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Title: A20 in inflammation and autoimmunity
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Review

A20 in inflammation and autoimmunity
Leen Catrysse1,2, Lars Vereecke1,2, Rudi Beyaert1,2, and Geert van Loo1,2
1
2

Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, VIB, B-9052 Ghent, Belgium
Department of Biomedical Molecular Biology, Ghent University, B-9052 Ghent, Belgium

Although known for many years as a nuclear factor (NF)kB inhibitory and antiapoptotic signaling protein, A20
has recently attracted much attention because of its
ubiquitin-regulatory activities and qualification by genome-wide association studies (GWASs) as a susceptibility gene for inflammatory disease. Here, we review
new findings that have shed light on the molecular and
biochemical mechanisms by which A20 regulates inflammatory signaling cascades, and discuss recent experimental evidence characterizing A20 as a crucial
gatekeeper preserving tissue homeostasis.

commensal bacteria was shown to be responsible for the
multiorgan inflammation and premature death of A20
knockout mice [8]. Next to its well-characterized role in
restricting NF-kB signaling and cell death, A20 has recently been shown to regulate other cellular signaling circuits,
such as the Wnt pathway, the autophagic response, and the
interferon regulatory factor (IRF) pathway [9–12].
In this review we discuss the molecular mechanisms by
which A20 regulates its different activities, and describe its
importance for tissue homeostasis based on human genetic
studies and experimental findings in animal models.

A20: the early years
The transcription factor NF-kB (Box 1) comprises a family
of transcription factors critical for inflammatory signaling,
and innate and adaptive immunity. NF-kB also controls
the expression of antiapoptotic genes important for cell
survival, and plays diverse roles in development, proliferation, cell differentiation, and metabolism [1]. NF-kB activation is central to many cellular processes, therefore,
tight regulation of NF-kB signaling pathways is an absolute requirement. Several regulatory mechanisms keep
NF-kB signaling in check in order to maintain tissue
homeostasis [2]. One of the proteins known to play a key
role in the termination of NF-kB signaling is A20.
A20, named after its cDNA clone number and also
referred to as tumor necrosis factor a-induced protein
(TNFAIP)3, was identified in endothelial cells as a primary
response gene induced upon treatment with TNF [3,4].
Although now widely recognized as an anti-inflammatory
protein, A20 was originally characterized as a protein
protecting cells from TNF-induced cytotoxicity [5]. Further
studies demonstrated a role for A20 as an inhibitor of not
only TNF-dependent NF-kB activation, but also of NF-kB
activation in response to interleukin (IL)-1, CD40, and of
signaling through pattern recognition receptors (PRRs),
and T cell and B cell antigen receptor activation [6]. The
first in vivo evidence for a role for A20 in tissue physiology
came from the phenotype of A20-deficient mice [7]. These
mice are hypersensitive to TNF and die prematurely due to
severe multiorgan inflammation and cachexia. Deregulated Toll-like receptor (TLR) signaling in response to

A20 inhibits activation of NF-kB via multiple
mechanisms
The molecular mechanisms by which A20 controls its
multiple activities are not fully understood and are the
subject of intense research. However, the ability of A20 to
modulate ubiquitin-dependent signaling cascades has
been shown to be of central importance to many of its
functions (Table 1).
NF-kB signaling cascades are heavily controlled by
ubiquitination, and several proteins including A20 may
interfere with these processes (Box 2) [13]. The first
evidence for a role for A20 in ubiquitin-dependent signaling came from a study by Dixit et al., wherein A20 was
shown to be a ubiquitin-editing enzyme containing an
amino-terminal deubiquitinating (DUB) activity mediated by its ovarian tumor (OTU) domain, and a carboxyterminal zinc finger (ZnF) domain that supports E3 ubiquitin ligase activity [14]. The first substrate identified
for the deubiquitinase activity of A20 was receptor interacting protein (RIP)1. Upon TNF receptor (TNFR) stimulation, RIP1 is polyubiquitinated at lysine63 (K63) by
the E3 ubiquitin ligases cellular inhibitor of apoptosis
protein (cIAP)1 and 2 [15]. A20 removes these K63-linked
polyubiquitin chains, preventing the interaction of RIP1
with NF-kB essential modulator (NEMO) (Figure 1).
Subsequently, A20 adds K48-linked polyubiquitin chains
to RIP1, targeting it for proteasomal degradation [14]. In
this way, A20 restricts TNF-induced NF-kB signaling
by sequential deubiquitination and ubiquitin-mediated
degradation of RIP1.
A20 can restrict NF-kB activity triggered by TLR4 and
nucleotide-binding oligomerization domain-containing
protein 2 (NOD2) by deubiquitinating TNF receptor associated factor (TRAF)6 and RIP2, respectively [16,17]
(Figure 1). A similar activity for A20 has recently been
reported in the IL-17 receptor signaling pathway; here,
A20 mediates feedback inhibition by removing K63-linked
polyubiquitin chains from TRAF6 [18] (Figure 1). A20 is

Corresponding author: van Loo, G. (Geert.vanLoo@dmbr.vib-UGent.be).
Keywords: A20; tumor necrosis factor a-induced protein 3; nuclear factor-kB;
inflammation; apoptosis; ubiquitination.
1471-4906/$ – see front matter
ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2013.10.005

