Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Sex-hormone-driven innate antibodies protect females and infants against EPEC infection

Nature Immunology volume 19, pages 1100–1111 (2018)Cite this article

Subjects

Abstract

Females have an overall advantage over males in resisting Gram-negative bacteremias, thus hinting at sexual dimorphism of immunity during infections. Here, through intravital microscopy, we observed a sex-biased difference in the capture of blood-borne bacteria by liver macrophages, a process that is critical for the clearance of systemic infections. Complement opsonization was indispensable for the capture of enteropathogenic Escherichia coli (EPEC) in male mice; however, a faster complement component 3–independent process involving abundant preexisting antibodies to EPEC was detected in female mice. These antibodies were elicited predominantly in female mice at puberty in response to estrogen regardless of microbiota-colonization conditions. Estrogen-driven antibodies were maternally transferrable to offspring and conferred protection during infancy. These antibodies were conserved in humans and recognized specialized oligosaccharides integrated into the bacterial lipopolysaccharide and capsule. Thus, an estrogen-driven, innate antibody-mediated immunological strategy conferred protection to females and their offspring.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Sex-biased differences in the capture of circulating E. coli by Kupffer cells.
Fig. 2: Antibody supports fast bacterial capture by Kupffer cells in females.
Fig. 3: Preexisting antibodies to E. coli IgM and IgG3 are prevalent in females.
Fig. 4: Preexisting anti–E. coli antibody is microbiota/exogenous antigen independent.
Fig. 5: Estrogen elicits production of innate anti–E. coli.
Fig. 6: Innate anti–E. coli IgG is maternally transferrable.
Fig. 7: Estrogen-driven anti–E. coli antibodies recognize specialized O antigens.
Fig. 8: Peritoneal B1 cells are the major anti-O127-producing cells.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Peleg, A. Y. & Hooper, D. C. Hospital-acquired infections due to gram-negative bacteria. N. Engl. J. Med. 362, 1804–1813 (2010).

    Article  CAS  Google Scholar 

  2. Laupland, K. B. Incidence of bloodstream infection: a review of population-based studies. Clin. Microbiol. Infect. 19, 492–500 (2013).

    Article  CAS  Google Scholar 

  3. Schroder, J., Kahlke, V., Staubach, K. H., Zabel, P. & Stuber, F. Gender differences in human sepsis. Arch. Surg. 133, 1200–1205 (1998).

    Article  CAS  Google Scholar 

  4. Flores-Mireles, A. L., Walker, J. N., Caparon, M. & Hultgren, S. J. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat. Rev. Microbiol. 13, 269–284 (2015).

    Article  CAS  Google Scholar 

  5. Klein, S. L. & Flanagan, K. L. Sex differences in immune responses. Nat. Rev. Immunol. 16, 626–638 (2016).

    Article  CAS  Google Scholar 

  6. Hickey, M. J. & Kubes, P. Intravascular immunity: the host-pathogen encounter in blood vessels. Nat. Rev. Immunol. 9, 364–375 (2009).

    Article  CAS  Google Scholar 

  7. Balmer, M. L. et al. The liver may act as a firewall mediating mutualism between the host and its gut commensal microbiota. Sci. Transl. Med. 6, 237ra266 (2014).

    Article  Google Scholar 

  8. Wong, C. H., Jenne, C. N., Petri, B., Chrobok, N. L. & Kubes, P. Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance. Nat. Immunol. 14, 785–792 (2013).

    Article  CAS  Google Scholar 

  9. Kubes, P. & Jenne, C. Immune responses in the liver. Annu. Rev. Immunol. 36, 247–277 (2018).

    Article  CAS  Google Scholar 

  10. Helmy, K. Y. et al. CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens. Cell 124, 915–927 (2006).

    Article  CAS  Google Scholar 

  11. Zeng, Z. et al. CRIg functions as a macrophage pattern recognition receptor to directly bind and capture blood-borne Gram-positive bacteria. Cell Host Microbe 20, 99–106 (2016).

