Maternal SARS-CoV-2 infection in pregnancy disrupts gene expression in Hofbauer cells with limited impact on cytotrophoblasts.

BACKGROUND: Hofbauer cells (HBCs) and cytotrophoblasts (CTBs) are major cell populations in placenta. The indirect impact of maternal SARS-CoV-2 disease on these cells that are not directly infected has not been extensively studied. Herein, we profiled gene expression in HBCs and CTBs isolated from placentae of recovered pregnant subjects infected with SARS-CoV-2 during all trimesters of pregnancy, placentae from subjects with active infection, SARS-CoV-2 vaccinated subjects, and those who were unexposed to the virus. METHODS: Placentae were collected within 4 h post-delivery and membrane-free tissues were enzymatically digested for the isolation of HBCs and CTBs. RNA extracted from HBCs and CTBs were sequenced using 150bp paired-end reads. Differentially expressed genes (DEGs) were identified by DESeq2 package in R and enriched in GO Biological Processes, KEGG Pathway, Reactome Gene Sets, Hallmark Gene Sets, and Canonical Pathways. Protein-protein interactions among the DEGs were modelled using STRING and BioGrid. RESULTS: Pregnant subjects (n = 30) were recruited and categorized into six groups: infected with SARS-CoV-2 in i) the first (1T, n = 4), ii) second (2T, n = 5), iii) third (3T, n = 5) trimester, iv) tested positive at delivery (Delivery, n = 5), v) never infected (Control, n = 6), and vi) fully mRNA-vaccinated by delivery (Vaccinated, n = 5). Compared to the Control group, gene expression analysis showed that HBCs from infected subjects had significantly altered gene expression profiles, with the 2T group having the highest number of DEGs (1,696), followed by 3T and 1T groups (1,656 and 958 DEGs, respectively). These DEGs were enriched for pathways involved in immune regulation for host defense, including production of cytokines, chemokines, antimicrobial proteins, ribosomal assembly, neutrophil degranulation inflammation, morphogenesis, and cell migration/adhesion. Protein-protein interaction analysis mapped these DEGs with oxidative phosphorylation, translation, extracellular matrix organization, and type I interferon signaling. Only 95, 23, and 8 DEGs were identified in CTBs of 1T, 2T, and 3T groups, respectively. Similarly, 11 and 3 DEGs were identified in CTBs and HBCs of vaccinated subjects, respectively. Reassuringly, mRNA vaccination did not induce an inflammatory response in placental cells. CONCLUSIONS: Our studies demonstrate a significant impact of indirect SARS-CoV-2 infection on gene expression of inner mesenchymal HBCs, with limited effect on lining CTB cells isolated from pregnant subjects infected and recovered from SARS-CoV-2. The pathways associated with these DEGs identify potential targets for therapeutic intervention.

The FOXP3+ Pro-Inflammatory T Cell: A Potential Therapeutic Target in Crohn's Disease.

BACKGROUND & AIMS: The incidence of Crohn's disease (CD) continues to increase worldwide. The contribution of CD4+ cell populations remains to be elucidated. We aimed to provide an in-depth transcriptional assessment of CD4+ T cells driving chronic inflammation in CD. METHODS: We performed single-cell RNA-sequencing in CD4+ T cells isolated from ileal biopsies of patients with CD compared with healthy individuals. Cells underwent clustering analysis, followed by analysis of gene signaling networks. We overlapped our differentially expressed genes with publicly available microarray data sets and performed functional in vitro studies, including an in vitro suppression assay and organoid systems, to model gene expression changes observed in CD regulatory T (Treg) cells and to test predicted therapeutics. RESULTS: We identified 5 distinct FOXP3+ regulatory Treg subpopulations. Tregs isolated from healthy controls represent the origin of pseudotemporal development into inflammation-associated subtypes. These proinflammatory Tregs displayed a unique responsiveness to tumor necrosis factor-α signaling with impaired suppressive activity in vitro and an elevated cytokine response in an organoid coculture system. As predicted in silico, the histone deacetylase inhibitor vorinostat normalized gene expression patterns, rescuing the suppressive function of FOXP3+ cells in vitro. CONCLUSIONS: We identified a novel, proinflammatory FOXP3+ T cell subpopulation in patients with CD and developed a pipeline to specifically target these cells using the US Food and Drug Administration-approved drug vorinostat.

