The Metalloprotease ADAM12 Regulates the Effector
Function of Human Th17 Cells
Angela X. Zhou1☯, Aimee El Hed1☯, Frances Mercer1, Lina Kozhaya1, Derya Unutmaz1,2,3*
1 Department of Microbiology, New York University School of Medicine, New York, New York, United States of America, 2 Department of Pathology, New York
University School of Medicine, New York, New York, United States of America, 3 Department of Medicine, New York University School of Medicine, New York,
New York, United States of America
A key modulator of immune homeostasis, TGFβ has an important role in the differentiation of regulatory T cells
(Tregs) and IL-17-secreting T cells (Th17). How TGFβ regulates these functionally opposing T cell subsets is not well
understood. We determined that an ADAM family metalloprotease called ADAM12 is specifically and highly
expressed in both Tregs and CCR6+ Th17 cells. ADAM12 is induced in vitro upon differentiation of naïve T cells to
Th17 cells or IL-17-secreting Tregs. Remarkably, silencing ADAM12 expression in CCR6+ memory T cells enhances
the production of Th17 cytokines, similar to suppressing TGFβ signaling. Further, ADAM12 knockdown in naïve
human T cells polarized towards Th17/Treg cells, or ectopically expressing RORC, greatly enhances IL-17-secreting
cell differentiation, more potently then inhibiting TGFβ signals. Together, our findings reveal a novel regulatory role
for ADAM12 in Th17 cell differentiation or function and may have implications in regulating their aberrant responses
during immune pathologies.
Citation: Zhou AX, El Hed A, Mercer F, Kozhaya L, Unutmaz D (2013) The Metalloprotease ADAM12 Regulates the Effector Function of Human Th17
Cells. PLoS ONE 8(11): e81146. doi:10.1371/journal.pone.0081146
Editor: Aftab A. Ansari, Emory University School of Medicine, United States of America
Received June 25, 2013; Accepted October 18, 2013; Published November 21, 2013
Copyright: © 2013 Zhou 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 study was funded by grants from National Institutes of Health (NIH) grant R01AI065303 and NIH R21AI087973 to DU and NIH training grant
5T32AI007647 to AZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: We have read the journal's policy and have the following conflicts: A patent application has been submitted by NYU Medical School
entitled "Methods for modulating TGF-beta signaling", serial number: 13/355,798. The application involves potential targeting of ADAM12 to modulate
signaling in Th17 cells. This does not alter our adherence to all the PLoS ONE policies on sharing data and materials as detailed in your guide for the
* E-mail: Derya@mac.com
☯ These authors contributed equally to this work.
Th17 cells are characterized by their secretion of IL-17, an
inflammation-inducing cytokine that is implicated in the
pathogenesis of several autoimmune processes, including
asthma, systemic lupus erythematosus, colitis and allograft
rejection . Th17 cell differentiation requires the combination
of TGFβ and pro-inflammatory cytokines including IL-6, IL-1β
and IL-23 . TGFβ is also crucial for generating induced
regulatory T cells, a portion of which secrete IL-17, and have
anti-inflammatory functions in controlling excessive immune
response [3-6]. How TGFβ signals regulate programming of
these functionally distinct IL-17-secreting T cell subsets are not
TGFβ is a pleiotropic cytokine involved in development,
fibrosis, wound healing, and immune regulation [7,8]. Part of
the regulation of TGFβ occurs through modulation of its
signaling. After secretion and processing, mature TGFβ is first
recognized by TGFβRII,
phosphorylates TGFβRI [8,9]. The receptor-ligand complex is
which then recruits and
typically endocytosed into early endosomes where it initiates
the downstream signaling cascade through R-Smads . A
recent study suggested that a member of the ADAM (a
disintegrin and metalloprotease) family of metalloproteases,
ADAM12, could interact with TGFβRII  and that this
interaction could enhance TGFβ signaling through control of
TGFβR localization and stability on early endosomes [11,12].
23 ADAMs have been identified in humans . All are cell-
surface proteins containing multiple domains that exert a
variety of effects on cell adhesion and migration . In
addition, a number of ADAMs, including ADAM12, contain
active metalloprotease domains that can mediate “shedding” of
cell surface proteins to activate and regulate their functions
[14,15]. ADAM12 is expressed primarily in mesenchymal
tissues that form skeletal muscle and bone and may have
functions during muscle regeneration after injury . In
addition, ADAM12 has been implicated in a number of
diseases, including musculoskeletal and neurological disorders
and cancer . Interestingly, ADAM12 was found to be
significantly upregulated on T cells infiltrating the spinal cords
PLOS ONE | www.plosone.org
1November 2013 | Volume 8 | Issue 11 | e81146
of mice in an EAE model, which is mediated by pathological
Th17 cell effector functions .
