Circulating monocytes from CF patients exhibit an Endotoxin Tolerant phenotype.

<p>Monocytes from healthy controls (HC, n = 11), CF patients (CF, n = 14), COPD patients (n = 8) and sepsis patients (n = 8) were isolated from circulation and cultured in the presence or not of 10 ng/ml LPS for 3 h. Then, cells were harvested and mRNA levels of IL10 (<i>A</i>), IL12p40 (<i>B</i>) and IL23p19 (<i>C</i>) were determined by real-time Q-PCR. The ratios [gene]/[β-actin] are depicted. *, <i>p</i><0.01, LPS vs. none. (<i>D</i>) Concentrations of TNFα were determined by ELISA in supernatants of cultures after 24 h of stimulation with 10 ng/ml LPS. *, <i>p</i><0.05 LPS <i>vs.</i> none. (<i>E</i>) Cultures of circulating Ms from healthy controls (HC, n = 11), CF (n = 14) and sepsis (n = 8) patients were exposed to GFP-labelled <i>E. coli</i> DH5α bacteria for 1 h according to the protocol described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029577#s4" target="_blank"><i>Materials and Methods</i></a>. Next, adherent cells were harvested and cell internalization was analyzed by flow cytometry. Percentage of green-positive cells is given, *, p<0,05 <i>vs.</i> HC. (<i>F</i>) Heterologous human lymphocytes isolated from healthy volunteers were labelled with the membrane stain PKH2 green fluorescent cell linker kit. Following <i>ex vivo</i> LPS stimulation of monocytes/macrophages isolated from healthy controls (HC, n = 11), CF (n = 14) and sepsis (n = 8) patients for 24 h, stained lymphocytes were added to the plates as responder cells in a relation of 1∶5. After three days, non adherent cells were harvested, and lymphocyte proliferation was assessed by flow cytometry as loss of green fluorescence intensity in the CD3+ gate. The fold induction is shown (*, <i>p</i><0.05 vs HC).