TFEB safeguards trophoblast syncytialization in humans and mice.

Nutrient sensing and adaptation in the placenta are essential for pregnancy viability and proper fetal growth. Our recent study demonstrated that the placenta adapts to nutrient insufficiency through mechanistic target of rapamycin (mTOR) inhibition-mediated trophoblast differentiation toward syncytiotrophoblasts (STBs), a highly specialized multinucleated trophoblast subtype mediating extensive maternal-fetal interactions. However, the underlying mechanism remains elusive. Here, we unravel the indispensable role of the mTORC1 downstream transcriptional factor TFEB in STB formation both in vitro and in vivo. TFEB deficiency significantly impaired STB differentiation in human trophoblasts and placenta organoids. Consistently, systemic or trophoblast-specific deletion of Tfeb compromised STB formation and placental vascular construction, leading to severe embryonic lethality. Mechanistically, TFEB conferred direct transcriptional activation of the fusogen ERVFRD-1 in human trophoblasts and thereby promoted STB formation, independent of its canonical function as a master regulator of the autophagy-lysosomal pathway. Moreover, we demonstrated that TFEB directed the trophoblast syncytialization response driven by mTOR complex 1 (mTORC1) signaling. TFEB expression positively correlated with the reinforced trophoblast syncytialization in human fetal growth-restricted placentas exhibiting suppressed mTORC1 activity. Our findings substantiate that the TFEB-fusogen axis ensures proper STB formation during placenta development and under nutrient stress, shedding light on TFEB as a mechanistic link between nutrient-sensing machinery and trophoblast differentiation.

Autophagy Determines Distinct Cell Fates in Human Amnion and Chorion Cells.

Human fetal membranes (amniochorion) that line the intrauterine cavity consist of two distinct cell layers; single-layer amnion epithelial cells (AEC) and multilayer chorion trophoblast cells (CTC). These layers are connected through a collagen-rich extracellular matrix. Cellular remodeling helps support membrane growth and integrity during gestation and helps to maintain pregnancy. Preterm prelabor rupture of the human amniochorionic (fetal) membrane (pPROM) is antecedent to 40% of all spontaneous preterm birth. Oxidative stress (OS) induced activation of the p38 MAPK due to various maternal risk exposures and the amniochorion cells' senescence are reported pathological features of pPROM. Our transcriptomics analysis implicated dysregulated autophagy and epithelial-mesenchymal transition (EMT) in fetal membranes from pPROM. The molecular interplay between OS-induced p38 MAPK activation, autophagy, and EMT was investigated in AECs and CTCs to better understand the involvement of autophagy and EMT. We report the differential impact of OS on the autophagic machinery in AECs and CTCs, resulting in distinct cell fates. In AECs, OS-induced p38 MAPK activation causes autophagosome accumulation and reduced autophagic flux mediated by decreased ULK1 activity and kinase activity, leading to senescence. In CTCs, induction of autophagy has a limited effect; however, inhibition of autophagy led to SQSTM1-mediated EMT of trophoblast cells. Autophagy, EMT, and senescence were associated with proinflammatory changes. Thus, AECs and CTCs respond differently to OS via differential autophagy response, partly mediated via p38 MAPK. Besides senescence, OS-induced autophagy dysregulation in amniochorion cells may play a mechanistic role in pPROM pathophysiology.

A new role for IFRD1 in regulation of ER stress in bladder epithelial homeostasis.

