Type I IFN promotes IL-10 production from T cells to suppress Th17 cells and Th17-associated autoimmune inflammation.

Abstract

Whereas the immune system is essential for host defense against pathogen infection or endogenous danger signals, dysregulated innate and adaptive immune cells may facilitate harmful inflammatory or autoimmune responses. In the CNS, chronic inflammation plays an important role in the pathogenesis of neurodegenerative diseases such as multiple sclerosis (MS). Our previous study has demonstrated a critical role for the type I IFN induction and signaling pathways in constraining Th17-mediated experimental autoimmune encephalomyelitis (EAE), an animal model of human MS. However, it remains unknown if self-reactive Th17 cells can be reprogrammed to have less encephalitogenic activities or even have regulatory effects through modulation of innate pathways. In this study, we investigated the direct effects of type I IFN on Th17 cells. Our data show that IFNβ treatment of T cells cultured under Th17 polarizing conditions resulted in reduced production of IL-17, but increased production of IL-10. We also found that IFNβ induced IL-10 production by antigen specific T cells derived from immunized mice. Furthermore, IFNβ treatment could suppress the encephalitogenic activity of myelin-specific T cells, and ameliorate clinical symptoms of EAE in an adoptive transfer model. Together, results from this study suggest that IFNβ may induce antigen-specific T cells to produce IL-10, which in turn negatively regulate Th17-mediate inflammatory and autoimmune response.

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Type I IFN Promotes IL-10 Production from T Cells to
Suppress Th17 Cells and Th17-Associated Autoimmune
Inflammation
Lixia Zhang1, Shunzong Yuan1, Genhong Cheng2*, Beichu Guo1*
1Department of Microbiology and Immunology, Hollings Cancer Center, Medical University of South Carolina (MUSC), Charleston, South Carolina, United States of
America, 2Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles (UCLA), Los Angeles, California, United States of
America
Abstract
Whereas the immune system is essential for host defense against pathogen infection or endogenous danger signals,
dysregulated innate and adaptive immune cells may facilitate harmful inflammatory or autoimmune responses. In the CNS,
chronic inflammation plays an important role in the pathogenesis of neurodegenerative diseases such as multiple sclerosis
(MS). Our previous study has demonstrated a critical role for the type I IFN induction and signaling pathways in constraining
Th17-mediated experimental autoimmune encephalomyelitis (EAE), an animal model of human MS. However, it remains
unknown if self-reactive Th17 cells can be reprogrammed to have less encephalitogenic activities or even have regulatory
effects through modulation of innate pathways. In this study, we investigated the direct effects of type I IFN on Th17 cells.
Our data show that IFNb treatment of T cells cultured under Th17 polarizing conditions resulted in reduced production of
IL-17, but increased production of IL-10. We also found that IFNb induced IL-10 production by antigen specific T cells
derived from immunized mice. Furthermore, IFNb treatment could suppress the encephalitogenic activity of myelin-specific
T cells, and ameliorate clinical symptoms of EAE in an adoptive transfer model. Together, results from this study suggest
that IFNb may induce antigen-specific T cells to produce IL-10, which in turn negatively regulate Th17-mediate
inflammatory and autoimmune response.
