Microbiota-targeted maternal antibodies protect neonates from enteric infection - PubMed (original) (raw)

Microbiota-targeted maternal antibodies protect neonates from enteric infection

Wen Zheng et al. Nature. 2020 Jan.

Abstract

Although maternal antibodies protect newborn babies from infection1,2, little is known about how protective antibodies are induced without prior pathogen exposure. Here we show that neonatal mice that lack the capacity to produce IgG are protected from infection with the enteric pathogen enterotoxigenic Escherichia coli by maternal natural IgG antibodies against the maternal microbiota when antibodies are delivered either across the placenta or through breast milk. By challenging pups that were fostered by either maternal antibody-sufficient or antibody-deficient dams, we found that IgG derived from breast milk was crucial for protection against mucosal disease induced by enterotoxigenic E. coli. IgG also provides protection against systemic infection by E. coli. Pups used the neonatal Fc receptor to transfer IgG from milk into serum. The maternal commensal microbiota can induce antibodies that recognize antigens expressed by enterotoxigenic E. coli and other Enterobacteriaceae species. Induction of maternal antibodies against a commensal Pantoea species confers protection against enterotoxigenic E. coli in pups. This role of the microbiota in eliciting protective antibodies to a specific neonatal pathogen represents an important host defence mechanism against infection in neonates.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Extended Data Fig. 1 |. Persistence and development of maternal antibodies.

The genotypes of mating pairs are indicated under the large mouse cartoons; the small mouse cartoons represent neonates born to the indicated dams and their genotypes. Red symbols denote the presence in neonates of antibodies that either were acquired transplacentally from μMT +/− mothers or, in the case of μMT +/− pups, were generated endogenously after 4 weeks of age. a, Reciprocal breeding scheme to study maternal antibody persistence and development. b, Serum IgG concentration in 1–8-week-old pups. Data are shown as μg ml−1. n = 5–15 mice in each breeding group for every week of 1–8 weeks. Further details are provided in the Source Data. c, IgA concentrations in small-intestine and colon (CO) homogenates from 1-week-old pups. Data are shown as μg per small intestine or colon. d, Faecal IgA concentration in 2–8-week-old pups. Data are shown as μg per g of faeces. n = 6–13 mice in each breeding group for every week of 2–8 weeks. Further details are provided in the Source Data. e, Serum IgM concentration in 1-week-old pups. Data are shown as μg ml−1. f, IgG concentration in small-intestine and colon homogenates from 1-week-old pups. Data are shown as μg per small intestine or colon. g, Faecal IgG concentration in 2–8-week-old pups. Data are shown as μg per g of faeces. n = 5–9 mice in each breeding group for every week of 2–8 weeks. Further details are provided in the Source Data. b–g, Data are mean ± s.e.m. Specific n numbers are shown in the figure.

Extended Data Fig. 2 |. Comparison of mNAb+ and mNab− pups.

a, Survival of 2-week-old mNAb+ and mNab− pups on day 1 after intraperitoneal challenge with 107 CFU of ETEC 6. mNab+ group, n = 6; mNab− group, n = 15. b, 16S rRNA gene analysis of the composition of the microbiota in 1-week-old reciprocally bred pups. Data are the average of 8 or 9 individual pups from each group; mNab+ group, n = 9; mNab− group, n = 8. c. Transcriptome analysis of small intestines of ETEC-6-infected mNab+ and mNab− pups using RNA sequencing. n = 8 mNab+ pups; n = 4 mNab− pups. Specific n numbers are shown in the figure.

Extended Data Fig. 3 |. Comparison of pups from the cross-fostering experiment.

For all panels, cross-fostering histories of neonates are denoted by horizontal arrows that provide the genotype of the pup followed by the genotype of the fostering dam. a. Cross-fostering experimental scheme. The genotypes of dams are indicated under the large mouse cartoons; the small mouse cartoons represent neonates that are born to those dams and have the same genotype as their mothers. Thicker arrows define the mother that fostered the indicated neonates. Red symbols denote the presence in neonates of antibodies that were acquired transplacentally from their μMT +/− mothers or, in the case of μMT −/− pups, from a μMT +/− fostering dam. b, Colon IgG concentration of 1-week-old cross-fostered pups. Data are shown as μg per colon. c, Colon IgA concentration of 1-week-old cross-fostered pups. Data are shown as μg per colon. d, Fostering scheme of μMT −/− pups cross-fostered by a μMT +/− dam. e, Survival of fostered μMT −/− pups at 20-h after ETEC infection. In the first experiment, n = 5 μMT −/− pups were fostered by μMT +/− dams; n = 5 μMT −/− pups were fostered by μMT −/− dams. In the second experiment, n = 6 μMT −/− pups were cross-fostered by μMT +/− dams; n = 8 μMT −/− pups were fostered by μMT −/− dams. b, c, Data are mean ± s.e.m. Specific n numbers are shown in the figure.

