In Utero Exposure to Simulated Complex Urban Air Pollution Disturbs Intestinal Suckling-to-Weaning Transition in Mice

Emerging data indicate that prenatal exposure to air pollution may lead to higher susceptibility to several non-communicable diseases. Limited research has been conducted due to diculties in modelling realistic air pollution exposure. In this study, pregnant mice were exposed from gestational day 10 to 17 to an atmosphere representative of a 2013 pollution event in Beijing, China. Intestinal homeostasis and microbiota were assessed in both male and female offspring during the suckling-to-weaning transition. Sex-specic differences were observed in progeny of gestationally-exposed mice. In utero exposed males exhibited decreased villus and crypt length, vacuolation abnormalities, and lower levels of tight junction protein ZO-1 in ileum. They showed an upregulation of absorptive cell markers and a downregulation of neonatal markers in colon. Cecum of in utero exposed male mice also presented a deeply unbalanced inammatory pattern. By contrast, in utero exposed female mice displayed less severe intestinal alterations, but included dysregulated expression of Lgr5 in colon, Tjp1 in cecum, and Epcam, Car2 and Sis in ileum. Moreover, exposed female mice showed dysbiosis characterized by a decreased weighted UniFrac β-diversity index, a higher abundance of Bacteroidales and Coriobacteriales orders, and a reduced Firmicutes/Bacteroidetes ratio.


Background
Air pollution has been estimated to be the single most important environmental health risk factor. It represents a greater disease burden than polluted water, soil contamination, and occupational exposures combined [1]. Air pollution causes a loss of life expectancy which rivals that of tobacco smoking [2]. It is well recognized as a major risk factor for many chronic non-communicable diseases such as cardiovascular, pulmonary and metabolic diseases [3][4][5]. Association studies also suggest a possible contribution of air pollution to the development of neurological illnesses such as Alzheimer's disease [6], chronic kidney disease and renal function decline [7], liver cirrhosis [8], and autoimmune diseases. For the latter, epidemiological studies have shown a relationship between exposure to air pollution and development and progression of multiple sclerosis [9], and exacerbation of rheumatoid disease [10] and systemic lupus erythematosus [11,12]. Furthermore, air pollution may also contribute to intestinal diseases [13,14].
There is a growing body of evidence which indicates that there is a prenatal window of susceptibility to adverse effects of air pollution. Exposure to air pollution early in life is directly linked to the development of major cardiovascular risks, including obesity, hypertension, and metabolic disorders [15,16]. Air pollution exposure in utero has been associated with childhood asthma and allergic diseases [17,18]. Regarding the intestinal tract, a population-based epidemiological study has highlighted that Ox exposure (as measured by redox-weighted oxidant capacity, a measure that takes into account the oxidative potential of both ozone (O 3 ) and NO 2 ) during the second trimester of pregnancy is associated with in ammatory bowel disease (IBD) development [19]. In mice, PM 2.5 exposure during gestation caused changes in the distribution and structure of gut microbiota of dams [20]. However, despite the emerging role of air pollution in intestinal pathologies, animal studies focusing on the intestinal burden induced by in utero exposure to air pollution are missing. Moreover, despite ambient air pollution consisting of both particulate matter (PM) and gaseous components including O 3 , volatile organic compounds (VOCs), carbon monoxide (CO), and nitrogen oxides (NOx), realistic experimental studies that simulate complex air pollution are lacking. In this work, we aimed to assess the early effects of gestational exposure to complex urban air pollution on intestinal tissues. For this purpose a more representative atmospheric model, based on a 2013 pollution event in Beijing, was generated. Pregnant mice were exposed to simulated Beijing-like air pollution from gestational day 10 (GD10) to GD17. Control mice were exposed to ltered Beijing-like air pollution during the same period. Effects of intrauterine exposure were assessed during the suckling-to-weaning transition (at postnatal day 17), which corresponds to a critical window for both structural and immune intestinal development [21,22].

Histomorphological analysis in male mice
In mice, as in humans, the neonatal intestine has several features that are distinct from adults. During the suckling-to-weaning period, gut growth and maturation accelerate involving both functional and structural changes in gut epithelium [23]. Thus, we rst performed an overall assessment of the gut mucosal structure. May Grünwald Giemsa (MGG)-stained sections of male colon and ileum were analyzed (Fig 1 A and D). In colon, the cell number in the submucosa and the mucosal surface area were measured (Fig 1 B and C). No variation was evident. In ileum, the villus length and the crypt depth were measured (Fig 1 E and F). They were both signi cantly decreased in Beijing-like air mice compared to control air mice. The villus/crypt ratio did not vary ( Fig 1G). Furthermore, during the suckling-to-weaning period, vacuolated fetal-type epithelium was replaced by non-vacuolated adult-type epithelium [23]. Since the small intestine of GD17 pups presented intense vacuolation of the villi, we developed a method for quantitative assessment of this vacuolation. The number of vacuoles per villi was determined and did not show variation between the 2 groups of mice ( Fig 1H). The mean vacuolated area per villus was signi cantly reduced in Beijing-like air mice (Fig 1I). Morphometric evaluation of the vacuoles also showed signi cant variations (Fig 1 J and K). In pups exposed in utero to air pollution, the vacuole circularity index was increased and the mean eccentricity index was decreased compared to control pups. Therefore, in males, postnatal exposure to air pollution induced histomorphological alterations in ileum, and notable disturbances of the vacuolation process.

