Maternal exposure to airborne particulate matter causes postnatal immunological dysfunction in mice offspring

Toxicology 306 (2013) 59–67

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Maternal exposure to airborne particulate matter causes postnatal immunological dysfunction in mice offspring Xinru Hong a,b,c,∗ , Chaobin Liu d,∗∗ , Xiaoqiu Chen e , Yanfeng Song a , Qin Wang f , Ping Wang g , Dian Hu h a

Department of Obstetrics and Gynecology, Fuzhou General Hospital, Fujian, China Fuzhou Clinic Medical College, Fujian Medical University, Fujian, China Dongfang Affiliated Hospital of Xiamen University, No. 156 North Xi’erhuan Road, Fuzhou 350025, Fujian, China d Department of Obstetrics and Gynecology, Fujian Provincial Maternal and Child Health Hospital, No. 18 Daoshan Road, Fuzhou 350001, Fujian, China e Fujian Central Station of Environmental Monitoring, No. 138 South Fufei Road, Fuzhou 350003, Fujian, China f Fuzhou Product Quality Monitoring Bureau, No. 83 Yangnan Street, Fuzhou 350025, Fujian, China g The Official Hospital of 92403 Unit of PLA, No. 34 Duihu Road, Fuzhou 350007, China h Department of Obstetrics and Gynecology, Changzheng Hospital, Second Military Medical University, No. 415 Fengyang Road, Shanghai 200003, China b c

a r t i c l e

i n f o

Article history: Received 30 November 2012 Received in revised form 15 January 2013 Accepted 1 February 2013 Available online 14 February 2013 Keywords: Airborne particulate matter In utero exposure Immunotoxicity Offspring

a b s t r a c t Evidence suggests that prenatal exposure to air pollution affects the ontogeny and development of the fetal immune system. The aim of this study was to investigate the effect of maternal exposure to airborne particulate matter (PM) on immune function in postnatal offspring. Pregnant female ICR mice were intralaryngopharyngeally administered with 30 ␮l of phosphate buffered solution (the control group) or resuspended PM of Standard Reference Material 1649a at 0.09 (low), 0.28 (medium), 1.85 (high) or 6.92 (overdose) ␮g/␮l once every three days from day 0 to 18 of pregnancy (n = 8–10). Offspring were sacrificed on postnatal day 30. Interleukin-4 and interferon-␥ levels in plasma and splenocytes, splenic lymphocyte proliferation, and expressions of GATA-3 and T-bet mRNA in the spleen were tested. The spleen and thymus were histopathologically examined. The offspring of the medium, high and overdose PM-exposed dams showed significantly suppressed splenocyte proliferation. Decreased interferon-␥ and increased interleukin-4 levels in the blood and splenocytes, and lowered T-bet and elevated GATA-3 mRNA expressions were found in the spleen in the medium, high and overdose groups when compared with the control or low dose group (P < 0.05). Histopathology revealed prominent tissue damage in the spleen and thymus in the overdose group. These results suggest that exposure of pregnant mice to PM modulates the fetal immune system, resulting in postnatal immune dysfunction by exacerbation of Thl/Th2 deviation. This deviation is associated with altered T-bet and GATA-3 gene expressions. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction As a result of extensive urbanization and an increasing number of vehicles, air pollution has become a severe problem in many cities within economically emerging countries (Zhang, 2011). Epidemiologic evidence has shown a significant increase in immunologic diseases in severely polluted areas (Nastos et al., 2010; Brauner et al., 2010). Additionally, the prevalence of

∗ Corresponding author at: Department of Obstetrics and Gynecology, Fuzhou General Hospital, No. 156 North Xihuan Road, Fuzhou 350025, Fujian, China. Tel.: +86 591 2285 9200; fax: +86 591 8789 3800. ∗∗ Corresponding author. Tel.: +86 591 8727 9622. E-mail addresses: [email protected] (X. Hong), [email protected] (C. Liu), [email protected] (X. Chen), [email protected] (Q. Wang), [email protected] (P. Wang), [email protected] (D. Hu). 0300-483X/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tox.2013.02.004

immunologic diseases in early life has been on the rise over the last few years (Price et al., 2012). The exposure to air pollution in utero has also been shown to be potentially devastating, as the first signs of some immune disorders, such as the infantile anaphylactic diseases, neonatal lupus erythematosus and some cancers, can develop in infants shortly after birth (Yu et al., 2006; Izmirly et al., 2010; Kozyrskyj et al., 2011). Thus, the high prevalence of immunologic diseases in children may be putatively associated, at least in part, with the consequent maldevelopment of the prenatally impaired immune system (Dietert, 2011). If this is true, it will suggest that exposure to air pollution in utero may well affect the development of the fetal immune system. Airborne particulate matter (PM) is one of the main contributors to ambient air pollution throughout the world, especially in developing countries (Kan et al., 2009). PM is a common air pollutant that could have a significant impact on the immune system during both pre- and postnatal periods of life, potentially resulting in system

