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ARTICLE
Histone demethylase KDM5B licenses macrophage-mediated
inflammatory responses by repressing Nfkbia transcription
✉
Yunkai Zhang1,2,6, Ying Gao3,4,6, Yuyu Jiang1,6, Yingying Ding1, Huiying Chen1, Yan Xiang1, Zhenzhen Zhan4,5 and
✉
Xingguang Liu 1,2

© The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare 2023

Macrophages play a critical role in the immune homeostasis and host defense against invading pathogens. However, uncontrolled
activation of inflammatory macrophages leads to tissue injury and even fuels autoimmunity. Hence the molecular mechanisms
underlying macrophage activation need to be further elucidated. The effects of epigenetic modifications on the function of
immune cells draw increasing attention. Here, we demonstrated that lysine-specific demethylase 5B (KDM5B), a classical
transcriptional repressor in stem cell development and cancer, was required for the full activation of NF-κB signaling cascade and
pro-inflammatory cytokine production in macrophages. KDM5B deficiency or inhibitor treatment protected mice from immunologic
injury in both collagen-induced arthritis (CIA) model and endotoxin shock model. Genome-wide analysis of KDM5B-binding peaks
identified that KDM5B was selectively recruited to the promoter of Nfkbia, the gene encoding IκBα, in activated macrophages.
1234567890();,:

KDM5B mediated the H3K4me3 modification erasing and decreased chromatin accessibility of Nfkbia gene locus, coordinating the
elaborate suppression of IκBα expression and the enhanced NF-κB-mediated macrophage activation. Our finding identifies the
indispensable role of KDM5B in macrophage-mediated inflammatory responses and provides a candidate therapeutic target for
autoimmune and inflammatory disorders.

Cell Death & Differentiation (2023) 30:1279–1292; https://doi.org/10.1038/s41418-023-01136-x

INTRODUCTION joints [6]. RA patients have increased number of active inflamma-

Macrophage is the master regulator of the induction and tory macrophages, which were widely known as the central
resolution of host inflammation in infection and autoimmunity players in the pathogenesis of RA [7, 8]. Therefore, increasing
[1, 2]. Generally, pattern recognition receptors (PRRs) on macro- studies focus on the therapeutic intervention for preventing the
phages specifically recognize and bind the pathogen-associated pathological effects of inflammatory macrophages.
molecular patterns (PAMPs) or damage-associated molecular Histone modification, mainly including histone methylation,
patterns (DMAPs) for launching the downstream signaling acetylation, phosphorylation, and ubiquitination, is well accepted
cascades, including nuclear factor kappa-B (NF-κB) pathway, as the important regulator of gene expression and cell destiny,
mitogen-activated protein kinase (MAPK) pathway, and etc [3]. whose dysfunction leads to pathological conditions and even
Upon activated, macrophages robustly produce pro-inflammatory severe diseases. Increasing researchers found that various histone
cytokine (e.g., IL-1β, IL-6, IL-12, and TNF-α) for the initiation of modifications participated in the regulation of macrophage
innate immune responses [4]. Otherwise, the co-stimulatory development and function, which might be the promising
signals and pro-inflammatory mediators produced by macro- therapeutic target for macrophage-associated diseases [9]. For
phages set up the immune environment for effective mobilization example, in the late phase of macrophage inflammatory polariza-
of adaptive immune responses, such as the potent T helper type 1 tion, cellular metabolism reprogramming in macrophages induced
(Th1)-Th17 response [5]. In this way, resident and infiltrating histone lactylation and the enhanced expression of homeostatic
macrophages in the inflamed tissues, together with T cells, B cells genes involving wound healing, such as arginase 1 (Arg1) [10].
and other inflammatory cells, disrupt the immune homeostasis Recent work found that mitochondrial glucose oxidation and ATP
and result in autoimmune disorders. Rheumatoid arthritis (RA) is a citrate lyase (ACLY)-dependent acetyl-CoA generation was the
chronic autoimmune disease involving joint and surrounding rate-limiting step in the histone 3 lysine 9 (H3K9) acetylation-
tissues with uncontrolled inflammatory responses. Synovial driven macrophage activation and inflammatory gene expression
hyperplasia and excessive immune cell accumulation cause the in vivo [11]. However, more biological functions of specific histone
undesirable tissue remodeling and reduced function of involved modifications, along with the “writer” or “erasers” for these

1
Department of Pathogen Biology, Naval Medical University, Shanghai 200433, China. 2National Key Laboratory of Medical Immunology, Naval Medical University, Shanghai
200433, China. 3Department of Rheumatology, Changhai Hospital, Naval Medical University, Shanghai 200433, China. 4Key Laboratory of Arrhythmias of the Ministry of Education
of China, Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China. 5Department of Liver Surgery,
Shanghai Institute of Transplantation, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China. 6These authors contributed equally: Yunkai
Zhang, Ying Gao, Yuyu Jiang. ✉email: [email protected]; [email protected]

Received: 22 September 2022 Revised: 9 February 2023 Accepted: 14 February 2023

Published online: 13 March 2023

Official journal of CDDpress

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modifications, in macrophages and autoimmune diseases remain American College of Rheumatology [22]. Compared with that in
to be further explored. HC, KDM5B expression was significantly repressed in ARA patients
Lysine-specific demethylase 5B (KDM5B), also named as Jumonji on both mRNA and protein levels (Supplementary Fig. 1H–J). The
AT-rich interactive domain 1B (JARID1B) or PLU-1, belongs to the above data imply that KDM5B has potential biological functions in
jumonji/ARAD (Jmjd) domain-containing family of histone the pathogenesis of these autoimmune diseases.
demethylases. KDM5B is highly expressed in a large variety of Next, we investigated the expression change of KDM5B in
cancers, especially breast cancer, colorectal cancer, gastric macrophages subjected to different stimuli. The data from
carcinoma and etc. [12]. KDM5B was reported to function as the GSE19315 showed that KDM5B expression was decreased in
transcription repressor through removing promoter-associated macrophage-like THP-1 cells treated with LPS, the ligand of Toll-
histone 3 lysine 4 trimethylation (H3K4me3) in kinds of cells. like receptor 4 (TLR4) (Supplementary Fig. 2A) [23]. As presented in
KDM5B regulated the expression of CC chemokine ligand 14 data from GSE5009, KDM5B expression was suppressed in IFN-γ-
(CCL14) in HT-29 cells and cyclin dependent kinase inhibitor 1A and LPS-treated macrophages but not in IL-4-treated murine
(CDKN1A) in MCF-7 cells through removing the H3K4me3 macrophages (Supplementary Fig. 2B) [24]. Our experimental data
modification at the gene promoters [13–15]. In addition, deletion also showed that the mRNA expression level of KDM5B was time-
of KDM5B in cancer cells was required for melanoma-induced dependently down-regulated in mouse BMDMs stimulated with
adaptive immune responses and enhanced responses to immune LPS, dsRNA analog poly(I:C) or gram-positive bacteria component
checkpoint blockade, which was not dependent on demethylase Pam3CSK4 (Supplementary Fig. 2C–E). Nevertheless, Kdm5b mRNA
activity but on the interaction with SET domain bifurcated histone expression in mouse BMDMs was hardly influenced by IL-4
lysine methyltransferase 1 (SETDB1) for silencing retroelements- treatment (Supplementary Fig. 2F). KDM5B protein expression was
triggered type I interferon (IFN-I) responses [16]. Hence KDM5B also suppressed in mouse BMDMs stimulated with LPS, poly(I:C) or
regulated gene expression in both demethylase-dependent or Pam3CSK4 but not IL-4 (Supplementary Fig. 2G–J). For exploring
-independent manner, according to different settings. However, the mechanisms underlying the decreased expression of KDM5B
the specific role of KDM5B in immune cells, especially in in LPS-activated macrophages, we analyzed the histone modifica-
macrophages, remains largely unknown. tion and RNA Pol II activity at the KDM5B promoter in BMDMs and
In this study, we found the significant decreased expression of found that LPS stimulation as long as 1 h (a short time) could
KDM5B in synovial macrophages from RA patients. KDM5B inhibit the H3K27ac and RNA Pol II binding as well as S2P activity
deficiency ameliorated the pathological injury and immune within the KDM5B gene locus, which were the markers of gene
disorder in collagen-induced arthritis (CIA) model and endotoxin transcriptional activation (Supplementary Fig. 2K) [25, 26]. In
shock model. KDM5B was essential for the production of pro- addition, we analyzed the newly synthesis Kdm5b mRNA by GRO-
inflammatory cytokines including IL-1β, IL-6 and TNF-α in seq to access the nascent RNA, and found that LPS stimulation
macrophages through maintaining the NF-κB signaling cascade resulted in the decreased Kdm5b nascent RNA production
activation. LPS stimulation induced the specific recruitment of (Supplementary Fig. 2K) [26]. These results suggest that KDM5B
KDM5B to the promoter of Nfkbia and mediated its transcriptional expression is suppressed by a variety of inflammatory stimuli
repression, while KDM5B deficiency led to the increased IκBα owing to the transcriptional inhibition, providing the potential
protein level that blocked NF-κB transcriptional activity. Our study explanation for the low expression in PBMCs or local inflamed
reveals that KDM5B functions as an indispensable epigenetic tissues in patients with autoimmune diseases.
regulator in macrophage activation and inflammatory disorders.
Loss of KDM5B ameliorates the immunologic injury in
experimental arthritis model
RESULTS To exploring the in vivo function of KDM5B, we generated the
The expression of histone demethylase KDM5B is extensively KDM5B knockout (KO) mice via CRISPR/Cas9 technology (Supple-
decreased in multiple autoimmune diseases mentary Fig. 3A, B). Compared with WT littermates, KDM5B-KO
To identify the regulatory protein involving in the inflammatory mice had normal physical development and comparable body
response and autoimmune diseases, we searched the Gene weight (Supplementary Fig. 3C, D), despite of the low fertility rate
Expression Omnibus (GEO) database and further analyzed the which was consistent with the previous studies [27, 28]. We further
differentially expressed genes (DEGs) in synovial macrophages observed the development of T lymphocytes in the thymus and
from health control and RA patients in GSE97779 [17] (Supple- peripheral immune organs from KDM5B-KO mice by FACS. KDM5B
mentary Fig. 1A). In synovial macrophages from RA group, the deficiency did not influence the percentages of double-negative
expression of several histone demethylase family members (DN), double-positive (DP) and single-positive CD4+ and CD8+ T
showed a dramatic difference from those in health control groups cell populations in thymus (Supplementary Fig. 3E). Meanwhile,
(Supplementary Fig. 1B). Among them, KDM5B that was sig- KDM5B deficiency had no effect on the populations of CD19+ B
nificantly down-regulated in RA patients drew our attention cells and CD3+ T cells, as well as CD4+ and CD8+ T cells in spleen
(Supplementary Fig. 1C). We next analyzed the expression of and lymphonodus (Supplementary Fig. 3F, G). Taken together,
KDM5B in other autoimmune diseases including multiple sclerosis KDM5B-deficient mice develop the functional immune system and
(MS), inflammatory bowel diseases (IBD) and cryopyrin-associated normal adaptive immune response, at least at the stable state.
periodic syndromes (CAPS), all of which involving the aberrant CIA model was taken to access the biological function of KDM5B
activation of macrophages and macrophage-derived excessive in vivo, which was a widely-accepted murine inflammatory model
pro-inflammatory responses. KDM5B expression was also signifi- with pathological characteristic similar to human RA patients [29].
cantly reduced in the PBMCs from MS patients, compared with Intriguingly, KDM5B-KO mice showed the relieved development of
that from health control (Supplementary Fig. 1D) [18]. Additionally, inflammatory arthritis, with delayed onset of disease, lower clinical
KDM5B was down-regulated in the PBMCs and inflamed colonic scores, less ankle joint diameter and milder joint swelling at the
mucosa samples from ulcerative colitis (UC) patients (Supplemen- front and hind paws, compared with WT mice (Fig. 1A–C;
tary Fig. 1E, F) [19, 20]. We also found the significant reduction of Supplementary Fig. 3H). Histological analysis of knee joints by
KDM5B expression in whole peripheral blood from CAPS patients H&E staining showed that the dramatic ameliorated disease
(Supplementary Fig. 1G) [21]. To get a more detail understanding severity with the reduced joint capsule swelling, attenuated
of KDM5B expression in RA patients, we collected the PBMCs from synovial hyperplasia and less inflammatory cell infiltration in
health control, stable RA (SRA) patients and active RA (ARA) KDM5B-KO mice (Fig. 1D). In addition, safranin O staining turned
patients, who were qualified for the diagnostic criteria set by out that cartilage and new bone destruction was prevented by

