IL-1 and TNF mediates IL-6 signaling at the maternal-fetal interface during intrauterine inflammation.

Introduction: IL6 signaling plays an important role in triggering labor and IL6 is an established biomarker of intrauterine infection/inflammation (IUI) driven preterm labor (PTL). The biology of IL6 during IUI at the maternal-fetal interface was investigated in samples from human subjects and non-human primates (NHP). Methods: Pregnant women with histologic chorioamnionitis diagnosed by placenta histology were recruited (n=28 term, n=43 for preterm pregnancies from 26-36 completed weeks of gestation). IUI was induced in Rhesus macaque by intraamniotic injection of lipopolysachharide (LPS, n=23). IL1 signaling was blocked using Anakinra (human IL-1 receptor antagonist, n=13), and Tumor necrosis factor (TNF) signaling was blocked by anti TNF-antibody (Adalimumab n=14). The blockers were given before LPS. All animals including controls (intraamniotic injection of saline n=27), were delivered 16h after LPS/saline exposure at about 80% gestation. Results: IUI induced a robust expression of IL6 mRNAs in the fetal membranes (chorion-amnion-decidua tissue) both in humans (term and preterm) and NHP. The major sources of IL6 mRNA expression were the amnion mesenchymal cells (AMC) and decidua stroma cells. Additionally, during IUI in the NHP, ADAM17 (a protease that cleaves membrane bound IL6 receptor (IL6R) to release a soluble form) and IL6R mRNA increased in the fetal membranes, and the ratio of IL6 and soluble forms of IL6R, gp130 increased in the amniotic fluid signifying upregulation of IL6 trans-signaling. Both IL1 and TNF blockade suppressed LPS-induced IL6 mRNAs in the AMC and variably decreased elements of IL6 trans-signaling. Discussion: These data suggest that IL1 and TNF blockers may be useful anti-inflammatory agents via suppression of IL6 signaling at the maternal-fetal interface.

Amnion responses to intrauterine inflammation and effects of inhibition of TNF signaling in preterm Rhesus macaque.

Intrauterine infection/inflammation (IUI) is a frequent complication of pregnancy leading to preterm labor and fetal inflammation. How inflammation is modulated at the maternal-fetal interface is unresolved. We compared transcriptomics of amnion (a fetal tissue in contact with amniotic fluid) in a preterm Rhesus macaque model of IUI induced by lipopolysaccharide with human cohorts of chorioamnionitis. Bulk RNA sequencing (RNA-seq) amnion transcriptomic profiles were remarkably similar in both Rhesus and human subjects and revealed that induction of key labor-mediating genes such as IL1 and IL6 was dependent on nuclear factor κB (NF-κB) signaling and reversed by the anti-tumor necrosis factor (TNF) antibody Adalimumab. Inhibition of collagen biosynthesis by IUI was partially restored by Adalimumab. Interestingly, single-cell transcriptomics, flow cytometry, and immunohistology demonstrated that a subset of amnion mesenchymal cells (AMCs) increase CD14 and other myeloid cell markers during IUI both in the human and Rhesus macaque. Our data suggest that CD14+ AMCs represent activated AMCs at the maternal-fetal interface.

The induction of preterm labor in rhesus macaques is determined by the strength of immune response to intrauterine infection.

Intrauterine infection/inflammation (IUI) is a major contributor to preterm labor (PTL). However, IUI does not invariably cause PTL. We hypothesized that quantitative and qualitative differences in immune response exist in subjects with or without PTL. To define the triggers for PTL, we developed rhesus macaque models of IUI driven by lipopolysaccharide (LPS) or live Escherichia coli. PTL did not occur in LPS challenged rhesus macaques, while E. coli-infected animals frequently delivered preterm. Although LPS and live E. coli both caused immune cell infiltration, E. coli-infected animals showed higher levels of inflammatory mediators, particularly interleukin 6 (IL-6) and prostaglandins, in the chorioamnion-decidua and amniotic fluid (AF). Neutrophil infiltration in the chorio-decidua was a common feature to both LPS and E. coli. However, neutrophilic infiltration and IL6 and PTGS2 expression in the amnion was specifically induced by live E. coli. RNA sequencing (RNA-seq) analysis of fetal membranes revealed that specific pathways involved in augmentation of inflammation including type I interferon (IFN) response, chemotaxis, sumoylation, and iron homeostasis were up-regulated in the E. coli group compared to the LPS group. Our data suggest that the intensity of the host immune response to IUI may determine susceptibility to PTL.