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malfunction in early life (Svendsen et al., 2007). In particular, particulate matter that is <2.5 ␮m in diameter (PM2.5 ) has been shown to influence fetal immune system development and appears to result in altered immunoglobulin E (IgE) (Herr et al., 2011) and lymphocyte distributions (Herr et al., 2010) in cord blood among neonates. Maternal exposure to air pollution before and during pregnancy can also alter immunity in offspring, thus increasing a child’s risk to develop health conditions like asthma and allergies later in life (Baïz et al., 2011). In summary, data from epidemiologic surveys and clinical observation suggests that the fetal immune system is highly vulnerable to environmental pollutants – much more so than the more developed systems of infants and older children. Exposure to ambient levels of PM generally causes no appreciable effects, but impalpable alteration that results from prenatal PM exposure may come to exert a more evident impact on later development throughout childhood or even adulthood (Corson et al., 2010). Nonetheless, the results are not always consistent regarding the immune system, in spite of the fact that infants who later develop immune disorders show some altered neonatal immune responses (Prescott, 2006). We hypothesized that in utero exposure to PM may affect the development of the fetal immune system, resulting in its abnormal phenotype in early life following birth. In the present work, we investigated in utero PM exposure on childhood immune function in a subacute murine model with administration of resuspended standard PM. We tested T-bet and GATA-3, both of which are the transcription factors that induce cell differentiation of T helper cell subgroup 1 (Th1) and subgroup 2 (Th2), in addition to systemic and local biochemical measurements and morphological changes in the immune organs. 2. Materials and methods 2.1. Animals Sixty females and 30 males specific pathogen free (SPF) ICR nonparous mice of 6-week-old were purchased from Fujian Center for Disease Control and Prevention (Certificate number: SCXK2011-0001, Fuzhou, China), of which the female mice weighed 29 ± 2 g and the male mice 32 ± 2 g for copulation. The mice were housed in a climate controlled room at 23 ± 1 ◦ C with 55 ± 7% humidity, in addition to a 12/12h light/dark cycle and standard rodent chow with water ad libitum. The experiments were performed in accordance with the guidelines of the “Principles of Laboratory Animal Care” by the National Society for Medical Research in China. The experimental protocol was approved by the Scientific Research Committee of Fuzhou General Hospital.

tongue pulled gently aside by a pair of forceps. A 30-␮l SRM 1649a suspension or the same volume of PBS alone was pipetted to the base of the tongue, in which the mouse was held steady for at least two deep breaths (less than 15 s). The particle suspension was administered beginning from day 0 and repeated at day 3, 6, 9, 12, 15 and 18 with a total of 18.9 (low), 109.2 (medium), 388.5 (high) or 1453.2 ␮g (overdose) of SRM 1649a per mouse. After the last SRM 1649a administration at day 18, the mice were housed individually and were allowed to deliver. Shortly after parturition, the pups, together with their dam in a cage, were transferred to an airborne particle-eliminated chamber for 30 days before the experiment was performed. Mice with the onset of delivery within day 20–22 of pregnancy and with number of pups within 8–12 per litter were allowed to enter the experiment. 2.3. Collection and storage of organ and blood samples After preclusion of the dams that did not match the condition, there were 8–10 dams (litters) enrolled in each group. Total of 40 fetal mice from the 8 to10 litters with each litter 4–5 fetuses in a group were randomized, weighed, and decapitated after eutherization. Blood samples from the randomly selected fetal mice in each litter, with each litter as one sample, were pooled into a heparin-containing tube (n = 8–10) and blood plasma was isolated and stored in −70 ◦ C. The thoracic gland and spleen were identified, isolated and weighed. Ratios of organ weights summed from the selected 4 to 5 fetal mice in each litter over their summed body weights were calculated and compared among the groups (n = 8–10). The randomly chosen 20 spleen organs and 20 thoracic gland organs from the total 40 organs with every litter 2–3 spleen or thoracic gland organs in a group were processed for histopathological examination. Half of the remaining 20 spleen organs, with every litter 2–3 organs pooled to form one sample (n = 8–10), were cut into small pieces about 2–3 mm3 in ice-cold saline and stored in RNA storage liquid in −70 ◦ C, and the other half in 37 ◦ C saline for preparation of spleen lymphocyte suspension. 2.4. Measurement of total immunoglobulin G1, G2a (IgG1, IgG2a) and IgE antibodies in plasma Plasma samples were thermometrically balanced to room temperature. Total IgE antibodies in the plasma were measured by a sandwich technique using the ELISA kit according to the manufacturer’s protocols (Yamasa Co., Chiba, Japan), in which two monoclonal rat anti-mouse IgE antibodies recognizing different epitopes on the Fc␧R fragment were used. Optical density at 450 and 550 nm was determined using a microplate reader. Total IgG1 and IgG2a antibodies in the plasma were also measured by ELISA. Anti-mouse Ig (BioLead Biology Sci & Tech Co. Ltd., Beijing, China) of 0.1 ml containing 10 ␮g/ml protein diluted by 0.05 mol/L coating buffer (pH = 9.6) was added to each reacting well in the microtiter plate and was incubated at 4 ◦ C overnight. The plate was washed four times in PBS containing 0.1% Tween-20, added by diluted plasma at 37 ◦ C for 2 h, and washed sequentially by PBS and PBS containing 0.1% Tween-20 (PBST) each for two times. One hundred microliters diluted horseradish peroxidase-labeled anti-mouse IgG1 or IgG2a (1:1000, BioLead Biology Sci & Tech Co. Ltd.) were added into each well, incubated at 37 ◦ C for 1 h, washed for 5 times, added by 100 ␮l substrate buffer, and then incubated away from light at 37 ◦ C for 30 min. The reaction was terminated by addition of 0.05 ml sulphuric acid (2 mol/L) and optical density was read at 450 nm with the microplate reader.

