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

Accepted Manuscript Title: Prenatal nicotine exposure induces thymic hypoplasia in mice offspring from neonatal to adulthood Authors: Wen Qu, Wen-hao Zhao, Xiao Wen, Hui-yi Yan, Han-xiao Liu, Li-fang Hou, Jie Ping PII: DOI: Reference:

S0378-4274(18)31859-9 https://doi.org/10.1016/j.toxlet.2018.12.015 TOXLET 10391

To appear in:

Toxicology Letters

Received date: Revised date: Accepted date:

9 September 2018 29 November 2018 28 December 2018

Please cite this article as: Qu W, Zhao W-hao, Wen X, Yan H-yi, Liu Hxiao, Hou L-fang, Ping J, Prenatal nicotine exposure induces thymic hypoplasia in mice offspring from neonatal to adulthood, Toxicology Letters (2018), https://doi.org/10.1016/j.toxlet.2018.12.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

TITLE PAGE Prenatal nicotine exposure induces thymic hypoplasia in mice offspring from neonatal to adulthood Wen Qu 1，2 a, Wen-hao Zhao1 a, Xiao Wen1, Hui-yi Yan1, Han-xiao Liu1, Li-fang

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Hou1 and Jie Ping 1,* Department of Pharmacology, Wuhan University School of Basic Medical Sciences,

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Wuhan 430071, China; [email protected]

Hubei Provincial Key Laboratory for Applied Toxicology, Hubei Provincial Center for Disease Control and Prevention, Wuhan 430079, China;

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[email protected]

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*Corresponding author: Jie Ping

Tel.: +86 27 6875 9310.

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Address: 185, East Lake Road, Wuhan 430071, China.

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Fax: +86 27 8733 1670.

authors contributed equally to this work.

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a These

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E-mail: [email protected]

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Highlights:  PNE induced persistent thymic hypoplasia in mice male offspring  PNE induced transient thymic hypoplasia in mice female offspring  PNE induced a decreased of CD4SP T cells proportion in thymocyte of both sexes

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 PNE male offspring showed a more serious thymus atrophy in the OVA-sensitized

 PNE induced excessive autophagy in fetal thymocytes

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ABSTRACT

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model

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Clinical study showed that smoking during pregnancy deceased the thymus size in

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newborns. However, the long-term effect remains unclear. This study was aimed to

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observe the effects of prenatal nicotine exposure (PNE) on the development of thymus

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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

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thymic hypoplasia in male offspring from neonatal to adult period and transient changes

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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

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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 1

autophagy in fetal thymocytes might be involved in these changes. All the results provided evidence for elucidating the PNE-induced programmed immune diseases.

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Keywords: Prenatal nicotine exposure, autophagy, thymus, thymic hypoplasia

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1. Introduction Immune system development, especially in the prenatal period, has profound impact for health during early childhood, even throughout life (Hertz-Picciotto et al., 2008). More and more evidences strongly support an association between prenatal

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intervention and the immune diseases of offspring later in life (Yang et al., 2014; Birnbaum and Miller, 2015; Thorburn et al., 2015). It was reported that prenatal

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nicotine exposure (PNE) could cause a long-term suppression of the proliferative response of offspring immune cells (Basta et al., 2000; Grieger et al., 2016; Singh et al.,

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2017).

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The mechanisms of thymus organogenesis and morphogenesis were only to be

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fully understood a few decades ago, and its function in establishing and maintaining an

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appropriate immunity was clearly delineated after the 1960s (Liu and Ellis, 2016). The

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detailed introduction for thymus development with age can be consulted in the review (Zdrojewicz et al., 2016). Age-associated thymic atrophy has been widely discussed

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(Abdul-Salam et al., 2000; Guo et al., 2017). However, aging is not the only cause for

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thymic atrophy. Alterations in the thymocyte numbers and thymic size have been observed in lots of different physiological and pathological states including puberty and pregnancy, inflammation, psychological conditions, bacterial and viral infections,

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environmental conditions, or exposure to toxic substances, etc (Taub and Longo, 2005). Many of these impacts are transient and reversible in contrast with age-associated thymic involution, but some impacts are persisted for a long time even after stressor cessation (Engler and Stefanski, 2003). Evidences have showed that nicotine is an 3

immunomodulator, and our previous study also indicated that PNE could cause increased apoptosis of total thymocytes and CD4 single-positive (SP) cells in fetus and adult offspring (Chen et al., 2016). However, to date, no studies investigated the longterm effects of PNE on thymus development in offspring at different life stages.