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Fig. 1 KDM5B deficiency ameliorates the immune injury and disease severity in mouse CIA model. A Clinical scores of littermate control
WT and KDM5B-deficient (KO) mice after the 2nd immunization of CIA model (n = 6 mice per group). B Diameter changes in ankle joints of WT
and KO mice on day 14 and day 28 after the 2nd immunization (n = 6 mice per group). C Morphology of the indicated paws from WT and KO
mice on day 28. H&E staining (D) or Safranin O staining (E) of the knee joints from WT and KO mice on day 28 after the 2nd immunization.
Scale bar = 200 μm. F ELISA analysis of the indicated cytokines in the sera of WT and KO mice after the 2nd immunization. G FACS of the
percentages of IFN-γ-producing CD4+ T cells (Th1 cells) and IL-17a-producing CD4+ T cells (Th17 cells) in spleens from WT and KO mice after
the 2nd immunization. **P < 0.01; ***P < 0.001; ****P < 0.0001. Two-way ANOVA (A), unpaired Student’s t test (B, F).

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KDM5B deficiency (Fig. 1E). The level of pro-inflammatory cytokine bone marrow and spleens between 8-week-old KO and WT mice
in sera including IL-1β, IL-6, IL-12p70 and TNF-α from KO mice was (Supplementary Fig. 4A, B). Meanwhile, KDM5B had no effect on
significantly lower than that from WT mice (Fig. 1F). These pro- the percentage of peritoneal macrophages or in vitro differen-
inflammatory mediators derived from innate immune cells such as tiated BMDMs induced by M-CSF (Supplementary Fig. 4C, D).
macrophages and dendritic cells (DCs) could result in the These data indicate that KDM5B does not influence the
proliferation and differentiation of CD4+ T cells, contributing to development and differentiation of macrophages. In response to
the disrupted immune homeostasis and joint inflammation in the LPS stimulation, KDM5B-deficient BMDMs exhibited lower mRNA
pathogenesis of RA [5, 30]. Therefore, we analyzed the pathogenic levels of cytokines including IL-1β, IL-6, IL-12β, TNF-α and IFN-β
and regulatory T cell population in CIA mice and found that (Fig. 3A). KDM5B deficiency also decreased mRNA levels of the
KDM5B-KO mice had the much lower percentages of pro- above cytokines in BMDMs stimulated with poly(I:C) and
inflammatory Th1 and Th17 cells (Fig. 1G), which might facilitate Pam3CSK4 (Fig. 3B). KDM5B-deficient BMDMs produced less IL-6,
bone erosion and destruction induced by collagen immunization. TNF-α and IFN-β in the cell culture medium after treatment with
However, there was no difference in the population of LPS, poly(I:C) or Pam3CSK4 (Fig. 3C). Meanwhile, the lower protein
CD25+FoxP3+ regulatory T (Treg) cells and CD4+CXCR5+PD-1+ level of pro-IL-1β was detected in KDM5B-KO BMDMs than that in
follicular helper T (Tfh) cells in the spleen between WT and KO WT BMDMs stimulated with LPS, poly(I:C) or Pam3CSK4 (Fig. 3D, E).
mice (Supplementary Fig. 3I, J). These data indicate that KDM5B We next investigated whether Kdm5b silencing affected cytokine
deficiency protects mice from the pathologic injury and joint production in the activated macrophages. The silencing of Kdm5b
inflammation with less pro-inflammatory cytokine production in medicated by the specific siRNA significantly inhibited LPS-
arthritis model. induced transcriptional expression of these pro-inflammatory
cytokine such as Il1b, Il6, Il12b and Tnf (Supplementary Fig. 4E–G).
KDM5B deficiency protects mice from endotoxin shock These data demonstrate that KDM5B deficiency or silencing
through inhibiting macrophage-mediated inflammatory impairs the production of pro-inflammatory cytokines and type I
responses interferon in macrophages upon different inflammatory stimuli.
Endotoxin shock models were performed subsequently to further
investigate the pathological function of KDM5B in inflammatory KDM5B is required for NF-κB activation and transcriptional
responses in vivo. Intraperitoneal (i.p.) injection of endotoxin such activity in macrophages
as LPS and poly(I:C) into mice results in the activation of innate PAMPs were recognized by TLR receptors in macrophages and
immune system and subsequent inflammatory tissue injury. launched the different signaling cascades including MAPK and NF-
KDM5B-KO mice produced much less pro-inflammatory cytokines κB pathway [33, 34]. Immunoblot assays showed that there was no
including IL-1β, IL-6, IL-12p70, TNF-α and IFN-β in the sera upon substantial difference in the phosphorylation levels of ERK, JNK
the non-lethal dose of LPS or poly(I:C) treatment (Fig. 2A). and p38 induced by LPS or Pam3CSK4 between KO and WT
Histological analysis by H&E staining showed that KDM5B macrophages, suggesting that KDM5B had no effect on MAPK
deficiency protected mice from the inflammatory tissue injury signaling activation (Fig. 4A, B). When it comes to NF-κB pathway,
with functional alveolar walls, less infiltration of inflammatory cells KDM5B did not affect the phosphorylation of IKKα/β. However, in
and reduced hemorrhage in lung tissues (Fig. 2B). KDM5B-KO mice KDM5B-deficient macrophages, the phosphorylation level of p65
showed pro-longed survival after i.p. injection of LPS with the was inhibited, which was might owing to the higher protein level
lethal dose, compared with WT littermates that all died within of IκBα (Fig. 4A, B). Previous studies confirm that IκBα is the major
3 days (Fig. 2C upper). Notably, KDM5B-KO mice were completely negative regulator of NF-κB activation through inhibiting p65
insensitive to the relative low dose of LPS (12 mg/kg body weight) phosphorylation and translocation into nucleus [35, 36]. Dual
while 50% survival rate on day 5 was observed for WT mice luciferase reporter gene assays of NF-κB further showed that
(Fig. 2C lower). Additionally, the LPS-induced peritonitis model KDM5B overexpression promoted both myeloid differentiation
was constructed for evaluating the role of KDM5B in macrophage primary response gene 88 (MyD88)-induced and TNF receptor-
activation and recruitment [31, 32]. FACS analysis showed that associated factor 6 (TRAF6)-induced NF-κB activation in a dose-
KDM5B-KO mice had the rather reduced percentage and absolute dependent manner, confirming that KDM5B facilitated NF-κB
number of F4/80+CD11b+ macrophage recruited to the peritoneal transcription activity (Fig. 4C). KDM5B was reported to remove
cavity (Fig. 2D, E). To address whether KDM5B regulated promoter-associated H3K4me3 modification with both Jmjc and
inflammatory responses mainly through myeloid cells such as Jmjn domains, the two classic demethylase activity domains as
macrophages, we performed bone marrow reconstruction experi- other family members [37, 38]. To determine whether KDM5B
ments (Fig. 2F; Supplementary Fig. 3K). CD45.1 mice reconstituted regulated NF-κB activation dependent on its demethylase activity,
with KDM5B-deficient bone marrow cells (KO → WT) produced less we constructed two demethylase activity mutants of KDM5B, one
pro-inflammatory cytokines including IL-1β, IL-6 and TNF-α in the lacking only Jmjc domain (called Jmjc-null) and the other lacking
endotoxin shock model, compared with those reconstituted with both Jmjc and Jmjn domains (called N terminal-null) (Fig. 4D).
WT bone marrow cells (WT → WT) (Fig. 2G). In addition, CD45.1 Interestingly, only full-length of KDM5B could promote MyD88-
mice reconstituted with KDM5B-deficient bone marrow cells had induced and TRAF6-induced NF-κB activation in HEK293T cells but
less tissue injury and inflammatory cell infiltration in their lungs in two demethylase activity mutants of KDM5B did not (Fig. 4E).
contrast to control mice (Fig. 2H). The above in vivo data These data indicate that KDM5B is essential for TLR-triggered NF-
demonstrate that KDM5B is essential for the pro-inflammatory κB activation dependent on its demethylase activity.
cytokine production in both acute and chronic inflammation
model, and KDM5B deficiency protects mice from inflammation- KDM5B inhibitor GSK467 restrains NF-κB signaling activation
associated tissue injury in the myeloid cell-intrinsic manner. and pro-inflammatory cytokine production in vitro and in vivo
Recent study identified a KDM5B enzyme activity inhibitor,
Impaired TLR-triggered pro-inflammatory cytokine GSK467, with a selective inhibition function on KDM5B but not
production in KDM5B-deficient macrophages KDM4C, KDM6 or other Jumonji family members [39]. Thus, we
Considering that macrophages are the major mediator of TLR- observed the effects of GSK467 on macrophage activation and
triggered pro-inflammatory cytokine production in vivo, we next inflammatory responses. GSK467 treatment (5 μM) resulted in the
explored whether KDM5B influenced the development or elevated H3K4me2 and H3K4me3 modification in macrophages,
differentiation of macrophages. FCAS analysis showed that the indicating GSK467 effectively inhibited KDM5B demethylase
proportion and number of macrophages were comparable in both activity (Fig. 5A). GSK467 significantly suppressed the