A healthy bladder requires the homeostatic maintenance of and rapid regeneration of urothelium upon stress/injury/infection. Several factors have been identified to play important roles in urothelial development, injury and disease response, however, little is known about urothelial regulation at homeostasis. Here, we identify a new role for IFRD1, a stress-induced gene that has recently been demonstrated to play a critical role in adult tissue proliferation and regeneration, in maintenance of urothelial function/ homeostasis in a mouse model. We show that the mouse bladder expresses IFRD1 at homeostasis and its loss alters the global transcriptome of the bladder with significant accumulation of cellular organelles including multivesicular bodies with undigested cargo, lysosomes and mitochondria. We demonstrate that IFRD1 interacts with several mRNA-translation-regulating factors in human urothelial cells and that the urothelium of Ifrd1-/- mice reveal decreased global translation and enhanced endoplasmic reticulum (ER) stress response. Ifrd1-/- bladders have activation of the unfolded protein response (UPR) pathway, specifically the PERK arm, with a concomitant increase in oxidative stress and spontaneous exfoliation of urothelial cells. Further, we show that such increase in cell shedding is associated with a compensatory proliferation of the basal cells but impaired regeneration of superficial cells. Finally, we show that upon loss of IFRD1, mice display aberrant voiding behavior. Thus, we propose that IFRD1 is at the center of many crucial cellular pathways that work together to maintain urothelial homeostasis, highlighting its importance as a target for diagnosis and/or therapy in bladder conditions.

D-Mannose reduces cellular senescence and NLRP3/GasderminD/IL-1ß-driven pyroptotic uroepithelial cell shedding in the murine bladder.

Aging is a risk factor for disease via increased susceptibility to infection, decreased ability to maintain homeostasis, inefficiency in combating stress, and decreased regenerative capacity. Multiple diseases, including urinary tract infection (UTI), are more prevalent with age; however, the mechanisms underlying the impact of aging on the urinary tract mucosa and the correlation between aging and disease remain poorly understood. Here, we show that, relative to young (8-12 weeks) mice, the urothelium of aged (18-24 months) female mice accumulates large lysosomes with reduced acid phosphatase activity and decreased overall autophagic flux in the aged urothelium, indicative of compromised cellular homeostasis. Aged bladders also exhibit basal accumulation of reactive oxygen species (ROS) and a dampened redox response, implying heightened oxidative stress. Furthermore, we identify a canonical senescence-associated secretory phenotype (SASP) in the aged urothelium, along with continuous NLRP3-inflammasome- and Gasdermin-D-dependent pyroptotic cell death. Consequently, aged mice chronically exfoliate urothelial cells, further exacerbating age-related urothelial dysfunction. Upon infection with uropathogenic E. coli, aged mice harbor increased bacterial reservoirs and are more prone to spontaneous recurrent UTI. Finally, we discover that treatment with D-mannose, a natural bioactive monosaccharide, rescues autophagy flux, reverses the SASP, and mitigates ROS and NLRP3/Gasdermin/interleukin (IL)-1ß-driven pyroptotic epithelial cell shedding in aged mice. Collectively, our results demonstrate that normal aging affects bladder physiology, with aging alone increasing baseline cellular stress and susceptibility to infection, and suggest that mannose supplementation could serve as a senotherapeutic to counter age-associated urothelial dysfunction.

The maternal gut microbiome in pregnancy: implications for the developing immune system.

The gut microbiome has important roles in host metabolism and immunity, and microbial dysbiosis affects human physiology and health. Maternal immunity and microbial metabolites during pregnancy, microbial transfer during birth, and transfer of immune factors, microorganisms and metabolites via breastfeeding provide critical sources of early-life microbial and immune training, with important consequences for human health. Only a few studies have directly examined the interactions between the gut microbiome and the immune system during pregnancy, and the subsequent effect on offspring development. In this Review, we aim to describe how the maternal microbiome shapes overall pregnancy-associated maternal, fetal and early neonatal immune systems, focusing on the existing evidence and highlighting current gaps to promote further research.

Morphological placental findings in women infected with SARS-CoV-2 according to trimester of pregnancy and severity of disease.

