The gut microbiota in infants of obese mothers increases inflammation and susceptibility to NAFLD

Abstract

Maternal obesity is associated with increased risk for offspring obesity and non-alcoholic fatty liver disease (NAFLD), but the causal drivers of this association are unclear. Early colonization of the infant gut by microbes plays a critical role in establishing immunity and metabolic function. Here, we compare germ-free mice colonized with stool microbes (MB) from 2-week-old infants born to obese (Inf-ObMB) or normal-weight (Inf-NWMB) mothers. Inf-ObMB-colonized mice demonstrate increased hepatic gene expression for endoplasmic reticulum stress and innate immunity together with histological signs of periportal inflammation, a histological pattern more commonly reported in pediatric cases of NAFLD. Inf-ObMB mice show increased intestinal permeability, reduced macrophage phagocytosis, and dampened cytokine production suggestive of impaired macrophage function. Furthermore, exposure to a Western-style diet in Inf-ObMB mice promotes excess weight gain and accelerates NAFLD. Overall, these results provide functional evidence supporting a causative role of maternal obesity-associated infant dysbiosis in childhood obesity and NAFLD.

Fig. 1

Diagrammatic representation of experimental design. Stool from three different 2-week-old infants born to normal-weight (NW) or obese (Ob) mothers was pooled in an anaerobic chamber. Pooled samples from each group, Inf-NWMB or Inf-ObMB, were gavaged into 3–5 GF mice per group and the inoculated mice were colonized for 21 days on a chow diet. This experiment was repeated three times using unique pooled samples of infant stools for each round of colonization. To study the effect of WSD, a fourth experiment was performed with 4 GF mice per group and following the 21-day colonization, mice were switched to a WSD for 6 weeks

Fig. 2

Inf-ObMB alters gut microbiome and increases intestinal permeability. Bacteroidetes to Firmicutes (B:F) ratio in the cecal microbiota depicted as percent of total of these two phyla (a) and class level relative abundance (b), with the percent of total highlighted for Gammaproteobacteria and Clostridia, after 21 days of colonization; n = 6 for both Inf-NWMB and Inf-ObMW groups. c Acetate, propionate, and butyrate SCFA concentrations measured in the cecum; n = 8 for Inf-NWMB, n = 7 for Inf-ObMB. d Relative mRNA expression of Tjp1 and Ocln measured in the colon, normalized to Gapdh and 18S rRNA; n = 8 for Inf-NWMB, n = 7 for Inf-ObMB. e FITC-dextran translocation into plasma; n = 4 for Inf-NWMB, n = 7 for Inf-ObMB. f Quantity of 16S DNA in liver; Cq, quantification cycle; n = 11 for Inf-NWMB and n = 14 for Inf-ObMB. c–f Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.0001 by Student’s t-test

Fig. 3

Inf-ObMB increases fecal BA loss and alters hepatic BA homeostasis genes. Concentration of primary, conjugated primary, secondary, conjugated secondary, and total BAs in the liver (a) and feces (b) in Inf-NWMB and Inf-ObMB mice; n = 3 for Inf-NWMB, n = 4 for Inf-ObMB for liver; n = 3 for both Inf-NWMB and Inf-ObMB for feces. c Hepatic gene expression of BA synthesis enzymes Cyp7a1 and Cyp8b1, exporter Bsep, importer Ntcp, BA receptor Fxr, and its downstream targets Shp and Hnf4a in Inf-NWMB and Inf-ObMB mice, normalized to Gapdh and 18S rRNA; n = 6 for Inf-NWMB, n = 7 for Inf-ObMB. Data are presented as mean ± SEM. *P < 0.05 and **P < 0.01 by two-tailed Student’s t-test

Fig. 4

Inf-ObMB increases subcutaneous fat and liver inflammation. Weight of subcutaneous white adipose tissue (scWAT) and body weight (a), and liver triglycerides (TG) (b); n = 8 for Inf-NWMB, n = 7 for Inf-ObMB. c Hepatic expression of lipid regulatory and inflammatory genes normalized to Gapdh and 18S rRNA; n = 6 for Inf-NWMB, n = 7 for Inf-ObMB (except Tnf, n = 3/group). d Hepatic resident, recruited, and total macrophage (Φ) quantities; n = 3 for Inf-NWMB, n = 4 for Inf-ObMB. a–d Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.005 by two-tailed Student’s t-test. e Representative photomicrographs of H&E staining in the liver from Inf-NWMB and Inf-ObMB mice; black bars represent 100 μm, black arrows indicate infiltrating lymphocytes; PV portal vein, BD bile duct. f Modified Pediatric NAFLD Histological Score (PNHS) for Inf-NWMB and Inf-ObMB mice. n = 3 for Inf-NWMB and n = 7 for Inf-ObMB. Data are presented as mean ± SEM. *P < 0.05 by two-tailed Mann–Whitney U-test

Fig. 5

BMDMs from Inf-ObMB mice have impaired function. a Cytokine relative gene expression in BMDMs with and without 20-h low-dose LPS treatment depicted as relative to the Inf-NWMB no treatment (No Tx) group, with endogenous normalization to Gapdh; n = 2 for both Inf-NWMB and Inf-ObMB. CD11b⁺/F4/80⁺ BMDMs for GFP-expressing Listeria (Lm) uptake (b) with representative flow diagrams (c); n = 4 for Inf-NWMB, n = 8 for Inf-ObMB for Lm-infected; n = 1 for Inf-NWMB, n = 2 for Inf-ObMB for uninfected control. Data are presented as mean ± SEM. *P < 0.05 and **P < 0.01 by two-tailed Student’s t-test

Fig. 6

Inf-ObMB phenotype is exacerbated by WSD. a Percent body fat before and after 6 weeks of WSD. Inf-NWMB and Inf-ObMB mice after 6 weeks on WSD for body weight (b), liver triglycerides (TG) (c), liver macrophage (Φ) quantity (d), and proinflammatory Il1b, Tnf, and anti-inflammatory Il10 gene expression in recruited macrophages from liver, normalized to Gapdh and 18S rRNA (e). a–e n = 4 for both Inf-NWMB and Inf-ObMB. Data are presented as mean ± SEM. *P < 0.05 by two-tailed Student’s t-test. f Representative photomicrographs of liver sections with H&E staining; black bars represent 100 μm, black arrows indicate infiltrating lymphocytes, white arrows indicate lipid droplets; CV central vein; PV portal vein; BD bile duct. g Modified Pediatric NAFLD Histological Score (PNHS) for Inf-NWMB and Inf-ObMB mice fed a WSD. n = 4 for both Inf-NWMB and Inf-ObMB. Data are presented as mean ± SEM. P = 0.086 by two-tailed Mann–Whitney U-test

Fig. 7

Inf-NWMB and Inf-ObMB gut microbiota are not different after 6 weeks on WSD. Cecal microbiota Bacteroidetes to Firmicutes (B:F) ratio (a), shown as percent of total of these two phyla, and Gammaproteobacteria and Clostridia relative abundances (b). c Acetate, propionate, and butyrate SCFA concentrations in the cecum in colonized mice fed a WSD; data are presented as mean ± SEM. a–c n = 4 for both Inf-NWMB and Inf-ObMB