Histomorphological analysis in female mice
We performed the same analyses in colon and ileum of female pups. In colon, the mucosal surface area and the submucosa cellularity did not show signi cant variation between air pollution-and controlexposed mice (Fig 2A, B, C). In ileum, there were no modi cation of the crypt depth, villus length, and villus/crypt ratio (Fig 2D, E, F, G). The morphometric analyses of the vacuoles showed no differences between the female pups in utero exposed to simulated Beijing-like polluted air compared to female control pups. Therefore, in females, no important histological impairments of colon and ileum were induced by the postnatal exposure of simulated Beijing-like air pollution.
Epithelium proliferation, differentiation, and maturation in male mice To further examine epithelium proliferation, immunohistochemical staining of the proliferative marker PCNA (proliferating cell nuclear antigen) was performed. In male colon, the number of PCNA-positive cells was counted and was similar between the polluted air-and the control air-exposed mice (Fig 3 A and B).
Transcript levels of several markers of intestinal cells were quanti ed by real-time polymerase chain reaction (PCR). The leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5), the best described intestinal stem cell marker, did not show signi cant variation (Fig 3C). The markers of absorptive cells alkaline phosphatase, intestinal (Alpi), epithelial cell adhesion molecule (Epcam), and carbonic anhydrase 2 (Car2) were all signi cantly upregulated in Beijing-like air mice compared to control air mice ( Fig 3D). The marker of goblet cells mucin 2 (Muc2) was signi cantly downregulated in Beijing-like air mice ( Fig  3E). The transcript levels of the markers of Paneth cells (lysozyme 1; Lyz1), of enterochroma n cells (chromogranin A; ChrgA), and of tuft cells (POU domain, class 2, transcription factor 3; Pou2f3), were not changed between the 2 groups of mice.
To assess the functional maturation of gut epithelium, we quanti ed mRNA levels of several neonatal and adult markers (Fig 3 F and G) [22]. During the suckling period, neonatal intestinal cells express disaccharidase lactase-phlorizin hydrolase (Lct) to digest milk lactose [1]. After the suckling period, enterocytes adapt to digest solid food that is rich in complex carbohydrates and low in fat. This is manifested by a switch in brush border disaccharidase expression from lactase to sucrase isomaltase (Sis) and trehalase (Treh) [2]. In colon of Beijing-like air mice compared to control air mice, expression of Lct was downregulated, expression of Treh was unchanged, and expression of Sis was upregulated. Furthermore, another metabolic switch is related to the low concentration of arginine in milk. To provide for the need for arginine, neonatal enterocytes express the rate limiting enzyme in arginine biosynthesis, argininosuccinate synthase-1 (Ass1) [3]. In contrast, adult enterocytes express arginase 2 (Arg2), an enzyme capable of catabolizing arginine that is abundant in solid foods. Colon expression of Ass1 and Arg2 were lower in mice exposed in utero to Beijing-like air pollution as compared with mice exposed to control air pollution. Furthermore, the neonatal intestinal epithelium expresses the neonatal Fc fragment of the IgG receptor and transporter (Fcgrt, also called FcRn), which mediates absorption of maternal IgG from milk into the bloodstream [24]. The expression of Fcgrt declines signi cantly during the suckling-toweaning transition [22]. Similarly, the expression of Prdm1 (PR domain containing 1, with ZNF domain; also called Blimp-1, or B lymphocyte-induced maturation protein-1) is lost at the suckling-to-weaning transition [25,26]. Prdm1 expression was not modi ed but Fcgrt transcript levels were signi cantly reduced in colon after Beijing-like air pollution exposure.
In ileum, the number of PCNA-positive cells was higher in Beijing-like air mice compared to control air mice, re ecting an increase of epithelial proliferation in these mice (Fig 3 H and I). The mRNA levels of the markers of stem, absorptive, and secretory cells were not modi ed (Fig 3 J-L). Moreover, there was no variation of expression of the neonatal and adult genes (Fig 3 M and N).
Altogether, these results tend to show that postnatal exposure to simulated Beijing-like air pollution in male pups led to alterations of differentiation and maturation biomarkers in colon epithelial cells, as well as to an increase of epithelial proliferation in ileum.

