CONTRIBUTION OF NEUTROPHILS TO THE CLEARANCE OF BACTERIA

LANDONI V.I.; CHIARELLA P; MARTIRE-GRECO D; SCHIERLOH P.; LABORDE E; BASILE J.I.; REARTE B.; ISTURIZ M.A.; FERNANDEZ G.C.

Congreso; First French - Argentine Immunology Congress, LVIII Reunión Anual de la Sociedad Argentina de Inmunología, XIII Jornada Científica del Grupo Rioplatense de Citometría de Flujo, 3º Jornadas Argentinas de Inmunodeficiencias ; 2010

The exposure to repetitive doses of LPS generates a refractory state known as tolerance to LPS. Despite impaired immune functions during tolerance, the clearance of infections is increased. Our aim was to address the contribution of neutrophils (PMN) in the enhanced clearance in a murine model of tolerance to LPS. Daily i.v. inoculation of LPS (5ƒÝg, Tol) or saline (Sn) was administered for 4 days. To determine the ability of PMN to eliminate an infection, we depleted peritoneal macrophages by chlodronate-loaded liposomes treatment prior to an i.p. polymicrobial bacterial challenge (5x106 colony formation units, CFU). The remaining CFU in the peritoneum 4h after challenge were lower in Tol (Sn=13125¡Ó1773;Tol=6822¡Ó1988;p<0.05). This was not due to an increased passage of bacteria to the bloodstream, as CFU in blood was also lower in Tol (Sn=209.1¡Ó62.1Tol=33.6¡Ó24.5; p<0.05). An increased number of migrated PMN was also found in peritoneum (PMNx106:Sn=5.3¡Ó0.5;Tol=10.4¡Ó1.3;p<0.05). Moreover, the number of PMN in blood was similar in both groups and did not change after bacterial challenge. However, the number of PMN in the marginal pool was increased in Tol as determined by perfusion of vessels (PMNx106:Sn=0.7¡Ó0.1;Tol=1.6¡Ó02;p<0.05). To determine the mechanisms involved, phagocytosis of i.p. inoculated FITC-bacteria was evaluated and FITC+PMN were determined by FACS. The amount of FITC+ bacteria/PMN was similar in both groups, but the number of FITC+PMN in Tol was higher (FITC+PMNx106:Sn=0.6¡Ó0.1; Tol=0.9¡Ó0.1;p<0.05). Although both groups showed by confocal microscopy the presence of neutrophil extracellular traps (NET) in situ after bacterial challenge, the contribution of NET in clearance was only significant in Tol, showing a two-fold increase in CFU when NET were degraded by S7 nuclease (p<0.05). Thus, the enhanced clearance in Tol is related to NET activity and the higher number of PMN that migrate to the infectious site as a result of an increased marginal pool.