Stress signaling boosts interferon-induced gene transcription in macrophages

SCIENCE SIGNALING | RESEARCH ARTICLE
I M M U N O LO G Y Copyright © 2022
The Authors, some
Stress signaling boosts interferon-induced gene rights reserved;
exclusive licensee
transcription in macrophages American Association
for the Advancement
of Science. No claim
Laura Boccuni1,2, Elke Podgorschek1,2, Moritz Schmiedeberg1,2, Ekaterini Platanitis1,2, to original U.S.
Peter Traxler3,4, Philipp Fischer1,2, Alessia Schirripa5, Philipp Novoszel6, Angel R. Nebreda7,8, Government Works
J. Simon C. Arthur9,10, Nikolaus Fortelny3,11, Matthias Farlik3,4, Veronika Sexl5, Christoph Bock3,12,
Maria Sibilia6, Pavel Kovarik1,2, Mathias Müller13, Thomas Decker1,2*
Promoters of antimicrobial genes function as logic boards, integrating signals of innate immune responses. One
such set of genes is stimulated by interferon (IFN) signaling, and the expression of these genes [IFN-stimulated
genes (ISGs)] can be further modulated by cell stress–induced pathways. Here, we investigated the global effect
of stress-induced p38 mitogen-activated protein kinase (MAPK) signaling on the response of macrophages to
IFN. In response to cell stress that coincided with IFN exposure, the p38 MAPK-activated transcription factors
CREB and c-Jun, in addition to the IFN-activated STAT family of transcription factors, bound to ISGs. In addition,
p38 MAPK signaling induced activating histone modifications at the loci of ISGs and stimulated nuclear trans-
location of the CREB coactivator CRTC3. These actions synergistically enhanced ISG expression. Disrupting this
synergy with p38 MAPK inhibitors improved the viability of macrophages infected with Listeria monocytogenes.
Our findings uncover a mechanism of transcriptional synergism and highlight the biological consequences of
coincident stress-induced p38 MAPK and IFN-stimulated signal transduction.
INTRODUCTION factors ISGF3 and GAF. ISGF3 consists of a tyrosine-phosphorylat-

Macrophages provide innate immunity through recognition, ed STAT1-STAT2 heterodimer in association with IFN-regulatory
phagocytosis, and killing of pathogens and through the synthesis factor 9 (IRF9). It is formed predominantly by the type I IFNR
of regulatory cytokines. The signaling environment of an immuno- and stimulates transcription by associating with IFN-stimulated re-
Downloaded from https://www.science.org on December 21, 2022

logically activated macrophage is generally determined by a multi- sponse elements (ISREs) of ISG promoters. On the other hand, GAF
tude of pathways that arise from the activation of pattern is a homodimer of tyrosine-phosphorylated STAT1 and is generated
recognition and cytokine receptors and that may reinforce or in larger quantities by IFN-γ receptor signaling. It activates the tran-
inhibit each other. The modular nature of control elements in scription of ISGs by binding to IFN-γ–activated sequences (GAS) of
target gene promoters provides an important means to integrate ISG promoters.
the output of signaling pathways through cooperativity of transcrip- ISG expression is modulated by additional signaling pathways.
tion factors. Among macrophage-activating cytokines, interferons For example, a large contingent of ISGs is regulated by cooperativity
(IFNs) play a major role (1, 2). Type I IFNs (IFN-I; including between STAT and nuclear factor κB (NF-κB) pathways (7–8). Ac-
IFN-α/β) are mainly antiviral, whereas type II IFNs (IFN-γ) increase tivity of the p38 mitogen-activated protein kinase ( p38 MAPK or
effector functions against nonviral pathogens. IFN receptors p38) pathway has also been linked to the control of ISG expression
(IFNRs) signal through Janus kinases (JAKs) and their tyrosine (9, 10). Pathways mediated by c-Jun N-terminal kinase (JNK) and
phosphorylation of signal transducers and activators of transcrip- p38 are activated through a wide variety of stimuli, such as environ-
tion (STATs) (3–6). IFNR signaling activates the transcription of mental stress, growth factors, inflammatory cytokines, or infection.
IFN-stimulated genes (ISGs) by generating the transcription Among the p38 family enzymes ( p38α, p38β, p38γ, and p38δ), p38α
is the only isoform expressed ubiquitously (11, 12). Dual Thr/Tyr
phosphorylation by MAP2K activates p38, which creates a
1
Max Perutz Labs, Vienna Biocenter Campus (VBC), Vienna 1030, Austria. complex signaling network by phosphorylation of several down-
2
University of Vienna, Center for Molecular Biology, Department of Microbiology,
Immunobiology and Genetics, Vienna 1030, Austria. 3CeMM Research Center for stream targets (13–15). Studies posit a direct involvement of p38
Molecular Medicine of the Austrian Academy of Sciences, Vienna 1090, Austria. in IFNR signaling and in the transcriptional activation of ISGs
4
Department of Dermatology, Medical University of Vienna, Vienna 1090, through ISRE and GAS promoter elements (10, 16).
Austria. 5Institute of Pharmacology and Toxicology, University of Veterinary Med-
icine, Vienna 1210, Austria. 6Center for Cancer Research, Medical University of
In this study, we systemically analyzed the interplay between
Vienna and Comprehensive Cancer Center, Vienna 1090, Austria. 7Institute for Re- stress and IFN pathways in macrophages. Our data show that p38
search in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technol- is not directly involved in IFN signaling but rather synergizes
ogy, Barcelona 08028, Spain. 8ICREA, Pg. Lluís Companys 23, Barcelona 08010, with IFN-induced JAK-STAT pathways when cells are simultane-
Spain. 9Division of Cell Signaling and Immunology and University of Dundee,
Dow Street, Dundee DD1 5EH, UK. 10Medical Research Council Protein Phosphor- ously exposed to IFN and stress. We defined the subset of stress-en-
ylation Unit, School of Life Sciences, Wellcome Trust Building, University of hanced ISGs and found that cooperativity between STAT and cAMP
Dundee, Dow Street, Dundee DD1 5EH, UK. 11Computational Systems Biology response element–binding protein (CREB) or c-Jun transcription
Group, Department of Biosciences and Medical Biology, Paris Lodron University
of Salzburg, Salzburg 5020, Austria. 12Institute of Artificial Intelligence, Medical factors was the basis for the synergy between p38 and IFN signaling.
University of Vienna, Vienna 1090, Austria. 13Institute of Animal Breeding and Ge- Analysis of macrophages exposed to Listeria monocytogenes under-
netics, University of Veterinary Medicine Vienna, Vienna 1210, Austria. pinned the importance of p38 and IFN pathway cooperation for the
*Corresponding author. Email: thomas.decker@univie.ac.at
Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 1 of 20

course of infection. Our data highlight the role of ISG promoters as BMDMs (TTPΔM) produced the same degree of Ifit3 mRNA en-
convergence points for the coordinated response to IFN and stress. hancement, and PH still abolished the anisomycin effect as in
wild-type (WT) cells (fig. S1I). The data rule out an involvement
of TTP-dependent mRNA decay in stress enhancement of ISG
RESULTS expression.
p38 MAPK selectively enhances IFN-γ– and IFN-β–induced
ISG expression p38 MAPK increases RNA polymerase II binding to stress-
To study the interaction of stress-induced MAPK and IFN pathways enhanced ISGs
without the confounding effects of NF-κB (8), we used the drug ani- The transcription cycle requires dynamic phosphorylation and de-
somycin in combination with IFN-β or IFN-γ, with or without one phosphorylation of the RNA polymerase II (Pol II) C-terminal
of two inhibitors of p38 MAPK activity, PH-797804 (PH) or LY- domain (CTD) (23). Whereas Ser5 phosphorylation of the Pol II
2228820 (LY), in bone marrow–derived macrophages (BMDMs) CTD is associated with promoter clearance and promoter-proximal
(Fig. 1A) (9, 17). Anisomycin is a bacterial pyrrolidine antibiotic pausing, Ser2 phosphorylation is indicative of progressive transcript
that can potently and reversibly inhibit eukaryotic protein synthesis. elongation. We considered the possibility that p38 signaling might
At the very low concentrations used here, anisomycin is known to selectively increase Pol II phosphorylation rather than promoting its
cause ribotoxic stress in cells, leading to the activation of JNKs and recruitment to ISGs. In line with pre-mRNA expression, chromatin
p38 MAPKs, through a mechanism not yet fully understood (18). immunoprecipitation (ChIP) analysis showed that anisomycin
As expected, anisomycin caused phosphorylation of both p38 and alone failed to recruit Pol II, but anisomycin and p38 enhanced
JNK but did not stimulate degradation of inhibitor of κB (IκB). PH the binding of both Pol II and its Ser5-phosphorylated form ( ph-
blocked p38 activity without consequences for JNK activation or Ser5 Pol II) to the Ifit3 and Ifit2 promoters compared with IFN-β
IκB degradation (fig. S1, A to C). Furthermore, neither IFN-γ nor alone. Relative increases in total Pol II and ph-Ser5 Pol II were
IFN-β promoted p38 phosphorylation, and anisomycin did not similar. Anisomycin had no impact on IFN-β–dependent Pol II re-
stimulate STAT1 Tyr701 phosphorylation (fig. S1D) (19). cruitment to the enhancement-resistant Mx1 promoter (Fig. 2, A
Treatment with IFN-γ increased the abundance of the STAT1 and B, and fig. S2A). The increases of Pol II and ph-Ser5 Pol II
homodimer–regulated pre-mRNAs Irf1, Socs3, and Gbp5 (20) inde- binding to the Irf1 and Socs3 promoters stimulated by IFN-γ or ani-
pendently of p38. The combined action of stress and IFN-γ further somycin + IFN-γ were comparably small (Fig. 2C and fig. S2B). This
enhanced Irf1 and Socs3 expression through p38-dependent signals. confirms findings that the Irf1 transcription start is preloaded with
In contrast, Gbp5 expression remained unaffected by either stress or Pol II, in line with the gene’s primary response to IFN-γ (24, 25).

both p38 inhibitors (Fig. 1, B and C, and fig. S1E). Likewise, IFN-β– Anisomycin did not further stimulate Pol II recruitment at the en-
induced expression of ISGs was unperturbed in the presence of hancement-resistant Gbp5 gene (Fig. 2D).
either p38 inhibitor, but anisomycin-induced enhancement of the In analyzing p38-dependent effects on elongation, we observed
Ifit2 and Ifit3 genes was dependent on p38. Expression of the Mx1 similar increases of stress-induced, p38-dependent increase of total
gene was not enhanced by anisomycin (Fig. 1, D and E, and fig. Pol II and Ser2-phosphorylated Pol II ( ph-Ser2 Pol II) at the Ifit3
S1F). The results demonstrate that these ISGs represented the fol- and Ifit2 gene bodies compared with IFN-β alone, and this effect
lowing categories: ISRE-controlled, stress-enhanced (Ifit2 and Ifit3); was not seen at the Mx1 gene body (Fig. 2, E and F, and fig. S2C).
ISRE-controlled, enhancement-resistant (Mx1); GAS-controlled, Contrasting the recruitment of Pol II and ph-Ser5 Pol II at the Irf1
stress-enhanced (Irf1 and Socs3); and GAS-controlled, enhance- and Socs3 promoters, anisomycin enhanced the IFN-γ–induced
ment-resistant (Gbp5). Deficiency in p38α ( p38αΔM) abolished presence of Pol II and ph-Ser2 Pol II at the bodies of these genes
both the anisomycin-dependent enhancement of Ifit2 stimulation but not at the Gbp5 gene (Fig. 2, G and H, and fig. S2D), confirming
by IFN-β and the enhanced expression of the Irf1 gene by IFN-γ. that IFN-γ increases Irf1 and Socs3 transcription by stimulating Pol
As expected, p38α deficiency did not reduce expression of the en- II elongation. Consistent with the pre-mRNA data (Fig. 1H), aniso-
hancement-resistant Mx1 and Gbp5 genes (Fig. 1, F and G). mycin did not enhance the IFN-β–dependent recruitment of phos-
These data show that anisomycin enhances the expression of a phorylated Pol II or total Pol II to the Irf1 promoter and gene body
subset of IFN-γ–induced genes controlled by STAT1 homodimers (fig. S2E).
and a subset of IFN-β–induced genes controlled by ISGF3. Thus, we In conclusion, the data show that binding of Pol II reflects the
next turned to study enhancement of STAT1 homodimer–con- control of ISGs pre-mRNA synthesis by IFN and stress, in line
trolled genes by IFN-β and that of ISGF3-controlled genes by with stress enhancement through augmented transcriptional initia-
IFN-γ. We found that IFN-β–stimulated expression of the Irf1 tion. However, the data do not support the notion of selective en-
and Socs3 genes lacked enhancement by anisomycin. In contrast, hancement of CTD phosphorylation through the p38 pathway,
induction of Ifit2 and Ifit3 genes with IFN-γ was enhanced by ani- because anisomycin-induced increases in recruitment were highly
somycin (Fig. 1, H and I). Thus, we conclude that induction via GAS similar for total Pol II and its phosphorylated forms.
or ISRE promoter elements determines whether ISG responses to
both IFN types are enhanced by stress. p38 MAPK signaling introduces activating histone
Kinetic studies showed that anisomycin + IFN-γ stimulation for modifications at ISG promoters
4 hours resulted in a larger enhancement of the Ifit2 and Ifit3 Stress signaling introduces activating histone modifications, partic-
mRNA compared with the pre-mRNA (Fig. 1, J and K, and fig. ularly histone 3 (H3) phosphorylation at Ser10 ( ph-H3S10) and
S1, G and H). Tristetraprolin (TTP) is an RNA binding protein Ser28 ( ph-H3S28) and its acetylation at Lys9 and Lys14 (H3K9/
that regulates mRNA decay of inflammatory genes, and its destabi- K14ac), thereby facilitating the transcription of stress-responsive
lizing activity is inhibited by p38 MAPK (21, 22). TTP-deficient promoters (26, 27). The amount of Ser28 phosphorylation after

Fig. 1. Selective ISG enhancement by p38 MAPK. (A) Experimental overview listing the genotypes used and their analysis. (B to E) Quantitative real-time polymerase
chain reaction (qPCR) showing pre-mRNA expression of the indicated genes in WT BMDMs upon stimulation with anisomycin (An; 100 ng/ml), IFN-γ (10 ng/ml for 30 min;
B and C), or IFN-β (250 IU/ml for 1.5 hours; D and E) either alone or after pretreatments with anisomycin (20 min) alone or with PH-797804 (PH; 1 μM; 1 hour) or LY-2228820
(LY; 200 nM; 1 hour). (F and G) qPCR-based mRNA expression of the indicated genes in p38αΔM BMDMs and their WT counterparts ( p38αfl/fl ) stimulated with anisomycin
(100 ng/ml) alone or (F) IFN-β (250 IU/ml for 2 hours) or (G) IFN-γ (10 ng/ml for 2 hours) alone or after a 20-min pretreatment with anisomycin. (H and I) qPCR-based
measurement of mRNA expression of the indicated genes in WT BMDMs stimulated as described in (B) and (C) and (D) and (E), respectively, minus LY. (J and K) Time course
of Ifit2 pre-mRNA and mRNA expression, analyzed by qPCR, in WT BMDMs treated as described in (H) and (I), respectively, minus the inhibitor. Data are means + SD of three
individual experiments, except (F) and (G), which are from triplicate samples from each of two mice. For (B) to (E) and (H) to (K), one-way analysis of variance (ANOVA)
corrected for multiple testing with Dunnett’s post hoc test was used. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.

