ARTICLE
Commensal bacteria promote type I interferon
signaling to maintain immune tolerance in mice
Adriana Vasquez Ayala
1
,Chia-YunHsu
1
, Renee E. Oles
1
,KazuhikoMatsuo
1,2
, Luke R. Loomis
1
, Ekaterina Buzun
1
,
Marvic Carrillo Terrazas
1
, Romana R. Gerner
3
, Hsueh-Han Lu
1
, Sohee Kim
4
, Ziyue Zhang
5
, Jong Hwee Park
6
, Paul Rivaud
6
,
Matt Thomson
6
, Li-Fan Lu
5
, Booki Min
4
,andHiutungChu
1,7,8
Type I interferons (IFNs) exert a broad range of biological effects important in coordinating immune responses, which have
classically been studied in the context of pathogen clearance. Yet, whether immunomodulatory bacteria operate through IFN
pathways to support intestinal immune tolerance remains elusive. Here, we reveal that the commensal bacterium,
Bacteroides fragilis
, utilizes canonical antiviral pathways to modulate intestinal dendritic cells (DCs) and regulatory T cell
(Treg) responses. Specifically, IFN signaling is required for commensal-induced tolerance as IFNAR1-deficient DCs display
blunted IL-10 and IL-27 production in response to
B. fragilis
. We further establish that IFN-driven IL-27 in DCs is critical in
shaping the ensuing Foxp3
+
Treg via IL-27R
α
signaling. Consistent with these findings, single-cell RNA sequencing of gut Tregs
demonstrated that colonization with
B. fragilis
promotes a distinct IFN gene signature in Foxp3
+
Tregs during intestinal
inflammation. Altogether, our findings demonstrate a critical role of commensal-mediated immune tolerance via tonic type I
IFN signaling.
Introduction
Type I interferons (IFNs) are involved in many essential im-
mune functions, influencing both innate and adaptive immune
responses (
Trinchieri, 2010
;
Ivashkiv and Donlin, 2014
;
McNab
et al., 2015
). Type I IFNs, namely IFN
α
and IFN
β
, are produced
upon sensing microbial products resulting in the expression of
interferon-stimulated genes (ISGs). While several hundred ISGs
with various known functions have been identified, type I IFN
has been primarily studied for its role in antiviral immunity
(
Schoggins et al., 2011
;
Schoggins, 2019
). This includes recent
work that revealed that commensal microbes are involved in
maintaining tonic type I IFN necessary to mount an effective
antiviral immune response (
Abt et al., 2012
;
Ganal et al., 2012
;
Yang et al., 2021
;
Bradley et al., 2019
;
Wirusanti et al., 2022
).
Apart from the induction of antiviral ISGs, type I IFN can pro-
mote dendritic cell (DC) activation and maturation to enhance
antigen presentation to prime adaptive immunity (
Honda et al.,
2003
;
Simmons et al., 2012
). Further, tonic type I IFN expression
is essential for effective T cell responses (
Aman et al., 1996
;
Levings et al., 2001
;
Bilsborough et al., 2003
;
Teijaro et al., 2013
;
Wilson et al., 2013
). These studies highlight a potential role for
microbial-induced IFN signaling in host immunity beyond an-
tiviral responses. Of particular interest is the divergent effect of
type I IFN on immune responses that depend on the context of
microbial exposure. Type I IFN responses to microbial patho-
gens generate a robust antimicrobial and proinflammatory re-
sponse via activation of distinct ISGs (
McNab et al., 2015
;
Boxx
and Cheng, 2016
). In contrast, detection of commensal products
during homeostatic conditions triggers type I IFN signaling to
support anti-inflammatory responses (
Lee and Ashkar, 2018
).
Previous studies also suggest that type I IFN may influence
regulatory T cell (Treg) function. Notably, signaling via inter-
feron-
α
/
β
receptor 1 (IFNAR1) is required for Treg expansion
and suppression of pathogenic T cells during colitis (
Lee et al.,
2012
;
Stewart et al., 2013
;
Kawano et al., 2018
). In humans,
several genes in the IFN pathway have been associated with
inflammatory bowel disease (IBD) susceptibility in genome-
wide association studies.