2.2. PM exposure Airborne PM of Standard Reference Material (SRM) 1649a was purchased from the National Institute of Standards and Technology (Gaithersburg, MD, USA). This particulate matter is an atmospheric material collected in an urban area with particle diameters ranging 6.7–100 ␮m (averaged 12.9 ␮m). Substances that deposit on the surface of these particle cores include polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCB) congeners and chlorinated pesticides, inorganic salts, heavy metals, bioactive components such as lipopolysaccharides, and other trace elements, many of which, especially the organic components that dominate the deposits, are noxious to human health (Benner, 1998; Chiu et al., 2001; Albinet et al., 2006). Prior to exposure, SRM 1649a was resuspended in sterile phosphate buffered saline (PBS) to obtain a final concentration of 0.09, 0.28, 1.85 or 6.92 ␮g/␮l and was stored at 4 ◦ C. The suspension, which contains the indissoluble (e.g., PAHs), poorly dissoluble (e.g., PCB and chlorinated pesticides) and dissoluble (e.g., inorganic constituents) components, was thoroughly mixed and sonicated for at least half an hour in an ultrasonic vibration instrument prior to administration. Mice were allowed one week adjustment prior to the experiments. The female mice were placed with the male mice (2:1) overnight and pregnancy was confirmed by the presence of vaginal plug valve (referred to as gestation day 0). The pregnant mice were weighed, numbered, and randomly divided into one of the five groups (12 mice per group): experimental groups, which were administered with SRM 1649a suspension at concentration of 0.09, 0.52, 1.85 or 6.92 ␮g/␮l (low, medium, high, or overdose group), and the control group, which received the same volume of PBS without particles. The instillation procedure to deliver resuspended particles to the mice was performed as described previously, by which 77.5–88.2% particles would enter the lung (Rao et al., 2003). The female mouse was briefly anesthetized in a glass container filled with isoflurane. The mouse’s mouth was opened using rubber bands with the

2.5. Spleen lymphocyte proliferation test Spleen homogenates were filtered through a 200-screen mesh grit made of stainless steel to prepare a single-cell suspension. The erythrocytes in the suspension were lyzed by addition of ammonium chloride. The lymphocytes were harvested by centrifugation and re-suspended in RPMI 1640 (Sigma–Aldrich Co., St. Louis, MO, USA) containing 10% calf serum. The vial lymphocytes, confirmed by trypan blue staining, were adjusted to a density of 1 × 106 /ml, and inoculated in 96-well culture plates with 200 ␮l in each well. Each sample was inoculated in 6 wells, 3 of which were added by 10 ␮l phytohemagglutinin and the other 3 were not to serve as the control. The mixture was cultured at 37 ◦ C for 72 h under 5% carbon dioxide and saturated humidity, and was added by 10 ␮l tetramethylthiazole blue (Sigma, St. Louis, MO, USA) prior to the end of the culture. Supernatant of 100 ␮l was gently removed from each well and replaced by the same amount of DMSO. The mixture was then vibrated for 30 min and tested at 570 nm for optical density. 2.6. Assay for interferon- (IFN-) and interleukin (IL)-4 contents The above spleen lymphocyte suspension preparation was inoculated in a 96well culture plate, followed by 10 ␮l phytohemagglutinin per well, and cultured at ◦ 37 C for 48 h under 5% carbon dioxide and saturated humidity. The suspension was then centrifuged at approximately 3000 × g at 4 ◦ C for 10 min and the supernatant was used for the assay. Contents of IFN-␥ and IL-4 in the supernatant and plasma were assayed by using a solid phase sandwich technique using a commercially available ELISA kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and were performed in duplicate. Results are expressed as pg/ml. Ratios of IL-4/IFN-␥ were calculated.

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Table 1 Primer sequences and fragment lengths of the targeted genes. Gene

Primer sequence

Fragment length (bp)

T-bet

Forward: 5 -GGT GTC TGG GAA GCT GAG AG-3 Reverse: 5 -TCT GGG TCA CAT TGT TGG AA-3