Fig. 2. p38 MAPK dependence of RNA Pol II binding and histone modification at ISG promoters. (A to H) BMDMs were stimulated with anisomycin (An; 100 ng/ml),
(A, B ,E, and F) IFN-β (250 IU/ml for 2 hours), or (C, D, G, and H) IFN-γ (10 ng/ml for 2 hours) alone or in combination with 20-min pretreatment in anisomycin with or
without PH (1 μM) for 1 hour and processed for site-directed ChIP with control IgG or antibodies to phospho-Ser5 or phospho-Ser2 Pol II, and total RNA-Pol II, analyzing
promoter and gene body association of (A, B, E, and F) Ifit3 and Mx1 and (C, D, G, and H) Irf1 andGbp5 as indicated. (I and J) BMDMs were stimulated as described in (A) and
(C), respectively, and processed for site-directed ChIP with ph-H3S28 antibody or control IgG, analyzing binding to (I) Ifit3 and Mx1 and (J) Irf1 and Gbp5 promoters. (K and
L) BMDMs were stimulated as described in (I) and (J), respectively, and processed for site-directed ChIP with H2A.Z antibody or control IgG, analyzing binding to the
indicated promoters. Data are means + SD of at least three independent experiments. Two-way ANOVA corrected for multiple testing with Dunnett’s post hoc test was
used. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

anisomycin or IFN-β treatment alone was slightly increased, coexpression network analysis (WGCNA) (31). A total of 26
whereas their combination strongly enhanced the p38-dependent WGCNA modules (clusters of highly coexpressed genes) were iden-
deposition of this mark. Anisomycin synergy with IFN was not se- tified. Among these, four modules (designated green, dark olive
lective for enhanced ISG expression, because it was observed at both green, gray, and midnight blue) were positively correlated with
the enhanced Ifit3 and the enhancement-resistant Mx1 promoters. the IFN and anisomycin with IFN conditions (fig. S3E). The coex-
Similar data were obtained for anisomycin and IFN-γ treatment pression network in the green and dark olive green modules was
(Fig. 2, I and J). Two further stress-associated H3 marks, Ser10 phos- associated with, respectively, IFN-β– and IFN-γ–induced genes
phorylation and Lys9/Lys14 acetylation, also occurred at all exam- that were either enhanced or not enhanced and significantly
ined ISG promoters (fig. S2, F and G). Contrasting the stress- [false discovery rate (FDR) ≤ 0.05] associated with gene ontology
induced histone marks, H3 Lys27 trimethylation (H3K27me3) (GO) terms related to general regulation of immune responses.
erasure and H3 Lys27 acetylation (H3K27ac), events that are more Most genes associated with the gray module consisted of ISGs en-
generally associated with gene activation (28), occurred in response hanced by anisomycin combined with IFN-γ that were enriched for
to IFN without further input by anisomycin (fig. S2, H and I). antiviral responses, whereas the midnight blue module consisted of
Deposition of the histone variant H2A.Z through p38 signaling enhancement-resistant genes enriched for metabolic processes,
is associated with some active promoters, but its removal occurs such as oxidative phosphorylation (OXPHOS) and mitochondrial
during transcriptional activation of ISGs by IFN-β (29, 30). The de- respiration (fig. S3F).
crease of H2A.Z at ISG promoters occurred in response to both Intersection of the RNA-seq data with available ChIP-sequenc-
IFN-β and IFN-γ, without an effect of cotreatment with anisomycin ing (ChIP-seq) datasets (32) allowed us to relate IFN-β– and IFN-γ–
or PH (Fig. 2, K and L). Considered together, these results indicate induced gene expression to promoter binding by STAT1, STAT2,
that p38 signaling increases typical stress-induced H3 modifications and IRF9 (fig. S3G and data files S2 and S3). Genes bound by
independently of enhancing ISG expression and that it does not STAT1 homodimers and/or ISGF3 were overlaid with the set of ani-
affect IFN-dependent H2A.Z removal, H3K27ac deposition, and somycin-enhanced ISGs. In accordance with the pre-mRNA data
H3K27me3 erasure. (Fig. 1), gene expression enhancement by anisomycin with IFN-γ
occurred at promoters associating with either STAT1 homodimers
Global analyses of stress-enhanced ISGs and their promoter or the ISGF3 complex (21 and 78 genes, respectively), whereas genes
configuration enhanced by anisomycin with IFN-β or either IFN type were bound
The data above generated with representative gene loci did not only by ISGF3 (57 and 66 genes, respectively). The data confirm our
reveal a selectivity factor deciding between enhancement and en- conclusions above (from Fig. 1) by clearly defining the subset of en-

hancement resistance of ISGs. We initiated our search for this hancement-responsive ISGs and by demonstrating the relevance of
factor by globally identifying ISGs affected by stress signaling GAS and ISRE elements for enhancement response to both
using bulk RNA sequencing (RNA-seq). Anisomycin treatment IFN types.
alone increased the expression of 2208 mRNAs, including those as-
sociated with inflammation (Il1a, Il1b, Nlrp3, and Il6), tumor ne- Stress signaling does not alter ISG promoter accessibility
crosis factor (TNF) signaling (Inhba, Nfkbiz, and Tnfsf18), and Further addressing the mechanism of p38-dependent enhancement
stress response pathways (Ptgs2, Nr4a3, Jun, and Fosl1) (fig. S3, A of ISG expression, dynamic changes of chromatin accessibility were
and B, and data file S1). One thousand four hundred sixty-seven explored by ATAC-seq (assay for transposase-accessible chromatin
IFN-γ–induced genes and 1757 IFN-β–induced genes included with high-throughput sequencing). We hypothesized that increased
gene sets either repressed or stimulated by anisomycin alone Pol II binding and transcription after anisomycin and IFN cotreat-
(Fig. 3, A and B, and data files S2 and S3). Among these, 182 ment might result from increased chromatin opening. For data
IFN-γ–induced ISGs and 116 IFN-β–induced ISGs were enhanced analysis, we divided ISGs according to RNA-seq into enhanced
by anisomycin but did not respond to the drug alone, whereas 103 and not-enhanced sets (data files S2 and S3). As expected (25),
genes showed enhancement after induction by either IFN type both enhanced and enhancement-resistant gene sets showed chro-
(magenta, royal blue, and aquamarine datasets in Fig. 3, A and B, matin opening upon stimulation with IFN-γ. Anisomycin alone
respectively). Further analysis revealed that, in line with our quan- slightly decreased the chromatin accessibility observed in untreated
titative polymerase chain reaction (qPCR) results, the group of BMDMs (Fig. 4, A and B, first three panels), and unexpectedly, the
GAS-controlled ISGs (Irf1, Socs3, and Cxcl9) was enhanced only drug failed to increase accessible chromatin at either enhanced or
by anisomycin combined with IFN-γ, whereas the group of ISRE- enhancement-resistant loci when combined with either IFN-γ or
controlled ISGs (Ifit1, Ifit2, Ifit3, and Mx2) was enhanced by aniso- IFN-β (Fig. 4, A to D). Inspection of individual gene loci confirmed
mycin combined with either IFN type. Conversely, Gbp5 and Mx1 chromatin opening by IFN and the absence of effects from aniso-
were not enhanced by the combination of anisomycin and either mycin or PH on enhanced (Irf1 and Ifit3) and not-enhanced gene
IFN type (Fig. 3, C to E, and data files S2 and S3). Representative loci (Gbp5 and Mx1) (Fig. 4, E and F). Chromatin accessibility at
genes enhanced by anisomycin combined with IFN-γ (Cxcl9), anisomycin-induced, stress response genes revealed by RNA-seq
IFN-β (Usp18), or either (Mx2) were validated by qPCR (Fig. 3F). (data file S1) remained similarly unchanged (Fig. 4G), suggesting
Principal components analysis (PCA) of the top 40 anisomycin- that such genes are primary response genes with preconfigured pro-
enhanced ISGs revealed a clear separation between IFN-induced moter chromatin (33). In conclusion, nucleosome remodeling is not
and enhanced ISGs, whereas anisomycin samples showed a a driving force behind stress-enhanced ISG or non-ISG expression,
similar expression profile to the untreated ones (fig. S3, C and D). suggesting a mechanism of action at subsequent steps of transcrip-
To identify the differentially expressed genes associated with the dif- tional activation.
ferent sample traits and phenotypes, we performed weighted gene

Fig. 3. Genome-wide analysis of

p38 MAPK-dependent ISG expres-
sion. (A to E) RNA-seq was performed
from three individual replicates of WT
BMDMs stimulated with anisomycin
(An; 100 ng/ml) alone or with IFN-γ
(10 ng/ml; A, C, and D) or IFN-β (250
IU/ml; B, C, and E) for 2 hours alone or
after pretreatment with anisomycin
for 20 min. Differentially expressed
genes were then categorized by re-
sponse into pie charts (A and B), as
indicated in the inset legends. For
clarity between the two “enhanced”
categories, the magenta slices (A)
and royal blue slices (B) represent
ISGs that were enhanced by aniso-
mycin only when combined with IFN
(annotated with “An < IFN < An +
IFN”); the dark/navy blue slices rep-
resent ISGs that also responded to
anisomycin alone (annotated with
“An ≥ IFN < An + IFN”). IFN log2-
FC ≥ 1, Padj ≤ 0.05; An + IFN versus
IFN or An ≥ 1. Details are provided in
data files S2 and S3. (C) Scatterplot
comparing the log2FC in mRNA
abundances from BMDMs described
in (A) and (B). Gray dots represent
insignificantly changed genes
(“N.S.”), and aquamarine and orange

dots represent ISGs whose expres-
sion changed in the same direction
(enhanced or not enhanced, respec-
tively) by anisomycin in the presence
of either IFN type (“common”; IFN
log2FC ≥ 1, Padj ≤ 0.05; An < IFN < An
+ IFN; An + IFN versus IFN log2FC ≥ 1).
Magenta, royal blue, pistachio green,
and pale blue dots represent genes
from the same categories described/
labeled in (A) and (B). (D and E) RNA-
seq heatmap of the top 40 enhanced
ISGs in BMDMs stimulated with ani-
somycin alone or IFN-γ (D) or IFN-β
(E) alone or in combination with ani-
somycin. Color key represents row z
score, and genes are clustered by
rows. (F) qPCR-based measurement
of Cxcl9, Usp18, and Mx2 mRNA ex-
pression after treatment with aniso-
mycin (100 ng/ml), IFN-β (250 IU/ml
for 2 hours), or IFN-γ (10 ng/ml for 2 hours) alone or after pretreatment with anisomycin (20 min) or with anisomycin and PH (1 μM; for 1 hour). Inset graphs display the
IFN-γ–specific data on a smaller y-axis scale. Data are means + SD of at least three independent replicates. For (F), one-way ANOVA corrected for multiple testing with
Dunnett’s post hoc test was used. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Enhanced ISG loci are highly enriched in AP-1 and CREB the ISRE at the Ifit3 and Mx1 promoters, but this was unaffected
binding motifs: Essential role for CREB in IFN and stress by PH. When combined with IFN-γ, anisomycin slightly augment-
cooperativity ed STAT1 homodimer binding to the GAS motifs in both the en-
To test the hypothesis that p38 enhances transcriptional initiation hanced Irf1 and not-enhanced Gbp5 promoters (fig. S4, A and B).
by recruitment and/or activation of transcription factors, we first These data do not support increased STAT binding as a cause for
examined promoter binding of STAT complexes. Anisomycin ISG enhancement by p38. To narrow down enhancement candi-
caused a slight increment of IFN-β–induced STAT1 binding to dates, we performed enrichment analysis for binding motifs based

Fig. 4. IFN, but not anisomycin, increases chromatin accessibility at ISG loci. (A to D) Summary profile plots and heatmap of chromatin accessibility generated by
using normalized read coverages in BMDMs treated with anisomycin (An; 100 ng/ml) alone, (A and B) IFN-γ (10 ng/ml for 2 hours), or (C and D) IFN-β (250 IU/ml for 2
hours) alone or after pretreatment with anisomycin (20 min) or anisomycin and PH (1 μM; 1 hour). The colored profile represents enhanced ISGs (A and C) and enhance-
ment-resistant ISGs (B and D) according to RNA-seq. Profiles and heatmaps represent regions from between −2 and +1 kb with regard to the TSS as indicated. Merged
data from three individual replicates are shown. (E and F) ATAC-seq genome browser tracks at respective gene loci in WT BMDMs treated with anisomycin alone, (E) IFN-γ,
or (F) IFN-β alone or in combination with anisomycin with or without PH as indicated. One representative replicate is shown. (G) Summary profile plots and heatmap of
chromatin accessibility similar to (A) to (D) showing An-induced genes according to the RNA-seq (log2FC ≥ 1) in BMDMs that were either untreated (black; UT) or treated
with anisomycin (gray; An). Data in (A) to (G) are derived from three individual replicates.

on RNA-seq data. As expected, both enhanced and not-enhanced response to anisomycin treatment and was partially inhibited by
gene loci were highly enriched in ISRE. Notably, activator the JNK inhibitor SP600125 (JNKi-II). PH also decreased c-Jun
protein-1 (AP-1) binding motifs were enriched specifically in en- phosphorylation, albeit to a lesser extent, and the combination of
hanced genes (Fig. 5A). The AP-1 subunit c-Jun is involved in the two inhibitors was slightly more effective (fig. S4, C and D).
stress and immune responses (34, 35, 36), is a constituent of the To assess the input of the c-Jun pathway into the enhancement of
type I IFN enhanceosome, and is required for induction of some ISG expression, we pretreated BMDMs with one of two JNK inhib-
ISGs by IFN-γ (37). c-Jun phosphorylation at Ser73 occurred in itors, JNKi-II and JNK-IN-8, that inhibit JNK activity and JNK
Fig. 5. ISG enhancement requires CRE/

AP-1 binding sites and CREB activity. (A)
Transcription factor prediction for ISGs
enhanced by anisomycin, representing the
most enriched motif (P ≤ 0.05). (B) Exper-
imental overview listing the genotypes
used and the timing of their treatment and
analysis. (C) qPCR analysis of pre-mRNA
expression in BMDMs that were untreated
(UT) or stimulated with anisomycin (An;
100 ng/ml) alone, IFN-β (Ifit3 and Ifit2; 250
IU/ml for 2 hours), or IFN-γ (Irf1 and Socs3;
10 ng/ml for 2 hours) alone or after a pre-
treatment with anisomycin, or with aniso-
mycin and SP600125 (JNKi-II; 20 μM; 1-
hour pretreatment) or JNK Inhibitor XVI
(JNK-IN-8; 10 μM; 1-hour pretreatment). (D)
qPCR showing mRNA expression in cJunfl/
fl
;Vav-iCre +/− compared with their WT
counterparts (cJunfl/fl ) upon stimulation
with anisomycin (100 ng/ml) alone, IFN-β