IFNAR1
has been implicated as an IBD
risk allele, as have single nucleotide polymorphisms that disrupt
the JAK/STAT pathway, resulting in defective IFN production
(
Jostins et al., 2012
). Collectively, these findings support the
...............................................................................................................................
..............................................
1
Department of Pathology, University of California, San Diego, La Jolla, CA, USA;
2
Division of Chemotherapy, Kindai University Faculty of Pharmacy, Higashi-osaka, Japan;
3
TUM School of Life Sciences Weihenstephan, ZIEL Institute for Food & Health, Freising-Weihenstephan, Germany;
4
Department of Microbiology and Immunology,
Northwestern University Feinberg School of Medicine, Chicago, IL, USA;
5
School of Biological Sciences, University of California, San Diego, La Jolla, CA, USA;
6
Division of
Biology, California Institute of Technology, Pasadena, CA, USA;
7
Chiba University-UC San Diego Center for Mucosal Immunology, Allergy and Vaccines, University of
California, San Diego, La Jolla, CA, USA;
8
Humans and the Microbiome Program, Canadian Institute for Advanced Research, Toronto, Canada.
Correspondence to Hiutung Chu:
hiuchu@ucsd.edu
.
© 2023 Vasquez Ayala et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at
https://creativecommons.org/
licenses/by/4.0/
).
Rockefeller University Press
https://doi.org/10.1084/jem.20230063
1of15
J. Exp. Med. 2024 Vol. 221 No. 1 e20230063
hypothesis that microbially induced type I IFN signaling plays a
role in the maintenance of mucosal homeostasis and immune
tolerance.
Clinical and experimental evidence has implicated the gut
microbiota in governing host immunity during steady-state and
disease (
Hooper et al., 2012
;
Belkaid and Hand, 2014
). In par-
ticular, the mechanism by which commensal bacteria regulate
type I IFN responses to maintain mucosal immunity is of sig-
nificant interest. Microbiota-induced IFN pathways have been
shown to be critical in mounting antiviral resistance in the lung
(
Steed et al., 2017
;
Bradley et al., 2019
). Moreover, glycolipids
from commensal
Bacteroides
have been reported to direct anti-
viral function via IFN
β
expression in DCs (
Stefan et al., 2020
). In
contrast, type I IFN is also involved in the maintenance of Tregs
in the gut (
Lee et al., 2012
;
Kole et al., 2013
;
Nakahashi-Oda et al.,
2016
). Whether the induction of type I IFN by immunomodu-
latory commensal bacteria is necessary to maintain intestinal
immune tolerance, in addition to driving antiviral responses, is
unknown.
Here, we establish that tonic type I IFN is maintained by
commensal bacteria and required for tolerogenic immune re-
sponses in the gut. Previous work from our group and others
established that
Bacteroides fragilis
prime DCs to promote Foxp3
+
Treg responses to control intestinal inflammation (
Shen et al.,
2012
;
Chu et al., 2016
). In the present study, we expand on these
findings by demonstrating that germ-free (GF) mice are indeed
deficient in IFN responses, and colonization with a single com-
mensal bacterium,
B. fragilis
, restores tonic type I IFN in the gut
comparable with specific pathogen
–
free (SPF) mice. We also
reveal that select commensal bacteria demonstrate variable in-
duction of IFN signaling in DCs, suggesting this immunomodu-
latory trait is specialized among certain commensal microbes.
Furthermore, our study highlights that
B. fragilis
triggers the
production of immunoregulatory cytokines, including IL-10 and
IL-27, by DCs through an IFN-dependent manner. This mecha-
nism intricately links
B. fragilis
–
induced tonic IFN production in
DCs to drive Treg responses via IL-27R
α
signaling. Indeed, while
investigating
B. fragilis
–
mediated gene signatures in Treg cells,
we discovered an enrichment of IFN-related genes among in-
testinal Foxp3
+
Treg cells, which importantly also include the
IL-27 signaling pathway. Our findings demonstrate that com-
mensal bacteria promote intestinal homeostasis through type I
IFN signaling.
