The m6A reader IMP2 directs autoimmune inflammation through an IL-17− and TNFα

-dependent C/EBP transcription factor axis

Rami Bechara, Nilesh Amatya, Rachel D. Bailey, Yang Li, Felix E. Y. Aggor, De-Dong Li, Chetan V. Jawale, Bianca M.
Coleman, Ning Dai, Nandan S. Gokhale, Tiffany C. Taylor, Stacy M. Horner, Amanda C. Poholek, Anita Bansal, Partha S.
Biswas and Sarah L. Gaffen

Downloaded from http://immunology.sciencemag.org/ at UNIVERSITY OF PITTSBURGH on July 2, 2021

Sci. Immunol. 6, eabd1287.
DOI: 10.1126/sciimmunol.abd1287

IL-17 makes its m6A marks

IL-17 and related cytokines not only support host defense against certain pathogens but also contribute to the
development of autoimmune diseases. A holistic understanding of how IL-17 initiates and propagates inflammation is
needed to identify additional therapeutic opportunities in autoimmunity. Bechara et al. probed the role of N6
-methyladenosine (m6A) modifications of IL-17−induced transcripts in enhancing their stability and translation.
Epitranscriptomic m 6A marks were detected on the mRNAs for the C/EBPβ and C/EBPδ transcription factors involved in
IL-17 signaling. The noncanonical m 6A reader protein IMP2 was implicated in binding these m6A marks. Mice lacking
IMP2 were less susceptible to an autoantibody-induced model of kidney disease driven by IL-17 signaling. These findings
establish posttranscriptional modifications of mRNAs involved in proinflammatory cytokine signaling as potential targets
for therapeutic intervention.

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CYTOKINES Copyright © 2021

The m6A reader IMP2 directs autoimmune

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inflammation through an IL-17– and TNF-dependent American Association
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C/EBP transcription factor axis of Science. No claim
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Rami Bechara1, Nilesh Amatya1, Rachel D. Bailey1, Yang Li1, Felix E. Y. Aggor1, De-Dong Li1,
Chetan V. Jawale1, Bianca M. Coleman1, Ning Dai2, Nandan S. Gokhale3, Tiffany C. Taylor1,
Stacy M. Horner3,4, Amanda C. Poholek5, Anita Bansal6, Partha S. Biswas1, Sarah L. Gaffen1*

Excessive cytokine activity underlies many autoimmune conditions, particularly through the interleukin-17 (IL-17)

Downloaded from http://immunology.sciencemag.org/ at UNIVERSITY OF PITTSBURGH on July 2, 2021

and tumor necrosis factor– (TNF) signaling axis. Both cytokines activate nuclear factor B, but appropriate
induction of downstream effector genes requires coordinated activation of other transcription factors, notably,
CCAAT/enhancer binding proteins (C/EBPs). Here, we demonstrate the unexpected involvement of a post-
transcriptional “epitranscriptomic” mRNA modification [N6-methyladenosine (m6A)] in regulating C/EBP and
C/EBP in response to IL-17A, as well as IL-17F and TNF. Prompted by the observation that C/EBP/-encoding
transcripts contain m6A consensus sites, we show that Cebpd and Cebpb mRNAs are subject to m6A modification.
Induction of C/EBPs is enhanced by an m6A methylase “writer” and suppressed by a demethylase “eraser.” The
only m6A “reader” found to be involved in this pathway was IGF2BP2 (IMP2), and IMP2 occupancy of Cebpd and
Cebpb mRNA was enhanced by m6A modification. IMP2 facilitated IL-17–mediated Cebpd mRNA stabilization and
promoted translation of C/EBP/ in response to IL-17A, IL-17F, and TNF. RNA sequencing revealed transcriptome-­wide
IL-17–induced transcripts that are IMP2 influenced, and RNA immunoprecipitation sequencing identified the subset
of mRNAs that are directly occupied by IMP2, which included Cebpb and Cebpd. Lipocalin-2 (Lcn2), a hallmark of
autoimmune kidney injury, was strongly dependent on IL-17, IMP2, and C/EBP/. Imp2−/− mice were resistant to
autoantibody-induced glomerulonephritis (AGN), showing impaired renal expression of C/EBPs and Lcn2. Moreover,
IMP2 deletion initiated only after AGN onset ameliorated disease. Thus, posttranscriptional regulation of C/EBPs
through m6A/IMP2 represents a previously unidentified paradigm of cytokine-driven autoimmune inflammation.

