The role of NF-kappaB activation in inflammasome activation and IL-1beta secretion is not straightforward. Studies have revealed an essential role for NF-kappaB activation in the production of pro-IL-1beta and inflammasome components such as NLRP3 [22], [23]. In contrast, deletion of IKKbeta, a kinase essential in NF-kappaB activation, increases IL-1beta secretion in murine macrophages [56], [57] and demonstrates a dual role for NF-kappaB in regulation of IL-1beta. To address this uncertainty in our experiments we also quantified IL-8, an important CF cytokine and marker of NF-kappaB activation [58], and found no differences between CF and control subjects. Similarly, levels of intracellular pro-IL-1beta in THP-1 cells were dependent on NF-kappaB activity and did not increase with CFTRinh172 treatment. Subsequent treatment of THP-1 cells and PBMCs with the NF-kappaB inhibitor Bay11-7082 significantly inhibited both IL-1beta and IL-8 secretion (Fig. 6d-h). Therefore, the IL-1beta and IL-8 responses observed were both dependent upon NF-kappaB activation. Priming with heat-killed P. aeruginosa, like LPS, is unable to induce a strong IL-1beta response as compared to live P. aeruginosa (Fig. 6c), but generated greater NF-kappaB/AP-1 activation (Fig. 6a) and IL-8 secretion (Fig. 6b) than live bacteria despite stimulation at equivalent MOIs. This may be indicative of the different degree and quality of the inflammatory response generated by live as opposed to dead bacteria [59].
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. 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. Consideration must also be given to the nature of P. aeruginosa infection and genotypic changes in P. aeruginosa as infection progresses. P. aeruginosa mediates inflammasome activation through its type III secretion system (T3SS) and the NLRC4 inflammasome [19], [20]. However, clones of P. aeruginosa established during chronic infection may accumulate mutations in virulence factors such as exsA [60]. By employing a deletion mutant in exsA, the key regulator in T3SS transcription, we confirmed that the T3SS is important for IL-1beta secretion (Fig. 5a-b), and that depending on the adaptation in type III secretion, the host IL-1beta response may be up or downregulated [19], [60], [61].
In conclusion, our data are consistent with a role for hematopoietic cells, not airway epithelial cells, as the major source of inflammasome-mediated IL-1beta production in the lungs in response to ATP and P. aeruginosa. Furthermore, we find little evidence to support an increased IL-1beta inflammatory response to NF-kappaB/Inflammasome stimulation in CF patients. 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.

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Ethics Statement
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. Subjects 7 years of age and older were required to provide informed assent as well. Protocols were approved by the Clinical Research Ethics Board (H09-01192).


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Cell Culture
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 deltaF508 and W1282X mutations and CuFi-1 were derived from a deltaF508 homozygous patient. 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. THP1-XBlue cells stably express a secreted embryonic alkaline phosphatase (SEAP) reporter inducible by NF-kappaB and AP-1 (Invivogen). Cells were cultured as recommended by their respective suppliers using standard protocols. 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. 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. 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). THP1-XBlue cells were cultured in complete RPMI with the addition of zeocin (100 microg/ml) to select for cells expressing the SEAP NF-kappaB/AP-1 reporter. Prior to stimulation, bronchial epithelial cell lines were plated in coated [62] 96-well plates (BD Biosciences) at 3x104 cells/well unless indicated, and allowed to adhere overnight. Plates for S9 and IB3-1 stimulations were coated in a mixture of bovine serum albumin (100 microg/ml), fibronectin (10 microg/ml), and bovine collage type I (30 microg/ml) (BD Biosciences). Plates for NuLi-1 and CuFi-1 were coated with collagen type IV (60 microg/ml) (Sigma Aldrich). PBMCs were plated in 96-well plates at a density of 1.5x105 cells/well in 200 microl (7.5x105 cells/mL). 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 1x105 cells/well in 200 microl (5x105 cells/ml). Cells were washed with PBS and allowed to rest a further 42 hours prior to stimulation.


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CF and control subject PBMCs
The diagnosis of CF was established by typical clinical features, increased sweat chloride concentrations (>60 mmol/l), and detection of CF-inducing mutations. 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. Control samples were provided by healthy adult volunteers. 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. Peripheral blood was collected in sodium heparin tubes (BD Biosciences) and PBMCs were isolated using density gradient centrifugation on Ficoll-Paque(TM) Plus (GE Healthcare). The layer containing PBMCs was isolated, washed twice in PBS and resuspended in complete RPMI. Cells were enumerated by trypan blue exclusion using the Countess automated cell counter (Invitrogen). 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. Monocytes were allowed to differentiate in RPMI 1640 supplemented with 10% human AB serum for 10 days.