TNF-Signaling Modulates Neutrophil-Mediated Immunity at the Feto-Maternal Interface During LPS-Induced Intrauterine Inflammation.

Accumulation of activated neutrophils at the feto-maternal interface is a defining hallmark of intrauterine inflammation (IUI) that might trigger an excessive immune response during pregnancy. Mechanisms responsible of this massive neutrophil recruitment are poorly investigated. We have previously showed that intraamniotic injection of LPS in rhesus macaques induced a neutrophil predominant inflammatory response similar to that seen in human IUI. Here, we demonstrate that anti-TNF antibody (Adalimumab) inhibited ~80% of genes induced by LPS involved in inflammatory signaling and innate immunity in chorio-decidua neutrophils. Consistent with the gene expression data, TNF-blockade decreased LPS-induced neutrophil accumulation and activation at the feto-maternal interface. We also observed a reduction in IL-6 and other pro-inflammatory cytokines but not prostaglandins concentrations in the amniotic fluid. Moreover, TNF-blockade decreased mRNA expression of inflammatory cytokines in the chorio-decidua but not in the uterus, suggesting that inhibition of TNF-signaling decreased the inflammation in a tissue-specific manner within the uterine compartment. Taken together, our results demonstrate a predominant role for TNF-signaling in modulating the neutrophilic infiltration at the feto-maternal interface during IUI and suggest that blockade of TNF-signaling could be considered as a therapeutic approach for IUI, the major leading cause of preterm birth.

Intraamniotic Zika virus inoculation of pregnant rhesus macaques produces fetal neurologic disease.

Zika virus (ZIKV) infection of pregnant women can cause fetal microcephaly and other neurologic defects. We describe the development of a non-human primate model to better understand fetal pathogenesis. To reliably induce fetal infection at defined times, four pregnant rhesus macaques are inoculated intravenously and intraamniotically with ZIKV at gestational day (GD) 41, 50, 64, or 90, corresponding to first and second trimester of gestation. The GD41-inoculated animal, experiencing fetal death 7 days later, has high virus levels in fetal and placental tissues, implicating ZIKV as cause of death. The other three fetuses are carried to near term and euthanized; while none display gross microcephaly, all show ZIKV RNA in many tissues, especially in the brain, which exhibits calcifications and reduced neural precursor cells. Given that this model consistently recapitulates neurologic defects of human congenital Zika syndrome, it is highly relevant to unravel determinants of fetal neuropathogenesis and to explore interventions.

IL-1 signaling mediates intrauterine inflammation and chorio-decidua neutrophil recruitment and activation.

Neutrophil infiltration of the chorioamnion-decidua tissue at the maternal-fetal interface (chorioamnionitis) is a leading cause of prematurity, fetal inflammation, and perinatal mortality. We induced chorioamnionitis in preterm rhesus macaques by intraamniotic injection of LPS. Here, we show that, during chorioamnionitis, the amnion upregulated phospho-IRAK1-expressed neutrophil chemoattractants CXCL8 and CSF3 in an IL-1-dependent manner. IL-1R blockade decreased chorio-decidua neutrophil accumulation, neutrophil activation, and IL-6 and prostaglandin E2 concentrations in the amniotic fluid. Neutrophils accumulating in the chorio-decidua had increased survival mediated by BCL2A1, and IL-1R blockade also decreased BCL2A1+ chorio-decidua neutrophils. Readouts for inflammation in a cohort of women with preterm delivery and chorioamnionitis were similar to findings in the rhesus macaques. IL-1 is a potential therapeutic target for chorioamnionitis and associated morbidities.

Extremely preterm fetal sheep lung responses to antenatal steroids and inflammation.

BACKGROUND: The efficacy of antenatal steroids for fetal lung maturation in the periviable period is not fully understood. OBJECTIVE: We sought to determine the lung maturational effects of antenatal steroids and inflammation in early gestation sheep fetuses, similar to the periviable period in human beings. STUDY DESIGN: Date-mated ewes with singleton fetuses were randomly assigned to 1 of 4 treatment groups (n = 8/group): (1) maternal intramuscular injection of betamethasone; (2) intraamniotic lipopolysaccharide; (3) betamethasone + lipopolysaccharide; and (4) intraamniotic + intramuscular saline (controls). Fetuses were delivered surgically 48 hours later at 94 days' gestation (63% term gestation) for comprehensive evaluations of lung maturation, and lung and systemic inflammation. RESULTS: Relative to controls, first, betamethasone increased the fetal lung air space to mesenchymal area ratio by 47% but did not increase the messenger RNAs for the surfactant proteins-B and -C that are important for surfactant function or increase the expression of pro-surfactant protein-C in the alveolar type II cells. Second, betamethasone increased expression of 1 of the 4 genes in surfactant lipid synthetic pathways. Third, betamethasone increased genes involved in epithelium sodium channel transport, but not sodium-potassium adenosine triphosphatase or Aquaporin 5. Fourth, lipopolysaccharide increased proinflammatory genes in the lung but did not effectively recruit activated inflammatory cells. Last, betamethasone incompletely suppressed lipopolysaccharide-induced lung inflammation. In the liver, betamethasone when given alone increased the expression of serum amyloid A3 and C-reactive protein messenger RNAs. CONCLUSION: Compared the more mature 125-day gestation sheep, antenatal steroids do not induce pulmonary surfactants during the periviable period, indicating a different response.