Vertical Transmission of SARS-CoV-2-Specific Antibodies and Cytokine Profiles in Pregnancy.

Despite intensive characterization of immune responses after COVID-19 infection and vaccination, research examining protective correlates of vertical transmission in pregnancy are limited. Herein, we profiled humoral and cellular characteristics in pregnant women infected or vaccinated at different trimesters and in their corresponding newborns. We noted a significant correlation between spike S1-specific IgG antibody and its RBD-ACE2 blocking activity (receptor-binding domain-human angiotensin-converting enzyme 2) in maternal and cord plasma (P < .001, R > 0.90). Blocking activity of spike S1-specific IgG was significantly higher in pregnant women infected during the third trimester than the first and second trimesters. Elevated levels of 28 cytokines/chemokines, mainly proinflammatory, were noted in maternal plasma with infection at delivery, while cord plasma with maternal infection 2 weeks before delivery exhibited the emergence of anti-inflammatory cytokines. Our data support vertical transmission of protective SARS-CoV-2-specific antibodies. This vertical antibody transmission and the presence of anti-inflammatory cytokines in cord blood may offset adverse outcomes of inflammation in exposed newborns.

Modeling gut neuro-epithelial connections in a novel microfluidic device.

The intestinal lumen is filled with diverse chemical and physical stimuli. Intestinal epithelial cells sense these stimuli and signal to enteric neurons which coordinate a range of physiologic processes required for normal digestive tract function. Yet, the neuro-epithelial connections remain poorly resolved, in part because the tools for orchestrating interactions between these cellular compartments are lacking. We describe the development of a two-compartment microfluidic device for co-culturing enteric neurons with intestinal epithelial cells. The device contains epithelial and neuronal compartments connected by microgrooves. The epithelial compartment was designed for cell seeding via injection and confinement of intestinal epithelial cells derived from human intestinal organoids. We demonstrated that organoids planarized effectively and retained epithelial phenotype for over a week. In the second chamber we dissociated and cultured intestinal myenteric neurons including intrinsic primary afferent neurons (IPANs) from transgenic mice that expressed the fluorescent protein tdTomato. IPANs extended projections into microgrooves, surrounded and frequently made contacts with epithelial cells. The density and directionality of neuronal projections were enhanced by the presence of epithelial cells in the adjacent compartment. Our microfluidic device represents a platform that may, in the future, be used to dissect structure and function of neuro-epithelial connections in the gut and other organs (skin, lung, bladder, and others) in health and disease.

Modeling Gut Neuro-Epithelial Connections in a Novel Micro uidic Device.

Organs that face external environments, such as skin and gut, are lined by epithelia, which have two functions - to provide a semi-permeable barrier and to sense stimuli. The intestinal lumen is filled with diverse chemical and physical stimuli. Intestinal epithelial cells sense these stimuli and signal to enteric neurons which coordinate a range of physiologic processes required for normal digestive tract function. Yet, the neuro-epithelial connections between intestinal epithelial cells and enteric neurons remain poorly resolved, which leaves us with limited mechanistic understanding of their function. We describe the development of a two-compartment microfluidic device for modeling neuro-epithelial interactions, and apply it to form the gut's neuro-epithelial connections. The device contains epithelial and neuronal compartments connected by microgrooves. The epithelial compartment was designed for cell seeding via injection and confinement of intestinal epithelial cells derived from human intestinal organoids. We demonstrated that organoids planarized effectively and retained epithelial phenotype for over a week. In the second chamber we dissociated and cultured intestinal myenteric neurons including intrinsic primary afferent neurons (IPANs) from transgenic mice that expressed the fluorescent protein tdTomato. IPANs extended projections into microgrooves, surrounded and frequently made contacts with epithelial cells. The density and directionality of neuronal projections were enhanced by the presence of epithelial cells in the adjacent compartment. Our microfluidic device represents a platform for dissecting structure and function of neuro-epithelial connections in the gut and other organs (skin, lung, bladder, and others) in health and disease.

