The intensity of the immune response to LPS and E. coli regulates the induction of preterm labor in Rhesus Macaques

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 lipopolysaccharyde (LPS) or live E. coli. PTL did not occur in LPS challenged Rhesus macaque 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 IL6 and prostaglandins, in the chorioamnion decidua and amniotic fluid. Neutrophil infiltration in the chorion 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. RNASeq analysis of fetal membranes revealed that specific pathways involved in augmentation of inflammation including type I interferon response, chemotaxis, sumoylation and iron homeostasis were upregulated in the E. coli group compared to the LPS group. Our data suggest that intensity of the host immune response to IUI may determine susceptibility to PTL.


INTRODUCTION
Intrauterine infection/inflammation (IUI), most commonly originates in the lower genitourinary tract and ascends to the uterine cavity including the chorio-decidual space and the amniotic cavity. About 40% of preterm labor (PTL) cases are associated with IUI (1). Importantly, the causal link between IUI and PTL is well-established (2). In a prospective preterm labor biomarker study, only 25% of women with increased amniotic fluid (AF) IL-6 had detection of microorganisms in the AF (3). Thus, both intrauterine infection and inflammation can cause PTL. Currently, the rate of prematurity remains stubbornly high at about 10% of all US births (4), causing 75% of perinatal mortality and 50% of the long-term morbidity (1). Apart from maternal morbidity, IUI causes fetal inflammation and increases the risk for fetal and newborn brain, gut, and lung injury in both clinical studies and in animal models (reviewed in (5)). Although IUI is a frequent cause of PTL, about 25-45% of pregnancies at risk for PTL (including cases with presumed IUI) do not delivery preterm within 14d of presentation (3,(6)(7)(8). Thus, PTL is not an invariable consequence of IUI.
However, large unbiased proteomic or genome wide association studies have not yielded clinical useful biomarkers of PTL (11,12). A caveat with transcriptomic studies in both humans and animal models of PTL is that the controls often have no or very little inflammation (13,14). Pathways leading to IUI tend to confound the analyses of PTL.
We previously reported that intraamniotic injection of LPS (from E. coli) or IL1b in pregnant Rhesus macaques caused IUI but not PTL (15)(16)(17). We now report that intraamniotic (IA) injection of live E. coli causes IUI and PTL. Importantly, live E. coli-induced PTL was not rescued by antibiotics, recapitulating the lack of efficacy of prenatal antibiotics in reducing PTL in humans (18,19).
Thus, our two models of LPS-induced IUI, and live E. coli-induced PTL with IUI offer unique opportunities to unravel the IUI specific pathways responsible for induction of PTL.
We tested the hypothesis that quantitative and qualitative thresholds in immune response exist in subjects with or without PTL.

New Rhesus model of IUI with PTL: Intraamniotic E. coli injection
E. coli was used for the studies since this organism is a prototypic invasive perinatal pathogen and a major cause of early neonatal sepsis resulting from maternal IUI (20). In our new model of IUI, pregnant Rhesus macaque were inoculated intraamniotically (IA) with live E. coli (1 x 10 6 CFU). PTL was monitored for 48h after LPS or live E. coli after which surgical delivery was performed. In contrast to IA LPS causing no PTL within 48h, infection with IA E. coli caused PTL in 5/5 dams within 48h of injection ( Table 1). To confirm lack of PTL in the IA LPS group, we extended the period of observation to 5d in another set and confirmed no PTL in 0 out of 9 animals ( Table 1). Furthermore, IA E. coli caused maternal bacteremia in 3/5 dams and fetal bacteremia in 100% of pregnancies ( Table 2).

LPS and E. coli induce similar immune infiltration in the chorioamnion decidua
To characterize IUI, we analyzed immune cell infiltration in the chorioamnion-decidua in different Rhesus models of IUI. Neutrophils were not detected in the controls but readily detected in the chorio-decidua after LPS or E. coli exposure ( Figure 1A). Additionally, E. coli  Figure 2). Neutrophil frequency and counts were higher after E. coli compared to LPS exposure. Macrophages and NK cell frequency decreased after either LPS or E. coli exposure but absolute counts were similar to controls. T-cell frequency was similar among all groups ( Figure 1B) but the absolute counts were higher in the IA E. coli group compared to saline controls (Supplementary Figure 1). NKT cells and B cell frequency or absolute counts did not significantly change in the LPS and E. coli groups ( Figure 1B). Overall, both models induced similar immune infiltration with a higher neutrophil frequency after IA E. coli compared to IA LPS exposure.