Citation: Zhang L, Yuan S, Cheng G, Guo B (2011) Type I IFN Promotes IL-10 Production from T Cells to Suppress Th17 Cells and Th17-Associated Autoimmune
Inflammation. PLoS ONE 6(12): e28432. doi:10.1371/journal.pone.0028432
Editor: Karen L. Mossman, McMaster University, Canada
Received May 6, 2011; Accepted November 8, 2011; Published December 6, 2011
Copyright: ? 2011 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported in part by a Special Fellowship from the Lymphoma and Leukemia Society, a startup fund from MUSC/HCC and a NIH/NIAID K22
AI87707 award to BG, and by NIH research grants R01 AI056154 and AI069120 to GC. No additional external funding received for this study. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: guobe@musc.edu (BG); genhongc@microbio.ucla.edu (GC)
Introduction
Accumulating evidence indicates that chronic Inflammation is
associated with a variety of human diseases. Therefore, constrain-
ing the inflammatory function of immune cells may provide a
novel strategy to treat or control many chronic diseases, such as
multiple sclerosis (MS) [1,2,3]. In response to pathogens, innate
immune cells quickly upregulate pro-inflammatory cytokines that
serve to initiate host defense against microbial invasion. However,
excessive inflammation may cause tissue damage and activation of
autoreactive T and B cells, which may have deleterious effects on a
host. To prevent collateral damage and autoimmunity, hosts also
develop a number of regulatory mechanisms, including generating
Tregs and production of IL-10, to maintain homeostasis of the
immune system. IL-10 is a potent anti-inflammatory cytokine with
broad effects on both innate and adaptive immune systems
[4,5,6,7,8,9]. During bacterial or viral infection, IL-10 is produced
by macrophages and DCs as a negative feedback mechanism to
dampen uncontrolled production of inflammatory cytokines. In
addition to innate cells, T cells, especially regulatory T cells, are
able to produce IL-10 to inhibit the activation of antigen-specific
cells and inflammatory response. Recently, studies from other and
our groups indicate that type I IFN is able to exert its anti-
inflammatory role through the induction of IL-10 and IL-27 from
macrophages and DCs [9,10,11,12].
When encountering specific antigens presented on APCs, naı ¨ve
T cells differentiate into distinct subsets of effector cells.
Depending upon cytokine milieu generated by macrophages and
DCs, CD4 T cells can become different T helper subsets such as
Th1, Th2, and Th17, or regulatory T cells such as Foxp3Treg and
Tr1 cells [13,14,15,16,17,18,19,20,21,22]. While Th1 cells are
required for the clearance of intracellular pathogens, Th17 is
involved in immune response against extracellular pathogens. On
the other hand, Th17 cells have been shown to associate with
pathogenesis of inflammatory autoimmune diseases, including
MS and experimental autoimmune encephalomyelitis (EAE)
[3,23,24,25,26,27,28]. Emerging evidence suggests that there is
significant flexibility or plasticity among different Th subsets or
between Th subsets and regulatory T cells [19,29,30,31,32,33,34].
MS and EAE are characterized by the infiltration of inflammatory
cells, including macrophages and self-reactive T cells, into the
central nervous system (CNS) that leads to neuron damage
[2,3,8,23,35,36,37,38,39]. Recent studies suggest that Th17 cells,
a novel subtype of CD4+T helper cells, play an important role in
the development of MS and EAE [3,40,41,42,43,44]. However,
experimental and clinical data indicate that CNS inflammation
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can result from over-activation of either Th1 or Th17, or both.
Despite extensive studies, the cellular and molecular events
triggering MS as well as regulatory mechanisms limiting the
initiation and progression of CNS inflammation are still not well
understood. To date, there are no curative treatments for MS.
Recent studies from other and our groups have demonstrated
that IFNb induction and signaling pathways play critical roles in
suppressing Th17-associated autoimmune and inflammatory
diseases including EAE [10,11,12,43,45]. The type I IFN,
consisting of a single IFNb and multiple IFNa members, is
induced by TLR or cytoplasmic RNA and DNA sensors. IFNa
and IFNb bind to a common receptor, the type I IFN receptor
(IFNAR), expressed on a wide variety of cell types, leading to
induction of a large set of genes important for antiviral responses
and other cellular functions. In addition to their antiviral
functions, types I IFNs are capable of exerting immunomodulatory
effects on both innate and adaptive immune cells. IFNa and IFNb
have been used to treat patients with cancer or autoimmune
diseases, particularly multiple sclerosis [35,45,46,47,48,49,50,51].
