through a 19-residue linker, which was only partially resolved in the structures and was observed
to associate with MHR4 and the C-terminal end
of the TRP domain.
A number of lipids were observed in our structures (fig. S9A). In addition to annular phospholipids, we identified three densities as CHS on
the basis of its characteristic shape (Figs. 3C
and 4B and fig. S9B). CHS is often used as a
cholesterol analog (27); thus, a cholesterol molecule is likely to bind at the same sites. One
CHS molecule fills a cleft at the back side of
the pore, interacting with the S6 helix and the
pore loop or the proposed selectivity filter of
one subunit and the pore helix of the neighboring subunit. This arrangement, together with
the relative rigidity of CHS over annular phospholipids, may stabilize the conformation of
the pore (Fig. 3C and fig. S8E). A second CHS
molecule is positioned at a location equivalent
to the vanilloid binding pocket in TRPV1 (6)
(fig. S9C). A third CHS is located at the pre-S1
elbow, which together with S1 and S4 creates
a cavity (Fig. 4B).
Although there is no noticeable change of the
pore between the two structures (Fig. 3B and
fig. S10, A and C), we observe subtle yet well-defined changes in the region surrounding the
Ca2+-binding site (fig. S10). In addition, CH2 is
horizontally displaced by ~2.5 Å around the
central coiled-coil, measured from the Ca of
Glu1116, in the distal end of CH2 (fig. S10D). The
rotation results in a slight tightening of the central coiled-coil (fig. S10E), likely stabilizing it, as
evident by the observation that more residues
in the C-terminal end of the coiled-coil were resolved in the CaCl2 structure (figs. S2G and 3G).
Given that the lower gate remains closed in both
structures, such conformational changes are insufficient for channel opening. However, as the
extensive soluble domains are implicated in interactions with various cofactors (28), such
conformational changes may be important functional features that enable the channel to better
detect or responds to its cofactors (28).
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We thank M. Diver, P. Dominik, A. Kintzer, and R. Stroud for
valuable discussions; M. Diver and E. Green for reading the
manuscript; and Y. Jiang for coordinating cosubmission of our
studies. H.E.A. is supported by a postdoctoral fellowship from
the Danish Council of Independent Research (grant DFF-5051-
00085). This work was supported by grants from the NIH
(R01NS047723 to D.J.; R01GM098672, S10OD020054, and
S10OD021741 to Y.C.). Y.C. is an investigator of the Howard
Hughes Medical Institute. Accession numbers for the human
TRPM4 structures in EDTA and CaCl2, respectively, are as follows:
6BQR and 6BQV (coordinates of atomic models; Protein Data
Bank), EMD-7132 and EMD-7133 (density maps; Electron
Microscopy Data Bank), and EMPIAR-10126 and EMPIAR-10127
(raw particle stacks; Electron Microscopy Public Image Archive).
Materials and Methods
Figs. S1 to S10
Tables S1 to S3
9 November 2017; accepted 27 November 2017
Published online 7 December 2017
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. Iliev1,2,6*
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.
Extensive studies on intestinal bacteria have demonstrated that alterations in the micro- biome have a dramatic impact on host im- munity 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 mam-
malian barrier surfaces (2). Intestinal fungal
dysbiosis has been shown to influence colitis, alco-
holic liver disease, and allergic lung disease (3–6),
providing evidence for its potential to influence
both local and distal inflammation. Moreover,
serum antibodies against Saccharomyces cerevisiae
mannan (ASCA) are elevated in several inflamma-
tory diseases, including Crohn’s disease (CD) (7–9).
Systemic ASCA can develop in response to intes-
tinal fungi (3, 7), providing a possible link between
the gut mycobiota and host immunity. Despite the
identification of receptors involved in the recog-
nition and immunity to intestinal fungi (3, 10), the
cell subsets that initiate and regulate mucosal im-
mune responses to the mycobiota remain unknown.
In the intestinal lamina propria (LP), several
subsets of phagocytes respond to bacterial infec-
tions or to fluctuations in the commensal bacterial
communities (11–13). Among those, mononuclear
phagocytes (MNPs), expressing the fractalkine re-
ceptor CX3CR1 (CX3CR1+ MNPs), and subsets
of dendritic cells (DCs) marked by differential
expression of the integrins CD11b and CD103 can
232 12 JANUARY 2018 • VOL 359 ISSUE 6372 sciencemag.org SCIENCE
1Gastroenterology 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.
*Corresponding author. Email: firstname.lastname@example.org