We demonstrated that intra-amniotic injection of IL-1 was sufficient to cause chorioamnionitis. However, contrary to our hypothesis, IL-1 was not an important mediator of LPS-induced chorioamnionitis in the preterm fetal sheep model. Interleukin 1 receptor antagonist is an endogenous inhibitor of IL-1 signaling and the recombinant human IL-1ra (Anakinra) used in the present study is an approved anti-inflammatory drug used for rheumatoid arthritis and other inflammatory conditions.12–14
The intra-amniotic dose of rhIL-1ra used in the present study blocked IL-1 signaling, because the inhibitor effectively suppressed IL-1 induced increases in pro-inflammatory cytokine expression in the chorioamnion. The dosing regimen and the same lot of rhIL-1ra also completely blocked intra-amniotic IL-1-induced lung inflammation in the same animals.24
Furthermore, intra-amniotic rhIL-1ra effectively blocked intra-amniotic LPS-induced lung and systemic inflammation.24
Therefore, the experiment is informative for interpreting the role of IL-1 signaling in LPS-induced chorioamnionitis. We did not analyze inflammation in the placenta, because we previously demonstrated that placental inflammation is not a part of the inflammatory response of fetal sheep to chorioamnionitis induced by intra-amniotic LPS.23
Fetal inflammatory response syndrome (FIRS) in the human is a unique systemic inflammatory response defined as chorioamnionitis associated with cord plasma IL-6 levels more than 11 pg/mL.2,3
Unlike the “cytokine storm” causing multi-organ dysfunction associated with the systemic inflammatory response syndrome in the adults,30
FIRS is a more subtle inflammatory response. Despite the modest inflammatory responses, FIRS was postulated to be the proximate cause of multiple adverse neonatal outcomes.31
Since less than 2% of the preterm infants exposed to chorioamnionitis have early onset bacteremia,32
systemic inflammation in the fetus must be initiated by inflammatory responses in the fetal organs in contact with infected amniotic fluid. Indeed, we and others have demonstrated that the fetal chorioamnion, lung, gut, and the skin contribute to the systemic inflammation induced by chorioamnionitis.33–36
We previously demonstrated that IL-1 signaling mediated most of the pulmonary inflammation and part of the systemic inflammation caused by intra-amniotic LPS.24
Taken together, these reports along with the findings of the present study indicate that mechanisms underlying fetal inflammatory responses to chorioamnionitis can be different in different fetal organs. An implication of the study is that chorioamnionitis-induced FIRS may be variably modulated by inhibitors in different fetal organs.
Lipopolysaccharide signals via toll-like receptor 4 (TLR4) whereas IL-1 signals via IL-1R, but both agonists share similar intracellular signaling pathways.37
Subsequent to TLR4 activation, an intracellular signaling cascade is activated culminating in the nuclear translocation and activation of NF-κB, which induces transcription of cytokines and other inflammatory genes. Several studies have examined the interactions between IL-1 and LPS signaling. Lipopolysaccharide-induced preterm labor in mice was not prevented by rhIL-1ra38
or in IL-1β knockout mice.39
Conversely, an IL-1 receptor antagonist reduced systemically administered LPS-induced lethality in adult rabbits and mice.40,41
In a preterm labor model induced by bacterial inoculation of uterine horns in mice, IL-1β signaling was not required but the combined signaling via IL-1β and TNFα receptors was necessary for initiation of preterm labor.21,42
In a nonhuman primate model, Sadowsky et al demonstrated that intra-amniotic infusions of IL-1β and TNFα, but not IL-6 or IL-8, induced preterm labor.18
In preterm fetal sheep, we demonstrated that fetal inflammation induced by intra-amniotic LPS was greatly decreased by inhibition of IL-1 signaling, but not IL-8 signaling,24,27
and intra-amniotic TNFα did not induce fetal inflammation.43
Lipopolysaccharide induced IL-1β expression in the inflammatory cells in the chorioamnion in fetal sheep.44
Collectively, these studies along with the findings of the present experiment demonstrate that different inflammatory signals can initiate fetal inflammation and preterm labor in the setting of chorioamnionitis and that IL-1β can contribute to preterm labor but is not invariantly required to initiate preterm labor.
In addition to IL-1β, other cytokines contribute to the inflammatory response. Increased amniotic IL-17 and the presence of IL-17-positive cells were associated with more advanced chorioamnionitis in preterm deliveries.45
Using a proteomic approach, Buhimschi et al46
demonstrated that the innate immune proteins, defensins 1 and 2, and calgranulins A and C were bonafide biomarkers of fetal inflammation associated with intra-amniotic inflammation. Romero et al47
demonstrated 39 unique (mostly previously unreported) proteomic “fingerprints” associated with chorioamnionitis and preterm labor. In a mouse model of preterm labor induced by intrauterine injection of LPS, proteomic discovery identified the acute phase reactants, SAA1 and 2, as consistently associated with preterm labor.48
Lipopolysaccharide-induced SAA3 expression was not inhibited by rhIL-1ra in the chorioamnion in the present study, while previously we demonstrated that the liver but not lung expression of LPS-induced SAA3 expression was inhibited by rhIL-1ra.24
These identify IL-1-dependent and independent pathways of fetal inflammation associated with chorioamnionitis.
Histopathological evaluation remains the cornerstone of the diagnosis of chorioamnionitis. The severity is based on the anatomic localization of the inflammatory cells (chorion vs. amnion), the numbers of inflammatory cells, and whether the umbilical cord inflammation is present.49
However, predominance of neutrophils (diagnosed by morphologic criteria) is a prerequisite for the diagnosis of acute chorioamnionitis.49
We also observed a predominance of neutrophils in the amniotic fluid or chorioamnion membrane after exposure to LPS and IL-1. Because of limited availability of antibodies for the sheep, a high-resolution evaluation of leukocyte subsets was not possible. Despite the limitation, expression of myeloperoxidase and MCP-1 in the inflammatory cells suggested activation of the inflammatory cells.29
The use of these and other activation markers may be helpful in further exploring the associations of chorioamnionitis with outcomes in preterm infants.
In conclusion, IL-1 can cause chorioamnionitis but may not be an essential mediator of LPS-induced chorioamnionitis. These results have implications for the pathogenesis of fetal inflammatory response syndrome in infants exposed to chorioamnionitis.