In this study, we determined that ADAM12 is highly and
specifically expressed in human IL-17-secreting T cells and
most Tregs. Knockdown of ADAM12 in primary human memory
T cells significantly enhanced the proportion of cells producing
IL-22, IL-17A, IL-17F, and both IL-17A and IFNγ, in both TGFβ-
dependent and -independent manners. Silencing ADAM12 in
naïve cells also greatly enhanced their differentiation into IL-17-
secreting T cells. Our results suggest that ADAM12 is an
important regulator of Th17 cell differentiation and effector
Materials and Methods
T cell purification
PBMCs from healthy individuals were prepared using Ficoll-
Paque plus (GE Healthcare) from discarded buffy coats
obtained anonymously from the New York Blood Center (New
York, NY). All donor samples were non-identifiable and did not
involve any donor-specific information for data analysis and
therefore consent forms were not required. All human material
was obtained and processed according to guidelines and
approval of NYU School of Medicine Institutional Human
Subjects Board. CD4+ T cells were isolated using Dynal CD4
Positive Isolation Kit (Invitrogen) and were >99% pure. CD4+
cells were sorted by flow cytometry (FACSAria; BD
Biosciences) on the basis of expression of CD45RO and CD25
for naïve T cells, memory T cells, naïve Tregs and memory
Tregs as described previously . Sorted subsets were >99%
pure and were kept at 37°C and 5% CO2 in Roswell Park
Memorial Institute 1640 medium with 10% fetal calf serum.
T cell activation and infection
CD4+ T cell subsets were stimulated using plate-bound anti-
CD3 antibody and soluble anti-CD28 or monocyte-derived
dendritic cells (MDDCs) and anti-CD3 (OKT3), and maintained
in IL-2-containing media. Activated cells were infected with
lentiviruses as indicated. The empty vector and RORC-cDNA
containing lentiviruses pseudotyped with VSVG envelope were
generated as previously described and express GFP as the
marker in place of the nef gene [20-23]. The lentivirus-encoding
RORC gene, RORC-IRES-GFP, was a gift from Dr. Dan
Littman (New York University School of Medicine, New York,
NY). ADAM12 and control lentiviral shRNAs, which encode
puromycin selection marker, were purchased from Sigma. For
shRNA transductions, 2ug/ml puromycin (Sigma) was added
on day 4 post-activation. For in vitro polarizations, Tn or naïve
Tregs were cultured in IL-1β (10ng/ml), IL-23 (100ng/ml) and
TGFβ (10ng/ml) and maintained in IL-2 for 12 days, as
previously described . For cytokine staining, cells were
reactivated for 5h with Phorbol 12-myristate 13-acetate (PMA
20 ng/mL; Sigma) and ionomycin (500 ng/mL; Sigma) in the
presence of GolgiStop (Brefeldin A; BD Biosciences). In the
experiments blocking TGFβ signals, cells were either treated
with SJN2511 (Tocris bioscience) or TGFβ neutralizing
antibody (clone 1D11; R&D) at the time of activation.
RNA isolation and quantitative RT-PCR
Purified T cells were flash frozen in liquid nitrogen. Total
RNA was isolated using RNeasy isolation kit (Qiagen)
according to the manufacturer’s protocol, reverse transcribed
into cDNA and quantified using an Applied Biosystems 7300
apparatus (Foster City, CA). Taqman primers and probe mixes
were purchased from Applied Biosystems, and their IDs are as
follows: Human ADAM12 (Hs01106101_m1), ADAM12S
(Hs00244640_ml), IL-17A (Hs99999082_ml) and β-Actin
Staining and FACS sorting analysis
Cells were stained with corresponding antibodies, as
previously described . For intracellular staining, fixation and
permeabilization was performed using FOXP3 staining Kit
(eBioscience) in accordance
instructions. Analyses were performed using LSRII flow
cytometer (BD Biosciences, San Jose, CA) and FlowJo
software (Tree Star Inc, Ashland, OR). The following anti-
human antibodies were used for staining: CD25, CD45RO,
interferon (IFN)-γ, IL-17A, IL-17F, IL-22 (Biolegend) and CCR6
with the manufacturer's
Ex vivo isolation of Th17 cells
Technique for isolation of Th17 cells from total CD4+ T cells
was adapted from Streeck et al. . Briefly, T cells were
activated for 3.5h using PMA and ionomycin as described
above. Cells were washed with ice-cold PBS containing 2%
fetal bovine serum and resuspended in PBS and 2% FBS. The
IL-17 capture complex containing anti-CD45-biotin combined
with anti-IL-17-biotin was prepared and vortexed thoroughly
before mixing in free avidin solution to link the two antibodies.