    Article  CAS  Google Scholar 

  12. Jalan, R. et al. Bacterial infections in cirrhosis: a position statement based on the EASL Special Conference 2013. J. Hepatol. 60, 1310–1324 (2014).

    Article  Google Scholar 

  13. Ochoa, T. J. & Contreras, C. A. Enteropathogenic Escherichia coli infection in children. Curr. Opin. Infect. Dis. 24, 478–483 (2011).

    Article  Google Scholar 

  14. Kotloff, K. L. et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382, 209–222 (2013).

    Article  Google Scholar 

  15. Welsher, K., Sherlock, S. P. & Dai, H. Deep-tissue anatomical imaging of mice using carbon nanotube fluorophores in the second near-infrared window. Proc. Natl Acad. Sci. USA 108, 8943–8948 (2011).

    Article  CAS  Google Scholar 

  16. Ramirez-Ortiz, Z. G. et al. The scavenger receptor SCARF1 mediates the clearance of apoptotic cells and prevents autoimmunity. Nat. Immunol. 14, 917–926 (2013).

    Article  CAS  Google Scholar 

  17. Taborda, C. P. & Casadevall, A. CR3 (CD11b/CD18) and CR4 (CD11c/CD18) are involved in complement-independent antibody-mediated phagocytosis of Cryptococcus neoformans. Immunity 16, 791–802 (2002).

    Article  CAS  Google Scholar 

  18. Koch, M. A. et al. Maternal IgG and IgA antibodies dampen mucosal T helper cell responses in early life. Cell 165, 827–841 (2016).

    Article  CAS  Google Scholar 

  19. Zeng, M. Y. et al. Gut microbiota-induced immunoglobulin G controls systemic infection by symbiotic bacteria and pathogens. Immunity 44, 647–658 (2016).

    Article  CAS  Google Scholar 

  20. Yurkovetskiy, L. et al. Gender bias in autoimmunity is influenced by microbiota. Immunity 39, 400–412 (2013).

    Article  CAS  Google Scholar 

  21. Markle, J. G. et al. Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity. Science 339, 1084–1088 (2013).

    Article  CAS  Google Scholar 

  22. Rickert, R. C., Rajewsky, K. & Roes, J. Impairment of T-cell-dependent B-cell responses and B-1 cell development in CD19-deficient mice. Nature 376, 352–355 (1995).

    Article  CAS  Google Scholar 

  23. Baumgarth, N. The double life of a B-1 cell: self-reactivity selects for protective effector functions. Nat. Rev. Immunol. 11, 34–46 (2011).

    Article  CAS  Google Scholar 

  24. Kearney, J. F., Patel, P., Stefanov, E. K. & King, R. G. Natural antibody repertoires: development and functional role in inhibiting allergic airway disease. Annu. Rev. Immunol. 33, 475–504 (2015).

    Article  CAS  Google Scholar 

  25. Pasare, C. & Medzhitov, R. Control of B-cell responses by Toll-like receptors. Nature 438, 364–368 (2005).

    Article  CAS  Google Scholar 

  26. Roopenian, D. C. & Akilesh, S. FcRn: the neonatal Fc receptor comes of age. Nat. Rev. Immunol. 7, 715–725 (2007).

    Article  CAS  Google Scholar 

  27. Georgountzou, A. & Papadopoulos, N. G. Postnatal innate immune development: from birth to adulthood. Front. Immunol. 8, 957 (2017).

    Article  Google Scholar 

  28. Tuanyok, A. et al. The genetic and molecular basis of O-antigenic diversity in Burkholderia pseudomallei lipopolysaccharide. PLoS Negl. Trop. Dis. 6, e1453 (2012).

    Article  CAS  Google Scholar 

  29. Raetz, C. R. & Whitfield, C. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635–700 (2002).

    Article  CAS  Google Scholar 

  30. Thomassin, J. L. et al. Both group 4 capsule and lipopolysaccharide O-antigen contribute to enteropathogenic Escherichia coli resistance to human α-defensin 5. PLoS One 8, e82475 (2013).