E. coli induces higher proinflammatory cytokines and prostaglandins in the AF compared to LPS
We determined the level of inflammation induced in the different IUI models. In comparison to controls, all of the mediators tested increased after LPS or E. coli exposure in the chorioamnion decidua with the exception of IL6, which only increased in the E. coli group (Figure 2A). IL1β, CCL2, IL6 and PTGS2 expression significantly increased in the chorioamnion-decidua of E. coli group compared to LPS (Figure 2A). Endotoxin levels in the amniotic fluid were significantly higher after IA E. coli compared to IA LPS ( Figure 2B). Given that neutrophils were the predominant immune cells in the inflamed chorioamnion-decidua, we next examined their levels in the amniotic fluid (AF). The numbers of AF neutrophils increased comparably in both E. coli and LPS groups ( Figure 2C). We then compared the cytokine responses in the AF of E. coli vs LPS groups. Levels of AF IL-1β, TNFα, IL-6 were all significantly higher in animals inoculated with E. coli and LPS groups compared to controls, with a trend towards higher levels in the E. coli compared to LPS group ( Figure 2D). Similarly, the prostaglandins PGE2 and PGF2α increased in both groups compared to controls but we observed 2-3 fold higher concentration in the AF from E. coli vs LPS animals ( Figure 2E). In contrast to large increases of cytokines in the AF, the changes in maternal plasma were much more modest. E. coli and LPS exposure increased IL-6 and CCL2 levels in the maternal plasma but did not change IL8 or TNFα levels (Supplementary Figure 3A). The fetal plasma cytokines increased slightly but significantly after LPS. Although we only had 2 samples available, E. coli exposure also increased fetal plasma cytokines (Supplementary Figure 3B).
Differential upregulation of mediators in the E. coli vs. LPS groups could be due to time dependent effects. We previously reported that IA LPS induction of intrauterine inflammation is higher at 16h compared to 48h (21). We therefore compared key genes differentially expressed in the E. coli group with LPS exposure of 16h (using samples archived from our previous study (21)). Similar to the results for the IA LPS 48h group, E. coli induction of chorioamnion-decidua expression of IL6 and CCL2 were higher compared to controls and AF prostaglandin levels were higher compared to IA LPS 16h (Supplementary Figure 4A-B).
These results suggest that higher induction of key mediators of IUI after IA E. coli compared to LPS may not be explained by temporal trajectories of gene expression.

E. coli drives higher levels of inflammation in the amnion
Because E. coli but not LPS induced neutrophil infiltration in the amnion ( Figure   1A), we analyzed the anatomic locations of inflammatory response. We surgically separated the amnion from the chorio-decidua. In comparison to controls, all of the mediators tested increased after LPS or E. coli exposure in the amnion with the exception of IL6 which did not increase after LPS exposure (Figure 3). In comparison to the LPS group, E. coli increased amnion expression of IL1b, IL8, TNFa, COX2, CCL2, IL6 and PTGS2 mRNA levels. Together, these data show that inflammation in the amnion is significantly more pronounced after E. coli compared to LPS exposure.

Antibiotic treatment does not decrease E. coli-driven inflammation and preterm birth
To control bacteremia and thus simulate clinical situations, we added antibiotic treatment in pregnant Rhesus infected with E. coli. Cefazolin + enrofloxacin starting 24h after IA E. coli effectively eradicated maternal bacteremia in 7/8 subjects. In 1 subject, there was a persistent amniotic and fetal bacteremia. Despite the clearance of organisms in the antibiotics group, PTL was observed in 6/8 subjects by day 3. To understand if anti-microbial therapy also reduced inflammation, we compared select mediators in different compartments between IA E. coli and IA E. coli + antibiotics (Abx) groups. The tested mediators were comparable between the two groups: neutrophil frequency and IL6 expression in the chorioamnion-decidua ( Figure 4A), IL6 expression in the amnion ( Figure 4B) and IL-6 and PGE2 levels in the amniotic fluid ( Figure 4C) were similar in the 2 groups.

