Abstract
ABSTRACT
Introduction
Preterm birth is often associated with fetal membrane (FM) inflammation. Opioid-use disorder during pregnancy is a major public health issue as all opioids carry a risk for preterm birth. While maintenance therapy with methadone improves maternal outcomes, it also elevates the risk of preterm birth. We previously reported that the opioid, buprenorphine, induced human FM sterile inflammation. The goal of this study was to investigate if methadone also induced FM inflammation and to determine the mechanisms involved.
Methods
Human FM explants were treated with or without methadone in the presence or absence of inhibitors to TLR4/MD2, TLR7, TLR8, NLRP3, the μ-opioid receptor, p65 NFκB, p38 MAPK, ERK or JNK. Supernatants were measured for cytokines/chemokines by ELISA. Tissues were analyzed by Western blot for NFκB and MAPK activity, and by RT-qPCR for the TLR7/8-activating miRs: miR-21a, miR-29a, miR-146a-3p, Let-7b. Pregnant
C57BL/6
mice were injected with PBS or methadone on E15.5 and 6hrs later FMs were analyzed by RT-qPCR.
Results
Methadone elevated human FM explant secretion of IL-1β, TNFα, MIP-1α, GM-CSF, and PGE2, and expression of miR-21a
. In vivo
, methadone elevated mouse FM expression of
MIP1A, KC,
and
COX2
. TLR4/MD2 inhibition had no effect on human FM inflammation in response to methadone. NLRP3 inhibition reduced methadone-induced FM IL-1β, TLR7 inhibition reduced methadone-induced FM TNFα, and inhibition of either TLR8 or the μ-opioid receptor reduced methadone-induced secretion of TNFα, MIP-1α, GM-CSF, and PGE2, but not IL-1β. Methadone increased human FM activity of p65 NFκB, p38 MAPK, JNK, and ERK and inhibition of these signaling pathways collectively reduced methadone-triggered FM inflammation. Finally, naloxone reduced most of the methadone-induced FM inflammatory response.
Conclusion
Methadone induces sterile inflammation in human and mouse FMs. In human FMs this inflammation is mediated through activation of innate immune TLR7, TLR8, and NLRP3; the μ-opioid receptor; and NFκB/MAPK signaling. These findings provide further insight into the mechanisms underlying the association between opioid use during pregnancy and preterm birth.