Maternal Monocytes Respond to Cell-Free Fetal DNA and Initiate Key Processes of Human Parturition.

Throughout gestation, the maternal immune system is tightly modulated to allow growth of a semiallogeneic fetus. During the third trimester, the maternal immune system shifts to a proinflammatory phenotype in preparation for labor. What induces this shift remains unclear. Cell-free fetal DNA (cffDNA) is shed by the placenta and enters maternal circulation throughout pregnancy. Levels of cffDNA are increased as gestation progresses and peak before labor, coinciding with a shift to proinflammatory maternal immunity. Furthermore, cffDNA is abnormally elevated in plasma from women with complications of pregnancy, including preterm labor. Given the changes in maternal immunity at the end of pregnancy and the role of sterile inflammation in the pathophysiology of spontaneous preterm birth, we hypothesized that cffDNA can act as a damage-associated molecular pattern inducing an inflammatory cytokine response that promotes hallmarks of parturition. To test this hypothesis, we stimulated human maternal leukocytes with cffDNA from primary term cytotrophoblasts or maternal plasma and observed significant IL-1ß and CXCL10 secretion, which coincides with phosphorylation of IFN regulatory factor 3 and caspase-1 cleavage. We then show that human maternal monocytes are crucial for the immune response to cffDNA and can activate bystander T cells to secrete proinflammatory IFN-γ and granzyme B. Lastly, we find that the monocyte response to cffDNA leads to vascular endothelium activation, induction of myometrial contractility, and PGE2 release in vitro. Our results suggest that the immune response to cffDNA can promote key features of the parturition cascade, which has physiologic consequences relevant to the timing of labor.

Increased cell-free fetal DNA release after apoptosis and sterile inflammation in human trophoblast cells.

PROBLEM: Cell-free fetal DNA (cffDNA) shed from the placenta can be detected in maternal blood and increases incrementally during gestation. Concentrations are further elevated with pregnancy complications. Specific activators of cffDNA release in such complications have not been identified. Here, we use trophoblast cells from early and term placenta to examine cffDNA release following apoptosis, infection, and sterile inflammatory stress. METHOD OF STUDY: HTR8/SVneo cells were used to model first-trimester trophoblasts, and term cytotrophoblasts (CTBs) were isolated from placentae collected after uncomplicated deliveries. Trophoblasts were treated with varying concentrations of doxorubicin (DOX), lipopolysaccharide (LPS), or high-mobility group box protein 1 (HMGB1) for 18 h. Cells or supernatants were quantified for caspase-3/7 cleavage, pro-inflammatory cytokine secretion, and cffDNA release. RESULTS: Both HTR8/SVneo and CTBs underwent caspase-3/7 cleavage following DOX treatment, with HTR8/SVneo cells more sensitive to apoptosis than term CTBs. Apoptotic cells released more cffDNA in a dose-dependent manner. Treatment with LPS resulted in an increase in pro-inflammatory IL-6 release, particularly in term CTBs compared to early trophoblasts; however, LPS did not affect cffDNA release. Lastly, while neither cell released more TNF-α following stimulation with HMGB1, both HTR8/SVneo and CTBs released significantly more cffDNA in the presence of HMGB1. CONCLUSIONS: These data show that apoptosis and sterile inflammation induced by DOX and HMGB1, respectively, cause an increase in cffDNA concentrations in both first-trimester and term trophoblasts. Understanding physiologic release of cffDNA during healthy and complicated pregnancy can identify new targets for the diagnosis and treatment of gestational complications.

Cytokinetic effects of Wee1 disruption in pancreatic cancer.