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Clarifying the long-term effect of PNE on thymus development in offspring can give us some clues on the increased allergic susceptibility in offspring of maternal smoking.

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Recent investigations showed that autophagy was involved in thymic T-cell

development and the thymus had considerably higher amounts of constitutive basal

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autophagy comparing with other tissues (Bronietzki et al., 2015). Autophagy is a

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lysosome-dependent process that degrades components of cells such as cellular

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molecules or whole organelles. In addition, autophagy promotes cell survival by

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eliminating damaged organelles and proteins aggregates (Mizushima, 2011). In the

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other side, autophagy has also been linked with a form of cell death, called autophagic or type II cell death (Das et al., 2012). Wang et al. (2015) indicated that highly

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pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) HuN4

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strain caused obvious thymic atrophy in piglets, which was related to a high frequency of thymocyte apoptosis and autophagy. It has been proven that nicotine as a main toxic component of cigarette can induce or trigger autophagy process (Kim et al., 2016; Du

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et al., 2017). As key regulators of autophagy, the autophagy related genes Beclin-1 and microtubule-associated protein light chain3 (LC3) were always measured to evaluate the levels of autophagy. With transmission electron microscope (TEM) technique and immunofluorescence staining, Du et al. (2017) observed that nicotine increased the 4

autophagy level of human periodontal ligament cells by up-regulating the expression of LC3. In addition, chronic nicotine treatment also enhanced both the protein and RNA expression levels of Beclin-1 and LC3 II (Xiao et al., 2018). Therefore, we speculated that nicotine might induce autophagic cell death which resulted in decreased number of

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lymphocytes and the thymus atrophy. To test our hypothesis, in the present study, we detected the thymus development

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in PNE offspring at 4 different life stages (postnatal day (PND) 4: neonatal period; PND21: prepuberal period; PND42: adolescent period; PND71: adulthood period). In

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addition, we observed the local inflammation response of the thymus in ovalbumin

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(OVA)-sensitized model in adult offspring. Finally, we took a preliminary mechanism

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exploration to see if PNE elevated the cell autophagy level of the fetal thymus. This

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work will be conducive to characterize the developmental toxicity of nicotine on the

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offspring thymus and provide evidence for the potential mechanisms for the developmental origin of immune diseases.

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2. Materials and methods

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2.1 Chemicals and reagents R Nicotine (C10H14N2, Product No.: N3876, purity ≥99%), OVA and Imject ○

Alum were purchased respectively from Sigma-Aldrich (St. Louis, MO, USA) and

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Thermo Fisher Scientific Inc. (Rockford, USA), and monoclonal fluorescently-labeled primary antibodies (anti-mouse CD4-APC, CD4-APC-Cy7, CD4-Percp-Cy5.5, CD8FITC) were purchased from eBioscience (San Diego, USA). Trizol was provided by Life Technologies (Gaithersburg, MD, USA). Reverse transcription and RT-qPCR kits 5

were provided by TaKaRa Biotechnology (Dalian, Liaoning, China). All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). All chemicals and reagents are analytical grade. 2.2 Animals and treatment

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Specific pathogen-free virginal female (22 ± 2 g) and male (25 ± 2 g) Balb/C mice were obtained from the Experimental Center of Hubei Medical Scientific Academy (No.

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2015-0018, Wuhan, Hubei, China). The animal experiments in this study were performed in the Center for Animal Experiment of Wuhan University (Wuhan, Hubei,

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China), which had been accredited by the Association for Assessment and

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Accreditation of Laboratory Animal Care International (AAALAC International). The

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protocol was approved by the Committee on the Ethics of Animal Experiments in the

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School of Medicine, Wuhan University (Permit number: 2016009). All animal

Laboratory Animals.