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Cell stimulation and cytokine quantification
Bronchial epithelial cells were plated and allowed to adhere overnight prior to stimulation. 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. PBMCs and THP-1 reporter cells were either rested or primed with LPS (Invivogen) or heat-killed PAO1 overnight (16 hours). The next day the cells were challenged with live PAO1, PAO1deltaexsA, ATP (Invivogen), or Poly(dA:dT) (Sigma Aldrich) for the times indicated (see Fig. 1). For stimulations with Poly(dA:dT), lipofectamine LTX was used at a 1:1 (w:v) ratio of microg of DNA to microl of lipofectamine and was mixed 30 minutes prior to stimulation. The NF-kappaB inhibitor Bay11-7082 (Invivogen) was added to cultures 1 hour prior to priming. If no priming was involved, inhibitor was added 1 hour prior to inflammasome stimulation. The CFTR inhibitor CFTRinh172 (Sigma Aldrich) was added to cultures 18 hours prior to inflammasome stimulation. The caspase-1 inhibitor z-YVAD-fmk (Biovision) was added to cultures 1 hour prior to inflammasome stimulation. Supernatants were collected and stored at -20degreesC. Cytokines released into supernatants from PBMCs stimulated with inflammasome activators were quantified using sandwich ELISA (eBioscience).


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Immunoblotting
1x106 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). Protein concentrations were determined by Bradford assay (Thermo Scientific). Lysates were resolved by electrophoreses on 10% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore). Blots were blocked for 1 hour at room temperature and probed overnight at 4degreesC for pro-IL-1beta (Santa Cruz), IkappaBalpha (Cell Signaling), or beta-actin (Cell Signaling). Blots were subsequently probed with fluorescently-labeled secondary antibodies, IRDye(R) 680 or 800CW (LI-COR Biosciences) for 1 hour. 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). Blots were imaged on a LI-COR Odyssey infrared imaging system (LI-COR Biosciences) and quantified using the included analysis software.


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Quantification of caspase-1 activity
Bronchial epithelial cells were plated in 6-well plates at 5x105 cells/well overnight. Cells were primed with LPS for 5 hours and stimulated with ATP for 1 hour or stimulated with live PAO1 for 3 hours. PBMCs were stimulated the same day as blood donation. PBMCs were seeded in a 96-well plate at a density of 4.5x105 cells/well (2.5x106 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. Caspase-1 activity was measured using FLICA (Immunochemistry Technologies), a cell-permeable fluorescent probe (FAM-YVAD-fmk) that binds active caspase-1. Cells were incubated 1 hour with FLICA at 37degreesC and stained with PE-Cy7-conjugated anti-CD14 antibodies (eBioscience) to identify monocytes. The gating strategy consisted of including live cells that were CD14 positive which were subsequently analyzed for the frequency of FLICA positive cells.


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NF-kappaB/AP-1 Activity Assay
Supernatants from THP-1 reporter cells were incubated with Quanti-Blue substrate (Invivogen) at 37degreesC and allowed to develop for 16-18 hours. Quanti-Blue contains a substrate for alkaline phosphatase and changes in the amount of NF-kappaB/AP-1 activity were quantified by optical density (lambda = 655) measured using a SpectraMax 384 Plus plate reader and SoftMax Pro software (Molecular Devices).


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Bacterial strains
P. aeruginosa laboratory strains PAO1 and the PAO1deltaexsA mutant were obtained from Dr. Robert Hancock. P. aeruginosa strains PAO1 and PAO1deltaexsA were grown from overnight cultures in Luria Bertani (LB) broth and LB+streptomycin (150 microg/ml) until mid-logarithmic phase. Cells were washed once in PBS and resuspended in PBS to an optical density of 0.5 (lambda = 600 nm). To prepare heat-killed bacteria, live PAO1 was resuspended in PBS to an optical density of 0.5 and heated at 60degreesC for 1 hour. 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. Heat-killed PAO1 was added in a volume equivalent to that used to achieve an MOI of 1 for live PAO1.


