REFERENCES AND NOTES

1. C.-H. Chen et al., Science 316, 597 (2007).

2. O. S. Akbari et al., Curr. Biol. 23, 671 (2013).
3. Y.-S. Chan, D. A. Naujoks, D. S. Huen, S. Russell, Genetics
188, 33 (2011).
4. K. M. Esvelt, A. L. Smidler, F. Catteruccia, G. M. Church, eLife
2014, e03401 (2014).
5. K. A. Oye et al., Science 345, 626 (2014).
6. V. M. Gantz, E. Bier, Science 348, 442 (2015).
7. A. Burt, Proc. R. Soc. London Ser. B 270, 921 (2003).
8. R. D. Henkel et al, Appl. Biosaf. 18, 171 (2012).
9. J. E. DiCarlo et al, bioRxiv 013896 (2015).
10. X. Ren et al., Proc. Natl. Acad. Sci. U.S.A. 110, 19012 (2013).
11. S. J. Gratz et al., Genetics 196, 961 (2014).
12. F. Port, H.-M. Chen, T. Lee, S. L. Bullock, Proc. Natl. Acad.
Sci. U.S.A. 111, E2967 (2014).
13. F. Port et al, G3 (Bethesda) 5, 1493 (2015).
14. S. Kondo, R. Ueda, Genetics 195, 715 (2013).
15. National Research Council, Gene Drive Research in NonHuman Organisms: Recommendations for Responsible
Conduct (DELS-BLS-15-06, National Academy of Sciences,
Washington, DC, 2015); http://bit.ly/CurrProjects-regul.
ACKNOWLEDGMENTS

The authors are grateful for conversations with T. Wu, J. Lunshof,

and A. Birnbaum. V.M.G., E.B., G.M.C., and K.M.E. are inventors
on relevant provisional and nonprovisional patents filed by the
University of California and Harvard University.
Published online 30 July 2015
10.1126/science.aac7932

MICROBIOME

Microbiota RORgulates
intestinal suppressor T cells
Gut microbes influence the balance of regulatory T cell
subtypes to control inflammation
By Ahmed N. Hegazy1,2 and Fiona Powrie1,2

he immune system in the intestine

is highly adapted to resist invading
pathogens while residing peacefully
with the abundant and diverse commensal bacteria that colonize the
gastrointestinal tract. In turn, bacterial signals shape immunity in the intestine, promoting intestinal homeostasis in
part by inducing and expanding specialized
regulatory T (Treg) cells that prevent aberrant
inflammatory responses to self and environmental stimuli (1). On pages 989 and 993 of
this issue, Ohnmacht et al. (2) and Sefik et
al. (3), respectively, report the development
and function of a subpopulation of Treg cells
found primarily in the large intestine, and
characterized by expression of the nuclear
hormone receptor retinoic acid receptorrelated orphan receptor t (RORt). This is
surprising because RORt classically promotes the differentiation of T helper 17
(TH17) cells, a population associated with
tissue inflammation in many inflammatory
diseases (4). Both studies show that microbiota-derived signals induce the expression of
RORt in Treg cells that control intestinal inflammation (see the figure). These findings
highlight the diversity of colonic Treg cells,
their complex transcriptional programs, and
their important role in the intestine.
Treg cells express the forkhead transcription factor Foxp3, which promotes their differentiation, maintenance, and function (5).
Alongside anti-inflammatory functions, they
control nonimmunological processes including tissue repair and metabolism in the
parenchyma (6). Treg cells also adapt to environmental cues by expressing canonical effector T cellassociated transcription factors
to control pathogenic immune responses (7).
Both Ohnmacht et al. and Sefik et al. found
that in mice, a large fraction of intestinal Treg
cells express RORt. These cells were distinct
from colonic Treg cells that express the transcription factor GATA3 and are poised to
respond to the cytokine interleukin (IL)33
after tissue damage (8, 9). However, RORtexpressing Treg cells had an activated phenotype similar to that of GATA3-expressing Treg
cells, and bore markers related to Treg cells

SCIENCE sciencemag.org

residing in lymphoid and non-lymphoid tissues (6). Strikingly, the microbiota was an
absolute requirement for the induction and
maintenance of RORt-expressing Treg cells
in these animals. This Treg cell population
was markedly reduced in germ-free mice,
and colonization with a diverse microbiota
or consortia of symbionts was sufficient for
the induction of RORt-expressing Treg cells.
Sefik et al. went further and recolonized
germ-free mice with 22 different bacterial
species, and showed that a number of them
(not belonging to any specific phylum or genus) elicited RORt-expressing Treg cells at
comparable frequencies to a diverse microbiota. Short-chain fatty acids, which are common bacterial metabolites, can selectively
expand intestinal Treg cells (10). Ohnmacht et
al. could increase RORt-expressing Treg cells
by feeding mice a diet rich in the short-chain
fatty acid butyrate.

