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CX3CR1+ mononuclear phagocytes control immunity to intestinal fungi

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Science  12 Jan 2018:
Vol. 359, Issue 6372, pp. 232-236
DOI: 10.1126/science.aao1503
  • Fig. 1 CX3CR1 mononuclear cells express antifungal receptors and recognize fungi in the intestine.

    (A) RNA-seq analysis was performed on sorted CD11b+ CD103+ dendritic cells and CX3CR1+ mononuclear phagocytes. Shown is a volcano plot of P value versus fold-change (FC) comparing gene expression in the two cell subsets; red dots indicate a false discovery rate (FDR) of <0.05. (B) Logarithmic count per million [log(cpm)] normalization of genes involved in antigen presentation (left) or fungal recognition (right). (C) The expression of antifungal CLRs was confirmed by means of quantitative reverse transcription–PCR. (D) Representative flow cytometry histogram of dectin-1, dectin-2, and Syk expression among CD11b CD103+, CD11b+ CD103+, and CD11b+ CX3CR1+ cells in colons of WT mice. (E) Representative confocal imaging of the intake of C. albicans–red fluorescent protein (RFP) (red) by CX3CR1+ MNPs [green; CX3CR1+, 4′,6-diamidino-2-phenylindole+ (DAPI+)] and other cell types (blue; CX3CR1, DAPI+) in the intestine. Bar graphs represent mean ± SEM of individual mice (n = 4 to 7 mice), representative of at least two independent experiments. *P < 0.05, **P < 0.01, one-way analysis of variance (ANOVA).

  • Fig. 2 CX3CR1+ MNPs control gut antifungal immunity.

    Colonic LP cells were collected from ΔIrf4 mice (orange bars) or littermates (litt, gray bars) fed (C.a) or not (NT) with 5 × 107 C. albicans at day 10. (A and B) Expression of RAR-related orphan receptor–γt (RORγt) and interleukin-17 (IL-17) by CD4+ T cells (pooled from two independent experiments). Cd11c-Cre+/− CX3CR1DTR mice (ΔCX3CR1, green bars) or Cd11c-Cre−/− CX3CR1DTR littermates (litt, gray bars) were treated with DT and fed with C. albicans. (C and D) RORγt and IL-17 expression by the CD4+ T cells in the colon (pooled from three independent experiments). (E) IgG against the commensal C. tropicalis and flagellin. (F) Systemic IgG responses after intraperitoneal injection with C. albicans at day 1, 2, and 5 (pooled from two independent experiments). (G) C. albicans in the feces of control, ΔIrf4, and ΔCX3CR1 mice at day 10 (dots represent individual mice). ΔCX3CR1 mice and littermates were transferred with purified CD4+ Thy1.1+ OT-II cells, fed C. albicans–OVA, and euthanized after 10 days. (H) Representative plots of CD4+ Thy1.1+ OT-II cells proliferation in the colon (pooled from three independent experiments). Cx3cr1-Cre-ERT+/− Sykfl/fl mice (ΔSyk) or littermates (Litt) treated with tamoxifen and fed with C. albicans. (I) RORγt expression by CD4+ T cells in the colon (pooled from two independent experiments). (J) C. albicans in the feces at day 10. Dots represent individual mice. (K) IgG responses against C. tropicalis at day 10 (n = 5 mice per group). (L) Quantification of proliferating CFSE CD4+ Thy1.1+ OT-II cells in the colon (pooled from two independent experiments). Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney Test in (E), (G), (J), (K), and (L) and one-way ANOVA in (B), (C), (D), and (F).

  • Fig. 3 Depletion of CX3CR1+ MNP affects gut mycobiota and results in exacerbated intestinal disease.

    Feces from ΔCX3CR1 or WT littermates (Litt) mice were collected 7 days after administration of the first DT dose. (A) Nonmetric multidimensional scaling (NMDS) plot of distance ordination based on Bray-Curtis dissimilarities in the colon for fungal OTUs (WT n = 5 mice; ΔCX3CR1 n = 6 mice). (B) α diversity (Simpson diversity index) among the Ascomycota (left) and Basidiomycota (right) phyla. Data are pooled from two independent experiments and show mean ± SEM. Each circle denotes one mouse. (C) Weight change during DSS colitis in ΔCX3CR1 mice or control littermates after short-term treatment with fluconazole (Fl) or no treatment (NT) (pooled from two independent experiments). (D) Representative plots of neutrophil infiltration (CD11b+ Gr-1high) in the colon after DSS administration. (E) Weight change during DSS colitis in ΔCX3CR1 mice or control littermates fed with C. tropicalis (C.t). Data show mean ± SEM (litt n = 5 mice; ΔCX3CR1 n = 5 mice). (F) C. tropicalis colony-forming units (cfu) per gram in the feces of ΔCX3CR1 or control littermates at day 7. Dots represent individual mice, mean ± SEM. Systemic IgG responses against C. tropicalis were assessed by means of enzyme-linked immunosorbent assay (ELISA). Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney test in (B) and (F) and two-way ANOVA in (C) and (E).

  • Fig. 4 Polymorphisms in the coding region of the CX3CR1 gene are associated with decreased antifungal IgG responses in CD patients.

    (A) Representative images of the intake of fungal species (colored) by CX3CR1+ MNPs (gray) in the colon. (B) Association between the missense mutation rs3732378 and the systemic serologic markers anti-neutrophil cytoplasmic antibodies (anca), flagellin (cbir), Pseudomonas fluorescens–associated sequence I-2 (i2), and antibodies to S. cerevisiae IgG (igg.asca) among 503 CD patients. FA, frequency affected; FU, frequency unaffected; L95 and U95, lower and upper 95th confidence interval. (C) IgG ASCA and antiflagellin (cbir) IgG responses were assessed in the sera from rs3732378 homozygous (AA), heterozygous (AG), and control (GG) CD patients by means of ELISA. Dots represent individual patients, and bars represent mean. (D) IgG responses against different commensal fungi. C. albicans, Pichia kudrazevii, S. cerevisiae, Aspergillus amstellodamii, Wallemia sebi, and Malassezia restricta were assessed. Dots represent individual patients, and bars represent mean. Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney test in (D) and one-way ANOVA in (C).

Supplementary Materials

  • CX3CR1+ mononuclear phagocytes control immunity to intestinal fungi

    Irina Leonardi, Xin Li, Alexa Semon, Dalin Li, Itai Doron, Gregory Putzel, Agnieszka Bar, Daniel Prieto, Maria Rescigno, Dermot P. B. McGovern, Jesus Pla, Iliyan D. Iliev

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods 
    • Figures S1 to S22 
    • Tables S1 and S2 
    • References

    Images, Video, and Other Media

    Movie S1
    A confocal microscopy video showing a 3D rendering of the colonic lamina propria of Cx3cr1GFP/+ mice fed with C. albicans-RFP, as shown in Figure 1D. CX3CR1+ cells are shown in green, DAPI nuclear staining in blue, C. albicans-RFP in red.

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