Metabolite-sensing receptor Ffar2 regulates colonic group 3 innate

lymphoid cells and gut homeostasis

Eunyoung Chun1, Sydney Lavoie1, Diogo Fonseca-Pereira1, Sena Bae1, Monia Michaud1, Graeme Fraser2, Wendy S. Garrett1,2,3,4
1Harvard T. H. Chan School of Public Health, Departments of Immunology and Infectious Diseases and Molecular Metabolism, MA, USA
2EPICS SA, Belgium, 3Dana-Farber Cancer Institute, MA, USA, 4Broad Institute of Harvard and MIT, MA, USA.

A B Ffar2fl/fl C Ffar2fl/fl D Ki-67+ ILC3 E BrdU+ ILC3

A
fl/fl DRorc
B C
Ffar2fl/fl
Abstract Ffar2 Ffar2 DRorc
ILC3 Ffar2DRorc DRorc Ffar2

RORγt+ILC3s (% of ILCs)
40 Ffar2
80 ** 30 Colonic Colonic Colonic Colonic

Colonic patch
Rorc

BrdU+ (% of ILC3s)
Ki-67+ (% of ILC3s)
30 * ROR𝛾t AHR 60
* patches SILTs patches SILTs
Ahr 20

Rorγt+ CD3- ILC3 number

Rorγt+ CD3- ILC3 number
20 6000 4 8 150 80
Group 3 innate lymphoid cells (ILC3s) sense environmental signals that 2500
*

(MFI of RORγt+ILC3s)
Ccr6 40

(MFI of RORγt+ILC3s)
*

(per colonic patch)

*

Number per colon

Number per colon

(per colonic SILT)

Ncr1 10 2000 10 3 6 60
are critical for gut homeostasis and host defense. However, the Il22
4000 20 100

RORγt
1500

Ahr
0 2 4 40
metabolite-sensing G-protein-coupled receptors that regulate colonic Il17a 50 1000 2000
0
30
**
0
40
50

RORγt+ILC3s (x103)

Colonic SILT
**

+/+

Ki-67+ cells (x103)

BrdU+ cells (x102)

500 1 2 20
*

-/-
40
ILC3s remain poorly understood. We found that colonic ILC3s expressed 30

Ffar2
Ffar2
30 0 0 20
0 0 0 0
Ffar2, a microbial metabolite-sensing receptor, and that Ffar2 agonism Expression (relative) 20
10
20

10
promoted ILC3 expansion and function. Deficiency of Ffar2 in ILC3s Row min Row max 10

0 0 0
Ffar2 influences colonic ILC3 expansion in colonic
decreased their in situ proliferation and ILC3-derived interleukin-22 (IL- F lymphoid tissues. (A) Distribution of colonic ILC3s in colonic
IL-22 IL-17A Ffar2fl/fl
80 *** 8 Ffar2DRorc patches and colonic SILTs from Ffar2∆Rorc or Ffar2fl/fl mice. (B)
22) production. This led to impaired gut epithelial function characterized

(% of RORγt+ILC3s)
(% of RORγt+ILC3s)
60 6 G H Number of colonic patches and SILTs. (C) Quantification of

IL17A+ILC3s
ILC3 subset IL-22+ ILC3

IL22+ILC3s
by altered mucus-associated proteins and anti-microbial peptides and 40 4
Live ILC3s gated
200 200
RORγt Ffar2 CD3
Ffar2-expressiong colonic ILC3s in colonic lymphoid
** * tissues.*p< 0.05, **p< 0.01
increased susceptibility to colonic injury and bacterial infection. Ffar2

IL-22+ ILC3s (x102)

NKp46+ 150
150

ILC3s (x102)
20 2

increased IL-22+CCR6+ ILC3s and influenced ILC3 abundance in colonic 0 0

CCR6+
100 100
A B

NKp46
40 2.0
*** Ffar2 agonist Propionate
lymphoid tissues. Ffar2 agonism differentially activated AKT or ERK 0.6 WT ILC3

IL-17A+ILC3s (x103)
50 50

IL-22+ILC3s (x103)
CCR6- 0.3

(Normalized to Actb)
0.4
30 1.5 ***

(Normalized to Actb)
(Normalized to Actb)
Il22 expression
signaling and increased ILC3-derived IL-22 via an AKT and STAT3 axis. ** CCR6 0 0 *** *

