Advanced glycation end products and lipopolysaccharides stimulate interleukin-6 secretion via the RAGE/TLR4-NF-κB-ROS pathways and resveratrol attenuates these inflammatory responses in mouse macrophages.

Macrophages are essential for regulating the physiology of pregnancy; however, excessive inflammatory responses to macrophages, induced by infection and/or endogenous danger signals, may potentially result in complications during pregnancy. Advanced glycation end-products (AGE) and lipopolysaccharides (LPS) are known to induce inflammation and are associated with adverse developmental outcomes. The aim of the present study was to examine the effect of AGE and LPS on cytokines in the J774 murine macrophage cell line and the potential effect of resveratrol on AGE- and LPS-induced inflammation in macrophages. AGE and LPS significantly increased IL-6 mRNA expression and secretion in J774 macrophages (P<0.05). Although AGE and LPS significantly stimulated IL-1ß mRNA expression (P<0.05), they had no significant effect on IL-1ß secretion. To assess the receptors for AGE and LPS, including receptor for AGE (RAGE) and Toll-like receptor (TLR4), blocking reagents (RAGE antagonist or TLR4 inhibitor) were added to the J774 macrophages. IL-6 secretion induced by AGE or LPS was significantly inhibited by pretreatment with RAGE antagonist (P<0.05) or TLR4 inhibitor (P<0.05). IL-6 secretion was dependent on nuclear factor (NF)-κB activation and the production of reactive oxygen species (ROS; P<0.05). Resveratrol suppressed mRNA expression and intracellular IL-6 production, resulting in significantly decreased IL-6 secretion after treatment with LPS or AGE (P<0.01). Furthermore, treatment with Ex527, which is a sirtuin-1 (SIRT1) inhibitor, significantly attenuated the anti-inflammatory effect of resveratrol (P<0.05), and treatment with 5-aminoimidazole-4-carboxamide ribonucleotide, which is a 5' adenosine monophosphate-activated protein kinase (AMPK) activator, resulted in a significant decrease in IL-6 secretion in J774 macrophages (P<0.05). The results of the present study indicated that AGE and LPS increase IL-6 secretion depending on NF-κB activation and ROS production through RAGE and/or TLR4 in the J774 murine macrophage cell line. Based on the present study, resveratrol appears to be an effective regulator of the inflammatory responses associated with SIRT1 and AMPK activation in macrophages. These results suggest that resveratrol may have therapeutic applications for controlling immune responses during pregnancy.

Age-associated mRNA expression changes in bovine endometrial cells in vitro.

BACKGROUND: Endometrial cells secrete various cytokines and the dysfunction of endometrial cells may directly lead to infertility. Interferon tau (IFNT) secreted by trophoblast cells, a well-known pregnancy recognition signal in ruminants, acts on the uterus to prepare for pregnancy. Aging causes cellular and organ dysfunction, and advanced maternal age is associated with reduced fertility. However, few studies have investigated age-dependent changes in the uterus. METHODS: Using next generation sequencing and real-time PCR, we examined mRNA expression in bovine endometrial cells in vitro obtained from young (mean 45.2 months) and aged (mean 173.5 months) animals and the effects of IFNT depending on the age. RESULTS: We showed that inflammation-related (predicted molecules are IL1A, C1Qs, DDX58, NFKB, and CCL5) and interferon-signaling (predicted molecules are IRFs, IFITs, STATs, and IFNs) pathways were activated in endometrial cells obtained from aged compared to young cows. Also, the activation of "DNA damage checkpoint regulation" and the inhibition of "mitotic mechanisms" in endometrial cells obtained from aged cows were evident. Moreover, we showed lower cell viability levels in endometrial cells obtained from aged compared to young cows. Although treatment with IFNT upregulated various types of interferon stimulated genes both in endometrial cells obtained from young and aged cows, the rate of increase by IFNT stimulus was obviously lower in endometrial cells obtained from aged compared to young cows. CONCLUSIONS: Endometrial cells obtained from aged cows exhibited higher levels of inflammatory- and IFN-signaling, and dysfunction of cell division compared with young cows. In addition, a high basal level of IFN-related genes in endometrial cells of aged cows is suggested a concept of "inflammaging".

