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Humoral regulation of iron metabolism by extracellular vesicles drives antibacterial response

Nature Metabolism volume 5, pages 111–128 (2023)Cite this article

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Abstract

Immediate restriction of iron initiated by the host is a critical process to protect against bacterial infections and has been described in the liver and spleen, but it remains unclear whether this response also entails a humoral mechanism that would enable systemic sequestering of iron upon infection. Here we show that upon bacterial invasion, host macrophages immediately release extracellular vesicles (EVs) that capture circulating iron-containing proteins. Mechanistically, in a sepsis model in female mice, Salmonella enterica subsp. enterica serovar Typhimurium induces endoplasmic reticulum stress in macrophages and activates inositol-requiring enzyme 1α signaling, triggering lysosomal dysfunction and thereby promoting the release of EVs, which bear multiple receptors required for iron uptake. By binding to circulating iron-containing proteins, these EVs prevent bacteria from iron acquisition, which inhibits their growth and ultimately protects against infection and related tissue damage. Our findings reveal a humoral mechanism that can promptly regulate systemic iron metabolism during bacterial infection.

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Fig. 1: Blockade of host EV release increases the susceptibility of mice infected with S. Typhimurium.
Fig. 2: Supplementation with serum EVs decreases S. Typhimurium infection severity in EV-release-deficient mice.
Fig. 3: Supplementation with EVs derived from BMDMs decreases infection severity in EV-deficient mice.
Fig. 4: TfR-, CD163- and CD91-bearing EVs derived from S. Typhimurium-infected serum or BMDMs bind iron.
Fig. 5: Host EV-induced hypoferremia protects against bacterial infection.
Fig. 6: S. Typhimurium infection induces host EV release via ERS-mediated lysosomal dysfunction.
Fig. 7: IRE1α-mediated lysosomal dysfunction enhances EV release during S. Typhimurium infection.
Fig. 8: A humoral mechanism that promptly regulates systemic iron metabolism during bacterial infection.

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Data availability

All the data supporting the findings of this study are available within this article and the Supplementary Information files. Other information of this study is available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant no. 82170925 to Shiyu Liu, no. 81991504 to Y.J. and no. 31800817 to Siying Liu) and National Key Research and Development Program of China (grant no. 2016YFC1101400 to Y.J.).

Author information

Author notes

  1. These authors contributed equally: Huijuan Kuang, Geng Dou.

Authors and Affiliations

  1. State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, Xi’an, PR China

    Huijuan Kuang, Geng Dou, Xiangdong Wang, Haokun Xu, Xuemei Liu, Feng Ding, Siying Liu, Lili Bao, Bei Li, Yan Jin & Shiyu Liu

  2. Department of Medical Microbiology and Parasitology, The Fourth Military Medical University, Xi’an, PR China

    Linfeng Cheng

  3. Department of Paediatric Dentistry, School of Stomatology, China Medical University, Shenyang, PR China

    Xuemei Liu & Yao Liu

  4. Stomatology Hospital, Southern Medical University, Guangzhou, PR China

    Xiaoshan Yang

  5. Department of Otolaryngology Head and Neck Surgery, Peking University Third Hospital, Beijing, PR China

    Huan Liu

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  1. Huijuan Kuang
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Contributions

H.K. and G.D. designed, performed and interpreted the experiments and wrote the manuscript. L.C. and X.W. performed bacterial experiments and characterized properties of liposome. H.X., X.L., F.D. and Y.L. assisted with animal experiments. X.Y. and Siying Liu performed the histopathological studies and collected data. L.B., H.L. and B.L. contributed to data analysis and interpretation. Shiyu Liu developed the original concept. Shiyu Liu and Y.J. conceived the study and supervised the experiments.

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Correspondence to Yan Jin or Shiyu Liu.

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Nature Metabolism thanks Frederik Verweij, Günter Weiss and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Yanina-Yasmin Pesch, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 The characterization of S.Tm-infected mice and serum EVs biodistribution in vivo.

a-c, The C57BL/6 J mice were intraperitoneally administrated with S.Tm and blood and tissues samples were collected for further analysis. a, The viable counts of S.Tm in the blood at 24 hours after infection. n = 6 mice. b, Serum (left), hepatic (middle), and splenic (right) iron levels in mice at 24 hours after infection. n = 6 mice. c, The concentration of EVs in serum within 24 hours after infection. n = 3 mice per time point. d, Ex vivo fluorescence images of various organs in mice systemically injected with DiR-labeled serum EVs. n = 3 mice. e, f, Confocal microscopy images showing the uptake of PKH26-labeled EVs (red) by F4/80+ cells (green) (e) or Macro+ cells (green) (f) in liver or spleen. Scale bar, 10 μm. n = 3 biologically independent samples. For a-c, data are presented as the mean ± s.d. For a-c, statistical significance was assessed by unpaired two-sided Student’s t-test.