22

Trends in Immunology, January 2014, Vol. 35, No. 1

Review

Trends in Immunology January 2014, Vol. 35, No. 1

Box 1. General principles of NF-kB signaling
The NF-kB transcription factor family consists of five family
members: NF-kB1 (p105), NF-kB2 (p100), RelA (p65), RelB, and cRel which are present in the cell as either homo- or heterodimers [1].
In resting conditions, NF-kB dimers are sequestered in the
cytoplasm by binding to IkB proteins. In general, NF-kB can be
activated either through the canonical or alternative, noncanonical
pathway. The common regulatory step in both cascades is the
activation of the IKK complex. In canonical signaling this consists of
two catalytic subunits IKK1 and IKK2 (also known as IKKa and IKKb)
and the regulatory subunit NEMO (also known as IKKg), whereas in
noncanonical signaling only IKK1 is present. The IKK complex
phosphorylates IkB proteins, leading to their polyubiquitination and
proteolytic processing by the proteasome. NF-kB can then translocate to the nucleus where it exerts its transcriptional activity. The
canonical (or classical) NF-kB signaling pathway is induced by
stimulation of specific receptors such as TNF-R1, TLRs, IL-1 receptor
(IL-1R), leading to the recruitment of adaptor proteins to their
intracellular domain and the activation of the IKK complex. In
canonical NF-kB signaling, both IKK2 and NEMO are necessary,
whereas IKK1 is dispensable. The noncanonical NF-kB pathway can
be activated by certain receptors such as the lymphotoxin (LT) b
receptor, B cell-activating factor receptor (BAFF-R) and CD40.
Ligand-induced activation of these receptors results in the activation
of NF-kB-inducing kinase (NIK), which specifically activates IKK1,
inducing the phosphorylation and proteolytic processing of NF-kB2
(p100) to p52, which now forms heterodimers with RelB.

also a key inhibitor of T and B cell-induced NF-kB signaling, acting as a DUB for K63-polyubiquitinated mucosaassociated lymphoid tissue lymphoma translocation protein (MALT)1, a scaffolding protein involved in the activation of NF-kB downstream of the T and B cell antigen
receptor [19] (Figure 1).
Several noncatalytic mechanisms of A20 action have
also been described. Upon lipopolysaccharide (LPS) or IL-1
stimulation, A20 is able to affect the E3 ligase activity of
TRAF6 by preventing its interaction with the E2 ubiquitinconjugating enzymes Ubc13 and UbcH5c [20]. In parallel,
A20 modifies Ubc13 and UbcH5c by the addition of K48linked ubiquitin chains, thus targeting these proteins for
proteasomal degradation [20]. Similarly, upon TNF stimulation, A20 interferes with the interaction between
Ubc13, and both TRAF2/5 and cIAP1/2 [20]. Finally, A20
can also inhibit TNF-induced NF-kB signaling through a
mechanism that involves binding to polyubiquitin chains
via its seventh zinc finger (ZnF7) [21–23] (Figure 1). In this
context, A20 was shown to bind linear polyubiquitin chains
attached to NEMO via its ZnF7 domain. This binding is
proposed to hinder the recruitment of other linear polyubiquitin binding proteins that are essential for productive

Table 1. Signaling pathways controlled by A20 through interaction with specific substratesa.
Pathway

Substrate
A20
RIP1

LUBAC–NEMO
Ubc13
TNF
ASK1
Ubc13/UbcH5c
IL-1

IL-17

TCR/CD28

TRAF6
TRAF6 and CBAD
domain of IL17RA
MALT1
TRAF6
Ubc13/UbcH5

TLR

DR4/5
NOD1/2
RIG-I

Beclin1
Caspase-8 (?)
RIP2
TRAF3–TBK1/IKKi

Wnt

Axin

Tight junction

Occludin

EDAR

TRAF6 (?)

Action A20
Homodimerization of A20 proteins
(independent of OTU and ZnF4)
Recognizes RIP1-K63Ub (ZnF4) Removes
K63Ub from RIP1 (OTU) Adds K48Ub on RIP1
leading to degradation (ZnF4)
Binding to linear Ub (ZnF7)
Prevents LUBAC–NEMO interaction
Prevents interaction of Ubc13 with TRAF2/
cIAPs (OTU)
Adds K48Ub on Ubc13 leading to degradation
(ZnF4)
Binding to and polyUb of ASK1 leading to
degradation (ZnF4)
Prevents interaction of Ubc13/UbcH5c with
TRAF6 (OTU)
Adds K48Ub on Ubc13/UbcH5c leading to
degradation (ZnF4)
Interacts with TRAF6
Binds to IL17RA-CBAD and prevents polyUb of
TRAF6 (OTU+ZnF4/ZnF5)
Removes K63Ub from MALT1 (OTU)
Removes K63Ub from TRAF6 (OTU)
Prevents interaction of Ubc13/UbcH5c with
TRAF6 (OTU)
Adds K48Ub on Ubc13/UbcH5c leading to
degradation (ZnF4)
Removes K63Ub from Beclin1
Removes polyubiquitin from caspase-8 (OTU)
Removes K63Ub from RIP2 (OTU)
Disrupts interaction between TRAF3 and
TBK1/IKKi, thereby disrupting K63Ub of TBK1/
IKKi
Binds to the b-catenin destruction complex
and adds K48Ub to Axin, leading to
degradation (OTU)
Deubiquitinates non-K48Ub occludin

Consequence
High-order oligomerization for efficient
inhibition of NF-kB signaling
Inhibition of TNF-induced NF-kB signaling

Refs
[30]

Inhibition of TNF-induced NF-kB signaling

[22,23]

Inhibition of TNF-induced NF-kB signaling

[20]

Inhibition of JNK-mediated apoptosis

[34]

Inhibition of IL1-induced NF-kB signaling

[20]

Inhibition
Inhibition
signaling
Inhibition
Inhibition
Inhibition

of IL1-induced NF-kB signaling
of IL17-induced NF-kB and MAPK

[6]
[18]

of TCR-induced NF-kB signaling
of LPS-induced NF-kB signaling
of LPS-induced NF-kB signaling

[19]
[17]
[20]

Inhibition
Inhibition
Inhibition
Inhibition

of
of
of
of

[10]
[32]
[16]
[40]

LPS-induced autophagy
DR-induced apoptosis
MDP-induced NF-kB signaling
virus-induced IRF signaling

[14,30]

Inhibition of Wnt signaling

[9]

Regulation of tight junction dynamics
promoting intestinal barrier function
Inhibition of EDA-A1-induced NF-kB signaling

[70]
[85]

a

Abbreviations: DR, death receptor; polyUb, polyubiquitination.