INTRODUCTION the CCAAT/enhancer binding proteins (C/EBPs). Most known cytokine-­

More than 20 million Americans live with an autoimmune disorder.
The estimated economic burden in medical expenses and loss of
productivity is enormous, exceeding even cancer care (1,  2). The
advent of anticytokine antibody (Ab) drugs three decades ago revo-
lutionized treatment of many autoimmune conditions, beginning
with biologics targeting tumor necrosis factor (TNF). More recently,
interleukin-17 (IL-17; IL-17A) and T helper 17 (TH17) cells were
found to be dysregulated in skin and joint conditions such as psoriasis,
psoriatic arthritis, and ankylosing spondylitis. IL-17 is also impli-
cated in other autoimmune settings such as multiple sclerosis and
autoantibody-induced glomerulonephritis (AGN) (3). In principle,
understanding the molecular basis of cytokine-induced autoimmune
pathology has potential to inform new therapeutic targets (4).
IL-17 signaling occurs predominantly in nonhematopoietic cell
types expressing the IL-17 receptor (5), whereas nearly all cells ex-
press TNF receptors and are responsive to this cytokine. TNF and
IL-17 mediate many overlapping signals, leading to similar, although
not identical, downstream gene profiles. Many transcription factors (TFs)
are activated by one or both of these cytokines, including nuclear factor
B (NF-B), inhibitor of NF-B zeta (IB), AP-1 (activator protein 1), and
1
Division of Rheumatology and Clinical Immunology, University of Pittsburgh,
Pittsburgh, PA, USA. 2Diabetes Unit, Massachusetts General Hospital, Boston, MA,
USA. 3Department of Molecular Genetics and Microbiology, Duke University Medical
Center, Durham, NC, USA. 4Department of Medicine, Duke University Medical Center,
Durham, NC, USA. 5Division of Pediatrics, University of Pittsburgh, Pittsburgh, PA,
USA. 6Department of Immunology, University of Pittsburgh, Pittsburgh, PA, USA.
*Corresponding author. Email: sarah.gaffen@pitt.edu
induced genes are regulated by combinations of these TFs (6–12).
C/EBPs are leucine zipper TFs described in the 1980s, yet their
regulation remains unexpectedly poorly understood, especially com-
pared with other immune TF families such as NF-B or STATs (signal
transducers and activators of transcriptions) (13–17). C/EBPs bind
to characteristic motifs on target promoters and enhancers and are
pleiotropic activators of inflammation in response to numerous im-
mune and microbial stimuli (14). C/EBPs form heterodimers and
homodimers and autoregulate their own promoters (14, 16). C/EBP
exists in multiple, alternatively translated isoforms and is also sub-
ject to inducible phosphorylation that tunes its activity in response
to cytokine signals (18–22). Early studies of IL-17 showed that this
cytokine induces both C/EBP and C/EBP, and both are required
for induction of downstream effector genes that underlie IL-17 bio-
logic activity (7, 8).
Whereas TNF is a potent activator of new transcription (23),
IL-17 is a major activator of posttranscriptional events through a
complex network of RNA binding proteins (RBPs) (5). RBPs bind
to respective client transcripts at cis-acting elements typically found
within 3′ or 5′ untranslated regions (UTRs). A variety of RBPs
[Regnase-1, Arid5a, ASF/SF2 (alternative splicing factor/splicing
factor 2), HuR (human antigen R), DDX3X (DEAD-box helicase 3
X-linked), Roquins, and Act1] influence downstream RNA stability
and/or translation in response to IL-17 (13, 24, 25). Moreover, there
is cross-talk between transcriptional and posttranscriptional events,
because several mRNAs encoding TFs are subject to posttranscriptional
control. This pheno­menon is well described for Nfkbiz (encoding

Bechara et al., Sci. Immunol. 6, eabd1287 (2021) 2 July 2021 1 of 17