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experiment procedures were complied with the Guidelines for the Care and Use of

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Mice were maintained under controlled temperature at 22-24°C and humidity at

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45%-65%. Food and water were provided ad libitum. A schematic of the maternal and offspring treatment procedures was shown in Figure 1. Briefly, after a one-week acclimation period, the timed pregnant mice were randomized into two groups: a PNE

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(prenatal nicotine exposure) group and a PSE (prenatal saline exposure, also named as control group) group. The day of a vaginal plug present was declared as gestational day (GD) 0. From GD9 to GD18, the mice in PNE group were subcutaneously injected with nicotine at 1.5 mg/kg twice daily. To avoid the sudden high concentration of nicotine 6

in vivo, the daily two doses were delivered at an interval of approximately 6 hours. The PSE mice were administrated with the same volume of saline. On GD18, a batch of pregnant mice were sacrificed under isoflurane anesthesia and then fetuses from 10 litters (each litter 6-8 fetuses) per group were obtained and weighed. Fetal thymuses

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from each littermate were collected and pooled into one sample, and 10 pooled samples of each group were stored at -80 °C for subsequent RT-PCR analysis. In addition, 3

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fetal thymuses from different litters were randomly selected for fixing in 2.5% glutaraldehyde solution for TEM analysis.

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The rest pregnant mice were kept until normal delivery (the day of delivery was

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designated as PND0). On PND4, 1 pup per sex per litter was selected randomly from

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10 different mothers in each group and euthanized for thymus weighing and following

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flow cytometry analysis or histopathology detection. To assure adequate and

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standardized nutrition, the rest pups were combined or transferred in the same group to keep 6~7 pups per litter and fed to the scheduled time-point. On PND21, PND42 and

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PND71, pups from different litters of each group were randomly selected, and

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euthanized. At the above each time-point, the thymuses were removed and weighed, and then prepared for the following flow cytometry and histopathology detection

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respectively.

2.3 OVA-sensitized adult offspring

Detailed schedule was indicated in Figure 1. When the offspring matured to 6 weeks of age, the animals were sensitized and challenged with OVA. Mice were divided into 4 groups (8-12 mice from different litters/sex/group): prenatal saline exposure and 7

saline challenge (PSE/NS), prenatal nicotine exposure and saline challenge (PNE/NS), prenatal saline exposure and OVA challenge (PSE/OVA), and prenatal nicotine exposure and OVA challenge (PNE/OVA). The PSE/OVA and PNE/OVA mice were immunized on days PND42, 56 by intraperitoneal (i.p.) injection of 20 μg of OVA

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R emulsified with 2 mg of Imject○ Alum in 200 μL of phosphate-buffered saline (PBS).

On PND67, 68 and 69, mice were challenged consecutively with intranasal OVA

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dosing (100 g/animal). Age- and sex-matched mice that had been sensitized and challenged with PBS at each time point were used as negative controls (PSE/NS and

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PNE/NS group). Mice were sacrificed at approximately 48 h after the last challenge.

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The thymuses were removed and weighed, and then prepared for the following

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histopathology detection.

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Figure 1. Experimental schedule for prenatal nicotine exposure and postnatal OVA sensitization and OVA challenges. GD, gestational day; i.p., intraperitoneal; OVA, ovalbumin.

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2.4 Cell preparation, staining and flow cytometry Thymuses of 4 mice per sex per group (each of the same sex born to different dams)

were collected respectively on PND4, PND21, PND42 and PND71. The harvested

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thymuses were placed in flow cytometry staining buffer (2% fetal bovine serum, 2mM EDTA and 0.05% NaN3 in PBS). Single cell suspensions were prepared with a plunger of a 2.5mL syringe and a 40 μm cell strainer in petri dishes. The number of thymocytes was counted with a cell counting chamber. Cells were then harvested following a series 8

of washing with staining buffer and centrifugation at 400× g, 4°C for 5 min. The cell pellet was re-suspended in 100 μL staining buffer containing appropriate amount of fluorochrome directly conjugated antibodies and incubated on ice for 20 minutes. AntiCD4 and anti-CD8 were used to identify double-negative (DN), double-positive (DP)

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and SP cell subpopulations. Data were acquired with a flow cytometer (BD FACSAriaTM III, BD) using FASCDiva Software in Research Center for Medicine

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and Structural Biology, Wuhan University. All analysis were performed using FlowJo

software (Tree Star Inc., Ashland, USA). The cell preparations from each pup were

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analyzed individually.

