TREM-1: una "puerta" a la inflamación perpetua en monocitos humanos circulantes

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímic

Translocated LPS Might Cause Endotoxin Tolerance in Circulating Monocytes of Cystic Fibrosis Patients

Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene codes of a chloride channel. The lungs of CF patients are chronically infected by several pathogens but bacteraemia have rarely been reported in this pathology. Besides that, circulating monocytes in CF patients exhibit a patent Endotoxin Tolerance (ET) state since they show a significant reduction of the inflammatory response to bacterial stimulus. Despite a previous description of this phenomenon, the direct cause of ET in CF patients remains unknown. In this study we have researched the possible role of microbial/endotoxin translocation from a localized infection to the bloodstream as a potential cause of ET induction in CF patients. Plasma analysis of fourteen CF patients revealed high levels of LPS compared to healthy volunteers and patients who suffer from Chronic Obstructive Pulmonary Disease. Experiments in vitro showed that endotoxin concentrations found in plasma of CF patients were enough to induce an ET phenotype in monocytes from healthy controls. In agreement with clinical data, we failed to detect bacterial DNA in CF plasma. Our results suggest that soluble endotoxin present in bloodstream of CF patients causes endotoxin tolerance in their circulating monocytes

Monocytes from cystic fibrosis patients are locked in an LPS tolerance state: down-regulation of TREM-1 as putative underlying mechanism.

Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene that codes for the chloride channel, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). CF patients are frequently colonized by several pathogens, but the mechanisms that allow colonization in spite of apparently functional immune systems are incompletely understood. In this paper we show that blood peripheral monocytes isolated from CF patients are found in an endotoxin tolerance state, yet this is not due to a deficient TLR activation. On the other hand, levels of the amplifier of inflammatory responses, TREM-1 (Triggering Receptor Expressed on Myeloid cells), are notably down-regulated in monocytes from patients, in comparison to those extracted from healthy volunteers. Furthermore, the soluble form of TREM-1 (sTREM-1) was not detected in the sera of patients. Additionally, and in strict contrast to patients who suffer from Chronic Obstructive Pulmonary Disease (COPD), CF monocytes challenged ex vivo with LPS neither up-regulated membrane-anchored TREM-1 nor sTREM-1. Finally, similar levels of PGE(2) expression and p65 translocation into the nucleus were found in both patients and healthy volunteers, thus suggesting that TREM-1 regulation is neither controlled by PGE(2) levels nor by p65 activation in this case. However, PU.1 translocation into the nucleus was significantly higher in CF monocytes than in controls, suggesting a role for this transcription factor in the control of TREM-1 expression. We conclude that down-regulation of TREM-1 expression in cystic fibrosis patients is at least partly responsible for the endotoxin tolerance state in which their monocytes are locked

CF but not COPD monocytes fail to induce TREM-1 expression after LPS stimulation.

<p>(A) Cultures of human monocytes from healthy volunteers (control, gray bars, n = 10) CF patients (CF, solid bars, n = 20) and COPD patients (COPD, hatched bars, n = 10) were treated with 10 ng/ml LPS for indicated times. Next, cells were harvested and stained with anti-TREM-1-FITC and analyzed by flow cytometry. The fraction of positive cells is given as % of total cells (A) and MFIs (B). The percentage of CD14 and TREM-1 positive cells was analyzed in monocytes from five of each of the studied groups (i.e., CF patients, COPD patients and healthy individuals; all samples were randomly selected), treated or not <i>ex vivo</i> with LPS for 1 hour. The flow cytometry analysis of these cells simultaneously stained with CD14-APC and TREM-1-FITC is shown (C). Supernatants from the same experiment (shown in panels A and B) were centrifuged at 400×g to remove detached cells and possible cellular debris. The concentration of sTREM-1 in the supernatants was analyzed with a commercial ELISA. The mean concentration of sTREM-1 in ng/ml is given (D). *, p<0.05 and **, p<0.01 with respect to similarly treated monocytes from healthy individuals.</p

Inflammatory responses are impaired in monocytes from CF patients.

<p>Monocytes from healthy volunteers (control, gray bars, n = 10) and from CF patients (CF, solid bars, n = 20) were cultured in the presence of 10 ng/ml LPS for indicated times. Cells were harvested, total RNA isolated and cDNA synthesized. TNFα (A) and IL-6 (B) mRNA levels were analyzed by real time Q-PCR. The ratios [TNFα]/[18S] and [IL-6]/[18S] are depicted. **, p<0.01 with respect to healthy controls (C) Concentrations of TNFα were determined in supernatants of cultures of human monocytes from healthy volunteers (control, gray bars, n = 10) and CF patients (CF, solid bars, n = 20), stimulated or not with 10 ng/ml LPS. *, p<0.05 with respect to healthy controls. (D) Monocytes isolated from healthy volunteers (control, gray bars, n = 10) and CF patients (CF, solid bars, n = 20) were stained with anti-CD14-PE, anti-CD16b or anti-CD1a antibodies and then analyzed by flow cytometry; the fraction of cells stained with each antibody is given.</p

Expression levels of TREM-1 and sTREM-1 are low in CF monocytes.

<p>(A) Monocytes isolated from healthy volunteers (controls, gray bars, n = 10) and CF patients (CF, solid bars, n = 20) were stained with anti–TREM-1-FITC and then analyzed by flow cytometry; the fraction of cells stained is given as % of total cells (left scale) and as <u>m</u>ean <u>f</u>luorescence <u>i</u>ntensity (MFI, right scale). *, p<0.05 with respect to healthy controls. (B) A typical histogram obtained from flow cytometric analysis of TREM-1 expression is shown (I, isotype; CF, CF patients; Control, healthy volunteers). (C) The concentration of sTREM-1 in the supernatants of the same cultures was analyzed with a commercial ELISA. The mean concentration of sTREM-1 is represented. (D) Supernatants from monocyte cultures (one from a healthy volunteer (control) and two from randomly selected CF patients) were centrifuged at 400×g to remove detached cells and possible cellular debris. A Western blot analysis of these supernatants was performed using an anti–TREM-1 commercial antibody that recognizes the extra-cellular domain of TREM-1. Human monocytes challenged with LPS for six hours were used as positive control (+). Notice detection of a band of approximately 27 kDa in this condition (marked with an arrow). Loading controls are also shown (membrane stained with Ponceau red, lower panel). The results of a triplicated experiment are shown.</p