23

Review
Box 2. Regulation of NF-kB signaling by ubiquitination
Ubiquitination, a post-translational modification of proteins, plays a
key role in the activation of NF-kB [13]. Ubiquitin is covalently
attached to other proteins in a highly regulated process involving
the stepwise activity of an E1 ubiquitin activating enzyme, E2conjugating enzymes and E3 ubiquitin ligases. The latter confer
substrate specificity and enable the attachment of ubiquitin to a
specific lysine in the target substrate. Each of the seven lysines (K6,
K11, K27, K29, K33, K48, and K63) in ubiquitin can themselves be
coupled to another ubiquitin, leading to a polyubiquitin chain on the
original target protein. Also linear polyubiquitination, in which
ubiquitin is linked via an amino-terminal methionine residue to
another ubiquitin molecule, has been demonstrated. The type of
polyubiquitin chain determines the fate of the conjugated target
substrate. Polyubiquitin chain formation through K48 of ubiquitin
directs proteasomal degradation of the modified protein. By
contrast, K63 polyubiquitin chain linkages or linear polyubiquitination normally do not lead to degradation of the substrate but
mediate a low-affinity binding of other proteins that contain specific
ubiquitin-binding domains. This can stabilize transient protein–
protein interactions important for driving downstream signaling.
K11 chains usually mark conjugated substrates for proteasomal
degradation, but may also be involved in the nondegradative
signaling to NF-kB. Ubiquitination is reversed by DUBs, which are
proteases that cleave ubiquitin chains from their substrate, making
ubiquitination a dynamic process. Several DUBs such as cylindromatosis (CYLD), A20, and Cezanne, have been demonstrated to
remove K63-linked ubiquitin chains from specific target substrates
in the NF-kB signaling pathway, thus negatively regulating NF-kB
activation [96]. A20 and CYLD regulate canonical NF-kB signaling,
whereas Cezanne (also known as Otud7b) was identified as a DUB
controlling noncanonical NF-kB activation [97]. Recently, OTULIN
[ovarian tumor (OTU) family linear chain deubiquitinase; also
known as Fam105b or Gumby] was described as a linear ubiquitin-specific DUB important for NF-kB regulation [98–100].

signaling downstream from the TNFR [23]. Linear polyubiquitination is mediated by LUBAC (linear ubiquitin
chain assembly complex), composed of HOIL-1, HOIP and
Sharpin, and is best characterized in TNF signaling [24].
Recently, LUBAC-mediated linear ubiquitination was
demonstrated in CD40 and NOD2 signaling [25,26], suggesting that these pathways may also be restricted by A20
in a noncatalytic manner.
The functional importance of the different A20 domains
and activities in the inhibition of NF-kB has been further
elucidated by a combination of structural, functional, and
genetic studies. In contrast to its DUB activity on K63polyubiquitinated proteins in vivo, A20 preferentially disassembles K48-linked polyubiquitin chains in vitro [27–
29]. Furthermore, A20 ZnF4 specifically recognizes K63linked polyubiquitin in vitro [29]. These observations suggest the involvement of other proteins or mechanisms
directing the DUB activity of A20 to K63-polyubiquitinated
proteins in a cellular context. To assess the physiological
impact of the DUB and the ZnF4 activity of A20 in vivo,
A20ZF4 and A20OTU knock-in mice displaying a disruptive
point mutation in the ZnF4 domain and in the DUB
domain, respectively, were recently generated [30]. Both
A20ZF4 and A20OTU mice are grossly normal [30], in contrast to A20 deficient mice which die perinatally [7]. A20ZF4
and A20OTU knock-in embryonic fibroblasts exhibit decreased NF-kB signaling in response to TNF, as compared
to A20-deficient cells, suggesting that neither the DUB
motif, nor the ZnF4 domain are singly responsible for
all the functions of A20. Although it is likely that mice
24