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Fig. 2 KDM5B deficiency inhibits LPS-induced systemic inflammation in vivo. A ELISA analysis of the indicated cytokines in the sera of WT
and KO mice 8 h after the intraperitoneal injection of LPS (12 mg/kg body weight) or Poly(I:C) (20 mg/kg body weight) (n = 6 mice per group).
B H&E staining of lung tissues from WT and KO mice treated as in A. Scale bar = 200 μm. C Survival rates of WT and KO mice after the
intraperitoneal injection of LPS (upper, 20 mg/kg body weight; lower, 12 mg/kg body weight) (n = 10-12 mice per group). D FACS of the
percentages of F4/80+CD11b+ macrophages in peritoneal lavage fluids from WT and KO mice 8 h after the intraperitoneal injection of LPS
(100 ng/per mouse) (n = 5 mice per group). E The percentage (left) and absolute number (right) of macrophages as in D. F The model of
reconstitution of bone marrows from KDM5B-KO or WT mice (CD45.2) into CD45.1 WT mice. G ELISA analysis of the indicated cytokines in the
sera of CD45.1 mice reconstituted with bone marrow cells from KDM5B-KO or WT mice 8 h after the intraperitoneal injection of LPS (12 mg/kg
body weight) (n = 5 mice per group). H H&E staining of lung tissues from WT and KO mice as in G. Scale bar = 100 μm. *P < 0.05; **P < 0.01;
***P < 0.001; ****P < 0.0001. One-way ANOVA (A), Log-rank test (C), unpaired Student’s t test (E, G).

transcriptional induction of Il1b, Il6, Il12b and Tnf in macrophages effects of GSK467 in endotoxin shock model. Firstly, we treated
upon LPS stimulation (Fig. 5B). In addition, the activation of LPS- mice with different doses of GSK467 to determine an appropriate
induced NF-κB signaling cascade was also impaired in GSK467- in vivo dosage without systemic toxicity. TUNEL staining of lung
treated macrophages with more IκBα protein expression and less tissues showed that the low dose of GSK467 did not induce cell
phosphorylation level of p65 (Fig. 5C). These in vitro data apoptosis in lung tissues except for mice from 20 mg/kg GSK467
confirmed the inhibitory function of GSK467 in NF-κB-triggered group with a little proportion of TUNEL-positive cells (Supple-
pro-inflammatory cytokine production. We next observed in vivo mentary Fig. 5A). Mice treated with 10 mg/kg or 20 mg/kg GSK467

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Fig. 3 KDM5B is required for TLR-triggered pro-inflammatory cytokine production. A Q-PCR analysis of the mRNA levels of pro-
inflammatory cytokines in WT and KDM5B-deficient (KO) BMDMs stimulated with LPS (100 ng/ml) for the indicated times. B Q-PCR analysis of
the mRNA levels of pro-inflammatory cytokines in WT and KO BMDMs stimulated with LPS, poly(I:C) (pI:C) or Pam3CSK4 (Pam3) (100 ng/ml,
20 μg/ml and 1 μg/ml, respectively) for 6 h or left untreated (UT). C ELISA analysis of pro-inflammatory cytokines in culture supernatants of WT
and KO BMDMs stimulated with LPS (100 ng/ml) or poly(I:C) (20 μg/ml) for 6 h. Immunoblot analysis of the indicated proteins in WT and KO
BMDMs stimulated with LPS for the indicated times (D) or stimulated with the indicated ligands for 6 h (E). **P < 0.01; ***P < 0.001; ****P <
0.0001. One-way ANOVA (A–C).

produced much less IL-6 in sera in LPS-induced endotoxin shock further explore whether KDM5B influenced the transcription level
model (Supplementary Fig. 5B). These data indicated that 10 mg/ of Nfkbia, the gene encoding IκBα. KDM5B deficiency markedly
kg GSK467 contributed to the immunosuppression condition increased the mRNA level of Nfkbia in BMDMs stimulated with LPS,
without toxicity. ELISA assays further showed that mice with poly(I:C) or Pam3CSK4 (Fig. 6A, B). To confirm the selective
GSK467 treatment (10 mg/kg) produced less pro-inflammatory regulatory function of KDM5B in Nfkbia mRNA expression, we also
cytokines in their sera 8 h after LPS challenge (Fig. 5D). Lung detected the expression of Nfkbib, Ikbka and Ikbkb, which
tissues from GSK467-treated mice exhibited more milder inflam- respectively encoded IκBβ, IKKα and IKKβ, and found that KDM5B
matory injury with relatively intact alveolar structure and less deficiency had no influence on these gene expression expect that
infiltration of inflammatory cells (Fig. 5E). These above findings of Nfkbia (Fig. 6C). Next, we analyzed the NFKBIA/Nfkbia expression
further confirm that KDM5B promotes inflammatory responses in autoimmune diseases and found in synovial macrophages from
in vivo and in vitro in demethylase activity-dependent manner. RA patients, the level of NFKBIA was much higher than health
control (Supplementary Fig. 6A). A more interesting thing was that
KDM5B represses the transcriptional induction of Nfkbia gene there existed a strong negative correlation between the expres-
Since our data found that IκBα protein level in KDM5B-KO sion levels of KDM5B and NFKBIA in these samples of synovial
macrophages was much higher than that in WT macrophages, we macrophages (Supplementary Fig. 6B). In addition, NFKBIA was

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Fig. 4 KDM5B selectively promotes the activation of TLR-triggered NF-κB signaling. Immunoblot analysis of the indicated proteins in WT
and KO BMDMs stimulated with LPS (100 ng/ml) (A) or Pam3CSK4 (1 μg/ml) (B) for the indicated times. C Dual luciferase reporter gene analysis
of NF-κB-luciferase activity in HEK293T cells 24 h after transfection with NF-κB-luciferase reporter plasmid (NF-κB-luci), the indicated amounts
of KDM5B-expressing plasmids and MyD88 (upper) or TRAF6 (lower). D Schematic diagram of the WT or mutants of KDM5B-expressing
plasmids. E Dual luciferase receptor gene analysis of NF-κB-luciferase activity in HEK293T cells 24 h after transfection with NF-κB-luci, the
indicated KDM5B mutants and MyD88 (left) or TRAF6 (right). **P < 0.01; ****P < 0.0001. One-way ANOVA (C, E).