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2.5 Histological examination

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For histological examination, thymuses of 3 mice per sex per group (each of the

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same sex born to different dams) were collected respectively on PND4, PND21,PND42

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and PND71. The collected thymus samples were fixed in 10% neutral buffered formalin at least for 24h. The fixed samples were processed in tissue processor (Leica TP 1020-

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1, Germany) and Tissue Embedding Console System (Leica EG1150H/0C) for

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dehydration, transparentizing, waxing and embedding. Then the embedded tissues were sectioned using rotary microtome (Leica RM2245, Germany) at 3 μm thick. The sliced tissue sections were stained with haematoxylin and eosin (HE). Two different visual

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field of each slide were randomly selected and the medulla area as well as the total area of the thymus section were measured with the image-analysis software HMIAS-2000. The ratio of medulla areas was calculated as following: medulla areas/total areas × 100%. For TEM, the selected fetal thymuses were excised into small pieces (< 1 mm3) 9

and fixed overnight in 2.5% glutaraldehyde in PBS. Samples were post-fixed with 1% osmium tetroxide after washed in 0.1 M sodium cacodylate buffer, and then dehydrated through a graded series of ethanol and embedded in EPON812 (SPI-CHEM, Structure Probe, Inc., West Chester, USA). Ultrathin sections (70 nm thick) were obtained with

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an EM UC7 ultramicrotome (Leica, Germany), dually stained with uranyl acetate and lead citrate, and examined with a HT-7700 transmission electron microscope (Hitachi,

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Tokyo, Japan). 2.6 RNA preparation and qPCR

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Total RNA were isolated from the pooled fetal thymuses of one litter (one litter of

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pooled thymuses weighing approximately 10 mg, 10 pooled samples per group in total)

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using Trizol reagent, according to the manufacturer’s protocol. The concentration and

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purity of the isolated RNA were determined by a micro-spectrophotometer (NanaDrop

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2000, Thermo), and the concentration of each sample was adjusted to 1 μg/μL. Singlestrand cDNA was prepared using the reverse transcription kit. All the primer sequences

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were designed by Primer Premier 5.0 from PREMIER Biosoft International (Palo Alto,

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CA, USA) and queried by NCBI BLAST database for homology comparison. PCR assays were performed using a QuantStudio 6 Flex from Applied Biosystems (Foster City, CA, USA) in a total volume of 20 μl reaction mixture containing 1 μl of cDNA

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template, 0.4 μl of 10 μM each primer, 10 μl of 2 × Premix Ex Taq, 0.4 μl of ROX and 7.8 μl of DEPC-H2O. The primers for each gene were listed in Table 1. PCR cycling conditions were as following: 30 s at 95°C for pre-denaturation, 5 s at 95°C for denaturation, 30 S at 60°C for annealing. Each gene was calculated by 10

ΔΔCt

method

using the ribosomal housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) as internal controls. Table 1 Oligonucleotide primers in quantitative real-time PCR.

Forward primer

Reverse primer

Product (bp)

GCTGGAGTTGGATGACGAA

GTGGCATTGAAGACATTGGTT

LC3b

CTCCCATCTCCGAAGTGT

TTGCTGTCCCGAATGTCT

GAPDH

AACTTTGGCATTGTGGAAGG

GGATGCAGGGATGATGTTCT

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Beclin 1

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Genes

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2.7 Statistical analyses

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All measurement data were expressed as mean ± SD. The difference between two

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groups was compared using the t-test. One-way ANOVA assay was used when more than two groups were compared. A 3-way ANOVA was used to compare the sex

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differences. Values of P<0.05 were considered statistically significant.