Epithelium proliferation, differentiation, and maturation in female mice
In females, PCNA immunostaining of colon showed no signi cant modi cation of proliferation in Beijinglike air mice compared to control air mice (Fig 4 A and B). The mRNA level of Lgr5 was signi cantly downregulated in Beijing-like air mice ( Fig 4C). The transcript abundance of absorptive and secretory cell markers did not show signi cant variation (Fig 4 D and E). The neonatal markers were not changed either ( Fig 4F). The transcription of Arg2 was signi cantly downregulated in Beijing-like air mice compared to control air mice ( Fig 4G).
In ileum, PCNA staining was similar between the Beijing-like air mice and the control air mice (Fig 4 H and  I). The level of Lgr5 was also unchanged ( Fig 4J). The expression of absorptive cell markers Epcam and Car2 were upregulated in Beijing-like air mice ( Fig 4K). Among the secretory cell markers, a relative overexpression of ChrgA transcripts was quanti ed in Beijing-like air mice as compared with control air mice ( Fig 4L). The maturation of ileal tissue was weakly altered, and an upregulation of Sis expression was detected in Beijing-like air mice (Fig 4 M and N). Because over-expression of ChgrA in Beijing-like airexposed mice ileum was observed, we quanti ed expression of other markers of enteroendocrine cells. Levels of tachykinin 1 (Tac1, or substance P)-producing enterochroma n cells, glucagon (Gcg)-and peptide YY (Pyy)-expressing L cells, gastric inhibitory protein (Gip)-producing K cells, neurotensin (Nts)and neuromedin-producing N cells, and secretin (Sct)-producing S cells were quanti ed [27] (Fig S2). An upregulation of Nts was found in the Beijing-like air-polluted group, re ecting an abnormal expression of neurotensin.

In ammatory pattern in male mice
In order to assess whether in utero exposure to air pollution induces impairments of the in ammatory process in GD17 pups, we quanti ed mRNA levels of transcription factors and in ammatory cytokines representative of the Th1, Th2, Th17, and Treg immune response. In proximal colon, expression of the immunomodulatory cytokine Il10 was strongly enhanced in Beijing-like air pups compared to control air pups ( Fig 5A). In cecum, the Th1 transcription factor Tbx21 (also called T-bet) was signi cantly higher in Beijing-like air mice compared to control mice ( Fig 5B). The Th2 cytokines Il4 and Il5 were also upregulated in Beijing-like air pups. The Th17 transcription factor Rorc was upregulated and the levels of Il17a and Il22 were downregulated in Beijing-like air mice compared to control air mice. Levels of Tgfb were also lower in cecum of Beijing-like air mice. In ileum, a signi cant upregulation of Rorc was induced by in utero exposure of simulated Beijing-like air pollution ( Fig 5C).

In ammatory pattern in female mice
In proximal colon of female mice, the transcript levels of Il13 and Il10 cytokines were greatly upregulated in Beijing-like air mice compared to control air mice ( Fig 6A). In addition, the mRNA expression of Il4 was enhanced in cecum by in utero Beijing-like air exposure ( Fig 6B). None of the other markers studied were modi ed in ileum ( Fig 6C).

Intestinal permeability markers in male and female mice
We assessed whether in utero exposure to air pollution disrupts the intestinal barrier. The transcript levels of 3 markers of intestinal permeability, occludin (Ocln), tight junction protein-1 (Tjp1; coding forzonula occludin 1 protein), and claudin-4 (Cldn4), were quanti ed. In male proximal colon there was no signi cant expression variation of these 3 markers (Fig 7A). In cecum of Beijing-like air-exposed males, we observed an upregulation of Ocln and a downregulation of Tjp1 and Cldn4 transcripts. Tjp1 expression was also strongly reduced in the ileum of males exposed to Beijing-like air. In females, in utero Beijing-like air pollution exposure induced an upregulation of Cldn4 and Tjp1 in proximal colon and cecum, respectively ( Fig 7B). Furthermore, we quanti ed the expression of ZO-1 protein by western blot and con rmed that ZO-1 protein levels were signi cantly reduced in ileum of male mice exposed in utero to Beijing-like air pollution compared to control air mice (Fig 7 C and D).