Palmitic acid stimulates interleukin-8 via the TLR4/NF-κB/ROS pathway and induces mitochondrial dysfunction in bovine oviduct epithelial cells.

PROBLEM: We investigated the effect of palmitic acid (PA), a major saturated fatty acid in NEFA, on bovine oviduct epithelial cells (OECs) during in vitro cell culture. METHOD OF STUDY: Bovine oviductal tissues ipsilateral to the corpus luteum were collected 1-3 days after ovulation; the OECs were isolated and cultured. RESULTS: PA increased lipid accumulation and activated caspase-3 in OECs, resulting in decreased cell proliferation. PA also stimulated the secretion of inflammatory cytokine interleukin (IL)-8 depending on TLR4, NF-κB activation, and reactive oxygen species (ROS) production. Moreover, PA induced mitochondrial dysfunction, including mitochondrial fission, ATP production, and mitochondrial ROS production. It also increased levels of LC3 and p62 proteins, suggesting autophagy induction in OECs. CONCLUSION: We suggest that bovine OECs recognize an excessive increase in endogenous and sterile danger signals, such as PA, which may contribute to chronic oviductal inflammation, resulting in infertility associated with oviductal dysfunction.

Age-dependent changes in inflammation and extracellular matrix in bovine oviduct epithelial cells during the post-ovulatory phase.

The mammalian oviduct is an essential site for sperm storage, the transport of gametes, fertilization, and embryo development-functions that are aided by cytokines secreted from oviduct epithelial cells (OECs). Aging leads to cellular and organ dysfunction, with infertility associated with advanced maternal age. Few studies have investigated age-dependent changes in the oviduct as a possible cause of infertility, so we compared OECs from young (30-50 months) versus aged (more than 120 months) cattle. Next-generation sequencing was first used to identify age-related differences in gene expression. Several proinflammatory-related genes (including IL1B, IL1A, IL17C, IL8, S100A8, S100A9, and TNFA) were activated in OECs from aged (more than 120 months) compare to young (30-50 months) individuals, whereas genes associated with extracellular matrix-related factors (COLs, POSTN, BGN, and LUM) were down-regulation in aged OECs. Indeed, IL1 B and IL8 abundance was higher in aged OECs than in young OECs. Young OECs also tended to proliferate faster, and the revolution frequency of young, ciliated OECs was higher than that of their aged counterparts. In contrast, aged OECs possessed more F-actin, an actin cytoskeleton marker associated with reduced elasticity, and contained high levels of reactive oxygen species, which are mediators of inflammation and senescence. These different functional characteristics of bovine OECs during the post-ovulatory phase support the emerging concept of "inflammaging," that is, age-dependent inflammation. Mol. Reprod. Dev. 83: 815-826, 2016 © 2016 Wiley Periodicals, Inc.

Palmitic acid induces interleukin-1ß secretion via NLRP3 inflammasomes and inflammatory responses through ROS production in human placental cells.