374

GATA-3

Forward: 5 -TCT GGA GGA GGA ACG CTA ATG G-3 Reverse: 5 -GAA CTC TTC GCA CAC TTG GAG ACT C-3

408

GAPDH

Forward: 5 -GAA GGG CTC ATG ACC ACA G-3 Reverse: 5 -GGA TGC AGG GAT GTT C-3

166

2.7. Histological examination The spleen and thoracic gland tissue samples were fixed with paraformaldehyde, dehydrated, embedded in paraffin, sliced at 5 ␮m, and stained with hematoxylin and eosin (H&E) for light microscopic examination (400×, Olympus BX-41, Japan). An experienced histopathologist who was blinded with the experimental design examined all the slides with reference to the pathological scoring standards, and the degree of each histopathological abnormality was evaluated in a semi-quantitative fashion and graded numerically from 0 (normal) to 3 (severest) (Xiping et al., 2007; Zhang et al., 2009). Ten micrographs from each specimen were taken from consecutive squares to define the degree of the anatomic abnormality. 2.8. T-bet and GATA-3 mRNA expressions Copy number of T-bet and GATA-3 mRNA in the spleen was estimated by relative quantification using real-time quantitative polymerase chain reaction (TaqMan Gene Expression assay) (Heid et al., 1996; Oomizu et al., 2006). The assay was performed with iCycler iQ real-time polymerase chain reaction detector (BioRad, Germany). The spleen samples from each litter of the young mice were pooled and homogenized by TRIzol Reagent (Invitrogen) methodology. The sequence of the primers and the length of the fragments for T-bet and GATA-3 were shown in Table 1. Reverse transcription was performed to synthesize the first-strand complementary DNA (cDNA) from 1 ␮g of total RNA using oligod (dT)18 primers according to the manufacturer’s instructions (Invitrogen). Applied Biosystems supplied the TaqMan minor groove binder probes for T-bet and GATA-3 genes, as well as the TaqMan Rodent GAPDH Control Reagents (Applied Biosystems, Carlsbad, CA, USA) for quantification of the internal standard glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA. The thermocycler program was set as 2 min at 50 ◦ C and 10 min at 95 ◦ C for one cycle, and 15 s at 95 ◦ C and 1 min at 60 ◦ C for 60 cycles. The gene copies were measured and calculated by correlating the polymerase chain reaction threshold cycle obtained from the spleen tissue sample to the amplicon-specific standard curve.

Fig. 1. Levels of total IgG1 and IgG2a, and IgE antibodies in the plasma in the mouse offspring with their dams receiving intralaryngopharyngeal SRM 1649a during the pregnancy. n = 8–10. CT: control, LD: low dose, MD: medium dose, HD: high dose, and OD: overdose. *P < 0.05 compared to the controls; # P < 0.05 compared to low dose; + P < 0.05 compared to overdose.

3.2. Total IgG1, IgG2a and IgE antibodies In the plasma, levels of IgG1 and IgE antibodies were significantly higher in the medium, high and overdose groups compared with the control or low dose group (P < 0.05). Levels of IgG2a antibodies were significantly lower in the overdose group compared with the control, low, medium or high dose group (P < 0.05) (Fig. 1). 3.3. Spleen lymphocyte proliferation Values of optical density from the spleen lymphocyte proliferation test was significantly lower in the medium, high and overdose groups compared with the control group, and significantly lower in the high and overdose groups compared with the low dose group (P < 0.05). No significant difference was found between the control group and the low dose group (P > 0.05) (Fig. 2). 3.4. IFN- and IL-4 levels

2.9. Statistical analysis Normally distributed data are expressed as mean ± standard deviation (SD) while abnormally distributed data expressed as median (interquartile range). One litter was considered one experimental unit in the statistic analysis. Normally distributed data were assessed by one-way ANOVA among the groups, and Newman–Keuls post hoc testing was applied for pair-wise comparisons between the means. Abnormally distributed data were analyzed by Kruskal–Wallis H test for non-parametric tests of multiple comparisons and Mann–Whitney U test for comparisons between two groups.

In the plasma and supernatant of the cultured spleen cells, levels of IFN-␥ were significantly lower in the medium, high and overdose groups when compared with the control or low dose group, and were significantly lower in the overdose group when compared with the medium or high dose group (P < 0.05). Levels of IL-4 and ratios of IL-4/IFN-␥ were significantly higher in the medium, high and overdose groups when compared with the control or the low dose group, and were significantly higher in the overdose group

3. Results 3.1. Organ weight All maternal mice survived throughout the experiment. Weights of the pregnant mice before the SRM 1649a challenge among the groups and the pooled weights of the selected fetuses from a litter (as one sample) were not significantly different (data not shown). For the controls, low, medium, high and overdose groups in the offspring, ratios of spleen weight over the body weight were 5.52 ± 0.30, 5.57 ± 0.29, 5.48 ± 0.27, 5.54 ± 0.21 and 5.38 ± 0.32, respectively, and were not significantly different among the five groups (F = 1.084, P > 0.05). Similar results were seen in ratios of thoracic gland weight over their body weight, which were 2.94 ± 0.19, 3.03 ± 0.14, 3.01 ± 0.14, 2.93 ± 0.16 and 2.94 ± 0.20, correspondingly (F = 1.493, P > 0.05).

Fig. 2. Values of optical density in the supernatants from the spleen lymphocyte proliferation test in the mouse offspring with their dams receiving intralaryngopharyngeal SRM 1649a during the pregnancy. n = 8–10. CT: control, LD: low dose, MD: medium dose, HD: high dose, and OD: overdose. *P < 0.05 compared to the controls; # P < 0.05 compared to low dose.