Results
Commensal bacteria direct intestinal type I IFN responses
Emerging evidence suggests commensal bacteria are important
regulators of tonic type I IFN signaling (
Sonnenburg et al., 2006
;
Yamamoto et al., 2012
;
Schaupp et al., 2020
;
Domizio et al., 2020
;
Lam et al., 2021
) and are required to mount an effective immune
response to pathogens (
Abt et al., 2012
;
Steed et al., 2017
;
Bradley
et al., 2019
;
Winkler et al., 2020
;
Erttmann et al., 2022
). Given
the pleiotropic effects of type I IFN, we examined the association
between the microbiota and type I IFN signaling in the gut
during steady state by assessing the expression of IFN-related
genes in colon tissue of GF and SPF C57BL/6J mice. The presence
of a commensal microbial community was required for the in-
duction of
Ifnb
and
Mx1
(an IFN-stimulated gene; ISG) expression
in the colon (
Fig. 1, A and B
). Particularly, monocolonization of
GF mice with the commensal bacterium,
B. fragilis
, was suffi-
cient to partially restore IFN-related gene expression in the
colon (
Fig. 1 A
). We next investigated the contribution of the
microbiota in priming local intestinal type I IFN responses upon
stimulation. Colon explants from GF and SPF mice were treated
with polyinosinic
–
polycytidylic acid (poly I:C), a Toll-like re-
ceptor 3 (TLR-3) agonist and potent inducer of IFN
β
. While poly
I:C induced a significant increase in IFN
β
production in SPF
colon explants, GF tissues remained unresponsive to poly I:C
(
Fig. 1 C
). We next investigated whether the presence of com-
mensal bacteria influenced IFNAR-dependent signaling in gut
DCs. Colonic lamina propria (cLP) cells were isolated from SPF
and GF mice and stimulated with recombinant IFN
β
ex vivo.
Colonic CD11c
+
cells from SPF mice responded to IFN
β
stimula-
tion via increased phosphorylation of signal transducer and
activator of transcription 1 (STAT1), as measured by flow
cytometry (
Fig. 1 D
). Conversely, pSTAT1 expression in colonic
CD11c
+
cells from GF mice remained unchanged in colonic CD11c
+
cells from GF mice following IFN
β
stimulation, suggesting im-
paired IFNAR signaling in mice lacking commensal bacteria de-
spite equivalent expression of
Ifnar1
(
Fig. 1 D
and
Fig. S1, A and
B
). Antibiotic cocktail treatment of SPF mice also led to dimin-
ished pSTAT1 expression (
Fig. S1 C
). These CD11c
+
cells encom-
pass both DCs and macrophages, and we speculate both cell
types play a role in sensing commensal bacteria in the gut. To-
gether, these findings establish that commensal bacteria direct
IFN signaling and upregulation of pSTAT1 in gut CD11c
+
immune
cells. To investigate whether this IFN defect in the intestinal
environment extends to systemic compartments, splenocytes
from GF and SPF mice were treated ex vivo with poly I:C. As
expected, at baseline, SPF splenocytes expressed higher levels of
type I IFN, as well as other cytokines and chemokines, compared
with GF (
Fig. S1 D
). In contrast, GF splenocytes failed to mount a
robust response to poly I:C stimulation compared with SPF.
Moreover, no induction of IFN
α
and IFN
ɣ
(a type II IFN) was
observed upon poly I:C treatment of GF splenocytes, while poly
I:C induced IFN
β
in GF splenocytes, secretion was limited
and equivalent to untreated SPF cells (
Fig. S1 D
). To confirm
the requirement of commensal bacteria for type I IFN response
in the intestinal environment in vivo, GF,
B. fragilis
–
monocolonized, and SPF mice were treated with poly I:C by
intraperitoneal (IP) injection and evaluated for IFN respon-
siveness. SPF mice treated with poly I:C demonstrated sig-
nificant expression of type I IFN
–
related genes (
Fig. 1, E
–
G
;
and
Fig. S1, F
–
H
). In contrast, GF mice injected with poly I:C
showed no response in comparison to the PBS control (
Fig. 1,
E
–
H
;and
Fig. S1, E
–
I
), consistent with ex vivo studies (
Fig. 1,
A
–
D
). To verify whether this IFN defect in GF mice can be
restored with commensal bacteria, we colonized GF mice with
B. fragilis
. Indeed, poly I:C treatment of
B. fragilis
mice led to a
significant induction of IFN
β
and IFN-related genes, indicat-
ing that the presence of commensal bacterium is sufficient to
restore homeostatic type I IFN responses (
Fig. 1, E
–
H
;and
Fig.