also highly expressed in the PBMCs from MS patients, which was driven transcriptional activity. KDM5B overexpression decreased
also negatively correlated with the expression level of KDM5B the reporter gene activity of the Nfkbia promoter but not Nfkbib,
(Supplementary Fig. 6C, D). We also compared the Nfkbia Ikbka or Ikbkb promoter in RAW264.7 cells stimulated with LPS
expression in colon tissues during different time period in a (Fig. 6D, E). To exclude the post-transcriptional effect of KDM5B on
mouse experimental colitis model induced by DSS treatment, it Nfkbia expression, we treated LPS-activated KO and WT macro-
turned out that Kdm5b expression was suppressed while Nfkbia phages with actinomycin D (ActD) for transcriptional inhibition,
level was elevated after DSS treatment, and there was a strong and found no difference in mRNA destabilization of Nfkbia
negative relationship between expression levels of these two between KO and WT macrophages (Fig. 6F). To validate the effect
genes (Supplementary Fig. 6E, F). Next, we analyzed the of IκBα overexpression on human macrophage activation, human
expression levels of KDM5B and NFKBIA in RA patients prior to monocyte-derived macrophages (hMDMs) were cultured and
or after anti-inflammatory or anti-rheumatic therapeutics. Current transfected with Myc-tagged NFKBIA (Fig. 6G). IκBα overexpres-
approved therapeutics for RA includes methotrexate (MTX), sion was sufficient to inhibit LPS-triggered the activation of NF-κB
tocilizumab (TCZ) and rituximab (RTX), which target proliferating signaling in macrophages, including the markedly impaired
adaptive immune cells, interleukin-6 receptor (IL-6R) and CD20+ B phosphorylation of p65 and the suppressed transcriptional
cells respectively. Although these current approved therapeutics induction of IL1B and IL6 (Fig. 6H, I). These data indicate that
did not significantly change the expression of KDM5B and NFKBIA, KDM5B plays an important role in the transcriptional repression of
the correlation analysis showed the significant negative relation- NFKBIA/Nfkbia gene in both human and mouse inflammatory
ship between KDM5B and NFKBIA expression levels (Supplemen- macrophages.
tary Fig. 6G–I). These results suggest that in autoimmune disease
settings, KDM5B may have an inhibitory role in NFKBIA mRNA KDM5B selectively binds to the Nfkbia promoter for erasing
expression. H3K4me3 modification and regulating chromatin accessibility
Dual luciferase reporter gene assay was next performed to Given that KDM5B was reported as the transcriptional suppressor
confirm whether KDM5B could directly repress Nfkbia promoter- in various settings [40], we hypothesized that KDM5B might

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Fig. 5 KDM5B-specific inhibitor GSK467 suppresses NF-κB-mediated inflammatory responses. A Immunoblot analysis of the indicated
proteins in BMDMs treated with GSK467 (5 μM) or DMSO overnight. Q-PCR analysis of the mRNA levels of pro-inflammatory cytokines (B) and
immunoblot analysis of the indicated proteins (C) in BMDMs treated as in A followed by stimulation with LPS (100 ng/ml) for the indicated
times. D ELISA analysis of pro-inflammatory cytokines in the sera of mice pre-treated with GSK467 (10 mg/kg per body weight) or DMSO
followed by intraperitoneal injection with LPS (12 mg/kg per body weight) for 8 h (n = 5 per group). E H&E staining of lung tissues from mice
treated as in D. **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way ANOVA (B), unpaired Student’s t test (D).

bound to the functional gene regions that were important in stimulation, indicating that KDM5B deficiency affected the
Nfkbia transcription. In this way, CUT&Tag followed by sequencing chromatin remodeling of the regulatory gene region around the
was done using KDM5B-specific antibody in BMDMs stimulated transcription start site (TSS) of Nfkbia (Fig. 7F). In addition, the
with LPS or not. Motif analysis by HOMER found that although two enrichment of RNA pol II in the promoter of Nfkbia was also
groups exhibited similar binding motif of KDM5B, there existed a elevated in KDM5B-KO BMDMs stimulated with LPS (Fig. 7G). The
quite different binding preference in LPS-activated macrophages treatment of KDM5B inhibitor GSK467 also led to the increased
(Fig. 7A). Next, we compared the target genes of KDM5B in H3K4me3 level, chromatin accessibility and RNA Pol II recruitment
macrophages stimulated with LPS or left untreated and found 252 in the Nfkbia promoter of BMDMs (Supplementary Fig. 8A–C). To
LPS-induced binding genes (UP genes) and 88 LPS-repressed sum up, KDM5B is selectively recruited to the promoter of Nfkbia
binding genes (DOWN genes) (Fig. 7B). With the gene functional gene, removing H3K4me3 and inhibiting the chromatin accessi-
annotation, we identified 13 immune-associated target genes (UP: bility for RNA Pol II-centered transcription machinery working
11 and DOWN: 2) (Fig. 7B; Supplementary Fig. 7A). Nfkbia gene (Supplementary Fig. 9).
was identified as the direct target of primary inflammatory
signaling factor regulated by KDM5B in BMDMs stimulated with
LPS, which was also confirmed by integrative genomics viewer DISCUSSION
(IGV) analysis (Fig. 7C). There existed two narrow peaks bound by In this work, we revealed the essential role of KDM5B in NF-κB
KDM5B within the Nfkbia promoter, and motif enrichment analysis activation and macrophage-mediated inflammatory responses
showed that transcription factor Sp1, NFAT and Nkx2.1 might be in vivo and in vitro. Recently, a work by Liu and his colleges
the downstream transcription factors influenced by KDM5B in LPS- found that in drosophila, KDM5 family transcriptionally regulated
stimulated macrophages (Supplementary Fig. 7B, C). A20, encoded component genes of the immune deficiency (IMD) signaling
by Tnfaip3, was also the widely-accepted negative regulator of NF- pathway and subsequent host-commensal bacteria homeostasis
κB activation [41]. As the negative control, there was no specific in a demethylase-dependent manner [42]. KDM5 inhibition also
binding peak in Tnfaip3 gene locus, or in pro-inflammatory restrained the inhibitory effect of PPARγ agonist on IL-17A
cytokine Il6 gene locus (Supplementary Fig. 7D, E). Then we expression in Th17 responses [43]. Although KDM5 family
performed ChIP assays to verify the direct enrichment of KDM5B removed the di/trimethylation of H3K4 in many cells, little work
at the promoter of Nfkbia gene, which was increasingly induced focuses their distinctive functions of different family members in
by LPS treatment during the very early period in macrophages immune regulation. Zhao et al. reported that KDM5A was required
(Fig. 7D). However, there was no difference of KDM5B enrichment for NK cell activation by inhibiting suppressor of cytokine signaling
at the promoter of Tnfaip3 or Il6 between macrophages stimulated 1 (SOCS1) expression. Mechanistically, KDM5A bound to the SOCS1
with LPS or left untreated (Fig. 7D). Considering the classical promoter in resting NK cells, leading to the elevated H3K4me3
function of KDM5B in erasing the histone methylation modifica- modification and improved chromatin configuration for SOCS1
tion of H3K4, we detected the H3K4me3 level at the promoter of transcription [44]. In addition, several studies identified the
Nfkbia gene, and observed the significant increase of H3K4me3 regulatory role of KDM5 in antiviral immunity. For example,
level in KDM5B-KO BMDMs (Fig. 7E). Notably, DNase I sensitivity H3K4 demethylase KDM5B and KDM5C were responsible to
assay showed that the chromatin accessibility in the Nfkbia repress STING transcription and KDM5 blockade triggered robust
promoter was increased in KDM5B-KO BMDMs after LPS interferon responses in breast cancer cells [45]. Respiratory

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Fig. 6 KDM5B selectively inhibits the transcription of Nfkbia gene. Q-PCR analysis of the Nfkbia mRNA expression in WT and KO BMDMs
stimulated with LPS (100 ng/ml) for the indicated times (A) or stimulated with the indicated ligands for 2 h (B). C Q-PCR analysis of the mRNA
levels of the indicated genes in WT and KO BMDMs stimulated with LPS (100 ng/ml) for 1 h. D Dual luciferase reporter gene analysis of Nfkbia
(−2000 to +121 region of the Nfkbia promoter) luciferase reporter activity in RAW264.7 cells transfected with the indicated amounts of mock
vector and KDM5B-expressing plasmid followed by stimulation with LPS for 2 h or left untreated. E Dual luciferase reporter gene analysis of
Nfkbia (IκBα), Nfkbib (IκBβ), Ikbka (IKKα) or Ikbkb (IKKβ) gene promoter-driven luciferase reporter activity in RAW264.7 cells transfected with
mock vector or KDM5B-expressing plasmid followed by stimulation with LPS for 2 h. F Q-PCR analysis of Nfkbia mRNA expression in WT and
KO BMDMs stimulated with LPS followed by treatment with ActD for the indicated times. G Immunoblot analysis of the indicated proteins in
hMDMs transfected with Myc-tagged NFKBIA plasmid. H Immunoblot analysis of the indicated proteins in hMDMs treated as in G followed by
stimulation with LPS (100 ng/ml) for the indicated times. I Q-PCR analysis of the mRNA levels of IL1B and IL6 genes in hMDMs treated as in
G followed by stimulation with LPS (100 ng/ml) for 4 h. *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way ANOVA (A, B, D, F, I),
unpaired Student’s t test (C, E).

