Asthma susceptibility in prenatal nicotine-exposed mice attributed to ß-catenin increase during CD4+ T cell development.

Cigarette smoke is a common global environmental pollutant. Asthma, the most frequent allergic airway disease, is related to maternal exposure to cigarette smoke. Our previous studies demonstrated that prenatal exposure to nicotine (PNE), the major active product of smoking, impairs fetal thymopoiesis and CD4+ T cell development after birth. This study aimed to investigate whether PNE contributes to asthma susceptibility through CD4+ T cell development alterations. First, A PNE model was established by administering 3 mg/kg/day nicotine to maternal mice, and then an ovalbumin-induced asthma model was established in the offspring. Further, ß-catenin and downstream pathways were inhibited in vitro to confirm the molecular mechanisms underlying the phenotype observed during the in vivo phase. The results showed that PNE induced Th2 and Th17 biases at developmental checkpoints and aggravated asthma symptoms in the offspring. In fetuses, PNE up-regulated α7 nAChR, activated PI3K-AKT, promoted ß-catenin level increase, and established potential Th2- and Th17-biased gene expression patterns during thymopoiesis, which persisted after birth. Similar results were also observed in 1 µM nicotine-treated thymocytes in vitro. Moreover, inhibiting PI3K-AKT by LY294002 abrogated nicotine-mediated ß-catenin level increase and thymopoiesis abnormalities, and an α7 nAChR antagonist (α-btx) also reversed nicotine-induced PI3K-AKT activation. Our findings provide strong evidence that PNE is a risk factor for T cell deviation and postnatal asthma, and revealed that nicotine-induced ß-catenin level increase induces thymopoiesis abnormalities.

Augmented autophagy suppresses thymocytes development via Bcl10/p-p65 pathway in prenatal nicotine exposed fetal mice.

Tobacco smoke is a common global environmental pollutant. Maternal tobacco smoke/nicotine exposure has long-term toxic effects on immune organs. We previously found that prenatal nicotine exposure (PNE)-induced programmed immune diseases caused by fetal thymic hypoplasia, but the mechanism still unknown. Autophagy has important functions in maintaining thymopoiesis, whether autophagy was involved in PNE-inhibited fetal thymocytes development is also obscure. Therefore, this study aimed to investigate how nicotine changed the development of fetal thymocytes from the perspective of autophagy in vivo and in vitro. PNE model was established by 3 mg/kg nicotine administration in Balb/c mice from gestational day 9 to 18. The results showed that PNE reduced the percentage and absolute number of CD69-CD4+SP cells, suggesting a block of fetal thymocytes mature. PNE promoted autophagosome formation, autophagy related proteins (Beclin1, LC3I/II) expression, and upregulated α7 nAChR as well as AMPK phosphorylation in fetal thymus. Moreover, PNE promoted Bcl10 degradation via autophagy-mediated proteolysis and inhibited p65 activation, blocking the transition of thymocytes between the DP to SP stage. Further, primary thymocytes were treated with nicotine in vitro and showed induced autophagy in a dose- and time-dependent manner. In addition, nicotine-inhibited CD69-CD4+SP cells and the Bcl10/p-p65 pathway have been reversed by an autophagy inhibitor. The α7 nAChR specific antagonist abrogated nicotine-induced AMPK phosphorylation and autophagy initiation. In conclusion, our findings showed that PNE repressed the Bcl10/p-p65 development pathway of CD4+SP cells by triggering autophagy, and illuminated the developmental origin mechanism of programmed immune diseases in PNE offspring.

Attenuated cholesterol metabolism pathway suppresses regulatory T cell development in prenatal nicotine exposed female mice.