(Ifit3 and Ifit2; 250 IU/ml for 2 hours), or
IFN-γ (Irf1 and Socs3; 10 ng/ml for 2 hours)
alone or after a 20-min treatment with
anisomycin. (E) Site-directed ChIP in
BMDMs stimulated with anisomycin (100
ng/ml) alone, IFN-β (left: 250 IU/ml for 2
hours), or IFN-γ (right: 10 ng/ml for 2
hours) alone or in combination with ani-
somycin (20 min before treatment) or with
anisomycin and PH (1 μM; 1-hour pre-
treatment) and processed for ChIP anti-
body targeting pSer133 CREB1 on the CRE
binding site of Ifit3 and Irf1. IgG isotype
was used as negative control. (F and G)
qPCR showing mRNA expression in
BMDMs transduced with sgRNA targeting
CREB1 or a nontargeting control (NTC)
upon stimulation with anisomycin (100
ng/ml; 2 hours and 20 min) alone, IFN-β
(Ifit2 and Mx1; 250 IU/ml for 2 hours), or
IFN-γ (Irf1 and Gbp5; 10 ng/ml for 2 hours)
alone or in combination with anisomycin
(20-min pretreatment). Data are
means + SD from at least three indepen-
dent replicates. For (C), one-way ANOVA
corrected for multiple testing with Dun-
nett’s post hoc test was used. For (D) to (G),
two-way ANOVA corrected for multiple
testing with Dunnett’s post hoc test was
used. *P < 0.05, **P < 0.01, ***P < 0.001,
and ****P < 0.0001.

activation, respectively (Fig. 5B) (38). Both inhibitors slightly de- RNA-seq data identifying IFN-γ– and IFN-β–induced genes, we
creased anisomycin-induced enhancement of IFN-β–stimulated discovered 575 ISG loci among the c-Jun and CREB1 co-bound
Ifit2 and Ifit3 expression, and of IFN-γ–stimulated Irf1 and Socs3 genes, and 240 or 132 ISG loci bound by, respectively, CREB1 or
expression, reaching statistical significance only for Ifit3. The de- c-Jun alone (Fig. 6, B and C, and data file S4). Whereas CREB1
crease in anisomycin-induced Ifit3 enhancement caused by JNK in- binding was generally constitutive, c-Jun binding was induced by
hibitors (Fig. 5C) appeared to be less than that caused by p38 IFN-γ on 43 genes, including 34 known ISGs (Fig. 6D). Gene set
inhibitors (Fig. 1D). Furthermore, the decrease of anisomycin-en- enrichment analysis (GSEA) of the ChIP-seq data (51) indicated
hanced ISG expression did not reach statistical significance in c- that genes bound by both CREB1 and c-Jun or by only c-Jun
Jun–deficient BMDMs (cJunfl/fl;Vav-iCre +/−) (Fig. 5D). IFN did were enriched for MAPK and JAK-STAT signaling pathways,
not increase c-Jun activation when provided with or without aniso- whereas these annotations were not found for genes bound by
mycin pretreatment (fig. S4E). These results suggest that c-Jun only CREB1 (fig. S5, A to C). The transcription factor binding
alone cannot account for the entire ISG enhancement by p38 signal- site prediction confirmed the GSEA by revealing that genes
ing, although it may be a contributing factor. bound by CREB and c-Jun or c-Jun only were enriched in STAT,
In addition to the AP-1 consensus element, enhanced ISGs were IRF1, IRF7, and ISRE binding sites (fig. S5, D and E), whereas
enriched in adenosine 3′,5′-monophosphate (cAMP) response genes bound by only CREB1 were devoid of ISRE or IRF binding
element (CRE) consensus sequences, which share core nucleotides motifs but enriched for GAS sequences annotated as STAT3
with the AP-1 element and represent binding sites for activating binding motifs (fig. S5F). The data suggest a predominance of pro-
transcription factor (ATF)/CREB family members (Fig. 5A). CRE- moters with CREB1 and c-Jun binding sites among enhanced ISGs
binding protein 1 (CREB1) is activated by p38 (39, 40) and is able to and, thus, associate c-Jun with IFN-γ signaling (37).
heterodimerize with c-Jun (41, 42). Anisomycin, but not IFN, acti-
vated CREB1 by phosphorylation at Ser133, and this was abolished CREB1 and c-Jun co-bind enhanced ISGs
by PH but not by JNKi-II (fig. S4, F and G). CREB activation occurs To corroborate the GSEA and motif discovery data correlating ISG
in its DNA-bound state (43, 44). Consistently, pSer133 CREB1 acti- promoter configuration with ISG enhancement, we combined
vation at the CRE of enhanced ISG promoters was increased by ani- RNA-seq and ChIP-seq data for a further integrated analysis.
somycin pretreatment of IFN-stimulated BMDMs, and this was Among the 182 genes enhanced by anisomycin in combination
blocked by PH (Fig. 5E and fig. S4H). After deletion of the Creb1 with IFN-γ, 71 were co-bound by CREB1 and c-Jun; these included
gene in BMDMs by CRISPR-Cas9 editing (fig. S4I) (45), stress en- Irf1 and Socs3. Thirty three of the 116 anisomycin-enhanced, IFN-
hancement of Irf1 and Ifit2 mRNAs after stimulation with, respec- β–induced ISGs and 49 of the 103 commonly enhanced genes—in-

tively, IFN-γ and IFN-β was abolished (Fig. 5F), but the expression cluding Ifit2, Ifit3, Mx2, Rsad2, and Isg20—were bound by both
of the enhancement-resistant Gbp5 and Mx1 genes was unabated transcription factors (Fig. 6E and data file S4). Among the enhance-
(Fig. 5G). Unexpectedly, the lack of CREB1 phosphorylation at ment-resistant, IFN-γ–induced ISGs, a smaller proportion (74 of
Ser133 was without consequences for the enhancement of IFN-γ– 280, which did not include Mx1 and Gbp5) showed co-binding of
induced Irf1 expression and IFN-β–induced Ifit2 mRNA expression the transcription factors, as did a smaller proportion of both the ani-
when assayed in BMDMs derived from CREB-S133A knock-in somycin-resistant, IFN-β–induced ISGs and the commonly not-en-
mice (fig. S4J) (46). Together, these results demonstrate that hanced ISGs (163 of 440 and 121 of 300, respectively, which also did
CREB1 is essential for the enhancement of ISG expression not include Mx1 and Gbp5) (Fig. 6F and data file S4). The data
through p38 signaling without strictly requiring its activation by confirm that ISG promoters with binding sites for both CREB1
phosphorylation at Ser133. and c-Jun are enriched among p38-enhanced genes but that
CREB1 acquires Ser133 phosphorylation-independent transcrip- binding of both transcription factors is not strictly necessary.
tional competence using the CREB-regulated transcription coacti- The genetic data (Fig. 5, D and F) suggest a dominant role of
vator family (CRTC) (47–49). In particular, CRTC3 is associated CREB1 and a minor role of c-Jun in Irf1 and Ifit2 enhancement,
with immunoregulatory genes in macrophages (50). Nuclear whose promoters were co-bound by both transcription factors.
access of CRTC is under control of kinase-dependent signaling However, a small number of enhanced genes showed binding of
pathways, prompting us to examine a potential role of p38. either CREB1 or c-Jun alone (fig. S5, G and H). The latter were in-
Nuclear translocation of CRTC3 was stimulated by anisomycin, vestigated in c-Jun–deficient BMDMs. Enhancement of Isg15 was
but not by IFN treatment, and a larger CRTC3 fraction remained unaffected, and that of the IFN-γ–induced Batf2 and Slamf7
in the cytoplasm in the presence of PH (fig. S4, K and L). Thus, genes was slightly decreased by c-Jun deficiency. In contrast, the
CRTC3 may contribute to p38-dependent CREB activity. loss of c-Jun completely abolished IFN-γ–induced expression and
enhancement of the Il2ra gene and abrogated the enhancement of
Genome-wide identification of CREB and c-Jun binding at IFN-β–induced Pdcd1 mRNA expression. In addition, Pdcd1
ISG loci mRNA enhancement was similarly reversed by pretreatment with
We then performed ChIP-seq to globally identify ISGs associating JNKi-II and JNK-IN-8 in WT BMDMs (fig. S5, I to L). These
with CREB and/or c-Jun, either constitutively or after treatment results confirm the impact of c-Jun on IFN-γ signaling (37) and
with anisomycin. Because c-Jun binding to an ISG subset is con- show that the expression of a small subset of ISGs is enhanced by
trolled by IFN-γ (37), treatment with the cytokine was included. c-Jun.
Eight thousand four hundred thirty constitutive CREB1 binding Browser tracking of the anisomycin-enhanced Irf1 and Ifit loci
sites and 5614 c-Jun binding sites were found in regions −2/+1 indicated binding sites for c-Jun and CREB1 close to STAT1 homo-
kb relative to the transcription start site (TSS), with an overlap of dimers and ISGF3 bound to GAS elements and ISRE sequences, re-
54.3% (Fig. 6A). By integrating these ChIP-seq data with our spectively. In addition, the RNA-seq tracks visually demonstrated

Fig. 6. ChIP-seq analysis of CREB1 and c-Jun

binding at ISG loci. (A to D) Venn diagrams of c-
Jun– and CREB1-bound genes detected by ChIP-
seq, wherein (A) shows genome-wide c-Jun–
and/or CREB1-bound genes, (B) shows the
CREB1/c-Jun co-bound ISGs derived from RNA-
seq data, (C) shows the ISGs that are bound only
by CREB1 or only by c-Jun, and (D) shows the
ISGs that are bound by c-Jun and which are
induced by IFN-γ (see data file S4). (E and F)
Scatterplot comparing the log2FCs in mRNA
abundances from BMDMs treated with IFN-γ or
IFN-β. Color code of the dots (genes) defined in
the inset keys. “N.S.” denotes not significantly
altered genes (IFN log2FC ≤ 1, Padj ≥ 0.05);
“common” denotes genes for which both IFNs
had a similar effect (E) or similar lack of effect (F).
(G and H) Genome browser tracks from ChIP-seq
and RNA-seq data. For ChIP-seq, WT BMDMs
were treated with An (100 ng/ml) alone, a
combination of SP600125 (JNKi-II) and PH alone
(20 μM and 1 μM, respectively), (G) IFN-γ (10 ng/
ml for 2 hours), or (H) IFN-β (250 IU/ml for 2
hours) alone or in combination with An (20-min
pretreatment) or with An and the combination
of the two inhibitors (1-hour pretreatment) as
indicated. Tracks are shown for c-Jun ( purple),
CREB1 (green), and IgG (negative control; black).
Merged data from three replicates are shown.
Previously published ChIP-seq (29) data were
used to show binding sites for STAT1, STAT2, and

IRF9, and RNA-seq tracks show IFN and An + IFN
samples, each one representative replicate.
stress-enhanced expression of the Irf1 gene and all clustered Ifit p38 MAPK enhances ISG expression and nitric oxide–
genes (Fig. 6, G and H). In contrast, browser tracks of the enhance- mediated cell death in macrophages infected with L.
ment-resistant Gbp5 and Mx1 promoters indicated binding of c-Jun monocytogenes
but not of CREB1 (fig. S5, M and N). To validate the relevance of our data for innate immunity to mi-
crobes, we infected macrophages with the intracellular bacterium
L. monocytogenes (Fig. 7A). Pattern recognition receptor signaling
in response to L. monocytogenes infection activates stress-

Fig. 7. p38 MAPK enhancement of Listeria

monocytogenes–induced ISG expression and
its blockade effect of macrophage viability.
(A) Overview of L. monocytogenes (Lm) infec-
tion and subsequent analysis. (B and C) qPCR of
pre-mRNA expression of the indicated genes in
WT BMDMs infected with Lm (MOI of 10; 4
hours) alone or after PH treatment (1 μM; 1-
hour pretreatment). A PH-alone (5 hours) con-
dition is also shown. (D) qPCR showing Nos2
pre-mRNA expression in WT BMDMs stimulated
with IFN-β (250 IU/ml) for 4 hours alone or after
1-hour pretreatment with PH (1 μM). (E) qPCR
assessing the expression of Nos2, Il1a, Il1m, and
Il6 pre-mRNA in Ifnar1 −/− BMDMs cultured in
the indicated conditions: stimulated with IFN-γ
(10 ng/ml, 4 hours), infected with Lm (MOI of
10; 4 hours), and treated with PH (1 μM; 5 hours
where alone, 1-hour pretreatment where com-
bined). UT, untreated. (F) Genome browser
tracks assessing c-Jun and CREB1 binding to
the Nos2 promoter in BMDMs treated with IFN-
γ for 2 hours. Tracks are representative of one of
three individual replicates. (G and H) Site-di-
rected ChIP with (G) pSer133 CREB1 or (H) c-Jun
antibodies (or control IgG) analyzing binding to
the CRE/AP-1 binding site of Nos2 in WT
BMDMs infected with Lm (MOI of 10) alone or in
combination with PH (1 μM; 1-hour pretreat-
ment) for 4 hours. (I and J) Colony-forming unit
(CFU) assay in WT BMDMs infected with Lm

(MOI of 10) alone or in combination with PH (1
μM; 1-hour pretreatment) for the indicated
time. (K) Griess assay analyzing the concentra-
tion of nitrite in the medium of WT BMDMs 24
hours after infection with Lm (MOI of 10) alone
or in combination PH (1 μM; 1-hour pretreat-
ment). PH alone condition as a control. (L) Cell
death assessed by propidium iodide (PI; 1 μg/
ml) staining and measured by FACS in WT
BMDMs 24 hours after infection and treatment
as in (K). Data are representative of one of four
biological replicates. FSC-A, forward scatter; PE-
Texas Red-A, PI fluorescence. Data are
means + SD of at least three independent rep-
licates. For (B) to (E), (G), (H), (K), and (L), one-
way ANOVA corrected for multiple testing with
Dunnett’s post hoc test was used. For (I) and (J),
two-tailed unpaired t test was used. *P < 0.05,
**P < 0.01, ***P < 0.001, and ****P < 0.0001.
responsive MAPK and IRF transcription factors, inducing IFN-β treatment (Fig. 7, C and D). Type I IFNR–deficient macrophages
synthesis and subsequent ISG expression (52, 53). Ifnb1 pre- (Ifnar1 −/−) lacking the background ISG transcription through en-
mRNA expression was inhibited by PH, indicating AP-1 family dogenous IFN-β synthesis responded with a moderate increase in
constituents of the IFN-β enhanceosome (Fig. 7B) (54). ISGs asso- antimicrobial ISG expression to L. monocytogenes infection or
ciated with activated macrophages, such as Nos2, Il1a, Il1rn, and Il6, IFN-γ treatment alone and demonstrated a pronounced synergism
were similarly induced by L. monocytogenes and inhibited by PH. In in the transcriptional induction by the combined treatment
contrast, PH did not reduce ISG expression in response to IFN-β (Fig. 7E). This IFN-γ and infection synergism was eliminated by