These studiesare an
important stepping stone
to deciphering the complex
dynamics of different tissueresident Treg cell subsets
Which signals promote RORt expression
in Treg cells? The TH17-favoring cytokines
IL-6 and IL-23 were required for accumulation of RORt-expressing Treg cells, which
raises the question of what tips the balance toward these T cells rather than TH17
cells. The vitamin A metabolite retinoic
acid promotes Treg cell generation in vivo
and RORt-expressing Treg cells in vitro (11,
12). Consistent with this, Ohnmacht et al.
show that vitamin A metabolism influences
the differentiation equilibrium by favoring
the development of RORt-expressing Treg
cells in vivo. Although both Treg cells and
TH17 cells express RORt, analysis of all the
transcripts expressed by each population revealed marked differences, suggesting that
the transcriptional footprint of RORt is
context-dependent in different T cells.
What is the function of RORt-expressing
28 AUGUST 2015 VOL 349 ISSUE 6251

Published by AAAS

929

Downloaded from www.sciencemag.org on August 27, 2015

it will reduce the probability of release to

a level that is acceptably low. This probability must be defined on a case-by-case
basis. The analyses necessary to confidently
predict the efficacy of confinement strategies for gene drive systems are in a nascent
form. Therefore, any proposal to use one
rather than multiple forms of confinement
requires even greater scrutiny and extensive
deliberation between regulatory authorities
and scientists.
3) Organisms carrying gene drive constructs that could spread if the reproductively capable life stages were to escape in
transit should not be distributed to other institutions until formal biosafety guidelines
are established. Whenever possible, laboratories should instead send DNA constructs
or information sufficient to reconstruct the
gene drive. Protocols for distributing materials should be established in discussion
with the wider research community and
other relevant stakeholders.
Broadly inclusive and ongoing discussions among diverse groups concerning safeguards, transparency, proper use, and public
involvement should inform expert bodies as
they develop formal research guidelines for
gene drive research in the laboratory and
potential transitions to open field trials. We
applaud the U.S. National Academy of Sciences for committing to provide recommendations for responsible gene drive research
(15). By recommending strong safeguards
and encouraging discussion of this technology, we hope to build a foundation of public trust for potential future applications in
public health, sustainable agriculture, and
ecological conservation.

INSIGHTS | P E R S P E C T I V E S

Metabolites

Lumen

Damage

Intestinal epithelium
Intestinal
epithelium

Retinoic
acid

IL-6
IL-23

IL-33
Macrophage

IL-23

Treg

ROR t
Blocks

Blocks

GATA3

Treg

Blocks

TH2

TH17

TH1

Reduced infammation
Fine-tuning intestinal homeostasis. Microbiota and tissue-derived factors regulate the balance between RORtexpressing and GATA3-expressing Treg cells in the mouse intestine. The microbiota promotes RORt expression in
intestinal Treg cells through multiple factors including bacterial metabolites, retinoic acid, and cytokines. Tissueresident Treg cells control effector T cell responses to promote intestinal homeostasis.

Treg cells in health and disease? Ohnmacht et

al. and Sefik et al. addressed this question
by conditional deletion of the Rorc gene in T
cells expressing Foxp3, producing mice specifically deficient in RORt-expressing Treg
cells. The results were, however, ambiguous,
perhaps reflecting differences in experimental models, animal housing, or the indigenous microbiota. Ohnmacht et al. observed a
pronounced increase in type 2 cytokines under steady-state conditions, with consequent
resistance to helminth infection. Inflammatory outcomes differed according to the
chemically induced colitis model used. In
oxazolone-induced colitis (a type 2 cytokine
driven model), mice developed severe and
1

Kennedy Institute of Rheumatology, Nufeld Department of

Orthopaedics, Rheumatology and Musculoskeletal Sciences,
University of Oxford, Headington, Oxford OX3 7FY, UK.
2
Translational Gastroenterology Unit, Nufeld Department
of Clinical Medicine, Experimental Medicine Division, John
Radclife Hospital, University of Oxford, Oxford OX3 9DU, UK.
E-mail: fona.powrie@kennedy.ox.ac.uk