Il22 expression
Il22 expression
20 1.0 CCR6+ CCR6- NKp46+ CCR6+ CCR6- NKp46+ 0.4 * 0.3
0.2 *
Our findings suggest that Ffar2 regulates colonic ILC3 proliferation and 10 0.5 Ffar2 agonist Propionate 0.2
0.2
function, and they identify an ILC3-receptor signaling pathway 0 0.0 0.1
0.1

Ffar2 regulates colonic ILC3 proliferation and ILC3-derived IL-22. (A) Heatmap represents
modulating gut homeostasis and pathogen defense. relative expression of select ILC3 signature genes. (B) Colonic ROR𝛾t+ ILC3s from Rorc-cre
0.0
Veh A P Ffar2 PTX
0.0
- + - PTX
0.0
- + -
agonist - - + YM-254890 - - +
Ffar2fl/fl (Ffar2∆Rorc) or control Ffar2fl/fl mice. (C) ROR𝛾t and AHR expression in colonic ILC3s from C YM-254890

Introduction Ffar2∆Rorc or Ffar2fl/fl mice. (D) Ki-67 expression in colonic ILC3s from Ffar2∆Rorc or Ffar2fl/fl mice. (E) 40 15 20 8
*
* * WT ILC3+Veh

pSTAT3+ cell (%)

pSTAT3+ cell (%)

BrdU+ colonic ILC3s from Ffar2∆Rorc or Ffar2fl/fl mice. (F) IL-22+ and IL-17A+ colonic ILC3s from

pERK+ cell (%)

pAKT+ cell (%)
30 15 6
10 * WT ILC3 +Ffar2 agonist
Ffar2∆Rorc or Ffar2fl/fl mice. (G) Colonic ILC3 subsets in Ffar2∆Rorc or Ffar2fl/fl mice. (H) IL-22+ CCR6+ IL-22
20 10 4
WT ILC3 +Propionate
ILC3s from Ffar2∆Rorc or Ffar2fl/fl mice.*p< 0.05, **p< 0.01, ***p< 0.001 5
10 5 2

SCFAs Ffar2 (GPR43) A Ffar2DRorc 0 0 0 0

1.0
Metabolites SCFAs

Fold change to Ffar2 fl/fl

Bile acids
Mucin Ffar2 regulates colonic ILC3-derived IL-22 via AKT and STAT3 activation or partially ERK and
AMPs 0.8
AHR ligands
Fiber 0.6 * * STAT3 activation. (A) Il22 mRNA expression in sorted ILC3s cultured with acetate, propionate or
* * Ffar2 agonist. (B) Il22 mRNA expression in sorted ILC3s cultured with Gi/o inhibitor (PTX) or Gq
0.4 *
* inhibitor (YM-254890) under the Ffar2 agonist or propionate activation. (C) AKT, ERK, or STAT3
0.2 *
activation in sorted ILC3s cultured with the Ffar2 agonist or propionate.*p< 0.05, ***p< 0.001
ILC3 0.0

R b

R α
3β
Ffar2 agonist

3γ
2

M 4
IL-22

3
uc

uc

uc
uc

eg
eg

eg
RORγt
?

R
DSS model Citrobacter rodentium model Conclusion
? ? Ffar2fl/fl
Gut homeostasis B Water
C Ffar2 fl/fl
DRorc
G H Ffar2DRorc
3% DSS Ffar2 0
Gut homeostasis ILC3 Host defense 5 5

Histologic colitis score

Weight change (%)
30
*
Histologic colitis score

Local or systemic • Ffar2 agonism regulates

Weight change (%)

** 4
0 -10
immunity 20 * 3
colonic ILC3 expansion and
RORγt -5 ** 2
-20
* ** ** function (IL-22 production)
IL-22/IL-17A -10
**
10
1

0
• Ffar2 regulates colonic ILC3
-15 0 -30
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 proliferation and ILC3-
Results D 100
Time (days)
Ffar2fl/fl
Ffar2DRorc I
Time after infection (days)
Ffar2
fl/fl
Ffar2DRorc derived IL-22 in a cell-
** 25 *** 100
*
40 intrinsic manner.
(% of RORγt+ILC3s)

(% of RORγt+ILC3s)
IL-22+ILC3s (x103)