Trends in Immunology January 2014, Vol. 35, No. 1

carrying both point mutations (A20ZF4/OTU knock-in mice)
more closely resemble A20 deficiency, these mice have not
yet been generated. Alternatively, other A20 functional
domains such as ZnF7 may be critically important
[21,23,31]. Although A20ZF4 and A20OTU mice do not develop spontaneous pathology, both mouse strains exhibit
increased responses to TNF injection and are hypersensitive to dextran sodium sulfate (DSS)-induced colitis, indicating that both the DUB and ZnF4 motifs of A20 do
contribute to the ability of A20 to restrict inflammatory
responses in vivo [30].
Varied roles for A20 in cell death processes
Independent from its role as a modulator of NF-kB signaling, A20 functions as an antiapoptotic protein in several
cell types. Whether these functions are dependent on
ubiquitin-dependent mechanisms similar to those described above remains elusive. Death receptors induce
apoptotic signaling by recruiting adaptor proteins, together assembling the so-called death-inducing signaling complex (DISC). Interestingly, A20 was identified as part of
the DISC, where it physically interacts with caspase-8
[32]. During apoptosis induced by stimulation of the
TNF-related apoptosis-inducing ligand (TRAIL) death receptor, caspase-8 is polyubiquitinated through a cullin3based E3 ligase. In overexpression studies, A20 reversed
cullin3-mediated ubiquitination of caspase-8 and blocked
the associated increase in caspase-8 activity, suggesting
that A20 inhibits apoptotic signaling through deubiquitination and inhibition of caspase-8 [32]. A20 has also been
shown to increase K63-linked polyubiquitination of RIP1
through mechanisms that remain largely unclear but have
been shown to be dependent on ZnF4. This enables RIP1
binding to the protease domain of caspase-8, which inhibits
caspase-8 dimerization and cleavage, and prevents TRAILinduced apoptosis [33]. Another study suggested that A20
blocks TNF-induced apoptosis through suppression of cJun N-terminal kinase (JNK) by targeting the upstream
apoptosis signal-regulating kinase (ASK)1 for proteasomal
degradation [34].
In pancreatic b-cells, dual antiapoptotic and anti-inflammatory functions have been reported for A20. Mice
engineered to overexpress A20 in b-cells in the islets of
Langerhans exhibited resistance to IL-1b- and IFNg-induced apoptosis, which is associated with decreased NFkB-induced nitric oxide (NO) production [35]. Also, proapoptotic functions have been reported for A20 in certain
cell types, and these activities are likely mediated through
the inhibition of NF-kB-dependent expression of antiapoptotic proteins such as Bcl-2 and Bcl-x [36,37]. Together,
these studies demonstrate that the effect of A20 on cell
death is highly cell type dependent and determined by the
balance between its antiapoptotic properties and its impact on NF-kB inhibition with consequences for the expression of antiapoptotic genes.
Modulation of the innate immune response to
pathogens
In addition to its role in the NF-kB pathway, A20 functions
to regulate IRF activation in response to pathogen recognition (Figure 1). Retinoic acid-inducible gene 1 M (RIG-I)

Review

Trends in Immunology January 2014, Vol. 35, No. 1

IL-17

(F)

(A) TNF

TRAF6

A20

TNFR complex I
TRADD
TRAF2/5 RIP1
RIP1

IL-17 receptor
complex

Act1

LPS, IL-1β, …

(B)

TRAF6

K48

cIAP1/2

Ubc13/H5

A20

A20

TLR/IL-1βR
complex

MyD88

K63

Ubc13

A20

K48

(C)

NEMO
N

NEMO
N
IKK2
IKK1

NEMO
N

TAB2/3 TAK1

NEMO
N
IKK2
IKK1

A20
CARMA1 TRAF6
Bcl-10

NF-κB
ac va on

NEMO

Proinflammatory
response

IKK2

PKC

NEMO
N
O

IKK2
IKK1

TAB2/3 TAK1
TA

LUBAC

A20
IKK2
IKK1

K63

cIAP Ubc13

TCR
complex

Ubc1 3

RIP2
TRAF

(D)

TTAB2/3 TAK1

IKK2
IKK1

NOD-1/2
receptor
complex

Co-receptor
s mula on

K63

MALT1
K63

A20

A20

(E)

NEMO
IKK2
IKK1

RIG-I/Mda5
receptor
complex

MAVS

TRAF3

IKKi

p

A20

TTBK1

IRF signaling
type I interferons

An -viral
response

TRENDS in Immunology

Figure 1. Nuclear factor (NF)-kB and interferon (IFN) regulatory factor (IRF) regulatory activities of A20. (A) Upon tumor necrosis factor receptor (TNFR) stimulation, several
adaptor proteins [TRADD, receptor interacting protein (RIP)1 and TRAF2/5] are recruited to the receptor (TNFR complex). With the aid of the E2–E3 enzymes Ubc13 and
cellular inhibitor of apoptosis protein (cIAP)1/2, RIP1 is K63-polyubiquitinated. These ubiquitin chains now act as a scaffold recruiting the TAK1 kinase complex, containing
the ubiquitin binding adaptor proteins TAB2 and TAB3, and the IkB kinase (IKK) complex via the ubiquitin-binding domain of NF-kB essential modulator (NEMO). TAK1
phosphorylates and activates IKK2, leading to downstream NF-kB activation. In addition, NEMO is linearly ubiquitinated by the linear ubiquitin chain assembly complex
(LUBAC), enhancing the stability of the IKK/LUBAC complex, which is important for efficient NF-kB activation. A20 acts at different levels in this pathway: it hydrolyzes K63linked polyubiquitin chains on RIP1 and adds K48-linked polyubiquitin chains to RIP1, leading to its proteasomal degradation. A20 also disrupts the interaction between
Ubc13 and cIAP1/2, and adds K48-linked polyubiquitin chains to Ubc13 targeting it for degradation. Recently, A20 was shown to compete for the binding of linear
ubiquitination chains to NEMO, destabilizing the NF-kB activating IKK/LUBAC complex. (B) Upon interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) binding, several
adaptor proteins including MyD88 and TRAF6 are recruited to the receptor. TRAF6 interacts with Ubc13 and UbcH5c, leading to the K63-linked polyubiquitination of TRAF6
and downstream TAK1 and IKK activation, with subsequent NF-kB activation. A20 inhibits NF-kB signaling by removing K63-linked polyubiquitin chains form TRAF6, and
disrupts the interaction of TRAF6 with Ubc13 and UbcH5c, followed by K48-linked polyubiquitination and degradation of Ubc13 and UbcH5c. (C) Upon NOD1/2 receptor
activation, RIP2 is recruited to the receptor followed by its K63-linked polyubiquitination by cIAP1/2, leading to downstream NF-kB activation. A20 inhibits NF-kB activation
by removing K63-linked polyubiquitin chains from RIP2. (D) Upon T cell receptor stimulation by antigen and co-stimulation, protein kinase (PK)Cu and the CARMA1/Bcl10/
mucosa-associated lymphoid tissue lymphoma translocation protein (MALT)1 (CBM) complex are recruited and activated, followed by MALT1 K63-linked polyubiquitination
by TRAF6, leading to the activation of the IKK complex and downstream NF-kB signaling. A20 removes the K63-linked polyubiquitin chains from MALT1, inhibiting NF-kB
activation. MALT1, however, also has proteolytic activity cleaving and inactivating A20. (E) Activation of RIG-I/Mda5 receptors by viral nucleic acid recruits and activates the
mitochondrial adaptor protein MAVS, leading to the assembly of a signaling complex containing TRAF3 and the noncanonical IkB kinases TBK1/IKKi, leading to
downstream IRF signaling and induction of Type I IFNs. MAVS can also initiate NF-kB signaling via IKK activation. A20 blocks IRF signaling by removing K63-linked ubiquitin
chains from TBK1/IKKi. (F) IL-17-induced activation of the IL-17 receptor recruits the E3 ligase Act1, which leads to the K63-linked polyubiquitination of TRAF6 and
downstream NF-kB signaling. A20 associates with the receptor and removes ubiquitin chains from TRAF6, blocking NF-kB activation. Abbreviations: CARMA, CARDcontaining MAGUK protein; MAVS, mitochondrial antiviral-signaling protein; Mda, melanoma differentiation-associated protein; MyD88, myeloid differentiation factor 88;
NOD, nucleotide-binding oligomerization domain-containing protein; RIG, retinoic acid-inducible gene; TAB, TAK1-binding protein; TAK, TGFbeta activated kinase; TBK,
TANK-binding kinase; TRADD, TNF receptor-associated death domain protein; TRAF, TNF receptor-associated factor.