930

lethal colitis accompanied by an increase in

type 2 cytokines, whereas no alteration in
pathology or TH1 and TH17 cell responses was
observed in dextran sulfate sodiuminduced
colitis (a type 1 and type 17 cytokinedependent model). Sefik et al. chemically blocked
RORt function and found that colonic Treg
cell frequency decreased, and interferon-
and IL-17 production by effector T cells
increased, under steady-state conditions.
These mice developed severe colitis in another chemically induced (trinitrobenzenesulfonic acid) colitis model.
The findings of Ohnmacht et al. and Sefik et al. show that microbe-induced expression of RORt by Treg cells contributes to
the control of intestinal inflammation. But
what is the role of distinct colonic Treg cell
subsets (RORt and GATA3) in intestinal homeostasis? Is there functional redundancy
or division of labor? It may be that RORtexpressing Treg cells, which produce increased amounts of cytotoxic T lymphocyte

antigen 4 (CTLA4) and IL-10 (both of which

dampen immune responses), decrease inflammation, whereas GATA3-expressing Treg
cells, which produce the tissue-remodeling
factor amphiregulin and respond to the
alarm signal (alarmin) IL-33, mediate tissue repair. It is certainly possible that locally
produced inflammatory and tissue-derived
factors might activate or antagonize different Treg cell subpopulations to coordinate the
anti-inflammatory and healing processes.
Another issue raised by these studies
is whether Treg cell subsets are specialized
in controlling particular effector T cell responses. Ohnmacht et al. propose that
RORt-expressing Treg cells are critical in
controlling aberrant type 2 responses and
that deficiencies in these microbiota-driven
Treg cells may contribute to increases in allergic disease. However, Sefik et al. observed
control of TH1 and TH17 cells by RORtexpressing Treg cells. It seems highly likely
that the relative activity and function of
distinct colonic Treg cell populations will be
highly context-dependent and influenced
by the microbiota. It is important to understand the ontogeny of RORt-expressing Treg
cells and GATA3-expressing Treg cells and
whether they represent distinct lineages
or can interconvert. Inducible labeling and
tracking of Treg cell subsets would provide
valuable insights into their interplay and stability under steady-state and inflammatory
conditions. It remains to be established why
only certain bacterial species induce RORt
expression in Treg cells and whether we can
identify similar Treg cell subsets in humans
and manipulate them in vivo. The studies
by Ohnmacht et al. and Sefik et al. are an
important stepping stone to deciphering
the complex dynamics of different tissueresident Treg cell subsets toward the understanding of how their dysregulation precipitates human disease.
REF ERENCES AND NOTES

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.

Y. Belkaid, T. W. Hand, Cell 157, 121 (2014).

C. Ohnmacht et al., Science 349, 989 (2015).
E. Sefik et al., Science 349, 993 (2015).
T. Korn, E. Bettelli, M. Oukka, V. K. Kuchroo, Annu. Rev.
Immunol. 27, 485 (2009).
S. Z. Josefowicz, L.-F. Lu, A. Y. Rudensky, Annu. Rev.
Immunol. 30, 531 (2012).
D. Burzyn et al., Nat. Immunol. 14, 1007 (2013).
D. J. Campbell, M. A. Koch, Nat. Rev. Immunol. 11, 119
(2011).
E. A. Wohlfert et al., J. Clin. Invest. 121, 4503 (2011).
C. Schiering et al., Nature 513, 564 (2014).
P. M. Smith et al., Science 341, 569 (2013).
D. Mucida et al., Science 317, 256 (2007).
M. Lochner et al., J. Exp. Med. 205, 1381 (2008).

ACKNOWL EDGMENTS

A.N.H. was supported by a European Molecular Biology

Organization fellowship (ALTF 116-2012) and currently is a Marie
Curie fellow (FP7-PEOPLE-2012-IEF, Proposal 330621). F.P. is
supported by a Wellcome Trust Investigator Award.
10.1126/science.aad0865

sciencemag.org SCIENCE

28 AUGUST 2015 VOL 349 ISSUE 6251

Published by AAAS

ILLUSTRATION: K. SUTLIFF/SCIENCE

Commensal microbiota

Microbiota RORgulates intestinal suppressor T cells

Ahmed N. Hegazy and Fiona Powrie
Science 349, 929 (2015);
DOI: 10.1126/science.aad0865

This copy is for your personal, non-commercial use only.

If you wish to distribute this article to others, you can order high-quality copies for your
colleagues, clients, or customers by clicking here.

The following resources related to this article are available online at

www.sciencemag.org (this information is current as of August 27, 2015 ):
Updated information and services, including high-resolution figures, can be found in the online
version of this article at:
http://www.sciencemag.org/content/349/6251/929.full.html
A list of selected additional articles on the Science Web sites related to this article can be
found at:
http://www.sciencemag.org/content/349/6251/929.full.html#related
This article cites 12 articles, 5 of which can be accessed free:
http://www.sciencemag.org/content/349/6251/929.full.html#ref-list-1
This article appears in the following subject collections:
Microbiology
http://www.sciencemag.org/cgi/collection/microbio

Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the
American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright
2015 by the American Association for the Advancement of Science; all rights reserved. The title Science is a
registered trademark of AAAS.

Downloaded from www.sciencemag.org on August 27, 2015

Permission to republish or repurpose articles or portions of articles can be obtained by

following the guidelines here.