Extended Data Fig. 4 |. Relative concentrations of all subclasses of IgG between dams and pups.

a–e, Serum IgG subclass concentrations in μMT −/− FcRn +/+ (or FcRn +/−) and μMT −/− FcRn −/− pups fostered by μMT +/− dams for 1 week. f–j, Relative concentrations of all subclasses of IgG between dam and pups. k, Adult (8-week-old) mice were orally gavaged with 5 mg of IgG, and serum IgG concentrations were quantified as ng ml−1. NS, no significant difference; calculated using a Mann–Whitney _U_-test. Specific n numbers are shown in the figure.

Extended Data Fig. 5 |. Serum from conventionally colonized (SPF) mice broadly recognizes human commensal bacteria and other enteric pathogens.

a, Total immunoglobulin titres against E. coli strain Nissle 1917 in germ-free and SPF mouse serum. b, Total immunoglobulin titres against Salmonella typhimurium in germ-free and SPF mouse serum. Specific n numbers are shown in the figure.

Extended Data Fig. 6 |. Characteristics of commensal-immunized and unimmunized serum.

a, Western blot analysis of serum IgG from pups born to _Pantoea_-1-immunized SPF mice shows epitopes of ETEC 6, Pantoea 1 and Enterobacter, similar in size to those in serum from pups born to _Pantoea_-1-immunized germ-free mice. b, Serum IgG of pups born to _Pantoea_-immunized germ-free dams cross-reacts with different Enterobacteriaceae isolates from different facilities. 1, Harvard SGM Pantoea; 2, ETEC 6; 3, Harvard SGM Enterobacter; 4, Bacteroides fragilis NCTC9343; 5, Charles River B6 Proteus mirabilis; 6, Charles River B6 E. coli isolate 1; 7, Charles River B6 E. coli isolate 2; 8, Charles River CD1 E. coli isolate 1; 9, Charles River CD1 E. coli isolate 2; 10, Taconic B6 E. coli isolate 1; 11, Taconic B6 E. coli isolate 2; 12, Taconic B6 Proteus isolate; 13, Taconic B6 Enterobacter isolate; 14, C. rodentium. c, Pronase-treated bacterial lysates blotted with serum IgG of pups born to _Pantoea_-immunized germ-free dams. The concentrations of pronase are specified in the figure. a–c, Proteins were detected using a goat anti-mouse IgG antibody. For gel source data, see Supplementary Fig. 1. d, Mouse milk IgG and IgA concentrations. Data are shown in μg ml−1. e. Mouse milk IgG titre against microbiota. Each line represents an independent mouse. d, Data are mean ± s.e.m.

Extended Data Fig. 7 |. Schematic summary of the findings in this study.

Top, mNabs induced by commensal microbiota in dams were transferred to neonates through the breast milk. Cross-reacting mNabs (especially IgG antibodies) were detected that bound to the pathogenic, non-indigenous bacterial species ETEC and correlated with protection against disease in pups challenged with ETEC. IgG antibodies were also shown to be transported from the milk to the bloodstream of pups by a process that we call IgG retro-transport. Bottom, mNabs react with many commensal species and among them an Enterobacteriaceae isolate (Pantoea) was found to induce antibodies that cross-react with ETEC. The immunogenicity of this commensal species is hypothesized to be a result of local antigen-sampling processes that involve dendritic cells and uptake by Peyer’s patch germinal centres. This ultimately leads to the induction of high-affinity IgGs directed against a Pantoea antigen that cross-reacts with ETEC. IgG was also shown to be transported from the blood stream to the intestinal lumen by FcRn in adult mice. Illustrations were created with BioRender (

https://biorender.com/

Fig. 1. mNabs protect neonates from an enteric bacterial pathogen.

a, Bacterial burdens of reciprocally bred mNab+ and mNab− pups (6–7 days old) orally challenged with 107 CFU of ETEC 6. Ig. Immunoglobulin; SI, small intestine. **P = 0.0004, two-tailed Mann–Whitney _U_-test. Data are representative of four independent experiments. b, Survival among reciprocally bred pups 20 h after oral–gastric challenge with 109 CFU of ETEC 6. Data are from three independent experiments (first experiment, n = 8 mNab+ mice, n = 5 mNab− mice; second experiment, n = 9 mNab+ mice, n = 9 mNab− mice; third experiment, n = 7 mNab+ mice, n = 6 mNab− mice). *P = 0.0011, two-tailed unpaired _t_-test. c, Serum IgG levels in ETEC-challenged reciprocally bred pups. ***P = 0.0002, two-tailed Mann–Whitney _U_-test. Data are representative of two independent experiments. d, Small-intestinal mucosal IgG levels in ETEC-challenged reciprocally bred pups. **P = 0.0022, two-tailed Mann–Whitney _U_-test. Data are representative of two independent experiments. SI, small intestine. e, Flow cytometry analysis of natural maternal IgG and IgA coating of commensal bacteria of 1-week-old mNab+ and mNab− pups. Data are representative of two independent experiments (n = 4–5 mice per group in each experiment). f, Flow cytometry analysis of natural maternal IgG and IgA coating of ETEC–GFP bacteria in mNab+ and mNab− pups 18 h after infection. IgG and IgA signals are gated on GFP+ population. Data are representative of two independent experiments (n = 4–7 mice per group in each experiment). g, Serum IgG levels after 1 week of cross-fostering. h, Small-intestinal IgG levels after 1 week of cross-fostering. i, Small-intestinal IgA levels after 1 week of cross-fostering. j, ETEC 6 bacterial burdens in the small intestine of pups cross-fostered for 1 week. ***P = 0.0002, two-tailed Mann–Whitney _U_-test. Data are representative of two independent experiments. a–d, g–j, Data are mean ± s.e.m. Specific n numbers are indicated in the figure.