The complex was then incubated for 10min at room
temperature before adding to cells. After 15 minutes on ice,
RPMI was added and cells were incubated at 37C with 5% CO2
for 1.5h. Cells were washed again and surface stained using
IL-17-PE antibody and sorted on FACS ARIA for IL17+ and
All statistical analyses were performed with GraphPad Prism
Software (GraphPad Inc., La Jolla, CA). The significance was
determined using One-Sample Student’s t-test.
Expression of ADAM12 is enriched in IL-17+ cells
During a microarray analysis of differential gene expression
in human T cell subsets [19,26], we observed that a member of
the ADAM family of metalloproteases, ADAM12 was
specifically expressed in CCR6+ memory T cells (Tm), in a
discovery based microarray analysis. We confirmed this
preferential and high expression of ADAM12 mRNA in highly
purified CCR6+ and CCR6- Tm cells (Figure 1A) through qRT-
PCR (Figure 1B). ADAM12 expression in CCR6+ Tm subsets
ADAM12 Regulates IL-17 Secretion
PLOS ONE | www.plosone.org2November 2013 | Volume 8 | Issue 11 | e81146
correlated with expression of the Th17 cell signature genes,
RORC and IL-17 (Figure 1B).
ADAM12 is also highly expressed on infiltrating T cells in the
CNS of an EAE mouse model, which is dependent on
dysregulated Th17 cell functions . Based on our finding of
Figure 1. ADAM12 expression in Th17 cells. (A) CD4+ T cells isolated from human PBMCs were stained for CCR6, CD25 and
CD45RO expression and sorted into CCR6+ and CCR6- Tm (CD25- CD45RO+) cells. Representative FACS plots showing gating
for isolation of CCR6+ and CCR6- Tm cells. Cells were subsequently activated by PMA and ionomycin and separated based on
secretion of IL-17 as described in materials and methods. (B) CCR6+ and CCR6- cells were activated by plate-bound anti-CD3 and
soluble anti-CD28 antibody for 16 hours and relative levels of RORC, IL-17 and ADAM12 were determined by qRT-PCR. (C)
Expression of RORC, IL-17 and ADAM12 in ex vivo isolated Th17 subsets was determined by qRT-PCR relative to expression on
CCR6- Tm cells. * p<0.05, ** p<0.01.
ADAM12 Regulates IL-17 Secretion
PLOS ONE | www.plosone.org3 November 2013 | Volume 8 | Issue 11 | e81146
preferential expression of ADAM12 in CCR6+ T cells, which
contain all Th17 cells, we hypothesized that ADAM12 may be
expressed at higher levels in IL-17-secreting Th17 cells. To test
this, we adapted a method  to isolate IL-17 secreting cells
ex vivo from CD4+CD45RO+CCR6+ T cells (Figure 1A).
Indeed, we found that ADAM12 expression was much higher in
IL-17-secreting T cells, and also correlated with RORC and
IL-17 mRNA expression (Figure 1C).
In order to verify that the preferential expression in CCR6+
Tm cells was unique to ADAM12, we also determined the
relative expression of two other ADAM genes: ADAM19 and
ADAM22. Like ADAM12, ADAM19 was shown to be
upregulated specifically on T cells in the spinal cords of EAE
mice, although to a much lower extent . In contrast to
ADAM12, we did not observe specific expression of ADAM19
and ADAM22 within CD4+ CCR6+ Tm cell subsets (Figure
Silencing ADAM12 expression in memory T cells
enhances Th17 cytokine secretion
To examine the role of ADAM12 in CCR6+ Tm cells, we
transduced purified CCR6+ cells with lentiviruses encoding
ADAM12 shRNA. Using qRT-PCR, we confirmed that the
shRNAs silenced gene expression of ADAM12 by 50-80%
(Figure S2A). We then reactivated these shRNA-expressing
cells through the TCR and assessed for changes in cytokine
and transcription factor expression (Figure S2B).
Based on previous reports describing the potential role of
ADAM12 in enhancing TGFβ signaling , we hypothesized
that the knockdown of ADAM12 would mimic the actions of
inhibiting TGFβ signals. We found that CCR6+ Tm cells
transduced with ADAM12 shRNA did not impact IFNγ secretion
or FOXP3 expression (Figure 2A and B). Strikingly, however,
ADAM12 knockdown caused significant increase in the
numbers of cells secreting Th17 cytokines, IL-17A, IL-17F, and
IL-22 (Figure 2C-E). In addition, populations of cells producing
both IL-17A and IFNγ were greatly increased upon silencing
ADAM12 expression (Figure 2F). Blocking TGFβ signals with
an inhibitor or neutralizing antibody also increased Th17
cytokine production, in line with previous reports that high
concentrations of TGFβ is inhibitory on IL-17-secreting cells
[27,28], but to a lower extent compared to ADAM12 knockdown
(Figure 2C-F). We also observed that blocking TGFβ signals on
cells where ADAM12 was silenced could further augment the
secretion of Th17 cytokines (Figure 2C-F). Together, these
findings suggest that reducing ADAM12 levels is either more
potent than blocking TGFβ signals via inhibitors or neutralizing
antibodies, or that ADAM12 can also function independent of
TGFβ in regulating Th17 cytokines.