Intra-amniotic Ureaplasma parvum-Induced Maternal and Fetal Inflammation and Immune Responses in Rhesus Macaques.

BACKGROUND: Although Ureaplasma species are the most common organisms associated with prematurity, their effects on the maternal and fetal immune system remain poorly characterized. METHODS: Rhesus macaque dams at approximately 80% gestation were injected intra-amniotically with 107 colony-forming units of Ureaplasma parvum or saline (control). Fetuses were delivered surgically 3 or 7 days later. We performed comprehensive assessments of inflammation and immune effects in multiple fetal and maternal tissues. RESULTS: Although U. parvum grew well in amniotic fluid, there was minimal chorioamnionitis. U. parvum colonized the fetal lung, but fetal systemic microbial invasion was limited. Fetal lung inflammation was mild, with elevations in CXCL8, tumor necrosis factor (TNF) α, and CCL2 levels in alveolar washes at day 7. Inflammation was not detected in the fetal brain. Significantly, U. parvum decreased regulatory T cells (Tregs) and activated interferon γ production in these Tregs in the fetus. It was detected in uterine tissue by day 7 and induced mild inflammation and increased expression of connexin 43, a gap junction protein involved with labor. CONCLUSIONS: U. parvum colonized the amniotic fluid and caused uterine inflammation, but without overt chorioamnionitis. It caused mild fetal lung inflammation but had a more profound effect on the fetal immune system, decreasing Tregs and polarizing them toward a T-helper 1 phenotype.

Neutrophil recruitment and activation in decidua with intra-amniotic IL-1beta in the preterm rhesus macaque.

Chorioamnionitis, an infection/inflammation of the fetomaternal membranes, is frequently associated with preterm delivery. The mechanisms of inflammation in chorioamnionitis are poorly understood. We hypothesized that neutrophils recruited to the decidua would be the major producers of proinflammatory cytokines. We injected intra-amniotic (IA) interleukin 1beta (IL-1beta) at ∼80% gestation in rhesus macaque monkeys, Macaca mulatta, delivered the fetuses surgically 24 h or 72 h after IA injections, and investigated the role of immune cells in the chorion-amnion decidua. IA IL-1beta induced a robust infiltration of neutrophils and significant increases of proinflammatory cytokines in the chorioamnion decidua at 24 h after exposure, with a subsequent decrease at 72 h. Neutrophils in the decidua were the major source of tumor necrosis factor alpha (TNFalpha) and IL-8. Interestingly, IA IL-1beta also induced a significant increase in anti-inflammatory indoleamine 2,3-dioxygenase (IDO) expression in the decidua neutrophils. The frequency of regulatory T cells (Tregs) and FOXP3 mRNA expression in the decidua did not change after IA IL-1beta injection. Collectively, our data demonstrate that in this model of sterile chorioamnionitis, the decidua neutrophils cause the inflammation in the gestational tissues but may also act as regulators to dampen the inflammation. These results help to understand the contribution of neutrophils to the pathogenesis of chorioamnionitis-induced preterm labor.

ChiS histidine kinase negatively regulates the production of chitinase ChiC in Streptomyces peucetius.

Computational analysis of sequence homology of the chiSRC gene cluster, encoding a chitinase in Streptomyces peucetius, showed that the gene cluster could be a two-component regulon comprising a sensor kinase (chiS) and a response regulator (chiR). To prove that the ChiSRC is an authentic two-component system, the chiS gene was cloned and expressed in E.coli and the purified protein was used for biochemical analysis. In this report, we provide biochemical evidence to show that the sensor kinase encoded by chiS gene indeed is a histidine kinase capable of autophosphorylation and the histidine 144 residue of the ChiS protein is the phosphate acceptor. An insertion mutation at the chiS locus led to overproduction chitinase protein in S. peucetius implying that the chiC gene is negatively regulated by the two-component system.