S1, H and I
). These data establish that commensal bacteria are
Vasquez Ayala et al.
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Commensal-induced type I IFN maintains tolerance
https://doi.org/10.1084/jem.20230063
critical in restoring and maintaining type I IFN responses in
intestinal tissues.
Select commensal bacteria induce IFN signaling to promote
tolerogenic responses in DCs
DCs establish and maintain the local gut immune milieu by
sampling luminal contents, including bacteria (
Rescigno and
Sabatino, 2009
). To gain insight into the impact of commensal-
derived type I IFN on DC responses, we first sought to assess the
levels of IFN
β
induction by commensal bacteria. We demonstrate
that DCs treated with
B. fragilis
and other
Bacteroides
species,
Bacteroides thetaiotaomicron
and
Bacteroides vulgatus
, drive IFN
β
secretion and pSTAT1 induction, while
Lactobacillus plantarum
induced modest IFN
β
production (
Fig. 2 A
and
Fig. S2 A
). In
contrast, other commensal bacteria tested did not support sig-
nificant IFN
β
induction, suggesting some degree of specificity in
their activity (
Fig. 2 A
and
Fig. S2 A
). Pathogenic bacteria, on the
other hand, induced pSTAT1 expression and IFN
β
production
(
Fig. S2, B and C
) at much greater levels than
B. fragilis
and other
commensals tested. These variations in type I IFN induction by
both commensal and pathogenic bacteria may underlie the
diverse immunomodulatory e
ffects observed downstream of
recognition.
Since monocolonization with
B. fragilis
effectively restored
type I IFN responses in vivo (
Fig. 1
) and yielded robust type I IFN
induction among DCs (
Fig. 2 A
), we employed
B. fragilis
as a
model commensal to delve deeper into the role of IFN responses
in mediating immune tolerance. Treatment of bone marrow
–
derived DCs (BMDCs) with
B. fragilis
induced expression of IFN
β
(
Fig. 2 B
), but not IFN
α
or IFN
ɣ
(
Fig. 2, C and D
), consistent with
in vivo findings (
Fig. 1
). As expected, this induction was lost in
Ifnar1
−
/
−
DCs, along with a decrease in pSTAT1 expression (
Fig. 2,
EandF
) in comparison with wild-type (WT) DCs. Additionally,
B. fragilis
significantly induced type I IFN
–
related genes in WT
DCs (e.g.,
Ifnb
,
Oas1
,and
Mx2
), whereas
Ifnar1
−
/
−
DCs remained
unresponsive to
B. fragilis
(
Fig. 2 E
and
Fig. S2, D and E
). Further,
B. fragilis
does not influence expression of other IFN-responsive
genes (e.g.,
Irf3
,
Irf9
)byWTDCs(
Fig. S2, F and G
). Next, we
leveraged the
IFNb
mob
EYFP reporter mouse to examine
B.
fragilis
–
induced IFN
β
among conventional and plasmacytoid
DCs. Treatment with
B. fragilis
elevated IFN
β
-YFP expression in
both DC subsets (
Fig. 2, G and H
). We then examined whether
colonization with
B. fragilis
induced IFN expression in colonic
DCs. To assess the extent of IFN
β
produced by intestinal DCs
primed by
B. fragilis
in vivo, we isolated colonic CD11c
+
cells from
GF and
B. fragilis
–
monocolonized mice and measured baseline
Figure 1.
Commensal bacteria maintain intestinal type I IFN responses. (A and B)
Expression of (A)
Ifnb
and (B)
Mx1
in colon tissue from GF,
B.
fragilis
–
monocolonized (Bf), and SPF as measured by qRT-PCR relative to
β
-actin. Each point represents a single mouse.
(C)
GF and SPF colon explants were
cultured with or without stimulation of poly I:C (pIC; 2
μ
g/ml) for 24 h. Supernatant was then collected and measured for IFN
β
secretion by ELISA. Each point
represents a single mouse.
(D)
cLP cells were isolated from GF and SPF mice and stimulated with IFN
β
(25 ng/ml). pSTAT1 was assessed by flow cytometry.
Each point represents colons pooled from multiple mice, with
n
= 10 per group.
