Mucosal immune stimulation after intragastrical inoculation of Shiga toxin-producing Escherichia coli (STEC) in mice immediately after weaning

FERNÁNDEZ BRANDO, RJ; BENTANCOR LV; RAMOS MV; FERNÁNDES GC; RIVAS M; PALERMO MS

Taos, New Mexico, USA.

Simposio; Keystone Symposia on Molecular and Cellular Biology; 2009

Keystone Symposia on Molecular and Cellular Biology.

Hemolytic Uremic Syndrome (HUS) is a rare but life-threatening complication of Shiga-toxin (Stx) Escherichia coli (STEC) infections and is characterized by acute renal failure, thrombocytopenia and hemolytic anemia. Since children up to 5 years old are mostly affected by HUS, it might be associated to an immature age-related immune response. Nevertheless, the STEC-induced immune response is still poorly understood and the existence of a specific and protective immune response to Stx is controversial. Because the mucosal associated lymphoid tissue (MALT) constitutes the first immune compartment met by STEC, the aim of this study was to investigate immune stimulation in Peyer’s Patches (PP) and mesenteric lymphoid nodes (MLN) by studying phenotypic alterations in lymphocytes after a clinical-isolated STEC strain sublethal inoculum. Immature weaned mice were i.g. inoculated with 1 x 109 CFU/kg of E. coli O157:H7 (In) or PBS (Ctrl). Twenty-four hours and 7 days later lymphocytes from PP and MLN were isolated. We evaluated T (CD3, CD4, CD8) and B (CD19) antigens and activation (CD62L) marker, by using FITC/PE-stained specific antibodies and flow cytometry. We found a decrease in PP B-cell percentage as soon as 24 hours post inoculation (mean+SD, Student test) (Ctrl=79+2.4, n=9 vs In=71+6.2, n=15, p<0.005), and it was sustained by the 7th day not only in PP but also in MLN (PP: Ctrl=73+4% vs In=68+6%, n=14, p<0.05; MLN: Ctrl=25+5% vs In=18+4%, n=15, p<0.001). This relative decrease in CD19+ cells was also evidenced as an enhanced CD3:CD19 ratio (PP: Ctrl=0.3+0.1 vs In=0.5+0.2, p<0.005; MLN: Ctrl= 3.1+0.8 vs In=4.7+1.4, p<0.001). In addition to a decreased B-cell absolute number (x103) (PP: Ctrl: 7.0+0.3 vs In=6.4+1.1, p<0.05; MLN: Ctrl=2.5+0.5 vs In=2.0+0.3, p<0.005), there was an increased T-cell absolute number (x103) (PP: Ctrl=2.2+0.5 vs In=2.7+0.7, p<0.05; MLN: Ctrl=7.5+0.5 vs In=8.1+0.5, p<0.005) while CD4+ and CD8+ T-cell percentages were conserved. Inoculated mice also showed a significantly diminished CD62L-mean fluorescence intensity in MLN B-cells at 7 days post inoculation (Ctrl=94.63+5.88 vs In=77.10+8.49, p<0.05). We conclude that STEC induce not only a local T and B cell activation but also selectively decrease B-cells and enhance T-cells from mucosal lymphoid tissues. However, the elucidation of the mechanism and the bacterial components responsible for these effects needs further investigation.