Microbiota DNA extraction and 16S rDNA gene amplicon sequencing analysis
To assess the impact of in utero exposure to air pollution on colon luminal bacterial content, we sequenced V3-V4 amplicons of 16S rRNA genes. After a denoising step performed with DADA2 software, we obtained a total of 884,707 reads. Exposure to air pollution did not signi cantly affect a-diversity (Chao1 diversity index, Fig 8A; Evenness and Simpson indices, Fig S3). The weighted UniFrac b-diversity index showed no signi cant difference in males, but a signi cant decrease in females (p=0.011; Fig 8B). After taxonomic assignment of amplicon sequence variants (ASVs), the effect of in utero air pollution exposure on the abundance of phyla was assessed. Taxonomic assignment at the phylum level of ASVs, with each color representing an individual bacterial phylum, is shown in Fig 8C. Bacterial composition was dominated by members of the Bacteroidetes phylum followed by Firmicutes, although mice presented important interindividual variations. A high abundance of Verrucomicrobia was found in only 4 control male and 2 control female mice, but no phylum was found to be signi cantly different between the exposed and control mice. Similarly, analyses at the class level showed important interindividual variability, but did not reveal signi cant variations between air pollution-and control-exposed groups (data not shown). At the order level, the main bacteria were Bacteroidales, Clostridiales, and Lactobacillales ( Fig 8D). Bacteroidales and Coriobacteriales orders were signi cantly more abundant in Beijing-like air-exposed mice than in control air-exposed female mice (p=0.001 and p=0.04, respectively; Fig 8E). Moreover, the Firmicutes/Bacteroides ratio, which is a widely used marker of intestinal dysbiosis, was calculated and found to be signi cantly reduced in females after in utero exposure to Beijing-like air (Fig 8F).