(E
–
H)
GF, Bf, and SPF mice were injected (IP) with 100 μg/ml pIC, and colon
tissues were harvested 4 h after injection. Gene expression for (E)
Mx2
,(F)
Irf7
,(G)
Ifit1
, and (H)
Ifnb
was measured. Each point represents a single mouse. Data
are representative of at least two independent experiments. Statistical significance was determined by Kruskal-Wallis, unpaired
t
test, and two-way ANOVA.
P<0.05(*),P<0.01(**),andP<0.0001(****).
Vasquez Ayala et al.
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Commensal-induced type I IFN maintains tolerance
https://doi.org/10.1084/jem.20230063
IFN
β
secretion. Indeed, colonic lamina propria CD11c
+
cells from
B. fragilis
–
monocolonized mice produced higher levels of IFN
β
compared with GF mice (
Fig. 2 I
).
It has been well documented that
B. fragilis
primes DCs to
foster immune tolerance (
Shen et al., 2012
;
Chu et al., 2016
).
However, the precise signaling mechanisms underlying this
immune regulation remain incompletely understood. Our ob-
servations thus far have revealed a distinct pattern of induc-
tion among type I IFN activity in
B. fragilis
–
primed DCs. To
investigate the key receptors that initiate this pathway, we
sought to determine which pattern recognition receptors
B.
fragilis
engages to drive type I IFN responses in BMDCs. Previous
studies with
B. fragilis
demonstrated that defects in TLR2 (
Round
et al., 2011
;
Shen et al., 2012
) and NOD2 (
Chu et al., 2016
) sig-
naling led to impaired Treg responses. We observed
B. fragilis
–
induced IFN
β
production required signaling via NOD2, but not
TLR2 (
Fig. S2, H and I
). Additionally, we examined the role of
TLR4 and revealed reduced IFN responses to
B. fragilis
in TLR4-
Figure 2.
Select commensal bacteria induce IFN signaling to promote tolerogenic responses in DCs. (A)
BMDCs from SPF mice were treated with
B.
fragilis
(Bf),
B. thetaiotaomicron
(Bt),
B. vulgatus
(Bv),
L. plantarum
(Lp),
A. caccae
(Ac),
B. producta
(Bp), and
C. ramosum
(Cr) for 18 h. Supernatant was collected
and IFN
β
production was measured by ELISA.
(B
–
D)
BMDCs were treated with Bf for 18 h and expression of (B)
Ifnb
,(C)
Ifna
, and (D)
Ifng
were measured
relative to
β
-actin by qRT-PCR.
(E and F)
WT and
Ifnar1
−
/
−
BMDCs were pulsed with Bf for 18 h. Cells were harvested and analyzed by qRT-PCR for gene
expression of (E) IFN
β
and stained for (F) flow cytometry analysis of pSTAT1 in CD11c
+
DCs.
(G and H)
Splenocytes from IFN
β
-YFP reporter mice were treated
with Bf ex vivo for 18 h and mean fluorescent intensity (MFI) of IFN
β
-YFP was assessed in (G) plasmacytoid dendritic cells (pDCs) and (H) conventional dendritic
cells (cDCs) by flow cytometry.
(I)
cLP cells were isolated and enriched for CD11c
+
cells from GF and Bf-monocolonized mice and cultured for 18 h. Supernatant
was collected and IFN
β
production was measured by ELISA. Each point represents colons pooled from five mice per point and represents
n
= 30 for each group.
(J
–
O)
WT and IFNAR-deficient BMDCs were pulsed with Bf for 18 h. Cells were harvested and analyzed by qRT-PCR for gene expression of (J)
Il10
,(L)
Il27p28
,
and (N)
Il1b
relative to
β
-actin. Supernatant from BMDC cultures was collected and cytokine secretion was measured for (K) IL-10, (M) IL-27p28, and (O) IL-1
β
by ELISA. Data are representative of at least two independent experiments. Statistical analysis was determined by unpaired
t
test and two-way ANOVA. P <
0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).
Vasquez Ayala et al.
