Research ArticleMACROPHAGES

IRF5 guides monocytes toward an inflammatory CD11c+ macrophage phenotype and promotes intestinal inflammation

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Science Immunology  22 May 2020:
Vol. 5, Issue 47, eaax6085
DOI: 10.1126/sciimmunol.aax6085
  • Fig. 1 IRF5 deficiency has limited impact on colon physiology at steady state.

    (A) Representative hematoxylin and eosin (H&E) sections of colons from WT (left) and Irf5−/− (right) mice at steady state. (B) Histopathology scoring of WT (n = 6) and Irf5−/− (n = 5) colons. (C) Number of cLP lymphocytes (cLPLs) retrieved from steady-state WT (n = 9) and Irf5−/− (n = 5) mice. (D) IRF5 expression in the steady-state cLP of WT mice (n = 3). MFI, mean fluorescence intensity. (E) The frequency of intestinal MNPs in the cLP of steady-state WT (n = 9) and Irf5−/− (n = 5) mice. (F) Quantification of early phase (annexin V+ Live/Dead) and late phase (annexin V+ Live/Dead+) cell death assessed by annexin V labeling combined with viability dye staining in WT (n = 3) and Irf5−/− (n = 3) cLP Ly6ChiMHCII+ (P2) monocytes and macrophages using flow cytometry immediately after cell isolation. (B, C, and E) Data are pooled from two independent experiments. (D and F) Data are representative of two independent experiments. (B and C) Mann-Whitney U test. (E and F) Two-way analysis of variance (ANOVA) with Sidak correction. Data presented are means ± SEM. ns, not significant. **P ≤ 0.01.

  • Fig. 2 IRF5 deficiency protects against intestinal inflammation.

    (A) Representative H&E sections of colons from WT (left) and Irf5−/− (right) mice at d21 Hh + αIL10R. (B) Histopathology scoring of WT and Irf5−/− colons. (C) Number of cLPLs retrieved from uninfected (u) and d21 Hh + αIL10R WT and Irf5−/− mice. (D) frequencies of IFNγ-, IL17A-, and IFNγ/IL17-producing CD4+ T cells in WT and Irf5−/− mice after 4 hours of culture with phorbol 12-myristate 13-acetate/ionomycin and brefeldin assessed by intracellular flow cytometry. (E) Spleen weights of WT and Irf5−/− mice at steady state and d21 Hh + αIL10R. (B and E) Data are representative of two independent experiments (u, n = 3; Hh + αIL10R n = 7). Two-way ANOVA with Tukey’s correction. (C and D) Data are representative of two independent experiments (WT u, n = 6; Irf5−/− u, n = 3; Hh + αIL10R, n = 7). Two-way ANOVA with Tukey’s correction. (F) The frequency of intestinal MNPs in the cLP at d21 Hh + αIL10R WT (n = 12) and Irf5−/− (n = 11) mice. (G) Representative H&E sections of colons from CX3CR1IRF5+ (left) and CX3CR1IRF5− (right) mice at d21 Hh + αIL10R. (H) Histopathology scoring of colons from CX3CR1IRF5+ (n = 11) and CX3CR1IRF5− (n = 11) mice at d21 Hh + αIL10R. (F and H) Data are pooled from two independent experiments. (F) Two-way ANOVA with Sidak correction and (H) unpaired t test. Data presented are means ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P < 0.0001.

  • Fig. 3 IRF5 has subtle effect on CD11c+intestinal macrophages at steady state.

    WT and Irf5−/− CD45+CD11b+SiglecFLy6GCX3CR1+ cells were sorted from the colons of five MBMC animals and subjected to scRNA-seq analysis. (A) Graph-based clustering (58) of equal numbers of WT and Irf5−/− cells (n = 4780 total) identified nine clusters of MNPs and one cluster of DCs. (B) The bar plots show the percentages of WT and Irf5−/− cells that were found in each cluster. KO, knockout. Panels (C) to (F) show the expression of DC cell type markers (C), genes expressed in Cd11c+ macrophages (D), genes expressed in Mrc1+ macrophages (E), and genes with associated with macrophage differentiation and activation (F).

  • Fig. 4 IRF5 promotes generation of CD11c+macrophages in inflamed colon.