syncytial virus (RSV) infection induced the up-regulated expression feedback for turning-off the NF-κB response and maintains the
of KDM5B in murine DCs, and KDM5B deficiency in DCs resulted in moderate activation of NF-κB pathway by the coordinated
higher production of IFN-γ and reduced IL-4 and IL-5, indicating degradation and synthesis of IκBα protein [50]. IκBα is degraded
KDM5B could regulate Th2 cytokines when RSV infection [46]. following exposure to inflammatory cytokines or microbial
However, our work found the down-regulation of KDM5B products and results in a decreased inhibitory effect on NF-κB,
expression in the setting of multiple autoimmune diseases mainly which is regulated by a variety of post-translational modifications
owing to the transcription inhibition, which might be the including the phosphorylation at Ser32, Ser36 and Tyr42,
protective manner of host immune to avoid excessive inflamma- ubiquitination at Lys21 and SUMOylation at Lys21 and Lys38
tory responses and tissues injury. Nevertheless, the difference [51–53]. Recent works also demonstrated that the expression of
between KDM5B and other KDM5 family members were largely IκBα was subjected to transcriptional regulation. The ChIP-seq of
not clear. high-mobility group box1 (HMGB1) in human retinal endothelial
Immune system and immune responses are generally in a cells identified NFKBIA as the binding site of HMGB1, which
precious equilibrium, involving kinds of regulatory mechanisms inhibited cell proliferation and promoted apoptosis [54]. Our
controlling the activation and inhibition of immune cell function, previous work also showed that ZBTB20 inhibited IκBα gene
and participating in the initiation and resolution of inflammation. transcription and promoted macrophage-mediated inflammatory
For example, the signal transduction of NF-κB is strictly regulated responses [55]. In addition, Wang et al. revealed H3K4 methyl-
on different levels. A20 removes K63-linked poly-ubiquitin from transferase Mixed lineage leukemia 1 (MLL1) was required for the
NEMO after TNF stimulation, whose activity is promoted by IKK2- increased H3K4 trimethylation level at the Nfkbia promoter
mediated phosphorylation of A20 at Ser381 [47, 48]. Kinase induced by TNF-α [56]. So, whether KDM5B functioned as the
calcium/calmodulin-dependent protein kinase II (CaMKII) pro- co-repressor of these transcriptional factors including zinc finger
motes NF-κB activation through directly binding and activating and BTB domain-containing 20 (ZBTB20), or influenced the
TAK1 in macrophages [49]. IκBα is the major player of negative recruitment of activating transcriptional regulators such as MLL1

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Fig. 7 KDM5B specifically binds to the Nfkbia promoter for regulating H3K4me3 modification and chromatin accessibility. A The motif
analysis of KDM5B-bound peaks in BMDMs stimulated with LPS for 1 h or left untreated. B The strategy for identifying the target immune
genes of KDM5B in macrophages. C IGV analysis of KDM5B (our work), H3K4me1 (GSE38377), H3K4me3 (GSE38377), ATAC (GSE108585) and
RNA Pol II (GSE17631) signals in the gene locus of Nfkbia. D ChIP-qPCR analysis of KDM5B enrichment at the promoter of the indicated genes
in BMDMs stimulated with LPS (100 ng/ml) for the indicated times. E ChIP-qPCR analysis of H3K4me3 enrichment at the promoter of Nfkbia
gene in WT and KO BMDMs stimulated with LPS for 1 h. F DNase I sensitivity analysis of the Nfkbia promoter in the nuclei of LPS-stimulated WT
and KO BMDMs. G ChIP-qPCR analysis of RNA Pol II enrichment at the promoter of Nfkbia gene in WT and KO BMDMs stimulated with LPS for
1 h or left untreated. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA (D–G).

Cell Death & Differentiation (2023) 30:1279 – 1292

 Y. Zhang et al.
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remained to be studied. Furthermore, beyond the NF-κB dogma, MATERIALS AND METHODS
IκBα has been found to associate with unclear proteins such as Reagents and antibodies
HDACs or nuclear co-repressors, and in primary keratinocytes it Complete Freund’s adjuvant (CFA) (F5881), Lipopolysaccharide (LPS)
bound the chromatin at specific genes and contributed to (Escherichia coli serotype 0111:B4), and poly(I:C) were purchased from
tumorigenesis [57, 58]. Here we found the strong negative Sigma-Aldrich. Pam3CSK4 was from Invivogen. Chicken Type II Collagen
(20012) was purchased from Chondrex. ChIP-grade protein G magnetic
correlation between KDM5B and NFKBIA expression in activated
beads (9006) and cell lysis buffer (9803) were from Cell Signaling Technology.
macrophages in the setting of autoimmune diseases. In this way, Mouse IL-1β, IL-6, IL-12p70 and TNF-α ELISA kits were from R&D. Mouse IFN-β
whether KDM5B-IκBα axis takes effects in other types of cells that ELISA kits were from PBL. Antibodies against IL-1β (12242), Tri-Methyl-Histone
lead to organ dysfunction or tumorigenesis is still largely H3 (Lys4) (C42D8), Di-Methyl-Histone H3 (Lys4) (C64G9), Histone H3 (1B1B2),
unknown. phospho-ERK (9106), phospho-JNK (4668), phospho-p38 (9211), p65 (8242),
The regulatory role of histone methylation has been implicated phospho-p65 (3033), IκBα (112B2) (9247), IKKα (D3W6N) (61294), phosphor-
in the development and function of immune cells, whose IKKα/β (2697), and β-Actin (3700) were from Cell Signaling Technology.
mutation or dysfunction might lead to immune deficiency or Antibody against KDM5B (A301-813) was from Bethyl Laboratories and
autoimmune diseases. For example, PBMCs from psoriasis patients Antibody against RNA Pol II (17-620) was from Millipore. Antibodies against
exhibited reduced levels of histone H3/H4 acetylation and CD45-BV605 (2D1), CD3-PerCP/Cy5.5 (17A2), CD19-APC (6D5), CD4-BV421
(GK1.5), CD8-BV510 (53-6.7), CD25-APC (PC61), PD-1-APC (29F.1A12), CXCR5-
increased H3K4 methylation, and another work found reduced PE (J252D4), IFN-γ-APC (XMG1.2), IL-17a-PE (TC11-18H10.1), CD11b-BV510
DNA methylation of gene including programmed cell death 1 (M1/70), and F4/80-FITC (QA17A29) were purchased from BioLegend.
ligand 2 (PDCD1LG2) in skin cells was related to immune activation Antibody against FoxP3-PE (FJK-16s) was purchased from eBioscience.
and autoimmunity [59, 60]. We previously identified the histone
demethylase KDM2B was indispensable for Il6 transcriptional
Mice
induction through Brg1-mediated chromatin remodeling, not Kdm5b-KO mice in C57BL/6 background were generated by CRISPR/Cas9
dependent on its H3K4me3 demethylase activity [61]. KDM5B technology via a 428-base pair (bp) fragment deletion and frameshift
deficiency was linked to the chromatin accessibility of Nfkbia gene mutation from the Kdm5b gene, resulting in the frameshift mutation and
regulatory regions, however, the epigenetic mechanisms under- gene knockout. B6.SJL-Ptprca Pepcb (B6 CD45.1) (Strain# 002014) mice were
lying the dynamic change of chromatin structure were not purchased from the Jackson Laboratory. Wild type C57BL/6 mice
elaborated. In the inducible transcription of Nfkbia gene, certain (6–8 weeks old) were obtained from Joint Ventures Sipper BK Experimental
methyltransferases or epigenetic readers could participate in Animal Company (Shanghai, China). Mice were bred in specific pathogen-
chromatin remodeling and promote transcriptional machinery free conditions. All animal experiments were undertaken in accordance
function, while KDM5B might functioned as the negative feedback with the National Institutes of Health’s Guide for the Care and Use of
Laboratory Animals.
or a transcription brake for this process.
Finally, emerging therapies and perspective drugs were
implicated in the treatment of autoimmune diseases like RA, Clinical specimens
including Tofacitinib, Baricitinib and other selective JAK inhibitors The peripheral blood mononuclear cells (PBMCs) of 38 subjects (28 RA cases
[62]. Meanwhile, monoclonal antibodies directed at pro- and 10 health control) were recruited and collected from Changhai Hospital
(Shanghai, China). Briefly, 10 ml anticoagulated whole blood was obtained
inflammatory cytokines such as Sirukumab and Tocilizumab were and then PBMCs were collected by Ficoll gradient centrifugation. All the RA
clinically proved to be effective. Although small-molecule patients met the 2010 criteria of the American College of Rheumatology and
inhibitors of epigenetic regulators were highlighted in enhancing the European Union League Against Rheumatism. Active RA was defined as
anti-tumor immune responses, little had been applied to the having a 28-joint disease activity score (DAS28) of 2.6 or higher. For health
treatment of autoimmune diseases [63]. Trichonstatin A (TSA) and control, subjects with severe cardiovascular diseases, liver and kidney
suberoylanilide hydroxamic acid (SAHA) are two broad spectrum dysfunction, malignant tumor and other autoimmune or musculoskeletal
inhibitors of classical HDACs, directly binding to their active site disorders were excluded. All samples were collected with written informed
for pharmacological inhibition. TSA-treated murine macrophages consent from the patients and health control.
led to downregulation of a set of LPS-induced genes participating
in multiple functions including inflammation, chemotaxis, antigen Collagen-induced arthritis model
presentation and tissue repair [64]. Other researches also Eight-week-old mice were used to establish CIA models as previously
demonstrated that inhibition of HDACs had opposing effects. described [69]. Bovine type II collagen emulsified in complete Freund’s
For example, Bode et al. and Halili et al. found that TSA and SAHA adjuvant (CFA) was injected into mouse tail base on day 0 for the first
strongly inhibited the transcription of IL-12 p40 in DCs and immunization. Then a booster immunization was administered on day 21. We
evaluated the severity of redness and swelling in the wrist and paw in a scale
macrophages respectively upon stimulation of TLRs [65, 66], while of 0–4. The maximum score is 16. All mice were scored blind to genotype.
Zhang et al. showed that despite of the general inhibitory role of Knee joints were fixed in paraformaldehyde for further H&E and Safranin O
TSA in pro-inflammatory gene transcription shortly after LPS staining. Sera were collected for cytokine measurement by enzyme-linked
stimulation, the mRNA of Il6 was enhanced during the late phase immunosorbnent assay (ELISA). Spleen was harvested for analyzing immune
in the presence of TSA [67]. A more recent work found that the cell clustering via fluorescence-activated cell sorter (FACS).
dual-functional engagement of HDAC3 activity governed inflam-
matory responses in a gene-specific manner by SAHA administra- LPS-induced systemic inflammation model
tion, which provided the potential explanation for these For non-lethal endotoxin shock model, 8-week-old mice were intraper-
contradictory functions. Given that the complexity of pharmaco- itoneally injected with LPS (12 mg/kg body weight) [33]. Sera were
logical effects, these epigenetic targets and inhibitors are being collected 8 h after injection for cytokine measurement via ELISA. Lung
evaluated for therapy in inflammatory and autoimmune diseases tissues were examined by H&E staining. For lethal endotoxin shock model,
[68]. In this work, we identified the therapeutic effects of KDM5B- 8-week-old mice were intraperitoneally injected with LPS (20 mg/kg body
specific inhibitor, GSK467 in LPS-induced systemic inflammation weight) and observed for survival rate, which was similar to previous study
[55]. For peritonitis model, 8-week-old mice were intraperitoneally injected
model with attenuation of pathogenic pro-inflammatory cytokine
with LPS (100 ng/mouse) [31]. The peritoneal exudate cells were collected
production in vivo. Nevertheless, the therapeutic results and the 8 h after injection and analyzed by FACS.
potential side-effects of GSK467 in autoimmune diseases such as
RA, are expected to be gradually revealed. To sum up, we identify
the protective mechanism of host immune system by inhibiting Cell culture
KDM5B expression and activity to avoid excessive inflammatory Murine bone marrow-derived macrophages (BMDMs) were prepared as
injury in autoimmune diseases. previously described [61]. Human monocyte-derived macrophages (hMDMs)