The Wee1 kinase, which is activated in response to DNA damage, regulates exit from G2 through inhibitory phosphorylation of Cdk1/Cdc2, and is an attractive drug target. However, recent work has highlighted effects of Cdk2 phosphorylation by Wee1 on movement through S-phase, suggesting the potential to sensitize to S-phase specific agents by Wee1 inhibitors. In this paper we applied multiparametric flow cytometry to patient-derived pancreatic cancer xenograft tumor cells to study the cell cycle perturbations of Wee1 disruption via the small molecule inhibitor MK-1775, and genetic knockdown. We find that in vitro treatment with MK-1775, and to a lesser degree, Wee1 RNA transcript knockdown, results in the striking appearance of S-phase cells prematurely entering into mitosis. This effect was not seen in vivo in any of the models tested. Here, although we noted an increase of S-phase cells expressing the damage response marker γH2AX, treatment with MK-1775 did not significantly sensitize cells to the cytidine analog gemcitabine. Treatment with MK-1775 did result in a transient but large increase in cells expressing the mitotic marker phosphorylated H3S10 that reached a peak 4 hours after treatment. This suggests a role for Wee1 regulating the progression of genomically unstable cancer cells through G2 in the absence of extrinsically-applied DNA damage. A single dose of 8Gy ionizing radiation resulted in the time-dependent accumulation of Cyclin A2 positive/phosphorylated H3S10 negative cells at the 4N position, which was abrogated by treatment with MK-1775. Consistent with these findings, a genome-scale pooled RNA interference screen revealed that toxic doses of MK-1775 are suppressed by CDK2 or Cyclin A2 knockdown. These findings support G2 exit as the more significant effect of Wee1 inhibition in pancreatic cancers.

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities.

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN(-/-) DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.

Essential gene profiles in breast, pancreatic, and ovarian cancer cells.

UNLABELLED: Genomic analyses are yielding a host of new information on the multiple genetic abnormalities associated with specific types of cancer. A comprehensive description of cancer-associated genetic abnormalities can improve our ability to classify tumors into clinically relevant subgroups and, on occasion, identify mutant genes that drive the cancer phenotype ("drivers"). More often, though, the functional significance of cancer-associated mutations is difficult to discern. Genome-wide pooled short hairpin RNA (shRNA) screens enable global identification of the genes essential for cancer cell survival and proliferation, providing a "functional genomic" map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting ~16,000 genes and a newly developed, dynamic scoring approach, we identified essential gene profiles in 72 breast, pancreatic, and ovarian cancer cell lines. Integrating our results with current and future genomic data should facilitate the systematic identification of drivers, unanticipated synthetic lethal relationships, and functional vulnerabilities of these tumor types. SIGNIFICANCE: This study presents a resource of genome-scale, pooled shRNA screens for 72 breast, pancreatic, and ovarian cancer cell lines that will serve as a functional complement to genomics data, facilitate construction of essential gene profiles, help uncover synthetic lethal relationships, and identify uncharacterized genetic vulnerabilities in these tumor types. SIGNIFICANCE: This study presents a resource of genome-scale, pooled shRNA screens for 72 breast, pancreatic, and ovarian cancer cell lines that will serve as a functional complement to genomics data, facilitate construction of essential gene profiles, help uncover synthetic lethal relationships, and identify uncharacterized genetic vulnerabilities in these tumor types.

Analysis of early C2C12 myogenesis identifies stably and differentially expressed transcriptional regulators whose knock-down inhibits myoblast differentiation.

Myogenesis is a tightly controlled process involving the transcriptional activation and repression of thousands of genes. Although many components of the transcriptional network regulating the later phases of myogenesis have been identified, relatively few studies have described the transcriptional landscape during the first 24 h, when myoblasts commit to differentiate. Through dense temporal profiling of differentiating C2C12 myoblasts, we identify 193 transcriptional regulators (TRs) whose expression is significantly altered within the first 24 h of myogenesis. A high-content shRNA screen of 77 TRs involving 427 stable lines identified 42 genes whose knockdown significantly inhibits differentiation of C2C12 myoblasts. Of the TRs that were differentially expressed within the first 24 h, over half inhibited differentiation when knocked down, including known regulators of myogenesis (Myod1, Myog, and Myf5), as well as 19 TRs not previously associated with this process. Surprisingly, a similar proportion (55%) of shRNAs targeting TRs whose expression did not change also inhibited C2C12 myogenesis. We further show that a subset of these TRs inhibits myogenesis by downregulating expression of known regulatory and structural proteins. Our findings clearly illustrate that several TRs critical for C2C12 myogenesis are not differentially regulated, suggesting that approaches that focus functional studies on differentially-expressed transcripts will fail to provide a comprehensive view of this complex process.