and melanoma differentiation-associated protein 5 (Mda5)
are PRRs that recognize viral nucleic acids and activate a
pathway involving the mitochondrial adaptor protein mitochondrial antiviral-signaling protein [MAVS; also known
as virus-induced signaling adapter IPS-1 (VISA), interferon-beta promoter stimulator 1 (IPS-1) or CARD adaptor
inducing IFN-b (Cardif)] and TRAF3, which couple to the
inhibitor of kB (IkB) kinase-related kinases TANK-binding
kinase 1 (TBK1) and IkB kinase (IKK)i (also known as
IKKe) [38]. Upon activation, these kinases specifically

phosphorylate the transcription factors IRF3 and IRF7,
leading to their dimerization, nuclear translocation, and
transcriptional activation of type I IFN genes. A20 inhibits
RIG-I-induced IRF activation and IFN responses by removing K63-linked polyubiquitin chains from TBK1/IKKi
[11,12,39,40]. By this, A20 may restrict antiviral signaling
responses.
Finally, A20 may also control autophagy in response to
TLR activation. Indeed, in macrophages, A20 was shown to
deubiquitinate Beclin-1, a protein essential for phagophore
25

Review
formation, limiting the induction of autophagy [10]. More
studies are however needed to confirm these findings in in
vivo conditions.
Mechanisms that regulate A20 function
A20 expression and activity are subject to tight regulation
[41]. Most cells express only low amounts of A20 under
basal conditions, but A20 expression is rapidly induced
upon NF-kB activation due to the presence of two NF-kB
binding sites in the A20 promoter. Lymphocytes, however,
constitutively express high levels of A20. Antigen receptor
stimulation leads to a rapid decrease and subsequent
reappearance of A20, suggesting that A20 removal is important in order to allow optimal NF-kB activation. Proteasomal degradation of A20, as well as its cleavage by the
paracaspase MALT1, contribute to the initial decrease in
A20 levels [19,42].
A20 activity is also regulated by phosphorylation. Upon
stimulation with TNF or LPS, A20 is phosphorylated by
IKK2, which augments – by currently unknown mechanisms – its ability to inhibit NF-kB signaling [43]. Interestingly, increasing evidence suggests that the
physiological state affects the stability of A20. In smooth
muscle cells cultured in high glucose conditions, A20 levels
were dependent on its post-translational O-glycosylation,
which regulated its ubiquitination and degradation [44].
Two recent studies demonstrated that DUBs, including
A20, can be (in)activated by reversible oxidation of a key
cysteine residue in the catalytic domain [45,46]. These
findings suggest that oxidative stress and the presence
of reactive oxygen species (ROS) may affect cellular physiology via effects on A20 and other DUBs.
A20 also exerts its anti-inflammatory activities through
collaboration with specific binding partners. A20-binding
inhibitor of NF-kB (ABIN) and Tax1-binding protein 1
(TAX1BP1) are A20 binding proteins that have been proposed to recruit A20 to its substrate by directly binding
polyubiquitin [47–50]. Also, the E3 ubiquitin ligases Itch
and RING finger protein 11 (RNF11) are involved in the
efficient inhibition of NF-kB signaling [51,52]. However,
the mechanisms by which these ligases function in restricting these signaling pathways remain unclear.
Finally, several studies have reported regulation of A20
expression by miRNAs (miRs). miR-125a and miR-125b
are overexpressed in diffuse large B cell lymphoma
(DLBCL), wherein they suppress A20 expression, contributing to constitutive NF-kB signaling [53]. The finding that
miR-125b is a direct NF-kB transcriptional target, further
suggests the existence of a self-regulatory circuit in which
termination of A20 function by miR-125 strengthens and
prolongs NF-kB activity [53]. miR-19b and miR-29c have
also been reported to target A20, at least in cell lines,
strengthening NF-kB signaling and apoptosis, respectively
[54,55]. A recent report describes a different effect of miR29 on A20 expression: in sarcoma tumor cells, miR-29 was
reported to act as a decoy for the RNA binding protein HuR,
protecting A20 transcripts from HuR-mediated degradation [56]. As a result, decreased miR-29 expression in
sarcoma cells is associated with absence of A20 expression
and constitutive NF-kB activity, which may contribute to
tumorigenesis. In conclusion, A20 expression and activities
26