ADAM12 expression in RORγt-overexpressing cells
RORγt is a master transcription factor regulator of Th17 cells
. Because ADAM12 expression was Th17 cell-specific and
correlated with RORC expression on Tm cells, we asked
whether RORγt was involved in inducing ADAM12 expression.
To test this, we transduced Tn cells with an RORC-lentivirus,
expanded cells in IL-2-and performed qRT-PCR for ADAM12
mRNA expression. We found that RORC-transduced cells had
also upregulated both IL-17 and ADAM12 expression (Figure
3A). Further, upon sorting IL-17-secreting RORC-transduced
cells, we found that ADAM12 was also preferentially
upregulated in the IL-17+ cells (Figure 3B), similar to our ex
vivo observations (Figure 1B and C). To determine the role of
ADAM12 on IL-17 expression in these cells, we silenced
ADAM12 expression in RORC-transduced cells using ADAM12
shRNA, as described above. Similar to CCR6+ Tm cells, the
knockdown of ADAM12 in RORC-transduced cells resulted in
increased expression of IL-17 (Figure 3C and D). Inhibiting
TGFβ signals with a TGFβ signaling inhibitor also increased
Th17 cytokine production in some donors, whereas treating
with exogenous TGFβ inhibited cytokine production completely
Regulatory T cells also express ADAM12
Because Tregs also require TGFβ for their differentiation ,
we next determined if ADAM12 was expressed in Tregs. We
found that, ADAM12 was enriched in all Tregs regardless of
CCR6 expression (Figure 4A). However, as expected, IL-17
expression was preferentially expressed in CCR6+ FOXP3+
Tregs (Figure 4A). A recent study demonstrated that IL-17-
secreting cells could be expanded from naïve Tregs using a
cytokine cocktail of IL-2, IL-23, IL-1β and TGFβ . To
determine if ADAM12 was induced during the course of Th17
polarization, we cultured naïve T cells and Tregs in these Th17
polarizing cytokines for 12 days and then extracted RNA for
qRT-PCR. We found that
upregulated 10-fold in Th17-polarized cells compared to non-
polarized cells in both Tregs and Teff cells (Figure 4B). Since
silencing ADAM12 enhanced Th17 cytokine secretion in Tm
and RORC-transduced cells, we asked whether ADAM12 also
regulated Th17 cell differentiation from naïve T cells to Th17
cells. To test this, we transduced naïve Tregs with control or
ADAM12 shRNA after activation through the TCR, and cultured
cells either in IL-2 media or in the polarizing cytokine cocktail.
Reducing ADAM12 gene expression, in the presence of
polarizing cytokines, further augmented generation of T cells
secreting Th17-cytokines (Figure 4C and D), most of which
also expressed FOXP3 (data not shown). Remarkably,
ADAM12 knockdown increased levels of IL-17 and IL-22 even
in the absence of Th17 polarizing cytokines (Figure 4C-E).
Similar to Tm cells, IFNγ levels were not affected (Figure 4F
and G), however, cells expressing both IL-17 and IFNγ were
significantly increased in polarized naïve Tregs (Figure 4F and
ADAM12 expression was
In this study, we determined that a surface metalloprotease,
ADAM12, is highly expressed in both Th17 cells and Tregs.
ADAM12 was induced by the Th17 master transcription factor
RORγt and Th17 polarizing cytokines, suggesting it is
upregulated as part of Th17 programming. We discovered that
silencing ADAM12 expression greatly enhanced the production
of Th17 cytokines.
We observed that CCR6+ Tm cells and both CCR6+ and
CCR6- Tregs expressed high levels of ADAM12. The
ADAM12 Regulates IL-17 Secretion
PLOS ONE | www.plosone.org4 November 2013 | Volume 8 | Issue 11 | e81146
preferential expression of ADAM12 in both Tregs and CCR6+
Tm cells supports proposed similarities in the development of
Th17 and Treg lineages . TGFβ, which is important for
development of both Th17 and Tregs, has also been reported
to induce expression of ADAM12 in hepatic stellate cells,
fibroblasts and epithelial cells [30-32]. Part of the TGFβ-
mediated ADAM12 induction
degradation of SnoN, which was shown to transcriptionally
repress ADAM12 in fibroblasts . TGFβ signals may underlie
the high levels of ADAM12 in both populations. It is possible
that TGFβ might also upregulate ADAM12 through RORγt,
which is induced by TGFβ , since overexpression of RORγt
was found to upregulate ADAM12 as well (Figure 3).