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deep-color lymphocytes in its cortex, and distinct blood vessels and reticulocytes with dispersively distributed lymphocytes in its medulla in the control group (Fig. 5a). In the overdose group, the thymic cortex was broadened with plenty of proliferated immature monocytoid cells whereas the thymic medulla was coarctate with a larger amount of apoptotic debris and apoptotic bodies. The boundaries between the cortex and medulla were unclear (Fig. 5b–d). The overall histopathology scores indicate that the histopathological changes were most severe in the spleen and thoracic gland tissues in the overdose group, moderate in the high dose group, and minor in the medium and low dose groups when compared with the control group. A detailed analysis of histological scores is shown in Table 2. 3.6. T-bet and GATA-3 mRNA expressions The medium, high and overdose groups had a significantly lower level of T-bet mRNA expression and a significantly higher level of GATA-3 expression compared to the control group (P < 0.05). Levels of GATA-3 and T-bet mRNA were not significantly different between the low dose group and the control group (P > 0.05) (Fig. 6). 3.7. Correlations between levels of IFN- and IL-4 and expressions of T-bet and GATA-3 IFN-␥ levels in the plasma and spleen were positively correlated with T-bet mRNA expression (rPlasma = 0.768, rSpleen = 0.757, P < 0.05) and were negatively correlated with GATA-3 mRNA expression (rPlasma = −0.797, rSpleen = −0.824, P < 0.05), whereas IL-4 levels were negatively correlated with T-bet mRNA expression (rPlasma = −0.755, rSpleen = −0.812, P < 0.05) and were positively correlated with GATA-3 mRNA expression (rPlasma = 0.801, rSpleen = 0.847, P < 0.05). 4. Discussion

Fig. 3. Interleukin-4 (IL-4), interferon-␥ (IFN-␥) and IL-4/IFN-␥ in the plasma and spleen tissue in the mouse offspring with their dams receiving intralaryngopharyngeal SRM 1649a during the pregnancy. n = 8–10. CT: control, LD: low dose, MD: medium dose, HD: high dose, and OD: overdose. *P < 0.05 compared to the controls; # P < 0.05 compared to low dose; † P < 0.05 compared to overdose.

when compared with the medium or the high dose group (P < 0.05) (Fig. 3). 3.5. Morphology of spleen and thoracic gland tissues The spleen tissue from the control group exhibited an intact structure of splenic corpuscle with intensive and hyperchromatic lymphocytes around its central arteries under light microscopy. In the splenic pulp area, distinct splenic cord and splenic sinus with sparsely distributed lymphocytes and phagocytes were seen (Fig. 4a). On the contrary, the spleen tissue from the overdose group revealed an unclear boundary between the white pulp and red pulp, where extrusive hyperplasy in the white pulp and laterally deviated central arteries were observed. The periarterial lymphatic sheaths with indistinct boundaries and heavy infiltration of lymphocytes were volumetrically increased 3–4 times larger than those in the control group, in addition to the locally hyalinized vessel wall that appeared to be much thicker in the splenic arterioles. The splenic sinus in the red pulp was dilated, congested, presented with heavily proliferated phagocytes, and surrounded by unevenly distributed and locally agglomerated lymphocytes (Fig. 4b–f). The thoracic gland tissue manifested intensive, evenly distributed and

Particulate air pollution has been shown to be associated with the induction of immune response (Miyata and van Eeden, 2011). This study intended to present findings on the capabilities and potentials of maternal exposure to different doses of standard PM to modulate immune responses in the offspring. Systemic and organic immune responses were observed in young offspring whose dams were exposed to standard PM SRM 1649a. Our data demonstrated that maternal exposure to PM resulted in modulation of Th1 and Th2 immune response in the postnatal offspring, as evidenced by decreased total IgG2a (Th1-dependent) and increased total IgG1 (Th2-dependent) levels in the plasma, decreased IFN-␥ and increased IL-4 levels in the plasma and the spleen, and decreased expression of transcription factor T-bet (Th1-related) and increased expression of GATA-3 (Th2-related) in the spleen in an exposure dose-dependent manner. Inhibited splenic lymphocyte proliferation suggested compromised cell immunity. Mounting evidence already exists for the involvement of airborne particulate pollutants in the exacerbation of immune response in the young and adults (Zhang and Smith, 2007). Our results may imply that altered immune response may exist in offspring that are prenatally challenged by airborne particles as well. PM alters the normal developmental pattern for the immune system that is constantly changing during pre- and postnatal growth. Susceptibility of the fetus to PM may be attributable to either direct or indirect hits on the immune system to alter cell differentiation, proliferation, maturation and/or possible secretion. T helper cells are the regulatory and effector cells in immune reaction. Th1 and Th2 are functionally balanced under normal conditions and the imbalance in Th1/Th2 is associated with immunofunctional disorders. Th2 induces differentiation of B cells and production

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Fig. 4. Representative photomicrographs of pathological changes in the spleen tissue in the young mice. Panel a shows normal morphological appearance in the spleen tissue from the control group. Panels b–f show various pathological alterations in the samples from overdose group. Opaque boundaries between white pulp and red pulp with deviated central arteries were observed in panel b. In panel c, periarterial lymphatic sheaths were enlarged and a large number of lymphocytes were present. In panel d, the vessel wall in the splenic arterioles was thickened with local hyalinization. In panel e, the splenic sinus in the red pulp was dilated and congested with agglomerated lymphocytes. In panel f, the splenic sinus exhibited massively increased macrophages. Arrows point to typical histopathological changes. Scale bars represent 2000 ␮m (a, b), 400 ␮m (c, d, f) or 800 ␮m (e). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Table 2 Comparison of histopathological severity scores for spleen and thoracic gland, M(QR ). Groups