PH, confirming enhancement of IFN-γ–induced ISGs by the infec- growth at later stages (56, 59). During autophagosome formation,
tion-induced p38 pathway. Similar results were obtained in WT the cytosolic form of the microtubule-associated protein 1A/1B
BMDMs, where Nos2 pre-mRNA expression resulted from L. mono- light chain 3 (LC3-I) is converted into LC3-II and recruited to au-
cytogenes infection–induced IFN-I synthesis (fig. S6A). tophagosomal membranes, and the ratio between the two forms
To verify the relevance of ISG enhancement for L. monocyto- represents the autophagic rate (60). We observed an increased
genes infection, we explored expression of the Nos2 gene, nitric LC3BII/LC3BI ratio in L. monocytogenes–infected BMDMs at
oxide (NO) production, and NO-dependent macrophage death both early and late stages of infection. However, neither IFN-γ pre-
(37, 55, 56). In agreement with Nos2 pre-mRNA enhancement by treatment nor p38 inhibition affected the autophagic rate, thus ex-
p38 signals, its promoter contains binding sites for CREB1 and c- cluding autophagy as a mechanism for the observed effect of p38
Jun (Fig. 7F). L. monocytogenes infection increased the binding of inhibition on bacterial growth (fig. S6C). We and others have pre-
both pSer133 CREB1 and c-Jun at the CRE/AP-1–bound DNA, and viously shown a linear relationship between NO production and cell
PH eliminated promoter-bound pSer133 CREB1 (Fig. 7, G and H). death by PANoptosis (a form of cell death involving key features of
Intramacrophage growth of L. monocytogenes, determined by pyroptosis, apoptosis, and necroptosis) in WT macrophages at these
colony-forming unit (CFU) assay, was slightly reduced by PH late stages of infection (56, 61, 62). The data suggest that by sustain-
between 1 and 8 hours but strongly diminished between 12 and ing their viability, PH increases the ability of macrophages to kill the
24 hours after infection (Fig. 7, I and J). Pretreatment with IFN-γ invading bacteria. Consistent with this assumption, both NO pro-
before infection confirmed the antimicrobial effect of this cytokine duction and macrophage death, assessed by propidium iodide (PI)
by decreasing L. monocytogenes growth at 12 and 24 hours, and staining, were significantly reduced by PH 24 hours after infection
blockade of p38 together with IFN-γ resulted in even stronger bac- (Fig. 7, K and L). Together, these findings demonstrate that p38 en-
terial clearance (fig. S6B) (57). To narrow down the beneficial effect hancement of ISG expression exacerbates consequences of L. mono-
of p38 blockade in the context of L. monocytogenes infection, we cytogenes infection, whereas p38 blockade is beneficial for the
sought to assess a potential role of p38 in restricting autophagy viability of host macrophages.
(58). L. monocytogenes–stimulated autophagy enhances phagoso-
mal escape early after infection while restricting cytoplasmic Stress and IFN synergism during VSV infection
To explore the IFN/stress synergism in the context of viral infection,
we infected BMDMs with green fluorescent protein–positive
(GFP+) vesicular stomatitis virus (VSV-GFP) (63). VSV-GFP infec-
tion resulted in increased antiviral gene expression, similar to IFN-β

stimulation. Pretreatment with IFN-β followed by VSV infection led
to enhancement of gene expression, which was decreased by block-
ade of JNK and p38 (fig. S6D). IFN-β treatment significantly de-
creased the levels of VSV viral RNA and the percentage of
infected cells, as assessed by the percentage of GFP+ cells. Pretreat-
ment with a combination of JNK and p38 inhibitors reverted the
IFN-β–dependent effect on VSV mRNA expression and increased
the percentage of infected cells (fig. S6, E and F).
Together, these data demonstrate that enhancement of antiviral
genes during VSV infection might contribute to a stronger IFN-I
response, leading to a decrease in VSV infectivity.
DISCUSSION
The co-occurrence of signaling by sensors of stress and IFN recep-
tors is a hallmark of innate responses to many viral and bacterial
pathogens. Here, we show that the enhancement of macrophage
ISG expression is mediated by cooperative action of the IFN and
p38 signaling pathways in the process of transcriptional initiation
Fig. 8. Working model of stress kinase–mediated control of ISG expression. (Fig. 8). In contrast to what has been found in mouse embryonic
Stress-induced p38-mediated control of ISG expression is triggered on a specific fibroblasts (10), p38 signaling in primary macrophages had no
subset of ISGs. In the “no enhancement” status, stress kinases do not alter the effect on transcriptional induction through ISRE or GAS sequences
IFN-γ– and IFN-β–induced transcription of genes controlled by STAT1 homo- by the IFNRs alone. Global definition of stress-enhanced ISGs for
dimers, such as Gbp5, or the ISGF3 (STAT1-STAT2-IRF9) complex, such as Mx1. both IFN types and compilation of their specific attributes com-
The presence of CRE/AP-1 binding sites at ISG promoters is characteristic of the
pared with stress-resistant ISGs revealed CRE/AP-1 promoter ele-
enhancement status, allowing stress kinases to enhance transcription. Stress-
ments as the cardinal elements of distinction. Reportedly, c-Jun
induced enhancement occurs at both STAT1 homodimer and ISGF3-controlled
ISGs when stimulated with IFN-γ but only at ISGF3-controlled ISGs when stimulat-
and CREB are transcription factors that are activated by the JNK
ed with IFN-β. L. monocytogenes infection of macrophages causes the production and p38 pathways, respectively (39, 40, 64), with a minor contribu-
of IFN-β and activation of p38 MAPK pathway. Together, they synergistically tion of p38 to c-Jun phosphorylation in some situations (65), as also
enhance ISG expression, thus amplifying the consequences of infection such as seen here (fig. S4D). Whereas CREB1 was essential for the en-
NO production and NO-dependent cell death. hanced expression of ISGs associating with both CREB1 and c-

Jun, the latter was largely dispensable. At the resolution of ChIP- enhancement may involve histone modification (26, 27, 29). Aniso-
seq, many binding sites for c-Jun and CREB were found to mycin-induced p38 had a profound effect on H3 modification at
overlap, but the experimental setup did not enable us to assess ISG promoters, increasing phosphorylation at Ser10 and Ser28.
whether the two transcription factors bind the same site and These effects were not specific for enhanced ISG but may yet be rel-
whether they interact physically in their promoter-associated evant for the transcriptional activities of CREB and c-Jun in en-
state. Generating this knowledge is required to understand the hancing ISG transcription. Stress-induced H3 modification may
lack of c-Jun requirement for the stress enhancement of co-bound create a transcription-permissive histone code at all ISG promoters,
ISGs. It might result either from redundancy with other members of but the realization of this potential requires stress-activated tran-
the AP-1 family that act as surrogates in a knockout situation (66) or scription factors such as CREB and c-Jun in addition to STAT1 ho-
from a lack of essential mechanistic input into transcriptional acti- modimers and/or ISGF3.
vation during stress enhancement of ISG. The latter possibility is The pleiotropic effects of the p38 pathway make it difficult to
suggested by the result that a small number of genes associating assess the specific contribution of ISG enhancement to immune re-
only with c-Jun require the transcription factor for pathway sponses with genetic or pharmacological tools. To circumvent this
synergy, demonstrating its inherent potential to cooperate with problem, we made use of the fact that the ISG Nos2 is subject to
STATs. Unexpectedly, promoters induced exclusively by STAT1 ho- stress enhancement and that IFN- and stress-dependent production
modimers failed to mediate stress input into gene control by IFN-β, of NO is a contributing factor to a complex form of L. monocyto-
whereas stress enhancement of IFN-γ inducibility took place. This genes–induced macrophage death referred to as PANoptosis (56,
difference between the IFN types may result from the increased 61). Inhibitors of p38 had little effect on the intracellular growth
presence of STAT1 homodimers in IFN-γ–treated cells or from a of L. monocytogenes at early stages of infection, but they enhanced
regulatory capacity of yet-undefined factors. bacterial killing while also protecting macrophages from PANop-
Given the profound transcriptome changes caused by anisomy- totic death at late stages of infection. The data suggest a role of
cin treatment alone, we were surprised to find a complete lack of stress-enhanced NO production in controlling the survival of L.
p38-dependent chromatin remodeling. Instead, our findings monocytogenes–infected macrophages. The reduced efficacy of
support the assumption that the transcriptional regulation by ani- IFN-β to curtail VSV replication in the presence of p38 inhibitors
somycin alone occurs largely at constitutively open promoters with is also in agreement with a blockade of ISG enhancement during
poised polymerases, a characteristic of primary response genes (33). viral infection; however, p38 signaling is likely to influence innate
This notion is further consistent with the unresponsiveness of ISGs immunity to VSV in various ways, including an impact on IFN-β
to treatment with anisomycin alone, which may in part be explained synthesis (69). Thus, the results reported here do not allow for com-

by an ability of STATs to increase accessibility of most ISG loci (25) prehensive conclusions about the role of p38 MAPK during VSV
that cannot be executed by stress-activated transcription factors. infection.
IFN-dependent promoter opening may be an important prerequi- Studies on murine severe acute respiratory syndrome coronavi-
site for CREB and c-Jun to unfold their activity in transcriptional rus 2 (SARS-CoV-2) infection identified inducible nitric oxide syn-
initiation, as also suggested by their constitutive or anisomycin- thase (iNOS)–dependent cell death as a contributing factor to severe
induced association with ISG promoters. disease (70). Studies in human patients indicate an important role
Although our study demonstrates the indispensable role of p38 for IFN in immunity against coronavirus disease 2019 (COVID-19)
signaling for the enhanced transcriptional induction of ISG, several (71) and attribute a role also in the cytokine storm associated with
possibilities remain of how it feeds into the mechanism of transcrip- severe disease (70). Likewise, p38 activity has been associated with
tional initiation. The most attractive of these, the phosphorylation severe COVID-19, and its inhibition is proposed as a therapeutic
of CREB at Ser133, was ruled out, because ISG enhancement was ob- strategy (72). Data corroborating a role for IFN and the p38
served in macrophages expressing a CREB-S133A mutant. p38 sig- MAPK pathway is shown in a multiomics study with blood leuko-
naling produced robust phosphorylation of promoter-bound cytes from patients with COVID-19, wherein the authors identified
CREB1 without an apparent contribution of the modification to STATs as associated factors and the p38 and AP-1 pathway as a key
ISG enhancement. The partial block of c-Jun phosphorylation by feature distinguishing severe COVID-19 from influenza virus infec-
PH may indicate a role for the kinase in the activation of AP-1 tion (73). On the basis of these and our studies, we propose that p38-
for the small group of ISGs showing enhancement via c-Jun. A po- enhanced ISG expression is a means to efficiently harness antimi-
tentially more general mechanism for the acquisition of phospho- crobial effector mechanisms while also carrying the risk of exagger-
Ser133–independent transcriptional CREB activity is to harness the ated, disease-associated inflammation.
CRTC3 coactivator (47–49). In support of a role for CRTC3, the
protein translocated to the nucleus after anisomycin treatment
and translocation was largely blocked by PH. One potential target MATERIALS AND METHODS
for the p38 effect in this scenario is salt-inducible kinase 2 (SIK2). Animal experiments
SIK2 inhibits nuclear translocation of CRTC3, but the inhibition C57BL6/N mice (WT) were purchased from Janvier Labs. p38αΔM
can be overcome by its phosphorylation (50, 67). A more likely pos- mice were previously described (74). cJunfl/fl;Vav-iCre +/− mice were
sibility is the direct phosphorylation of CRTC3 by p38, because the housed and bred at the University of Veterinary Medicine of
protein contains several MAPK phosphorylation sites and was Vienna, Vienna, Austria. Knock-in CREB-S133A mice were gener-
shown to be a substrate of extracellular signal–regulated kinase ated as previously described (46). Rosa26-Cas9 knock-in mice were
(ERK). Phosphorylation of these sites increases recruitment of purchased from the Jackson Laboratory. WT, Ifnar1 −/−, and Ttp fl/
fl
protein phosphatase 2A (PP2A) and CRTC3 dephosphorylation– ;LysM-Cre mice in C57BL6/N background were housed and bred at
dependent nuclear translocation (68). Last, stress-induced ISG the Max Perutz Labs.

Mice were housed under identical conditions in a specific path- were from Cell Signaling Technology: p38 phospho-Thr180/Tyr182
ogen–free (SPF) facility according to the Federation of European (catalog no. 9211, used at 1:1000), total p38 (catalog no. 9212,
Laboratory Animal Science Association (FELASA) guidelines and 1:1000), stress-activated protein kinase (SAPK)/JNK phospho-
additionally monitored for being norovirus negative. Mice were Thr183/Tyr185 (catalog no. 9251, 1:1000), total SAPK/JNK (catalog
bred under the approval of the institutional ethics and animal no. 9252, 1:1000), MSK1 phospho-Thr581 (catalog no. 9595,
welfare committee of the University of Veterinary Medicine of 1:1000), total MSK1 (catalog no. 3489, 1:1000), CREB1 phospho-
Vienna and the national authority Federal Ministry Republic of Ser133 (catalog no. 9198, 1:1000), total CREB (catalog no. 4820,
Austria Education Science and Research section 8ff of the Animal 1:1000), c-Jun phospho-Ser73 (catalog no. 9164, 1:1000), total c-
Science and Experiments Act [Tierversuchsgesetz (TVG), BMWF- Jun (catalog no. 9165, 1:1000), total IκBα (catalog no. 9242,
68.205/0068-WF/V/3b/2015 and GZ 2020-0.200.397]. The study 1:1000), and LC3B (catalog no. 2775, 1:1000). The horseradish per-
did not involve animal experiments as defined in the TVG and oxidase–coupled secondary antibodies used were purchased from
did not require ethical approval according to the local and national Jackson ImmunoResearch Inc. (catalog nos. 111-035-003 and
guidelines. Mice were used at an age of 8 to 12 weeks for the isola- 115-035-144, each used at 1:6000). For development of protein
tion of bone marrow. The study did not involve animal experiments signals, SuperSignal West Pico PLUS (Thermo Fisher Scientific)
as defined in the TVG and did not require ethical approval accord- was used. For signal detection, a Bio-Rad ChemiDoc imaging
ing to the local and national guidelines. system was used. For LC3BII/LC3BI ratio, the intensities of the dif-
ferent lanes were analyzed using ImageJ (76), normalized on the
Cell cultures housekeeping gene control, and expressed as a ratio of the two pro-
All cell lines used were grown at 37°C, 5% CO2. BMDMs were dif- teins of interest.
ferentiated from bone marrow isolated from femurs and tibias of 8-
to 12-week-old mice from both sexes that were pooled for each ex- Immunofluorescence
periment. Femur and tibia were flushed with Dulbecco’s modified BMDMs (70 × 105) were seeded on Millicell EZ SLIDE eight-well
Eagle’s medium (DMEM; Sigma-Aldrich), and cells were cultured glass chamber slides (Thermo Fisher Scientific) with 500 ml of
and differentiated for 9 to 10 days in DMEM supplemented with medium per well. The next day, the cells were stimulated for 2
10% of fetal bovine serum (FBS; Sigma-Aldrich), human recombi- hours with anisomycin (100 ng/ml) alone, IFN-γ (10 ng/ml), or
nant macrophage colony-stimulating factor (M-CSF) (500 ng/ml; a IFN-β (250 IU/ml) alone or pretreated with anisomycin for 20
gift from L. Ziegler-Heitbrock, Helmholtz Center, Munich, min or with anisomycin and 1 μM PH (1 hour pretreatment).
Germany), penicillin (100 U/ml), and streptomycin (100 ng/ml) Cells were fixed with 3% paraformaldehyde for 15 min at room tem-