In response to a knockdown of endogenous ADAM12,
CCR6+ Tm cells increased secretion of IL-17A, IL-17F and
IL-22, which was similar to blocking TGFβ using either a
signaling inhibitor or a neutralizing antibody (Figure 2C-F). This
potential link between ADAM12 and TGFβ signaling in
regulating Th17 function is consistent with previous reports that
ADAM12 interacts with TGFβRII, enhancing TGFβ signaling
through increasing receptor endocytosis and decreasing its
is through proteasomal
ubiquitination . It is conceivable that, in T cells, ADAM12
localizes and stabilizes endosomal TGFβRII, leading to
amplification of TGFβ signals, as a mechanism to
downregulate production of pro-inflammatory cytokines. Since
ADAM12 is also upregulated by TGFβ, this would suggest a
positive feedback relationship between TGFβ and ADAM12.
However, using the TGFβ inhibitors alone did not significantly
enhance cytokine secretion in Tm cells, suggesting that
ADAM12 may also act through signaling pathways independent
of those blocked by TGFβ inhibitors in Tm cells. Future studies
dissecting the molecular mechanisms of ADAM12 functions in
T cells could provide an important framework for understanding
how the Treg-Th17 equilibrium is maintained.
Knockdown of ADAM12 in polarized naive cells either
through RORC transduction or culture in Th17 polarizing
cytokines also enhanced Th17 cytokine secretion. Inhibition of
TGFβ signals in these RORC-transduced Tn cells, also
enhanced their cytokine production, particularly secretion of
IFNγ. This finding suggests a phenotypic difference in the
sensitivity of naïve versus memory T cells to TGFβ-inhibitor
mediated enhancement of cytokine production and might be
Figure 2. Effect of ADAM12 knockdown on Th17 cytokine production by CCR6+ Tm cells. (A) After transduction with control
(shCont.) or ADAM12 (shA12) shRNA, CCR6+ Tm cells were activated in media alone, with a TGFβ signaling inhibitor or with TGFβ
neutralizing antibodies. Graphs show fold change in percentage of cells that were FOXP3+, (B) IFNγ+, (C) IL-17A+, (D) IL-17F+, (E)
IL-22+ or (F) IL-17A+ IFNγ+ on day 4 post-activation compared to shCont.-tranduced cells in media. Statistical significance **
p<0.01, *** p<0.001.
ADAM12 Regulates IL-17 Secretion
PLOS ONE | www.plosone.org5 November 2013 | Volume 8 | Issue 11 | e81146
caused by the suppressive effects of higher levels of TGFβ on
T cell differentiation . It has recently been shown that IL-23
potentiates inflammatory function of Th17 cells [33,34]. In the
absence of IL-23 signals, RORγt-expressing cells have limited
Figure 3. Regulation of Th17 cytokines by ADAM12 expression induced in RORC-transduced Tn cells. (A) Expression of
RORC, IL-17 and ADAM12 was determined in Tn cells transduced with EV or RORC lentiviruses by qRT-PCR. (B) IL-17 expressing
Tn cells transduced with RORC lentivirus were sorted as described in materials and methods and expression of IL-17 and ADAM12
was determined by qRT-PCR. (C, D) Tn cells were transduced with both RORC-lentivirus and ADAM12 (shA12-1) or control shRNA
(shCont.). After selection, cells were reactivated through the TCR and assessed for expression of IFNγ and IL-17. (C)
Representative FACS plots of intracellular cytokine-expression. (D) Percent comparison of IL-17+ cells transduced with shCont or
shA12. Statistical significance: * p<0.05, ** p<0.01.
ADAM12 Regulates IL-17 Secretion
PLOS ONE | www.plosone.org6 November 2013 | Volume 8 | Issue 11 | e81146
Figure 4. Expression and effects of ADAM12 on polarized Tregs. (A) Tm cells and Tregs (CD45RO+ CD25+) were sorted
based on surface CCR6 and analyzed for expression of FOXP3, IL-17 and ADAM12 by qRT-PCR. (B) Teff and Tregs were
activated with or without Th17 polarizing cytokines for 12 days before analyzing for expression of FOXP3, RORC, and ADAM12 by
qRT-PCR. (C-H) Percent of IL-22, IL-17 and IFNγ production in Tregs activated in IL-2 media or Th17 polarizing cytokines after
transduction with either a control (shCont.) or ADAM12 shRNA (shA12-1) was determined by flow cytometry. (C and F)
Representative FACS plots of cytokine expression. Percent comparison of : (D) IL-17+ cells (E) IL-22+ cells (G) IFNγ+ cells (H)
IL17+IFNγ+. Statistical significance: * p<0.05, ** p<0.01, *** p<0.001.
