Suhas G. Kallapur

Introduction: Decidual macrophages (DMs) are strategically located at the maternal-fetal interface to orchestrate innate host defense while balancing tolerance to the allogenic fetus. An important pregnancy complication is chorioamnionitis characterized by infection/inflammation in the fetal membranes and the amniotic fluid with upregulation of TNFα and other pro-inflammatory cytokines. The goal of this study was to determine maternal vs fetal origin of DMs and to investigate whether TNF signaling play a role in DM response to chorioamnionitis. Methods: Decidua tissue from pregnant Rhesus macaques given intraamniotic injection of lipopolysaccharide/saline with or without the TNF inhibitor Adalimumab (n=33) and human subjects with/out chorioamnionitis (n=11) were used for bulk RNAseq, scRNAseq, and flow cytometry experiments. Results: In both human and Rhesus macaques, DMs coordinately upregulate both pro- and anti-inflammatory cytokines during chorioamnionitis. Although DMs did not express TNF during chorioamnionitis, inhibition of TNF signaling downregulated 50% of LPS induced genes. About 4% of the DMs were of male fetus origin. These fetal origin DMs selectively upregulated IL6 mRNA during chorioamnionitis, suggesting a potential role in fetal immune priming. Discussion: We suggest that DMs orchestrate a carefully balanced host defense with a restrained pro-inflammatory profile during chorioamnionitis to potentially prevent adverse outcomes while protecting the mother-fetus dyad. Activation of fetal origin macrophages during chorioamnionitis may have implications for shaping neonatal immune responses.

Intrauterine infection and inflammation are major causes of preterm labor, yet antibiotics alone often fail to prevent preterm birth despite achieving microbial clearance. Interleukin1 (IL1) is a key mediator of intra-amniotic inflammation and functional progesterone withdrawal, processes linked to labor initiation. We tested whether IL1 blockade with the clinically approved IL1 receptor antagonist, anakinra, could reduce inflammation and preterm labor in a Rhesus macaque model of intra-amniotic Escherichia coli infection followed by delayed antibiotic treatment. Here we show that anakinra reduced the incidence of preterm labor by 40% (p=0.07), attenuated inflammation in the fetal membranes and cervix, and preserved progesterone receptor-B abundance in the fetal membranes. These effects were most pronounced in animals protected from preterm labor. Our findings in a relevant model support the translational potential of combining IL1 blockade with antibiotics for infection-associated preterm labor. One sentence summary: Recombinant human IL1 receptor antagonist (anakinra) decreased E. coli infection induced preterm labor and partially decreased intrauterine inflammation in a non-human primate model of inflammation mediated preterm labor.

Chorioamnionitis or infection of the amniotic fluid and membranes surrounding the fetus can cause fetal systemic inflammation and adverse neurological outcomes. To investigate the pathogenesis of chorioamnionitis-induced neurological injury, we developed a non-human primate model by infusion of live Escherichia coli . A micro-osmotic pump was placed in pregnant Rhesus macaques at ~ 85% term gestation via hysterotomy. Live E. coli (10 ⁵ CFU/h, 1µL/h, n = 7) or sterile broth (Controls, 1µL/h, n = 7) infusions were given over 72 h followed by necropsy. A third group of pregnant macaques received microbicidal antibiotics 24 h after the start of E. coli infusion ( n = 5). Amniotic fluid, cord plasma, and fetal brain regions were carefully preserved for comparative analysis of fetal systemic and brain inflammation. E. coli exposure caused fetal systemic inflammation without bacteremia and induced pro-inflammatory cytokines in the periventricular white matter (PVWM) and thalamus. A diffuse inflammation characterized by aberrant proliferation and activation of IBA1 ⁺ microglia and GFAP ⁺ astrocytes was observed in multiple brain regions. There was a selective vulnerability of the DCX expressing immature neurons with relative sparing of cortical mature neurons expressing MAP2 or NeuN. Periventricular and entorhinal white matter regions were particularly vulnerable with relative sparing of the cortex. These pathogenic changes of fetal brain injury were observed after 72 h exposure. Maternal antibiotics cleared the bacteremia but did not decrease neuroinflammation or neuropathology. These results highlight the potential need for anti-inflammatory adjunctive therapy in addition to antibiotics for optimal outcomes in chorioamnionitis.