    Article  Google Scholar 

  31. Okabe, Y. & Medzhitov, R. Tissue-specific signals control reversible program of localization and functional polarization of macrophages. Cell 157, 832–844 (2014).

    Article  CAS  Google Scholar 

  32. Ng, L. G. et al. BAFF costimulation of Toll-like receptor-activated B-1 cells. Eur. J. Immunol. 36, 1837–1846 (2006).

    Article  CAS  Google Scholar 

  33. Morrow, A. L. & Rangel, J. M. Human milk protection against infectious diarrhea: implications for prevention and clinical care. Semin. Pediatr. Infect. Dis. 15, 221–228 (2004).

    Article  Google Scholar 

  34. Perlmann, P., Hammarström, S., Lagercrantz, R. & Campbell, D. Autoantibodies to colon in rats and human ulcerative colitis: cross reactivity with Escherichia coli O:14 antigen. Proc. Soc. Exp. Biol. Med. 125, 975–980 (1967).

    Article  CAS  Google Scholar 

  35. Bryson, S. et al. Structures of preferred human IgV genes-based protective antibodies identify how conserved residues contact diverse antigens and assign source of specificity to CDR3 loop variation. J. Immunol. 196, 4723–4730 (2016).

    Article  CAS  Google Scholar 

  36. Thomson, C. A., Little, K. Q., Reason, D. C. & Schrader, J. W. Somatic diversity in CDR3 loops allows single V-genes to encode innate immunological memories for multiple pathogens. J. Immunol. 186, 2291–2298 (2011).

    Article  CAS  Google Scholar 

  37. Kim, K. S. et al. Dietary antigens limit mucosal immunity by inducing regulatory T cells in the small intestine. Science 351, 858–863 (2016).

    Article  CAS  Google Scholar 

  38. Mickiewicz, B. et al. Development of metabolic and inflammatory mediator biomarker phenotyping for early diagnosis and triage of pediatric sepsis. Crit. Care 19, 320 (2015).

    Article  Google Scholar 

  39. Shrum, B. et al. A robust scoring system to evaluate sepsis severity in an animal model. BMC Res. Notes 7, 233 (2014).

    Article  Google Scholar 

  40. Quan, S., Hiniker, A., Collet, J. F. & Bardwell, J. C. Isolation of bacteria envelope proteins. Methods Mol. Biol. 966, 359–366 (2013).

    Article  CAS  Google Scholar 

  41. Westphal, O. Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5, 83–91 (1965).

    CAS  Google Scholar 

Download references

Acknowledgements

We thank T. Nussbaumer, W.-Y. Lee and K. Poon for technical assistance; J. Deniset, S. Fassl and C. Deppermann for critical reading and advice; J. Kearney and P. Patel for providing valuable information and help; T. Mak (University of Toronto, Toronto, Canada) for providing Fcmr−/− mice; R. M. Medzhitov (Yale University) for providing Lyz2-Cre × Gata6-floxed mice; B. Yipp (University of Calgary) for providing CD19−/− mice; Z. Tian and Q. Zhang (University of Science and Technology of China, Hefei, China) for providing Tlr2−/−; Tlr4−/−; Tlr9−/− serum samples; K. Poon (University of Calgary) for providing S. Typhimurium; S. Lewenza (University of Calgary) for providing P. aeruginosa; and J. Wong, the Critical Care Epidemiologic and Biologic Tissue Resource (CCEPTR tissue bank), Alberta Sepsis Network (ASN) and Snyder Biobanking Resource Laboratory for assistance in obtaining human serum. This work was supported by the Snyder Mouse Phenomics Resources Laboratory and Live Cell Imaging Facility, both of which were funded by the Snyder Institute for Chronic Diseases at the University of Calgary. This work was funded by grants from Alberta Innovates Health Solutions (Z.Z., B.G.J.S. and P.K.), the Canadian Institutes of Health Research (P.K.) and the Canada Research Chairs Program (C.N.J. and P.K.).