ADAM12 Regulates IL-17 Secretion
PLOS ONE | www.plosone.org7 November 2013 | Volume 8 | Issue 11 | e81146
inflammatory capacity, despite upregulation of IL-17 . One
mechanism that TGFβ uses to block generation of Th17 cells is
by inhibiting expression of IL-23R . In contrast, Tm cells
have already completed the differentiation program and may
therefore be more resistant to modulatory effects of TGFβ
Both CCR6+ and CCR6- Tregs express high levels of
ADAM12, which is further enriched in the IL-17-secreting
Tregs. This is likely due to TGFβ signals, which are involved in
differentiation of both natural and induced Tregs [4-6,36].
ADAM12 may be one factor that limits the capacity of Tregs to
produce effector cytokines and is therefore necessary in all
Treg subsets . Our findings suggest that ADAM12
expression in plays a similar role in regulating IL-17-secretion
in a subset of Tregs that can express this cytokine. However, it
is also plausible that ADAM12 plays additional role in Treg
function given its more broad expression in Tregs.
In summary, our results suggest that ADAM12 functions to
repress secretion of Th17 cytokines and thereby act as
negative feedback modulator of IL-17 secretion to prevent the
potentially destructive nature of inflammatory cytokines on host
tissue. It will be important to determine the levels of ADAM12 in
T cells in pathologies where Th17 cells are implicated, such as
multiple sclerosis patients, psoriasis and other inflammatory
disorders. Future approaches to target ADAM12 expression
could be an important therapeutic strategy during Th17-
associated pathologies. In addition, ADAM12 could act through
a novel mechanism to regulate IL-17-secreting Treg and Tm
cell differentiation and functions, thus is a potential target to
modulate Treg and Th17 cell actions in the context of
inflammation and autoimmunity.
Figure S1. Specificity of ADAM12 expression in CCR6+ Tm
cells. Expression of ADAM12, ADAM19 and ADAM22 in
CCR6+ and CCR6- Tm cells was determined by qRT-PCR.
Figure S2. Knockdown of ADAM12 through shRNA
expression. (A) Levels of ADAM12 mRNA in CCR6+ Tm cells
transduced with shADAM12 (shA12-1 or shA12-2) as a
percentage of ADAM12 mRNA from cells transduced with
control shRNA (shCont.) was determined by qRT-PCR. (B)
Purified CCR6+ Tm cells were transduced with shRNA against
ADAM12 at the time of activation. After 4 days, puromycin was
added to select for cells expressing the lentivirus. Live cells
were purified and reactivated by MDDCs and anti-CD3
antibody before assessing cytokine and FOXP3 expression.
Conceived and designed the experiments: AZ DU. Performed
the experiments: AZ AE FM LK. Analyzed the data: AZ AE DU.
Wrote the manuscript: AZ AE DU.
1. Korn T, Bettelli E, Oukka M, Kuchroo VK (2009) IL-17 and Th17 Cells.
Annu Rev Immunol 27: 485-517. doi:10.1146/annurev.immunol.
021908.132710. PubMed: 19132915.
2. Zúñiga LA, Jain R, Haines C, Cua DJ (2013) Th17 cell development:
from the cradle to the grave. Immunol Rev 252: 78-88. doi:10.1111/imr.
12036. PubMed: 23405896.
3. Ayyoub M, Deknuydt F, Raimbaud I, Dousset C, Leveque L et al.
(2009) Human memory FOXP3+ Tregs secrete IL-17 ex vivo and
constitutively express the T(H)17 lineage-specific transcription factor
RORgamma t. Proc Natl Acad Sci U S A 106: 8635-8640. doi:10.1073/
pnas.0900621106. PubMed: 19439651.
4. Chen W, Jin W, Hardegen N, Lei KJ, Li L et al. (2003) Conversion of
peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells
by TGF-beta induction of transcription factor Foxp3. J Exp Med 198:
1875-1886. doi:10.1084/jem.20030152. PubMed: 14676299.
5. Fantini MC, Becker C, Monteleone G, Pallone F, Galle PR et al. (2004)
Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25-
T cells through Foxp3 induction and down-regulation of Smad7. J
Immunol 172: 5149-5153. PubMed: 15100250.