Discussion
Although exposure to air pollution during pregnancy is linked to high risk of adverse pregnancy outcomes and long-term postnatal health, limited mechanistic data exists to assess these impacts under controlled exposure conditions. This limitation of our knowledge is mainly attributed to the complexity of the polluted atmosphere, and to the great di culty in modelling the impact of realistic exposures. Using CESAM, an atmospheric simulation chamber, we have capitalized on a totally innovative platform for exposing mice to more realistic atmospheric conditions. Among the constituents of air pollution associated with deleterious effects on health, we considered gaseous pollutants (O 3 , SO 2 , CO, NOx, and VOCs) and particles (PM 10 , PM 2.5 , and ultra ne PM). In order to simulate atmospheric mixtures in all their complexity in the laboratory, environmental chemists have developed photo-reactors that are equipped to reproduce and control atmospheric processes such as solar radiation, concentrations of species, and the timely injection of aerosols and gases. These atmospheric simulation chambers thus offer the possibility of studying the myriad of products resulting from the atmospheric oxidation of primary compounds [28].
This innovative experimental approach allowed us to mimic the effects of "real life" exposure of urban air pollution on the suckling-to-weaning intestinal tissues. In mice intestinal development begins right before birth and intestinal maturation is completed at approximately 3 weeks postnatally during the suckling-toweaning transition [29]. In female offspring, in utero exposure to Beijing-like air pollution induced mild modi cations of intestinal development. Enhanced expression of Epcam and Car2 absorptive epithelial cell markers and ChrgA enteroendocrine cell marker were observed in small intestine. In colon, the only alterations were decreased expression of Lgr5 stem cell marker and Arg2 adult epithelial cell marker. Therefore, our results argue in favor of weak effects of postnatal air pollution exposure on intestinal differentiation and maturation in female young offspring. By contrast, in males, several parameters of intestinal development were affected by air pollution exposure. Notably, in small intestine, both the villus length and the crypt depth were reduced. This was associated with increased epithelial proliferation, as well as decreased size and morphological alterations of the vacuoles. This latter observation may re ect a trend to replacement of vacuolated fetal-type epithelium with non-vacuolated adult-type epithelium and therefore more precocious maturation in in utero pollution-exposed mice. However, the markers of neonatal cells were not changed, indicating the absence of a functional impact of exposure to air pollution on intestinal maturation. Moreover, our results show that in utero exposure to Beijing-like air pollution led to disturbances of proliferation processes. It is known that villus and crypt morphogenesis occurs during embryogenesis and postnatally, respectively; crypts are the architectural unit of the stem cell niche [30]. Villus and crypt morphogenesis are complex processes which are essential for normal intestinal physiology. A precedent for imprinting of skin epithelial stem cells has been reported [31]. Therefore, the abnormalities observed in male small intestine may re ect disorders of intestinal development which deserve further study.
We also studied whether in utero air pollution exposure could affect the maturation of the immune system. The expression of the immunomodulatory cytokine IL-10 was exacerbated in colon by in utero exposure to Beijing-like air, both in males and females. In neonates, as in adults, IL-10 can have antiin ammatory properties and can be produced by macrophages and T cells [32]. IL-10R signaling in macrophages is pivotal in con ning a microbiota-driven in ammatory response beginning at the third week of life [33]. IL-10 can also be produced by neonatal type 1 conventional dendritic cells (cDC1) before their differentiation into IL-12p40-producing cDC1 [34]. Therefore, if IL-10 overexpression derives from cDC1, it could re ect a defect in maturation of these cells. Apart from this nding, the cytokine pro le in the colon was not altered. In ileum, the cytokine pattern was very similar between Beijing-like air mice and control air exposed mice. By contrast, in cecum, and only in males, we observed an increase of 2 major immune transcription factors (Tbx21 and Rorc), an overexpression of Th2 cytokines (Il4 and Il5), and decreased expression of cytokines Il17a, Il22, and Tgfβ. These alterations are numerous but they do not make it possible to demonstrate a clear defect in the immune response. Association of the upregulation of Th17 transcription factor Rorc and downregulation of Th17 cytokines Il17a and Il22 is paradoxical, and to date the data in the literature is not su cient to fully explain this apparent discrepancy. It is known that immune homeostasis in the gut is normally maintained by the production of low levels of IL-17A and IL-22 by resident Th17 lymphocytes [35] and ILC3 [36]. In neonates IL-17A can also be produced by γδT cells and has an essential role in host defense against C.di cile infection [37].
Another important parameter of intestinal immune response is the development of an e cient mucosal barrier including a tailored regulation of tight junctions which seal the epithelial cell-cell contacts and regulate the paracellular passage of solutes [38]. The modi cations of permeability markers that we observed, particularly downregulation of ZO-1 at the transcript and protein levels in male ileum, could lead to a gut barrier defect. The leakiness of the gut epithelium has been associated with the development of allergic and autoimmune diseases, especially when it is associated with a dysbiotic microbiota that cross the damaged barrier [39].
From birth, the normal gut microbiota contributes to the development of gut function, educates the immune system, contributes to the regulation and maintenance of intestinal barrier function, provides protection against infection, and promotes tolerance of foods. In addition, the early life microbiota has a crucial role in the risk of acquiring diseases such as asthma, atopic dermatitis, diabetes, allergic diseases, obesity, cardiovascular diseases, and neurological disorders [40]. In our study, in utero exposure to air pollution did not modify the α-diversity (within sample diversity) and especially the Chao1 species richness index, indicating that the number of different species was similar between the groups. By contrast, and only in females, the bacterial composition showed few signi cant variations. At the phylum level, the ratio of Firmicutes/Bacteroidetes was reduced. In adults, this ratio is a hallmark of low-grade in ammation [41]. It has also been proposed as a marker of intestinal maturation during aging (in the second year of adulthood) [42]. In female mice exposed in utero to air pollution, the relative abundance of Coriobacteriales was higher. This order is reduced in colitic mice [43] and enriched in acute liver failure [44]. By contrast, Coriobacteriales are more abundant in diseased mucosal ileum tissues from Crohn's disease patients compared to control mucosa from non-IBD patients [45]. Coriobacteriales are also more abundant in fecal microbiota of multiple sclerosis patients [46]. Moreover, female mice exposed in utero to air pollution presented higher abundance of Bacteroidales. The Bacteroidales (or S24-7 family) are numerically dominant intestinal organisms that associate with the mucosal surface and have properties that both positively and negatively affect the host. The Bacteroidales showed a higher abundance in dextran sulfate sodium-induced mice [43,47]. In adult mice, bacteria from the order Bacteroidales are su cient to promote appearance of intraepithelial lymphocytes in the colon, which are important for the maintenance of a healthy intestinal barrier [48]. In humans, Bacteroidales produce bacteroidetocins, a family of broad-spectrum peptide toxins that kill members of the Bacteroidetes phylum, including Bacteroides, Parabacteroides, and Prevotella gut species, as well as pathogenic Prevotella species [49]. A decrease in adherent Bacteroidales diversity (i.e., the number of different Bacteroides species per biopsy) has been found at sites with increased in ammation in IBD subjects [50]. Therefore, current knowledge shows that bacteria of the orders Bacteroidales and Coriobacteriales have a role in the regulation of intestinal homeostasis and are deregulated in certain pathological conditions, but this does not allow us to predict the consequences of their deregulation in females having been exposed in utero to air pollution.
Most of the effects that we observed in the gut as consequences of maternal air pollution exposure are sex-speci c. This gender dependency is a classical feature of air pollution impact. For instance, prenatal air pollution exposure has been shown to "program" offspring for increased susceptibility to diet-induced weight gain and neuroin ammation in adulthood in a sex-speci c manner [51]. Moreover, the effects induced on the immune system of offspring by maternal exposure to diesel exhaust particles (DEP) are also sex-dependent. Indeed, exposure to DEP in utero decreased the frequency of CD1d high CD5 + B cells in female mice and IFN-γ production by splenocytes in both sexes. Male mice had elevations in macrophage and lymphocyte numbers in response to DEP whereas female mice only had elevated IL-6, MCP-1, and MIP-2 levels [52]. Epidemiological studies have also highlighted gender-speci c effects of air pollution on respiratory health. Overall, studies of children suggest stronger effects among boys in early life and among girls in later childhood [53].
Despite accumulating evidence of sex-dependent adverse effects of maternal air pollution exposure, the mechanisms involved are only now starting to be identi ed. Firstly, prenatal air pollution exposure induces epigenetic modi cations in placenta and in cord blood. Notably, ambient PM causes signi cant epigenomic changes, including alterations in DNA methylation, miRNA regulation, and histone modi cations. Birth cohort studies have shown that PM 2.5 exposure during the last trimester of pregnancy was positively associated with placental methylation of the promoter regions of regulatory genes in the circadian pathway and key DNA repair genes [54]. PM 10 exposure during the rst 2 trimesters of pregnancy was positively associated with placental methylation of HSD11B2 (i.e., genes involved in the glucocorticoid metabolism and fetal growth). Moreover, speci c PM 2.5 pollution exposure windows were associated with altered placental miR-20a, miR-21, miR-146a, and miR-222 expression [55]. Also, prenatal PM 2.5 exposure was positively associated with cord plasma histone H3 modi cations [56]. This altered biomolecular functioning of the placenta may contribute to early and even later-life health consequences.

