Gestational Diabetes Exacerbates Intrauterine Microbial Exposure in Offspring PDF

Nutrition & Diabetes www.nature.com/nutd ARTICLE OPEN Gestational diabetes exacerbates intrauterine microbial...

Nutrition & Diabetes www.nature.com/nutd ARTICLE OPEN Gestational diabetes exacerbates intrauterine microbial exposure induced intestinal microbiota change in offspring contributing to increased immune response ✉ 2,6,7 ✉ Juncheng Liu1,2,3,7, Yan Chen2,4,7, Irakoze Laurent1,2, Ping Yang5, Xiaoqiu Xiao1,2,7 and Xinyu Li © The Author(s) 2024 BACKGROUND: maternal health during pregnancy can affect the intestinal microbial community of offspring, but currently the impact of intrauterine environmental changes resulting from gestational diabetes mellitus (GDM) on the microbiota of offspring as well as its interaction with the immune system remains unclear. AIMS: to explore the impact of intrauterine microbial exposure during pregnancy of gestational diabetes mellitus on the development of neonate’s intestinal microbiota and activation of immune responses. METHODS: Levels of lipopolysaccharides in cord blood from GDM and expression of microbial recognition-related proteins in the 1234567890();,: placenta were measured. To evaluate embryonic intestinal colonization, pregnant mice with GDM were administered with labeled Escherichia coli or Lactobacillus. The intestinal colonization of pups was analyzed through 16S rRNA gene sequencing and labeled microbial culture. Additionally, memory T lymphocyte and dendritic cell co-culture experiments were conducted to elucidate the immune memory of intestinal microbes during the embryonic stages. RESULT: Gestational diabetes mellitus led to elevated umbilical cord blood LPS level and increased GFP labeled Escherichia coli in the offspring’s intestine after gestational microbial exposure. The mouse model of GDM exhibited increased immune markers including TLR4, TLR5, IL-22 and IL-23 in the placenta and a recall response from memory T cells in offspring’s intestines, with similar observations found in human experiments. Furthermore, reduced intestinal microbiome diversity and an increased ratio of Firmicutes/Bacteroidetes was found in GDM progeny, with the stability of bacterial colonization been interfered. CONCLUSIONS: Our investigation has revealed a noteworthy correlation between gestational diabetes and intrauterine microbial exposure, as well as alterations in the neonatal microbiota and activation of immune responses. These findings highlight the gestational diabetes’s role on offspring’s gut microbiota and immune system interactions with early-life pathogen exposure. Nutrition and Diabetes (2024)14:87 ; https://doi.org/10.1038/s41387-024-00346-7 INTRODUCTION the intestines, can translocate to the maternal bloodstream from a The human microbiota encompasses a rich ecosystem that form a leaky gut and adversely affect pregnancy outcomes such as stable symbiotic relationship with the host and substantially impairing spermatogenesis and developing ASD-like behavior in impacts host’s metabolism and physiology. In recent years, the next generation [8, 9]. extensive attention has been focused on the microbiota develop- Gestational diabetes mellitus (GDM) is prevalent worldwide. Its ment during pregnancy and early life, as disruptions of the high prevalence, along with associated transgenerational con- microbiome have been linked to chronic diseases and mental sequences and serious complications, necessitates new disorders in the next generation [2–4]. Contrary to the past belief approaches to understand its pathophysiology [10, 11]. It has that the fetus remains germ-free during gestation, some studies been suggested that GDM significantly alters the composition of have reported traces of microbes in human placental and fetal the intestinal microbiota in offspring, potentially leading to health samples [5–7], emphasizing the potential for maternal transmis- risks such as obesity, insulin resistance, and even diabetes in the sion of microbes. Fetal exposure to maternally derived microbial next generation [12–14]. However, the association between GDM, components was also found which associated with early-life microbial signaling in the embryonic stage, postpartum micro- immune system development. Lipopolysaccharide(LPS), a typical biota construction, and early-life immune responses remains proinflammatory molecule produced by gram-negative bacteria in unclear. 1 Department of Endocrinology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China. 2The Chongqing Key Laboratory of Translational Medicine in Major Metabolic Diseases, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China. 3Department of Gastroenterology, Chongqing University Cancer Hospital, School of Medicine, Chongqing University, Chongqing, China. 4Department of Endocrinology and Nephrology, Chongqing Emergency Medical Center, Chongqing University Central Hospital, Chongqing, China. 5Yongchuan Hospital of Traditional Chinese Medicine, Chongqing, China. 