Author information

Authors and Affiliations

  1. Calvin Phoebe & Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

    Zhutian Zeng, Bas G. J. Surewaard, Connie H. Y. Wong, Christopher Guettler, Regula Burkhard, Madeleine Wyss, Kathy D. McCoy, Craig N. Jenne & Paul Kubes

  2. Department of Physiology and Pharmacology Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

    Zhutian Zeng, Bas G. J. Surewaard, Connie H. Y. Wong, Christopher Guettler, Kathy D. McCoy & Paul Kubes

  3. Department of Medical Microbiology, University Medical Centre, Utrecht, the Netherlands

    Bas G. J. Surewaard

  4. Department of Microbiology, Immunology & Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

    Bjӧrn Petri, Regula Burkhard, Madeleine Wyss, Rebekah Devinney, Kathy D. McCoy, Craig N. Jenne & Paul Kubes

  5. Mouse Phenomics Resource Laboratory, Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

    Bjӧrn Petri

  6. Department of Microbiology & Immunology, Microbiome and Disease Tolerance Centre, Faculty of Dentistry, McGill University, Montreal, Quebec, Canada

    Hervé Le Moual

  7. Department of Pediatrics and Emergency Medicine, University of Calgary, Calgary, Alberta, Canada

    Graham C. Thompson & Jaime Blackwood

  8. Division of Pediatric Critical Care Medicine, Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada

    Ari R. Joffe

Authors
  1. Zhutian Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bas G. J. Surewaard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Connie H. Y. Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christopher Guettler
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bjӧrn Petri
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Regula Burkhard
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Madeleine Wyss
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hervé Le Moual
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Rebekah Devinney
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Graham C. Thompson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jaime Blackwood
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ari R. Joffe
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Kathy D. McCoy
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Craig N. Jenne
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Paul Kubes
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Z.Z. designed and performed the experiments, analyzed the data and wrote the manuscript. B.G.J.S. performed serum immunoglobulin and LPS separation and assisted in experiments. C.H.Y.W. and C.G. performed some intravital imaging experiments. B.P. contributed to the whole-body-imaging experiments. R.B., M.W. and K.D.M. contributed to the study of GF and antigen-free mice. G.C.T., J.B. and A.R.J. contributed to the study of human infant serum samples. H.L.M. and R.D. contributed to the construction of bacterial strains. C.N.J. cosupervised the study. P.K. supervised the study and wrote the manuscript.

Corresponding authors

Correspondence to Craig N. Jenne or Paul Kubes.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Antibody supports fast bacterial capture by Kupffer cells.

(a) Intravital liver imaging analysis of E. coli capture by Kupffer cells in male WT mice treated with either clodronate-liposome or PBS-liposome 24 hours prior to infection. n = 3 mice per group. (b) Intravital liver imaging analysis of E. coli capture by Kupffer cells in male WT mice (n = 4), male C3−/− mice (n = 2) or male C3−/− mice that were pre-immunized with E. coli Xen14 (n = 4). (c) Intravital liver imaging analysis of capture of serum pre-coated E. coli Xen14 in C3−/− mice (n = 2 mice per group, except the C3−/− male serum group, where n = 1 as negative control). Each symbol represents an individual mouse (c), small horizontal lines (a, b) indicate the mean (±s.e.m.).

Supplementary Figure 2 Profiling the immunoglobulin subtypes of preexisting anti–E. coli antibodies.