The recession of regulatory T cells (Tregs) contributes to development of autoimmune disease. Our previous study suggested that prenatal nicotine exposure (PNE) inhibited Tregs frequency in offspring, but the mechanisms are still uncertain. This study aimed to explore the molecular mechanisms of PNE-induced Tregs inhibition from the perspective of cellular cholesterol homeostasis both in vivo and in vitro. PNE mice model were established by 3 mg/kg/d nicotine administration in Balb/c strain from gestational day (GD) 9 to GD 18. The results showed that PNE significantly decreased thymic Tregs frequency in neonatal offspring. The activation of mTOR and downregulation of p-STAT5/Foxp3 pathway of Tregs were observed in PNE offspring. Mechanism study found that PNE elevated ATP-binding cassette transporter G1 (ABCG1) expression and decreased intracellular cholesterol content of Tregs in offspring, indicating impaired intracellular cholesterol homeostasis. Similar results were observed in 1 µM nicotine-treated primary thymocytes in vitro. Further, cholesterol-replenishment can abrogate nicotine-induced mTOR activation and the following suppression of p-STAT5/Foxp3 pathway and Tregs frequency. In addition, Abcg1 siRNA transfection can partly reverse the nicotine-decreased intracellular cholesterol content and cell frequency of Tregs. In conclusion, this study showed that PNE could suppress Tregs development in female mice by up-regulating ABCG1-dependent cholesterol efflux, and suggested that PNE-induced thymic Tregs recession of offspring at early life was the developmental origin mechanism of immune dysfunction in later life.

Attenuated Tregs increase susceptibility to type 1 diabetes in prenatal nicotine exposed female offspring mice.

The recession of regulatory T cells (Tregs) is pivotal for type 1 diabetes (T1D) progressing. Our previous study observed the decreased Tregs in prenatal nicotine exposure (PNE) offspring, but whether this led to the onset of T1D remains uncertain. Thus, this study aimed to investigate the effects of PNE on T1D susceptibility and the role of PNE-suppressed Tregs in T1D of female offspring. The decreased body weights and elevated blood glucose levels from postnatal day (PND) 21 to PND 42 indicated that PNE caused persistent impaired glucose homeostasis in offspring. The elevated serum glutamic acid decarboxylase autoantibody, the "Gold Standard" for the detection of T1D, was observed on PND 42, suggesting the early stage of T1D in PNE offspring during adolescence. The reduced pancreatic islet areas and beta cells number in PNE offspring were observed at neonatal period and became more severe during adolescence. In addition, PNE caused immune dysfunction in offspring, manifested as suppressed thymic Tregs percentage from PND 4 to PND 42 and splenic Tregs/Th17 ratio on PND 42. In conclusion, PNE resulted in metabolic changes of offspring that were consistent with T1D characteristics, which could be the consequence of Tregs recession from early life to adolescence.

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The effects of single and combined pollution of enrofloxacin and Cu on the digestive enzymes of earthworms were studied, based on the actual pollution of caused by the application of livestock feces in farmland soil. Results showed that single enrofloxacin (0.1-4 mg·kg-1, 28 d) did not affect protease, but inhibited cellulase and alkaline phosphatase, with an induced effect on acid phosphatase. Single Cu pollution (20-200 mg·kg-1, 28 d) had inhibitory effects on the four digestive enzymes in earthworms. The effects of combined exposures on the digestive enzymes were mainly negative, showing a synergistic increasing character of toxicity in cellulase and acid phosphatase activities. The response dynamics of digestive enzymes to exposure duration was regulatory response (3 d)-intense response (7 d)-reaction recovery (14 d)-chronic exposure (28 d). Chronic exposure results showed that the combined treatments containing high-dose pollutant (200 mg·kg-1 Cu or 4 mg·kg-1 ENR) had more ecological risk.

Inhibition of thymocyte autophagy-associated CD4+T thymopoiesis is involved in asthma susceptibility in mice exposed to caffeine prenatally.