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Commensal-induced type I IFN maintains tolerance
https://doi.org/10.1084/jem.20230063
deficient BMDCs (
Fig. S2 J
). These findings are consistent with
published reports demonstrating TLR4 agonists, but not TLR2,
stimulate IFN
β
-induced pSTAT1 expression (
Fig. S2, I and J
;
Toshchakov et al., 2002
;
Stefan et al., 2020
). We also previously
reported a shift in the cytokine milieu in DCs defective in mi-
crobial sensing (
Chu et al., 2016
), demonstrating a significant
decrease in expression of IL-10, which is required for induction
of tolerogenic Tregs (
Shen et al., 2012
). Thus, we investigated
how type I IFN deficiency may alter downstream cytokine pro-
duction. We observed a significant reduction in IL-10 expression
and production among
Ifnar1
−
/
−
BMDCs compared with
B.
fragilis
–
treated WT BMDCs (
Fig. 2, J and K
). The induction of
IL-10 by
B. fragilis
has been well established (
Round et al., 2011
;
Shen et al., 2012
;
Chu et al., 2016
); however, this data reveals the
essential role of commensal-induced IFN signaling among DCs to
promote tolerogenic responses. We further demonstrate here
that
B. fragilis
also stimulates the production of IL-27 in DCs, and
this response is abrogated upon IFNAR1 deletion (
Fig. 2, L and
M
). Moreover, we observed a significant reduction in IL-10
production in
Il27
−
/
−
DCs treated with
B. fragilis
, suggesting IFN-
induced IL-27 is necessary for commensal-mediated immune
homeostasis (
Fig. S2 K
). Indeed, IL-27 is documented as an im-
munoregulatory cytokine that targets Tregs to mediate anti-
inflammatory activity (
Kim et al., 2019
;
Nguyen et al., 2019
),
while suppressing IL-17
–
producing CD4
+
T cells (
Batten et al.,
2006
). Given that the signaling of IL-27 and type I IFN are in-
tricately linked (
Stumhofer et al., 2007
;
Amsden et al., 2022
), we
investigated the requirement of IL-27 in
B. fragilis
–
mediated type
I IFN activity among DCs. We observed
B. fragilis
–
induced ex-
pression of pSTAT1 (
Fig. S2 L
)andsecretionofIFN
β
(
Fig. S2 M
)
relied upon IL-27 production in DCs. These data suggest induc-
tion of IFN by
B. fragilis
drives IL-27 expression, which further
reciprocates type I IFN signaling. Considering the reduction in
IL-10 and IL-27 upon type I IFN deficiency, we investigated how
proinflammatory responses may be affected. Notably, the de-
creased anti-inflammatory response observed is paired with an
increase in IL-1
β
upon
B. fragilis
treatment of
Ifnar1
−
/
−
BMDCs
(
Fig. 2, N and O
).
B. fragilis
treatment of BMDCs did not alter
levels of proinflammatory IFN
ɣ
, indicating selectivity in cyto-
kine regulation by commensal microbes (
Fig. 2 D
;and
Fig. S3, A
and B
). Consistent with these observations, we report extensive
dysregulation of cytokine and chemokine production in
B.
fragilis
–
treated
Ifnar1
−
/
−
BMDCs (
Fig. S3, C
–
H
). Collectively, these
data establish a critical role for IFN signaling in DCs to induce
immunoregulatory responses upon sensing commensal bacteria.
B. fragilis
induces type I IFN expression in DCs to coordinate
Treg responses
We hypothesize that this skewed proinflammatory environment
driven by IFNAR-deficient DCs would restrain downstream Treg
responses. To test this hypothesis, WT and
Ifnar1
−
/
−
BMDCs were
treated with
B. fragilis
and cocultured with WT CD4
+
Tcells.
Indeed,
B. fragilis
–
pulsed WT DCs supported the induction of
CD4
+
Foxp3
+
Tregs (
Fig. 3 A
) and production of IL-10 (
Fig. 3 B
).
However,
B. fragilis
–
treated
Ifnar1
−
/
−
BMDCs resulted in a sig-
nificant reduction of IL-10
+
Treg populations, indicating IFN
signaling in DCs is necessary for the induction of anti-
inflammatory T cell. We next assessed whether neutralization
of type I IFN phenocopies the Treg defect observed in
B.
fragilis
–
treated
Ifnar1
−
/
−
DCs. The induction of Treg and IL-10
production by
B. fragilis
was abrogated upon addition of neu-
tralizing antibodies for IFN
α
and IFN
β
in DC:T cell cocultures
compared with isotype controls (
Fig. 3, A and B
). This suggests
IFN
α
and/or IFN
β
function are critical signals in mediating
B.
fragilis
–
induced Tregs.
