Preterm delivery of dead fetuses induced by Shiga toxin type 2 is mediated by TNF-α and nitric oxide.

BURDET JULIANA; CELLA MAXIMILIANO; ZOTTA ELSA; FRANCHI ANA MARÍA; IBARRA CRISTINA

Amsterdam

Simposio; VTEC2012: 8th International Symposium on Shiga Toxin (Verocytotoxin)-producing Escherichia coli infections.; 2012

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:"Times New Roman"; mso-ansi-language:ES; mso-fareast-language:ES;} @page Section1 {size:612.0pt 792.0pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Introduction: Shiga toxin-producing Escherichia coli (STEC) infections could be one of the causes of fetal morbimortality in pregnant women. The main virulence factor of STEC is Shiga toxin type 1 or 2 (Stx1, Stx2), although strains that express only Stx2 are highly prevalent in Argentina.  We have previously reported that Stx2 induced preterm delivery of dead fetuses in rats in the late stage of pregnancy. Stx2 causes vascular injury that involves production of proinflammatory agents such as tumor necrosis factor (TNF)-a, nitric oxide (NO). Both factors are able to stimulate the prostaglandins (PGs) biosynthesis catalyzed by cyclooxygenase (COX)-2, an important key in preterm delivery. The aim of this study was to determine whether TNF, NO and PGs are involved in the mechanisms by which Stx2 induced preterm delivery. Methods: Pregnant rats on days 14-16 of gestation were i.p. injected with 0.5 ml of culture supernatant from recombinant E. coli containing 0.4 µg/ml Stx2 and 30 ng/ml LPS (sStx2). At different times, NO was measured in placenta by conversion of L-[14C]arginine into L-[14C]citruline, PGs in decidua and placentas by RIA, and TNF-a in rat serum by ELISA. Western blot analyses were performed to determine inducible NO synthase (iNOS) and COX expression in placental tissues. Aminoguanidine (AG), meloxicam (Melo) and etanercept (ETA) were used alone or combinated to inhibit NO, PGs and TNF-a, respectively. Stx2-induced preterm delivery induction was evaluated in the presence of these inhibitors. Results: Pregnant rats treated with sStx2 showed an increase of NO, PGE and PGF2a and TNF-a levels (p < 0.05). In addition, a higher expression of iNOS and COX-2 was observed in placentas from Stx2-treated rats than in those from controls. Delivery time and pups status of rats treated with sStx2 and different inhibitors are shown in the table below.       Preterm delivery (%)   Term delivery (%)   Treatments n 17 gd 18 gd 19 gd 20 gd  21 gd   22 gd 23 gd Live /dead pups (%) Control 6 0 0 0 0 0   0 100 100/0 sStx2 8 100 0 0 0 0   0 0 0/100 AG + sStx2 6 100 0 0 0 0   0 0 0/100 Melo + sStx2 8 100 0 0 0 0   0 0 0/100 AG-Melo + sStx2 6 0 0 50 25 25   0 0 0/100 ETA + sStx2 10 30 30 0 0 10   0 30 30/70 ETA-AG + sStx2 10 0 0 0 0 30   0 70 70/30   AG combinated with Melo delayed the delivery time but did not prevent the Stx2-induced preterm delivery. Instead, ETA alone was able to prevent the Stx2 effect on the pregnancy (30%) and this prevention was higher when ETA is administrated with AG (70%).   Conclusions: Our results have demonstrated for the first time that the increase of TNF-a and NO levels produced by sStx2 are mostly responsible for the preterm delivery of dead fetuses induced by Stx2.