Our previous studies demonstrated that prenatal caffeine exposure (PCE) caused thymopoiesis inhibition, immune disorders, and airway remodeling in offspring, which raises the question of whether PCE is a risk factor for postnatal asthma. Meanwhile, the mechanism of PCE-induced thymopoiesis inhibition is not clear yet. Considering caffeine's pro-autophagy effects (lacking evidence in thymus) and the important role of autophagy in maintaining thymopoiesis, this study aimed to investigate whether PCE contributes to asthma susceptibility, and further explore the molecular mechanisms of thymopoiesis inhibition from the perspective of pro-autophagy effects of caffeine both in vivo and in vitro. The PCE mouse model was established by 96 mg/kg/day caffeine administration from gestational day (GD) 9-GD 18, and an asthma model was established on the offspring by ovalbumin sensitization and challenge. The results confirmed our hypothesis that PCE could suppress pulmonary CD4+T development and aggravate allergen-induced asthma symptoms in the offspring. In fetuses, PCE significantly suppressed A2AR-PKA signaling, upregulated Beclin1-LC3II autophagy, promoted Bcl10 degradation, reduced A20 expression, and inhibited CD4+T thymopoiesis. Similar results were also observed in 4 µM caffeine-treated thymocytes in vitro. Moreover, inhibiting A2AR by antagonist (SCH 58261) performed the same downstream biological effects as caffeine treatment, and autophagy inhibitor (BafilomycinA1) clearly abolished the caffeine-induced Bcl10 degradation and A20 suppression. In conclusion, our findings, for the first time, showed that PCE could attenuate CD4+T thymopoiesis and suppress pulmonary CD4+T development by directly enhancing autophagy in thymocytes, and provided a firm experimental evidence that PCE is a risk factor for postnatal asthma.

Prenatal nicotine exposure induces thymic hypoplasia in mice offspring from neonatal to adulthood.

Clinical study showed that smoking during pregnancy deceased the thymus size in newborns. However, the long-term effect remains unclear. This study was aimed to observe the effects of prenatal nicotine exposure (PNE) on the development of thymus and the T-lymphocyte subpopulation in mice offspring from the neonatal to adulthood. Both the thymus weight and cytometry data indicated that PNE caused persistent thymic hypoplasia in male offspring from neonatal to adult period and transient changes in female offspring from neonatal to prepuberal period. Flow cytometry analysis disclosed a permanent decreased proportion and number of mature CD4 single-positive (SP) T cells in thymus of both sex. In addition, the PNE male offspring showed a more serious thymus atrophy in the ovalbumin (OVA)-sensitized model. Moreover, increased autophagic vacuole and elevated mRNA expression of Beclin 1 were noted in PNE fetal thymus. In conclusion, PNE offspring showed thymus atrophy and CD 4 SP T cell reduction at different life stages. Mechanically, PNE induced excessive autophagy in fetal thymocytes might be involved in these changes. All the results provided evidence for elucidating the PNE-induced programmed immune diseases.

α7 nAChR mediated Fas demethylation contributes to prenatal nicotine exposure-induced programmed thymocyte apoptosis in mice.

This study aimed to investigate the effects of prenatal nicotine exposure (PNE) on thymocyte apoptosis and postnatal immune impairments in vivo and further explore the epigenetic mechanisms of the pro-apoptotic effect of nicotine in vitro. The results showed that PNE caused immune impairments in offspring on postnatal day 49, manifested as increased IL-4 production and an increased IgG1/IgG2a ratio in serum. Enhanced apoptosis of total and CD4+SP thymocytes was observed both in fetus and in offspring. Further, by exposing thymocytes to 0-100 µM of nicotine in vitro for 48 h, we found that nicotine increased α7 nicotinic acetylcholine receptor (nAChR) expression, activated the Fas apoptotic pathway, and promoted thymocyte apoptosis in concentration-dependent manners. In addition, nicotine could induce Tet methylcytosine dioxygenase (TET) 2 expression and Fas promoter demethylation, which can be abolished by TET2 siRNA transfection. Moreover, the α7 nAChR specific antagonist α-bungarotoxin can abrogate nicotine-induced TET2 increase, and the following Fas demethylation and Fas-mediated apoptosis. In conclusion, our findings showed, for the first time, that α7 nAChR activation could induce TET2-mediated Fas demethylation in thymocytes and results in the upregulation of Fas apoptotic pathway, which provide evidence for elucidating the PNE-induced programmed thymocyte apoptosis.

