Interaction of peptidoglycans recognition proteins (PGRPs) with monocytes/macrophages cells

TODONE M; PARRADO C; ROMASANTA P; FERNANDEZ LYNCH MJ; NOLI TRUANT S; ANTONOGLOU B; SARRATEA B; FERNANDEZ M; DE MARZI MC; MALCHIODI EL

Mar del Plata, Buenos Aires

Congreso; LIX Reunión Científica Anual, SAIC, LXII Reunión Anual SAI; 2014

Sociedad Argentina de Inmunología-Sociedad Argentina de Investigación Clínica

Ver página 293 del adjunto PGRPs are pattern recognition receptor that binds peptidoglycan (PGN). Previously, we have determined that PGRPs bind to monocytes/macrophages (THP-1) cell membrane increasing their capacity to phagocyte bacteria and promoting a potent inflammatory response. In the present work, we incubated THP-1 cells with PGRP-S, PGRP-Iα or PGRP?Iβ conjugated with FITC at 37°C and analyzed the results, in the presence of Trypan Blue to quenching the extracellular fluorescence, by flow cytometry (FC) and fluorescence microscopy. We found an increase of 250-300% in FITC fluorescence signal in cells treated with PGRP-FITC respect to controls, suggesting that internalization is effectively occurring after binding. The presence of wortmannin in culture cells did not reduce the incorporation of PGRPs. These results suggest that macropinocytosis would not play a major role in PGRPs uptake. Moreover, PGRP-PGN or PGRP increases 30-100% the level of NF-κB expression respect their respective controls of cells treated or not with PGN. These results suggest that a cellular receptor would be able to recognize PGRPs. Moreover, preliminary results indicated that anti-TLR-2 antibodies reduce PGRPs binding to THP-1 cells. We observed that PGRPs not only diminish apoptotic process produced by PGN, but they also reduce apoptotic process of cells in cultures without PGN. In order to verify this phenomenon we use the LIVE/DEAD Cell Vitality Assay Kit to determine membrane integrity and THP-1 cell viability observing that PGRP or PGRP-PGN decrease ~50% the incorporation of Sytox Green by THP-1 cells respect to controls. Moreover, PGRPs or PGN-PGRPs increased 20-30% the metabolic activity of THP-1 cells respect controls determined as resarzurin incorporation by FC. In all, these results suggest that the PGRPs mediated bacteria/PGN phagocytosis after been recognized by a cell receptor and protect cells from apoptosis induced by PGN or bacterial infection increasing their metabolic activity.