6Department of Pharmacy, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China. 7These authors contributed equally: Juncheng Liu, Yan Chen, Xiaoqiu Xiao, Xinyu Li. ✉email: [email protected]; [email protected] Received: 10 September 2023 Revised: 20 September 2024 Accepted: 9 October 2024  J. Liu et al. 2 In this study, using samples from GDM women and animal section in a sterile environment, and the pups were nursed by normal model, we first aimed to confirm whether GDM led to elevated female mice. Placenta were collected immediately after cesarean section to intrauterine microbial signaling after gestational microbial expo- detect immune related indicators. All the animal experiments conformed sure. The presence of intestinal microbes in offspring was also to guidelines for the Care and Use of Animals published by Institutional examined, with further study on innate and adaptive immune Animal Ethical Committee. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Chongqing Medical responses. Finally, whether maternal GDM affects the intestinal University. all methods were performed in accordance with the relevant microbial composition and colony stability of offspring were guidelines and regulations. examined. For animal models, sterile cesarean section delivery of fetuses and fostering by normal female mice were conducted to minimize postnatal microbial exposure. Bacteria preparation Lactobacillus (GMCC0460.2) were first grown and expanded in MRS broth at 37 °C and 5% CO2, and resuspended in modified Hank’s balanced saline solution (HBSS) supplemented with 0.04 M MgSO4, 0.03 M MnSO4, 1.15 M METHODS K2PO4, 0.36 M sodium acetate, 0.88 M ammonium citrate, 10% polysorbate Subject recruitment and 20% dextrose. Bacteria then were propagated overnight at 37 °C, 5% Pregnant women and neonates were recruited at The First Affiliated CO2 under non-agitating conditions to 2 × 109 cfu/ml. Hospital of Chongqing Medical University. The study was approved by the E. coli Nissle 1917 were engineered to eGFP labeled and confer Medicine Ethics Committee at The First Affiliated Hospital of Chongqing resistance to Kanamycin. Before oral administrations, E.coli were grown for Medical University. All participants provided written informed consent for 6 h in LB broth supplemented with Kanamycin (50 µg/mL) at 37 °C with themselves and the neonates. Pregnant women with GDM were diagnosed shaking. This culture was diluted 1:100 in LB broth without antibiotics and by specialized doctors according to the results of oral glucose tolerance cultured overnight at 37 °C with shaking. Bacterial pellets from this test (OGTT) and were recruited as a case group. The diagnosis of GDM is overnight culture were diluted in sterile PBS to the concentration of 109 based on the GDM diagnostic guidelines formulated by the World Health cfu/ml. Organization. The diagnosis of GDM was performed by the 75 g OGTT at 24–28th gestational weeks with at least one of the following criteria: fasting ≥ 5.1 mmol/L, 1 h ≥ 10.0 mmol/L, or 2 h ≥ 8.5 mmol/L [15, 16]. In this Fostering study, subjects with pre-existing diabetes, pre-existing metabolic diseases, On embryonic day 19, mice were anaesthetized and euthanized via antibiotics usage within 3 months, alcohol or substance abuse, period- cervical dislocation. The pups were delivered via sterile c-section, moved to ontitis, bacterial vaginosis, and chronic diseases requiring medication were a preheated, sterile, damp gauze and carefully monitored for onset of excluded. Women with normal pregnancies were matched for maternal regular breathing. Next, mice were brought into contact with bedding from the cage from the dam that they would be fostered to the age- age, BMI and medical history. Placenta and umbilical cord blood were matched pups of the foster mother were marked via clipping of the tail to collected immediately after cesarean section to detect immune related indicators. Patient sample and data were collected under approval by the distinguish them from the fostered pups that were placed in the same Medicine Ethics Committee at The First Affiliated Hospital of Chongqing cage. Cages were left undisturbed for at least 2 days to facilitate Medical University (2020–565). Written informed consent was obtained acceptance of foster pups. Pups were weaned at 3 weeks of age. Some from all participants. All methods were performed in accordance with the pups are sacrificed to collect intestinal immune cells after weaning. The remaining pups were treated by oral gavage with 109 cfu of E. coli or relevant guidelines and regulations. Lactobacillus in 100 µl/day of PBS or 100 µl/day of PBS as negative control within three days. Animals and diet C57BL/6 J mice (6 weeks old) were purchased from the animal core facility Sample preparations and DNA extractions of Chongqing Medical University. Mice were housed in a stable facility with Fecal samples were collected from colons at sacrifice and stored at −20 °C. 12 h light/12 h dark cycles at 25 °C. All mice were stabilized for a week For bacterial DNA extractions, we