Trends in Immunology January 2014, Vol. 35, No. 1

are regulated by diverse mechanisms in order to adapt
rapidly to a specific cellular condition restricting excessive
proinflammatory signaling. Understanding these mechanisms of regulation will help to clarify the specific role of
A20 in controlling tissue physiology and may help to
explain why specific conditions lead to the development
of inflammatory pathology.
A20 as a disease susceptibility gene
Multiple studies over the past few years, mostly based on
GWASs on genetic material from large cohorts of patients,
have identified A20/TNFAIP3 as a susceptibility locus for
human inflammatory and autoimmune pathology, including rheumatoid arthritis (RA) and juvenile idiopathic
arthritis, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), coeliac disease, psoriasis, type I
diabetes, Sjogren’s syndrome, coronary artery disease,
rheumatic heart disease, and systemic sclerosis [57].
The majority of single nucleotide polymorphisms (SNPs)
in the A20 locus are located up- or downstream of the A20
coding regions or in intronic regions, suggesting that they
interfere with regulatory elements affecting A20 expression. The SLE-associated TT>A polymorphic dinucleotide
results in reduced NF-kB binding and reduced A20 mRNA
expression [58]. Only two of the reported SNPs induce
nonsynonymous mutations (rs5029941/A125 V and
rs2230926/F127C). These SNPs are both located in exon
3 and affect the DUB domain of A20 impairing its NF-kB
inhibiting potential [59,60]. Two A20 polymorphisms
linked with psoriasis (rs2230926/F127C and rs610604)
have been associated with responsiveness to anti-TNF
therapy and may serve as potential prognostic markers;
patients harboring these alleles respond positively to TNF
blockade [61].
Genetic studies have also identified mutations and
deletions in A20 in multiple B cell lymphomas, including
MALT lymphoma, Hodgkin’s lymphoma, activated B cell
like DLBCL, and follicular lymphoma; these genetic aberrancies are associated with constitutive activation of the
NF-kB pathway [62–65]. In other cell types, A20 has been
ascribed protumorigenic activities, likely connected to its
antiapoptotic functions. A20 is highly expressed in glioma
stem cells, and its depletion attenuates glioma stem cell
survival and tumor growth [66]. A20 is also highly expressed
in estrogen-resistant breast cancer cell lines and in aggressive breast carcinomas [67]. Together, these studies suggest
that depending on the cell type and tumor stage, A20 may
act as a tumor suppressor or a tumor enhancer.
Given that A20 may have different roles in different cell
types, conditional deletion studies in mice are important to
obtain a clear understanding of the diverse physiological
functions of A20.
Conditional gene targeting studies in mice
Mice deficient for A20 die prematurely due to severe multiorgan inflammation [7], impeding the in vivo study of A20
in adults and in specific disease pathologies. In order to
assess the function of A20 in different cell types and in
various models of human disease, we and others developed
mice with a conditional A20 allele, allowing tissue-specific
deletion of A20 [37,68,69] (Figure 2).

Review

Trends in Immunology January 2014, Vol. 35, No. 1

SLE ?

Polyarthri s
Inflamma on

Protec on
against influenza

Hyperac vated
macrophages

SLE
Autoan body produc on
B and T cell autoimmunity

↑ Prolifera on and survival
Spontaneous ac va on

Spontaneous ac va on and matura on
↑ Survival

↑ An viral response
Influenza
B cells
Macrophages

CNS

No effect

Cerebral
ischemia

DCs

Tissue-specific
A20 deficiency

Intes nal epithelial cells

Kera nocytes

APC+/–

↑ Wnt signaling
Abnormal epidermal
extremi es

Hyperprolifera on
No inflamma on

DSS

↑ Apoptosis
↑ Intes nal barrier permeability
Bacterial infiltra on

Colon adenoma
IBD

Psoriasis ?
TRENDS in Immunology

Figure 2. Cell type-specific A20 deficiency and consequences for autoimmune disease pathology. Several tissue-specific A20 knockout mouse lines were generated and
phenotypically analyzed. The specific deletion of A20 in the intestinal epithelium sensitizes mice to dextran sodium sulfate (DSS)-induced colitis and tumor necrosis factor
(TNF) lethality due to intestinal epithelial cell (IEC) apoptosis and loss of barrier integrity. When crossed with APC+/min mice, IEC-specific A20 knockout mice are more prone
to develop colon tumors as a result of excessive Wnt signaling. A20 deficiency in keratinocytes induces hyperkeratosis and ectodermal organ abnormalities, but not
psoriasis. In the central nervous system (CNS), A20 deficiency does not affect the progression of cerebral ischemia. Macrophages lacking A20 are hyperactivated and
spontaneously produce inflammatory cytokines, leading to the development of polyarthritis. In addition, myeloid A20 deficiency enhances the antiviral immune response,
protecting mice from influenza lethality. Genetic deletion of A20 in B cells triggers the production of autoantibodies and the development of an autoimmune syndrome with
characteristics of systemic lupus erythematosus (SLE). Deletion of A20 in dendritic cells (DCs) causes severe autoimmunity similar to SLE characterized by the production of
autoantibodies, lupus nephritis, the antiphospholipid syndrome, and lymphosplenomegaly. Abbreviation: IBD, inflammatory bowel disease.

