Abstract
Maternal immune activation (MIA) contributes to behavioural abnormalities associated with neurodevelopmental disorders in both primate and rodent offspring1,2,3,4. In humans, epidemiological studies suggest that exposure of fetuses to maternal inflammation increases the likelihood of developing autism spectrum disorder5,6,7. In pregnant mice, interleukin-17a (IL-17a) produced by T helper 17 (TH17) cells (CD4+ T helper effector cells involved in multiple inflammatory conditions) induces behavioural and cortical abnormalities in the offspring exposed to MIA8. However, it is unclear whether other maternal factors are required to promote MIA-associated phenotypes. Moreover, the underlying mechanisms by which MIA leads to T cell activation with increased IL-17a in the maternal circulation are not well understood. Here we show that MIA phenotypes in offspring require maternal intestinal bacteria that promote TH17 cell differentiation. Pregnant mice that had been colonized with mouse commensal segmented filamentous bacteria or human commensal bacteria that induce intestinal TH17 cells were more likely to produce offspring with MIA-associated abnormalities. We also show that small intestine dendritic cells from pregnant, but not from non-pregnant, females secrete IL-1β, IL-23 and IL-6 and stimulate T cells to produce IL-17a upon exposure to MIA. Overall, our data suggest that defined gut commensal bacteria with a propensity to induce TH17 cells may increase the risk of neurodevelopmental disorders in the offspring of pregnant mothers undergoing immune system activation owing to infections or autoinflammatory syndromes.
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Acknowledgements
We thank S. Hang, D. Paik and N. Silverstein for valuable discussions and Y. Yang, M. Xu, N. Geva-Zatorsky, D. Kasper, C. Benoist and D. Mathis for reagents. We thank M. Trombly, N. Silverstein and A. Park for critical reading of the manuscript. We also thank E. Bridge, E. Jaskolski and other staff members at the Department of Animal Medicine at University of Massachusetts Medical School. This work was supported by the Simons Foundation Autism Research Initiative 274443 (D.R.L.) and 402005 (J.R.H.), the Simons Foundation to the Simons Center for the Social Brain at MIT (Y.S.Y., J.R.H and G.B.C.), Hock E. Tan and K. Lisa Yang Center for Autism Research (G.B.C.), the Howard Hughes Medical Institute (D.R.L.), Robert Buxton (G.B.C.), the National Research Foundation of Korea grants MEST-35B-2011-1-E00012 (S.K.) and NRF-2014R1A1A1006089 (H.K.), the Searle Scholars Program (J.R.H.), the Pew Scholar for Biomedical Sciences (J.R.H.), the Kenneth Rainin Foundation (J.R.H.), and the National Institutes of Health grants R01DK106351 and R01DK110559 (J.R.H.).
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S.K., T.G.T., R.S.L., K.H., D.R.L., G.B.C. and J.R.H. designed the experiments and/or provided advice, reagents and technical expertise. S.K., H.K., Y.S.Y., S.H. and K.A. performed the experiments. S.K., G.B.C. and J.R.H. wrote the manuscript with inputs from the co-authors.
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Extended data figures and tables
Extended Data Figure 1 Maternal vancomycin-treatment prevented induction of behavioural abnormalities in MIA offspring.
a, USV index (n = 27, 29 (PBS; male, female); n = 28, 21 (poly(I:C); male, female); 6 independent experiments). b, c, Total investigation time (b) and total distance travelled (c) during the sociability test (n = 13, 15 (vehicle; PBS, poly(I:C)); n = 12, 16 (vancomycin; PBS, poly(I:C)); 3–4 independent experiments). d, Schematic of the experimental design. e, f, Quantification of SATB2+ cells (e) in the cortex divided into ten equal bins representing different depths of the cortex or of the cortical patch size (f) in the primary somatosensory cortex (S1) (n = 3, 4 (PBS; vehicle, vancomycin); n = 3, 4 (poly(I:C); vehicle, vancomycin); 2 independent experiments). g, Flow cytometry of CD4+ T cells (gated on TCRβ+CD4+) stained intracellularly for IL-17a and RORγt. Mononuclear cells were collected at E14.5 from the ilea of poly(I:C)-treated mice with/without vancomycin treatment; representative FACS plot from 3 independent experiments. h, qPCR analysis measuring relative SFB levels in C57BL/6 mice before/after vancomycin treatments (n = 4–5 per group). i, Representative s.e.m. images of epithelial surfaces in the ilea of the vehicle-/vancomycin-treated mice from 2 independent experiments. Scale bars, 30 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 calculated by two-way (a, e) and one-way (b, c, f) ANOVA with Tukey post hoc tests. NS, not significant. Graphs indicate mean ± s.e.m.
