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.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
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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.).
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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.
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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
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
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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
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DOI: https://doi.org/10.1038/s41590-018-0211-2
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