(both from Sigma-Aldrich). perature and then permeabilized with 0.1% Triton X-100 in phos-
Lenti-X 293T cells (Takara Bio) were cultured in DMEM supple- phate-buffered saline (PBS) for 10 min. Blocking was carried out in
mented with 10% FBS, penicillin (100 U/ml), and streptomycin 0.1% Triton X-100 and 3% bovine serum albumin (BSA) in PBS for
(100 ng/ml) (each from Sigma-Aldrich). Transfection of cells was 1 hour. The primary antibody (TORC3/CRTC3, Cell Signaling
performed using Lipofectamine 3000 (Thermo Fisher Scientific) ac- Technology, catalog no. 2720, 1:100) was diluted in a blocking
cording to the manufacturer’s instructions. buffer and incubated for 1 hour at room temperature. Secondary
goat anti-rabbit Alexa Fluor 488 immunoglobulin G (IgG; H + L,
Reagents Thermo Fisher Scientific, catalog no. A-11008, 1:500) was incubated
Anisomycin (Sigma-Aldrich, catalog no. 176880) was used at a con- for 30 min in a blocking buffer. Samples were then stained with 4′,6-
centration of 100 ng/ml. Murine IFN-γ (a gift from G. Adolf, Boeh- diamidino-2-phenylindole (DAPI; 1 μg/ml) in PBS for 10 min.
ringer Ingelheim, Vienna) was used at a concentration of 10 ng/ml, Samples were mounted in ProLong Gold Antifade Mountant
and murine IFN-β (PBL Assay Science, catalog no. 12400-1) was (Thermo Fisher Scientific) and left overnight at 4°C before image
used at a concentration of 250 IU/ml. PH (Selleckchem, catalog acquisition. Images were acquired using a Zeiss Axio Imager Z2
no. S2726) was used at a concentration of 1 μM, LY (Selleckchem, with a 40× oil objective and ×1.6 magnification. Images were pro-
catalog no. S1494) at a concentration of 200 nM, JNK inhibitor II cessed using ImageJ software (76). The background range in all
(SP600125, Sigma-Aldrich, catalog no. 420119) at a concentration images for DAPI was adjusted to 584 to 7834, and for GFP, it was
of 20 μM, and JNK-IN-8 (JNK Inhibitor XVI, Selleckchem, adjusted to 943 to 16,383. The color depth was changed to 16 bit. A
catalog no. S4901) at a concentration of 10 μM. Lipopolysaccharide composite picture was made, and DAPI was set to magenta and GFP
(Sigma-Aldrich, catalog no. L2637) was used at a concentration of was set to green. The color space of the picture was converted to red,
100 ng/ml. VSV-GFP was grown on Vero cells for 42 hours, after green, and blue (RGB), and a scale bar displaying 10 μm was insert-
which supernatants were clarified and titrated on Madin-Darby ed. For statistics, at least 20 cells per condition were analyzed in four
canine kidney (MDCK) cells by plaque assay as previously described individual replicates. The ratio between CRTC3 abundance in the
(75). nucleus and in the cytoplasm was obtained by dividing the mean
intensity values (integrated intensity) of the GFP signal in the
Immunoblotting nucleus (DAPI channel) by the mean intensity values (integrated
Cell lysis and immunoblotting were performed as previously de- intensity) of the GFP signal in the surrounding cytoplasm (GFP sig-
scribed (32). Primary antibodies to α-tubulin (catalog no. T9026, nal–DAPI signal) using CellProfiler software v. 4.2.1 (77).
used at 1:5000) and vinculin (V9131, 1:5000) were purchased
from Sigma-Aldrich. Primary antibody to glyceraldehyde-3-phos- RNA preparation and RT-qPCR
phate dehydrogenase (GAPDH) was purchased from Millipore RNA was isolated using the NucleoSpin RNA II Kit (Macherey-
(catalog no. ABS16, 1:3000). The following primary antibodies Nagel, catalog no. 740955). The complementary DNA (cDNA)

synthesis was performed by reverse transcription of 400 ng of total BMDM transduction

RNA using oligo(dT)18 primers for mRNA, Random Hexamer Rosa26-Cas9 knock-in BMDMs (Cas9 BMDMs) (45) were differen-
Primer for pre-mRNA, and RevertAid Reverse Transcriptase tiated for a total of 11 days. On day 2, 5 × 106 cells were seeded into
(Thermo Fisher Scientific) according to the manufacturer’s instruc- 10-cm nontreated tissue culture plates with 5 ml of medium supple-
tions. When a low amount of RNA was obtained, the cDNA synthe- mented with recombinant M-CSF (500 ng/ml). After 4 hours, Cas9
sis was carried out with the LunaScript RT SuperMix Kit (NEB, BMDMs were transduced with 5 ml of lentiviral supernatant sup-
catalog no. E3010). Real-time qPCR (RT-qPCR) was run on Master- plemented with Polybrene (8 μg/ml) (Sigma-Aldrich). The next day,
cycler (Eppendorf ) using the Luna Universal qPCR Master Mix the cells were harvested, centrifuged for 5 min at 500g, seeded onto
(NEB, catalog no. M3003). Primers for RT-qPCR are listed in 10-cm nontreated tissue culture plates with 5 ml of medium, and
table S1. transduced for the second time with 5 ml of lentiviral supernatant
supplemented with Polybrene (8 μg/ml). Two days after the first
Generation of knockout BMDMs using CRISPR-Cas9 transduction, the medium was discarded and replaced with fresh
gene editing medium supplemented with recombinant M-CSF (500 ng/ml)
Constructs for gRNA expression and transduced Cas9 BMDMs were differentiated for five more
Per target gene, one guide RNA (gRNA) was designed using the days. At day 9 of differentiation, the mCherry+ (sgRNA) GFP+
VBC Score (www.vbc-score.org/) for each two complementary (Cas9) cells were fluorescence-activated cell sorting (FACS)–
oligos ordered (table S1). Constructs for gRNA expression were sorted using FACSAria (BD Biosciences). Sorted cells were cultured
cloned as previously described (78) with minor modifications. Non- for two additional days in medium supplemented with recombinant
targeting gRNA sequences were previously published (79). In short, M-CSF (500 ng/ml). Transduced BMDMs were analyzed by FACS.
1 μg of CROPseq-Guide-Puro-mCherry2 plasmid (will be shared For RNA isolation, the RNeasy Micro Kit (Qiagen, catalog no.
via Addgene upon publication) was digested in a mixture with 1 74004) was used according to the manufacturer’s instructions. Pro-
μl of BsmBI-v2, 3 μl of NEBuffer r3.1 (NEB), and double-distilled teins were precipitated with acetone from cell lysates prepared using
water to 30 μl total volume. The reaction was incubated at 55°C for the RNA lysis buffer RTL according to the kit supplementary pro-
60 min, after which 2 μl of rSAP (NEB) enzyme was added and the tocol and boiled at 95°C for 7 min before loading onto a 10% SDS-
reaction was further incubated at 37°C for 60 min and heat-inacti- polyacrylamide gel.
vated at 80°C for 20 min. Meanwhile, corresponding forward and
reverse gRNA oligos were mixed and resuspended to 100 μM in nu- RNA-sequencing
clease-free water (Thermo Fisher Scientific). To anneal and phos- BMDMs (10 × 106) were seeded after 10 days of differentiation in

phorylate the oligos, we added 1 μl of the mixture to 1 μl of 10× 15-cm dishes with 20 ml of medium. The day after, the cells were
T4 ligation buffer (NEB), 7.5 μl of water, and 0.5 μl of T4 PNK stimulated for 2 hours with IFN-γ (10 ng/ml) or IFN-β (250 IU/ml)
enzyme (NEB). The reaction was incubated at 37°C for 20 min, alone or after pretreatment with anisomycin (100 ng/ml) for 20
95°C for 5 min, and then ramped down to 25°C at 5°C/min. One min. Total RNA was isolated using an AllPrep DNA/RNA Mini
microliter of the annealed oligo mixture was mixed with 199 μl kit (Qiagen, catalog no. 80004) according to the manufacturer’s in-
of water. structions. The quality controls, RNA-seq, and preliminary analysis
For the ligation, 1.6 μl of digested and rSAP-treated plasmid was were performed by the Biomedical Sequencing Facility (CeMM,
mixed with 1 μl of diluted oligo duplex, 5 μl of 2× Quick ligase Vienna, Austria; www.biomedical-sequencing.org/). The experi-
buffer, 2.4 μl of water, and 1 μl of Quick ligase (NEB). This 11-μl ment was carried out as three biological replicates per condition.
reaction was incubated for 15 min at 25°C, and 2 μl was heat-trans-
formed into 10 μl of NEB Stable Competent Escherichia coli (NEB) RNA-seq analysis
according to the manufacturer’s protocol. Colonies were grown RNA-seq data were processed and quality-controlled using estab-
overnight on LB plates (Merck Millipore) with carbenicillin (100 lished bioinformatics software. Raw reads were trimmed using trim-
μg/ml; Carl Roth) at 32°C and thereafter incubated in 2 μl of momatic (v.0.32) (80) and aligned to the mouse reference genome
shaking liquid LB culture with carbenicillin at 32°C overnight. Plas- (mm10) using STAR (v.2.7.1) (81). Gene expression was quantified
mids were isolated using the Qiagen Plasmid Mini Prep Kit by counting uniquely aligned reads in exons using the function
(Qiagen), and correct ligation was confirmed via Sanger sequencing summarizeOverlaps from the GenomicAlignments package
using the primer 5′-GAGGGCCTATTTCCCATGATTCC-3′. (v.1.6.3) in R. Gene annotations were based on the Ensembl
Lentiviral production GENCODE Basic set (genome build GRCm38 release 93). Differ-
Lenti-X 293T cells (Takara Bio, catalog no. 632180) were seeded the ential expression analysis was carried out using DESeq2 (v.1.24.0)
day before transfection at 8 × 106 in 8 ml of medium in 10-cm (82), with an FDR threshold of 0.05. For comparison between treat-
dishes. The day after, they were transfected with 10,148.04 ng of ments and for establishment of enhanced and not-enhanced genes,
single-guide RNA (sgRNA) plasmid and the packaging plasmid a threshold of log2 fold change (log2FC) ≥ 1 expression and an ad-
pMDLg/pRRE, pRSV-Rev, and pMD2.G (Addgene) in a 1:1:1 justed P value (Padj ) ≤ 0.05 were considered. GSEA analysis was per-
ratio using Lipofectamine 3000 (Thermo Fisher Scientific) accord- formed with GSEA (v.4.0.3) using log2FC values (83). Volcano plots
ing to the manufacturer’s instructions. One day after transfection, were generated with the ggplot2 package of the R software (v.4.0.2)
the supernatant was discarded and replaced with 7.5 ml of fresh using the log2-transformed FC and −log10-transformed Padj for
medium. The transfected cells were cultured for another 24 gene expression. Scatterplots were generated with the ggplot2
hours. The lentiviral supernatant was collected 48 and 72 hours package of the R software (v.4.0.2) comparing the log2FCs in
after transfection and sterile-filtered with a 0.45-mm sterile mRNA abundances from IFN-β– and IFN-γ–treated BMDMs.
syringe filter to remove cell debris. Venn diagrams were generated using a Venn tool () with the gene

lists obtained from the DESeq2 analysis. Heatmaps were generated the Mastercycler (Eppendorf ). Primers for ChIP-qPCR are listed in
using pheatmap tool (v.1.0.12) with the normalized counts from the table S1.
gene lists obtained from the DESeq2 analysis and clustered by row. For ChIP-seq, a minimum of three IPs per condition were
The PCA plot was generated using the function prcomp() from the pooled to obtain enough starting material. Library preparation,
base package stats in R (v.4.2.0). WGCNA (v.1.71) was performed in quality check, and sequencing were performed by the Vienna Bio-
R (v.4.2.0) using as input DESeq2 normalized count data (31). Hi- center Core Facilities NGS Unit (www.viennabiocenter.org/vbcf/
erarchical clustering with the function hclust() was performed on next-generation-sequencing/). Libraries were sequenced on a
the 1-TOM (Topological Overlap Matrix) dissimilarity using NovaSeq S1 PE100.
average as the agglomeration method. Branch pruning of the den-
drogram was carried out using the cutreeDynamic() function. The ChIP-seq analysis
module clustered genes of cutreeDynamic(), and the expression ChIP-seq data were processed using the ChIP-seq pipeline from the
data were then used to compute the principal components of the nf-core framework (nfcore/ChIP-seq v.1.2.2; zenodo.org/record/
modules using the moduleEigengenes(). Spearman correlations of 3529400#.YA8YGmRKjZk). Reads were aligned against the Illumi-
the moduleEigengenes and the traits/conditions are color-coded na iGenome Mus musculus GRCm38 reference genome.
in the heatmap. Correlations below 0 are in blue tones and those Downstream analysis of ChIP-seq data was performed using
above 0 are in red tones (fig. S3C). GO enrichment analysis of the MACS2 narrow-peak calling (included in nf-core/atacseq pipeline).
WGCNA modules was performed with ShinyGO v. 0.76 (http:// Volcano plots were obtained using ggplot2. PygenomeTracks (84,
bioinformatics.sdstate.edu/go/). For defining the groups of en- 85) was used for visualization of ChIP-seq tracks and previously
hanced and not-enhanced ISGs in Fig. 3 (A to C), the significant published ChIP-seq tracks (29). Biological replicates were merged
(Padj ≤ 0.05) genes (An + IFN versus IFN sorted by log2FC ≥ 1 when an overlap was found in at least two replicates using
and An versus UT sorted by log2FC ≤ 2) and the significant (Padj BEDOPS (v2.4.40), using BED files after narrow-peak calling. All
≤ 0.05) genes (An + IFN versus IFN sorted by 1 < log2FC > −1 and different treatments were merged with the same tool, and each
An versus UT sorted by log2FC ≤ 2) were filtered, respectively. For peak was annotated to a gene using the HOMER script annotate-
identification of STAT1- or ISGF3-bound enhanced genes, previ- Peaks.pl (86). Venn diagrams were generated using a Venn tool
ously published ChIP-seq data were used (29). PygenomeTracks (https://bioinformatics.psb.ugent.be/webtools/Venn/) with the
(84, 85) was used for RNA-seq tracks visualization. The list of differ- peak lists obtained from the annotated merged BED files and
entially expressed genes upon anisomycin stimulation (volcano plot from the gene lists obtained from the DESeq2 analysis of the
in fig. S3B) is provided in data file S1; the list of differentially ex- RNA-seq data merging data from both IFN types (IFN versus UT;