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3. Results

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3.1 PNE disturbed the thymus developmental curve in offspring

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Figure 2 The effect of PNE on the absolute and relative weight of the offspring’s thymus. PSE: prenatal saline exposure; PNE: prenatal nicotine exposure. Data were indicated as Mean±SD, n=7-11 per sex per group. *P<0.05, ** P<0.01, compared with PSE

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To observe the long-term effect of PNE on the thymus of the offspring, we monitored the offspring’s thymus weight at 4 different life stages: PND4, PND21,

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PND42 and PND71. As shown in Figure 2, both the absolute and the relative thymus

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weight reached the peak value on PND21. For male offspring, as indicated in Figure 2A and 2B, from PND4 to PND71 the thymus weight and thymus index in PNE

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offspring decreased markedly when compared to the control. The thymus weight in male PNE offspring decreased by 34%, 14%, 13% and 32% at the above 4 time points

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respectively, as compared with the control. And the same level of changes were observed in the thymus index. As for females, the thymus weight and index in PNE offspring were significantly lower than that of control only at PND4 and PND21 (P<0.01 or P<0.05, Figure 2C and 2D). By comparison with the control, the thymus 12

weight and index in female PNE offspring decreased by 33% and 12% at PND4, 25% and 17% at PND21 respectively.

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3.2 PNE induced the reduction of thymus medullar areas in offspring

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Figure 3 Representative light micrographs of thymus and the ratio of medullar areas measured by image-analysis software HMIAS-2000 in offspring at different life stages. PSE: prenatal saline exposure; PNE: prenatal nicotine exposure; PND: postnatal day. The scale bar is 100μm. The values are expressed as Mean±SD, n=6. *P < 0.05; **P < 0.01 as compared with that of current control. The magnification multiple is 100×.

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Furthermore, the thymus sections were examined at a lower magnification using a digital microimaging device (image-analysis software HMIAS-2000) to evaluate relative medulla areas (medulla areas/total areas × 100%). As indicated in Figure 3,

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compared with PSE offspring, the PNE offspring showed significantly decreased medulla area as compared with that of the concurrent control. This kind of lesions were lasted from neonatal to adult period for males and only were presented in the neonatal and prepuberty for females, which was consistent with the changes of the thymus 13

weight.

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3.3. PNE altered the phenotypes and counts of thymocytes in the offspring

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Figure 4 PNE altered the phenotypes and counts of thymocytes in the offspring at different time points. PSE: prenatal saline exposure; PNE: prenatal nicotine exposure; PND: postnatal day; TC: Total thymocytes counts; DP: CD4+CD8+ double-positive thymocytes, CD4SP: CD4+CD8single-positive thymocytes, CD8SP: CD4-CD8+ single-positive thymocytes, DN: CD4-CD8double-negative thymocytes. The values are expressed as Mean±SD, n=4. *P < 0.05; **P < 0.01, as compared with that of concurrent control.

We further analyzed the thymocyte phenotypes at the above mentioned 4

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timepoints PND4, PND21, PND42 and PND71. At the same time, we counted the cell number of the entire thymus and subpopulations. As shown in Figure 4, PNE offspring exhibited significantly lower percentages and absolute numbers of CD4SP thymocytes when compared with the concurrent control PSE offspring (P<0.05 or P<0.01). Both 14

the male and female offspring showed increased proportions of DP thymocytes in all the tested timepoints with or without statistic significance. Consistent with the changes of thymus weight, the cell number of total thymocytes and subpopulations in PNE offspring decreased significantly (P<0.05 or P<0.01) when compared with the control.

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3.4. OVA-induced serious thymic atrophy in both PSE and PNE offspring

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Figure 5 PNE altered the weight and histopathology of adult offspring’s thymus in OVAsensitized model. A: The absolute weight of thymus in OVA-sensitized model; B: The relative weight of thymus in OVA-sensitized model. The values are expressed as Mean±SD, n=8-12. *P < 0.05; **P < 0.01 as compared with the relevant control; C: Representative light micrographs of thymus in OVA-sensitized model. The scale bar is 50μm and the magnification multiple is 400×.

To observe the thymus alteration during antigen stimulation in PSE and PNE

offspring, we immunized the mice with OVA. As shown in Figure 5A and 5B, after OVA stimulation, the thymus shrinked remarkably. Both the thymus weight and index in PNE or PSE offspring decreased significantly after OVA stimulation, and the male 15

PNE offspring had a much smaller thymus than that of either the OVA-sensitized PSE offspring or PNE offspring without OVA stimulation, although no significance were observed between them. Compared with the thymus weight from PSE offspring without OVA challenge, the thymus weight decreased by 32% and 51% respectively for PSE

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and PNE offspring with OVA challenge. However, the thymus weight of PSE and PNE offspring with OVA challenge decreased by 32% and 28% when compared with their

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own counterpart without OVA challenge. As indicated in Figure 5C, the histological

profiles showed the normal thymic architecture in PSE/NS, however, for the thymus of

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OVA-sensitized mouse from PSE offspring, the gap between cells became widened and

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a large number of infiltrated macrophage around the vessels were observed. Notably,

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the scarcity of the lymphocyte cells, numerous granule cells and erythrocyte were

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present in the cortico-medullary zone in the OVA-sensitized male PNE offspring. The

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infiltration of inflammatory cells were more serious in PNE male offspring than in females.

