Research ArticleMicrobiome

Commensal bacteria contribute to insulin resistance in aging by activating innate B1a cells

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Science Translational Medicine  14 Nov 2018:
Vol. 10, Issue 467, eaat4271
DOI: 10.1126/scitranslmed.aat4271
  • Fig. 1 Aging induces accumulation and activation of monocytes in the PeC.

    (A) Shown is the gating strategy for myeloid cells (without granulocytes) in the PeC of young and aged mice. (B to H) Quantification of (B) monocytes, (C) dendritic cells (DC), (D) macrophages (Mφ), and (E) CCR2+Ly6C+ monocytes. (F to H) Aged mouse monocytes up-regulated key factors involved in 4BL cell conversion. Y axes show flow cytometry cell counts in individual mice (n = 8 to 10 per group, with each representative experiment reproduced at least three times). (I) Z-score heatmap of the change in the mRNA expression of selected genes in PeC Mφ, DC, and monocytes in aged compared to young mice (n = 4 per group; see also fig. S1, H and I). Only monocytes converted B1a cells into 4BL cells, as inferred by up-regulated surface expression of 4-1BBL and membrane (m) TNFα in CD5+CD19+ cells. (J to L) Sort-purified PeC Mφ, DC, and monocytes were cultured overnight with eFluor450-labeled B1 cells from young mice at a 1:1 ratio (n = 4 to 6 per group; the experiment was reproduced twice). Shown are representative flow cytometry data, with numbers showing the percentage of B1a cells expressing both 4-1BBL and TNFα (n = 5) (J) and its summary result for expression of 4-1BBL and TNFα in B1a cells (K and L). Data are represented as means ± SEM. P < 0.05, **P < 0.001, and ***P < 0.0001 [Mann-Whitney U test and Kruskal-Wallis test in (B) to (D), Dunn’s test for multiple corrections in (K) and (L)]. n.s., not significant.

  • Fig. 2 CCR2+ monocytes induce 4BL cell conversion in the omentum.

    (A) B1a cells expressed more 4-BBL than B2 and B1b cells. Shown is a representative histogram for 4-1BBL expressing B cell types in aged omentum (left) and absolute numbers of 4-1BBL+ B1a cells in the omentum of young and aged mice (right). Each symbol is for an independent mouse; n = 7 to 8 mice per group. (B) B220+ (B cells) and 4-1BBL+ cells colocalized with CD115+ cells (monocytes) in a whole-mount immunofluorescence staining of aged omentum. (C) Compared to young omentum, aged mouse omentum efficiently converted eFluor450-labeled B1 cells from young mice into 4BL cells in vitro. Shown is a representative flow cytometric plot (left) and summary result (right) of B1 cells stimulated with phosphate-buffered saline (PBS), young omenta, or aged omenta (n = 3 mice per group; the experiment was reproduced twice). (D and E) The 4BL cell conversion was induced by CCR2+ monocytes. After a 12-day treatment with control immunoglobulin G (IgG) or CCR2+ monocyte–depleting Ab (αCCR2), aged mice were adoptively intravenously transferred with 5 × 106 GFP+ B1 cells from young mice on day 13. Shown are (D) the number of host monocytes in PeC and omentum and (E) the number of donor GFP+CD19+ cells in PeC and omentum 4 days after B1 cell transfer (n = 5 mice per group; the experiment was reproduced three times). *P < 0.05 and **P < 0.001 (Kruskal-Wallis test with Dunn’s test for multiple corrections in (C) to (E) and Mann-Whitney test for all others).

  • Fig. 3 Enrofloxacin (Abx) treatment eliminates the accumulation of monocytes and 4BL cells in aged mice.

