PMC:3359311 / 12380-15231 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/3359311","sourcedb":"PMC","sourceid":"3359311","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3359311","text":"NF-κB activation is required for IL-1β and IL-8 responses to P. aeruginosa \nWe 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. 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). 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. However, IL-1β 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-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. 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\u003c0.001) (Fig. 6d) and the subsequent production of IL-8 (P\u003c0.01) (Fig. 6e) and IL-1β (P\u003c0.001) (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\u003c0.001) (Fig. 6g–h). 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.\n10.1371/journal.pone.0037689.g006 Figure 6 NF-κB activation potentiates the degree of IL-1β production and secretion upon inflammasome activation.\nTHP-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-κB/AP-1 activity, (B) IL-8, and (C) IL-1β secretion (n = 3–6 experiments). 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. Supernatants were assayed at 24 hours for (D) NF-κB/AP-1 activity, (E) IL-8, and (F) IL-1β secretion (n = 3–5). 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. Statistical analysis was performed using two way ANOVA with Bonferroni correction for multiple comparisons. *, **, and *** signify P\u003c0.05, 0.01, and 0.001.\n\nB","divisions":[{"label":"Title","span":{"begin":0,"end":75}},{"label":"Figure 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