    WT and Irf5−/− CD45+CD11b+SiglecFLy6GCX3CR1+ cells were sorted from the colons of three MBMC animals at d21 of Hh + αIL10R colitis and subjected to droplet-based single-cell transcriptomic analysis. (A) Graph-based clustering (58) of equal numbers of WT and Irf5−/− cells (n = 1106 total) identified four clusters of MNPs and four clusters of DCs. (B) The violin plots show the expression levels (x axes) of selected known cLP MNP and DC subpopulation markers in each of the identified clusters (y axes). (C) The bar plots show the percentages of WT and Irf5−/− cells that were found in each cluster.

  • Fig. 5 IRF5 defines an inflammatory MNP signature during colitis.

    WT and Irf5−/− P1 monocytes, P2 monocytes, and macrophages were sorted from three MBMC animals at d21 Hh + αIL10R. (A) The dot plot shows the expression [mean transcripts per million (TPM), n = 3 biological replicates] of selected genes found to be differentially expressed between WT and Irf5−/− cells at one or more stages of the monocyte waterfall. The significant changes (|fc| > 2, BH-adjusted P < 0.05) are indicated by the gray triangles. (B) Selected GO biological process categories that showed a significant enrichment (colored dots, gene set enrichment analysis, BH-adjusted P < 0.1) in at least one of the three Irf5 KO versus WT small-bulk RNA-seq comparisons. (C to E) Intracellular or extracellular flow cytometry labeling was used to quantify the expression of (C) TNFα, (D) pro–IL-1β, and (E) MHCII on WT versus Irf5−/− macrophages in MBMC uninfected (n = 3) and at d21 Hh + αIL10R colitis (n = 4). Two-way ANOVA with Sidak correction. Data from one representative experiment presented are means ± SEM. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

  • Fig. 6 IRF5 promotes monocyte to Cd11c macrophage differentiation during intestinal inflammation.

    WT and Irf5−/− monocytes and macrophages from the inflamed intestine (Fig. 4) were reclustered at higher resolution (fig. S10) and subjected to pseudotime analysis. (A) Embedding of the cells in the first three dimensions of a diffusion map shows the three differentiation trajectories (solid lines) identified by the Slingshot pseudotime algorithm (with the Ly6c2 monocytes assumed to represent the root state). (B) Expression of selected cell-type marker genes and genes associated with Cd11c (Itgax) and Mrc1 (Cd206) macrophages. (C) The bar plots show the percentages of WT and Irf5−/− cells that were found in each cluster. (D) The violin plots show the progression of the WT and Irf5−/− (KO) cells through pseudotime along the three identified trajectories [as shown in (A)]. Differences in the distribution of cells in pseudotime between the genotypes were assessed with a Kolmogorov-Smirnov tests (P values adjusted using the Bonferroni correction). The position of the cells in pseudotime is shown on top of the violin plots [cells colored by cluster as in (A)]. The position of the 50th quantiles is indicated by the bold vertical lines.

  • Fig. 7 CD11c+macrophages occupy a distinct colonic niche.

    (A) Representative images of immunofluorescent labeling of colonic sections at steady state. Individual channels visualize the distribution of F4/80+ (blue), CD206+ (green), and CD11c+ (red) cells within the structure of the colon. CD206+ macrophages (white arrow) and CD11c+ macrophages (*), as well as single-positive F4/80+ cells (#), can be detected in the merged image. Cell nuclei are labeled with Sytoxblue (gray). Scale bars, 50 μM. (B) Localization of double-positive CD11c+ F4/80+ (cyan) and CD206+ F4/80 (yellow) cells in steady state (n = 6) and colitic mouse colons (n = 6) by immunohistofluorescence. Separate channels based on overlap of staining were created. Cell nuclei were labeled with SYTOX blue (gray). A minimum of five sections per mouse were evaluated. Macrophages at the base of the crypts (white arrow), at the tips of the villi (*), interspersed within the villi (†), and at the muscularis mucosae membrane (#). Scale bars, 100 μM (in the overview images) and 50 μM (in the enlargement). (C) Schematic depiction of image quantification analysis. Localization of macrophages is assessed by their minimal distance (black arrow) to the tip of the villi (artificial luminal surface depicted in green), muscularis mycosae (red), and serous membrane (blue). (D) Quantification of minimal distance of CD206+ F4/80+ and CD11c+ F4/80+ cells in steady state and d21 Hh + αIL10R to the luminal surface. Minimal distance is presented as a percentage of the distance to the tip of the villi to the total distance between the luminal surface and serous membrane. Two-way ANOVA with Tukey’s multiple comparisons test. Data presented as means ± SEM. P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 8 IRF5 controls phenotype of CD11c+macrophages.