    (A to G) Young and aged mice (n = 7 to 9 per group) were treated with Abx (170 mg/liter) in drinking water for 3 to 4 months; representative results of experiments reproduced at least three times. The frequency (top) and absolute counts (bottom) of (A) monocytes, (B) 4BL cells, and (C) GrB+CD8+T cells in PeC were quantified. (D) and (E) show absolute counts of Ly6C+ monocytes and 4BL cells, respectively, in the omentum of aged mice. (F) Abx-treated mice were intravenously injected with GFP+ B1 cells from young mice to assess 4BL cell conversion in vivo after 5 days (n = 8 to 10 per group). (G) To evaluate the importance of monocytes in the 4BL cell conversion, flow-sorted PeC myeloid LinCD11b+ cells from mock- or Abx-treated young or aged mice were cultured overnight with eFluor450+ PeC B cells from young mice at a 1:1 ratio (n = 6 to 8 per group). (H) Abx treatment decreased 4-1BBL+CD20+ B cells in the peripheral blood of elderly macaques (each symbol represents one individual; n = 6 to 7 per group). (I) Abx treatment impaired the monocyte-mediated 4BL cell conversion in macaques. CD14+ monocytes from saline (mock)- or Abx-treated aged macaques were cultured overnight with eFluor450+ CD20+ cells from young macaques (n = 6 to 7 primates per group). *P < 0.05 and **P < 0.001 (Kruskal-Wallis test, Dunn’s test for multiple corrections).

  • Fig. 4 Aging gut microbiota initiates the monocyte–4BL cell induction.

    (A) The composition of gut microbiota was changed in aged mice. 16S rRNA gene sequencing results are shown as percent abundance of selected microbiota genera in murine feces (n = 8 mice per group). Note that y-axes lengths differ across plots. (B) A. muciniphila abundance inversely correlated with 4BL cells in PeC. Spearman correlations and associated P values are shown for each sample (n = 7 to 8 per group). (C) Gut microbiota of aged mice induced 4BL cells in 20-week-old germ-free mice. Shown are 4BL cells in the omentum and PeC of germ-free mice after weekly oral gavage with 25 μg of fecal suspension from SPF aged or young mice for 4 weeks (n = 4 to 5 mice per group, experiment was reproduced three times). (D) A representative image (left) and quantification (right) show that the thickness of colonic inner mucus layer was reduced in aged mice, which was reversed after replenishment with A. muciniphila (1 × 108 colony-forming units, with oral gavage every second day for 20 days). The yellow bar indicates the inner mucus layer. (E and F) Aged mice showed increased gut permeability, which was restored by gavage with A. muciniphila, as shown by serum leakage of FITC-dextran (E) or endotoxin (F; n = 5 to 7 per group; the experiment was reproduced twice). The increase in 4BL cells (G) and their downstream GrB+CD8+ T cells (H) in aged mice was not observed after A. muciniphila treatment (n = 6 to 8 per group; the experiment was reproduced three times). (I) Enrofloxacin (Abx) treatment (as in Fig. 3A) reversed gut permeability, as shown by serum endotoxin levels and (J) leakage of orally gavaged FITC-dextran (n = 8 per group). *P < 0.05, **P < 0.001, and ***P < 0.0001 (Kruskal-Wallis test with Dunn’s test for multiple corrections). EU, endotoxin unit.

  • Fig. 5 The role of butyrate in 4BL conversion.

    Compared to young mice, SCFAs (micromolar) in (A) cecum and (B) butyrate in PeC were markedly decreased in aged mice and after Abx treatment (n = 7 to 9 mice per group). Note that y-axes lengths differ across panels. Supplementation with butyrate (BA; oral gavage, 2.5 g/kg every second day for 4 weeks) decreased monocytes (C), 4BL cells (D), and GrB+CD8+ T cells (E) in PeC (n = 8 per group; the experiment was reproduced twice). (F and G) Two days after the termination of BA [as in (C) to (E)], mice were intravenously transferred with PeC GFP+ B1 cells from young mice to evaluate their conversion into 4BL cells after 5 days in PeC (F) and omentum (G; n = 6 to 9 per group; the experiment was reproduced twice). Data are represented as means ± SEM. *P < 0.05, **P < 0.001, and ***P < 0.0001 (Mann-Whitney test and Kruskal-Wallis test with Dunn’s test for multiple corrections).

  • Fig. 6 IR is increased in aged mice.

    (A to C) Measurements of (A) fasting plasma insulin concentrations (n = 38 to 46), (B) starting glucose in ITTs (n = 38 to 46), and (C) blood glucose concentrations in oral OGTTs (n = 38 to 46); each symbol indicates a single female C57BL/6 mouse. Plasma insulin levels were measured by enzyme-linked immunosorbent assay after overnight fasting. (D) Mice were injected with insulin, after which phosphorylation of the insulin receptor [pTyr, using immunoprecipitation (IP)] and AKT (pThr308 and pSer473) were quantified in liver lysates. (E to G) IR in aged mice is reversed by treatment with enrofloxacin (Abx), as measured by ITT (E) and OGTT (F; n = 7 to 9 per group, as in Fig. 3A) or (G) butyrate (BA; n = 8 to 10 per group, as in Fig. 5, C to G). Y axis in (E) to (G) shows area under the curve (AUC). Data are means ± SEM. *P < 0.05, **P < 0.001, and ***P < 0.0001 (Mann-Whitney test and Kruskal-Wallis test with Dunn’s test for multiple corrections).