Maternal Glucocorticoid Elevation and Associated Fetal Thymocyte Apoptosis are Involved in Immune Disorders of Prenatal Caffeine Exposed Offspring Mice.

Our previous study showed that prenatal caffeine exposure (PCE) could induce intrauterine growth retardation (IUGR) and glucocorticoid elevation in the fetus. Researchers suggested that IUGR is a risk factor for T helper cell (Th)1/Th2 deviation. However, whether PCE can induce these immune disorders and the underlying mechanisms of that induction remain unknown. This study aimed to observe the effects of PCE on the Th1/Th2 balance in offspring and further explore the developmental origin mechanisms from the perspective of glucocorticoid overexposure-induced thymocyte apoptosis. An IUGR model was established by caffeine administration from gestational day (GD) 9 to GD 18, and the offspring were immunized on postnatal day (PND) 42. The results show that maternal glucocorticoid overexposure increased fetal thymocyte apoptosis by activating both the Fas-mediated and the Bim-regulated apoptotic pathways. After birth, accelerated thymocyte apoptosis and Th1 suppression were also found in the PCE offspring at PND 14 and PND 49. Moreover, the PCE offspring showed immune disorders after immunization, manifesting as increased IgG1/IgG2a ratio and IL-4 production in the serum. In conclusion, PCE could induce fetal overexposure to maternal glucocorticoids and increase thymocyte apoptosis, which could persist into postnatal life and be implicated in Th1 inhibition and further immune disorders.

Prenatal caffeine ingestion increases susceptibility to pulmonary inflammation in adult female rat offspring.

This study aimed to investigate the association between prenatal caffeine ingestion (PCI) and risk of postnatal pulmonary inflammation. Pregnant Wistar rats were administered 60mg/kg/d caffeine intragastrically from gestational day (GD) 7 to GD 20. The results showed that PCI obviously increased intrauterine growth retardation rate to 39.2% and suppressed weight growth of the offspring. PCI also enhanced the expression of transforming growth factor ß, α-smooth muscle actin, interleukin (IL)-1ß, and IL-8 in lungs and caused pulmonary interstitial thickening in the offspring. Further, with lipopolysaccharide stimulation on postnatal day 77, PCI offspring showed more serious inflammatory infiltration, higher injury scores, and higher levels of IL-6 and tumor necrosis factor-α in lungs than those of the control. Our findings showed, for the first time, that PCI is a certainly threat to postnatal pulmonary inflammation. The potential mechanism is that PCI alter the expression of pulmonary interstitial thickening-associated genes in the offspring.

Enhanced thymocyte apoptosis induced by maternal undernutrition in late gestation results in declined mature T cells in rat fetal thymus.

This study was designed to observe the effects of maternal food restriction (MFR) on the development of fetal thymus in different gestation periods. Timed pregnant rats were randomized into 3 groups: CN (free access to standard chow throughout gestation), MFR0-21 (50% MFR throughout gestation), MFR0-14 (50% MFR from gestational day (GD) 0 to GD14, early-mid gestation). Results showed that MFR during early-mid period had few impact on the fetal thymus and T cell subpopulations. However, MFR throughout gestation resulted in thymic atrophy, deceased frequency of both CD4+ and CD8+ single positive (SP) T cells and enhanced thymocyte apoptosis in fetus. Our results suggest the fetal thymus is more vulnerable to the adverse intrauterine environments in the late gestation period, and the decreased number of SP T cells could result from the enhanced thymocyte apoptosis.

Reproductive Toxicity of T Cells in Early Life: Abnormal Immune Development and Postnatal Diseases.