Source data

Extended Data Fig. 2 The effect of EVs release blockade or EVs supplementation on the host defense response to S.Tm infection.

a-c, To evaluate the effects of EVs release blockade on iron homeostasis, uninfected or S.Tm-infected mice were pretreated with GW4869 to block EVs release. a, Western blot analysis of FPN1 and FTH1 expressions in liver or spleen. Experiments were repeated three times and representative images are shown. b, The iron levels in liver or spleen at 12 hours after S.Tm infection. n = 6 mice. c, The viable count of S.Tm in liver and spleen at 12 hours after S.Tm infection. n = 6 mice. d,e, GW4869-pretreated S.Tm-infected mice were injected with EVs derived from uninfected mouse serum (Serum EVs group) or EVs derived from S.Tm-infected mouse serum [Serum(S.Tm)-EVs group]. d, The viable count of S.Tm in the liver and spleen. n = 5 mice. e, Representative fluorescence images of LPS (red) in the liver (upper) and spleen (bottom) and quantitative analysis of the percentage of LPS + area in liver or spleen cells. Scale bar, 50 μm. n = 3 biologically independent samples. For b-e, data are represented as the mean ± s.d. For b-e, statistical significance was assessed by one-way ANOVA with Tukey’s post-hoc test.

Source data

Extended Data Fig. 3 S.Tm infection affects iron homeostasis in BMDM.

a, Flow cytometric analysis of the expressions of macrophage surface marker F4/80 and CD11b on BMDM. b, c, BMDM were treated with S.Tm in the absence or presence of GW4869 for 24 hours to determine the iron homeostasis. b, Western blot analysis of expressions of iron-related proteins TfR, CD91, CD163, FTH1, and FPN1. Experiments were repeated three times and representative images are shown. c, The iron content in BMDM. n = 5 biologically independent samples. For c, data are represented as the mean ± s.d. For c, statistical significance was assessed by one-way ANOVA with Tukey’s post-hoc test.

Source data

Extended Data Fig. 4 EVs derived from S.Tm-infected BMDM decreased iron level and bacterial number in blood.

a,b, The infected BMDM-derived EVs [BMDM(S.Tm)-EVs group] and infected BMDM-derived EVs pretreated with excess holo-transferrin [BMDM(S.Tm)-EVs+holo-Tf group] were injected into GW4869-pretreated infected mice respectively. a, The iron level in serum. n = 4 mice. b, Viable count of S.Tm in blood. n = 4 mice. For a,b, data are represented as the mean ± s.d. For a,b, statistical significance was assessed by one-way ANOVA with Tukey’s post-hoc test.

Source data

Extended Data Fig. 5 TfR-, CD163-, CD91-bearing EVs derived from S.a-infected mice or BMDM bind iron.

a-c, Wild-type mice was intraperitoneally injected with S.a and the serum were collected after 24 hours. BMDM were infected with S.a for 24 hours at a MOI of 25 and supernatant was collected. EVs were isolated from the serum or supernatant. a, Western blot analysis of TfR, CD163, CD91 and TSG101 expression in EVs derived from uninfected and infected mouse serum (left), and in EVs released from uninfected or infected BMDM (right). Experiments were repeated three times and representative images are shown. b, Immunoelectron microscopy detection for TfR, CD163, and CD91 antibodies in EVs derived from infected mouse serum (upper) or infected BMDM (bottom). Scale bar, 50 nm. Experiments were repeated twice and representative images are shown. c, Schematic diagram of the overall design of the experiments to test the ability of EVs to bind iron in serum. EVs derived from uninfected mouse serum (Serum EVs group) or S.a-infected mouse serum [Serum (S.a)-EVs group] were added to the serum respectively and then removed by ultracentrifugation. The serum supernatant was collected for further analysis. d, e, Total iron levels (d) and transferrin levels (e) in the serum supernatant after incubation with EVs derived from uninfected or infected mouse serum. n = 3 biologically independent samples. f, Growth of S.a in serum supernatant. n = 5 biologically independent samples. For d-f, data are presented as the mean ± s.d. For d and e, statistical significance was assessed by one-way ANOVA with Tukey’s post-hoc test. For f, statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test and Kruskal-Wallis test.

Source data

Extended Data Fig. 6 HKS.Tm treatment enhances EVs release and expression of iron-related receptors.

a, b, Wild-type mice were treated with HKS.Tm for 24 hours, and serum EVs were isolated. a, The concentration of EVs in serum. n = 6 mice. b, Western blot analysis of iron-related receptors TfR, CD91, and CD163 expressions on EVs. Experiments were repeated three times and representative images are shown. For a, data are presented as the mean ± s.d. For a, statistical significance was assessed by unpaired two-tailed Student’s t-test.