Spleen

Thoracic gland

Control Low dose Medium dose High dose Overdose H P

0.0 (0.0) 0.0 (1.0) 0.5 (1.0) 1.5 (2.0)* 2.0 (1.0)*,# 11.585 <0.05

0.0 (0.0) 0.0 (0.0) 0.0 (1.0) 1.0 (1.5)* 2.0 (2.0)*,# 9.647 <0.05

Note: * P < 0.05 versus control or low dose group. # P < 0.05 versus low dose group.

of immunoglobulins by secretion of cytokines including IL-4. The enhancement of Th2 cellular response includes increased IL-4 and 5 production, elevated serum levels of total IgE, IgG1, and decreased levels of IgG2a (Zhu et al., 2004). An activity and Th2-skewing capacity of airborne particles have been shown in different animal models and in humans (Porter et al., 2007; He et al., 2010; Zhao et al., 2012). Results from the present study suggested that intrauterine exposure to PM exaggerates Th2 immune response in the offspring postnatally, as indicated by elevated plasma levels of IgG1 and IgE, and plasma and spleen levels of IL-4 in the offspring of the PM-administered dams. Maternal exposure to overdose PM reduced the level of plasma total IgG2a in the offspring compared with the controls; the immune response induced by the presence of PM dosed in the other three doses was, though less powerful to the overdose, of the Th2 type. On the other hand, decreased IFN␥ production in plasma and spleen may imply a suppressed Th1

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Fig. 5. Representative photomicrographs of pathologic changes in the thoracic gland in the young mice. Panel a shows normal morphological appearance in the thoracic gland tissue from the control group. Broaden thymic cortex and narrowed thymic medulla were seen (b) with opaque boundaries between cortex and medulla (c). Relic debris in the cytoplasm was present (d). Arrows point to typical histopathological changes. Scale bars represent 2000 ␮m (a, b), 800 ␮m (c) or 200 ␮m (d).

function. These results imply that maternal exposure to PM possibly modulates both Th1- and Th2-responses in the offspring. The existence of discrete developmental processes within the immune system is likely to influence differential developmental susceptibility to toxicants, which may result in lifelong toxicological changes (Dietert et al., 2000). Hamada et al. (2007) found that prenatal exposure to aerosolized leachate of residual oil fly ash resulted in an increasing susceptibility to the development of asthma in early life, in which the offspring were challenged by the pollutant prenatally and postnatally coupled with sensitization by ovalbumin. In the present study, the offspring were challenged by PM prenatally, which may further imply that the prenatal effect on the immune system could be “memorized” and presented later in life.

Fig. 6. T-bet and GATA-3 mRNA expressions in the spleen tissue in the young mice with their dams receiving intralaryngopharyngeal SRM 1649a during the pregnancy. n = 8–10. CT: control, LD: low dose, MD: medium dose, HD: high dose, and OD: overdose. *P < 0.05 compared to the controls.

In contrast to the extensive reports on induction of Th2 responses, such as IgG1 and IgE antibody and IL-4 cytokine by exposure to environmental chemicals, few have referred to transcription factor involvement. T-bet and GATA-3 are two transcription factors that have been found to play a critical role in differentiation of Th1 or Th2 cells. T-bet promotes Th1 cell production by inducing the expression of cytokines, which are required for Th1 function, such as IFN-␥, and suppressing the expression of GATA-3 (Agnello et al., 2003). Mice with an inactivation of the T-bet gene possess an increased number of Th2 cells and an absence of Th1 cells, which makes them susceptible to Th2-mediated immune disorders (Szabo et al., 2002). GATA-3, whose expression is modulated by IL-4, affects the gene locus of IL-4 and promotes Th2 differentiation (Zhou and Ouyang, 2003). Studies regarding differentiation of helper T cells reveal that GATA-3 functions as the transactivator of the IL-4 gene and as the determinant for Th2 lineage (Yoh et al., 2003; Pai et al., 2004). By exposing adult Wistar–kyoto rats to PM2.5 , Zhao et al. reported decreased T-bet and increased GATA-3 expressions in the heart after PM exposure, suggesting a link between PM exposure and cardiac injury (Zhao et al., 2012). Our results in a maternal–fetal–filial model showed similar reversely changed expressions of T-bet and GATA-3 mRNA in an exposure dose-dependent manner. Although the reasons remain unclear, the present data may indicate that prenatal exposure to PM causes modulation of both Th1 and Th2 related-transcription factors in young mice. Significantly decreased T-bet and increased GATA-3 expressions in the spleen, coupled with suppressed production of IFN-␥ and exaggerated production of IL-4 in plasma and the spleen support both Th1- and Th2-response paradigm in the offspring. This was further supported by a mathematic correlation between changes in T-bet and GATA-3 gene expressions and IFN␥ and IL-4 cytokine levels. The effector cytotoxic T cells revealed

