Urinary neutrophils are a hallmark of urinary tract infection (UTI), yet the mechanisms governing their activation, function, and efficacy in controlling infection remain incompletely understood. Tamm-Horsfall glycoprotein (THP), the most abundant protein in urine, uses terminal sialic acids to bind an inhibitory receptor and dampen neutrophil inflammatory responses. We hypothesized that neutrophil modulation is an integral part of THP-mediated host protection. In a UTI model, THPdeficient mice showed elevated urinary tract bacterial burdens, increased neutrophil recruitment, and more severe tissue histopathological changes compared with WT mice. Furthermore, THPdeficient mice displayed impaired urinary NETosis during UTI. To investigate the effect of THP on NETosis, we coupled in vitro fluorescence-based NET assays, proteomic analyses, and standard and imaging flow cytometry with peripheral human neutrophils. We found that THP increases proteins involved in respiratory chain, neutrophil granules, and chromatin remodeling pathways; enhances NETosis in an ROS-dependent manner; and drives NET-associated morphologic features including nuclear decondensation. These effects were observed only in the presence of a NETosis stimulus and could not be solely replicated with equivalent levels of sialic acid alone. We conclude that THP is a critical regulator of NETosis in the urinary tract, playing a key role in host defense against UTI.

Hematologic side effects are associated with prolonged antibiotic exposure in up to 34% of patients. Neutropenia, reported in 10-15% of patients, increases the risk of sepsis and death. Murine studies have established a link between the intestinal microbiota and normal hematopoiesis. We sought to identify predisposing factors, presence of microbiota-derived metabolites, and changes in intestinal microbiota composition in otherwise healthy pediatric patients who developed neutropenia after prolonged courses of antibiotics. In this multi-center study, patients with infections requiring anticipated antibiotic treatment of two or more weeks were enrolled. Stool samples were obtained at the start and completion of antibiotics and at the time of neutropenia. We identified 10 patients who developed neutropenia on antibiotics and 29 controls matched for age, sex, race, and ethnicity. Clinical data demonstrated no association between neutropenia and type of infection or type of antibiotic used; however intensive care unit admission and length of therapy were associated with neutropenia. Reduced intestinal microbiome richness and decreased abundance of Lachnospiraceae family members correlated with neutropenia. Untargeted stool metabolomic profiling revealed several metabolites that were depleted exclusively in patients with neutropenia, including members of the urea cycle pathway, pyrimidine metabolism and fatty acid metabolism that are known to be produced by Lachnospiraceae . Our study confirms a relationship between intestinal microbiota disruption and abnormal hematopoiesis and identifies taxa and metabolites likely to contribute to microbiota-sustained hematopoiesis. As the microbiome is a key determinant of stem cell transplant and immunotherapy outcomes, these findings are likely to be of broad significance. Key Points:Neutropenia occurred in 17% of patients receiving prolonged antibiotic therapy.We found no association between neutropenia and type of infection or class of antibiotic used. Development of neutropenia after prolonged antibiotic treatment was associated with decreased prevalence of Lachnospiraceae and Lachnospiraceae metabolites such as citrulline.

Group B Streptococcus (GBS) is a pervasive perinatal pathogen, yet factors driving GBS dissemination in utero are poorly defined. Gestational diabetes mellitus (GDM), a complication marked by dysregulated immunity and maternal microbial dysbiosis, increases risk for GBS perinatal disease. Using a murine GDM model of GBS colonization and perinatal transmission, we find that GDM mice display greater GBS in utero dissemination and subsequently worse neonatal outcomes. Dual-RNA sequencing reveals differential GBS adaptation to the GDM reproductive tract, including a putative glycosyltransferase ( yfhO ), and altered host responses. GDM immune disruptions include reduced uterine natural killer cell activation, impaired recruitment to placentae, and altered maternofetal cytokines. Lastly, we observe distinct vaginal microbial taxa associated with GDM status and GBS invasive disease status. Here, we show a model of GBS dissemination in GDM hosts that recapitulates several clinical aspects and identifies multiple host and bacterial drivers of GBS perinatal disease.