Cell Death & Differentiation (2023) 30:1279 – 1292

 Y. Zhang et al.
1290
were collected from PBMCs of healthy volunteers and cultured by elutriation Assay Kit (Beyotime). The antibodies against KDM5B (2 μL/sample) and
and GM-CSF-induced differentiation (50 ng/ml; Peprotech) as described RNA Pol II (1 μL/sample) were from Bethyl Laboratories and Millipore
before [5]. HEK293T cell lines and RAW264.7 cell lines were obtained from respectively. Purified DNA was detected by Q-PCR and then normalized to
the American Type Culture Collection (ATCC), and cultured in DMEM medium the input DNA for each sample. The primers used for amplification and
or RPMI 1640 medium with 10% FBS (Gibco) as described [70]. quantification in ChIP-qPCR were listed as follows: Nfkbia (Forward: 5′-
AAAGTTCCCTGTGCATGACC-3′; Reverse: 5′-CTGGCAGGGGATTTCTCAG-3′),
Tnfaip3 (Forward: 5′-TTGAATGGTGGTGGTCTTCA-3′; Reverse: 5′-TGAGGAGG
Flow cytometry and intracellular cytokine staining AGGGGAATAACC-3′) and Il6 (Forward: 5’-CCTGCGTTTAAATAACATCAGC
Single cell suspensions were prepared from the indicated tissues or TTTG CTT-3’; Reverse: 5’-GCACAATGTGACGTCGTTTAGCATCGAA-3’).
peritoneal cavity and subjected to FACS using SONY ID7000. FACS data
were analyzed by FlowJo software. Cells were stained with fluorochrome-
labeled antibodies after incubation with anti-CD16/CD32 to block non- Cleavage under targets & tagmentation (CUT&Tag)
specific antibody binding. For intracellular cytokine detection of IFN-γ and CUT&Tag was performed using Hyperactive Universal CUT&Tag Assay Kit
IL-17a, cells were stimulated with Cell Activation Cocktail (with Brefeldin A) for Illumina (Vazyme, Shanghai) according to the product instructions.
(BioLegend) for 4–6 h. After incubation, cells were stained with CD16/32 Briefly, BMDMs were stimulated with LPS and then harvested after PBS
and other antibodies against surface molecules. After surface staining, cells wash. Cells (1 × 105) were bound with concanavalin A-coated magnetic
were fixed and permeabilized with the fixation/permeabilization kit (BD beads and then incubated with antibody against KDM5B (Bethyl
Biosciences) and stained for intracellular cytokines. Intracellular Laboratories) at 4 °C overnight. On next day, samples were incubated
FoxP3 staining was performed using Foxp3/Transcription Factor Staining with second antibody and then with Hyperactive pA/G-Transposon for 1 h.
Buffer Set (Invitrogen) without cell stimulation. DNA was treated with TTBL at 37 °C for 1 h and extracted, following by
purification for library amplification. The Next Generation Sequencing
(second-generation high-throughput sequencing) technique was applied
RNA isolation and quantitative-PCR (Q-PCR) assays to sequence DNA. Genome mapping was performed using Bowtie (version:
Total RNA was extracted from cells using TRIzol reagent (ThermoFisher 0.12.8) and clean reads were compared to reference genome mm10. MACS
Scientific) according the instruction. Q-PCR assays were performed using a (version 2.2.7.1) was applied to call peak. The bam file and R package
LightCycler 480 (Roche) and SYBR RT-PCR kit (Takara). Data were
DiffBind were used to analyze the differences between the peaks of the
normalized to Actb expression.
specified two samples. The default parameter was set as fold change ≥ 2 or
≤ 1/2 and P value < 0.05, and the differences between the obtained peaks
Immunoblot analysis were annotated. The annotation program was the same as the above peak
Immunoblot analysis was performed as described previously [71, 72]. annotation program, including different peak location information,
Briefly, total proteins from cells were extracted with cell lysis buffer (Cell annotation information, etc. The motif analysis was conducted using
Signaling Technology) with addition of a protease inhibitor cocktail and “HOMER” tool. And the result of HOMER motif is the motif information
PMSF (Merck). The protein concentration was measured with a BCA protein predicted based on the existing database within the peak. The
assay kit (ThermoFisher Scientific) and subject to immunoblot assays using findmotifsGenome.pl tool for HOMER was used to analyze the specific
BIO-RAD electrophoresis system. location of Motif at peak. The sequencing data are available in the GEO
database under accession number GSE212808.
Plasmid construction
Vectors encoding mouse Kdm5b (NM_152895.2) and human NFKBIA DNase I sensitivity assays
(NM_020529.3) were constructed by PCR-based amplification from cDNA DNase I sensitivity assays were performed as described before [61]. Briefly,
of mouse macrophages or hDMDs and then cloned into the pcDNA3.1-V5- nuclei of BMDMs stimulated with LPS for 1 h were extracted by nuclear
His or pcDNA3.1-Myc eukaryotic expression vector (Invitrogen) as extraction Kit (Biotechnology, Shanghai) and then treated with DNase I (0.1
described [61]. Mutants of KDM5B were constructed by PCR-based U/μL, Promega) at 37 °C for 30 min followed by termination with EDTA
amplification from the wild type plasmid and the primers used were (50 mM). Genomic DNA was extracted and purified using QIAamp DNA
listed as follows: KDM5B-WT (Forward: 5′-GGGGTACCGTGATGGAGC micro kit (QIAGEN) and subjected to Q-PCR assays.
CGGCCA CCACGCTGCCCCCAG-3′; Reverse: 5′-GCYCYAGACTCTTTCGGCTT
GGTGCGT CCTTCCCAGTAC-3′), Jmjc-null (Forward: 5′-CCTAAGTGTTTGGCTC Statistical analysis
AAGAATG TAATGTTGATTGGCTGCC-3′; Reverse: 5’-GGCAGCCAATCAACATT
The statistical significance of differences between the two groups was
ACATTCTT GAGCCAAACACTTAGG-3′), N terminal-null (Forward: 5′-GGG
determined by unpaired Student’s t-test. For comparison of more than two
GTACCGTGATGC TGGATGATCTCTATCCAATGATG-3′; Reverse: 5′- GCYCYAG groups, one- or two-way ANOVA with the least significant difference t-test
ACTCTTTCGGCTT GGTGCGTCCTTCCCAGTAC-3′). was performed. The statistical significance of survival curves was estimated
according to the method of Kaplan and Meier, and curves were compared
RNA interference and plasmid transfection with the generalized Wilcoxon test. P values of less than 0.05 were considered
SiRNA targeting Kdm5b (siGENOME SMART pool siRNA M-058167-01) and statistically significant. The statistical tests were justified as appropriate
control siRNA (siGENOME non-targeting siRNA Pool #1) were from according to assessment of normality and variance of the distribution of the
Dharmacon. The siRNA duplexes were transfected into macrophages data. No randomization or exclusion of data points was used.
using Lipofectamine RNAiMax (ThermoFisher Scientific) according to the
manufacturer’s protocol. The indicated plasmids were transfected into Reporting summary
HEK293T cells or RAW264.7 cells using jetPEI (Polyplus-transfection) or Further information on research design is available in the Nature Research
FuGene HD (Promega) respectively. Reporting Summary linked to this article.