included an additional enzymatic lysis before initiating any experimental procedures. Before assigning the procedure before using the Powersoil Isolation Kit (Mo Bio Laboratories). animals, it is crucial to ensure that the mice are uniform in terms of litter, Briefly, 50 μL lysozyme, 6 μL mutanolysin, and 3 μL lysostaphin were added gender, and age. Then, the mice are to be paired based on weight, forming to 100 μL aliquot of cell suspension followed by incubation for 1 h at 37 °C. a basic group of four mice with similar weights. Subsequently, the animals The lysate was then subjected to further DNA isolation and purification within each group will be randomly assigned to either the experimental or using the Powersoil DNA Isolation Kit (Mo Bio Laboratories) as the control groups, ensuring that the groups have similar litter, gender, age, manufacturer’s instructions. The final DNA concentration was determined and weight composition. Throughout the subsequent experiments, the by the Quanti-It Picogreen dsDNA HS assay kit (Invitrogen, UK). animals will be cared for by personnel who are unaware of the mice’s grouping, thus guaranteeing uniform care across all groups. A GDM mice model was established by feeding mice with a high-fat diet (HFD, 60% kcal, Oral glucose tolerance tests D12492, Research Diet, USA) for 4 weeks before pregnancy and was Following fasting (12 h deprivation of diet: 8:00 p.m. to 8:00 a.m.), these maintained with HFD until delivery. Normal pregnant (CON) mice were fed mice were intraperitoneally injected with a solution of D-glucose (2 g/kg a normal chow diet throughout pregnancy. Female mice were mated with body weight). We obtained blood samples from the tail vein of mice at male mice overnight at a proportion of 2:1 per cage. Pregnancy was 0 minutes (just before glucose load), as well as 15, 30, 60, 90, and 120 min determined by the presence of vaginal plugs the next morning, which was after glucose administration. Blood glucose levels were measured using identified as gestational day 0. The next day, the mice in GDM group were the glucometer, followed by the calculation of the area under curve (AUC). intraperitoneally injected with STZ (25 mg/kg, dissolved in 0.1 mmol/L citrate buffer, pH 4.2–4.5, S0130, Sigma–Aldrich, USA), followed by 3 Isolation of CD4+ T Cell injections every 24 h. The control mice were injected with a similar amount Disrupt spleen in phosphate-buffered saline (PBS) containing 2% fetal of citrate buffer. Random blood glucose was tested from the tail by bovine serum (FBS). Remove clumps and debris by passing the cell glucometer (ACCU-CHEK Guide, Roche, USA) 72 h after the first STZ suspension through a 70-μm mesh nylon strainer. Centrifuge at 300 g for injection. Mice with random blood glucose ≥ 11.1 mmol/L were regarded 10 min and resuspend in medium (PBS containing 2% FBS and 1 mM as qualified GDM mice. The GDM mice were then randomly divided into EDTA). CD4+ T Cells were subsequently isolated using the Mouse CD4 two groups: the GDM- group and the labelled bacteria group (GDM + ). Positive Selection Kit (Beaver Bio Ltd). The purpose is to clarify the colonization of microorganisms from different sources in their offspring under different maternal health conditions. The Control mice also were then randomly divided into two groups: the Isolation of mononuclear cells Control group (CON-) and the labelled bacteria group (CON + ). Disrupt spleen in phosphate-buffered saline (PBS) containing 2% fetal On gestational day 1, the CON+ and GDM+ group were fed 0.1 ml of bovine serum (FBS). Remove clumps and debris by passing the cell preparation with 109 colony forming unit (CFU) / ml of labeled E. coli daily suspension through a 70-μm mesh nylon strainer. Adding the same until gestational day 19, the CON- and GDM- group were fed same volume amount of separation solution as the organ tissue single-cell suspension of sterile PBS. On gestational day 21, all mice underwent cesarean (P6340, Solarbio). Carefully aspirate the single-cell suspension and add it to Nutrition and Diabetes (2024)14:87  J. Liu et al. 3 the surface of the separation solution. Centrifuge at 500 g for 25 min. Carefully pipet the second layer of mononuclear cells into another sterile Table 1. characteristics among women with GDM and their age- 15 mL centrifuge tube, add 10 ml of cell washing solution to the centrifuge matched controls. tube to wash, and then centrifuge at 250 g for 10 min. Discard the Characteristic GDM Non-GDM P supernatant, resuspend the cells in 5 mL of PBS or cell washing solution, (n = 15) (n = 15) and centrifuge at 250 g for 10 min. Age, year 30.93 ± 0.74 29.93 ± 0.77 0.37 Monocytes differentiate into dendritic cells Gestational week at 38.47 ± 0.25 39.2 ± 0.14 0.13 Isolating mononuclear cells from peripheral blood by Blood Mononuclear sample collection, Cell Separation Kit (P6340, Solarbio, China), which were induced in vitro by week colony stimulating factor (P00184, Solarbio, China) and recombinant Prenatal BMI, kg/m2 30.19 ± 0.84 26.64 ± 0.54