Prolonged antibiotic exposure causes dangerous hematologic side effects, including neutropenia, in up to 34% of patients. Murine studies established a link between the intestinal microbiota and hematopoiesis. To identify factors that predispose to neutropenia in pediatric patients, we evaluated changes in microbiota-derived metabolites and intestinal microbiota composition after prolonged courses of antibiotics. In this multi-center study, patients with infections requiring anticipated antibiotic treatment of two or more weeks were enrolled. Stool samples were obtained at the start and completion of antibiotics or at neutropenia onset (prospective arm). Some patients were enrolled in a retrospective arm in which a stool sample was collected at the time of neutropenia during antibiotic therapy and 2-4 weeks after completion of antibiotics with recovery of blood counts. We identified 10 patients who developed neutropenia on antibiotics and 29 controls matched for age, sex, race, and ethnicity. Clinical data demonstrated no association between neutropenia and the type of infection or antibiotic used; however, patients with neutropenia were admitted to the intensive care unit more often and received longer courses of antibiotics. Reduced intestinal microbiome richness and, specifically, decreased abundance of Lachnospiraceae family members correlated with neutropenia. Untargeted stool metabolomic profiling revealed several metabolites that were depleted exclusively in patients with neutropenia, including members of the urea cycle pathway, pyrimidine metabolism, and fatty acid metabolism that are known to be produced by Lachnospiraceae. Our study shows a relationship between intestinal microbiota disruption and abnormal hematopoiesis and identifies taxa and metabolites likely to contribute to microbiota-sustained hematopoiesis.

Preterm birth is the leading cause of infant mortality resulting in over one million neonatal deaths annually. Maternal urinary tract infection (UTI) during pregnancy increases risk for preterm birth; however, biological processes mediating UTI-associated preterm birth are not well-described. We established a murine maternal UTI model in which challenge with uropathogenic E. coli resulted in preterm birth in about half of dams. Dams experiencing preterm birth displayed excessive bladder inflammation and altered uteroplacental T cell polarization compared to non-laboring infected dams, with no differences in bacterial burdens. Additional factors associated with preterm birth included higher proportions of male fetuses and lower maternal serum IL-10. Furthermore, exogenous maternal IL-10 treatment absolved UTI-associated preterm birth but contributed to fetal growth restriction in this model. Using urine samples from a cohort of human pregnancies with or without UTI, we correlated urinary cytokines with birth outcomes and urine culture status. These analyses yielded a non-invasive, highly predictive three-model system for evaluating preterm birth risk implicating cytokines IL-10, IL-15, IL-1β, and IL-1RA. Our unique bimodal murine model coupled with patient samples provides a platform to investigate immunological and microbial factors governing UTI-associated preterm birth, revealing novel therapeutic opportunities to predict or prevent preterm birth.

Microorganisms colonizing the human vaginal mucosa are associated with healthy states, as well as conditions such as bacterial vaginosis and infection-associated preterm birth. Here, we report complete genome sequences of 37 bacterial isolates from the human vaginal tract.

Group BStreptococcus(GBS) is a pervasive neonatal pathogen accounting for a combined half a million deaths and stillbirths annually. The most common source of fetal or neonatal GBS exposure is the maternal microbiota.

Vaginal microbial composition is associated with differential risk of urogenital infection. Although Lactobacillus spp. are thought to confer protection against infection, the lack of in vivo models resembling the human vaginal microbiota remains a prominent barrier to mechanistic discovery. Using 16S rRNA amplicon sequencing of C57BL/6J female mice, we found that vaginal microbial composition varies within and between colonies across three vivaria. Noting vaginal microbial plasticity in conventional mice, we assessed the vaginal microbiome of humanized microbiota mice (HMbmice). Like the community structure in conventional mice, HMbmice vaginal microbiota clustered into community state types but, uniquely, HMbmice communities were frequently dominated by Lactobacillus or Enterobacteriaceae. Compared to conventional mice, HMbmice were less susceptible to uterine ascension by urogenital pathobionts group B Streptococcus (GBS) and Prevotella bivia. Although Escherichia and Lactobacillus both correlated with the absence of uterine GBS, vaginal pre-inoculation with exogenous HMbmouse-derived E. coli, but not Ligilactobacillus murinus, reduced vaginal GBS burden. Overall, HMbmice serve as a useful model to elucidate the role of endogenous microbes in conferring protection against urogenital pathogens.