6. Fu S, Zhang N, Yopp AC, Chen D, Mao M et al. (2004) TGF-beta
induces Foxp3 + T-regulatory cells from CD4 + CD25 - precursors. Am
J Transplant 4: 1614-1627. doi:10.1111/j.1600-6143.2004.00566.x.
7. Weaver CT, Hatton RD (2009) Interplay between the TH17 and TReg
cell lineages: a (co-)evolutionary perspective. Nat Rev Immunol 9:
883-889. doi:10.1038/nri2660. PubMed: 19935807.
8. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA (2006)
Transforming growth factor-beta regulation of immune responses. Annu
Rev Immunol 24: 99-146.
24.021605.090737. PubMed: 16551245.
9. Rubtsov YP, Rudensky AY (2007) TGFbeta signalling in control of T-
cell-mediated self-reactivity. Nat Rev Immunol 7: 443-453. doi:10.1038/
nri2095. PubMed: 17525753.
10. Hayes S, Chawla A, Corvera S (2002) TGF beta receptor
internalization into EEA1-enriched early endosomes: role in signaling to
Smad2. J Cell Biol 158: 1239-1249. doi:10.1083/jcb.200204088.
11. Atfi A, Dumont E, Colland F, Bonnier D, L'Helgoualc'h A et al. (2007)
The disintegrin and metalloproteinase ADAM12 contributes to TGF-
beta signaling through interaction with the type II receptor. J Cell Biol
178: 201-208. doi:10.1083/jcb.200612046. PubMed: 17620406.
12. Gruel J, Leborgne M, LeMeur N, Théret N (2009) In silico investigation
of ADAM12 effect on TGF-beta receptors trafficking. BMC Res Notes 2:
193. doi:10.1186/1756-0500-2-193. PubMed: 19778441.
13. White JM (2003) ADAMs: modulators of cell-cell and cell-matrix
interactions. Curr Opin Cell Biol 15: 598-606. doi:10.1016/j.ceb.
2003.08.001. PubMed: 14519395.
14. Reiss K, Saftig P (2009) The "a disintegrin and metalloprotease"
(ADAM) family of sheddases: physiological and cellular functions.
Semin Cell Dev Biol 20: 126-137. doi:10.1016/j.semcdb.2008.11.002.
15. Loechel F, Gilpin BJ, Engvall E, Albrechtsen R, Wewer UM (1998)
Human ADAM 12 (meltrin alpha) is an active metalloprotease. J Biol
Chem 273: 16993-16997. doi:10.1074/jbc.273.27.16993. PubMed:
16. Kurisaki T, Masuda A, Osumi N, Nabeshima Y, Fujisawa-Sehara A
(1998) Spatially- and temporally-restricted expression of meltrin alpha
(ADAM12) and beta (ADAM19) in mouse embryo. Mech Dev 73:
211-215. doi:10.1016/S0925-4773(98)00043-4. PubMed: 9622634.
17. Kveiborg M, Albrechtsen R, Couchman JR, Wewer UM (2008) Cellular
roles of ADAM12 in health and disease. Int J Biochem Cell Biol 40:
1685-1702. doi:10.1016/j.biocel.2008.01.025. PubMed: 18342566.
18. Toft-Hansen H, Nuttall RK, Edwards DR, Owens T (2004) Key
metalloproteinases are expressed by specific cell types in experimental
autoimmune encephalomyelitis. J Immunol 173: 5209-5218. PubMed:
19. Wang R, Wan Q, Kozhaya L, Fujii H, Unutmaz D (2008) Identification of
a regulatory T cell specific cell surface molecule that mediates
suppressive signals and induces Foxp3 expression. PLOS ONE 3:
e2705. doi:10.1371/journal.pone.0002705. PubMed: 18628982.
ADAM12 Regulates IL-17 Secretion
PLOS ONE | www.plosone.org8 November 2013 | Volume 8 | Issue 11 | e81146
20. Unutmaz D, KewalRamani VN, Marmon S, Littman DR (1999) Cytokine Download full-text
signals are sufficient for HIV-1 infection of resting human T
lymphocytes. J Exp Med 189:
189.11.1735. PubMed: 10359577.
21. Oswald-Richter K, Grill SM, Leelawong M, Tseng M, Kalams SA et al.
(2007) Identification of a CCR5-expressing T cell subset that is
resistant to R5-tropic HIV infection. PLoS Pathog 3: e58. doi:10.1371/
journal.ppat.0030058. PubMed: 17465678.
22. Oswald-Richter K, Grill SM, Shariat N, Leelawong M, Sundrud MS et al.
(2004) HIV infection of naturally
reprogrammed human regulatory T-cells. PLoS Biol 2: E198. doi:
10.1371/journal.pbio.0020198. PubMed: 15252446.