Our previous study shows that that type I IFN is required for LPS-
induced IL-10 upregulation in macrophages [9]. Moreover, our
recent studies revealed a novel immunoregulatory or immuno-
suppressive function of IFN induction pathway in innate and
antigen-specific immune response. We found that mice lacking
IFNa/b receptor (IFNAR) had enhanced development of Th17
cells in vivo and developed much more severe EAE than wild type
mice. Experimental results from our laboratory and others have
also provided critical links between IFNa/b and the induction of
IL-27 from innate immune cells in the inhibition of Th17 cell
differentiation. Recent studies suggest that IL-27-induced produc-
tion of IL-10 may contribute to its immunosuppressive effects
[52,53,54,55,56,57].
Although our previous studies indicate that IFNb is able to exert
its anti-inflammatory role through macrophages and DCs, the
direct effect of type I IFN on Th17 cell differentiation and
plasticity remains less understood. As our previous studies show
that IFNa/b can directly upregulate IL-10 expression in
macrophages, we hypothesize that IFNb may directly induce IL-
10 production from T cells, consequently, inhibiting IL-17
production. Results from this study show that IL-10 expression
was significantly upregulated in CD4 T cells upon IFN
stimulation. Furthermore, IFN-treated encephalitogenic T cells
generated less EAE in vivo in an adaptive transfer model. Data
from this study support a novel mechanism in which IFNb-
induced IL-10 production may contribute to IFN-mediated
inhibition of Th17-associated inflammation.
Results
Type I IFN directly acts on Th17 cells
Our previous studies have demonstrated that type I IFN
induction and signaling pathways in innate immune cells play an
important role in limiting Th17-mediated autoimmune inflam-
mation. This promoted us to determine if self-reactive Th17 cells
can be modulated directly by IFN pathways. To test this
hypothesis, we firstly examined the direct effect of type I IFN on
Th17 differentiation and activity in vitro. Highly purified naı ¨ve
CD4 T cells cultured under Th17 differentiation condition (IL-6
and TGFb) were treated with IFNa or IFNb, and then cytokine
production and gene expression were analyzed. As shown in
Figure 1A, while naı ¨ve T cells in the Th17 polarizing condition
produced a significant amount of IL-17, adding exogenous IFNb
into the T cell culture led to decreased production of IL-17 in a
dosage-dependent manner. In contrast to wt T cells, the
production of IL-17 from IFNAR deficient T cultured under
Th17 condition was not affected by type I IFN. Furthermore,
intracellular cytokine staining reveled that IFNb reduced the
frequency of IL-17-positive T cells cultured in Th17-polarizing
conditions (Fig. 1B).
Differentiation of different CD4 T cell subsets is controlled by
distinct transcriptional factors. Th17 differentiation program is
orchestrated by the transcriptional factor RORct, which itself is
induced by TGFb and IL-6. To determine the effect of IFNb on
the levels of gene expression in Th17 cells, the expression of
mRNA encoding IL-17 and RORc in Th17 cells treated with
IFNb was measured by Quantitative RT-PCR. Figure 2 shows
that IFNb could significantly inhibit RORct expression in T cells
stimulated with TGFb and IL-6. Consequently, a direct treatment
of naı ¨ve T cells cultured in Th17-polarizing condition with type I
IFN led to reduced expression of IL-17 (Fig. 2).
Type I IFN inhibits Th17 cells via both IL-10-dependent
and -independent mechanisms
Next, we would like to determine the mechanisms by which
IFNb directly inhibits IL-17 production from T cells. We
hypothesized that IFNb could potentially induce T cells under
Th17 polarizing conditions to produce IL-10, which in turn limits
IL-17 production. To test this, naı ¨ve CD4 T cells were cultured in
Th17 culture conditions in the presence of recombinant IFNa or
IFNb for 72 hours, induction of IL-10 was measured by ELISA
and intracellular cytokine staining. As shown in Figure 3A and 3B,
IFNa and IFNb induced IL-10 production but inhibited IL-17
production in Th17-polarized cells. Accordingly, FACS analysis
revealed that IFNb reduced the percentage of IL-17+cells and
increased IL-10+T cell populations (Figure 4). We also found that
treatment of IFNa/b increased percentage of IL-17 and IL-10
double positive T cells.