Fig. 2 |. FcRn mediates postnatal IgG retro-transport.

a, Breeding and fostering strategy to specifically study the postnatal milk IgG transfer process. All pups discussed in this figure are μMT −/−. b, Serum IgG levels in 1-week-old FcRn-deficient or FcRn-sufficient μMT −/− pups after 1 week of fostering by a μMT +/− dam. **P = 0.0013, two-tailed Mann–Whitney _U_-test. Data are representative of two independent experiments. c, Titres in pups of IgG specific to the microbiota of the foster dam. Data are representative of two independent experiments. d, Adult (8-week-old) mice were intraperitoneally (i.p.) injected with 5 mg of IgG, and faeces samples were collected 1 day later. Faecal IgG levels are shown as μg per gram of faeces. *P = 0.0357, two-sided Mann–Whitney _U_-test. Data are representative of two independent experiments. e, IgG treatment scheme of dams treated with IgG, data for the pups were tested are shown in f and g. f, Comparison of ETEC 6 bacterial burden in the small intestine of pups from untreated μMT −/− dams and from μMT −/− dams treated with IgG. **P = 0.0043, two-sided Mann–Whitney _U_-test. Data are representative of two independent experiments. g, Serum IgG levels of pups from untreated μMT −/− dams compared with μMT −/− dams treated with IgG. Data are representative of two independent experiments. b–d, f, g, Data are mean ± s.e.m. Specific n numbers are indicated in the figure.

Fig. 3 |. The commensal microbiota elicits antibodies that cross-react with ETEC 6.

a, Total immunoglobulin titres against ETEC 6 in serum from germ-free (GF) and SPF adult female mice as well as in serum from SPF mice absorbed by mouse microbiota. Data are representative of four independent experiments. OD405, optical density at 405 nm. b, IgG titres against ETEC 6 in serum from germ-free and SPF mice. c, Total immunoglobulin titres against ETEC 6 in serum from 1-week-old neonatal mNab+ and mNab− mice obtained by reciprocal breeding. d, IgG titres against ETEC 6 in serum from 1-week-old neonatal mNab+ and mNab− mice obtained by reciprocal breeding. e, Total immunoglobulin titres against ETEC 6 in serum from germ-free mice, serum from SPF mice and _Pantoea-_1-absorbed serum from SPF mice. f, IgG titres against ETEC 6 in serum from germ-free mice, serum from SPF mice and _Pantoea-_1-absorbed serum from SPF mice. Data are mean ± s.e.m. Specific n numbers are indicated in the figure.

Fig. 4 |. Immunization of dams with commensal microorganisms conveys neonatal protection against pathogens.

a, Survival of pups born to ETEC-6- or _Pantoea-_1-immunized dams or unimmunized dams. Data are from two individual experiments (first experiment, ETEC n = 12 mice, unimmunized (non) n = 7 mice, Pantoea n = 4 mice; second experiment, ETEC n = 5 mice, unimmunized n = 5 mice, Pantoae n = 4 mice). b, Liver total bacterial burdens 3 days after intraperitoneal ETEC 6 challenge. **P = 0.0025, one-way analysis of variance (ANOVA) with Bonferroni post-test. Data are from two independent experiments. c, Spleen bacterial burdens 3 days after intraperitoneal ETEC 6 challenge. **P = 0.0041, one-way ANOVA with Bonferroni post-test. Data are from two independent experiments. d, Cross-reactivity against ETEC 6 of serum IgG from pups born to germ-free dams with or without Pantoea 1 immunization. e, Western blot showing that serum IgG of pups born to a _Pantoea-_1-immunized dam recognizes antigens in cellular lysates of members of the Enterobacteriaceae family (ETEC 6, Pantoea 1 and Enterobacter). Lane 1, Staphylococcus; lane 2, ETEC 6; lane 3, Pantoea 1; lane 4, Enterobacter. Blot is detected with goat anti-mouse IgG antibody. Data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1. b–d, Data are mean ± s.e.m.

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Microbiota-targeted maternal antibodies protect neonates from enteric infection - PubMed (original) (raw)