Anatomical localization of IL6 and PTGS2 genes in the fetal membranes
Since IL6 and prostaglandins were the key differentially expressed genes between E. coli +/-Abx and LPS groups, we colocalized IL6 or PTGS2 with MPO, a marker of activated neutrophils. We used dual RNAscope® fluorescence in situ hybridization to visualize the IL6 or PTGS2 mRNA expression coupled with immunofluorescence to detect MPO + cells. Sample from E. coli +/-Abx were combined because they did not show any significant differences (Figure 4). MPO + cells infiltrated the chorio-decidua tissue similarly in both LPS and E. coli +/-Abx groups ( Figure 5A-B), confirming the previous H&E findings ( Figure 1A). However, in the amnion, MPO + cells were rarely detected in the LPS group but abundant in the E. coli +/-Abx group (Figure 5A-B) again confirming the H&E findings ( Figure 1A). The majority (>80-90%) of PTGS2 + cells were also MPO + both in the LPS and E. coli groups, respectively, suggesting that chorio-decidua neutrophils are a major source of prostaglandin production in the fetal membranes during IUI ( Figure   5B). MPO + cells in the amnion of the E. coli exposed animals also expressed PTGS2, with far fewer MPO + PTGS2 + cells in the amnion of the LPS group. In contrast to the chorio-decidua being a major source of PTGS2 expression, IL6 + cells were exclusively present in the amnion of the E. coli +/-Abx group ( Figure 5C) consistent with our rtPCR data (Figure 3). Notably, MPO + cells did not express IL6. Rather, IL6 + cells morphologically appeared to be amnion epithelial cells and the amnion mesenchymal cells. Taken together, these observations indicate that neutrophil recruitment and PTGS2 expression in the chorion is a common feature to both LPS and E. coli +/-Abx immune response. However, neutrophilic PTGS2 expression and IL6 expression in the amnion is differentially induced by E. coli infection.

E. coli induces specific pathways in chorioamnion-decidua involved in exacerbation of inflammation
To gain transcriptomic insights associated with PTL, we compared RNA sequencing (RNA-seq) analysis using the chorioamnion-decidua from the different animal groups. For these studies, we combined RNA-seq analyses from E. coli (n=3) and E. coli+Abx (n=2) since both had similar inflammatory response (Figure 4).

Pathways for preterm labor induction
Within the E. coli exposed group, only 2/8 animals did not undergo PTL (both  Figure 7C).

