IL-1b Production in Cystic Fibrosis Figure 8. Disruption of CFTR activity does not increase IL-1b production in PBMCs or macrophages. PBMCs from CF patients (n = 15) and controls (n = 13) were treated with CFTRinh172 (10 mM) for 18 hours prior to stimulation with live PAO1 (MOI = 1). (A) IL-1b 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-1b production at 24 hours. THP-1 reporter cells were treated with CFTRinh172 24 hours prior to stimulation with PAO1 and measured for (D) IL-1b secretion, (E) IL-8, and (F) NF-kB/AP-1 activity at 24 hours (n = 4). doi:10.1371/journal.pone.0037689.g008 different degree and quality of the inflammatory response generated by live as opposed to dead bacteria [59]. Potential shortcomings of these experiments include its trans- latability 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 modula- tion 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-1b secretion (Fig. 5a–b), and that depending on the adaptation in type III secretion, the host IL-1b 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-1b production in the lungs in response to ATP and P. aeruginosa. Furthermore, we find little evidence to support an increased IL-1b inflammatory response to NF-kB/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. Materials and Methods 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). 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 DF508 and W1282X mutations and CuFi-1 were derived from a DF508 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-kB and AP- 1 (Invivogen). Cells were cultured as recommended by their PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e37689