Extended Data Figure 2 MIA in SFB-absent Jax mothers does not induce changes in the total activity of the adult offspring, properties of the litter and maternal cytokine production.
a, b, Total investigation time (a) and total distance travelled (b) during the sociability test of adult offspring described in Fig. 2b–d. c, Litter size upon weaning (n = 59, 125 (Tac; PBS, poly(I:C)); n = 51, 50 (Jax; PBS, poly(I:C)); n = 55, 81 (co-housed Jax; PBS, poly(I:C)); n = 55, 89 (SFB-gavaged Jax; PBS, poly(I:C)). d, Weight of male offspring from the groups described in c (n = 32, 50 (Tac; PBS, poly(I:C)); n = 29, 27 (Jax; PBS, poly(I:C)); n = 29, 29 (co-housed Jax; PBS, poly(I:C)); n = 33, 30 (SFB-gavaged Jax; PBS, poly(I:C)). Data in c and d are from 7–8 independent experiments. e, f, Quantification of SATB2+ cells (e) in the cortex divided into ten equal bins representing different depth and of patch size (f) in the S1 (n = 4 (Tac; PBS); n = 3, 3, 4, 3 (Tac, Jax, co-housed Jax, SFB-gavaged Jax; poly(I:C)). g, Maternal plasma concentrations of TNFα and IFNβ at 3 h after PBS, poly(I:C) injection into Tac/Jax dams at E12.5; n = 4 per group. *P < 0.05, **P < 0.01, ***P < 0.001 calculated by two-way (e) and one-way ANOVA (a–d, f) with Tukey post hoc tests and Student’s t-test (g). Graphs indicate mean ± s.e.m.
Extended Data Figure 3 SFB colonization leads to increased levels of gut TH17 cells in Jax pregnant mice.
a, Schematic of the experimental design. b, Flow cytometry of CD4+ T cells (gated on TCRβ+CD4+) stained intracellularly for IL-17a and RORγt. Mononuclear cells were collected at E14.5 from the ilea of poly(I:C)-treated Tac, Jax, co-housed Jax, SFB-gavaged Jax mothers. c, Representative s.e.m. images of epithelial surfaces in the ilea of Tac, Jax, co-housed Jax, SFB-gavaged Jax mothers. Scale bars, 30 μm. Data representative of 3 (b) and 2 (c) independent experiments. d, qPCR analysis for SFB levels in the faecal samples of the groups described in (a) (n = 4–5 per group). ****P < 0.0001 calculated by one-way (d) ANOVA with Tukey post hoc test. Graphs indicate mean ± s.e.m.
Extended Data Figure 4 Poly(I:C)-induced inflammation during pregnancy, not after giving birth, is critical in inducing MIA-associated behavioural abnormalities in offspring.
a, Schematic of the experimental design for cross-fostering experiments. b, USV index (n = 21, 20 (PBS dams; PBS, poly(I:C) pups); n = 22, 15 (poly(I:C) dams; PBS, poly(I:C) pups); 2–4 independent experiments). c–g, Marble burying index (c), time spent in the centre of an open field (d), and percentage of interaction (e), total investigation time (f), and total distance travelled (g) during the sociability test (n = 9, 14 (PBS dams; PBS, poly(I:C) pups); n = 12, 10 (poly(I:C) dams; PBS, poly(I:C) pups); 2 independent experiments). **P < 0.01, ****P < 0.0001 calculated by one-way (b–d, f, g) and two-way (e) ANOVA with Tukey post hoc tests. NS, not significant. Graphs indicate mean ± s.e.m.
Extended Data Figure 5 Composition of maternal gut microbiota during pregnancy, not after giving birth, is critical in inducing MIA-associated behavioural abnormalities in offspring.
a, Schematic of the experimental design for cross-fostering experiments. b, USV index (n = 9, 36 (Tac pups with Jax dams; PBS, poly(I:C)); n = 10, 24 (Jax pups with Tac dams; PBS, poly(I:C)); 2–4 independent experiments). c–g, Marble burying index (c), time spent in the centre of an open field (d), and percentage of interaction (e), total investigation time (f), and total distance travelled (g) during the sociability test (n = 7, 22 (Tac pups with Jax dams; PBS, poly(I:C)); n = 7, 21 (Jax pups with Tac dams; PBS, poly(I:C)); 2 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 calculated by one-way (b–d, f, g) and two-way (e) ANOVA with Tukey post hoc tests. NS, not significant. Graphs indicate mean ± s.e.m.