Discussion
Viviparous species have evolved a strategy of inflammation inducing labor (22,23). A corollary of the inflammation hypothesis is that there is a threshold of inflammation that triggers labor (14). Whether these concepts extend to inflammation-mediated preterm labor are not known. The peculiar anatomy of the female genital tract lends itself to vulnerability for ascension of the lower genital organisms in the upper genital space. An exuberant immune response to invading pathogens can incur collateral tissue injury and preterm labor (24). Therefore, the host immune system must balance the risk of inflammation induced preterm labor with protection from infectious organism. To gain insights into the pathogenesis of preterm labor, we used nonhuman primate models of IUI with and without PTL simulating human chorioamnionitis. We suggest that both the magnitude of inflammation and activation of certain pathways (e.g. IL-6, CCL2, prostaglandins) play a key role in triggering preterm labor (Figure 8).
Although a number of pro-inflammatory cytokines are upregulated both by LPS and live E. coli, IL-6 and prostaglandins levels increased significantly more after E. coli. Mice with IL6 genetic knock out have delayed increases in prostaglandins, delayed onset of normal parturition despite on time progesterone withdrawal, and resistance to LPS induced preterm delivery (25,26). In humans, higher amniotic fluid IL-6 predicted earlier preterm delivery (3,27). However, exogenous administration of IL-6 alone, did not induce preterm labor in either mice or Rhesus macaques (28,29). Thus, IL-6 signaling in the context of an inflammatory response seems to be required to trigger PTL. A number of different knock outs for genes in the prostaglandin signaling pathway in mice demonstrates the critical need for prostaglandin signaling in lysis of the corpus luteum of the ovary leading to progesterone withdrawal and normal parturition in mice (30). In humans, prostaglandin levels increase prior to parturition and administration of prostaglandins can induce preterm labor (31,32). Thus, IL-6 and prostaglandin differentially upregulated by E. coli being potential factors for causing preterm labor is strongly supported by biologic rationale.
Differential responses to inflammatory stimuli may include different cell/tissues participating and/or different quantitative or qualitative host response. We observed that neutrophil infiltration in the amnion and IL6 expression in the amnion was present after live E. coli stimulation but not after LPS injection in the amniotic fluid. Histological demonstration of neutrophils in the amnion denotes a higher stage of chorioamnionitis in humans compared to neutrophils only in the chorio-decidua (33). Although amnion cells can express PTGS2 during normal labor (34), the source of prostaglandins during inflammation mediated preterm labor is not well understood. Both in our LPS and live E. coli models, we demonstrated that activated neutrophils expressing MPO were the major cells expressing inducible PTGS2 expression in the fetal membranes. Interestingly, IL6 expression in the amnion was detected after live E. coli but not after LPS exposure. The cellular source of IL6 was amnion epithelial cells and amnion mesenchymal cells with no contribution from the infiltrating neutrophils.
The quality and quantity of signals that trigger different inflammatory response at the maternal fetal interface are not well explored. Innate immune cells are known to modulate their responses based on input stimuli. As an example, the dynamics of NFkB activation in macrophages depends on which pattern recognition receptor (PRR) signaling is triggered and whether a combination of PRRs are signaled (35). Consistently, in our study, live E. coli with a combination of different PRRs induced more NFkB activation in the fetal membranes compared to LPS alone. Another mechanism that might explain a higher inflammatory response with live organism is that bacterial mRNA is known to induce a more potent innate immune response (36). Effector cells in the host can mount a tiered inflammatory response by switching on or off specific modules of transcription factors in response to different signals (37). Important classes of transcription factor differentially regulated by live E. coli include regulation of chemotaxis, type I IFN axis, sumoylation and iron homeostasis (Figure 7).
Using genetic knock out mice and antibody neutralization, we previously demonstrated that Type IFN axis primes LPS responses on maternal hematopoetic cells, increase the expression of IL6 and TNF and increase susceptibility to preterm birth (26).
Furthermore, we observed that type I interferon priming of LPS responses are conserved in non-human primates and humans (26). Type I interferon signaling can also increase the chemoattractant CXCL10-driven neutrophil recruitment to the site of inflammation (38). Thus, the higher type I interferon response in live E. coli may be an important driver of PTL in our study.
Sumoylation is a post-translational modification by small ubiquitin like modifiers (SUMO) proteins, that regulates innate immune response predominantly by negative regulation of IRF, type I interferon responses and inflammation (39)(40)(41). Our data suggest that E. coli increased the SUMO gene expression in the fetal membranes, which would be expected to decrease inflammation. This is counter to the increased inflammation observed in our study. However, SUMO expression is increased in the placenta in pre-eclampsia and chorioamnionitis (42,43). Since sumoylation regulates function of a vast array of proteins, more work is needed to precisely understand how SUMO genes regulate inflammation during preterm labor.
Inflammation has a potent effect on iron homeostasis. Known as hypoferremia of inflammation, the cytokine-driven increase in hepcidin, largely mediated by IL6, decreases iron transport into plasma (44). The net effect is to decrease availability of nontransferrin bound free iron, which stimulates the growth of certain pathogenic bacteria.
We recently demonstrated that in a Rhesus macaque model of IUI-induced by LPS, the fetus responded by rapidly upregulating hepcidin and lowering iron in fetal blood, without altering amniotic fluid iron status (45). The present study demonstrates a differential regulation of genes involved in iron homeostasis after live E. coli exposure. Overall, the findings suggest that the effects of iron homeostasis are likely due to bacterial infection from an invasive organism and innate host defense response.
A relatively large number of genes are differentially regulated (both up and down) in the chorioamnion-decidua of LPS vs. live E. coli exposed Rhesus macaques. However, in both screens (LPS vs. live E. coli and animals undergoing PTL vs no PTL within the E. coli exposed group), only a handful of differentially regulated pathways were represented. In both screens, IL6, NFkB, type I interferon, and neutrophil mediated immunity genes were represented. In sum, our findings potentially provide possible explanations for the role of both qualitative and quantitative thresholds of inflammation in induction of preterm labor. Our study provides further insights into the pathogenesis of intrauterine inflammation driven PTL. After delivery, fetuses were euthanized with pentobarbital, and fetal tissues were collected. Some control and LPS animals were used in a previous study (21). The animals used for each experiment are listed in Supplementary Table 1.