23. Wan Q, Kozhaya L, ElHed A, Ramesh R, Carlson TJ et al. (2011)
Cytokine signals through PI-3 kinase pathway modulate Th17 cytokine
production by CCR6+ human memory T cells. J Exp Med 208:
1875-1887. doi:10.1084/jem.20102516. PubMed: 21825017.
24. Valmori D, Raffin C, Raimbaud I, Ayyoub M (2010) Human
RORgammat+ TH17 cells preferentially differentiate from naive
FOXP3+Treg in the presence of lineage-specific polarizing factors.
Proc Natl Acad Sci U S A 107: 19402-19407. doi:10.1073/pnas.
1008247107. PubMed: 20962281.
25. Streeck H, Cohen KW, Jolin JS, Brockman MA, Meier A et al. (2008)
Rapid ex vivo isolation and long-term culture of human Th17 cells. J
Immunol Methods 333: 115-125.
26. Mercer F, Kozhaya L, Unutmaz D (2010) Expression and function of
TNF and IL-1 receptors on human regulatory T cells. PLOS ONE 5:
e8639. doi:10.1371/journal.pone.0008639. PubMed: 20066156.
27. Evans HG, Suddason T, Jackson I, Taams LS, Lord GM (2007)
Optimal induction of T helper 17 cells in humans requires T cell
receptor ligation in the context of Toll-like receptor-activated
monocytes. Proc Natl Acad Sci U S A 104: 17034-17039. doi:10.1073/
pnas.0708426104. PubMed: 17942669.
28. Manel N, Unutmaz D, Littman DR (2008) The differentiation of human
T(H)-17 cells requires transforming growth factor-beta and induction of
occurring and genetically
the nuclear receptor RORgammat. Nat Immunol 9: 641-649. doi:
10.1038/ni.1610. PubMed: 18454151.
29. Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A et al. (2006)
The orphan nuclear receptor RORgammat directs the differentiation
program of proinflammatory IL-17+ T helper cells. Cell 126: 1121-1133.
doi:10.1016/j.cell.2006.07.035. PubMed: 16990136.
30. Le Pabic H, Bonnier D, Wewer UM, Coutand A, Musso O et al. (2003)
ADAM12 in human liver cancers: TGF-beta-regulated expression in
stellate cells is associated with matrix remodeling. Hepatology 37:
1056-1066. doi:10.1053/jhep.2003.50205. PubMed: 12717386.
31. Le Pabic H, L'Helgoualc'h A, Coutant A, Wewer UM, Baffet G et al.
(2005) Involvement of the serine/threonine p70S6 kinase in TGF-beta1-
induced ADAM12 expression in cultured human hepatic stellate cells. J
Hepatol 43: 1038-1044. doi:10.1016/j.jhep.2005.05.025. PubMed:
32. Solomon E, Li H, Duhachek Muggy S, Syta E, Zolkiewska A (2010) The
role of SnoN in transforming growth factor beta1-induced expression of
metalloprotease-disintegrin ADAM12. J Biol Chem 285: 21969-21977.
doi:10.1074/jbc.M110.133314. PubMed: 20457602.
33. McGeachy MJ, Chen Y, Tato CM, Laurence A, Joyce-Shaikh B et al.
(2009) The interleukin 23 receptor is essential for the terminal
differentiation of interleukin 17-producing effector T helper cells in vivo.
Nat Immunol 10: 314-324. doi:10.1038/ni.1698. PubMed: 19182808.
34. Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ et al.
(2010) Generation of pathogenic T(H)17 cells in the absence of TGF-
beta signalling. Nature 467: 967-971. doi:10.1038/nature09447.
35. Zhou L, Ivanov II, Spolski R, Min R, Shenderov K et al. (2007) IL-6
programs T(H)-17 cell differentiation by promoting sequential
engagement of the IL-21 and IL-23 pathways. Nat Immunol 8: 967-974.
doi:10.1038/ni1488. PubMed: 17581537.
36. Liu Y, Zhang P, Li J, Kulkarni AB, Perruche S et al. (2008) A critical
function for TGF-beta signaling in the development of natural
CD4+CD25+Foxp3+ regulatory T cells. Nature immunology 9: 632-640.
37. Sakaguchi S, Yamaguchi T, Nomura T, Ono M (2008) Regulatory T
cells and immune tolerance. Cell 133: 775-787. doi:10.1016/j.cell.
2008.05.009. PubMed: 18510923.
ADAM12 Regulates IL-17 Secretion
PLOS ONE | www.plosone.org9 November 2013 | Volume 8 | Issue 11 | e81146