A20 deficiency in intestinal epithelial cells (IECs)
To study A20 in intestinal homeostasis, inflammation and
the development of IBD, we generated mice that are
specifically deficient for A20 in IECs [68]. These A20IECKO
mice develop normally, but are hypersensitive to DSSinduced colitis that is associated with increased IEC apoptosis. A20IEC-KO mice are also highly sensitive to TNF, a
cytokine associated with IBD pathology, leading to enterocyte apoptosis, complete loss of intestinal tissue integrity,
and infiltration of commensal intestinal bacteria leading to
systemic toxicity and lethality [68]. These data are supported by complementary findings using transgenic mice
with IEC-specific A20 overexpression [70]. In contrast to
wild type control mice, these mice are protected from
increases in intestinal permeability upon LPS exposure,
indicating a role for A20 in supporting epithelial tight
junctions, which was shown to relate to A20-mediated
deubiquitination of the polyubiquitinated tight junction
protein occludin [70]. Together, these findings identify
A20 as a protective factor essential for epithelial barrier
integrity and tissue homeostasis, and indicate that defects
in A20 might contribute to IBD in humans.
One of the consequences of chronic intestinal inflammation is the promotion of tumorigenesis, and patients with
IBD have a higher risk of developing colitis-associated
cancer [71]. A20 may also be important in protecting

against the development of colitis-induced colon cancer.
The expression of A20 was shown to be significantly lower
in samples of human adenomatous colonic polyps compared to the surrounding tissue [9], in agreement with
reports describing reduced expression of A20 in human
colorectal cancer samples [72,73]. Mice harboring a mutation in the adenomatous polyposis coli (APC) gene, which
spontaneously develop multiple small intestinal tumors
but infrequent colonic tumors, develop many colonic adenomas when crossed in an A20IEC-KO background, suggesting that A20 is capable of restricting colonic tumorigenesis
[9]. Mechanistically, this finding was explained as the
result of A20-mediated inhibition of Wnt signaling by
the binding of A20 to the b-catenin destruction complex,
supporting ubiquitination and degradation of b-catenin
[9].
A20 deficiency in myeloid cells
GWAS studies identified A20 as a susceptibility gene for
RA [74,75]. To address experimentally the role of A20 in
the development of RA, we generated mice that specifically
lack A20 in all cells of myeloid origin [76]. These A20myel-KO
mice develop spontaneous polyarthritis, with many features
of human RA, including the presence of type II collagen
autoantibodies and inflammatory cytokines in serum. Primary macrophages from these mice have sustained NF-kB
27

Review

Trends in Immunology January 2014, Vol. 35, No. 1

signaling and proinflammatory cytokine production in response to LPS. Interestingly, myeloid A20 deficiency also
promotes the expansion of osteoclast precursor cells and
enhanced osteoclast differentiation, suggesting that A20
may also directly control receptor activator of nuclear factor
kappa-B (RANK)-induced NF-kB responses [76], as previously suggested [77]. Remarkably, the hyperinflammatory
phenotype of myeloid A20-deficient mice is restricted to the
joints, implying that factors other than systemic immune
elements, such as local physical stress and tissue damage
releasing endogenous ‘danger’ signals, might contribute to
the RA phenotype in these mice [76].
In contrast to its susceptibility to RA, A20myel-KO mice
are protected against lethal acute influenza A virus (IAV)
infection, although with increased cytokine and chemokine
production, challenging the general belief that an excessive
host proinflammatory response is associated with IAVinduced lethality [39]. These results suggest that boosting
the immune response by intranasal administration of A20
siRNA or treatment with small compound inhibitors of A20
might be a valid therapeutic approach for the treatment of
influenza and potentially other viral infections.

proliferation and survival, and autoimmune pathology, but
not spontaneous development of B cell lymphomas
[37,69,83]. According to Tavares et al., A20-deficient B cell
survival results from the resistance of B cells to Fasinduced apoptosis due to increased NF-kB-dependent expression of the antiapoptotic protein Bcl-x [37]. These mice
develop a lupus-like autoimmune pathology characterized
by elevated numbers of germinal center B cells, autoantibodies and glomerular immunoglobulin deposits [37]. By
contrast, another A20B-KO mouse strain shows the development of a progressive inflammatory phenotype, leading
to an autoimmune syndrome only in old mice [83]. These
mice do not display significant levels of antibodies against
nuclear self-antigens, which are the most common autoantibodies observed in SLE, but a general IgG autoreactivity to cardiolipin, a common autoantigen in autoimmune
disease [83]. The fact that A20B-KO mice do not develop B
cell lymphomas in naive conditions [37,69,83], suggests
that A20 deficiency may sensitize to lymphomagenesis
only in cooperation with other oncogenes. Future studies
should provide more insight into this aspect to clarify the
role of A20 as a tumor suppressor in B cells.

A20 deficiency in dendritic cells (DCs)
Two groups independently generated a DC-specific A20
knockout with different phenotypes [36,78]. Both A20DC-KO
strains develop massive splenomegaly and lymphadenopathy due to accumulation of DCs and myeloid and lymphoid
cells, and their DCs spontaneously mature and are hyperresponsive to activating stimuli. A20-deficient DCs show
enhanced survival through the expression of the antiapoptotic proteins Bcl-2 and Bcl-x [36]. This suggests that A20dependent NF-kB inhibition preventing the expression of
pro-survival signals has a more important impact on cell fate
then the antiapoptotic capacities of A20 in this cell type.
However, both DC A20-deficient mouse strains also show
remarkable differences. One strain develops SLE-like symptoms, including the presence of double-stranded DNA-specific autoantibodies, glomerulonephritis, antiphospholipid
syndrome, and arthritis [36]. By contrast, the other strain
spontaneously develops lymphocyte-dependent colitis, seronegative ankylosing arthritis, and enthesitis; conditions
typical of human IBD [78]. Different genetic backgrounds,
genetic differences related to the targeting strategy by
which mice were generated, and/or differences in microbiota
in the two colonies may explain the differences between the
two A20DC-KO strains [79]. Nevertheless, both studies clearly demonstrate a central role for A20 in DC activation and
immune homeostasis.
A20 silencing might provide a strategy to increase the
efficiency of DC-based vaccination against cancer and perhaps infectious diseases. Indeed, A20-deficient DCs induce
far better antigen-specific immune responses than wild type
DCs [78]. Also, RNAi-mediated downregulation of A20 in
DCs was shown to enhance T cell stimulatory capacity and
suppress regulatory T cell activity boosting antitumoral and
anti-HIV cellular immune responses [80–82].