Chorioamnion-decidua dissection
Extra-placental membranes were dissected away from the placenta as previously described (17,21). After scraping decidua parietalis cells with the attached chorion, the amnion tissue was peeled away from the chorion with forceps. Chorio-decidua cells were washed, and digested with Dispase II (Life Technologies, Grand Island, NY) plus collagenase A (Roche, Indianapolis, IN) for 30 min followed by DNase I (Roche) treatment for another 30 min. Cell suspensions were filtered, the red blood cells lysed and prepared for flow cytometry or FACS-sorting. Viability was >90% by trypan blue exclusion test.
Tissues of fetal membranes were used for RNA analyses described below.

Flow cytometry of chorio-decidua cells
Monoclonal antibodies (mAbs) used for multiparameter flow cytometry (LSR Fortessa 2, BD Biosciences, San Diego, CA) and gating strategy to identify the different leukocyte subpopulations was done as previously described (21)

Chorioamnion-decidua tissue cytokine Quantitative RT-PCR
Total RNA was extracted from snap-frozen chorioamnion-decidua and amnion biopsies after homogenizing in TRIzol (Invitrogen). RNA concentration and quality were measured by Nanodrop spectrophotometer (Thermo-Scientific). Reverse transcription of the RNA and quantitative RT-PCR were performed using qScript One-Step RT-qPCR Kit (Quanta Biosciences), following the manufacturer's instructions and with Rhesus-specific TaqMan gene expression primers (Life Technologies). Eukaryotic 18S rRNA (Life Technologies) was endogenous control for normalization of the target RNAs.

RNA sequencing and analysis
Total RNA was purified, treated with DNase using RNeasy mini kit following manufacturer's recommendation (Qiagen, Valencia, CA, USA). After purification, the concentration of total RNA was measured using Nanodrop ND-1000 spectrophotometer The counts for each gene were obtained using quantMode GeneCounts in the STAR commands, and only counts for the featured genes were reserved. Differential expression analyses were carried out using DESeq2. PCA analysis were performed on the rlog counts from DESeq2 using the R function prcomp. Volcano plots were made for the results from the differential expression analyses. Heatmaps were plotted on the log2 value of the normalized counts. Inference of biological processes were generated using Enrichr (46).

Cytokines and Prostaglandins ELISA
Cytokine/chemokine concentrations in AF, fetal, and maternal plasma were determined by Luminex using non-human primate multiplex kits (Millipore). Lipids were extracted from the AF using methanol to measure prostaglandins PGE2 (Oxford Biomedical Research, Oxford, MI) and PGF2a (Cayman Chemical, Ann Arbor, MI).

Endotoxin assay
Endotoxin level in the AF was determined by Limulus amebocyte lysate assay (LAL; Lonza) according to the test procedure recommended by the manufacturer's instructions.

Histology of fetal membranes for chorioamnionitis
H&E staining for both Rhesus and human fetal membrane was performed, and pictures were taken. H&E-stained sections of human fetal membranes were scored in a blinded manner (by SGK) for chorioamnionitis using criteria outlined by Redline et al (47).

Statistics
Prism version 7 software (GraphPad) was used to analyze data. Values were expressed as means ± SEM. Two-tailed Mann-Whitney U tests (for nonnormally distributed continuous variables) and Fisher's exact test for categorical variables were used to determine differences between groups. Results were considered significant for p ≤ 0.05.

Study approval
All animal procedures were approved by the IACUC at the University of California Davis.

DISCLOSURE
The authors have declared that no conflict of interest exists.