To determine if IFNb-mediated inhibitory effects on Th17 cells
is completely dependent on IL-10, we examined the effect of type I
IFN on T cells from IL-10 KO mice in Th17 culture. While IL-10
deficient T cells produced more IL-17 compared with wt T cells,
IFNb could still exert certain degree of inhibition on Th17 cells,
albeit in a less efficient manner (Fig. 3C). These results imply that
IFN-induced IL-10 contributes to the negative regulatory function
of IFNb, but IL-10-independent mechanisms also exist in IFN-
mediated inhibition of Th17 responses.
Synergy between IFNa/b and IL-27 in the induction of IL-
10
Our previous results show that IFNb could induce IL-27
production from innate immune cells. Consistent with studies
reported by other groups, we also found that IL-27 induced
production of IL-10 in T-cells (data not shown). We further
examined if IFNb and IL-27 could cooperate in the modulation of
Th17 cells. T cells in Th17 culture were treated with either IFNb
or IL-27 alone or together at different concentrations, the
induction of IL-17 and IL-10 was measured by intracellular
cytokine staining and ELISA. Interestingly, our data demonstrate
that IFNb and IL-27 acted in synergy to inhibit the production of
IL-17, and to promote the induction of IL-10 in Th17 polarizing
culture (Fig. 5A and 5B).
Effects of IFNb on Th17 proliferation and IL-10
production
The altered profile in IL-10/IL-17 production by type I IFN
might be related to cytokine skewing or caused by preferentially
promoting growth of IL-10 producing cells during T cell
Type I IFN Reprograms Th17 Cells to Produce IL-10
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differentiation. To test these possibilities, we evaluated prolifera-
tion of IL-10 and IL-17 producing CD4 T cells by a CFSE
labeling assay. T cells were labeled with the cytosolic dye CFSE,
then cultured at Th17 condition in the presence of type I IFN. As
shown in Figure 6A, both IL-10 and IL-17 producing T cells were
actively dividing. Moreover, the rate of T cell proliferation in
either IL-10+or IL-17+populations was generally comparable,
although IL-10+T cells divided slightly less than IL-17 positive T
cells. These results indicate that IFN most likely promoted the
expression of IL-10 from T cells, rather than selectively induced
Figure 1. IFNb directly acts on Th17 cells. (A) Naive CD4+T cells from wt and IFNAR KO mice were purified by fluorescence-activated cell sorting,
and stimulated with solid-phase anti-CD3 (5 mg/ml) and soluble anti-CD28 (2 mg/ml) under Th17 polarizing condition (TGFb plus IL-6, and anti-IL-4/
anti-IFNc). IFNb at different concentration was added at the beginning of culture. After 72 hours, IL-17 production in the culture supernatants was
measured by ELISA. Results are reported as mean6SD of triplicate samples from one representative experiment. (B) Flow cytometry analysis of Th17
development in CD4 T cells cultured in the presence of IFNb. Naive CD4 T cells were cultured in the Th17 condition in the presence of IFNb (0, 250 U,
500 U/ml). After 72 hours, cells were stained for surface CD4 and intracellular IL-17. Plots were gated on CD4+T cells. Data are representative of five
independent experiments with similar results.
doi:10.1371/journal.pone.0028432.g001
Type I IFN Reprograms Th17 Cells to Produce IL-10
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the expansion of IL-10 expressing T cells in Th17-polarizing
conditions.
To further confirm if type I IFN could influence the fully
differentiated Th17 cells, we cultured T cells for three rounds in
Th17 polarizing conditions. In this experiment, naı ¨ve T cells were
cultured in the presence of TGFb and IL-6 for 3 days, then
differentiated T cells were collected, washed and re-cultured under
Th17 polarizing conditions for two more rounds. In the third round
of Th17 culture, we treated differentiated Th17 cells with IFNb.
When these in vitro generated Th17 cells were cultured again in the
Th17-polarizign condition (IL-6/TGFb), IL-17 production was
maintained. Consistent with our observations on naı ¨ve T cells, IFNb
treatment induced IL-10 production, but inhibited IL-17 production
by T cells in the third round of Th17 polarizing culture (Figure 6B).