Previous studies have demonstrated a critical role for IL-27 in
supporting IL-10 expression among Tregs (
Awasthi et al., 2007
;
Pot et al., 2009
;
Kim et al., 2019
;
Nguyen et al., 2019
). In par-
ticular, IL-27 has been shown to initiate a transcriptional net-
work that facilitates IL-10 expression among CD4
+
T cell subsets
(
Zhang et al., 2020
). Further corroborating these studies, we
observed marked upregulation of IL-10 expression among
Foxp3
+
Tregs upon IL-27 stimulation (
Fig. S4 A
). Notably,
treatment with commensal bacteria yielded a similar trend in the
production of IL-27 and IL-10 among BMDCs and Tregs, re-
spectively, suggesting that commensal-induced IL-27 may play a
role in directing Treg responses (
Fig. S4, B and C
). Given the
IFN-dependent induction of IL-27 by
B. fragilis
(
Fig. 2, L and M
),
we hypothesized that IL-27 signals directly on Foxp3+ Tregs to
promote tolerogenic function. The IL-27 receptor consists of two
subunits, IL-27R
α
and glycoprotein 130 (gp130). To elucidate the
significance of IL-27 signaling in Foxp3
+
Tregs, we employed
Il27ra
fl/fl
× Foxp3-Cre (
Il27ra
Δ
Foxp3
)mice(
Do et al., 2017
). As ex-
pected, coculture of
Il27ra
fl/fl
CD4
+
Tcellswith
B. fragilis
–
pulsed WT DCs led to the induction of IL-10 among CD4
+
Foxp3
+
Tregs. However, this induction was markedly diminished when
cocultured with
Il27ra
Δ
Foxp3
CD4
+
T cells (
Fig. 3, C and D
). Fur-
thermore, when we performed these studies with
Ifnar1
−
/
−
DCs,
we again observed a significant reduction in IL-10
–
producing
Foxp3+ Tregs in response to
B. fragilis
,withboth
Il27ra
fl/fl
and
Il27ra
Δ
Foxp3
CD4
+
Tcells(
Fig. 3, C and D
). Comparable levels of
IL-27 were detected across the various DC:T cell coculture con-
ditions, although a reduction in IL-27 was observed in CD4
+
Tcellscoculturedwith
B. fragilis
–
pulsed
Ifnar1
−
/
−
DCs (
Fig. S4 D
).
This finding aligns with our observation that the induction of
IL-27 by
B. fragilis
was lost in
Ifnar1
−
/
−
BMDCs (
Fig. 2, L and M
).
Altogether, these data suggest a model wherein commensal
bacteria promote the production of IL-27 from DCs through an
IFN-dependent pathway, which in turn signals via IL-27R
α
in
Foxp3
+
Tregs to orchestrate immune tolerance.
Commensal colonization drives IFN gene signature in
intestinal Tregs
Given the requirement of type I IFN signaling for
B. fragilis
–
induced Treg responses in vitro, we investigated the conse-
quence of IFNAR1 deficiency in SPF mice in vivo. Proportions of
Foxp3
+
Tregs in the cLP were reduced in
Ifnar1
−
/
−
mice (
Fig. 4 A
),
consistent with previous work that reported loss of Treg in-
duction in
Rag1
−
/
−
recipients upon transfer of
Ifnar1
−
/
−
Tregs (
Lee
et al., 2012
). As expected,
B. fragilis
treatment of WT SPF mice
induced IL-10
–
producing Foxp3
+
Tregs compared with PBS-
treated controls (
Fig. 4 B
). In contrast,
B. fragilis
–
induced Treg
responses were abolished in
Ifnar1
−
/
−
SPF mice (
Fig. 4 B
). This
dysregulation of Treg function is often associated with an
Vasquez Ayala et al.