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Statistics
All graphs display the mean +/- SEM and were generated with Prism 5 (Graphpad). Statistical significance was determined by performing one or two-way ANOVA and the Bonferroni post-test where applicable.


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Cell stimulation and inhibitor schedule.
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. Inhibitor treatments and stimulations were carried out as described in the Materials and Methods section.


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Airway epithelial cells do not significantly contribute to IL-1beta production in response to inflammasome stimuli.
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-1beta (10 ng/ml), for the indicated times (n = 3 individual experiments). Cells were primed with LPS (100 ng/ml) for 4 hours where appropriate. Cell culture supernatants were assayed for IL-1beta and IL-8 production by ELISA. Insert shows IL-8 secretion in response to stimulation with IL-1beta (10 ng/ml).

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Airway cells do not strongly upregulate caspase-1 activity in response to inflammasome stimuli.
S9 and IB3-1 cells were examined for caspase-1 activation following inflammasome stimulation with P. aeruginosa (MOI = 10) and ATP (5 mM). Cells were primed with LPS for 5 hours where appropriate. 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 (5x104 cells/well) were pre-treated for 1 hour with increasing concentrations of z-YVAD-fmk (10-30 microM) prior to stimulation with P. aeruginosa (MOI = 50). Cell culture supernatants were collected after 6 hours and assayed for IL-6 by ELISA (n = 3).


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PBMCs from CF patients and controls show similar increases in caspase-1 activity upon inflammasome activation.
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 microg/ml) for 3 hours. PBMCs were stimulated with P. aeruginosa strain PAO1 for 3 hours. A representative histogram of the % caspase-1-active cells is shown in (A) with the averaged values shown in (B).


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PBMCs from CF patients do not produce increased amounts of IL-1beta.
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 microg/ml) for 24 hours. P. aeruginosa lacking exsA was used as a type III secretion control in comparison with wild-type P. aeruginosa. Supernatants were assayed for (A) IL-1beta and (B) IL-8.

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NF-kappaB activation potentiates the degree of IL-1beta production and secretion upon inflammasome activation.
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. Cell culture supernatants were assayed for (A) NF-kappaB/AP-1 activity, (B) IL-8, and (C) IL-1beta secretion (n = 3-6 experiments). Using the same stimulation method, THP-1 reporter cells were treated with Bay11-7082 (20 microM) for 1 hour prior to priming with heat-killed PAO1 or live PAO1. Supernatants were assayed at 24 hours for (D) NF-kappaB/AP-1 activity, (E) IL-8, and (F) IL-1beta secretion (n = 3-5). PBMCs from CF patients (n = 11-15) and controls (n = 10-13) were treated with z-YVAD-fmk (20 microM) or Bay11-7082 (10 microM) and stimulated with live PAO1 (MOI = 1), ATP (5 mM), or Poly(dA:dT) (1 microg/ml) according to the schedule in Figure 1. (G) IL-1beta and (H) IL-8 levels were measured at 24 hours. Statistical analysis was performed using two way ANOVA with Bonferroni correction for multiple comparisons. *, **, and *** signify P<0.05, 0.01, and 0.001.

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Bay11-7082 inhibits pro-IL-1beta production in response to P. aeruginosa.
PMA-differentiated THP-1 cells were treated with 10 microM CFTRinh172 or 20 microM Bay11-7082 and harvested after (A) 4 hours (n = 3) or (B) 0.5, 1, and 1.5 hours (n = 3) stimulation with PAO1. One representative blot is shown with a graph of the averaged fluorescence intensity values over 3 experiments.


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Disruption of CFTR activity does not increase IL-1beta production in PBMCs or macrophages.
PBMCs from CF patients (n = 15) and controls (n = 13) were treated with CFTRinh172 (10 microM) for 18 hours prior to stimulation with live PAO1 (MOI = 1). (A) IL-1beta and (B) IL-8 production was measured at 24 hours. (C) Monocytes from controls (n = 3) were differentiated into macrophages. Macrophages were treated with CFTRinh172, stimulated as per monocytes, and measured for IL-1beta production at 24 hours. THP-1 reporter cells were treated with CFTRinh172 24 hours prior to stimulation with PAO1 and measured for (D) IL-1beta secretion, (E) IL-8, and (F) NF-kappaB/AP-1 activity at 24 hours (n = 4).

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Input: Inflammasome Activators.