INTRODUCTION: Placental morphology findings in SARS-CoV-2 infection are considered nonspecific, although the role of trimester and severity of infection are underreported. Therefore, we aimed to investigate abnormal placental morphology, according to these two criteria. METHODS: This is an ancillary analysis of a prospective cohort study of pregnant women with suspected SARS-CoV-2 infection, managed in one maternity, from March 2020 to October 2021. Charting of clinical/obstetric history, trimester and severity of COVID-19 infection, and maternal/perinatal outcomes were done. Placental morphological findings were classified into maternal and fetal circulatory injury and acute/chronic inflammation. We further compared findings with women with suspected disease which tested negative for COVID-19. Diseases' trimester of infection and clinical severity guided the analysis of confirmed COVID-19 cases. RESULTS: Ninety-one placental discs from 85 women were eligible as a COVID-19 group, and 42 discs from 41 women in negative COVID-19 group. SARS-CoV-2 infection occurred in 68.2% during third trimester, and 6.6% during first; 16.5% were asymptomatic, 61.5% non-severe and 22.0% severe symptomatic (two maternal deaths). Preterm birth occurred in 33.0% (one fetal death). Global maternal vascular malperfusion (MVM) were significant in COVID-19 group whether compared with negative COVID-19 tests group; however, fetal vascular malperfusion lesions and low-grade chronic villitis were not. Three placentas had COVID-19 placentitis. Decidual arteriopathy was associated with infection in first/mid trimester, and chorangiosis in asymptomatic infections. DISCUSSION: Placental abnormalities after an infection by COVID-19 were more frequent after first/mid-trimester infections. Extensive placental lesions are rare, although they may be more common upon underlying medical conditions.

Loss of stearoyl-CoA desaturase 2 disrupts inflammatory response in macrophages.

Macrophages are innate immune cells that patrol tissues and are the first responders to detect infection. They orchestrate the host immune response in eliminating invading pathogens and the subsequent transition from inflammation to tissue repair. Macrophage dysfunction contributes to age-related pathologies, including low-grade inflammation in advanced age that is termed "inflammaging." Our laboratory has previously identified that macrophage expression of a fatty acid desaturase, stearoyl-CoA desaturase 2 (SCD2), declines with age. Herein, we delineate the precise cellular effects of SCD2 deficiency in murine macrophages. We found that deletion of Scd2 from macrophages dysregulated basal and bacterial lipopolysaccharide (LPS)-stimulated transcription of numerous inflammation-associated genes. Specifically, deletion of Scd2 from macrophages decreased basal and LPS-induced expression of Il1b transcript that corresponded to decreased production of precursor IL1B protein and release of mature IL1B. Furthermore, we identified disruptions in autophagy and depletion of unsaturated cardiolipins in SCD2-deficient macrophages. To assess the functional relevance of SCD2 in the macrophage response to infection, we challenged SCD2-deficient macrophages with uropathogenic Escherichia coli and found that there was impaired clearance of intracellular bacteria. This increased burden of intracellular bacteria was accompanied by increased release of pro-inflammatory cytokines IL6 and TNF but decreased IL1B. Taken together, these results indicate that macrophage expression of Scd2 is necessary for maintaining the macrophage response to inflammatory stimuli. This link between fatty acid metabolism and fundamental macrophage effector functions may potentially be relevant to diverse age-related pathologies. IMPORTANCE Macrophages are immune cells that respond to infection, but their dysfunction is implicated in many age-related diseases. Recent evidence showed that macrophage expression of a fatty acid enzyme, stearoyl-CoA desaturase 2, declines in aged organisms. In this work, we characterize the effects when stearoyl-CoA desaturase 2 is deficient in macrophages. We identify aspects of the macrophage inflammatory response to infection that may be affected when expression of a key fatty acid enzyme is decreased, and these findings may provide cellular insight into how macrophages contribute to age-related diseases.

MV140 mucosal bacterial vaccine improves uropathogenic E. coli clearance in an experimental model of urinary tract infection.

MV140 is a mucosal vaccine of inactivated whole bacteria (E. coli, K. pneumoniae, E. faecalis, P. vulgaris) with clinical efficacy against recurrent urinary tract infections (UTIs). Here, MV140 was evaluated in a murine model of acute uropathogenic E. coli (UPEC)-induced UTI using the UTI89 strain. MV140 vaccination resulted in UPEC clearance, concomitant with increased influx of myeloid cells in urine, CD4+ T cells in the bladder, and a systemic adaptive immune response to both MV140-containing E. coli and UTI89.

Bladder Mucosal Cystitis Cystica Lesions Are Tertiary Lymphoid Tissues That Correlate With Recurrent Urinary Tract Infection Frequency in Postmenopausal Women.

