Nature - USA (2020-09-24)

594 | Nature | Vol 585 | 24 September 2020

Article

characterized the effects of HVx on the gut and the spleen. We observed
a significant reduction in the proportion of pTreg cells among CD4+
T cells as well as the expression and activity of aldehyde dehydrogenase
in APCs obtained from the large intestine of HVx and VGx mice com-
pared with sham-operated mice (Fig. 2e, Extended Data Fig. 4a–d). This
HVx-induced decrease in colonic pTreg cells occurred rapidly, by day 2
after surgery, and consistently across rodent sexes, strains and spe-
cies (Extended Data Fig. 4e–i). Since in vivo-generated pTreg cells show
demethylated Treg-specific demethylation regions, the rapid reduction
in pTreg cells in HVx mice could be attributed to the epigenetic effects
of the vagus nerve on the maintenance and stability of gut pTreg cells
caused by changing DNA methylation status at Treg-specific demethyla-
tion regions^29 –^34. Consistently, HVx impaired pTreg cell differentiation
and stability in T cell-reconstituted mice and unleashed the retinoic
acid-mediated repression of the T helper 17 (TH17) cell differentiation
program (Extended Data Fig. 4j–l). The key role of hepatic sensory
afferents in the left NG in maintaining the reservoir of gut pTreg cells
is further supported by the finding that perturbation of hepatic vagal
sensory afferents to the left NG by perivagal capsaicin treatment, but
not deafferentation of DRGs by intrathecal capsaicin or resiniferatoxin
treatment, resulted in reductions in colonic Treg cell numbers and alde-
hyde dehydrogenase activity in APCs (Extended Data Fig. 5). Indeed, left
vagal stimulation—but not right vagal stimulation—was essential for
maintaining intestinal pTreg cells and aldehyde dehydrogenase activity
in colonic APCs, suggesting functional asymmetry in the vagal afferents
(Fig. 2f, Extended Data Fig. 6a–c). Maintenance of colonic Treg cells is
less dependent on the sympathetic nervous system than maintenance
of muscularis macrophages and group 2 innate lymphoid cells (ILC2s),
as previously reported in the context of viral and parasitic infection^35 ,^36
(Extended Data Fig. 6d–h). In contrast to the effects in the small and
large intestines, HVx mice exhibited normal frequencies of splenic Treg
cells, whereas surgical ablation of the coeliac and superior-mesenteric

ganglion (CG-SMG) complex and chemical blockade of the β2 adren-

ergic receptor or the α7-nicotinic acetylecholine receptor (α7-nAchR)

resulted in a significant reduction in splenic Treg cells (Extended Data

Fig. 6i–n). As the splenic nerve, which mainly comprises adrenergic

fibres arising from CG-SMG, has previously been reported to supress

T cell activation and inhibit systemic cytokine production from the

splenic macrophages through α7-nAchR^37 –^42 , ablation of CG-SMG or

the splenic nerve rather than vagotomy itself was predicted to affect

the splenic Treg cell population. These results support the proposal that

the liver–brain–gut neural arc monitors the gut microenvironment and

tunes the levels of gut pTreg cells by sending acetylecholine signalling

and controlling colonic APCs.

This prompted us to investigate the role of muscarinic acetylecholine

signalling in intestinal APCs in vivo. Genetic ablation of mAChRs was

associated with decreased Aldh1a1 and Aldh1a2 expression in colonic

APCs, resulting in reduced numbers of pTreg cells in the colon (Fig. 3a–d).

In VGx and HVx mice, we observed fewer c-Fos+ enteric neurons in the

colonic myenteric plexus than in sham-operated mice, whereas the

expression of Hand2—which encodes a transcription factor required

for terminal differentiation of enteric neurons—was not affected

(Extended Data Fig. 7a–f ). In addition, intestinal small-molecule

and peptide neurotransmitters for parasympathetic (acetylecho-

line), but not sympathetic (noradrenaline) and sensory (calcitonin

gene-related peptide) systems, was decreased in VGx and HVx mice

compared with sham-operated mice (Extended Data Fig. 7g, h). Given

that hepatic-selective vagotomy and VGx primarily reduced local acety-

lecholine levels in the gut, we tested whether mAChR activation could

restore aldehyde dehydrogenase expression and activity in gut APCs.

Treatment with bethanechol, a mAChR agonist, restored Aldh1a1 and

Aldh1a2 expression in colonic APCs in HVx mice, whereas an α7-nAchR

agonist (GTS-21) and antagonist (MLA) had minimal effects (Extended

Data Fig. 8a–j), similar to genetic deletion of α7-nAchR^43. Consistently,

KO FOXP

3

KO ROR

γT

CD4

WT

23. 6

Sham HVx

21.1

c

39. 
    
    
    
    23. 5 25
    
    
    
    

50

75

0

WT KO

WT

FOXP3 in

CD4+ cells (%)

FOXP3 in

CD4+ cells (%)

RORγT+ in

Treg cells (%)

P = 0.003

P = 0.021

P = 0.891

d

Sham

WT

Helios

35. 04 8. 1

30.2 28. 0

47.1 47. 9

Sham HVx

47.8 31. 1

HVx

KO

HVx + bethanechol

WT

Sham

HVx

Sham

HVx

Sham

WT KO

Aldh1a2

P = 0.003

e

HVx

WT KO KO

P = 0.891

P = 0.001

KO

Aldh1a1

P < 0.001

P < 0.001

100

0

20

40

60

80

100

0

20

40

60

80

P < 0.001

P = 0.979

P = 0.993

P < 0.001

P = 0.020

P = 0.521

P = 0.999

P < 0.001

HVx

0

0.5

1.0

1.5

2.0

2.5

0

0.5

1.0

1.5

2.0

2.5

Relative mRNA level

P = 0.942

b

WT

Sham

P < 0.001

P < 0.001

P = 0.852

20

30

40

50

10

0

ALDH

+ in

30 MHC-II cells (%)

60

90

120

0

50

100

150

0

Count

a

12.2

HVx

12. 
    

Sham

mAchRTKO

GatedonAPCs: CD4 5 +CD3–B220–NK1. 1 – MHC-II+

Sham HVx

WT

27. 61 3. 9

Alde“uor

HVx-DEAB (–)

HVx-DEAB (+)

Sham-DEAB (–)

Sham-DEAB (+)

Fig. 3 | The liver–brain–gut axis regulates colonic Treg cell homeostasis
through muscarinic signalling in APCs. a–d, Wild-type and mAChR TKO (KO)
mice were subjected to HVx or sham surgery. Colonic T cell phenotypes were
analysed 2 days later (n = 8 per group). Dashed line, DEAB treated; solid line,
vehicle only. e, Wild-type and mAchR TKO mice were subjected to HVx or sham
surgery and additionally treated with bethanechol (intraperitoneal injection,
300 μg per day) daily. Phenotypes of colonic immune cells were analysed at day

2 after surgery (n = 5 per group). a, Frequency of ALDH+ cells among MHC-II+

colonic APCs. The percentage of Aldef luor+ cells is shown above the horizontal

line indicating the positive gate. Histograms of ALDH+ cells in colonic APCs

(left) and quantification (right). b, Aldh1a1 and Aldh1a 2 mRNA expression in

colonic APCs. c, e, Frequency of Foxp3+ cells among CD4+ cells in the colon.

d, Frequency of RORγt+ cells among colonic Foxp3+ Treg cells. Data are

mean ± s.e.m. P values by unpaired one-way ANOVA with Tukey’s post hoc test.