Conclusions
In mice, postnatal exposure to simulated Beijing-like air pollution induced sex-speci c effects on intestinal proliferation, maturation, permeability, immune response, and microbiota composition.
According to the "Developmental Origins of Health and Disease" (DOHaD) concept, early-life alterations induced by intrauterine pollutant exposure may have long-term effects in uencing offspring susceptibility to diseases later in adulthood. Therefore, precocious disturbances that we observed in intestinal development following prenatal exposure to air pollution may underlie a higher susceptibility for developing diseases later in life.

Generation of complex air pollution
The innovative approach developed for this study was to realistically simulate the atmospheric mixture in its whole complexity, while maintaining the ability to control, reproduce, and fully characterize the experimental conditions. The CESAM chamber (described at https://cesam.cnrs.fr/; a 4.2 m 3 stainless steel atmospheric simulation chamber; evaceable down to a few 10 -7 atm; temperature controlled between +15°C and +60°C) was used to study the myriad of products arising from the atmospheric oxidation of primary organic compounds. The experimental protocol included a continuous injection of relevant mixtures of primary pollutants (mainly nitrogen oxides, organic compounds from a representative mix of anthropogenic emissions, sulphur dioxide, soot, inorganic salts, and mineral dust particles from the Gobi Desert to simulate a Beijing-like atmosphere from 2013) at low concentrations (ppb levels) in air with the CESAM simulation chamber operating as a slow ow reactor. The residence time of simulated air parcels in the experimental volume was xed to 4 h in order to represent air masses of regional scale. During this time the synthetic mixture was exposed to arti cial solar irradiation, allowing secondary pollutants such as O 3 , nitric acid, formaldehyde, peroxyacetyl nitrates (PANs) as well as complex polyfunctional organics including secondary organic aerosols, to be produced and to reach their chemical steady state. Mice were exposed to constant ows of this mixture.
Simulated atmospheric environment at the laboratory The 2013 China smog event was an extreme air pollution episode that affected East China, initiated by slow air carrying industrial emissions and meteorological conditions, that took place in December of that year. High levels of PM 2.5 (generated from coal combustion and industrial sources) and low visibility were observed, leading to the closure of airports, highways, and schools. For the simulation of a Beijing-like atmosphere, VOCs were introduced into the atmospheric chamber as precursors, which were the most abundant and commonly emitted particulates in the urban atmosphere of Beijing at the time: isopentane (alkane), propene (alkene), acetylene (alkyne), acetaldehyde (aldehyde), benzene, toluene, and m,p-xylene (aromatic hydrocarbons). The concentrations were determined in order to have stable levels of pollutants in the chamber, and representative of the pollution episode [28]. By the end of 20 h of VOC injection, nitrogen monoxide (NO) was introduced in the chamber and then the lights were turned on, simulating the sunlight irradiation in the troposphere. Additionally, seeds of ammonium sulfate particles were injected in the chamber. They formed contact surfaces for the condensation of oxidized products resulting in the formation of secondary organic aerosols. These particles were produced from the nebulization of an ammonium sulfate solution through an atomizer. In order to realistically simulate an atmosphere representative of Beijing, soot particles were generated using a soot generator (miniCAST Series 5200, which produces combustion soot particles by using a well-de ned ame that simulates the combustion in modern combustion engines), based on the combustion of propane. These particles were transferred into a smaller chamber (soot reservoir chamber) and were injected once a day into the CESAM chamber.
Moreover, also once a day, mineral dust particles were injected, produced through a shaking process from a Gobi Desert sample, into the chamber (simulating a desert storm that impacted Beijing with a mineral dust plume). Table 1 gives an overview of the pollutants de ned as reference to qualify a "Beijing-like" atmosphere simulation. This shows how the atmospheric environment to which the mice were exposed is complex (tens of pollutants, both in aerosol and gaseous phases), and representative of the urban situation of Beijing in December 2013 (most of the reference pollutants are present in a relative abundance that is similar to the targeted range). Table 1: Expected and observed ranges of December 2013 "Beijing-like" atmospheric pollutants.