PURPOSE: We identify correlates and clinical outcomes of cystitis cystica, a poorly understood chronic inflammatory bladder change, in women with recurrent urinary tract infections. MATERIALS AND METHODS: A retrospective, observational cohort of women with recurrent urinary tract infections who underwent cystoscopy (n=138) from 2015 to 2018 were identified using electronic medical records. Cystitis cystica status was abstracted from cystoscopy reports and correlations were identified by logistic regression. Urinary tract infection-free survival time associated with cystitis cystica was evaluated by Cox proportional hazards regression. Exact logistic regression was used to identify factors associated with changes to cystitis cystica lesions on repeat cystoscopy. Biopsies of cystitis cystica lesions were examined by routine histology and immunofluorescence. RESULTS: Fifty-three patients (38%) had cystitis cystica on cystoscopy. Cystitis cystica was associated with postmenopausal status (OR: 5.53, 95% CI: 1.39-37.21), pelvic floor myofascial pain (6.82, 1.78-45.04), having ≥4 urinary tract infections in the past year (2.28, 1.04-5.09), and a shorter time to next urinary tract infection (HR: 1.54, 95% CI: 1.01-2.35). Forty-two patients (82%) demonstrated improvement or resolution of lesions. Ten/11 (91%) biopsied cystitis cystica lesions were tertiary lymphoid tissue with germinal centers and resembled follicular cystitis. CONCLUSIONS: Cystitis cystica lesions were associated with postmenopausal status, pelvic floor myofascial pain, and number of urinary tract infections in the prior year and predicted worse recurrent urinary tract infection outcomes. Cystitis cystica lesions are tertiary lymphoid tissue/follicular cystitis that may improve or resolve over time with treatment. Identifying cystitis cystica in recurrent urinary tract infection patients may be useful in informing future urinary tract infection risk and tailoring appropriate treatment strategies.

COVID-19 and pregnancy: clinical outcomes; mechanisms, and vaccine efficacy.

As the COVID-19 pandemic continues into its third year, emerging data indicates increased risks associated with SARS-CoV-2 infection during pregnancy, including pre-eclampsia, intrauterine growth restriction, preterm birth, stillbirth, and risk of developmental defects in neonates. Here, we review clinical reports to date that address different COVID-19 pregnancy complications. We also document placental pathologies induced by SARS-CoV-2 infection, entry mechanisms in placental cells, and immune responses at the maternal-fetal interface. Since new variants of SARS-CoV-2 are emerging with characteristics of higher transmissibility and more effective immune escape strategies, we also briefly highlight the genomic and proteomic features of SARS-CoV-2 investigated to date. Vector and mRNA-based COVID-19 vaccines continue to be rolled out globally. However, because pregnant individuals were not included in the vaccine clinical trials, some pregnant individuals have safety concerns and are hesitant to take these vaccines. We describe the recent studies that have addressed the effectiveness and safety of the current vaccines during pregnancy. This review also sheds light on important areas that need to be carefully or more fully considered with respect to understanding SARS-CoV-2 disease mechanisms of concern during pregnancy.

Role of biomarkers (sFlt-1/PlGF) in cases of COVID-19 for distinguishing preeclampsia and guiding clinical management.

OBJECTIVES: To analyze soluble fms-like tyrosine kinase 1 (sFlt-1) and placental growth factors (PlGF) concentrations and their ratio in pregnant and postpartum women with suspected COVID-19, and further investigate conditions associated with an increased ratio (sFlt-1/PlGF > 38), including preeclampsia (PE) and severe acute respiratory syndrome (SARS). STUDY DESIGN: The present study is a secondary analysis of a prospective cohort. Blood samples were collected at time of COVID-19 investigation and the serum measurements of sFlt-1 and PlGF were performed. Clinical background, SARS-CoV-2 infection characteristics, maternal and perinatal outcomes were further analyzed. MAIN OUTCOME MEASURES: Serum measurements of sFlt-1 and PlGF; obstetrics and clinical outcomes. RESULTS: A total of 97 SARS-CoV-2 unvaccinated women with suspected infection were considered, 76 were COVID-19 positive cases and 21 COVID-19 negative. Among COVID-19 positive cases, 09 presented with SARS and 11 were diagnosed with PE, of which 6 had SARS-CoV-2 infection in first and second trimester (04 with sFlt-1/PlGF ≥ 38) and 05 with PE and COVID-19 diagnosed at the same time, during third trimester (03 with sFlt-1/PlGF ≥ 38). Five presented with PE with severe features. sFlt-1/PlGF ratio was significantly higher in the COVID-19 positive/PE positive group compared to COVID-19 positive/PE negative group (p-value = 0.005), with no increase in cases complicated by SARS. CONCLUSIONS: sFlt-1/PlGF ratio could be a useful tool for differential diagnosis and adequate counseling among cases of COVID-19 and PE, especially if severe disease. COVID-19 early in pregnancy could potentially be a risk factor for PE later during gestation.