IL-22+ILC3s (x103)
80 20 80
30 ** • Ffar2-deficient ILC3s
IL22+ILC3s

IL22+ILC3s

A Colon C 60 15 60
0.020 ****
Control (NaCl)
SCFAs (A,P, B)
Control
Ffar2 agonist 20 enhance susceptibility to
(Normalized to Actb)

40 10 40
colonic inflammation and
Ffar2 expression

80 100
0.015 ** * 10
(% of RORγt+ILC3s)

(% of RORγt+ILC3s)

20 5 20
60 * 80 infection.
IL22+ ILC3s

IL22+ ILC3s

0 0 0 0
0.010 **** 40
60
Control Control • Ffar2 agonism controls ILC3-
0.005 20
40
E Ffar2 agonist F J Ffar2 agonist K derived IL-22 via AKT and
20 3% DSS Water
0
P = 0.05
*
5 40 4 STAT3 activation, or
Weight change (%)

0 0
Histologic colitis score

Histologic colitis score

0.000 *
Weight change (%)

-5
NK Gr Mac DC ILC ILC3 50 80
* 0 30 3 * partially ERK and STAT3
IL-22+ ILC3s (x103)

IL-22+ILC3s (x103)

B Control (NaCl) Control

40
** 60 * * -10
activation.
SCFAs (A,P, B) Ffar2 agonist 30 -5 20 2
-15
C: sodium chloride
RORγt+ILC3s (% of ILCs)

50 80 40
RORγt+ILC3 (% of ILCs)

** *** 20 -10 10 -20 1

40 A: sodium acetate
60 10 20
P: sodium propionate -15 0 -25 0
30
B: sodium butyrate 0 1 2 3 4 5 6 7
References
40 0 0 0 1 2 3 4 5 6 7
20 C A P B Control Control
Time (days) Time after infection (days) Ffar2 agonist
Ffar2 agonist
20 Ffar2 agonist(-D3) Ffar2 agonist(-D3)
10

0 0 Ffar2 agonism selectively promotes colonic ILC3 Ffar2-expressing ILC3s contribute to protection against colonic inflammation and bacterial
200
** 100
infection. (A) Gene expression in epithelial cells from Ffar2∆Rorc or Ffar2fl/fl mice. (B-F) DSS model • Chun E, Lavoie S, Fonseca-Pereira D, Bae S, Michaud M, Hoveyda HR, Fraser GL, Gallini Comeau CA,
expansion and function. (A) Ffar2 mRNA expression in
RORγt+ILC3s (x103)
RORγt+ILC3s (x103)

*** 80 *** Glickman JN, Fuller MH, Layden BT, Garrett WS. Metabolite-sensing receptor Ffar2 regulates colonic
150 mouse colonic immune cells. (B) Colonic ROR𝛾t+ ILC3s in Ffar2∆Rorc or Ffar2fl/fl mice. (B) Body weight changes. (C) Colitis score. (D) Colonic IL-22+ ILC3s.
60 group 3 innate lymphoid cells and gut immunity. Immunity. 2019, 51, 5, 871-884, e6.
100 from mice fed SCFAs or a synthetic Ffar2 agonist. (C) IL- (E-F) DSS model in WT mice treated with Ffar2 agonist. (E) Body weight changes. (F) Colitis score. • Tan, J.K., McKenzie, C., Marin ̃o, E., Macia, L., and Mackay, C.R. Metabolite-Sensing G Protein-Coupled
40
50
22 production in colonic ILC3s from mice fed SCFAs or (G-K) Citrobacter rodentium model in Ffar2∆Rorc or Ffar2fl/fl mice. (G) Body weight changes. (H) Receptors-Facilitators of Diet- Related Immune Regulation. Annu. Rev. Immunol. 2017, 35, 371–402.
20
the synthetic Ffar2 agonist.*p< 0.05, **p< 0.01, ***p< Colitis score. (I) Colonic IL-22+ ILC3s. (J-K) Citrobacter rodentium model in WT mice treated with • Vivier, E., Artis, D., Colonna, M., Diefenbach, A., Di Santo, J.P., Eberl, G., Koyasu, S., Locksley, R.M.,
0 0
C A P B 0.001, **** p< 0.0001 Ffar2 agonist. (J) Body weight changes. (K) Colitis score. *p< 0.05, **p< 0.01, ***p< 0.001 McKenzie, A.N.J., Mebius, R.E., et al. Innate Lymphoid Cells: 10 Years On. Cell. 2018, 174, 1054–1066.