Nature - USA (2020-09-24)

592 | Nature | Vol 585 | 24 September 2020

Article

FOXP3+ T helper cells, in particular Helios−RORγt+ pTreg cells, in the
colon compared with sham-operated mice (Fig. 1c, d, Extended Data
Fig. 1e, f ). In addition to the reduction of colonic pTreg cells, there was
a marked decrease in the levels of Aldh1a1 and Aldh1a2, which encode
the retinoic acid-synthesizing enzymes RALDH1 and RALDH2, and in
aldehyde dehydrogenase activity in colonic APCs (Fig. 1e, f).
To identify the neurotransmitter responsible for conveying
signals from the enteric neurons to colonic APCs, we performed
RNA-sequencing analysis on APCs obtained from the spleen and
intestine. The gut APCs exhibited higher levels of Chrm1 mRNA, which
encodes the muscarinic acetylecholine receptor (mAchR), than splenic

APCs, suggesting a tissue-specific role for neurotransmitters in regulat-

ing intestinal APCs (Fig. 1g, Extended Data Fig. 1g). Of note, APC fractions

enriched in CX3CR1+ mononuclear phagocytes and CD103+ dendritic

cells share increased expression of Chrm1 as well as Aldh1a1 and Aldh1a2

compared with genes that define prototypical APC subsets, such as Itgae

(which encodes CD103), Cx3cr1 and Irf8 (Fig. 1h, i, Extended Data Fig. 1h).

We confirmed this finding by quantifying the expression of Aldh1a1 and

Aldh1a2 in colonic APCs stimulated with multiple neurotransmitters,

including acetylecholine, muscarine, adrenaline, neuropeptide Y, sub-

stance P, serotonin and neuromedin U (Fig. 1j). Furthermore, muscarine

and enteric neurospheroids induced Aldh1a1 and Aldh1a2 expression in

a

l

Gated on APCs:

CD45+CD3–B220–NK1.1–MHC-II+

b

c d

gj

k

TPM

06

MHC-II CD11c

CD45.2

68.9

CX3CR1–GFP

18.0 35.5

32.5

CD45+TCRβ–CD3–

B220–NK1.1– APC

f

ColonSpleen

Adra1a

Adra1b

Adra1d

Adra2a

Adra2b

Adra2c

Adrb1

Adrb2

Adrb3

Chrm1

Chrm2

Chrm3

Chrm4

Chrm5

Chrna2

Chrna3

Chrna4

Chrna5

Chrna6

Chrna7

Chrnb2

Chrnb3

Chrnb4

Htr1a

Htr1b

Htr1d

Htr1f

Htr2a

Htr2b

Htr2c

Htr3a

Htr3b

Htr4

Htr7

Npy1r

Npy2r

Npy4r

Npy5r

Npy6r

CX3CR1

β-tubulin III

e

Sham VGx

24.3 16.7

Aldeuor

Sham + DEAB

Sham

VGx + DEAB

VGx

h

Itgae

(CD103)

Chrm1

Irf8

Itgam

(CD11b)

Cx3cr1

Adrb2

Aldh1a1

Aldh1a2

CD11c SP

DP

CD11b SP

CD11b

Count

80

60

40

20

0

60

40

20

0

SP

Row min Row max

Cx3cr1

Itgam (CD11b)

Itgax (CD11c)

Itgae (CD103)

Irf4

Irf8

Batf3

DPCD11cSP

i

Human

Mouse WT mAChR TKO

ShamVGx ShamVGx ShamVGx ShamVGx

0

10

20

30

40

50

FOXP3

+ in CD4 cells (%)

P < 0.001

0

20

40

60

80

ROR

γt

+ in T

reg

cells (%)

P < 0.001

ShamVGx

0

10

20

30

40

ALDH

+ in MHC-II cells (%)