    (A to C) Comparison of IL-12p40, TNF, and IL-1β inflammatory cytokine expression in CD11c+ versus CD11c cLP macrophages assessed by intracellular flow cytometry in uninfected MBMC (n = 3) and d21 Hh + αIL10R (n = 4). One experiment. Two-way ANOVA with Tukey’s correction. Data presented are means ± SEM. *P ≤ 0.05, **P ≤ 0.01, and ****P < 0.0001. (D) The frequency of parent WT and Irf5−/− macrophages expressing CD11c or CD206 at d21 Hh + αIL10R colitis. Two-way ANOVA with Sidak correction. Data presented are means ± SEM from two independent experiments. ****P < 0.0001. (E) IRF5 expression in CD11c+ versus CD11c macrophages in MBMC assessed by intracellular flow cytometry. One representative experiment, uninfected n = 3, Hh + αIL10R n = 4. (F) Heat map of expression of selected genes in WT and Irf5−/− Cd11c macrophages from the inflamed cLP of the MBMCs (see Fig. 5). All of the genes shown were found to be significantly differentially expressed between the WT and Irf5−/− cells of this cluster (Wilcoxon tests, BH-adjusted P < 0.05). Asterisks (*) denote significant differential expression between the genotypes in the macrophage small-bulk RNA-seq data.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/47/eaax6085/DC1

    Materials and Methods

    Fig. S1. IRF5 deficiency and cecum physiology at steady state.

    Fig. S2. IRF5 deficiency and cecum in inflammation.

    Fig. S3. MBMC: Monocyte development in the bone marrow and blood.

    Fig. S4. scRNA-seq: Monocyte development in blood.

    Fig. S5. scRNA-seq: MNP populations in cLP at steady state.

    Fig. S6. scRNA-seq: IRF5 effect on blood monocytes and cLP MNPs at steady state.

    Fig. S7. scRNA-seq: MNP populations in inflamed colon.

    Fig. S8. scRNA-seq and bulk RNA-seq: Comparison of IRF5-dependent genes.

    Fig. S9. IRF5 in monocyte to macrophage differentiation.

    Fig. S10. scRNA-seq: Monocyte and macrophage populations in inflamed colon.

    Fig. S11. scRNA-seq: Gene expression in Cd11c versus Cd206 macrophages.

    Table S1. List of antibodies used for surface staining.

    Table S2. List of antibodies used for intracellular staining.

    Table S3. Raw data in Excel spreadsheet.

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. IRF5 deficiency and cecum physiology at steady state.
    • Fig. S2. IRF5 deficiency and cecum in inflammation.
    • Fig. S3. MBMC: Monocyte development in the bone marrow and blood.
    • Fig. S4. scRNA-seq: Monocyte development in blood.
    • Fig. S5. scRNA-seq: MNP populations in cLP at steady state.
    • Fig. S6. scRNA-seq: IRF5 effect on blood monocytes and cLP MNPs at steady state.
    • Fig. S7. scRNA-seq: MNP populations in inflamed colon.
    • Fig. S8. scRNA-seq and bulk RNA-seq: Comparison of IRF5-dependent genes.
    • Fig. S9. IRF5 in monocyte to macrophage differentiation.
    • Fig. S10. scRNA-seq: Monocyte and macrophage populations in inflamed colon.
    • Fig. S11. scRNA-seq: Gene expression in Cd11c versus Cd206 macrophages.
    • Table S1. List of antibodies used for surface staining.
    • Table S2. List of antibodies used for intracellular staining.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S3. Raw data in Excel spreadsheet.

    Files in this Data Supplement:

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