Cytometry is an advanced technique for simultaneously identifying and quantifying many cell surface and intracellular proteins at a single-cell resolution. Analyzing high-dimensional cytometry data involves identifying and quantifying cell populations based on their marker expressions. This study provided a quantitative review and comparison of various ways to phenotype cellular populations within the cytometry data, including manual gating, unsupervised clustering, and supervised auto-gating. Six datasets from diverse species and sample types were included in the study, and manual gating with two hierarchical layers was used as the truth for evaluation. For manual gating, results from five researchers were compared to illustrate the gating consistency among different raters. For unsupervised clustering, 23 tools were quantitatively compared in terms of accuracy with the truth and computing cost. While no method outperformed all others, several tools, including PAC-MAN, CCAST, FlowSOM, flowClust, and DEPECHE, generally demonstrated strong performance. For supervised auto-gating methods, four algorithms were evaluated, where DeepCyTOF and CyTOF Linear Classifier performed the best. We further provided practical recommendations on prioritizing gating methods based on different application scenarios. This study offers comprehensive insights for biologists to understand diverse gating methods and choose the best-suited ones for their applications.

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.

Preterm birth, defined as birth before 37 weeks of gestation, is the leading cause of death of children under the age of five years worldwide. Globally there has been no change in the preterm birth rate between 2010 and 2020, when rates were calculated to be 9.8% and 9.9% of all livebirths, respectively. The analysis of amniotic fluid has been advocated by many to identify the mechanisms driving threatened preterm labor. Amniocentesis is largely considered to be safe, but care provider concerns about complications in the setting of threatened preterm labor (tPTL) exist. This systematic review will critically review the data regarding safety and outcomes of amniocentesis in cases of PPROM and tPTL and help in the counseling and clinical decision-making of patients and care providers alike. This systematic review was conducted in accordance with the preferred reporting items for systematic reviews and meta-analyses (PRISMA). All English language, peer-reviewed human studies where amniocentesis was used to sample the amniotic fluid of patients presenting with tPTL or PPROM and published between January 1990 and March 2022 were included. Data on the uptake rates, success rates, and safety profile associated with amniocentesis in this context of tPTL or PPROM were analyzed as part of the systematic review. Included studies compromised of randomized control trials, cohort studies, and case-control studies. Conference abstracts and abstracts with no full text were excluded. 10,215 studies were returned after searches were conducted in MEDLINE, EMBASE, EMCARE, Web of Science, and SCOPUS databases using free text and Medical Subject Headings (MESH). 399 studies were assessed for eligibility with 15 studies being included in the final review. The main reason for exclusion from the review was an absence of safety data. Four studies gave information on uptake rates of amniocentesis in cases of PPROM and tPTL, with a range of 55% in an observational study to >99% in centers offering it as part of routine care. Eleven studies detailed success rates of amniocentesis, with all centers reporting >90% success rates. However, in some centers, a "successful" amniocentesis was deemed to be a retrieval of >0.5 ml. Three studies reported risks associated with amniocentesis. There were four reported complications (all transitory) in a series of 1119 cases (0.35%). This systematic review found that amniocentesis in cases of PPROM or threatened preterm labor is a safe and feasible procedure. These data should give care providers confidence to counsel patients appropriately.

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 liveEscherichia coli. PTL did not occur in LPS challenged rhesus macaques, whileE.coli–infected animals frequently delivered preterm. Although LPS and liveE.coliboth 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 andE.coli. However, neutrophilic infiltration andIL6andPTGS2expression in the amnion was specifically induced by liveE.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 theE.coligroup compared to the LPS group. Our data suggest that the intensity of the host immune response to IUI may determine susceptibility to PTL.

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.