Effects of cytokine-suppressive anti-inflammatory drugs on inflammatory activation in ex vivo human and ovine fetal membranes

Abstract

Intrauterine infection and inflammation are responsible for the majority of early (!32 weeks) spontaneous preterm births (PTBs). Anti-inflammatory agents, delivered intra-amniotically together with antibiotics, may be an effective strategy for preventing PTB. In this study, the effects of four cytokine-suppressive anti-inflammatory drugs (CSAIDs: N-acetyl cysteine (NAC), SB239063, TPCA-1 and NEMO binding domain inhibitor (NBDI)) were assessed on human and ovine gestational membrane inflammation. Full-thickness membranes were collected from healthy, term, human placentas delivered by Caesarean section (nZ5). Using a Transwell model, they were stimulated ex vivo with g-irradiation-killed Escherichia coli applied to the amniotic face. Membranes from near-term, ovine placentas were stimulated in utero with lipopolysaccharide, Ureaplasma parvum or saline control and subjected to explant culture. The effects of treatment with CSAIDs or vehicle (1% DMSO) on accumulation of PGE 2 and cytokines (human interleukin 6 (IL6), IL10 and TNFa; ovine IL8 (oIL8)) were assessed in conditioned media at various time points (3-20 h). In human membranes, the IKKb inhibitor TPCA-1 (7 mM) and p38 MAPK inhibitor SB239063 (20 mM) administered to the amniotic compartment were the most effective in inhibiting accumulation of cytokines and PGE 2 in the fetal compartment. NAC (10 mM) inhibited accumulation of PGE 2 and IL10 only; NBDI (10 mM) had no significant effect. In addition to the fetal compartment, SB239063 also exerted consistent and significant inhibitory effects in the maternal compartment. TPCA-1 and SB239063 suppressed oIL8 production, while all CSAIDs tested suppressed ovine PGE 2 production. These results support the further investigation of intra-amniotically delivered CSAIDs for the prevention of inflammation-mediated PTB.

N,N-Dimethylacetamide Significantly Attenuates LPS- and TNFα-Induced Proinflammatory Responses Via Inhibition of the Nuclear Factor Kappa B Pathway

“…This could be explained by the absence of iNOS in human trophoblast cells, as previously reported (51). In another study, the presence implicated as a valuable therapeutic approach for various conditions including cancer (73,74), chronic kidney disease (75), metabolic diseases (76), airway inflammatory disease (77), arthritis (78), and, in line with our findings in previous and current work, PTB (79). Interestingly, our data do not support any significant effect of DMA on the MAPK pathway.…”

Maternal Intravenous Treatment with either Azithromycin or Solithromycin Clears Ureaplasma parvum from the Amniotic Fluid in an Ovine Model of Intrauterine Infection

“…Thus, additional anti-inflammatory therapy may be needed to prevent PTB. We have recently demonstrated the significant suppressive effect of IA administration of polymyxin B (49) and cytokine-suppressive anti-inflammatory drugs (50) against intrauterine inflammation. In addition, Gravett et al re- ported significantly delayed age at birth and reduction in IA inflammatory cytokine/chemokine levels due to a combination of antibiotics, glucocorticoids, and nonsteroidal anti-inflammatory drugs (47).…”

Preclinical evaluation of drugs to block inflammation-driven preterm birth

“…Six replicate Transwells were established from each membrane and placed into six-well culture plates as previously described (Figure 1b). 20 The outer 'maternal' or decidual-facing compartment contained 3.5 ml supplemented media [DMEM/Ham's Nutrient Mixture F-12, phenol red-free, supplemented with 15 mM HEPES, pH 7.3 (Sigma Aldrich Pty Ltd, Castle Hill, NSW, Australia. ), 0.5% endotoxin/fatty acid-free BSA (Bovogen Biologicals Pty Ltd, East Keilor, VIC, Australia) and 4 mg/ml azithromycin (Pfizer, New York, NY, USA)].…”

“…Shahin et al 18 found that the addition of N-acetyl cysteine (NAC), a free-radical scavenger and non-specific inhibitor of NF-kB and MAPK signalling, in conjunction with progestogen treatment for high-risk women with bacterial vaginosis in mid-pregnancy, greatly reduced the rates of PTB and neonatal morbidity/mortality. NAC has also been shown to prevent pro-inflammatory mediator production in vitro 19,20 and in vivo models of IUI. 21 NAC is not always well tolerated in pregnancy, 18,22 and alternate drugs with similar or improved effects are still being sought.…”

“…a MAPK inhibitor successfully used to block inflammation in animal models and in human fetal membranes; 29,30 and 5 z-7-oxozeaenol (OxZ), a selective inhibitor of TGF-b activated kinase 1 (TAK1, also known as MAP3K7) that has been shown to inhibit pro-inflammatory mediator production in murine fibroblasts, 31 primary cortical neurons, 32 dermal fibroblasts 33 and ear swelling models. 31 We recently compared the effects of NAC and three of these CSAIDs (SB239063, TPCA1 and iNBD) on human and ovine gestational membranes stimulated ex vivo with g-irradiation-killed Escherichia coli and showed that TPCA1 and SB239063, and to a lesser extent NAC, effectively suppressed cytokine and prostaglandin production within just 3 h. 20 Furthermore, we have shown in a large animal model of LPS-induced chorioamnionitis that intra-amniotic administration of a single dose of TPCA1 or OxZ can suppress LPSinduced intra-amniotic inflammatory responses, while fetal effects were minimal. 34 In the present study, we assessed the abilities of five CSAIDs, administered in a manner to replicate intraamniotic delivery, to inhibit pro-inflammatory mediator production at both the amniotic and decidual faces of human, preterm extraplacental membranes delivered after spontaneous labour, both in the presence and absence of HCA and positive bacterial culture (infected; INF).…”