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3.5. PNE increased the thymocytes autophagy in fetal thymus

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Figure 6 PNE induced thymocytes autophagy in fetal thymus as indicated in representative TEM graph and RT-PCR results. PSE: prenatal saline exposure; PNE: prenatal nicotine exposure. A and C:Rare autophagic vacuole was observed in PSE offspring thymocyte. (A: 1000; C: 5000); B and D: Lots of autophagic vacuoles were presented in the cytoplasm in PNE offspring thymocyte as shown by the black arrow. (B: 1000; D: 5000); E: the magnification of autophagic vacuole in Figure 6D. (E: 10000); F: the effect of PNE on the expression of Beclin1 and LC3b mRNA level. Data were indicated as Mean±SD, n=9-10, *P<0.05, compared with PSE

As indicated in Figure 6B and 6D, the autophagic vacuole in PNE offspring

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increased notably when compared with that of PSE (Figure 6A and Figure 6C). And the

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mRNA level of autophagy related gene Beclin 1 was increased to almost 1.5-fold of the control (Figure 6F, p < 0.05). No obvious changes were observed for the mRNA level of LC3b between the PNE and PSE groups.

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4. Discussion

In this study, we selected the dosage of nicotine at 3 mg/kg/d which could equate to a moderate smoker during pregnancy (exposure to 6-8 cigarettes per day) (Benowitz et al., 1998; Chen et al., 2016) . The amounts of nicotine and the administration method 17

of subcutaneous injection selected in this study were previously widely used for nicotinic toxicity studies in rodent and the effects had been confirmed (Mohsenzadeh et al., 2014; Liu et al., 2017). The subcutaneous injection method provides better control of blood nicotine levels compared to cigarette smoke inhalation because of individual

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differences in smoking/breathing pattern or the uptake of nicotine by the airways (Keith, 1988). Compared with the dosing route of inhalation, subcutaneous injection is more

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practicable and the internal exposure is more controllable. The exposure time window from GD9 to GD18 covered all the period of fetal thymus organogenesis and

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development and also avoided the adverse effect of nicotine on the embryo implantation.

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With this PNE model, we focused on the observation of thymus development in

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offspring at different life stages. Therefore, we chose four time points PND4, PND21,

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PND42 and PND71, which covered the stages from neonatal, prepuberal, adolescent to

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adult periods (Dicken et al., 2012; Schneider, 2013). The immune system of animals and humans changes with age, which have been

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reported to be especially remarkable in the thymus. In murine, the absolute weight of

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the thymus starts to decline around 40-50 days of age (Takeoka et al., 1996; Dominguez-Gerpe and Rey-Mendez, 1998). Our results showed that both the absolute and relative weight of the thymus reached its maximum at PND21 and started to decline

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at PND42, which was almost consistent with the aforementioned result. A clinical study reported that the thymus index of the newborns from the smoking mothers were significantly lower than that of the controls (Zeyrek et al., 2008). In our study, we found a decreased thymus weight on PND4 and PND21 both in female and male PNE 18

offspring, which correlated with the aforementioned clinical results. Nevertheless, after adolescence, PNE male offspring showed a still reduced thymus size on PND 71. Our previous research showed that the growth rates of PNE offspring were higher than that of the concurrent control, but the body weight of PNE offspring were still lower than

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or almost equal to that of control (Chen et al., 2016). The similar results were observed in this study, which meant the reduced thymus weight or thymus index of PNE offspring

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were not due to the changes of body weight. The thymus is a very sensitive target organ

to immunotoxicants, and a decrease in its weight or size is often regarded as one of the

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characterization of toxicity in early stage (Schuurman et al., 1992). Consistent with the

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thymus weight changes, the cell count of the thymus showed the same decreased trends.