Intersectin 1 forms a complex with adaptor protein Ruk/CIN85 in vivo independently of epidermal growth factor stimulation.

Intersectin 1 (ITSN1) is an adaptor protein involved in clathrin-mediated endocytosis, apoptosis, signal transduction and cytoskeleton organization. Here, we show that ITSN1 forms a complex with adaptor protein Ruk/CIN85, implicated in downregulation of receptor tyrosine kinases. The interaction is mediated by the SH3A domain of ITSN1 and the third or fourth proline-rich blocks of Ruk/CIN85, and does not depend on epidermal growth factor stimulation, suggesting a constitutive association of ITSN1 with Ruk/CIN85. Moreover, both proteins colocalize in MCF-7 cells with their common binding partner, the ubiquitin ligase c-Cbl. The possible biological role of the interaction between ITSN1 and Ruk/CIN85 is discussed.

Significant conservation of synthetic lethal genetic interaction networks between distantly related eukaryotes.

Synthetic lethal genetic interaction networks define genes that work together to control essential functions and have been studied extensively in Saccharomyces cerevisiae using the synthetic genetic array (SGA) analysis technique (ScSGA). The extent to which synthetic lethal or other genetic interaction networks are conserved between species remains uncertain. To address this question, we compared literature-curated and experimentally derived genetic interaction networks for two distantly related yeasts, Schizosaccharomyces pombe and S. cerevisiae. We find that 23% of interactions in a novel, high-quality S. pombe literature-curated network are conserved in the existing S. cerevisiae network. Next, we developed a method, called S. pombe SGA analysis (SpSGA), enabling rapid, high-throughput isolation of genetic interactions in this species. Direct comparison by SpSGA and ScSGA of approximately 220 genes involved in DNA replication, the DNA damage response, chromatin remodeling, intracellular transport, and other processes revealed that approximately 29% of genetic interactions are common to both species, with the remainder exhibiting unique, species-specific patterns of genetic connectivity. We define a conserved yeast network (CYN) composed of 106 genes and 144 interactions and suggest that this network may help understand the shared biology of diverse eukaryotic species.

Multiple domains of Ruk/CIN85/SETA/CD2BP3 are involved in interaction with p85alpha regulatory subunit of PI 3-kinase.

Ruk/CIN85/SETA/CD2BP3 and CD2AP/CMS/METS-1 comprise a new family of proteins involved in such fundamental processes as clustering of receptors and rearrangement of the cytoskeleton in regions of specialised cell-cell contacts, ligand-activated internalisation and targeting to lysosome degradation pathway of receptor tyrosine kinases, and apoptotic cell death. As typical adapter proteins they execute these functions by interacting with other signalling molecules via multiple protein-protein interaction interfaces: SH3 domains, Pro-rich region and coiled-coil domain. It has been previously demonstrated that Ruk is able to interact with the p85alpha regulatory subunit of PI 3-kinase and that the SH3 domain of p85alpha is required for this interaction. However, later observations hinted at a more complex mechanism than simple one-way SH3-Pro-rich interaction. Because interaction with p85alpha was suggested to be important for pro-apoptotic activity of the long isoform of Ruk, Ruk(l)/CIN85, we carried out detailed studies of the mechanism of this interaction and demonstrated that multiple domains are involved; SH3 domains of Ruk are required and sufficient for efficient interaction with full-length p85alpha but the SH3 domain of p85alpha is vital for their "activation" by ousting them from intramolecular interaction with the Pro-rich region of Ruk. Our data also suggest that homodimerisation via C-terminal coiled-coil domain affects both intra- and intermolecular interactions of Ruk proteins.