Ecology of Danger-dependent Cytokine-boosted Spontaneous Abortion in the CBA × DBA/2 Mouse Model. I. Synergistic Effect of LPS and (TNF-α + IFN-γ) on Pregnancy Loss
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PROBLEM: Previous data have shown "danger" signals, such as bacterial lipopolysaccharide (LPS) acting via toll-like (tlr) receptors are required for early pregnancy failure in several murine abortion models. Indeed, the abortion rate increased in the CBA x DBA/2 model after a gestation day (gd) 7.5 injection of tumour necrosis factor (TNF)-alpha + interferon (IFN)-gamma only if the LPS-tlr signalling pathway was intact. High rates of cytokine-boosted abortion >80% loss can be achieved in certain animal colonies, that have a high endogenous (spontaneous) rate of resorption (30-50%). A specific role for LPS has been postulated to determine both the endogenous and cytokine-boosted losses. METHODS: To test the role of LPS in spontaneous and cytokine-boosted abortions, recombinant TNF-alpha + IFN-gamma, and LPS were injected in different doses and sequences intraperitoneally (i.p.) into CBA x DBA/2 mated mice in the Toronto General Research Institute animal facility where the endogenous abortion rate is <30%. The effects of poly IC, a tlr3 agonist that induces IFN-gamma that can reverse LPS-induced tolerance, and effects of anti-MD-1 on TNF-alpha induction by LPS, poly IC, CPG, or HSP in vitro were also examined. RESULTS: A high endogenous rate of loss similar to that seen in Clamart could be achieved by increasing exposure to LPS on the morning after mating (gd 0.5). The magnitude by which the abortion rate could be increased by an i.p. injection of 2000 u TNF-alpha + 1000 u IFN-gamma on gd 7.5 was independent of the endogenous rate of loss, and could not be increased by doubling the dose. One microgram of LPS given on day 7.5 achieved a similar rate of loss, and if given with the cytokines, synergistically boosted the rate of loss to near Clamart rates. LPS given 1 day prior to the cytokines abrogated the cytokine effect, whereas LPS given day 0.5 had no significant effect on the response to day 7.5 cytokine injection. Blocking MD-1 inhibited TNF-alpha stimulation by poly IC, LPS, CPG, or HSP in vitro, and reduced abortion rates. Poly IC did not avert LPS-type tolerance effects in vivo. CONCLUSIONS: High endogenous rates of abortion in the CBA x DBA/2 model may be explained by exposure to LPS at the time of mating. Increased rates of loss triggered by cytokines later in pregnancy may depend on increased absorption of LPS from intestinal flora.
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