Our data also show that IFNb could still enhance the percentage of
IL-17/IL-10 doublepositive T cells insuchculture system.Although
T cells after multiple rounds of differentiation culture may still
contain mixed subpopulations, our data imply that IFNb was able to
modulate IL-17 and IL-10 production in well differentiated Th17
cells. These results also indicate that at least some of IL-10 could be
produced by fully differentiating Th17 cells. However, further
studies will be required to define the cell populations responsible for
IFN-induced IL-10 production in T cells.
Induction of IL-10 in antigen-specific T cells
Our results demonstrate that treatment of naı ¨ve T cells under
Th17 polarizing condition with IFNb could result in the increased
Figure 3. Type I IFN upregulates IL-10 production by Th17
cells. (A) and (B) Naı ¨ve CD4 T cells were cultured in Th17 culture
conditions in the presence of purified IFNa or IFNb (500 U/ml) for
72 hours. Production of IL-17 and IL-10 by CD4 T cells was determined
by ELISA. (C) IL-102/2naive CD4 T cells were cultured in Th17 culture
condition with IFNb for 72 hours, the concentration of IL-17 in the
supernatants was measured by ELISA. Results are reported as mean6SD
of triplicate samples from one representative experiment. Data are
representative of five (A, B) and three (C) experiments with similar
results.
doi:10.1371/journal.pone.0028432.g003
Figure 2. IFN-mediated inhibition of gene expression in Th17
cells. (A) The expression of IL-17A mRNA in Th17 cells treated with
IFNb. Naive T cells were cultured under Th17 condition in the presence
of IFNb (500 U/ml) for 3 days, the expression level of IL-17 mRNA was
measured by Quantitative RT-PCR. Values are normalized to their
average beta-actin values, and are presented as relative expression
units. Error bars indicate 6 SD among duplicate samples from one
experiment. (B) Quantitative RT-PCR of the expression of mRNA
encoding RORct in Th17 cells treated with IFNb. Values are normalized
to their average beta-actin values and are presented as relative
expression units. Error bars indicate 6 SD among duplicate samples
from one experiment. Data are representative of three independent
experiments with similar results.
doi:10.1371/journal.pone.0028432.g002
Type I IFN Reprograms Th17 Cells to Produce IL-10
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expression and production of IL-10. We next investigated if IFNb
could induce IL-10 production from antigen specific Th17 cells.
To test this, spleen cells isolated from MOG peptide-immunized
mice were re-stimulated with the peptide ex vivo in the presence of
IFNb, and then the induction of IL-17 and IL-10 by T cells was
measured by ELISA or FACS analysis. As reported previously,
IFNb inhibited IL-17 production from antigen-specific T cells
(Figure 7A). Particularly, we found that IFNb could induce IL-10
production from antigen-specific T cells generated from in vivo
immune response (Figure 7B and 7C). Results from these studies
suggest that IFNb may induce antigen-specific T cells, possibly
MOG-specific Th1 and Th17 cells, into IL-10-producing T cells.
IFNb decreases the encephalitogenic activity of antigen-
specific Th17 cells and ameliorates symptoms of EAE
We next investigated effects of type I IFN on the pathogenic
potential of self-reactive T cells. To test if IFNb treatment can
reduce the encephalitogenic activity of antigen-specific T cells, we
utilized adoptive transfer experiments in which MOG-specific
CD4 T cells were transferred into naı ¨ve wt mice. Spleen and
Figure 4. Upregulation of IL-10 expression in T cells by IFNb. Naı ¨ve CD4 T cells were cultured in Th17 polarizing conditions as described
above in the presence of IFNb for 72 hours. Expression of IL-17 and IL-10 in CD4 T cells was determined by intracellular cytokine staining. Data are
representative of five experiments with similar results.
doi:10.1371/journal.pone.0028432.g004
Type I IFN Reprograms Th17 Cells to Produce IL-10
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