67 experiments in placentas without any pathology, infectious villitis (i.e., cytomegalovirus) or chronic villitis. Results demonstrated an expansion of T cell clones that recognized viral epitopes in our infectious cohort, but in the non-infectious chronic villitis cases there were no shared T cell receptors between cases. This indicated that the antigen being recognized by T cells in chronic villitis are unique to each pregnancy, providing further evidence that this is an anti-fetal, not anti-pathogen, response. Recently, we have transitioned to using spatial methods such as imaging mass cytometry and digital spatial profiling to better understand immune responses in the placenta. The benefit with these technologies is that you can look at multiple targets within spatial confines. Using spatial technologies in the human placenta, we have begun to look more closely at the T cell phenotypes and their relationship with fetal Hofbauer cells in inflamed and normal tissues. Defining these intricate interactions will provide deeper insights into T cell activation during villitis which can allow for the development of novel strategies to help predict and prevent it in the future. S16.2 Uncovering the role of maternal and fetal T cells in preterm labor and birth Nardhy Gomez-Lopez Wayne State University School of Medicine, Detroit, Michigan, USA Preterm birth is the leading cause of neonatal morbidity and mortality worldwide, which is preceded by spontaneous preterm labor, a syndrome of multiple etiologies. Intra-amniotic infection is a recognized cause of spontaneous preterm labor; however, the remaining etiologies (i.e. idiopathic preterm labor and birth) are poorly understood. To fill this gap in knowledge, we have performed a series of investigations based on the hypothesis that a tight balance between regulatory (Tregs) and effector (Teffs) T cells is required for successful pregnancy, and that a conflict between these adaptive immune cells can lead to preterm labor and birth. First, we showed evidence supporting fetal Tcell activation as a newly described trigger for preterm labor and birth in a subset of cases categorized as idiopathic in nature. These findings represent an exciting area of future research focused on further elucidating the fetal immune mechanisms implicated in such a response. Recently, we also provided evidence of a potential maternal immune mechanism responsible for a subset of preterm births formerly considered to be idiopathic. Specifically, we have shown that the impairment of maternal Tregs leads to preterm birth, likely due to the loss of immunosuppressive activity resulting in unleashed effector T-cell responses. Collectively, these findings implicate maternal and fetal T cells in the pathological processes underlying preterm labor and birth and accentuate the importance of the adaptive limbof immunity during pregnancy. S16.3 Heterogeneity, function and specificity of human decidual CD8+ T cells ShwetaMahajan, Aria Alexander, Zachary Koenig, Nicolas Saba, Sandra Andorf, Tamara Tilburgs Cincinnati Children’s Hospital, Cincinnati, OH, USA Problem: To establish a healthy pregnancy the maternal immune systemmust tolerate fetal allo-antigens and remain competent to respond to infections in placental tissues. Maternal decidual CD8+ T cells are key cells that candirectly recognize fetalMHCclass I including the fetal HLA-C antigens expressed by invading fetal extravillous trophoblasts (EVT). In addition decidual CD8+ T cells are also predominant immune effector cells to provide immunity to placental infections. Our previous studies demonstrated that decidual CD8+ T cells are highly differentiatedeffector-memory (EM)Tcells and compared tobloodCD8+Tcells have increased protein and RNA signatures of dysfunction, activation, and effector function. These features may provide temporary CD8 T cell inactivation, permissive of fetal and placental growth, while decidual CD8+ T cells retain capacity to reactivate and respond to infection. However, no information is present on decidual CD8+ T cell heterogeneity and whether separate activated, cytolytic, suppressed or dysfunctional CD8+ T cell types exist that relates to their specificity for fetalor viralantigens. Methods: Here we used high dimensional (21 parameter) spectral flow cytometric analysis to identify decidual CD8+ T cells subpopulations based on their expression of T cell differentiation markers, activation markers, inhibitory molecules and cytolytic molecules. The data was analyzed by a series of high-dimensional analysis tools including FlowSOM, which uses self-organizing maps followed by hierarchical consensus metaclustering to separate phenotypically distinct clusters. Distinct CD8+ T cell subpopulations were purified by FACS sort and assessed for their ability to degranulate, secrete cytokines (e. g. IFNγ, TNFα and IL-2) and capacity to resist apoptosis as measures of their (dys)functionality. Results: The high-dimensional analysis using FlowSOM identified the presence of 14 distinct CD8+ T cell clusters including i) one cluster of CCR7+CD45RA+ Naïve CD8+ T cells which were highly enriched in peripheral blood; ii) two clusters of CCR7-CD45RA+ effector T cells; And iii) 11 clusters of CCR7-CD45RAeffector-memory CD8+ T cells with high phenotypic diversity in their expression of T cell differentiation, activation and inhibitory markers. Five of these Tem clusters were present in both blood and decidua while six clusters were unique to decidual tissues. Functional analysis of purified decidual CD8+ Tem clusters determined that clusters associated with CD39 expression had limited survival capacity and low ability to secrete cytokines and to degranulate. In contrast several purified CD8+ Tem cell clusters expressing several combinations of CD103, CD69 and PD1 had high survival capacity and

UTI is one of the most common causes of outpatient antibiotic use, and rising antibiotic resistance threatens the ability to control UTI unless alternative treatments are developed. Bacteriophage (phage) therapy is gaining renewed interest; however, much like with antibiotics, bacteria can readily become resistant to phages.