  • Fig. 7 Gut dysbiosis in aging promotes IR via 4BL cells.

    (A) ITTs, (B) OGTTs, and (C) fasting plasma insulin show that IR in aged mice is reversed by the depletion of monocytes and B cells (n = 7 to 8 per group, as in Fig. 2D; “*,” #,” and “&” show for P < 0.05 for comparisons of the following groups of mice: young versus aged + IgG, aged + IgG versus aged + αCCR2, or aged + αCD20, respectively). (D) Sort-purified or B1a cells from young or aged mice were intravenously transferred into B cell–deficient JHT mice fed with an HFD for 4 months. ITT was performed 1 week after intravenous transfer (n = 10 per group; “#” is for P < 0.05 for comparisons of PeC B cells from young versus aged mice). (E and F) Aged mice were gavaged with A. muciniphila for 20 days, but at day 14, mice were intravenously transferred with sort-purified B1a cells from the PeC of WT or 4-1BBL KO aged mice (Aged + Akk + WT B1 or Aged + Akk + KO B1, respectively) to evaluate IR at days 20 to 22 (n = 8 per group, with the experiment reproduced three times; “*,” “#,” “&,” and “%” are for P < 0.05 for comparisons of the following groups of mice: young versus aged + mock, aged + mock versus aged + Akk, aged + Akk versus aged + Akk + WT B1, or aged + Akk + KO B1, respectively). (G) At day 24, mice in (E) and (F) were injected with insulin to quantify pTyr induction of the liver insulin receptor (as in Fig. 6D). Numbers show the average pTyr increase normalized to β-tubulin. (H) IR was also increased in aged macaques (M. mulatta, n = 6 to 7 per group), which was reversed by Abx treatment (as in Fig. 3, H and I). Data are means ± SEM. *P < 0.05, **P <0.001, and ***P < 0.0001 (Mann-Whitney test and Kruskal-Wallis test with Dunn’s test for multiple corrections).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/467/eaat4271/DC1

    Materials and Methods

    Fig. S1. Aging induces monocyte accumulation and activation in the PeC.

    Fig. S2. The PeC and ometum of aged mice are inflamed.

    Fig. S3. Aging induced 4BL cells in primates.

    Fig. S4. Aging substantially changed the gut microbiome.

    Fig. S5. A. muciniphila loss in aging induced gut leakiness and 4BL conversion.

    Fig. S6. Aging changed fatty acids in the intestine and feces.

    Fig. S7. Chronic LPS injection imitated aging-induced 4BL cell accumulation.

    Fig. S8. 4BL cells increased IR in aging.

    Fig. S9. Schema summarizing our findings.

    Table S1. Operational taxonomic units for several butyrate inducers were markedly reduced in aging of C57BL/6 mice.

    Table S2. Compared to middle-aged mice, aged C57BL/6 mice increase IR.

    Table S3. Raw data for the experiments.

    References (5874)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Aging induces monocyte accumulation and activation in the PeC.
    • Fig. S2. The PeC and ometum of aged mice are inflamed.
    • Fig. S3. Aging induced 4BL cells in primates.
    • Fig. S4. Aging substantially changed the gut microbiome.
    • Fig. S5. A. muciniphila loss in aging induced gut leakiness and 4BL conversion.
    • Fig. S6. Aging changed fatty acids in the intestine and feces.
    • Fig. S7. Chronic LPS injection imitated aging-induced 4BL cell accumulation.
    • Fig. S8. 4BL cells increased IR in aging.
    • Fig. S9. Schema summarizing our findings.
    • Table S1. Operational taxonomic units for several butyrate inducers were markedly reduced in aging of C57BL/6 mice.
    • Table S2. Compared to middle-aged mice, aged C57BL/6 mice increase IR.
    • References (5874)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Raw data for the experiments.

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