pressed genes upon IFN-γ stimulation and STAT1/ISGF3-bound log2FC ≥ 1; Padj ≤ 0.05). The genes from the RNA-seq were filtered
ISG (Fig. 3A and fig. S3F) is provided in data file S2, and the list from the IFN list for Padj ≤ 0.05 and log2FC ≥ 1. GSEA was per-
of differentially expressed genes upon IFN-β stimulation and formed using ChIP-Enrich (48) (Bioconductor) R tool with the hy-
ISGF3-bound ISG (Fig. 3B and fig. S3F) is provided in data file S3. bridenrich method. All enriched pathways or transcription factor
binding sites were considered significant with an FDR P value of
ChIP and ChIP-seq ≤0.05. The built-in gene sets used in this analysis are Kegg_pathway
In total, 1.5 × 107 BMDMs were seeded on a 15-cm dish with 20 ml [Kyoto Encyclopedia of Genes and Genomes v.3.2.3 (genome.jp/
of medium supplemented with M-CSF (500 ng/ml). The next day, kegg)] and transcription_factors [Transcription Factors (MSigDB)
cells were stimulated for 2 hours with IFN-γ (10 ng/ml) or IFN-β v.6.0; software.broadinstitute.org/gsea/msigdb/collections.jsp]. A
(250 IU/ml) alone or after pretreatment with anisomycin (100 ng/ list of ISGs constitutively bound by both c-Jun and CREB1, only
ml) for 20 min. To block JNK and p38 MAPK activity, we added 1 by c-Jun, or only by CREB1 is provided in data file S4.
μM PH together with 20 μM JNK inhibitor II 1 hour before aniso-
mycin stimulation. Cells were processed with the ChIP protocol as ATAC-seq
previously described (32), using the following antibodies: RNA Pol BMDMs (3 × 106) were seeded onto 6-cm nontreated tissue culture
II CTD repeat YSPTSPS phospho-Ser5 (Abcam, catalog no. ab5408, plates on day 7 of differentiation. The next day, cells were stimulated
5 μl), RNA Pol II phospho-Ser2 (Bethyl, catalog no. A300-654A, 7 for 2 hours with IFN-γ (10 ng/ml) or IFN-β (250 IU/ml) alone or
μl), RNA Pol II CTD repeat YSPTSPS (Abcam, catalog no. ab817, 10 pretreated with anisomycin (100 ng/ml) for 20 min. To block p38
μl), histone H3 acetyl-Lys27(Cell Signaling Technology, catalog no. MAPK activity, we added 1 μM PH for 1 hour before anisomycin
8173, 5 μl), histone H3K27me3 (Active Motif, catalog no. 39155, 5 stimulation. Cells were processed as previously described (25). The
μl), histone H3 acetyl-Lys9/Lys14 (Cell Signaling Technology, quality of the libraries was confirmed on a bioanalyzer to further
catalog no. 9677, 5 μl), H2A.Z (Cell Signaling Technology, catalog determine the size distribution. Libraries were sequenced on a
no. 50722, 2 μl), histone H3 phospho-Ser28 (Abcam, catalog no. NovaSeq SP PE50.
ab32388, 5 μl), histone H3 phospho-Ser10 (Abcam, catalog no.
ab5176, 10 μl), histone H3 (Abcam, catalog no. ab1791, 2 μl), ATAC-seq analysis
STAT1 (Cell Signaling Technology, catalog no. 14995, 10 μl), ATAC-seq data were processed using the ATAC-seq pipeline from
CREB phospho-Ser133 (Cell Signaling Technology, catalog no. the nf-core framework (nfcore/atacseq v1.2.1; zenodo.org/record/
9198, 5 μl), CREB (Cell Signaling Technology, catalog no. 4820, 5 3965985#.YA8a8GRKjZk). Reads were aligned against the Illumina
μl), c-Jun (Cell Signaling Technology, catalog no. 9165, 10 μl), and iGenome Mus musculus GRCm38 reference genome. Downstream
rabbit monoclonal IgG isotype control (clone DA1E, Cell Signaling analysis of ATAC-seq data was performed using MACS2 narrow-
Technology, catalog no. 3900, 1 μl). RT-qPCR assays were run on peak calling and differential chromatin accessibility analysis (in-
cluded in nf-core/atacseq pipeline).

For the generation of summary profile plots and heatmaps, hours. For flow cytometry, BMDMs were harvested by incubation
density information (bigwig) for gene regions and surrounding for 5 min in citric saline (0.135 M potassium chloride and 0.015 M
regions (2 kb upstream of the TSS) and 1 kb downstream of the tran- sodium citrate), collected by centrifugation together with the
scription end site (TES) was plotted using deeptools (v3.4.3) culture medium to include cells in suspension, and then washed
(zenodo.org/record/3965985#.Yh-DPejMJPZ). The list of genes twice in PBS. Cell pellets were resuspended in FACS buffer (1×
used for summary profile plots and heatmaps was obtained accord- PBS + 2% BSA) and stained with PI (Thermo Fisher Scientific) at
ing to the RNA-seq data. The computeMatrix command was used a final concentration of 1 μg/ml immediately before FACS analysis.
in the scale-regions mode with the option missingDataAsZero. Sub- Unstained cells and untreated cells were used as negative control,
sequently, plotHeatmap was used for the generation of heatmaps and cells killed by 95°C incubation were used as positive control.
and summary profiles. PygenomeTracks was used for track visual- FACS analysis was carried out with a BD LSRFortessa Cell Analyzer
ization (84, 85). at the Max Perutz Labs FACS facility (www.maxperutzlabs.ac.at/
research/facilities/biooptics-facs). Analysis of the percentage of
BMDM infection with L. monocytogenes PI-positive cells was performed with FlowJo (www.flowjo.com/).
For BMDM infection, the LO28 strain of L. monocytogenes was A minimum of 10,000 cells were acquired for each dot plot. The per-
grown overnight in brain heart infusion (BHI) medium at 37°C centage of positive cells in the FACS analysis represents the percent-
with continuous shaking. After reaching stationary phase, bacterial age of PI-positive cells over the live-cell population. This was
concentration was measured with a spectrophotometer and the calculated using a forward and side-scatter gate to exclude dead
correct volume of bacterial culture medium (1 OD600nm = 1 × 109 cells and debris from the analysis.
viable bacteria, where OD600nm is the optical density measured at
600 nm) was transferred into a tube and pelleted by centrifugation. VSV-GFP infection and flow cytometry analysis
Bacteria were washed twice with PBS and resuspended in DMEM WT BMDMs were infected with VSV-GFP at MOI of 5 for 4 hours
supplemented with 10% FBS. BMDMs were seeded in DMEM sup- for RT-qPCR and for 24 hours for analysis of GFP expression by
plemented with 10% FBS and M-CSF (500 ng/ml) and infected with FACS. For flow cytometry, BMDMs were harvested by incubation
a multiplicity of infection (MOI) of 10 for the indicated time. After for 5 min in citric saline (0.135 M potassium chloride and 0.015
1 hour, the medium was changed to DMEM supplemented with M sodium citrate), collected by centrifugation together with the
10% FBS and gentamicin (50 μg/ml; MP Biomedicals). One hour culture medium to include cells in suspension, and then washed
later, the medium was replaced with DMEM supplemented with twice in PBS. Cell pellets were resuspended in FACS buffer (1×
10% FBS and gentamicin (10 μg/ml), which was kept until the PBS + 2% BSA), and GFP expression was analyzed using a BD

end of the experiment. Primers for RT-qPCR are listed in table S1. LSRFortessa Cell Analyzer at the Max Perutz Labs FACS facility.
Analysis of the percentage of GFP-positive cells was performed
CFU assay with FlowJo. A minimum of 8000 cells were acquired for each dot
For in vitro CFU assays, 5 × 105 BMDMs were seeded in 200 μl of plot. The percentage of positive cells in the FACS analysis represents
DMEM supplemented with 10% FBS and M-CSF (500 ng/ml) in 96- the percentage of VSV-positive cells over the live-cell population.
well plates and infected with L. monocytogenes at different time This was calculated using a forward and side-scatter gate to
points. After each time point, cells were washed twice with PBS exclude dead cells and debris from the analysis.
and lysed in 50 μl of sterile nuclease-free water twice for 5 min at
37°C. For quantifying bacterial loads, four 1:10 serial dilutions of Quantification and statistical analysis
these lysates were plated on plates containing BHI medium and in- Experiments were repeated at least three times with the following
cubated for 1 day at 37°C. exceptions: RT-qPCR of BMDMs derived from p38αΔM mice was
performed on two biological replicates per condition, and RT-
Measurement of NO production qPCR of BMDM derived from knock-in CREB-S133A mice was
For measurement of NO production, 1 × 106 BMDMs were seeded performed with cells from two mice differentiated into three differ-
in 500 μl of DMEM supplemented with 10% FBS and M-CSF (500 ent plates. All data are presented as the mean values with the SD.
ng/ml) in 24-well plates. After 24 hours of L. monocytogenes infec- Differences in mRNA/pre-mRNA expression data, NO measure-
tion (MOI of 10), NO production was measured indirectly by assay- ment, and PI staining were compared using one-way analysis of var-
ing the concentration of nitrite in the cell culture medium with the iance (ANOVA) and corrected for multiple testing with Dunnett’s
Griess method. One hundred microliters of cell supernatant was post hoc test. Differences in ChIP data and in mRNA expression
transferred into a 96-well plate in triplicates to which 100 μl of sul- data between WT and knockout cells were compared using two-
fanilic acid solution (1% sulfanilamide in 5% phosphoric acid) and way ANOVA and corrected for multiple testing with Dunnett’s
100 μl of NEDD solution [0.1% N-(1-naphthyl)ethylenediamine in post hoc test. CFU assay significance was assessed with two-tailed
double-distilled water] were added, and the absorbance was imme- unpaired t test. All statistical analyses were performed using Graph-
diately measured at 545 nm with a plate reader. Serial dilutions of Pad Prism V7 (GraphPad) software. Statistical significance is noted
sodium nitrite (NaNO2) were prepared and used as standard curve in the figures as follows: ns, P > 0.05, *P ≤ 0.05, **P ≤ 0.01,
to assess the NO concentration in the samples. ***P ≤ 0.001, and ****P ≤ 0.0001. Each dot represents one biological
replicate. The number of independent biological replicates (n) for
PI staining and flow cytometry all experiments is indicated in the figure legends. For immunoblots
For assessment of cell death, 1 × 106 BMDMs were seeded in 2 ml of and immunofluorescence, the reported images are representative of
DMEM supplemented with 10% FBS and M-CSF (500 ng/ml) in at least three independent experiments.
six-well plates. Cells were infected with L. monocytogenes for 24

Supplementary Materials Tristetraprolin-driven regulatory circuit controls quality and timing of mRNA decay in
This PDF file includes: inflammation. Mol. Syst. Biol. 7, 560 (2011).
Figs. S1 to S6 22. J. D. O’Neil, A. J. Ammit, A. R. Clark, MAPK p38 regulates inflammatory gene expression via
tristetraprolin: Doing good by stealth. Int. J. Biochem. Cell Biol. 94, 6–9 (2018).
Other Supplementary Material for this 23. H. P. Phatnani, A. L. Greenleaf, Phosphorylation and functions of the RNA polymerase II
manuscript includes the following: CTD. Genes Dev. 20, 2922–2936 (2006).
Table S1 24. I. Steinparzer, V. Sedlyarov, J. D. Rubin, K. Eislmayr, M. D. Galbraith, C. B. Levandowski,
Data files S1 to S4 T. Vcelkova, L. Sneezum, F. Wascher, F. Amman, R. Kleinova, H. Bender, Z. Andrysik,
MDAR Reproducibility Checklist J. M. Espinosa, G. Superti-Furga, R. D. Dowell, D. J. Taatjes, P. Kovarik, Transcriptional re-
sponses to IFN-γ require mediator kinase-dependent pause release and mechanistically
distinct CDK8 and CDK19 functions. Mol. Cell 76, 485–499.e8 (2019).
View/request a protocol for this paper from Bio-protocol.
25. E. Platanitis, S. Gruener, A. R. S. J. Geetha, L. Boccuni, A. Vogt, M. Novatchkova, A. Sommer,
I. Barozzi, M. Müller, T. Decker, Interferons reshape the 3D conformation and accessibility of
macrophage chromatin. iScience 25, 103840 (2022).
REFERENCES AND NOTES 26. S. Saccani, S. Pantano, G. Natoli, p38-dependent marking of inflammatory genes for in-
1. J. D. MacMicking, Interferon-inducible effector mechanisms in cell-autonomous immunity. creased NF-κB recruitment. Nat. Immunol. 3, 69–75 (2002).
Nat. Rev. Immunol. 12, 367–382 (2012). 27. A. Sawicka, D. Hartl, M. Goiser, O. Pusch, R. R. Stocsits, I. M. Tamir, K. Mechtler, C. Seiser,
2. W. M. Schneider, M. D. Chevillotte, C. M. Rice, Interferon-stimulated genes: A complex web H3S28 phosphorylation is a hallmark of the transcriptional response to cellular stress.
of host defenses. Annu. Rev. Immunol. 32, 513–545 (2014). Genome Res. 24, 1808–1820 (2014).
3. D. E. Levy, J. E. Darnell Jr., STATs: Transcriptional control and biological impact. Nat. Rev. 28. A. A. Igolkina, A. Zinkevich, K. O. Karandasheva, A. A. Popov, M. V. Selifanova, D. Nikolaeva,
Mol. Cell Biol. 3, 651–662 (2002). V. Tkachev, D. Penzar, D. M. Nikitin, A. Buzdin, H3K4me3, H3K9ac, H3K27ac, H3K27me3 and
4. C. Schindler, D. E. Levy, T. Decker, JAK-STAT signaling: From interferons to cytokines. J. Biol. H3K9me3 histone tags suggest distinct regulatory evolution of open and condensed
Chem. 282, 20059–20063 (2007). chromatin landmarks. Cell 8, 1034 (2019).
5. G. R. Stark, J. E. Darnell, The JAK-STAT pathway at twenty. Immunity 36, 503–514 (2012). 29. A. Cuadrado, N. Corrado, E. Perdiguero, V. Lafarga, P. Muñoz-Canoves, A. R. Nebreda, Es-
6. L. B. Ivashkiv, L. T. Donlin, Regulation of type I interferon responses. Nat. Rev. Immunol. 14, sential role of p18Hamlet/SRCAP-mediated histone H2A.Z chromatin incorporation in
36–49 (2014). muscle differentiation. EMBO J. 29, 2014–2025 (2010).
7. M. Farlik, B. Reutterer, C. Schindler, F. Greten, C. Vogl, M. Müller, T. Decker, Nonconventional 30. N. Au-Yeung, C. M. Horvath, Histone H2A.Z suppression of interferon-stimulated tran-
initiation complex assembly by STAT and NF-κB transcription factors regulates nitric oxide scription and antiviral immunity is modulated by GCN5 and BRD2. iScience 6, 68–82 (2018).
synthase expression. Immunity 33, 25–34 (2010). 31. P. Langfelder, S. Horvath, WGCNA: An R package for weighted correlation network analysis.
8. S. Wienerroither, P. Shukla, M. Farlik, A. Majoros, B. Stych, C. Vogl, H. Cheon, G. R. Stark, BMC Bioinform. 9, 559 (2008).
B. Strobl, M. Müller, T. Decker, Cooperative transcriptional activation of antimicrobial genes 32. E. Platanitis, D. Demiroz, A. Schneller, K. Fischer, C. Capelle, M. Hartl, T. Gossenreiter,
by STAT and NF-κB pathways by concerted recruitment of the mediator complex. Cell Rep. M. Müller, M. Novatchkova, T. Decker, A molecular switch from STAT2-IRF9 to ISGF3 un-
12, 300–312 (2015). derlies interferon-induced gene transcription. Nat. Commun. 10, 2921 (2019).