Extended Data Figure 6 CD11c+ DCs stimulate gut TH17 cells to produce high levels of IL-17a ex vivo.
a–f, Flow cytometry of CD4+ T cells (gated on TCRβ+CD4+) stained intracellularly for IL-17a and RORγt. Mononuclear cells were collected at E14.5 from the gut ilea, spleens, and mesenteric lymph nodes (mLN) of PBS-, poly(I:C)-treated mice (n = 5 per group (a, c, e); n = 3 per group (b, d, f)). MFI denotes mean fluorescence intensity. g–i, Supernatant concentrations of IL-17a from mononuclear cells of the ilea in poly(I:C)-treated Tac dams (g) (n = 3 per group), from co-cultures of CD4+ and non-CD4+ cells of the ilea in PBS-, poly(I:C)-treated Tac dams (h) (n = 3 per group), or from co-cultures of CD4+ and CD103−CD11b+, CD103+CD11b+ and CD103+CD11b− (gated on MHCII+CD11c+) cells of the ilea in poly(I:C)-treated dams (i) (n = 7 per group). All cultures were isolated at E14.5 and stimulated ex vivo with poly(I:C) for 18 h (g, h) or for 48 h (i). Data are pooled from 2 (g, h) or 3 (i) independent experiments. j, USV index (n = 16, 17 (poly(I:C); wild type or TLR3-KO); 2 independent experiments). k, Supernatant concentrations of IL-6, IL-1β and IL-23 from cultures of CD11c+ isolated at E14.5 from the ilea of poly(I:C)-treated non-pregnant or pregnant mice (n = 5 per group; 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 calculated by Student’s t-test (a–f, j, k) and one-way ANOVA (g–i) with Tukey post hoc tests. NS, not significant. Graphs indicate mean ± s.e.m.
Extended Data Figure 7 SFB-specific 7B8-Tg CD4+ T cells produce IL-17a upon transfer to MIA-exposed pregnant mothers.
a, Schematic of the experimental design. b, c, Both TCRα-KO and IL-17a-KO females, with or without adoptive transfers of 7B8-Tg-derived CD4+ T cells, were crossed with wild-type C57BL/6 males to produce heterozygous wild-type offspring. USV index (n = 16, 30 (TCRα-ΚΟ; poly(I:C), 7B8-Tg T cell transfer); n = 23, 23 (IL-17a-KO; poly(I:C), 7B8-Tg T cell transfer + poly(I:C)), marble burying index, time spent in the centre of an open field, and percentage interaction and total distance travelled during the sociability test of TCRα-KO (b) or IL-17a-KO (c) offspring (n = 12, 15 (TCRα-ΚΟ; poly(I:C), 7B8-Tg T cell transfer); n = 12, 14 (IL-17a-KO; poly(I:C), 7B8-Tg T cell transfer + poly(I:C)). Data pooled from 2–3 independent experiments. d, e, Representative SATB2 staining in the cortex of the animals prepared as in a. Arrowheads indicate cortical patches. Scale bars, 100 μm. f, g, Quantification of SATB2+ cells (n = 7, 6 (TCRα-KO; poly(I:C), 7B8-Tg T cell transfer); n = 6, 7 (IL-17a-KO; poly(I:C), 7B8-Tg T cell transfer + poly(I:C)). h, Cortical patch size (n = 5, 5 (TCRα-KO; poly(I:C), 7B8-Tg T cell transfer); n = 4, 4 (IL-17a-KO; poly(I:C), 7B8-Tg T cell transfer + poly(I:C)). i, j, IL-17a concentrations in maternal plasma collected at E14.5. k, Flow cytometry of ileal CD4+ T cells (gated on CD4+TCRβ+) stained intracellularly for IL-17a. Mononuclear cells were collected from small intestines of poly(I:C)-treated IL-17a-KO mothers transferred with 7B8-Tg CD4+ T cells. CD45.1+ cells refer to donor cells and CD45.2+ to recipient cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 calculated by Student’s t-test (b, c, h–j) and one-way (f, g) ANOVA with Sidak post hoc tests. Graphs indicate mean ± s.e.m.
Extended Data Figure 8 A mix of twenty human commensals induces colonic TH17 cell differentiation in SFB-absent Jax mice.
a, Schematic of the experimental design. b, Flow cytometry of CD4+ T cells (gated on CD4+TCRβ+) stained intracellularly for IL-17a and RORγt. Mononuclear cells were collected from colons of poly(I:C)-treated Jax mothers with/without human bacteria gavage. c, Representative scanning electron microscopy images of epithelial surfaces in the ilea from 2 independent experiments. d, e, Total interaction time (d), and total distance travelled (e) during the sociability test of adult offspring described in a (n = 23, vehicle-gavaged only; 22, human-bacteria-gavaged + isotype control antibody; 13, human-bacteria-gavaged + anti-IL-17a antibody; 4 independent experiments). f–g, Quantification of SATB2+ cells (n = 5 per group) and cortical patch size (n = 7, 6, 5 (poly(I:C); vehicle-treated Jax, human-bacteria-gavaged Jax with isotype control antibody, human-bacteria-gavaged Jax with anti-IL-17a antibody). *P < 0.05 calculated by one-way (d, e, g) and two-way (f) ANOVA with Tukey post hoc test. Graphs indicate mean ± s.e.m.