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a decreased IFN-␥ production in all except low dose PM-treated groups, whereas a higher level of IL-4 is parallel to a larger dose of exposed PM. Therefore, the IL-4/IFN-␥ ratio in T helper cells in intrauterinely PM-exposed animals was markedly changed during the immune response, further suggesting a deviation of Th1/Th2. There are physiological coherences among these observed parameters, e.g., altered T-bet and GATA-3 gene expressions resulting in cytokine disequilibrium, triggering the Th2 pathway, resulting in immune responses by IgG1, IgG2a, IgE, and histopathological lesions. Bezemer et al. (2011) found that respirable pollutants activated the innate immune response with Th2-immune responsiveness, but exposure of mice to environmental PM dampened IFN-␥ secretion. Our results indicated a significantly suppressed IFN-␥ production, which seems to be more pronounced than that of Bezemer’s. Although there is difference regarding PM doses, administration route, animal strains, and measurement parameters, the greater vulnerability of the fetal immune system to PM exposure may elevate susceptibility to immunologically mediated disorders postnatally. The spleen and thoracic gland are the most important peripheral and central immune system organs involved in T and B lymphocyte differentiation and maturation. The spleen and thoracic gland develop largely during the prenatal and early postnatal periods and their development is highly sensitive to immunotoxic chemical substances (Holladay and Smialowicz, 2000). Little is known regarding the effects of environmental toxicants on the development of immune organs during the prenatal period. We exposed pregnant mice to PM for the whole gestational duration. Although prenatal exposure to PM did not alter the immune organ weight and its ratio over the body weight in the offspring, the evaluation of histopathological changes in the spleen and thoracic gland reflected dose-related small to severe lesions. Biological measurements, including spleen lymphocyte proliferation, indicated a compromised lymphocyte proliferation capacity in medium, high and overdose groups, suggesting an impaired cellular immune response in the spleen. Splenic lymphocyte proliferation plays an important role in immunological regulation, anti-viral infection and anti-oncogenesis. Thus, immunologic damage in the offspring of PM-treated mothers was morphologically and functionally confirmed in the present study. Significant immunologic damage may suggest a greater sensitivity and susceptibility to immune disorders at a young stage of development. A predominance of Th2 in early life has been associated with some disorders including allergic diseases in children and later life. Clinical evidences revealed that a high circulating Th2/Th1 ratio at birth, as reflected by Th1- and high Th2-associated chemokine levels, was associated with the development of eczema, wheeze and sensitization in children (Sandberg et al., 2009; Abrahamsson et al., 2011). Rothers et al. (2011) reported that total IgE levels and active asthma through age 5 years correlate with IL-4, IL-5, IFN-␥ and other cytokines regulations in early life. Using a murine model of childhood asthma, Siegle et al. reported that administration of neutralizing antibodies against IL-4 or IL-25 prevents development of some key features in asthma, suggesting an association between Th2 response and immune disease during neonatal period or later in childhood (Siegle et al., 2011). We may suggest a raised susceptibility to the immune-related diseases in the offspring mice with higher Th2/Th1 ratio resulting from prenatal exposure to PM. Heterogeneity of the monocytes/macrophages has long been recognized and, in part, is a result of the specialization, especially in function, of tissue macrophages in particular microenvironments (Mantovani et al., 2005, 2008). “M1” cells, or classically activated macrophages, are inflammatory cells and may be initiated by stimulating their progenitors with inflammatory stimuli, such as lipopolysaccharides and granulocyte macrophage

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colony-stimulating factor. “M2” cells, or alternatively activated macrophages, display a more regulatory function and may be generated when progenitors are cultured with IL-4 and IL-13. However, the heterogeneity of the mononuclear phagocyte system is poorly recapitulated, and some models and experiments do not represent a model to study the specialized functions of the diverse cell types that are present in vivo, or the regulation of their development and functions by the tissue microenvironment (Geissmann et al., 2010). Due to the complexity of the mechanisms involved in the heterogeneity of the tissue macrophages and the limited scope of our work, we intentionally did not show any data about the phenotypic changes in M1/M2 at both circulatory and tissues levels in order to prevent any misleading or misinterpreted data. The role and its heterogeneity of tissue macrophages in air pollution exposure-induced diseases have been studied by other researchers recently. Sun et al. (2009) found that PM2.5 exposure increased systemic increased the numbers of F4/80+ adipose tissue macrophages (ATMs), increased markers of classically activated (M1) ATMs, and decreased markers of alternatively activated (M2) ATMs in rodent models. In an early exposure rodent model, Xu et al. (2010) found that PM2.5 exposure led to a significant increase in the expression of proinflammatory genes (M1) tumor necrosis factor-␣, nitric oxide synthase-2 and IL-6, no changes in integrin ␣X expression, and downregulation of IL-10 expression, demonstrating that PM2.5 exposure downregulated genes correlated with an anti-inflammatory M2 phenotype while inducing a proinflammatory phenotype. However, this categorization should not be simply extrapolated in studies of macrophage polarization states as more details and factors should be considered in terms of in vivo versus in vitro models and animal versus human studies (Geissmann et al., 2010). To assess a Th2 response to PM2.5 , Deiuliis and his team measured IL-4 expression in CD4+ T cells isolated from the lung and found no difference compared with the controls, suggesting that PM2.5 exposure has little physiological effect on Th2 populations, while a Th1 response may dominate (Deiuliis et al., 2012). In the same study, three major CXCR3binding chemokines (CXCL9, CXCL10, and CXCL11) were induced in a variety of cells, including lung macrophages and dendritic cells, in response to other cytokine mediators such as IFN-␥, providing an amplification loop for Th1 immune responses by attracting more CXCR3-expressing Th1 cells that was evidenced by a marked increase in CXCR3+ CD4+ cells in response to PM2.5 exposure. Therefore, both macrophages and dendritic cells have crucial roles in air pollution exposure-induced immunological response and disease, which merits further mechanistic investigation. In the present study, the exposure model was prepared in pregnant mice whose offspring were experimentally processed at postnatal day 30, which is equivalent to childhood around 5–10 years of age in humans (Downing et al., 2009). PM doses used in our study were calculated with reference to the U.S. National Ambient Air Quality Standard 26–534 ␮g/m3 for environmental inhalable particulate matters with the airborne particulate matters with aerodynamic diameters less than 10 ␮m (PM10 ). Particular emphasis was given to real world scenarios, with PM10 in large cities and industrial regions of developing countries like China averaging around 80 ␮g/m3 (Xie et al., 2005; Kan et al., 2007; Xu et al., 2008; van Donkelaar et al., 2010; The U.S. Embassy in Beijing, 2011; Yang et al., 2012), and with a peak value of up to 2000 ␮g/m3 under extreme conditions (Wang et al., 2009; Kan et al., 2012). As the vital capacity for an adult mouse is 24 ml/min, the total calculated amount of air inhaled into the body over 24 h is about 0.035 m3 . Moreover, the daily inhaled amount of PM10 for a mouse is about 0.9 ␮g and 18.5 ␮g according to the standard 26 and 534 ␮g/m3 , which is the reference for the low dose and high dose we used. The medium dose and overdose amounts were determined with reference to 80 ␮g/m3 and 2000 ␮g/m3 , respectively. We did not

