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CX3CR1+ mononuclear phagocytes control immunity to intestinal fungi Irina Leonardi,1,2 Xin Li,1,2 Alexa Semon,1,2 Dalin Li,3 Itai Doron,1,2 Gregory Putzel,2 Agnieszka Bar,1,2 Daniel Prieto,4 Maria Rescigno,5 Dermot P. B. McGovern,3 Jesus Pla,4 Iliyan D. Iliev 1,2,6* Intestinal fungi are an important component of the microbiota, and recent studies have unveiled their potential in modulating host immune homeostasis and inflammatory disease. Nonetheless, the mechanisms governing immunity to gut fungal communities (mycobiota) remain unknown. We identified CX3CR1+ mononuclear phagocytes (MNPs) as being essential for the initiation of innate and adaptive immune responses to intestinal fungi. CX3CR1+ MNPs express antifungal receptors and activate antifungal responses in a Syk-dependent manner. Genetic ablation of CX3CR1+ MNPs in mice led to changes in gut fungal communities and to severe colitis that was rescued by antifungal treatment. In Crohn’s disease patients, a missense mutation in the gene encoding CX3CR1 was identified and found to be associated with impaired antifungal responses. These results unravel a role of CX3CR1+ MNPs in mediating interactions between intestinal mycobiota and host immunity at steady state and during inflammatory disease.
Leonardi et al., Science 359, 232–236 (2018)
Cytl1 Tmem60
Cox6a2 Il22ra2 Ly6d Samsn1 Lyz1 Cd74
5
0
4
CD11b+ CX3CR1+ CD103+
CX3CR1+ Cd74 H2−Eb1 H2−Aa H2−Ab1 Fcgr2b Ctse Traf6 H2−Oa Unc93b1 H2−DMb2 Fcer1g H2−DMb1 H2−DMa Ifi30
0 −4
*Corresponding author. Email: iliev@med.cornell.edu
8
1.5
Bcl10 Syk Clec4e Clec7a Malt1 Bak1 Tlr2 Atg5 Clec4n Slc22a19 Btk log(cpm) 0
Clec7a / Rlp32
Clec7a
Gastroenterology and Hepatology Division, Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medicine, New York, NY 10021, USA. 2The Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, New York, NY 10021, USA. 3The F. Widjaja Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. 4Faculty of Pharmacy, Department of Microbiology II, Universidad Complutense de Madrid, 28040 Madrid, Spain. 5Department of Experimental Oncology, European Institute of Oncology, I-20141 Milan, Italy. 6Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY 10065, USA.
10
logFC
ns
** *
1.0
0.5 0.0 CD11b- CD11b+ CX3 CD103+ CD103+ CR1+ 0.7
Clec4n / Rlp32
10
1
Fungal recognition
CD11b+ CD103+
Lyz2
FDR<0.05
Antigen presentation
0.6
ns
** **
0.5 0.4 0.3 0.2 0.1 0.0 CD11b- CD11b+ CX3 CD103+ CD103+ CR1+
Dectin-1
ns
Dectin-2
20μm Syk
12 January 2018
CX3CR1+
DAPI
5μm C. albicans-RFP
Clec4e / Rlp32
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).
providing evidence for its potential to influence both local and distal inflammation. Moreover, serum antibodies against Saccharomyces cerevisiae mannan (ASCA) are elevated in several inflammatory diseases, including Crohn’s disease (CD) (7–9). Systemic ASCA can develop in response to intestinal fungi (3, 7), providing a possible link between the gut mycobiota and host immunity. Despite the identification of receptors involved in the recognition and immunity to intestinal fungi (3, 10), the cell subsets that initiate and regulate mucosal immune responses to the mycobiota remain unknown.
−log10(PValue)
E
xtensive studies on intestinal bacteria have demonstrated that alterations in the microbiome have a dramatic impact on host immunity and contribute to several diseases of inflammatory origin. Fungi are present in the mammalian intestine (1–5), yet little is known about their ability to influence immune homeostasis. Recent advances in deep sequencing technologies have redefined our understanding of fungal communities (mycobiota) colonizing mammalian barrier surfaces (2). Intestinal fungal dysbiosis has been shown to influence colitis, alcoholic liver disease, and allergic lung disease (3–6),
0.0002
ns
ns
0.0001 0.0001 0.0000 0.0000 CD11b- CD11b+ CX3 CD103+ CD103+ CR1+
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In the intestinal lamina propria (LP), several subsets of phagocytes respond to bacterial infections or to fluctuations in the commensal bacterial communities (11–13). Among those, mononuclear phagocytes (MNPs), expressing the fractalkine receptor CX3CR1 (CX3CR1+ MNPs), and subsets of dendritic cells (DCs) marked by differential expression of the integrins CD11b and CD103 can initiate immunity and prime T helper 17 (TH17) responses to both commensal and pathogenic bacteria in the gut (11, 12, 14). Despite their well-described ability to respond to gut bacteria, their role in mucosal immunity to gut fungi remains unknown. To assess the in vivo ability of gut resident phagocytes to respond to fungi, we colonized mice with the opportunistic human fungal commensal Candida albicans and analyzed the changes in the surface expression of the costimulatory molecules. We found that colonization with C. albicans altered the surface expression of CD40 and CD86 among CX3CR1+ MNPs but not among the other subsets (fig. S1, A and B). We thus assessed the ability of CX3CR1+ MNPs to recognize intestinal fungi. We purified CX3CR1+ MNPs from the intestinal LP and compared their RNA-sequencing (RNA-seq) expression profile to those of CD11b+
GUT IMMUNITY