Experimental design
Primiparous pregnant C57/BL6J mice (Janvier labs) were exposed to simulated Beijing-like air (n = 8) or ltered Beijing-like air (n = 8) from GD10 to GD17. Water and food were given ad libitum. Then, the mice were housed under standard conventional conditions (20°C, stable ambient humidity of 65%, and imposed daylight cycle of 12 h of light and 12 h of darkness). The delivery occurred between GD19 and Histological analysis and image processing Proximal colon and ileum were xed in 4% paraformaldehyde overnight, processed, and embedded in para n wax by an automatic sample preparation system (LOGOS One, Milestone). Serial histological sections of 4 μm thickness were cut, depara nized, rehydrated, and stained with MGG (Carlo Erba reagents, Val de Reuil, France, ref. E460583 and E453612). Images were acquired with a DM5500B microscope (Leica Microsystems, Nanterre, France) and intestinal layers were photographed at a magni cation of 20x. Histomorphometric analyses were performed using Image J software. Submucosal cellularity and mucosal surface area in the colon were measured, as well as villus height and crypt depth in the ileum. At least 100 well-oriented mucosa, villi, and crypts were measured in at least 5 individual mice from each group. For vacuole morphologic analyses, an image processing algorithm developed by Lustig et al. was adapted [57]. This algorithm converts a microscope image from a red-green-blue image to a grayscale image: the vacuoles are colored white and all other components are blackened. Based on the overlay of the binary image and the grayscale-processed image, we selected clearly visible vacuoles and Image J allowed the measurement of 1) vacuole number; 2) vacuole area, automatically calculated in pixels and converted to µm 2 ; 3) vacuole circularity, a measurement of how closely the shape of the vacuoles approaches that of a circle (circularity can be valued between 0 and 1 inclusively, where 1 is the circularity value of an ideal circle); and 4) cell eccentricity, a measurement of how close the shape of the marked region approaches that of a line or a circle (eccentricity varies between 0 and 1 inclusively, where 0 is the eccentricity value of an ideal circle shape and 1 is the eccentricity value of a line segment).
Immunohistochemical PCNA staining and quanti cation Serial histological sections of 4 µm thickness were cut, depara nized, and rehydrated. For antigen unmasking, sections were placed in 10 mM sodium citrate buffer (pH 6.0) and incubated in a heatinduced antigen retrieval chamber for 20 min at 121°C. After washing, sections were blocked for 30 min with 5% bovine serum albumin in PBS. PCNA primary antibody (NB300-524, Novus Biologicals) was then incubated overnight at 4°C at a 1:1000 concentration. After washing, tissue sections were incubated for 1 h at room temperature with an Alexa uor 647-conjugated anti-rabbit PCNA antibody (Thermo Fisher Scienti c). Nuclear staining with Hoechst was performed before adding a uorescent mounting medium.
Microscopy was performed using a Leica DM5500 B microscope and data was processed with Leica LAS V3.8 software. Cells positive for PCNA were counted blindly by 2 investigators (5 crypts/slide, 1 slide/animal).
Quantitative reverse transcription-PCR Small intestine, cecum, and colon tissue samples were homogenized with ceramic beads using Precellys lysing equipment (Bertin Technologies). Total RNA was extracted with the NucleoSpin RNA kit (Macherey-Nagel). Transcript levels of genes were quanti ed with the StepOne TM Real-Time PCR System using a SYBR Green PCR master mix (Thermo Fisher Scienti c). The primer sequences were designed using Primer Express 3 and are available upon request. Melting curve analyses were performed for each sample and gene. The relative expression of each target gene was normalized to the relative expression of the Polr2a housekeeping gene. Quanti cation of target gene expression was based on the comparative cycle threshold (Ct) value. Fold changes in target genes were analyzed by the 2 −ΔΔCt method.

Western blot
Proteins were extracted from male ileum samples and homogenized in TRI Reagent® lysis buffer (Sigma-Aldrich) according to the manufacturer's instructions. Twenty micrograms of total protein lysate was separated by SDS-PAGE and electroblotted on nitrocellulose membranes using an Amersham Semi Dry Transfer Unit at 0.8 mA/cm 2 (Amersham Pharmacia Biotech). Membranes were blocked for 1 h in StartingBlock™ T20 (TBS) with 5% milk blocking buffer (Thermo Fisher Scienti c) at room temperature.
Immunoreactivity was visualized with SuperSignal™ West Pico Plus Chemiluminescent HRP Substrate (Thermo Fisher Scienti c). Images were taken using the iBright FL1500 Imaging System (Thermo Fisher Scienti c). For relative quanti cation, the volume intensity of the bands was obtained using iBright software.
Bacterial DNA extraction and Illumina MiSeq sequencing Genomic DNA was extracted from the colon luminal content using a DNA stool kit (Macherey Nagel, France). The quantity and purity of DNA (expressed as the ratio of absorbance at 260 nm and 280 nm (A260/A280)) were assessed using a Nanodrop® spectrophotometer. The sequencing library was generated by amplifying the V3-V4 region of the bacterial 16S-rRNA gene using 16S rRNA amplicon generation for MiSeq with the primers Bact-0341 (CCTACGGGNGGCWGCAG) and Bact-0785 (GACTACHVGGGTATCTAATCC). Individual samples were barcoded, pooled to construct the sequencing library, and sequenced using an Illumina MiSeq (Illumina, San Diego, CA) to generate paired-end 2x300 bp reads.