Source data

Extended Data Fig. 7 EVs derived from macrophages protect against bacterial infection.

a, Representative dot plots of mouse spleen macrophages analyses at 48 hours after treatment with liposome-encapsulated clodronate or PBS. The graph showing the quantitative analysis of the percentage of F4/80+/MHCII+ macrophages in the spleen. n = 3 mice. b-e, To explore the role of macrophage EVs release in host EVs level regulation during infection, macrophage-depleted mice were injected intravenously with an equal volume of PBS, BMDM, or Rab27a shRNA-transfected BMDM for 36 hours, followed by S.Tm infection. b, The concentration of EVs in serum at 12 hours after S.Tm infection. n = 5 mice. c, The iron level in serum at 12 hours after S.Tm infection. n = 5 mice. d, Viable count of S.Tm in the blood at 12 hours after S.Tm infection. n = 5 mice. e, Survival rates of mice. n = 6 mice. f, To determine whether macrophage is the major source of serum EVs induced by HKS.Tm, macrophage-depleted mice were treated with an equal volume of PBS or BMDM for 36 hours, followed by HKS.Tm treatment. The graph showed the concentration of EVs in serum. n = 5 mice. For a-d and f, data are presented as the mean ± s.d. For a, statistical significance was assessed by unpaired two-tailed Student’s t-test. For b-d and f, statistical significance was assessed by one-way ANOVA with Tukey’s post-hoc test. For e, statistical significance was performed using the log-rank test.

Source data

Extended Data Fig. 8 S.a infection induces EVs release via endoplasmic reticulum stress.

a, Representative fluorescence images of ROS in BMDM infected with S.a at a MOI of 25, and quantitative analysis of the ROS levels in cells. Scale bar, 50 μm. n = 3 biologically independent samples. (b-g), BMDM were infected with S.a in the absence or presence of 4-PBA to clarify the association between ERS and EVs release. b, TEM images showed the ultrastructural morphology of the endoplasmic reticulum in BMDM. Scale bar, 1 μm. High-magnification images of the area marked by yellow boxes are arrayed at the lower panel. White arrow indicated endoplasmic reticulum. c, Western blot analysis of the expression of IRE1α, ATF4, ATF6 and GRP78 in BMDM. In b and c, experiments were repeated three times and representative images are shown. d, Representative fluorescence images of GRP78 in BMDM, and quantitative analysis of the fluorescence intensity Scale bar, 10 μm. n = 3 biologically independent samples. e, Representative fluorescence images of intracellular Ca2+ level in cells, and quantitative analysis of the Ca2+ levels. Scale bar, 4 μm. n = 3 biologically independent samples. f, Intracellular Ca2+ levels measured by fluorescence intensity. n = 3 biologically independent samples. g, The concentration of EVs in supernatant. n = 3 biologically independent samples. For a and d-g, data are presented as the means ± s.d. For a, statistical significance was analyzed by unpaired two-tailed Student’s t-test. For d-g, statistical significance was analyzed by one-way ANOVA with Tukey’s post-hoc test.

Source data

Extended Data Fig. 9 The biodistribution of 4-PBA-encapsulated liposome.

a, Ex vivo fluorescent images of various organs in mice injected with RhB-labeled 4-PBA-encapsulated liposome. n = 3 mice. b,c, The uptake of RhB -labeled liposome (red) by macrophages in liver (b) or in vitro cultured BMDM (c) stained with Hoechst (nucleus; blue) and F4/80 (cell membrane; green). Scale bar, 10μm. n = 3 biologically independent sample.

Extended Data Fig. 10 Rab31 regulates the enrichment of iron-related receptors onto EVs.

a, BMDM were treated with S.Tm in the absence or presence of 4-PBA for 24 hours, then EVs were isolated. The TfR, CD91, CD163, and TSG101 expressions on EVs were detected by Western blot. b-d, BMDM were pretreated with Rab31-siRNA and then infected with S.Tm. EVs from BMDM were isolated at 24 hours after S.Tm infection. The Rab31 expressions in BMDM (b) and TfR, CD91, CD163, and TSG101 expressions on EVs (c) were detected by Western blot. d, The concentration of EVs released by BMDM. n = 3 biologically independent samples. e, BMDM were treated with S.Tm in the absence or presence of 4-PBA for 24 hours. The IRE1α and Rab31 expressions in BMDM were detected by Western blot. f-i, BMDM were pretreated with IRE1α-siRNA (f,h) or toyocamycin (g,i) respectively and then were infected with S.Tm. The IRE1α and Rab31 expressions in BMDM (f, g), and TfR, CD91, CD163, and TSG101 expressions on EVs from BMDM (h,i) were analyzed by Western blot. For d, data are presented as the means ± s.d. For d, statistical significance was analyzed by one-way ANOVA with Tukey’s post-hoc test. All western blots were repeated three times and representative images are shown.

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Kuang, H., Dou, G., Cheng, L. et al. Humoral regulation of iron metabolism by extracellular vesicles drives antibacterial response. Nat Metab 5, 111–128 (2023). https://doi.org/10.1038/s42255-022-00723-5

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