Extended Data Figure 9 The IL-17a pathway promotes abnormal behavioural phenotypes in MIA offspring born to mice colonized with human commensal bacteria.
a, Schematic representation of the experimental design. b, Quantification of bacterial colonization levels through colony forming unit (CFU) counts or qPCR analyses. c, USV index (n = 13, 12, 28, 16, 17, 14 (poly(I:C); vehicle, SFB, Listeria monocytogenes, Bacteroides fragilis, Bifidobacterium adolescentis, CD-SpA 2A). d, e, Maternal plasma concentrations of IL-17a and IFNγ at E14.5 (n = 4, 4, 3, 6, 3 (poly(I:C); vehicle, Listeria monocytogenes, Bacteroides fragilis, Bifidobacterium adolescentis, CD-SpA 2A). f, qPCR analysis measuring relative SFB levels in Jax mice gavaged with various bacteria; from two independent experiments. *P < 0.05, ****P < 0.0001 calculated by one-way (c–f) ANOVA with Tukey post hoc tests and Student’s t-test (b). ND, not determined. NS, not significant. Graphs indicate mean ± s.e.m.
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Kim, S., Kim, H., Yim, Y. et al. Maternal gut bacteria promote neurodevelopmental abnormalities in mouse offspring. Nature 549, 528–532 (2017). https://doi.org/10.1038/nature23910
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DOI: https://doi.org/10.1038/nature23910
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Kim et al. (ref. 1) demonstrate that pregnant mice are more likely to produce offspring with aspects of autism phenotype when their gut is colonized with mouse commensal segmented filamentous bacteria or human commensal bacteria, and they are in maternal immune activation (MIA) state. Specifically, they show that MIA phenotypes in offspring require maternal intestinal bacteria that promote TH17 cell differentiation. These findings extend their prior work that showed that MIA offspring exhibit cortical patches that resemble brain lesions described in patients with autism spectrum disorder (ref.2,3).
Yim et al (4) identify a cortical region primarily centred on the somato-sensory cortex (SIDZ) as the major node of a neural network that mediates behavioural abnormalities observed in offspring exposed to maternal inflammation. These findings extend the prior work of Casanova et al. (ref.5) which delineated focal cortical dysplasias and of Stoner et al. (6) which described patches of disorganization in the neocortex of individuals with the autism spectrum disorder.
The findings of Kim, Yim, and other investigators provide deep insights into biology of autism and its relationships with maternal microbiota, and gut ecology, immune dynamics, structural brain changes, nodes of neural networks, and autism-like phenotypes. Beyond this, the work is crucial for parents, physicians, and public health policy makers. For parents and public health community, it definitively dispels the notion that vaccination is responsible for the epidemic increases in the prevalence of ASD, which prevents vaccination protection for children in many regions. The other side of this coin concerns physicians who generally dismiss nutritional and indigenous therapies of empirical benefits for children with ASD and related neurodevelopmental challenges. It is noteworthy that most children with such clinicopathologic entities suffer from IgE-mediated disorders such as eczema, inhalant allergy, brochospastic diathesis, and symptom-complexes suggestive altered states of bowel ecology (ref. 7). Among the common metabolic disruptions in this population are mitochondrial dysfunction (ref. 8) and hyperinsulinism (ref.9).
In 1980, work in gut immunopathology of this writer (ref. 10,11) led to his publication Altered States of Bowel Ecology (ref.7) to focus on the matters of: (a) gut flora; (b) food allergy and related adverse effects; (c) digestive-absorptive dysfunctions; and increased disruptions of neuro-enteric dynamics. He put forth a ?seed-feed-weed approach? for restoring bowel ecology; seeding here is done with probiotics, feeding with foods and spices that favor healthful bowel flora, and weeding with selected food elimination, antifungal herbs, spices, and judicious use of antifungal pharmacologic agents, notably Nystatin. Clinical applications of this ecologic approach were described in detail in Darwin, Dysox, and Integrative Protocols, the 12th volume of The Principles and Practice of Integrative Medicine (2009), (ref.12).
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