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It had also been shown that the histological findings in the thymus was associated well

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with thymus weight and peripheral lymphocyte counts in both the rat and dog (Elmore,

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2006). Other studies, also conducted by our research group, have investigated the longterm consequences of prenatal nicotine exposure (Dwyer et al., 2008; Chen et al., 2016),

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but few have observed the potential sex differences in exposure outcomes (Cross et al.,

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2017). However, the clinical studies on maternal smoking suggested that such differences occurred in offspring (Weissman et al., 1999). It was also reported that the thymus involution started earlier in males than in females, which might be mediated by

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gonadal hormones (Dominguez-Gerpe and Rey-Mendez, 1998). This indicated that male sex hormones were stronger than female sex hormones as an immunosuppressant for thymus development. Therefore, the weight and histopathology alterations of thymus were lasted from neonatal to adult period in males and only were presented in 19

the neonatal and prepuberty stages for females. It has been reported that gestational nicotine exposure delays the onset of puberty in female animals (Meyer and Carr, 1987), whereas pubertal milestones occurs at an earlier age among the male offspring of cigarette smokers than that of nonsmokers (Fried et al., 2001). Therefore, PNE induced

males and females of thymus development observed in our study.

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gonadal hormones alteration in offspring might contribute to the different changes in

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As T cells are developed and differentiated in thymus, the changes of microenvironment in the thymus may impact the differentiation of T cells (Qu et al.,

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2017). During T cell development in the thymus, CD4−CD8− DN cells differentiate to

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CD4+CD8+ DP cells, which develop to either CD4SP or CD8SP populations by positive

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selection. Many study models have been described how positive selection ensures that

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selected CD4 SP and CD8 SP populations express TCR precisely restricted by MHC-II

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and MHC-I, respectively (Carpenter and Bosselut, 2010). In this study, we successively observed thymocyte phenotype from neonatal to adult period. Consistently, PNE not

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only changed the thymus morphology, but also induced a permanent lower CD4 SP

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proportion and slightly increased DP proportion in both sex. Middlebrook et al (2002) took fetal thymus organ culture (FTOC) as an in vitro model for T cell maturation, and they showed that nicotine altered the positive selection of immature T cells and arrested

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the T cell maturation, which meant nicotine could directly impede the transition from DP to SP stage. Another research reported that developmental changes within the thymic microenvironment at GD15 are critical for the positive selection of a mature MHC-II -restricted T-cell repertoire (Fairchild and Austyn, 1995). It meant that the 20

prenatal nicotine exposure around GD15 resulted crucial impact on the CD4 SP T cells maturation. Our previous study also manifested that the apoptosis level of fetal CD4 SP in PNE group were higher than those of control and the increased apoptosis percentage of CD4 SP was also observed in PNE offspring (Chen et al., 2016). Both previous data

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and our current results indicated that CD4 SP were more vulnerable to PNE. Besides, another study showed that more sustained signaling was required for selection into the

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CD4 compartment than that of CD8 generation (Fu et al., 2014), however, such signaling may be impeded by PNE. Hereby, we demonstrated that PNE decreased both

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the percentage and number of CD4SP in mice thymus of offspring from neonatus to

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adults.

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The changes of thymocyte phenotype might alter the local thymic inflammation

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during immune response. It was reported that marked atrophy, severe degeneration of

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the lymphocytes and infiltration of macrophage in the thymus were noted in the rats with acute experimental allergic encephalomyelitis (Hara et al., 1986). In current study,

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we observed the thymus alterations in OVA-induced allergic response. OVA derived

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from chicken egg is widely used as an allergen, which can induce allergic pulmonary inflammation in the laboratory rodents (Fuchs and Braun, 2008; Sun et al., 2010). Our study showed that sensitization with i.p. injection at a lower dose of OVA (20μg/animal)

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followed by a higher dose of OVA intranasal challenging (100μg/animal) could effectively arouse allergic response in animals. Most of studies with the OVA induced allergic model were focused on observing the response in secondary lymph organs and lung (Yamashita et al., 2006; Penido et al., 2008). Rare studies concerned the thymus 21

changes in OVA-induced allergic response. A recent study showed the percentage of CD3+CD4+ T cells in the mice thymus was increased after OVA sensitization and challenging when compared to the group without OVA-immunization (de Castro et al., 2017). These results could indicate that thymus also might play an active role in