Immunity is a balanced status with adequate biological defenses to recognize and fight "non-self", as well as adequate tolerance to recognize "self". To maintain this immune homeostasis, a well-organized T cell immune network is required, which in part depends on the well-controlled development of alternative effector T cells, with different cytokine repertoires. Recent researches have pointed that developing fetal T cells network is a remarkably sensitive toxicological target for adverse factors in early life. Epidemiological and experimental studies showed an inseparable relationship between T cell developmental toxicity and immune diseases in adults. Considering that the inflammatory and immune disorders have become a growing health problem worldwide, increasing attention is now being paid to the T cell developmental toxicity. We propose that adverse factors may have programming effects on the crucial functions of immune system during early life which is critical for fetal T cell development and the establishment of the distinct T cell repertoires balance. The permanently disturbed intrathymic or peripheral T cell development may in turn lead to the immune disorders in later life. In this manuscript, we reviewed how adverse factors affected T cell development in early-life with the consequence of the immune dysfunction and immune diseases, and further elucidate the mechanisms. These mechanisms will be helpful in prevention and treatment of the increased prevalence of immune diseases by interfering those pathways.

Increased Fetal Thymocytes Apoptosis Contributes to Prenatal Nicotine Exposure-induced Th1/Th2 Imbalance in Male Offspring Mice.

Nicotine, a definite risk factor during pregnancy, is an immunomodulator. This study was designed to investigate the effects of prenatal nicotine exposure (PNE) on the balance of Th1/Th2 in offspring, and further explore the developmental origin mechanisms from the perspective of fetal thymocytes apoptosis. Pregnant Balb/c mice were administered 1.5 mg/kg nicotine subcutaneously twice per day from gestational day (GD) 9 to GD18. Results showed that PNE could cause a Th2 shift in male offspring, manifested as increased ratio of IgG1/IgG2a, IL-4 production in serum, and IL-4/IFN-γ expression ratio in spleen. Increased apoptosis of total thymocytes and CD4SP and reduced cell proportion of CD4SP were found in PNE male offspring on postnatal day (PND) 14 and PND 49. In the fetuses, decreased body weight and organ index of fetal thymus, histological changes in fetal thymus, reduced CD4SP proportion and increased fetal thymocyte apoptosis were observed in nicotine group. The increased mRNA expression of genes involved in Fas-mediated apoptotic pathway and protein expression of Fas were also detected. In conclusion, PNE could cause a Th2 shift in male offspring mediated by reduced CD4+ T cells output, which may result from the increasing apoptosis of total thymocytes and CD4SP.

Developmental origins of inflammatory and immune diseases.

Epidemiological and experimental animal studies show that suboptimal environments in fetal and neonatal life exert a profound influence on physiological function and risk of diseases in adult life. The concepts of the 'developmental programming' and Developmental Origins of Health and Diseases (DOHaD) have become well accepted and have been applied across almost all fields of medicine. Adverse intrauterine environments may have programming effects on the crucial functions of the immune system during critical periods of fetal development, which can permanently alter the immune function of offspring. Immune dysfunction may in turn lead offspring to be susceptible to inflammatory and immune diseases in adulthood. These facts suggest that inflammatory and immune disorders might have developmental origins. In recent years, inflammatory and immune disorders have become a growing health problem worldwide. However, there is no systematic report in the literature on the developmental origins of inflammatory and immune diseases and the potential mechanisms involved. Here, we review the impacts of adverse intrauterine environments on the immune function in offspring. This review shows the results from human and different animal species and highlights the underlying mechanisms, including damaged development of cells in the thymus, helper T cell 1/helper T cell 2 balance disturbance, abnormal epigenetic modification, effects of maternal glucocorticoid overexposure on fetal lymphocytes and effects of the fetal hypothalamic-pituitary-adrenal axis on the immune system. Although the phenomena have already been clearly implicated in epidemiologic and experimental studies, new studies investigating the mechanisms of these effects may provide new avenues for exploiting these pathways for disease prevention.