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 . Thus, we first 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 significantly 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 . 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 significantly reduced in Beijing-like air mice (Fig 1I). Morphometric evaluation of the vacuoles also showed significant 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 significant variation between air pollution- and control-exposed mice (Fig 2A, B, C). In ileum, there were no modification 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 quantified 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 significant variation (Fig 3C). The markers of absorptive cells alkaline phosphatase, intestinal (Alpi), epithelial cell adhesion molecule (Epcam), and carbonic anhydrase 2 (Car2) were all significantly upregulated in Beijing-like air mice compared to control air mice (Fig 3D). The marker of goblet cells mucin 2 (Muc2) was significantly downregulated in Beijing-like air mice (Fig 3E). The transcript levels of the markers of Paneth cells (lysozyme 1; Lyz1), of enterochromaffin 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 quantified mRNA levels of several neonatal and adult markers (Fig 3 F and G) . During the suckling period, neonatal intestinal cells express disaccharidase lactase-phlorizin hydrolase (Lct) to digest milk lactose . 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) . 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) . 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 . The expression of Fcgrt declines significantly during the suckling-to-weaning transition . 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 modified but Fcgrt transcript levels were significantly 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, reflecting 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 modified (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 significant modification of proliferation in Beijing-like air mice compared to control air mice (Fig 4 A and B). The mRNA level of Lgr5 was significantly downregulated in Beijing-like air mice (Fig 4C). The transcript abundance of absorptive and secretory cell markers did not show significant variation (Fig 4 D and E). The neonatal markers were not changed either (Fig 4F). The transcription of Arg2 was significantly 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 over-expression of ChrgA transcripts was quantified 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 air-exposed mice ileum was observed, we quantified expression of other markers of enteroendocrine cells. Levels of tachykinin 1 (Tac1, or substance P)-producing enterochromaffin 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 quantified  (Fig S2). An upregulation of Nts was found in the Beijing-like air-polluted group, reflecting an abnormal expression of neurotensin.
Inflammatory pattern in male mice
In order to assess whether in utero exposure to air pollution induces impairments of the inflammatory process in GD17 pups, we quantified mRNA levels of transcription factors and inflammatory 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 significantly 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 significant upregulation of Rorc was induced by in utero exposure of simulated Beijing-like air pollution (Fig 5C).
Inflammatory 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 modified 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 quantified. In male proximal colon there was no significant 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 quantified the expression of ZO-1 protein by western blot and confirmed that ZO-1 protein levels were significantly 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 significantly affect a-diversity (Chao1 diversity index, Fig 8A; Evenness and Simpson indices, Fig S3). The weighted UniFrac b-diversity index showed no significant difference in males, but a significant 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 significantly different between the exposed and control mice. Similarly, analyses at the class level showed important interindividual variability, but did not reveal significant 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 significantly 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 significantly reduced in females after in utero exposure to Beijing-like air (Fig 8F).