Journal of Experimental Medicine
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Commensal-induced type I IFN maintains tolerance
https://doi.org/10.1084/jem.20230063
increased frequency of pathogenic T cell subsets, such as Th1 or
Th17. Given the significant reduction in Treg populations ob-
served in
Ifnar1
−
/
−
mice, we further investigated whether Th17
subsets were reciprocally elevated due to this imbalance. Indeed,
we observed a significant induction of CD4
+
IL-17A
+
Tcellsin
Ifnar1
−
/
−
mice treated with
B. fragilis
compared with their WT
counterparts (
Fig. 4 C
), underscoring the critical role of type I
IFN signaling in maintaining Treg/Th17 balance in response to
the commensal bacteria. To further demonstrate the role of
commensal bacteria in IFN responses among Tregs, WT SPF
mice were treated with an antibiotic cocktail for 2 wk and we
observed a significant reduction of pSTAT1 expression within
Foxp3
+
Tregs and decreased IL-10
+
among Tregs (
Fig. S4, E
and F
). Consistent with this finding, the depletion of com-
mensal bacteria led to decreased IL-10
+
among CD11c
+
cells in
the colon, further supporting
the notion that intestinal com-
mensals are required for DC-directed Treg responses (
Fig.
S4 G
). Together, this data demonstrates a pivotal role for
commensal bacteria and type I IF
N signaling in orchestrating
T cell responses.
We next aimed to elucidate the specific contribution of IFN
signaling directly within Foxp3
+
Tregs in vivo. Using
Ifnar1
fl/fl
×
Foxp3-Cre (
Ifnar1
Δ
Foxp3
),
Ifnar1
fl/fl
× CD11c-Cre (
Ifnar1
Δ
CD11c
), and
Ifnar1
fl/fl
control mice, we orally administered live
B. fragilis
or
PBS for 2 wk and examined intestinal Treg responses. While
Foxp3
+
Treg proportions remained consistent between the
groups (
Fig. 4 D
), we did observe a decrease in proportions of
Foxp3
+
RORgt
+
intestinal Tregs in
Ifnar1
Δ
Foxp3
and
Ifnar1
Δ
CD11c
mice compared with floxed controls (
Fig. S4 H
). As expected,
B.
fragilis
–
treated WT control mice exhibited a marked induction of
IL-10
+
Tregs compared with PBS-treated groups (
Fig. 4 E
), in line
with previous findings (
Round et al., 2011
;
Shen et al., 2012
;
Chu
et al., 2016
). In contrast, the induction of IL-10 by
B. fragilis
was
completely abrogated in
Ifnar1
Δ
Foxp3
mice (
Fig. 4 E
), demon-
strating a critical role for IFN signaling in Foxp3 Tregs. Notably,
when we performed these experiments using
Ifnar1
Δ
CD11c
mice,
Figure 3.
Type I IFN signaling in DCs is required to promote Treg responses.
WT and
Ifnar1
−
/
−
BMDCs were untreated (
−
) or treated with
B. fragilis
(Bf)
cocultured with WT CD4
+
Tcells.
(A and B)
Cells were treated with 10 μg/ml of IFN
α
/
β
neutralizing antibodies or isotype control (IgG2a) every 24 h and then
stained and analyzed by flow cytometry for (A) Foxp3
+
Tregs and (B) IL-10
–
producing Foxp3
+
Tregs.
(C and D)
WT and
Ifnar1
−
/
−
BMDCs were untreated (
−
)or
treated with Bf cocultured with
Il27ra
fl/fl
or
Il27ra
Δ
Foxp3
CD4
+
T cells. Cells were stained and analyzed by flow cytometry for IL-10
–
producing Foxp3
+
Tregs. (C)
Representative plots are shown and (D) proportions of IL-10
–
producing Foxp3
+
Tregs are quantified. Data are representative of at least two independent
experiments. Statistical significance was determined by two-way ANOVA. P < 0.05 (*), P < 0.01 (**), and P < 0.0001 (****).
Vasquez Ayala et al.
Journal of Experimental Medicine
6of15
Commensal-induced type I IFN maintains tolerance
https://doi.org/10.1084/jem.20230063
Figure 4.
Bacterial colonization induces type I IFN signature in intestinal Tregs. (A
–
C)
WT and
Ifnar1
−
/
−
SPF mice were orally gavaged with either sterile
PBS or live
B. fragilis
(Bf; 2 × 10
8
CFUs) for 2 wk and proportions of (A) CD4
+
Foxp3
+
Tregs, (B) CD4
+
Foxp3
+
IL10
+
Tregs, and (C) CD4
+
Foxp3
−
IL17A
+
Tcellsin
Vasquez Ayala et al.
Journal of Experimental Medicine
7of15
Commensal-induced type I IFN maintains tolerance
https://doi.org/10.1084/jem.20230063