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immune response. Hence, for the first time, we observed the size and histology changes of thymus in adult offspring after OVA stimulation and detected if PNE would alter

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these response. We found the thymus size decreased significantly both in PNE and PSE offspring compared to the group without OVA stimulation, and notably, the PNE male

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offspring had a much smaller thymus. This reminded us that there might be an altered

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immune response in PNE offspring during antigen stimulation.

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Autophagy has been proven to play an important role in thymic development.

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When using CD4Cre mice with conditionally deleted Atg (autophagy related genes)

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genes at later stages of thymic development, thymocyte numbers were not affected (Willinger and Flavell, 2012; Parekh et al., 2013). However, when deletion occurred

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early in the thymus development, total thymocyte numbers were reduced modestly to

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severely (Pua et al., 2009; Stephenson et al., 2009; Arsov et al., 2011; Jia and He, 2011). It means disturbed autophagy in early life affect the number of thymocytes and the proportion of subpopulations. Furthermore, the autophagy process was initiated and

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regulated by autophagy related genes such as LC3 or Beclin 1 (Khoso et al., 2017). LC3b (one of the isoforms of LC3) initiates autophagosome biogenesis, which is essential for the extension of autophagosomal isolation membrane (Huang and Liu, 2015). Beclin1 serving as an interaction hub or scaffold targets proteins to specific 22

membranes for autophagosome formation (Mei et al., 2016). In our study, we found PNE increased autophagy in fetal thymus, which manifested as increased autophagic vacuole and elevated mRNA level of Beclin 1. The mRNA level of LC3b was not influenced by PNE, which might indicate a different regulation mechanism of PNE on

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these two genes. Thus, we speculated that increased autophagy might be involved in the thymocyte atrophy and the change of subsets proportion. Numerous studies have

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confirmed that autophagy promote cells survival by exemption from apoptosis under

conditions of oxidative stress. In contrast, some studies demonstrated that autophagy

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induced apoptosis by priming autophagosome formation (Joshi-Barr et al., 2014; Wu

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et al., 2015; Moon et al., 2016). Another recent study showed that hypoxia-induced

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autophagy facilitated apoptosis and resulted in muscle atrophy (Chen et al., 2017). Our

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previous study revealed that PNE could increase the apoptosis of thymocyte in fetus

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and adult offspring (Chen et al., 2016), whether the autophagy facilitated the thymocyte apoptosis need to be further investigated.

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In summary, we observed the thymus development in PNE offspring from

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neonatal to adulthood stages and found that PNE could induce the reduction of thymus size and decrease the quantities and proportions of mature CD4 SP cells. We also demonstrated excessive autophagy in PNE fetal thymocytes and proposed it might be

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involved in the thymus atrophy and the reduction of CD4 SP T cells proportion. In addition, the thymus from PNE male offspring showed a more serious atrophy when encountered with OVA immunization. Our studies showed for the first time a systematical observation for PNE induced thymus alterations in offspring at different 23

life stages, and the results could be valuable for exploring the developmental origin of immune disease susceptibility in PNE offspring. ABBREVIATIONS DN: CD4-CD8- double-negative thymocytes; DP: CD4+CD8+ double positive

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thymocytes; FBS: fetal bovine serum; FCS: flow cytometry staining; GAPDH:

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glyceraldehyde phosphate dehydrogenase; GD: gestational day; OVA: ovalbumin; PBS: phosphate-buffered saline; PND: postnatal day; PNE: prenatal nicotine exposure; PSE: prenatal saline exposure; RT-PCR: reverse-transcription PCR; SP: single positive

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thymocytes.

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ACKNOWLEDGEMENTS

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This work was supported by Grants from the National Natural Science Foundation of China [grant numbers 81673215, 81273107]; the Applied Fundamental Research

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Project of Wuhan [grant number 2017060201010199]; and the youth scholar of Luojia.

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CONFLICTS OF INTEREST

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No potential conflicts of interest were disclosed. REFERENCES

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