(a) Flow-cytometric plot of serum anti-E. coli Xen14 IgM and IgG3 in WT mice. (b) ELISA showing the IgG1, IgG2a/c, IgG2b and IgG3 antibodies against E. coli Xen14 in the serum of WT mice. n = 5 mice per group. (c) Flow-cytometric plot of serum anti-E. coli IgA in female mice. (d) Flow-cytometry showing serum anti-E. coli IgM and IgG in age-matched adult female Balb/c and C57BL/6 mice; or (e) in age-matched adult female C57BL/6 mice that were housed in the SPF facility of the U of Calgary or of the Jackson Laboratories. (f) Flow-cytometric validation of IgG and IgM serum fractions that were isolated from sera of E. coli Xen14 immunized mice. (g) Intravital liver imaging analysis of E. coli Xen14 capture by Kupffer cells in C3−/− male mice that received either IgG or IgM serum fraction transfer. n = 3 mice per group. Intravital liver imaging analysis of E. coli capture by Kupffer cells in (h) C3−/−Fcegr1−/−Fcmr−/− female mice (n = 2), female mice treated with anti-Fcα/µR plus anti-CD16/32 blocking antibodies (n = 3) or C3−/− female controls (n = 2); (i) C3−/−C1q−/− mice (n = 3 of each sex) or C3−/− female mice. n = 3 (j) C3−/− female mice that were treated with anti-CD18 (block CR3/CR4) or isotype IgG. n = 5 mice. Each symbol represents an individual mouse (b, h-j); small horizontal lines (b, g) indicate the mean (±s.e.m.); *p<0.05, **p<0.01 (unpaired two-sided student t test). Pool data from two experiments in (i, j). Data are representative of at least three independent experiments (a-g).

Supplementary Figure 3 Preexisting anti–E. coli antibodies are T cell independent but B1 cell and TLR dependent.

Flow-cytometry showing serum anti-E. coli Xen14 IgM and IgG in (a) 8 week-old female WT or Tcrb−/− mice; (b) 8-10 week-old female WT or CD19−/− mice. (c) Flow-cytometric quantification of serum anti-E. coli IgM and IgG in 8-10 week old WT and Myd88−/− mice. n = 3 per group for each sex. (d) ELISA analysis showing serum anti-E. coli Xen14 lgG and IgM in male WT mice (n = 5), female WT mice (n = 3), Tlr2−/− mice (n = 5), Tlr4−/− mice (n = 4), Tlr9−/− mice (n = 4) and Tlr2−/−Tlr4−/−Tlr9−/− mice (n = 5). Each symbol represents an individual mouse (c, d); small horizontal lines (c, d) indicate the mean (±s.e.m.); ***p<0.01 (One-way ANOVA followed by Tukey’s test). Data are representative of three independent experiments (a, b).

Supplementary Figure 4 Innate anti–E. coli antibodies specifically recognize LPS-O127.

Flow-cytometry showing anti-E. coli Xen14 IgG and IgM in serum samples from WT female mice that were (a) pre-absorbed with EPEC 2348/69, EHEC, UPEC, C. rodentium (CR), S. typhimurium (ST) or P. aeruginosa (PA), or with PBS as a control; or (b) pre-absorbed with LPS extracted from EPEC Xen14 or EHEC strain. (c) ELISA showing anti-E. coli Xen14 IgG and IgM in serum samples from WT female mice that were pre-absorbed with LPS containing different O antigens. n = 5 mice. (d) Flow-cytometric quantification of serum IgG and IgM against WT EPEC E2348/69, E2348/69 ΔwaaL, E2348/69 ΔgfcA and E2348/69ΔwaaLΔgfcA in WT female mice. n = 4. (e) Immunoblot of membrane extracts from WT EPEC E2348/69 or EPEC E2348/69ΔwaaLΔgfcA using C3−/− female serum and anti-mouse IgG. Each symbol represents an individual mouse (c, d); *p<0.05, **p<0.01, ***p<0.001 (One-way ANOVA followed by Tukey’s test).

Supplementary Figure 5 Anti–LPS-O127 antibody recapitulates the anti–E. coli antibody phonotype.

(a) ELISA showing serum IgM and IgG against LPS O127 and LPS O111 in indicated mice. n = 5 per group. (b) ELISA showing serum IgM and IgG against LPS O127 in 8-10 week old female WT and Esr1−/− mice, n = 3 per group; or in (c) female C3−/− mice (n = 6) or ovariectomized C3−/− mice (n = 4). (d) ELISA analysis of serum anti-LPS-O127 and anti-LPS-O111 IgM (left panel) and IgG (right panel) in 4-week old WT mice that were fed with normal drinking water (n = 7 mice per sex) or LPS-O111 containing drinking water (1mg/L, n = 5 male mice, n = 4 female mice) for 4 consecutive weeks. Each symbol represents an individual mouse (a-d); small horizontal lines (a-d) indicate the mean (±s.e.m.); N.S., no significance, *p<0.05, **p<0.01, ***p<0.001 (unpaired two-sided student t test (b, c); One-way ANOVA followed by Tukey’s test (d)).