Repeated exposure to intra-amniotic LPS partially protects against adverse effects of intravenous LPS in preterm lambs.

Histologic chorioamnionitis, frequently associated with preterm births and adverse outcomes, results in prolonged exposure of preterm fetuses to infectious agents and pro-inflammatory mediators, such as LPS. Endotoxin tolerance-type effects were demonstrated in fetal sheep following repetitive systemic or intra-amniotic (i.a.) exposures to LPS, suggesting that i.a. LPS exposure would cause endotoxin tolerance to a postnatal systemic dose of LPS in preterm sheep. In this study, randomized pregnant ewes received either two i.a. injections of LPS or saline prior to preterm delivery. Following operative delivery, the lambs were treated with surfactant, ventilated, and randomized to receive either i.v. LPS or saline at 30 min of age. Physiologic variables and indicators of systemic and lung inflammation were measured. Intravenous LPS decreased blood neutrophils and platelets values following i.a. saline compared to that after i.a. LPS. Intra-amniotic LPS prevented blood pressure from decreasing following the i.v. LPS, but also caused an increased oxygen index. Intra-amniotic LPS did not cause endotoxin tolerance as assessed by cytokine expression in the liver, lung or plasma, but increased myeloperoxidase-positive cells in the lung. The different compartments of exposure to LPS (i.a. vs i.v.) are unique to the fetal to newborn transition. Intra-amniotic LPS incompletely tolerized fetal lambs to postnatal i.v. LPS.

Intra-amniotic IL-1ß induces fetal inflammation in rhesus monkeys and alters the regulatory T cell/IL-17 balance.

Very low birth weight preterm newborns are susceptible to the development of debilitating inflammatory diseases, many of which are associated with chorioamnionitis. To define the effects of chorioamnionitis on the fetal immune system, IL-1ß was administered intra-amniotically at ~80% gestation in rhesus monkeys. IL-1ß caused histological chorioamnionitis, as well as lung inflammation (infiltration of neutrophils or monocytes in the fetal airways). There were large increases in multiple proinflammatory cytokine mRNAs in the lungs at 24 h postadministration, which remained elevated relative to controls at 72 h. Intra-amniotic IL-1ß also induced the sustained expression of the surfactant proteins in the lungs. Importantly, IL-1ß significantly altered the balance between inflammatory and regulatory T cells. Twenty-four hours after IL-1ß injection, the frequency of CD3(+)CD4(+)FOXP3(+) T cells was decreased in lymphoid organs. In contrast, IL-17A-producing cells (CD3(+)CD4(+), CD3(+)CD4(-), and CD3(-)CD4(-) subsets) were increased in lymphoid organs. The frequency of IFN-γ-expressing cells did not change. In this model of a single exposure to an inflammatory trigger, CD3(+)CD4(+)FOXP3(+) cells rebounded quickly, and their frequency was increased at 72 h compared with controls. IL-17 expression was also transient. Interestingly, the T cell profile alteration was confined to the lymphoid organs and not to circulating fetal T cells. Together, these results suggest that the chorioamnionitis-induced IL-1/IL-17 axis is involved in the severe inflammation that can develop in preterm newborns. Boosting regulatory T cells and/or controlling IL-17 may provide a means to ameliorate these abnormalities.

Identification of maternally regulated fetal gene networks in the placenta with a novel embryo transfer system in mice.

The mechanisms for provisioning maternal resources to offspring in placental mammals involve complex interactions between maternally regulated and fetally regulated gene networks in the placenta, a tissue that is derived from the zygote and therefore of fetal origin. Here we describe a novel use of an embryo transfer system in mice to identify gene networks in the placenta that are regulated by the mother. Mouse embryos from the same strain of inbred mice were transferred into a surrogate mother either of the same strain or from a different strain, allowing maternal and fetal effects on the placenta to be separated. After correction for sex and litter size, maternal strain overrode fetal strain as the key determinant of fetal weight (P < 0.0001). Computational filtering of the placental transcriptome revealed a group of 81 genes whose expression was solely dependent on the maternal strain [P < 0.05, false discovery rate (FDR) < 0.10]. Network analysis of this group of genes yielded highest statistical significance for pathways involved in the regulation of cell growth (such as insulin-like growth factors) as well as those involved in regulating lipid metabolism [such as the low-density lipoprotein receptor-related protein 1 (LRP1), LDL, and HDL], both of which are known to play a role in fetal development. This novel technique may be generally applied to identify regulatory networks involved in maternal-fetal interaction and eventually help identify molecular targets in disorders of fetal growth.