Supraphysiologic Vaginal Estrogen Therapy in Aged Mice Mitigates Age-Associated Bladder Inflammatory Response to Urinary Tract Infections.

IMPORTANCE: Bladder diseases characterized by chronic inflammation are highly prevalent in older women, as are recurrent urinary tract infections (rUTIs). Recurrent urinary tract infections lead to chronic inflammation of the bladder mucosa and cause lower urinary tract symptoms that persist even after the infection is cleared. Vaginal estrogen therapy (VET) has long been used for the treatment of rUTIs; however, its mechanism of action remains unclear. OBJECTIVES: The objective of this study was to examine the mechanism(s) by which VET affects bladder inflammation and response to rUTIs. STUDY DESIGN: Here, we induced surgical menopause in aged (18 months old) mice followed by VET. Mice were then infected with uropathogenic Escherichia coli , and course of infection was investigated. Inflammatory cytokine response was assessed before and during infection using enzyme-linked immunosorbent assay. RNA sequencing analysis was used to compare the inflammatory status of the young versus aged bladder and principal changes confirmed via quantitative reverse transcriptase-polymerase chain reaction to determine the effects of VET on bladder inflammation. Impact on age-associated bladder tertiary lymphoid tissue formation was evaluated histologically. RESULTS: In the ovariectomized aged model, VET not only mitigated uterine atrophy but was also associated with reduced rUTIs, number of bacterial reservoirs, dampened immune response, and promotion of terminal differentiation of urothelial cells. Bladder tertiary lymphoid tissue lesions were also reduced with VET, with an associated decrease in signals important for bladder tertiary lymphoid tissue formation. Finally, we determined that VET reverses age-associated upregulation of inflammatory genes and pathways. CONCLUSIONS: Our data suggest that VET is effective by reducing age-associated hyperinflammatory conditions in bladder mucosa and in enhancing the host response to infection.

Effects of aging on urinary tract epithelial homeostasis and immunity.

A global increase in older individuals creates an increasing demand to understand numerous healthcare challenges related to aging. This population is subject to changes in tissue physiology and the immune response network. Older individuals are particularly susceptible to infectious diseases, with one of the most common being urinary tract infections (UTIs). Postmenopausal and older women have the highest risk of recurrent UTIs (rUTIs); however, why rUTIs become more frequent after menopause and during old age is incompletely understood. This increased susceptibility and severity among older individuals may involve functional changes to the immune system with age. Aging also has substantial effects on the epithelium and the immune system that led to impaired protection against pathogens, yet heightened and prolonged inflammation. How the immune system and its responses to infection changes within the bladder mucosa during aging has largely remained poorly understood. In this review, we highlight our understanding of bladder innate and adaptive immunity and the impact of aging and hormones and hormone therapy on bladder epithelial homeostasis and immunity. In particular, we elaborate on how the cellular and molecular immune landscape within the bladder can be altered during aging as aged mice develop bladder tertiary lymphoid tissues (bTLT), which are absent in young mice leading to profound age-associated change to the immune landscape in bladders that might drive the significant increase in UTI susceptibility. Knowledge of host factors that prevent or promote infection can lead to targeted treatment and prevention regimens. This review also identifies unique host factors to consider in the older, female host for improving rUTI treatment and prevention by dissecting the age-associated alteration of the bladder mucosal immune system.