Problem : Group B Streptococcus (GBS) is a leading cause of neonatal morbidity, mortality, and preterm birth. GBS is a resident of the maternal vaginal microbiota in approximately 1 in 4 women and can be transmitted to the neonate during vaginal delivery. Alterna-tively, GBS can ascend the reproductive tract to cause in utero fetal infection. Neonates born to mothers with gestational diabetes melli-tus (GDM) have 3-5-fold greater risk of GBS sepsis but mechanistic insight into this increased susceptibility is lacking. Factors required for GBS uterine ascension in healthy or GDM pregnancy are cur-rentlyunknownandunderstandingmicrobialandhostfactorsthatcon- tributetoGBSvirulenceinGDMmayrevealnoveltherapeuticavenues. We hypothesize that GDM renders the host more susceptible to GBS by perturbing maternal immunity and altering GBS virulence and fitness. Method of Study :ToelucidatehostandGBSfactorsthatdriveheight-ened susceptibility seen in GDM, we have developed an in vivo model of GBS reproductive tract ascension in mice with gestational diabetes induced by a high-fat-high-sucrose diet. GDM and pregnant control mice were vaginally inoculated with GBS on days 14.5 and 15.5 of pregnancy. We measured maternal reproductive tract colonization and in utero dissemination to fetal tissues by plating on GBS selec-tive agar. Maternal and fetal cytokines were quantified by 23-plex cytokine assay. In a separate cohort of mice, we assessed pup survival, weight, and specific between and mucins remain unknown. Here we demonstrate for the first time that and promotes by inhibiting both bacterial attachment to human epithelial cells and ascension from the vagina to the uterus in a murine model of GBScolonization.RNAsequencinganalysisofGBSexposedtoMUC5B identified 128 differentially expressed GBS genes, including upregulation of the pilus island-2b (PI-2b) locus. We subsequently show that PI-2b is important for GBS attachment to reproductive cells, binding to immobilized mucins, and vaginal colonization in vivo. Our results suggest that while MUC5B plays an important role in host defense, within the vaginal tract, illustrating the dynamic interplay between pathogen and host. ABSTRACT experiments in placentas without any pathology, infectious villitis (i.e., cytomegalovirus) or chronic villitis. Results demonstrated an expan-sion of T cell clones that recognized viral epitopes in our infectious cohort, but in the non-infectious chronic villitis cases there were no shared T cell receptors between cases. This indicated that the anti-gen being recognized by T cells in chronic villitis are unique to each pregnancy, providing further evidence that this is an anti-fetal, not anti-pathogen, response. Recently, we have transitioned to using spatial methods such as imaging mass cytometry and digital spatial profil-ing to better understand immune responses in the placenta. The ben-efit with these technologies is that you can look at multiple targets within spatial confines. Using spatial technologies in the human placenta, we have begun to look more closely at the T cell phenotypes and their relationship with fetal Hofbauer cells in inflamed and nor-mal tissues. Defining these intricate interactions will provide deeper insights into T cell activation during villitis which can allow for the development of novel strategies to help predict and prevent it in the future.

During pregnancy, GBS ascension into the uterus can cause fetal infection or preterm birth. In addition, GBS exposure during labor creates a risk of serious disease in the vulnerable newborn and mother postpartum.

ABSTRACTUrinary tract infections (UTIs) are among the most common infections treated worldwide each year and are primarily caused by uropathogenicE. coli(UPEC). Rising rates of antibiotic resistance among uropathogens have spurred consideration of alternative strategies such as bacteriophage (phage) therapy; however, phage-bacterial interactions within the urinary environment are poorly defined. Here, we assess the activity of two phages, HP3 and ES17, against clinical UPEC isolates usingin vitroandin vivomodels of UTI. In both bacteriologic medium and pooled human urine, we identified phage resistance arising within the first 6-8 hours of coincubation. Whole genome sequencing revealed that UPEC resistant to HP3 and ES17 harbored mutations in genes involved in lipopolysaccharide (LPS) biosynthesis. These mutations coincided with severalin vitrophenotypes, including alterations to adherence to and invasion of human bladder epithelial HTB-9 cells, and increased biofilm formation. Interestingly, these phage-resistant UPEC demonstrated reduced growth in pooled human urine, which could be partially rescued by nutrient supplementation, and were more sensitive to several outer membrane targeting antibiotics than parental strains. Additionally, these phage-resistant UPEC were attenuated in a murine UTI model. In total, our findings suggest that while resistance to phages, such as LPS-targeted HP3 and ES17, may readily arise in the urinary environment, phage resistance is accompanied by fitness costs rendering UPEC more susceptible to host immunity or antibiotics.IMPORTANCEUTIs are one of the most common causes of outpatient antibiotic use, and rising antibiotic resistance threatens the ability to control these infections unless alternative treatments are developed. Bacteriophage (phage) therapy is gaining renewed interest, however, much like antibiotics, bacteria can readily become resistant to phage. For successful UTI treatment, we must predict how bacteria will evade killing by phage and identify the downstream consequences of phage-resistant bacterial infections. In our current study, we found that while phage-resistant mutant bacteria quickly emerged, these mutations left bacteria less capable of growing in human urine and colonizing the murine bladder. These results suggest that phage therapy poses a viable UTI treatment if phage resistance confers fitness costs for the uropathogen. These results have implications for developing cocktails of phage with multiple different bacterial targets, each of which is only evaded at the cost of bacterial fitness.