NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl- concentration.

The impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) activity induces intracellular chloride (Cl- ) accumulation. The anion Cl- , acting as a second messenger, stimulates the secretion of interleukin-1ß (IL-1ß), which starts an autocrine positive feedback loop. Here, we show that NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) are indirectly modulated by the intracellular Cl- concentration, showing maximal expression and activity at 75 mM Cl- , in the presence of the ionophores nigericin and tributyltin. The expression of PYD and CARD domain containing (PYCARD/ASC) remained constant from 0 to 125 mM Cl- . The CASP1 inhibitor VX-765 and the NLRP3 inflammasome inhibitor MCC950 completely blocked the Cl- -stimulated IL-1ß mRNA expression and partially the IL-1ß secretion. DCF fluorescence (cellular reactive oxygen species, cROS) and MitoSOX fluorescence (mitochondrial ROS, mtROS) also showed maximal ROS levels at 75 mM Cl- , a response strongly inhibited by the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase (NOX) inhibitor GKT137831. These inhibitors also affected CASP1 and NLRP3 mRNA and protein expression. More importantly, the serum/glucocorticoid regulated kinase 1 (SGK1) inhibitor GSK650394, or its shRNAs, completely abrogated the IL-1ß mRNA response to Cl- and the IL-1ß secretion, interrupting the autocrine IL-1ß loop. The results suggest that Cl- effects are mediated by SGK1, in which under Cl- modulation stimulates the secretion of mature IL-1ß, in turn, responsible for the upregulation of ROS, CASP1, NLRP3 and IL-1ß itself, through autocrine signalling.

Epidermal growth factor receptor activity upregulates lactate dehydrogenase A expression, lactate dehydrogenase activity, and lactate secretion in cultured IB3-1 cystic fibrosis lung epithelial cells.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It has been postulated that reduced HCO3- transport through CFTR may lead to a decreased airway surface liquid pH. In contrast, others have reported no changes in the extracellular pH (pHe). We have recently reported that in carcinoma Caco-2/pRS26 cells (transfected with short hairpin RNA for CFTR) or CF lung epithelial IB3-1 cells, the mutation in CFTR decreased mitochondrial complex I activity and increased lactic acid production, owing to an autocrine IL-1ß loop. The secreted lactate accounted for the reduced pHe, because oxamate fully restored the pHe. These effects were attributed to the IL-1ß autocrine loop and the downstream signaling kinases c-Src and JNK. Here we show that the pHe of IB3-1 cells can be restored to normal values (∼7.4) by incubation with the epidermal growth factor receptor (EGFR, HER1, ErbB1) inhibitors AG1478 and PD168393. PD168393 fully restored the pHe values of IB3-1 cells, suggesting that the reduced pHe is mainly due to increased EGFR activity and lactate. Also, in IB3-1 cells, lactate dehydrogenase A mRNA, protein expression, and activity are downregulated when EGFR is inhibited. Thus, a constitutive EGFR activation seems to be responsible for the reduced pHe in IB3-1 cells.

Impairment of CFTR activity in cultured epithelial cells upregulates the expression and activity of LDH resulting in lactic acid hypersecretion.

Mutations in the gene encoding the CFTR chloride channel produce cystic fibrosis (CF). CF patients are more susceptible to bacterial infections in lungs. The most accepted hypothesis sustains that a reduction in the airway surface liquid (ASL) volume favor infections. Alternatively, it was postulated that a reduced HCO3- transport through CFTR leads to a decreased ASL pH, favoring bacterial colonization. The issue is controversial, since recent data from cultured primary cells and CF children showed normal pH values in the ASL. We have reported previously a decreased mitochondrial Complex I (mCx-I) activity in cultured cells with impaired CFTR activity. Thus, we hypothesized that the reduced mCx-I activity could lead to increased lactic acid production (Warburg-like effect) and reduced extracellular pH (pHe). In agreement with this idea, we report here that cells with impaired CFTR function (intestinal Caco-2/pRS26, transfected with an shRNA-CFTR, and lung IB3-1 CF cells) have a decreased pHe. These cells showed increased lactate dehydrogenase (LDH) activity, LDH-A expression, and lactate secretion. Similar effects were reproduced in control cells stimulated with recombinant IL-1ß. The c-Src and JNK inhibitors PP2 and SP600125 were able to increase the pHe, although the differences between control and CFTR-impaired cells were not fully compensated. Noteworthy, the LDH inhibitor oxamate completely restored the pHe of the intestinal Caco-2/pRS26 cells and have a significant effect in lung IB3-1 cells; therefore, an increased lactic acid secretion seems to be the key factor that determine a reduced pHe in these epithelial cells.

Outer membrane vesicles obtained from Bordetella pertussis Tohama expressing the lipid A deacylase PagL as a novel acellular vaccine candidate.

In an effort to devise a safer and effective pertussis acelullar vaccine, outer membrane vesicles (OMVs) were engineered to decrease their endotoxicity. The pagL gene from Bordetella bronchiseptica, which encodes a lipid A 3-deacylase, was expressed in Bordetella pertussis strain Tohama I. The resulting OMVs, designated OMVs(BpPagL), contain tetra- instead of penta-acylated LOS, in addition to pertussis surface immunogens such as pertactin and pertussis toxin, as the wild type OMVs. The characterized pertussis OMVs(BpPagL) were used in murine B. pertussis intranasal (i.n.) challenge model to examine their protective capacity when delivered by i.n. routes. Immunized BALB/c mice were challenged with sublethal doses of B. pertussis. Significant differences between immunized animals and the PBS treated group were observed (p<0.001). Adequate elimination rates (p<0.005) were observed in mice immunized either with OMVs(BpPagL) and wild type OMVs. All OMV preparations tested were non toxic according to WHO criteria; however, OMVs(BpPagL) displayed almost no weight loss at 3 days post administration, indicating less toxicity when compared with wild type OMVs. Induction of IL6- and IL1-expression in lung after i.n. delivery as well as neutrophil recruitment to airways showed coincident results, with a lower induction of the proinflammatory cytokines and lower recruitment in the case of OMVs(BpPagL) compared to wild type OMVs. Given their lower endotoxic activity and retained protective capacity in the mouse model, OMVs(BpPagL) obtained from B. pertussis seem as interesting candidates to be considered for the development of novel multi-antigen vaccine.

Determination of a large number of kinase activities using peptide substrates, P81 phosphocellulose paper arrays and phosphor imaging.

To perform phosphoproteomics and signal transduction studies, a number of protein kinase activities and levels must be simultaneously analyzed in different cell samples and correlated with phosphoprotein patterns to obtain conclusions with regard to the regulation of kinase networks. We describe here a miniaturized format of the classical phosphocellulose (P81) paper binding assay with which up to 594 kinase reactions can be simultaneously analyzed. Kinase peptide substrates possessing a minimum of three consecutive basic residues were subjected to phosphorylation in 96-well plates and aliquots of the phosphorylation reactions were spotted on arrays printed on P81 papers. Phosphorylation levels were quantified using a storage phosphor system imager. The versatility of the procedure was validated by analyzing casein kinase 2, protein kinase C, and p34cdc2/cyclin B in cell extracts and testing the effect of known inhibitors and activators on kinase activities. This improved, miniaturized version of the classical P81 paper method combines simplicity, high sensitivity, high reproducibility, high reliability, and optimal Z factors and takes into account possible sources of background signals. We discuss the possibility of automation and the advantages over other methods.