Dual luciferase reporter gene assay

HEK293T cells or RAW264.7 cells were co-transfected with a mixture of the DATA AVAILABILITY
indicated luciferase reporter plasmids, pRL-TK-Renukka-luciferase plasmid Data supporting the present study are available from the corresponding author upon
and KDM5B wild type or mutant plasmids. After 24–36 h, luciferase reasonable request. Original images of unprocessed immunoblot and tissue staining
activities were measured using the Dual Luciferase Reporter Assay System are available at Supplementary information.
(Promega) according to the manufacturer’s instructions. Data are normal-
ized for transfection efficiency by dividing Firefly luciferase activity with the
activity of Renilla luciferase. REFERENCES
1. Na YR, Stakenborg M, Seok SH, Matteoli G. Macrophages in intestinal inflam-
Chromatin immunoprecipitation (ChIP) mation and resolution: a potential therapeutic target in IBD. Nat Rev Gastro-
BMDMs were firstly stimulated with LPS for the indicated times and cross- enterol Hepatol. 2019;16:531–43.
linked with 1% (v/v) methanol-free formaldehyde for 10 min, terminated 2. Watanabe S, Alexander M, Misharin AV, Budinger GRS. The role of macrophages
with glycine solution, and then subjected to ChIP assays using the ChIP in the resolution of inflammation. J Clin Investig. 2019;129:2619–28.

Cell Death & Differentiation (2023) 30:1279 – 1292

 Y. Zhang et al.
1291
3. Brubaker SW, Bonham KS, Zanoni I, Kagan JC. Innate immune pattern recogni- 29. Brand DD, Latham KA, Rosloniec EF. Collagen-induced arthritis. Nat Protoc.
tion: a cell biological perspective. Annu Rev Immunol. 2015;33:257–90. 2007;2:1269–75.
4. Yao X, Huang J, Zhong H, Shen N, Faggioni R, Fung M, et al. Targeting interleukin- 30. Teng MW, Bowman EP, McElwee JJ, Smyth MJ, Casanova JL, Cooper AM, et al. IL-
6 in inflammatory autoimmune diseases and cancers. Pharmacol Ther. 12 and IL-23 cytokines: from discovery to targeted therapies for immune-
2014;141:125–39. mediated inflammatory diseases. Nat Med. 2015;21:719–29.
5. Krausgruber T, Blazek K, Smallie T, Alzabin S, Lockstone H, Sahgal N, et al. IRF5 31. De Filippo K, Henderson RB, Laschinger M, Hogg N. Neutrophil chemokines KC
promotes inflammatory macrophage polarization and TH1-TH17 responses. Nat and macrophage-inflammatory protein-2 are newly synthesized by tissue mac-
Immunol. 2011;12:231–8. rophages using distinct TLR signaling pathways. J Immunol. 2008;180:4308–15.
6. Udalova IA, Mantovani A, Feldmann M. Macrophage heterogeneity in the context 32. Yu L, Zhang B, Deochand D, Sacta MA, Coppo M, Shang Y, et al. Negative
of rheumatoid arthritis. Nat Rev Rheumatol. 2016;12:472–85. elongation factor complex enables macrophage inflammatory responses by
7. Ross EA, Devitt A, Johnson JR. Macrophages: the good, the bad, and the gluttony. controlling anti-inflammatory gene expression. Nat Commun. 2020;11:2286.
Front Immunol. 2021;12:708186. 33. Liu X, Zhang P, Zhang Y, Wang Z, Xu S, Li Y, et al. Glycolipid iGb3 feedback
8. Tardito S, Martinelli G, Soldano S, Paolino S, Pacini G, Patane M, et al. Macrophage amplifies innate immune responses via CD1d reverse signaling. Cell Res.
M1/M2 polarization and rheumatoid arthritis: a systematic review. Autoimmun 2019;29:42–53.
Rev. 2019;18:102397. 34. Oeckinghaus A, Hayden MS, Ghosh S. Crosstalk in NF-kappaB signaling pathways.
9. Daskalaki MG, Tsatsanis C, Kampranis SC. Histone methylation and acetylation in Nat Immunol. 2011;12:695–708.
macrophages as a mechanism for regulation of inflammatory responses. J Cell 35. Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat Rev Immunol.
Physiol. 2018;233:6495–507. 2002;2:725–34.
10. Zhang D, Tang Z, Huang H, Zhou G, Cui C, Weng Y, et al. Metabolic regulation of 36. Viatour P, Merville MP, Bours V, Chariot A. Phosphorylation of NF-kappaB and
gene expression by histone lactylation. Nature. 2019;574:575–80. IkappaB proteins: implications in cancer and inflammation. Trends Biochem Sci.
11. Lauterbach MA, Hanke JE, Serefidou M, Mangan MSJ, Kolbe CC, Hess T, et al. Toll- 2005;30:43–52.
like receptor signaling rewires macrophage metabolism and promotes histone 37. Fu YD, Huang MJ, Guo JW, You YZ, Liu HM, Huang LH, et al. Targeting histone
acetylation via ATP-citrate lyase. Immunity. 2019;51:997–1011. demethylase KDM5B for cancer treatment. Eur J Med Chem. 2020;208:112760.
12. Xhabija B, Kidder BL. KDM5B is a master regulator of the H3K4-methylome in 38. Xiang Y, Zhu Z, Han G, Ye X, Xu B, Peng Z, et al. JARID1B is a histone H3 lysine 4
stem cells, development and cancer. Semin Cancer Biol. 2019;57:79–85. demethylase up-regulated in prostate cancer. Proc Natl Acad Sci USA.
13. Yan G, Li S, Yue M, Li C, Kang Z. Lysine demethylase 5B suppresses CC chemokine 2007;104:19226–31.
ligand 14 to promote progression of colorectal cancer through the Wnt/beta- 39. Johansson C, Velupillai S, Tumber A, Szykowska A, Hookway ES, Nowak RP, et al.
catenin pathway. Life Sci. 2021;264:118726. Structural analysis of human KDM5B guides histone demethylase inhibitor
14. Wong PP, Miranda F, Chan KV, Berlato C, Hurst HC, Scibetta AG. Histone deme- development. Nat Chem Biol. 2016;12:539–45.
thylase KDM5B collaborates with TFAP2C and Myc to repress the cell cycle 40. Zheng YC, Chang J, Wang LC, Ren HM, Pang JR, Liu HM. Lysine demethylase 5B
inhibitor p21(cip) (CDKN1A). Mol Cell Biol. 2012;32:1633–44. (KDM5B): a potential anti-cancer drug target. Eur J Med Chem. 2019;161:131–40.
15. Wang X, Gu M, Ju Y, Zhou J. Overcoming radio-resistance in esophageal squa- 41. Vereecke L, Beyaert R, van Loo G. The ubiquitin-editing enzyme A20 (TNFAIP3) is
mous cell carcinoma via hypermethylation of PIK3C3 promoter region mediated a central regulator of immunopathology. Trends Immunol. 2009;30:383–91.
by KDM5B loss. J Radiat Res. 2022;63:331–41. 42. Chen K, Luan X, Liu Q, Wang J, Chang X, Snijders AM, et al. Drosophila histone
16. Zhang SM, Cai WL, Liu X, Thakral D, Luo J, Chan LH, et al. KDM5B promotes demethylase KDM5 regulates social behavior through immune control and gut
immune evasion by recruiting SETDB1 to silence retroelements. Nature. microbiota maintenance. Cell Host Microbe. 2019;25:537–52.
2021;598:682–7. 43. Miao Y, Zheng Y, Geng Y, Yang L, Cao N, Dai Y, et al. The role of GLS1-mediated
17. Kang K, Park SH, Chen J, Qiao Y, Giannopoulou E, Berg K, et al. Interferon-gamma glutaminolysis/2-HG/H3K4me3 and GSH/ROS signals in Th17 responses coun-
represses M2 gene expression in human macrophages by disassembling teracted by PPARgamma agonists. Theranostics. 2021;11:4531–48.
enhancers bound by the transcription factor MAF. Immunity. 2017;47:235–50. 44. Zhao D, Zhang Q, Liu Y, Li X, Zhao K, Ding Y, et al. H3K4me3 demethylase Kdm5a
18. Kemppinen AK, Kaprio J, Palotie A, Saarela J. Systematic review of genome-wide is required for NK cell activation by associating with p50 to suppress SOCS1. Cell
expression studies in multiple sclerosis. BMJ Open. 2011;1:e000053. Rep. 2016;15:288–99.
19. Burczynski ME, Peterson RL, Twine NC, Zuberek KA, Brodeur BJ, Casciotti L, et al. 45. Wu L, Cao J, Cai WL, Lang SM, Horton JR, Jansen DJ, et al. KDM5 histone
Molecular classification of Crohn’s disease and ulcerative colitis patients using demethylases repress immune response via suppression of STING. PLoS Biol.
transcriptional profiles in peripheral blood mononuclear cells. J Mol Diagn. 2018;16:e2006134.
2006;8:51–61. 46. Ptaschinski C, Mukherjee S, Moore ML, Albert M, Helin K, Kunkel SL, et al. RSV-
20. Olsen J, Gerds TA, Seidelin JB, Csillag C, Bjerrum JT, Troelsen JT, et al. Diagnosis of induced H3K4 demethylase KDM5B leads to regulation of dendritic cell-derived
ulcerative colitis before onset of inflammation by multivariate modeling of innate cytokines and exacerbates pathogenesis in vivo. PLoS Pathog.
genome-wide gene expression data. Inflamm Bowel Dis. 2009;15:1032–8. 2015;11:e1004978.
21. Stojanov S, Lapidus S, Chitkara P, Feder H, Salazar JC, Fleisher TA, et al. Periodic 47. Wertz IE, Newton K, Seshasayee D, Kusam S, Lam C, Zhang J, et al. Phosphor-
fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) is a disorder of ylation and linear ubiquitin direct A20 inhibition of inflammation. Nature.
innate immunity and Th1 activation responsive to IL-1 blockade. Proc Natl Acad 2015;528:370–5.
Sci USA. 2011;108:7148–53. 48. Mauro C, Pacifico F, Lavorgna A, Mellone S, Iannetti A, Acquaviva R, et al. ABIN-1
22. Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Bingham CO 3rd, et al. 2010 binds to NEMO/IKKgamma and co-operates with A20 in inhibiting NF-kappaB. J
rheumatoid arthritis classification criteria: an American College of Rheumatology/ Biol Chem. 2006;281:18482–8.
European League Against Rheumatism collaborative initiative. Ann Rheum Dis. 49. Liu X, Yao M, Li N, Wang C, Zheng Y, Cao X. CaMKII promotes TLR-triggered
2010;69:1580–8. proinflammatory cytokine and type I interferon production by directly binding
23. Leyva-Illades D, Cherla RP, Galindo CL, Chopra AK, Tesh VL. Global transcriptional and activating TAK1 and IRF3 in macrophages. Blood. 2008;112:4961–70.
response of macrophage-like THP-1 cells to Shiga toxin type 1. Infect Immun. 50. Hoffmann A, Levchenko A, Scott ML, Baltimore D. The IkappaB-NF-kappaB sig-
2010;78:2454–65. naling module: temporal control and selective gene activation. Science.
24. Martinez FO, Helming L, Milde R, Varin A, Melgert BN, Draijer C, et al. Genetic 2002;298:1241–5.
programs expressed in resting and IL-4 alternatively activated mouse and human 51. Wang X, Peng H, Huang Y, Kong W, Cui Q, Du J, et al. Post-translational mod-
macrophages: similarities and differences. Blood. 2013;121:e57–69. ifications of IkappaBalpha: the state of the art. Front Cell Dev Biol. 2020;8:574706.
25. Nicodeme E, Jeffrey KL, Schaefer U, Beinke S, Dewell S, Chung CW, et al. Suppression 52. Hendriks IA, Lyon D, Young C, Jensen LJ, Vertegaal AC, Nielsen ML. Site-specific
of inflammation by a synthetic histone mimic. Nature. 2010;468:1119–23. mapping of the human SUMO proteome reveals co-modification with phos-
26. Cuartero S, Weiss FD, Dharmalingam G, Guo Y, Ing-Simmons E, Masella S, et al. phorylation. Nat Struct Mol Biol. 2017;24:325–36.
Control of inducible gene expression links cohesin to hematopoietic progenitor 53. Komives EA. Consequences of fuzziness in the NFkappaB/IkappaBalpha interac-
self-renewal and differentiation. Nat Immunol. 2018;19:932–41. tion. Adv Exp Med Biol. 2012;725:74–85.
27. Albert M, Schmitz SU, Kooistra SM, Malatesta M, Morales Torres C, Rekling JC, et al. 54. Liang WJ, Yang HW, Liu HN, Qian W, Chen XL. HMGB1 upregulates NF-kB by
The histone demethylase Jarid1b ensures faithful mouse development by protecting inhibiting IKB-alpha and associates with diabetic retinopathy. Life Sci.
developmental genes from aberrant H3K4me3. PLoS Genet. 2013;9:e1003461. 2020;241:117146.
28. Backe MB, Jin C, Andreone L, Sankar A, Agger K, Helin K, et al. The lysine 55. Liu X, Zhang P, Bao Y, Han Y, Wang Y, Zhang Q, et al. Zinc finger protein ZBTB20
demethylase KDM5B regulates islet function and glucose homeostasis. J Diabetes promotes Toll-like receptor-triggered innate immune responses by repressing
Res. 2019;2019:5451038. IkappaBalpha gene transcription. Proc Natl Acad Sci USA. 2013;110:11097–102.