9. K. Ramsauer, I. Sadzak, A. Porras, A. Pilz, A. R. Nebreda, T. Decker, P. Kovarik, p38 MAPK 33. K. Adelman, M. A. Kennedy, S. Nechaev, D. A. Gilchrist, G. W. Muse, Y. Chinenov, I. Rogatsky,
enhances STAT1-dependent transcription independently of Ser-727 phosphorylation. Proc. Immediate mediators of the inflammatory response are poised for gene activation through
Natl. Acad. Sci. U.S.A. 99, 12859–12864 (2002). RNA polymerase II stalling. Proc. Natl. Acad. Sci. U.S.A. 106, 18207–18212 (2009).
10. Y. Li, A. Sassano, B. Majchrzak, D. K. Deb, D. E. Levy, M. Gaestel, A. R. Nebreda, E. N. Fish, 34. J. V. Falvo, B. S. Parekh, C. H. Lin, E. Fraenkel, T. Maniatis, Assembly of a functional beta
L. C. Platanias, Role of p38α map kinase in type I interferon signaling. J. Biol. Chem. 279, interferon enhanceosome is dependent on ATF-2–c-Jun heterodimer orientation. Mol. Cell.
970–979 (2004). Biol. 20, 4814–4825 (2000).
11. A. Cuadrado, A. R. Nebreda, Mechanisms and functions of p38 MAPK signalling. Biochem. J. 35. A. G. Papavassiliou, A. M. Musti, The multifaceted output of c-Jun biological activity: Focus
429, 403–417 (2010). at the junction of CD8 T cell activation and exhaustion. Cell 9, 2470 (2020).
12. B. Canovas, A. R. Nebreda, Diversity and versatility of p38 kinase signalling in health and 36. P. Novoszel, B. Drobits, M. Holcmann, C. D. S. Fernandes, R. Tschismarov, S. Derdak,
disease. Nat. Rev. Mol. Cell Biol. 22, 346–366 (2021). T. Decker, E. F. Wagner, M. Sibilia, The AP-1 transcription factors c-Jun and JunB are es-
13. D. Faust, C. Schmitt, F. Oesch, B. Oesch-Bartlomowicz, I. Schreck, C. Weiss, C. Dietrich, sential for CD8α conventional dendritic cell identity. Cell Death Differ. 28,
Differential p38-dependent signalling in response to cellular stress and mitogenic stimu- 2404–2420 (2021).
lation in fibroblasts. Cell Commun. Signal. 10, 6 (2012). 37. D. J. Gough, K. Sabapathy, E. Y.-N. Ko, H. A. Arthur, R. D. Schreiber, J. A. Trapani, C. J. P. Clarke,
14. A. Cuenda, S. Rousseau, p38 MAP-kinases pathway regulation, function and role in human R. W. Johnstone, A novel c-Jun-dependent signal transduction pathway necessary for the
diseases. Biochim. Biophys. Acta 1773, 1358–1375 (2007). transcriptional activation of interferon γ response genes. J. Biol. Chem. 282,
15. J. M. Kyriakis, J. Avruch, Mammalian MAPK signal transduction pathways activated by stress 938–946 (2007).
and inflammation: A 10-year update. Physiol. Rev. 92, 689–737 (2012). 38. T. Zhang, F. Inesta-Vaquera, M. Niepel, J. Zhang, S. B. Ficarro, T. Machleidt, T. Xie, J. A. Marto,
16. D. Saleiro, S. Mehrotra, B. Kroczynska, E. M. Beauchamp, P. Lisowski, B. Majchrzak-Kita, N. Kim, T. Sim, J. D. Laughlin, H. Park, P. V. LoGrasso, M. Patricelli, T. K. Nomanbhoy,
T. D. Bhagat, B. L. Stein, B. McMahon, J. K. Altman, E. M. Kosciuczuk, D. P. Baker, C. Jie, P. K. Sorger, D. R. Alessi, N. S. Gray, Discovery of potent and selective covalent inhibitors of
N. Jafari, C. B. Thompson, R. L. Levine, E. N. Fish, A. K. Verma, L. C. Platanias, Central role of JNK. Chem. Biol. 19, 140–154 (2012).
ULK1 in type I interferon signaling. Cell Rep. 11, 605–617 (2015). 39. T. Zarubin, J. Han, Activation and signaling of the p38 MAP kinase pathway. Cell Res. 15,
17. M. Macias-Silva, G. Vazquez-Victorio, J. Hernandez-Damian, Anisomycin is a multifunc- 11–18 (2005).
tional drug: More than just a tool to inhibit protein synthesis. Curr. Chem. Biol. 4, 40. V. Di Giacomo, S. Sancilio, L. Caravatta, R. A. Rana, R. Di Pietro, A. Cataldi, Regulation of CREB
124–132 (2010). activation by P38 mitogen activated protein kinase during human primary erythroblast
18. J. D. Laskin, D. E. Heck, D. L. Laskin, The ribotoxic stress response as a potential mechanism differentiation. Int. J. Immunopathol. Pharmacol. 22, 679–688 (2009).
for MAP kinase activation in xenobiotic toxicity. Toxicol. Sci. 69, 289–291 (2002). 41. D. M. Benbrook, N. C. Jones, Heterodimer formation between CREB and JUN proteins.
19. P. Kovarik, D. Stoiber, P. A. Eyers, R. Menghini, A. Neininger, M. Gaestel, P. Cohen, T. Decker, Oncogene 5, 295–302 (1990).
Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated 42. J. M. Rozenberg, P. Bhattacharya, R. Chatterjee, K. Glass, C. Vinson, Combinatorial recruit-
protein kinase whereas IFN-γ uses a different signaling pathway. Proc. Natl. Acad. Sci. U.S.A. ment of CREB, C/EBPβ and c-Jun determines activation of promoters upon keratinocyte
96, 13956–13961 (1999). differentiation. PLOS ONE 8, e78179 (2013).
20. A. Michalska, K. Blaszczyk, J. Wesoly, H. A. R. Bluyssen, A positive feedback amplifier circuit 43. S. Naqvi, K. J. Martin, J. S. C. Arthur, CREB phosphorylation at Ser133 regulates transcription
that regulates interferon (IFN)-stimulated gene expression and controls type I and type II via distinct mechanisms downstream of cAMP and MAPK signalling. Biochem. J. 458,
IFN responses. Front. Immunol. 9, 1135 (2018). 469–479 (2014).
21. F. Kratochvill, C. Machacek, C. Vogl, F. Ebner, V. Sedlyarov, A. R. Gruber, H. Hartweger, 44. A. Steven, M. Friedrich, P. Jank, N. Heimer, J. Budczies, C. Denkert, B. Seliger, What turns
R. Vielnascher, M. Karaghiosoff, T. Rülicke, M. Müller, I. Hofacker, R. Lang, P. Kovarik, CREB on? And off? And why does it matter? Cell. Mol. Life Sci. 77, 4049–4067 (2020).

45. R. J. Platt, S. Chen, Y. Zhou, M. J. Yim, L. Swiech, H. R. Kempton, J. E. Dahlman, O. Parnas, 68. J. Ostojić, Y.-S. Yoon, T. Sonntag, B. Nguyen, J. M. Vaughan, M. Shokhirev, M. Montminy,
T. M. Eisenhaure, M. Jovanovic, D. B. Graham, S. Jhunjhunwala, M. Heidenreich, R. J. Xavier, Transcriptional co-activator regulates melanocyte differentiation and oncogenesis by in-
R. Langer, D. G. Anderson, N. Hacohen, A. Regev, G. Feng, P. A. Sharp, F. Zhang, CRISPR-Cas9 tegrating cAMP and MAPK/ERK pathways. Cell Rep. 35, 109136 (2021).
knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014). 69. S. S. Mikkelsen, S. B. Jensen, S. Chiliveru, J. Melchjorsen, I. Julkunen, M. Gaestel, J. S. Arthur,
46. A. D. Wingate, K. J. Martin, C. Hunter, J. M. Carr, C. Clacher, J. S. C. Arthur, Generation of a R. A. Flavell, S. Ghosh, S. R. Paludan, RIG-I-mediated activation of p38 MAPK is essential for
conditional CREB Ser133Ala knockin mouse. Genesis 47, 688–696 (2009). viral induction of interferon and activation of dendritic cells: Dependence on TRAF2 and
47. M. D. Conkright, G. Canettieri, R. Screaton, E. Guzman, L. Miraglia, J. B. Hogenesch, TAK1. J. Biol. Chem. 284, 10774–10782 (2009).
M. Montminy, TORCs. Mol. Cell 12, 413–423 (2003). 70. D. S. Simpson, J. Pang, A. Weir, I. Y. Kong, M. Fritsch, M. Rashidi, J. P. Cooney, K. C. Davidson,
48. M. A. Bittinger, E. McWhinnie, J. Meltzer, V. Iourgenko, B. Latario, X. Liu, C. H. Chen, C. Song, M. Speir, T. M. Djajawi, S. Hughes, L. Mackiewicz, M. Dayton, H. Anderton, M. Doerflinger,
D. Garza, M. Labow, Activation of cAMP response element-mediated gene expression by Y. Deng, A. S. Huang, S. A. Conos, H. Tye, S. H. Chow, A. Rahman, R. S. Norton, T. Naderer,
regulated nuclear transport of TORC proteins. Curr. Biol. 14, 2156–2161 (2004). S. E. Nicholson, G. Burgio, S. M. Man, J. R. Groom, M. J. Herold, E. D. Hawkins, K. E. Lawlor,
49. J. Y. Altarejos, M. Montminy, CREB and the CRTC co-activators: Sensors for hormonal and A. Strasser, J. Silke, M. Pellegrini, H. Kashkar, R. Feltham, J. E. Vince, Interferon-γ primes
metabolic signals. Nat. Rev. Mol. Cell Biol. 12, 141–151 (2011). macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell
50. K. F. MacKenzie, K. Clark, S. Naqvi, V. A. McGuire, G. Nöehren, Y. Kristariyanto, M. van den death pathway. Immunity 55, 423–441.e9 (2022).
Bosch, M. Mudaliar, P. C. McCarthy, M. J. Pattison, P. G. Pedrioli, G. J. Barton, R. Toth, 71. P. Bastard, A. Gervais, T. Le Voyer, J. Rosain, Q. Philippot, J. Manry, E. Michailidis, H.-
A. Prescott, J. S. Arthur, PGE2 induces macrophage IL-10 production and a regulatory-like H. Hoffmann, S. Eto, M. Garcia-Prat, L. Bizien, A. Parra-Martínez, R. Yang, L. Haljasmägi,
phenotype via a protein kinase A-SIK-CRTC3 pathway. J. Immunol. 190, 565–577 (2013). M. Migaud, K. Särekannu, J. Maslovskaja, N. de Prost, Y. Tandjaoui-Lambiotte, C.-E. Luyt,
51. R. P. Welch, C. Lee, P. M. Imbriano, S. Patil, T. E. Weymouth, R. A. Smith, L. J. Scott, B. Amador-Borrero, A. Gaudet, J. Poissy, P. Morel, P. Richard, F. Cognasse, J. Troya,
M. A. Sartor, ChIP-Enrich: Gene set enrichment testing for ChIP-seq data. Nucleic Acids Res. S. Trouillet-Assant, A. Belot, K. Saker, P. Garçon, J. G. Rivière, J.-C. Lagier, S. Gentile,
42, –e105 (2014). L. B. Rosen, E. Shaw, T. Morio, J. Tanaka, D. Dalmau, P.-L. Tharaux, D. Sene, A. Stepanian,
B. Megarbane, V. Triantafyllia, A. Fekkar, J. R. Heath, J. L. Franco, J.-M. Anaya, J. Solé-Violán,
52. T. Decker, M. Müller, S. Stockinger, The yin and yang of type I interferon activity in bacterial
L. Imberti, A. Biondi, P. Bonfanti, R. Castagnoli, O. M. Delmonte, Y. Zhang, A. L. Snow,
infection. Nat. Rev. Immunol. 5, 675–687 (2005).
S. M. Holland, C. Biggs, M. Moncada-Vélez, A. A. Arias, L. Lorenzo, S. Boucherit, B. Coulibaly,
53. K. Hansen, T. Prabakaran, A. Laustsen, S. E. Jørgensen, S. H. Rahbæk, S. B. Jensen, R. Nielsen,
D. Anglicheau, A. M. Planas, F. Haerynck, S. Duvlis, R. L. Nussbaum, T. Ozcelik, S. Keles,
J. H. Leber, T. Decker, K. A. Horan, M. R. Jakobsen, S. R. Paludan, Listeria monocytogenes
A. A. Bousfiha, J. E. Bakkouri, C. Ramirez-Santana, S. Paul, Q. Pan-Hammarström,
induces IFNβ expression through an IFI16-, cGAS- and STING-dependent pathway. EMBO J.
L. Hammarström, A. Dupont, A. Kurolap, C. N. Metz, A. Aiuti, G. Casari, V. Lampasona,
33, 1654–1666 (2014).
F. Ciceri, L. A. Barreiros, E. Dominguez-Garrido, M. Vidigal, M. Zatz, D. van de Beek,
54. D. Panne, T. Maniatis, S. C. Harrison, An atomic model of the interferon-β enhanceosome. S. Sahanic, I. Tancevski, Y. Stepanovskyy, O. Boyarchuk, Y. Nukui, M. Tsumura, L. Vidaur,
Cell 129, 1111–1123 (2007). S. G. Tangye, S. Burrel, D. Duffy, L. Quintana-Murci, A. Klocperk, N. Y. Kann, A. Shcherbina, Y.-
55. L. Boscà, M. Zeini, P. Través, S. Hortelano, Nitric oxide and cell viability in inflammatory cells: L. Lau, D. Leung, M. Coulongeat, J. Marlet, R. Koning, L. F. Reyes, A. Chauvineau-Grenier,
A role for NO in macrophage function and fate. Toxicology 208, 249–258 (2005). F. Venet, G. Monneret, M. C. Nussenzweig, R. Arrestier, I. Boudhabhay, H. Baris-Feldman,
56. H. Zwaferink, S. Stockinger, S. Reipert, T. Decker, Stimulation of inducible nitric oxide D. Hagin, J. Wauters, I. Meyts, A. H. Dyer, S. P. Kennelly, N. M. Bourke, R. Halwani, N. S. Sharif-
synthase expression by β interferon increases necrotic death of macrophages upon Listeria Askari, K. Dorgham, J. Sallette, S. M. Sedkaoui, S. A. Khater, R. Rigo-Bonnin, F. Morandeira,
monocytogenes infection. Infect. Immun. 76, 1649–1656 (2008). L. Roussel, D. C. Vinh, S. R. Ostrowski, A. Condino-Neto, C. Prando, A. Bonradenko,