Maternal obesity, a major risk factor for adverse pregnancy complications, results in inflammatory cytokine release in the placenta. Levels of free fatty acids are elevated in the plasma of obese human. These fatty acids include obesity-related palmitic acids, which is a major saturated fatty acid, that promotes inflammatory responses. Increasing evidence indicates that nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasomes mediate inflammatory responses induced by endogenous danger signals. We hypothesized that inflammatory responses associated with gestational obesity cause inflammation. To test this hypothesis, we investigated the effect of palmitic acid on the activation of NLRP3 inflammasomes and inflammatory responses in a human Sw.71 trophoblast cell line. Palmitic acid stimulated caspase-1 activation and markedly increased interleukin (IL)-1ß secretion in Sw.71 cells. Treatment with a caspase-1 inhibitor diminished palmitic acid-induced IL-1ß release. In addition, NLRP3 and caspase-1 genome editing using a CRISPR/Cas9 system in Sw.71 cells suppressed IL-1ß secretion, which was stimulated by palmitic acid. Moreover, palmitic acid stimulated caspase-3 activation and inflammatory cytokine secretion (e.g., IL-6 and IL-8). Palmitic acid-induced cytokine secretion were dependent on caspase-3 activation. In addition, palmitic acid-induced IL-1ß, IL-6, and IL-8 secretion was depended on reactive oxygen species (ROS) generation. In conclusion, palmitic acid caused activation of NLRP3 inflammasomes and inflammatory responses, inducing IL-1ß, IL-6, and IL-8 secretion, which is associated with ROS generation, in human Sw.71 placental cells. We suggest that obesity-related palmitic acid induces placental inflammation, resulting in association with pregnancy complications.

AGEs and HMGB1 Increase Inflammatory Cytokine Production from Human Placental Cells, Resulting in an Enhancement of Monocyte Migration.

PROBLEM: Advanced glycation end products (AGEs) and high-mobility group box-1 (HMGB1) are considered contributing to placental inflammation. We examined the effect of AGEs and HMGB1 on cytokines from Sw.71 human trophoblast cell lines and the interactions between Sw.71 cells and THP-1-monocytes. METHODS OF STUDY: Sw.71 cells were cultured with/without AGEs or HMGB1. We examined the role of AGEs or HMGB1 on THP1 migration and effect of AGEs on IL-6 from Sw.71 cells using co-cultures or conditioned medium from THP-1 cells. RESULTS: AGEs and HMGB1 increased interleukin (IL)-6, IL-8, and chemokine C-C motif ligand 2 (CCL2) secretion from Sw.71 cells. The secretion of IL-6 was dependent on reactive oxygen species (ROS) and NF-κB. AGEs stimulated IL-6 secretion through receptor RAGE and TLR4, whereas HMGB1 stimulated it through TLR4. AGEs, but not HMGB1, increased monocyte migration via IL-8 and CCL2 from Sw.71 cells. THP-1 monocytes induced IL-6 secretion from Sw.71 cells, and AGEs further stimulated it. CONCLUSIONS: AGEs and HMGB1 may promote sterile placental inflammation cooperating with monocytes/macrophages.

Moderate Hypoxia Down-Regulates Interleukin-6 Secretion and TLR4 Expression in Human Sw.71 Placental Cells.

BACKGROUND/AIMS: The placenta is a vital organ for pregnancy. Many in vitro placental experiments are conducted under 21% O2; however, O2 tension could influence cellular functions, including cytokine secretion. We investigated the effects of oxygen tension between moderate hypoxia (5% O2) and normoxia (21% O2) by testing the hypothesis that moderate hypoxia regulates cellular phenotypes differently from normoxia in human trophoblast cells. METHODS AND RESULTS: Sw.71 trophoblast cells were incubated under normoxic or moderately hypoxic conditions. Cells were also treated with lipopolysaccharide (LPS) as a Toll-like receptor 4 (TLR4) ligand inducing inflammation. Interleukin-6 (IL-6) as an inflammatory cytokine was determined, and TLR4, hypoxia-induced factor-1α (HIF1α), and reactive oxygen species (ROS) production were detected. Moderate hypoxia increased HIF1α expression and cell proliferation and acted by two different mechanisms to decrease IL-6 secretion compared with normoxia: it limits the TLR4 expression and ROS production. Treatment with cobalt chloride as an HIF1 activator inhibited IL-6 secretion and TLR4 expression; this effect was reversed on treatment with PX-12 as an HIF1 suppressor. CONCLUSION: IL-6 secretion, TLR4 expression, and ROS production, classical markers of inflammation, are down-regulated by moderate hypoxia, and HIF1α and ROS have a potential to regulate these responses in human trophoblast cells.