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increase the administered PM doses with increasing murine body weight during the experimental period because we were aiming to investigate the effect of PM under analogous environmentally relevant levels, which were not associated with the increase in the body weight. There are several limitations in the present study. First, although the exposure model is frequently used in similar studies (Riva et al., 2011; Musah et al., 2012) for its ease of manipulation and reproducibility (Rao et al., 2003), the route of instillation is not equivalent to inhalation. The SRM 1649a particles we used in the present study are total suspended particulate matters with many possessing large aerodynamic diameters that cannot enter the alveoli of the lung or translocation across the blood-gas barrier in terms of the entire particles as a whole but it is still possible, and, it is likely, that the chemical components from those PM10 or bigger particles could penetrate into the circulation and reach remote organs and tissues (Hertz-Picciotto et al., 2008) and on the fetus (Ravindra et al., 2001), so as the fine components (Mills et al., 2009). Although we calculated the doses based on real world exposure levels in locations where humans may be exposed (especially in major cities in developing countries like China) the lack of a modern, sophisticated exposure system that allows inhalation exposure in our study limits scientifically the data interpretation and significance. Second, the variability of the particles’ recovery does not precisely reflect the local situation. Although SRM 1649a is a standardized, well-studied particle reference that has been used in several experimental observations (Sun et al., 2008; Ulrich et al., 2010; Wong et al., 2011), it is different in property from other ambient particles like diesel exhaust particles (Wong et al., 2011). We believe that using particles collected from the local area would best represent the impact on the residents who live there. To best serve this purpose, an exposure system that has a capability to suck the ambient air into the system, and allows certain sizes of particulate matters (such as PM2.5 ) into the chambers is under construction in our laboratory. Third, although the current study investigated at multiple levels, especially associated with the T-bet and GATA-3 expressions, there is a lack of evidence and pathways regarding the mechanisms by which PM exposure ultimately induces immunologic responses. Besides, locally deposited substances may affect indirectly the body by inducing local biological reaction such as inflammation, oxidative stress, profibrotic and allergic processes (van Berlo et al., 2012), with the mechanisms of hemodynamic alterations in placental blood flow and reduction of nutrient transfer into the fetus (Proietti et al., 2012). It is reasonably postulated that SRM 1649a may affect the immune function in the offspring via both direct and indirect pathways in the present exposure model. In summary, the present study demonstrated that intrauterine exposure to PM resulted in a deviation of Th1/Th2 that skewed immune responses toward the Th2 phenotype by exacerbating systemic IgG1, IgE and IL-4 productions and splenic GATA-3 expression, and by attenuating systemic IgG2a and IFN-␥ productions and splenic T-bet expression. The components in PM are complex, and the attached substances that are present on PM, which consist mostly of polyaromatic hydrocarbons exerting as proallergic effects (Lubitz et al., 2010), allergens, and some bacterial components like endotoxin attaching on the particles (Inoue et al., 2007), are often suggested for being responsible for the side-effect of PM on health. Whether the attached substances of PM are involved in this skewing toward the Th2 side, as shown by other reporters (Braun et al., 2010; Lubitz et al., 2010), remains to be further elucidated.

Conflict of interest statement There are no conflicts of interest.

Acknowledgements This work was supported by research grants from the Chinese National Natural Science Fund (#81172677), Fujian Science and Technology Bureau Unode Project (#2007Y0017), and Medical Science Technology Project of Nanjing Command (#07M093), China. The authors would like to thank Prof. Hongyu Yu for his histopathological evaluation and Mr. Geoffrey Gatts for his critical reading and editing of the manuscript.

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