Analysis of sequencing data
Bioinformatic analyses were performed using the QIIME2 pipeline (version 2020.2) [58]. The Divisive Amplicon Denoising Algorithm plug-in (DADA-2) in QIIME2 was used to lter, dereplicate, identify chimeric sequences, and merge PE reads to obtain a set of ASVs for each sample [59]. Then the representative sequences were picked for each ASV. The classify-sklearn plug-in in QIIME2, with the Silva database (version 132), was applied to assign a taxonomic annotation to each representative ASV sequence. In the next step, ASVs identi ed as eukaryotic contamination (3 ASVs; 12 reads) and external contamination, identi ed with the decontam package (3 ASVs; 3119 reads), were ltered out [60]. The diversity metrics (alpha and beta diversity) were obtained with the QIIME2 core-metrics-phylogenetic plug-in, with psampling depth parameter xed to 13781 reads which corresponded to the total frequency that each sample should be rare ed to prior to computing diversity metrics. This sampling depth allowed retention of more than 61% of reads and only one sample was discarded. Tests for differential relative abundance were performed with corncob at the order, family, and genus levels [61].

Statistics
Results are expressed as mean ± standard error of the mean. Except for metagenomic data, the statistical signi cance of differences between experimental groups was calculated using the Mann-Whitney nonparametric U test (GraphPad Prism software, USA). Statistical signi cance was de ned as p<0.05. For all experiments, * = p<0.05, ** = p<0.01, *** = p<0.005, and = ****p<0.001.

Declarations
Ethics approval and consent to participate Not applicable Consent for publication

Not applicable
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.   Histomorphological analysis in female mice. A Representative pictures of MGG-stained proximal colon from control air-and Beijing-like air-exposed mice, scale bar 500 µm (n=10/group). B Submucosa cellularity. C Mucosal surface area. D Representative pictures of MGG-stained ileum control air-and Beijing-like air-exposed mice, scale bar 200 µm (n=10/group). E Villus length. F Crypt depth. G Villus/crypt ratio. H Vacuole number per villus. I Mean vacuolated area per villus. J Vacuole circularity index. K Mean eccentricity index. Figure 3 Epithelium proliferation, differentiation, and maturation in male mice. A Representative pictures of PCNAstained proximal colon from control air-and Beijing-like air-exposed mice, scale bar 50 µm (n=10/group). In ammatory pattern in male mice. Transcript levels of Th1 (Tbx21, Tnfα, Ifnγ), Th2 (Gata3, Il4, Il5, Il13), Th17 (Rorc, Il17a, Il22), and Treg (Foxp3, Il10, Tgfβ) genes in proximal colon (A), cecum (B), and ileum (C) from control air-and Beijing-like air-exposed male mice (n=10/group). * p<0.05, ** p<0.01, *** p<0.005 as determined by the Mann-Whitney U test. In ammatory pattern in female mice. Transcript levels of Th1 (Tbx21, Tnfα, Ifnγ), Th2 (Gata3, Il4, Il5, Il13), Th17 (Rorc, Il17a, Il22), and Treg (Foxp3, Il10, Tgfβ) genes in proximal colon (A), cecum (B), and ileum (C) from control air-and Beijing-like air-exposed male mice (n=10/group). ** p<0.01 as determined by the Mann-Whitney U test. Tight junction expression in male and female mice. A-B Transcript levels of Ocln, Tjp1 and Cldn4 in proximal colon, cecum, and ileum of male (A) and female (B) mice exposed to control air or Beijing-like air (n=10/group). C ZO-1 and β-actin western blot analysis of male ileum. D Densitometric analysis of ZO-1 western blots. * p<0.05, ** p<0.01 as determined by the Mann-Whitney U test. Luminal content microbiota analysis in male and female mice exposed in utero to control air or Beijinglike air (n=10/group). A Chao1 α-diversity index. B Weighted UniFrac β-diversity index. C Overview of the relative abundance of gut bacteria depicted at the phylum level. D Overview of the relative abundance of gut bacteria depicted at the order level. E The differential abundance of signi cantly changed bacterial