Timing is everything: impact of development, ageing and circadian rhythm on macrophage functions in urinary tract infections.

The bladder supports a diversity of macrophage populations with functional roles related to homeostasis and host defense, including clearance of cell debris from tissue, immune surveillance, and inflammatory responses. This review examines these roles with particular attention given to macrophage origins, differentiation, recruitment, and engagement in host defense against urinary tract infections (UTIs), where these cells recognize uropathogens through a combination of receptor-mediated responses. Time is an important variable that is often overlooked in many clinical and biological studies, including in relation to macrophages and UTIs. Given that ageing is a significant factor in urinary tract infection pathogenesis and macrophages have been shown to harbor their own circadian system, this review also explores the influence of age on macrophage functions and the role of diurnal variations in macrophage functions in host defense and inflammation during UTIs. We provide a conceptual framework for future studies that address these key knowledge gaps.

Gut-bladder axis enters the stage: Implication for recurrent urinary tract infections.

The gut microbiome is a critical modulator of systemic physiology, including infectious disease susceptibility. Although this niche is a reservoir for uropathogenic Escherichia coli, knowledge of its role in urinary tract infections (UTIs) is limited. We discuss two recent studies, Thänert et al. (2022) and Worby et al. (2022), that interrogate the roles of the gut-bladder axis in UTIs.

Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens.

Trophoblast organoids derived from placental villi provide a 3D model system of human placental development, but access to first-trimester tissues is limited. Here, we report that trophoblast stem cells isolated from naive human pluripotent stem cells (hPSCs) can efficiently self-organize into 3D stem-cell-derived trophoblast organoids (SC-TOs) with a villous architecture similar to primary trophoblast organoids. Single-cell transcriptome analysis reveals the presence of distinct cytotrophoblast and syncytiotrophoblast clusters and a small cluster of extravillous trophoblasts, which closely correspond to trophoblast identities in the post-implantation embryo. These organoid cultures display clonal X chromosome inactivation patterns previously described in the human placenta. We further demonstrate that SC-TOs exhibit selective vulnerability to emerging pathogens (SARS-CoV-2 and Zika virus), which correlates with expression levels of their respective entry factors. The generation of trophoblast organoids from naive hPSCs provides an accessible 3D model system of the developing placenta and its susceptibility to emerging pathogens.

The Impact of Methenamine Hippurate Treatment on Urothelial Integrity and Bladder Inflammation in Aged Female Mice and Women With Urinary Tract Infections.

IMPORTANCE: Antibiotics are commonly used to treat and prevent urinary tract infection (UTI), but resistance is growing. Nonantibiotic prophylaxis such as methenamine hippurate (MH) shows clinical promise, but its impact on bladder factors influencing recurrent UTIs (rUTIs) is not well described. OBJECTIVE: The aim of the study was to examine the effect of MH on bladder inflammation and barrier function in aged mice and women with rUTI. STUDY DESIGN: This study included urine samples from an experimental study involving aged female mice with and without methenamine treatment as well as women with rUTI who received either no prophylaxis, MH alone, vaginal estrogen therapy and/or d-mannose alone, or MH in addition to vaginal estrogen therapy and/or d-mannose. We performed a comprehensive cytopathological analysis, which included enzyme-linked immunosorbent assay for immunoglobulin A (IgA), interleukin 6 (in human samples), and fluorescein isothiocyanate-conjugated-dextran permeability assay (in mice) to assess for urothelial permeability. RESULTS: In the aged mice model, there was a decreased urothelial permeability (as seen by retention of fluorescein isothiocyanate-conjugated-dextran fluorescence in superficial cells) and increased urinary IgA in mice treated with MH compared with controls. There was no significant difference in urothelial shedding (P > 0.05). In human samples, there was significantly increased urinary IgA in those taking MH alone compared with no prophylaxis (830.1 vs 540.1 ng/mL, P = 0.04), but no significant difference in interleukin 6. CONCLUSIONS: Methenamine hippurate seems to enhance barrier function as evidenced by decreased urothelial permeability and increased urinary IgA levels, without worsening inflammation. This may reflect another beneficial mechanism by which MH helps prevent rUTI.