Supplementary Figure 6 Protective role of anti-O127 antibodies during EPEC bacteremia.

(a) Representative liver images of WT mice infected with E. coli Xen14 for 24 h. (b) The necrotic areas in the largest lobe of liver were quantified as the percentage to total area of that liver lobe. n = 11 per sex. (c) Serum ALT levels at 24 hours post-infection. n = 9 per sex. (d) Representative liver images of WT mice infected with E. coli E2348/69 for 24 hours. (e) Representative liver images of WT male mice infected with E2348/69 WT or E2348/69 ΔwaaLΔgfcA. The percentages of necrotic areas in the largest lobe of liver were quantified. n = 5 mice per group. (f) Serum ALT levels in WT mice at 24 hours post E2348/69 ΔwaaLΔgfcA infection (n = 11 per sex), or (g) post EHEC EDL933 infection (n = 4 per sex). (h) Survival analysis of WT female mice injected i.v. with 3.5 mg/kg body weight LPS-O111 or LPS-O127. n = 13 per group. (i) Survival analysis of C3−/− male (n = 4) and female mice (n = 5) infected with EHEC EDL933 (1×108CFU). (j) Survival analysis of WT mice infected with 1×108CFU (n = 5 per sex), 2×108CFU (n = 10 per sex) or 4×108CFU (n = 5) EPEC Xen14. Each symbol represents an individual mouse (b, c, e-g); small horizontal lines (b, c, e-g) indicate the mean (±s.e.m.); N.S., no significance, **p<0.01, ***p<0.001 (unpaired two-sided student t test (b, c, f, g), two-sided Logrank test (h, i, j). Pooled data from two (b, c, f, j) and three (h) independent experiments. Representative of two independent experiments were shown in a, d.

Supplementary Figure 7 Peritoneal macrophages promote anti-O127 production.

(a) Flow-cytometry and (b) ELISA showing serum anti-E. coli Xen14 IgG and IgM or anti-LPS O127 IgG levels in female Lyz2Cre+Gata6fl/fl mice (n = 6) and Cre- littermates (n = 5). (c) Flow-cytometry and (d) ELISA showing serum anti-E. coli Xen14 or anti-LPS O127 IgG and IgM levels in WT mice that were treated i.p. with clodronate-liposome at 5-week of age. Assay were performed at 9-week of age. n = 5 mice. (e) Cytometric bead array (CBA) assay showing IL-4, IL-5, IL-6, IL-10 and IL-13 levels in the supernatant of peritoneal macrophage cultures in the absence or presence of β-estradiol (E2) for 24 h. Cells were pooled from 5 female mice, n = 3 technical replicates. (e) CBA assay showing IL-10 levels in the supernatant of peritoneal cell or spleen cell cultures. Cells were pooled from 3 female mice, n = 3 technical replicates. (g) ELISA showing serum anti-LPS O127 IgM and IgG in C3−/− male mice that were treated with E2 plus anti-IL10 antibody, E2 plus isotype antibody or without treatment. n = 3 mice per group. (h) qPCR detection of Tnfsf13b and Tnfsf13 mRNA expression in peritoneal macrophages isolated from WT mice (left, n = 3 mice per sex), or in ex vivo E2-stimulated female peritoneal macrophages (right, n = 4 cell samples from vehicle treatment, n = 6 cell samples from E2 treatment). Each symbol represents an individual mouse (a, b, d, g) or individual replicate (e, f); small horizontal lines (a, b, d, g, h) indicate the mean (±s.e.m.); *p<0.05, **p<0.01, ***p<0.001 (unpaired two-sided student t test. Data are representative of two independent experiments (c, e, f).