57. E. M. Eshleman, N. Bortell, D. S. McDermott, W. J. Crisler, L. L. Lenz, Myeloid cell respon- A. N. Spaan, L. Gilardin, J. Fellay, S. Lyonnet, K. Bilguvar, R. P. Lifton, S. Mane; HGID Lab;
siveness to interferon-γ is sufficient for initial resistance to Listeria monocytogenes. Curr. COVID Clinicians; COVID-STORM Clinicians; NIAID Immune Response to COVID Group; NH-
Opin. Immunol. 1, 1–9 (2020). COVAIR Study Group; Danish CHGE; Danish Blood Donor Study; St. James’s Hospital; SARS
58. T. Matsuzawa, B.-H. Kim, A. R. Shenoy, S. Kamitani, M. Miyake, J. D. Macmicking, IFN-γ elicits Co V Interest group; French COVID Cohort Study Group; Imagine COVID-Group; Milieu
macrophage autophagy via the p38 MAPK signaling pathway. J. Immunol. 189, Intérieur Consortium; Co V-Contact Cohort; Amsterdam UMC Covid-19; Biobank Investi-
813–818 (2012). gators; COVID Human Genetic Effort; CONSTANCES cohort; 3C-Dijon Study; Cerba Health-
59. G. Mitchell, M. I. Cheng, C. Chen, B. N. Nguyen, A. T. Whiteley, S. Kianian, J. S. Cox, Care; Etablissement du Sang study group, M. S. Anderson, B. Boisson, V. Béziat, S.-Y. Zhang,
D. R. Green, K. L. McDonald, D. A. Portnoy, Listeria monocytogenes triggers noncanonical E. Vandreakos, O. Hermine, A. Pujol, P. Peterson, T. H. Mogensen, L. Rowen, J. Mond,
autophagy upon phagocytosis, but avoids subsequent growth-restricting xenophagy. S. Debette, X. de Lamballerie, X. Duval, F. Mentré, M. Zins, P. Soler-Palacin, R. Colobran,
Proc. Natl. Acad. Sci. U.S.A. 115, E210–E217 (2018). G. Gorochov, X. Solanich, S. Susen, J. Martinez-Picado, D. Raoult, M. Vasse, P. K. Gregersen,
60. Y.-K. Lee, J.-A. Lee, Role of the mammalian ATG8/LC3 family in autophagy: Differential and L. Piemonti, C. Rodríguez-Gallego, L. D. Notarangelo, H. C. Su, K. Kisand, S. Okada, A. Puel,
compensatory roles in the spatiotemporal regulation of autophagy. BMB Rep. 49, E. Jouanguy, C. M. Rice, P. Tiberghien, Q. Zhang, A. Cobat, L. Abel, J.-L. Casanova, Auto-
424–430 (2016). antibodies neutralizing type I IFNs are present in ~4% of uninfected individuals over 70
years and account for ~20% of COVID-19 deaths. Sci. Immunol. 6, eabl4340 (2021).
61. R. Karki, B. R. Sharma, S. Tuladhar, E. P. Williams, L. Zalduondo, P. Samir, M. Zheng,
B. Sundaram, B. Banoth, R. K. S. Malireddi, P. Schreiner, G. Neale, P. Vogel, R. Webby, 72. J. M. Grimes, K. V. Grimes, p38 MAPK inhibition: A promising therapeutic approach for
C. B. Jonsson, T.-D. Kanneganti, Synergism of TNF-α and IFN-γ triggers inflammatory cell COVID-19. J. Mol. Cell. Cardiol. 144, 63–65 (2020).
death, tissue damage, and mortality in SARS-CoV-2 infection and cytokine shock syn- 73. COvid-19 Multi-omics Blood ATlas (COMBAT) Consortium, A blood atlas of COVID-19
dromes. Cell 184, 149–168.e17 (2021). defines hallmarks of disease severity and specificity. Cell 185, 916–938.e58 (2022).
62. M. S. Diamond, T.-D. Kanneganti, Innate immunity: The first line of defense against SARS- 74. C. Youssif, M. Cubillos-Rojas, M. Comalada, E. Llonch, C. Perna, N. Djouder, A. R. Nebreda,
CoV-2. Nat. Immunol. 23, 165–176 (2022). Myeloid p38α signaling promotes intestinal IGF-1 production and inflammation-associated
63. R. K. Kandasamy, G. I. Vladimer, B. Snijder, A. C. Müller, M. Rebsamen, J. W. Bigenzahn, tumorigenesis. EMBO Mol. Med. 10, e8403 (2018).
A. Moskovskich, M. Sabler, A. Stefanovic, S. Scorzoni, M. Brückner, T. Penz, C. Cleary, 75. R. Hai, L. Martínez-Sobrido, A. F. Kathryn, J. Ayllon, A. García-Sastre, P. Palese, Influenza B
R. Kralovics, J. Colinge, K. L. Bennett, G. Superti-Furga, A time-resolved molecular map of virus NS1-truncated mutants: Live-attenuated vaccine approach. J. Virol. 82,
the macrophage response to VSV infection. NPJ Syst. Biol. Appl. 2, 16027 (2016). 10580–10590 (2008).
64. G. L. Johnson, K. Nakamura, The c-Jun kinase/stress-activated pathway: Regulation, func- 76. C. A. Schneider, W. S. Rasband, K. W. Eliceiri, NIH Image to ImageJ: 25 years of image
tion and role in human disease. Biochim. Biophys. Acta 1773, 1341–1348 (2007). analysis. Nat. Methods 9, 671–675 (2012).
65. Y. J. Kang, J. Chen, M. Otsuka, J. Mols, S. Ren, Y. Wang, J. Han, Macrophage deletion of p38α 77. D. R. Stirling, M. J. Swain-Bowden, A. M. Lucas, A. E. Carpenter, B. A. Cimini, A. Goodman,
partially impairs lipopolysaccharide-induced cellular activation. J. Immunol. 180, CellProfiler 4: Improvements in speed, utility and usability. BMC Bioinformatics 22,
5075–5082 (2008). 433 (2021).
66. R. Eferl, E. F. Wagner, AP-1: A double-edged sword in tumorigenesis. Nat. Rev. Cancer 3, 78. P. Datlinger, A. F. Rendeiro, C. Schmidl, T. Krausgruber, P. Traxler, J. Klughammer,
859–868 (2003). L. C. Schuster, A. Kuchler, D. Alpar, C. Bock, Pooled CRISPR screening with single-cell
67. M. Muraoka, A. Fukushima, S. Viengchareun, M. Lombès, F. Kishi, A. Miyauchi, transcriptome readout. Nat. Methods 14, 297–301 (2017).
M. Kanematsu, J. Doi, J. Kajimura, R. Nakai, T. Uebi, M. Okamoto, H. Takemori, Involvement 79. D. W. Morgens, M. Wainberg, E. A. Boyle, O. Ursu, C. L. Araya, C. K. Tsui, M. S. Haney,
of SIK2/TORC2 signaling cascade in the regulation of insulin-induced PGC-1α and UCP- G. T. Hess, K. Han, E. E. Jeng, A. Li, M. P. Snyder, W. J. Greenleaf, A. Kundaje, M. C. Bassik,
1gene expression in brown adipocytes. Am. J. Physiol. Endocrinol. Metab. 296, Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput
E1430–E1439 (2009). screens. Nat. Commun. 8, 15178 (2017).

80. A. M. Bolger, M. Lohse, B. Usadel, Trimmomatic: A flexible trimmer for Illumina sequence CRISPR-Cas9 gene editing and for the VSV-GFP. We thank A. Aszódi from the VBCF Core Facilities
data. Bioinformatics 30, 2114–2120 (2014). GmbH (www.viennabiocenter.org/vbcf/computational-biology-training/) for critical advice on
81. A. Dobin, C. A. Davis, F. Schlesinger, J. Drenkow, C. Zaleski, S. Jha, P. Batut, M. Chaisson, statistical data analysis. Solexa sequencing was performed by the VBCF NGS Unit (www.vbcf.ac.
T. R. Gingeras, STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013). at). Funding: Funding was provided by the Austrian Science Fund (FWF) through projects SFB
82. M. I. Love, W. Huber, S. Anders, Moderated estimation of fold change and dispersion for F6101, F6102, F6103, F6106, and F6107 to T.D., C.B., V.S., and M.M and by the European Research
RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). Council Advanced grant ERC-2015-AdG TNT-Tumors 694883 and the FWF-funded doctoral
83. A. Subramanian, P. Tamayo, V. K. Mootha, S. Mukherjee, B. L. Ebert, M. A. Gillette, program DK W1212 "Inflammation and Immunity" (PN) to M. Sibilia. L.B. was supported by the
A. Paulovich, S. L. Pomeroy, T. R. Golub, E. S. Lander, J. P. Mesirov, Gene set enrichment FWF through the doctoral program W1261 "Signaling Mechanisms in Cell Homeostasis."
analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Author contributions: L.B. and T.D. conceived and designed experiments. L.B., E. Podgorschek,
Proc. Natl. Acad. Sci. U.S.A. 102, 15545–15550 (2005). M. Schmiedeberg, and E. Platanitis performed experiments and analyzed the data. L.B., P.F., and
N.F. performed bioinformatics/statistical analysis. P.T., A.S., P.N., A.R.N., J.S.C.A., M.F., V.S., C.B.,
84. F. Ramírez, V. Bhardwaj, L. Arrigoni, K. C. Lam, B. A. Grüning, J. Villaveces, B. Habermann,
M. Sibilia, P.K., and M.M. provided critical reagents, essential mice, and expert advice. L.B. and
A. Akhtar, T. Manke, High-resolution TADs reveal DNA sequences underlying genome or-
T.D. wrote the manuscript. T.D., P.K., M.F., and M.M. supervised the studies. T.D., M.M., and V.S.
ganization in flies. Nat. Commun. 9, 189 (2018).
took care of funding acquisition. Competing interests: The authors declare that they have no
85. L. Lopez-Delisle, L. Rabbani, J. Wolff, V. Bhardwaj, R. Backofen, B. Grüning, F. Ramírez,
competing interests. Data and materials availability: All data needed to evaluate the
T. Manke, pyGenomeTracks: Reproducible plots for multivariate genomic datasets. Bioin-
conclusions in the paper are present in the main text or the Supplementary Materials. Publicly
formatics 37, 422–423 (2021).
available raw data used in this paper are available under the accession number GEO: GSE115435
86. S. Heinz, L. Texari, M. G. B. Hayes, M. Urbanowski, M. W. Chang, N. Givarkes, A. Rialdi, (STAT1, STAT2, and IRF9 ChIP-seq; www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE115435).
K. M. White, R. A. Albrecht, L. Pache, I. Marazzi, A. García-Sastre, M. L. Shaw, C. Benner, All the sequencing data generated for this publication have been deposited in NCBI’s Gene
Transcription elongation can affect genome 3D structure. Cell 174, 1522–1536.e22 (2018). Expression Omnibus and are available under the accession number GEO: GSE199166 (www.
ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE199166). The paper does not report original code.
Acknowledgments: We thank B. Strobl (University of Veterinary Medicine, Vienna) for critical
comments on the manuscript. C. Pecoraro and Physalia Courses (www.physalia-courses.org) are Submitted 14 April 2022
thanked for the next-generation sequencing (NGS) data analysis training. We are especially Accepted 16 November 2022
grateful to F. Comoglio for sharing expertise and enthusiasm for data visualization, ATAC-seq, Published 13 December 2022
and ChIP-seq analysis. We thank S. Grüner for fundamental help in NGS data analysis and 10.1126/scisignal.abq5389
D. Martin for help in the RNA-seq data analysis. We thank the Max Perutz Labs BioOptics FACS
Facility for help with FACS analysis. We thank S. Scinicariello and G. Versteeg for help with

Stress signaling boosts interferon-induced gene transcription in macrophages
Laura BoccuniElke PodgorschekMoritz SchmiedebergEkaterini PlatanitisPeter TraxlerPhilipp FischerAlessia
SchirripaPhilipp NovoszelAngel R. NebredaJ. Simon C. ArthurNikolaus FortelnyMatthias FarlikVeronika SexlChristoph
BockMaria SibiliaPavel KovarikMathias MüllerThomas Decker
Sci. Signal., 15 (764), eabq5389. • DOI: 10.1126/scisignal.abq5389
Stressed into boosting interferon signaling

Interferon signaling stimulates the innate immune response to infection and is regulated in part by stress signaling
mediated by the kinase p38. Boccuni et al. clarified the functional interaction between these pathways and the
consequences of their integration. In macrophages stimulated with interferon, coincident stress signaling induced
a p38-dependent recruitment of additional factors to interferon-stimulated genes, synergistically enhancing their
expression. In Listeria-infected cultured macrophages, this boost elicited greater production of pathogen-fighting
factors like cytokines and nitric oxide, but it also increased cell death. Blocking p38 signaling preserved macrophage
viability without sacrificing function. The findings indicate that p38 signaling enhances not only the beneficial effects,
but also the detrimental effects of interferon-mediated immune responses.-LKF

View the article online
https://www.science.org/doi/10.1126/scisignal.abq5389
Permissions
https://www.science.org/help/reprints-and-permissions
Use of this article is subject to the Terms of service
Science Signaling (ISSN ) is published by the American Association for the Advancement of Science. 1200 New York Avenue NW,
Washington, DC 20005. The title Science Signaling is a registered trademark of AAAS.
Copyright © 2022 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim
to original U.S. Government Works

Stress signaling boosts interferon-induced gene transcription in macrophages

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Stress signaling boosts interferon-induced gene transcription in macrophages

Uploaded by

Copyright:

Available Formats

SCIENCE SIGNALING | RESEARCH ARTICLE

INTRODUCTION factors ISGF3 and GAF. ISGF3 consists of a tyrosine-phosphorylat-

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 1 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 2 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 3 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 4 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 5 of 20

Fig. 3. Genome-wide analysis of

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 6 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 7 of 20

Fig. 5. ISG enhancement requires CRE/

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 8 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 9 of 20

Fig. 6. ChIP-seq analysis of CREB1 and c-Jun

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 10 of 20

Fig. 7. p38 MAPK enhancement of Listeria

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 11 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 12 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 13 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 14 of 20

synthesis was performed by reverse transcription of 400 ng of total BMDM transduction

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 15 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 16 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 17 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 18 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 19 of 20

Downloaded from https://www.science.org on December 21, 2022

Boccuni et al., Sci. Signal. 15, eabq5389 (2022) 13 December 2022 20 of 20

Sci. Signal., 15 (764), eabq5389. • DOI: 10.1126/scisignal.abq5389

Stressed into boosting interferon signaling

Downloaded from https://www.science.org on December 21, 2022

Use of this article is subject to the Terms of service

You might also like