A20 deficiency in the skin
A20 polymorphisms have been identified in psoriasis
patients [84]. Epidermis-specific A20-deficient mice were
generated to define experimentally the role of A20 in skin
homeostasis and psoriasis pathology [85]. These mice,
however, do not develop spontaneous skin inflammation.
Instead, A20E-KO mice display keratinocyte hyperproliferation, and develop ectodermal organ abnormalities, including dishevelled hair, longer nails, and sebocyte
hyperplasia, resembling mice overexpressing ectodysplasin-A1 (EDA-A1) or its receptor EDAR, important for the
development of ectodermal appendages. In vitro studies
further characterized A20 as an inhibitor of EDAR-induced
NF-kB signaling, independent from its DUB activity, assuring proper skin homeostasis and epidermal appendage
development [85]. Further studies are needed to assess if
A20 is involved in inflammatory reactions associated with
skin inflammation and psoriasis.

A20 deficiency in B cells
Three independent B cell-specific A20 knockout strains
have been generated; all three exhibiting enhanced B cell
28

A20 in other tissues
Studies with overexpression of A20 have provided important information on the role of A20 in other cell types and
tissues in vivo. Gene transfer of A20 in adult rats and in
hippocampal cultures has been reported to exert a neuroprotective effect in middle cerebral artery occlusion
(MCAO) [86], a model of cerebral ischemia that is strongly
associated with NF-kB activation. A20 is also strongly
upregulated in conditions of permanent MCAO in mice.
However, mice in which A20 is deleted exclusively in
neurons or in all CNS cell types, exhibited no difference
in disease outcome as compared to control animals [87].
Overexpression of A20 in pancreatic islets of Langerhans confers resistance to cytokine-mediated activation of
NF-kB, protecting them from apoptosis in the early posttransplantation period [88]. Moreover, direct A20 gene
transfer into the pancreas also protects mice from streptozotocin-induced diabetes [89]. A20 overexpression in
liver has been shown to be protective in conditions of

Review
hepatectomy [90–92] and acute toxic hepatitis [93] through
combined antiapoptotic, anti-inflammatory, and pro-proliferative functions. In lung, adenoviral delivery of A20 was
shown to attenuate allergic airway inflammation through
suppression of inflammatory cytokine production [94]. Finally, adenoviral overexpression of A20 in smooth muscle
cells of rats subjected to a vascular injury model prevents
neointimal hyperplasia through combined anti-inflammatory and antiproliferative activities leading to accelerated
re-endothelialization of the injured vessels [95].
In conclusion, these studies in mice show that A20 may
have very different functions in different cell types and
depending on the specific disease context. Reduced expression of A20 may sensitize to the development of inflammatory pathology possibly in collaboration with additional
genetic defects or in specific environmental conditions.
Concluding remarks
A20 is now widely accepted as a key regulator of inflammation and immunity based on its activities on the NF-kB
and IRF pathways, as well as its role in protecting from
apoptosis. It is likely that future studies will reveal additional roles for A20 in the regulation of signaling pathways.
Recent in vivo gene targeting studies have clearly demonstrated an important function for A20 in multiple cell types
including myeloid cells, DCs, B cells, keratinocytes, and
IECs. These have provided insights as to how genetic
variants in the A20/TNFAIP3 gene may predispose to
various inflammatory and autoimmune pathologies. Future cell-specific gene targeting studies in other cell types
such as in T cells, pancreatic b-cells, hepatocytes, or mast
cells, should provide important new information on the
relation between the identified genetic variants and the
development of inflammatory and autoimmune disease.
Human genetic studies as well as experimental studies in
mice clearly suggest that A20 expression directly correlates with disease susceptibility and in some cases, response to therapy, therefore, A20 may serve as both a
predictive and prognostic biomarker. Moreover, enhancing
the expression and/or function of A20 may be a promising
strategy to treat inflammatory autoimmune pathology,
although therapeutic targeting of A20 may be less obvious
and should imply approaches to increase intracellular A20
levels locally in the affected cell type.
Acknowledgments
L. Catrysse is a PhD fellow with the ‘‘Institute for the Promotion of
Innovation by Science and Technology’’ (IWT) and L. Vereecke is a
postdoctoral researcher with the ‘‘Fund for Scientific Research of
Flanders’’ (FWO). Research in the authors’ laboratory is supported by
an FWO Odysseus Grant and by research grants from the ‘‘Geneeskundige Stichting Koningin Elisabeth’’ (GSKE), the Charcot Foundation, the
‘‘Interuniversity Attraction Poles program’’ (IAP7/32), the FWO, the
‘‘Belgian Foundation against Cancer’’, the ‘‘Strategic Basis Research
program’’ of the IWT and the ‘‘Group-ID MRP’’, ‘‘GOA’’ and ‘‘Hercules’’
initiatives of the Ghent University.

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