Supplementary Information

Supplementary Text and Figures

Supplementary Figures 1–7

Reporting Summary

Supplementary Video 1 Kupffer cells play a dominant role in clearing circulating E. coli.

WT mice were treated i.v. with clodronate-liposome to deplete Kupffer cells or with PBS-liposome as control. Intravital liver imaging was performed in these mice to show the capture of E. coli Xen14 (Red, syto60 labeled, pseudocolor) by Kupffer cells (Blue, anti-F4/80 labeled, pseudocolor) in real time. Kupffer cell depletion resulted in completely absence of E. coli Xen14 capture in the liver. Videos were recorded from -1 to 20 minutes post E. coli Xen14 i.v. injection. Hepatocytes showed green autofluorescence. Scale bars, 100 µm. Experiments were repeated three times with similar results

Supplementary Video 2 Complement is indispensable for E. coli capture in males but not females.

Intravital liver imaging was performed in 8-week old female or male C3−/− mice to show the capture of E. coli (Red, syto60 labeled, pseudocolor) by Kupffer cells (Blue, anti-F4/80 labeled, pseudocolor) in real time. Whereas Kupffer cells in female C3−/− mice caught E. coli Xen14 very efficiently, Kupffer cells in male mice did not catch at all. Videos were recorded from -1 to 15 minutes post E. coli Xen14 i.v. injection. Scale bars, 100 µm. Experiments were repeated at least five times with similar results

Supplementary Video 3 Systemic dissemination of E. coli in C3−/− males.

Whole-body imaging of E. coli distribution in C3−/− female or male mice at 1 hour post E. coli Xen14 i.v. injection using MARS (multimodal animal rotation system). Bacteria accumulated mostly in the liver of C3−/− female mice but disseminated in male mice. E. coli Xen14 was bioluminescent and visualized as blue signals in the video. Experiments were repeated twice with similar results

Supplementary Video 4 Female serum transfer restores E. coli capture in C3−/− males.

C3−/− male mice were transferred i.v. with heat inactivated serum from C3−/− male mice, C3−/− female mice, WT male mice or WT female mice respectively. Intravital liver imaging was performed in recipient mice to show the capture of E. coli Xen14 (Red, syto60 labeled, pseudocolor) by Kupffer cells (Blue, anti-F4/80 labeled, pseudocolor) in real time. Female but not male serum transfer efficiently restored the bacterial capture in C3−/− mice. Scale bars, 100 µm. Experiments were repeated twice with similar results

Supplementary Video 5 Pre-pubertal female C3−/− mice do not capture E. coli by Kupffer cells.

Intravital liver imaging was performed in 8-week old or 4-week old female C3−/− mice to show the capture of E. coli Xen14 (Red, syto60 labeled, pseudocolor) by Kupffer cells (Blue, anti-F4/80 labeled, pseudocolor) in real time. Pre-pubertal female mice lost the ability to catch E. coli by Kupffer cells. Videos were recorded from -1 to 15 minutes post E. coli Xen14 i.v. injection. Scale bars, 100 µm. Experiments were repeated twice with similar results

Supplementary Video 6 Ovariectomy abolishes the bacterial capture ability of C3−/− females.

C3−/− female mice were subjected to sham or ovariectomy at 3-week old. Intravital liver imaging was performed at age of 8 weeks to show the capture of E. coli Xen14 (Red, syto60 labeled, pseudocolor) by Kupffer cells (Blue, anti-F4/80 labeled, pseudocolor) in real time. Ovariectomized C3−/− female mice totally lost the ability to catch E. coli by Kupffer cells when compared to sham control. Videos were recorded from -1 to 15 minutes post E. coli Xen14 i.v. injection. Scale bars, 100 µm. Experiments were repeated twice with similar results

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zeng, Z., Surewaard, B.G.J., Wong, C.H.Y. et al. Sex-hormone-driven innate antibodies protect females and infants against EPEC infection. Nat Immunol 19, 1100–1111 (2018). https://doi.org/10.1038/s41590-018-0211-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41590-018-0211-2

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing