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Fibroblastic reticular cells initiate immune responses in visceral adipose tissues and secure peritoneal immunity

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Science Immunology  10 Aug 2018:
Vol. 3, Issue 26, eaar4539
DOI: 10.1126/sciimmunol.aar4539
  • Fig. 1 Impact of FRC-specific Myd88 ablation on omental FALC organization.

    (A) Confocal microscopic analysis of omental FALCs in 8- to 10-week-old Ccl19-EYFP mice (top) and mice lacking MYD88 expression in FRCs (bottom) using the indicated markers. Scale bar, 100 μm. (B) Flow cytometric analysis of canonical FRC marker expression on CD45PDPN+EYFP+ cells from the omentum of the indicated mouse strains; isotype control staining is indicated in gray. ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule. (C to E) Enumeration of FRCs by flow cytometry. Representative dot plots of PDPN+EYFP+ cells (C) and frequency (D) and absolute numbers (E) of PDPN+EYFP+ cells in the omentum of the indicated mouse strains. (F) Quantification of omental FALC numbers and size in whole mount stains; number of FALCs per square millimeter (left) and FALC-covered area in the omentum (right). Enumeration of hematopoietic cells (G) and B cell subsets (H) in mice lacking MYD88 expression in FRCs or in Cre-negative littermates (Ctrl) by flow cytometric analysis. Data are from one representative sample with five to eight mice (A to C) or pooled data from two independent experiments [n = 5 to 8 mice per group, with bars in (D) to (H) representing mean values ± SEM]. Statistical analysis was performed using Student’s t test with *P < 0.05 and **P < 0.01.

  • Fig. 2 Expansion of omental FRCs and FALC remodeling after intraperitoneal exposure to bacterial antigen.

    (A to C) Confocal microscopic analysis of FALCs on day 4 after immunization with S. Typhi OmpC/F. (A) Microscopic structure of omental FALCs from Ccl19-EYFP mice (top) and mice lacking MYD88 expression in FRCs (bottom) using the indicated markers. Representative image from one of four mice per group. Scale bar, 100 μm. Number of FALCs per square millimeter (B) and percentage of FALC-covered area in the omentum (C). Relative frequency (D) and absolute numbers (E) of PDPN+EYFP+ cells in the omentum of the indicated mouse strains on day 4 after immunization with S. Typhi OmpC/F or in untreated mice (naive); bars represent mean values ± SEM. Enumeration of CD11b+ myeloid cells (F), B220+ B cells (G), and B cell populations (H) on day 4 after immunization in mice lacking MYD88 expression in FRCs or in Cre-negative littermates (Ctrl). Data in (B) to (H) represent mean values ± SEM and are from four to nine mice per group from two to three independent experiments; statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparison test (D and E) or Student’s t test (B, C, and F to H). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 RNA-seq transcriptional profiling of FRCs in the omentum.

    (A) MDS plot of the logCPM values of differentially expressed genes in at least one pairwise comparison between Myd88-proficient and Myd88-deficient Ccl19-EYFP+ FRCs under naive conditions and on day 4 after S. Typhi OmpC/F immunization. (B) Volcano plot indicating 110 differentially expressed genes between denoted FRC populations under naive conditions. Number of up- and down-regulated genes is indicated. Thresholds for differential gene expression, |FC| ≥ 2 and adjusted P ≤ 0.05, are indicated on a log2 and log10 scale, respectively. FC, fold change. (C) Heat map of genes related to innate sensing/inflammation, activation, and antigen presentation for indicated FRC populations under naive conditions. (D) Gene set enrichment analysis of FRC populations denoted with [A] and [B] after OmpC/F immunization. Nominal P values and false discovery rate (FDR)–corrected P values are shown for indicated gene sets with significance level FDR ≤ 0.1. (E) Heat maps of gene expression levels related to enriched pathways in (D). Data are pooled from two to five mice per group. Data represent logCPM values that have been row mean–centered and row-normalized (C and E). Statistical analysis was performed using gene-wise likelihood ratio tests for a negative binomial generalized linear model using edgeR (B) and competitive gene set tests (D), with a Benjamini-Hochberg (FDR) multiple comparison correction.

  • Fig. 4 Mechanisms of FRC-dependent myeloid cell recruitment.

    Flow cytometry–based enumeration of CD11b+F4/80Ly6G-Ly6C+ inflammatory monocytes in the omentum (A) and peritoneal cavity (B) in mice lacking MYD88 expression in FRCs or in Cre-negative littermates (Ctrl) at the indicated days after intraperitoneal immunization with S. Typhi OmpC/F. (C) Production of the inflammatory mediators TNF (left) and the chemokine CCL2 (right) by fibroblastic stromal cells isolated from the omentum of Myd88-proficient or Myd88-deficient mice. Data are representative of one of three independent experiments with at least triplicate measurements. nd, nondetected. (D and E) Recruitment of CD11b+ myeloid cells (D) and CD11b+F4/80Ly6G-Ly6C+ inflammatory monocytes (E) into the omentum of the indicated bone marrow chimeric mice on day 4 after immunization with S. Typhi OmpC/F. (F) Flow cytometric analysis of CD11b+ Ly6C+ CCR2+ monocytes in circulation of the indicated mice under naive conditions with representative dot plots is shown on the left. Data are shown as mean values ± SEM and are pooled from six to nine mice from three independent experiments (A and B) and from six to eight mice per group from two independent experiments (D to F); statistical analysis was performed using Student’s t test (A, B, and F) or one-way ANOVA with Dunnett’s multiple comparison test (C to E) with *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 Regulation of antibacterial B cell responses in the peritoneal cavity via MyD88 signaling in FRCs.

    Enumeration of IgM or IgG ASCs in the omentum (A) and peritoneal cavity (B) in mice lacking MYD88 expression in FRCs or in Cre-negative littermates (Ctrl) using ELISPOT analysis at the indicated days post-intraperitoneal (p.i.) immunization with S. Typhi OmpC/F. (C) Flow cytometry–based assessment of germinal center B cells in the indicated mouse strains after OmpC/F intraperitoneal immunization. Representative dot plot analysis on day 4 after immunization (left) and time course analysis of pooled data (right). (D and E) Serum titers of S. Typhi OmpC/F-specific IgM (D) and IgG (E) antibodies on day 4 after immunization. (F to H) C57BL/6 mice were treated with CCR2-specific depleting antibody (MC-21) or isotype control antibody (isotype), and B cell responses were analyzed at day 4 after immunization. (F) Flow cytometry–based assessment of germinal center B cells and enumeration of IgM or IgG ASC in the omentum (G) and peritoneal cavity (H). (I) ASC in the omentum of the indicated bone marrow chimeric mice on day 4 after immunization with S. Typhi OmpC/F. (J) Bacterial load in blood and liver of mice lacking MYD88 expression in FRCs or in Cre-negative littermates (Ctrl) on day 4 after intraperitoneal infection with 5 × 105 CFU of attenuated S. Typhimurium. Data are shown as mean values ± SEM and are pooled from 4 to 10 mice per group from two (D to H) or three (A to C, and I and J) independent experiments; statistical analysis was performed using Student’s t test (A to H, and J) or one-way ANOVA with Dunnett’s multiple comparison test (I) with *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 TNF-dependent communication between FALC FRCs and myeloid cells.

    C57BL/6 (B6) (A, B, and E to G) or Cc19-EYFP (C and D) mice were treated with the indicated fusion proteins 1 day before immunization with S. Typhi OmpC/F. (A and B) Accumulation of CD11b+F4/80Ly6GLy6C+ inflammatory monocytes in the omentum; relative frequency (A) and absolute numbers (B) of inflammatory monocytes in the omentum. (C) Frequency of PDPN+EYFP+ cells in the omentum after TNFR-Ig treatment as determined by flow cytometric analysis. (D) Number of FALCs per square millimeter (left) and percentage of FALC-covered area in the omentum (right). Enumeration of IgM and IgG ASCs in the omentum (E) and the peritoneal cavity (F) and flow cytometry–based analysis of germinal center–like B cells (G) on day 4 after immunization in TNFR-Ig-treated mice. (H to K) Assessment of CD11b+ myeloid cell (H) and inflammatory monocyte recruitment (I) and accumulation of germinal center B cells (J) and IgG-producing B cells (K) in the omentum of the indicated bone marrow chimeric mice on day 4 after S. Typhi OmpC/F immunization. Data are shown as mean values ± SEM and are pooled from 7 to 8 mice from three independent experiments (B, C, and E to G), from 4 to 6 mice from two independent experiments (C and D), and from 7 to 12 mice from three independent experiments (H to K); statistical analysis was performed using Student’s t test (C to G) or one-way ANOVA with Dunnett’s multiple comparison test (A, B, and H to K) with *P < 0.05, **P < 0.01, and ***P < 0.001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/26/eaar4539/DC1

    Fig. S1. Impact of FRC-specific MYD88 ablation on mesenteric FALCs.

    Fig. S2. OmpC/F immunization–induced immune responses in the omentum.

    Fig. S3. Quality control of FRC sorting for transcriptional profiling.

    Fig. S4. Mechanisms of FRC-dependent myeloid cell recruitment.

    Fig. S5. Regulation of antibacterial B cell responses in the peritoneal cavity via MYD88 signaling in FRCs.

    Fig. S6. Myeloid cell recruitment to omental FALCs under conditions of TNF and LT ablation.

    Table S1. Antibodies used in this study.

    Table S2. Raw data sets.

    Movie S1. Organization and interaction of FRCs in omental FALCs.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Impact of FRC-specific MYD88 ablation on mesenteric FALCs.
    • Fig. S2. OmpC/F immunization–induced immune responses in the omentum.
    • Fig. S3. Quality control of FRC sorting for transcriptional profiling.
    • Fig. S4. Mechanisms of FRC-dependent myeloid cell recruitment.
    • Fig. S5. Regulation of antibacterial B cell responses in the peritoneal cavity via MYD88 signaling in FRCs.
    • Fig. S6. Myeloid cell recruitment to omental FALCs under conditions of TNF and LT ablation.
    • Table S1. Antibodies used in this study.
    • Legend for movie S1

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    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Raw data sets.
    • Movie S1 (.mov format). Organization and interaction of FRCs in omental FALCs.

    Files in this Data Supplement:

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