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

FERNANDEZ-BRANDO R.J; BENTANCOR L.; MILIWEBSKY E; RAMOS M.V; FERNÁNDEZ G.C; PANEK C.A; RIVAS M; PALERMO M.S

Buenos Aires

Simposio; 7th International Symposium on Shiga Toxin (Verocytotoxin)-producing Escherichia coli Infections; 2009

Sociedad Argentina de Microbiología

Hemolytic Uremic Syndrome (HUS) is a rare but life-threatening complication of Shiga-toxin (Stx) Escherichia coli (STEC) infection 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 encountered 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. Methods: Immature weaned mice were i.g. inoculated with 1 x 109 CFU/kg of E. coli O157:H7 Stx+ (125/99), 1 x 109 CFU/kg of E. coli O157:H7 Stx- (605/03) or PBS (Ctrl). Twenty-four hours and 7 days after inoculation, 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. Results: A decreased B-cell percentage in PP was found in both E. coli-inoculated mice as soon as 24 hours post inoculation (mean+SD, ANOVA) (Ctrl=78+3, n=13, 605/03=70+6, n=4, 125/99=67+9, n=21, p<0.001). This decrease was still observed after 7 days (Ctrl=74+6%, n=21, 605/03=66+3, n=4, 125/99=70+4%, n=21, p<0.05). The relative decrease in CD19+ cells was also evidenced as an enhanced CD3:CD19 ratio (24hs: Ctrl=0.27+0.04, 605/03=0.41+0.14, 125/99=0.53+0.24, p<0.005; 7d: Ctrl=0.33+0.09, 605/03=0.52+0.07, 125/99=0.42+0.09, p<0.005). Analyzing the percentage of activated B-cells by CD62L expression we observed a significant decrease only in 125/99-inoculated mice in PP at 24h post inoculation (Ctrl=82+2, 605/03=81+3, 125/99=77+3; p<0.05, 125/99 vs Ctrl). On the other hand, 24h post inoculation, the B-cell percentage increased in MLN only in those mice inoculated with 125/99 strain (Ctrl=28+6, n=12, 605/03=27+2, n=4, 125/99=33+6, n=13, p<0.05). In spite of this early increase, 7 days post inoculation a decrease in the B-cell percentage was observed in 125/99- and 605/03-inoculated mice (Ctrl=26+5%, n=20, 605/03=22+3, n=4, 125/99=19+4%, n=20, p<0.001). The relative decrease in CD19+ cells was also evidenced as an enhanced CD3:CD19 ratio (Ctrl=2.98+0.75, 605/03=3.70+0.52, 125/99=4.52+1.31, p<0.05). CD4+ and CD8+ T-cell percentages were conserved. Conclusions: Both E. coli Stx+ and Stx- strains induce, as soon as 24h, a decrease in B-cell percentage from PP which is also observed after 7 days post inoculation. Nevertheless, only E. coli Stx+ strain generates an early drop in PP CD62L+ B-cell percentage and a transient MLN B-cell percentage increase. Although the two E. coli strain shared some virulence factors and cause local stimulation, these results suggest that Stx2 lead to a specific pattern of MALT activation.