P < 0.001

ContAchMusAdreNPYSub P5HTNMUContAchMusAdreNPYSub P5HTNMU

0

10

20

Relative mRNA level

Aldh1a1

P < 0.001

P < 0.001

0

5

10

15

20

Aldh1a2

P < 0.001

P < 0.001

0

5

10

15

20

25

0

5

10

15

20

25

Relative mRNA level

Aldh1a1

P = 0.998

P = 0.005

P = 0.014

Aldh1a2

P = 0.998

P = 0.015

P = 0.018

Mus:+– –+

Mus:+– –+

0

10

20

Relative mRNA level

ALDH1A1

P = 0.008

0

5

10

15

ALDH1A2

P = 0.014

0

0.5

1.0

1.5

2.0

2.5

Aldh1a1

Relative mRNA level

P = 0.023

0

1

2

3

Aldh1a2

P = 0.006

Concentration

High

Low

Fig. 1 | Potential interaction between APCs and neurons in the gut.
a, Representative immunof luorescence of CX3CR1–GFP (green) and β-tubulin
III (red) in the mouse colon. Scale bar, 50 μm. b, Representative CD11c and
MHC-II staining of CD45.2+TCRβ− CD3−B220−NK1.1− colonic lamina propria
mononuclear cells from C x 3 cr1g fp mice. c–f, Eight-week-old male wild-type B6
mice were subjected to VGx or sham surgery. Colonic T cell phenotypes
and colonic gene expression were analysed 2 days later (n = 12 per group).
c, Frequency of FOXP3+ (Treg) cells among CD4+ T cells in colonic lamina propria.
d, Expression of RORγt in colonic FOXP3+ Treg cells. e, Expression of Aldh1a1 and
Aldh1a 2 mRNA in colonic APCs. f, Frequency of ALDH+ cells among MHC-II+
APCs (CD 45+TCRβ−CD3−B220−NK1.1−MHC-II+) in the colon. Left, histograms
of ALDH+ cells in APCs. Colonic mononuclear cells were incubated with
Aldef luor in the absence (filled) or presence (dotted line) of the ALDH inhibitor
diethylaminobenzaldehyde (DEAB). The percentage of Aldef luor+ cells
is shown above the horizontal line indicating the positive gate. Right,
quantification of ALDH+ cells. g, Heat map of the expression of genes encoding
neurotransmitter receptors, classified by sorted colonic and splenic APCs, as
determined by RNA-sequencing analysis. TPM, transcripts per million. h, Heat
map of macrophage and dendritic cell marker genes (gene product in

parentheses) for colonic CD11b+CD11c− (CD11b SP), CD11b+CD11c+ (DP) and

CD11b−D11c+ (CD11c SP) cells. The sorting strategy for the experiment is shown

in Extended Data Fig. 1h. Max, maximum; min, minimum. i, Ternary plot of

gene expression in colonic CD11b SP, DP and CD11c SP cells. The colour

scale indicates mRNA concentration. Neurotransmitter receptors and

representative markers for macrophage and dendritic cell are shown. j, Aldh1a1

and Aldh1a 2 mRNA expression in colonic APCs treated with PBS (control),

10 μM acetylcholine (Ach), 10 μM muscarine (Mus), 100 nM adrenaline (Adre),

100 μM neuropeptide Y (NPY), 100 nM substance P (Sub P), 10 μM serotonin

(5-HT) or 100 ng ml−1 neuromedin U (NMU) for 12 h (n = 5 per group). k, Aldh1a1

and Aldh1a 2 expression in wild-type (WT) and mAChR TKO colonic APCs.

Colonic APCs were isolated from wild-type or mAChR TKO mice and treated

with 10 μM muscarine or untreated for 12 h (n = 6 per group). l, ALDH1A1 and

ALDH1A2 mRNA levels in human colonic APCs. Colonic APCs were treated with

10 μM Mus or untreated for 12 h (n = 7 per group). Representative of three (a, b,

j–l) independent experiments or pooled from three independent experiments

(c–f). Data are mean ± s.e.m. P values by unpaired two-tailed Student’s t-test

(c–f, k) or one-way ANOVA with Tukey’s post hoc test (j–l).