Cell Death & Differentiation (2023) 30:1279 – 1292

 Y. Zhang et al.
1292
56. Wang X, Zhu K, Li S, Liao Y, Du R, Zhang X, et al. MLL1, a H3K4 methyltransferase, AUTHOR CONTRIBUTIONS
regulates the TNFalpha-stimulated activation of genes downstream of NF- YZ, YG, YJ, YD, HC and YX performed the experiments, analyzed the data, and
kappaB. J Cell Sci. 2012;125:4058–66. interpreted the results. YZ drafted the manuscript. XL and ZZ conceived the idea,
57. Mulero MC, Bigas A, Espinosa L. IkappaBalpha beyond the NF-kB dogma. designed the experiments, assisted data analyses, revised the manuscript, and
Oncotarget. 2013;4:1550–1. provided funding for this study. All authors read and approved the final manuscript.
58. Aguilera C, Hoya-Arias R, Haegeman G, Espinosa L, Bigas A. Recruitment of
IkappaBalpha to the hes1 promoter is associated with transcriptional repression.
Proc Natl Acad Sci USA. 2004;101:16537–42.
59. Roberson ED, Liu Y, Ryan C, Joyce CE, Duan S, Cao L, et al. A subset of methylated
FUNDING
CpG sites differentiate psoriatic from normal skin. J Investig Dermatol.
This work was supported by the National Key R&D Program of China
2012;132:583–92.
(2019YFA0801502), the National Natural Science Foundation of China (82071790,
60. Ovejero-Benito MC, Reolid A, Sanchez-Jimenez P, Saiz-Rodriguez M, Munoz-
82070415, 82271797), the Shuguang Program sponsored by the Shanghai Education
Aceituno E, Llamas-Velasco M, et al. Histone modifications associated with bio-
Development Foundation and Shanghai Municipal Education Commission (18SG33,
logical drug response in moderate-to-severe psoriasis. Exp Dermatol.
19SG17), and the China National Postdoctoral Program for Innovative Talents
2018;27:1361–71.
(BX2021046).
61. Zhou Q, Zhang Y, Wang B, Zhou W, Bi Y, Huai W, et al. KDM2B promotes IL-6
production and inflammatory responses through Brg1-mediated chromatin
remodeling. Cell Mol Immunol. 2020;17:834–42.
62. Ferro F, Elefante E, Luciano N, Talarico R, Todoerti M. One year in review 2017:
novelties in the treatment of rheumatoid arthritis. Clin Exp Rheumatol. COMPETING INTERESTS
2017;35:721–34. The authors declare no competing interests.
63. Hogg SJ, Beavis PA, Dawson MA, Johnstone RW. Targeting the epigenetic reg-
ulation of antitumour immunity. Nat Rev Drug Discov. 2020;19:776–800.
64. Serrat N, Sebastian C, Pereira-Lopes S, Valverde-Estrella L, Lloberas J, Celada A.
The response of secondary genes to lipopolysaccharides in macrophages ETHICS APPROVAL
depends on histone deacetylase and phosphorylation of C/EBPbeta. J Immunol. All experiments with animals were approved by the Animal Ethics Committee of
2014;192:418–26. Naval Medical University. All studies involving human PBMC samples were approved
65. Bode KA, Schroder K, Hume DA, Ravasi T, Heeg K, Sweet MJ, et al. Histone by the Medical Ethics Committee of Naval Medical University.
deacetylase inhibitors decrease Toll-like receptor-mediated activation of proin-
flammatory gene expression by impairing transcription factor recruitment.
Immunology. 2007;122:596–606.
66. Halili MA, Andrews MR, Labzin LI, Schroder K, Matthias G, Cao C, et al. Differential ADDITIONAL INFORMATION
effects of selective HDAC inhibitors on macrophage inflammatory responses to Supplementary information The online version contains supplementary material
the Toll-like receptor 4 agonist LPS. J Leukoc Biol. 2010;87:1103–14. available at https://doi.org/10.1038/s41418-023-01136-x.
67. Zhang Q, Zhao K, Shen Q, Han Y, Gu Y, Li X, et al. Tet2 is required to resolve
inflammation by recruiting Hdac2 to specifically repress IL-6. Nature. Correspondence and requests for materials should be addressed to Zhenzhen Zhan
2015;525:389–93. or Xingguang Liu.
68. Nguyen HCB, Adlanmerini M, Hauck AK, Lazar MA. Dichotomous engagement of
HDAC3 activity governs inflammatory responses. Nature. 2020;584:286–90. Reprints and permission information is available at http://www.nature.com/
69. Xia M, Liu J, Wu X, Liu S, Li G, Han C, et al. Histone methyltransferase Ash1l reprints
suppresses interleukin-6 production and inflammatory autoimmune diseases by
inducing the ubiquitin-editing enzyme A20. Immunity. 2013;39:470–81. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims
70. Huai W, Liu X, Wang C, Zhang Y, Chen X, Chen X, et al. KAT8 selectively inhibits in published maps and institutional affiliations.
antiviral immunity by acetylating IRF3. J Exp Med. 2019;216:772–85.
71. Liu X, Zhan Z, Li D, Xu L, Ma F, Zhang P, et al. Intracellular MHC class II molecules
promote TLR-triggered innate immune responses by maintaining activation of Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to
the kinase Btk. Nat Immunol. 2011;12:416–24. this article under a publishing agreement with the author(s) or other rightsholder(s);
72. Wang Y, Wang P, Zhang Y, Xu J, Li Z, Li Z, et al. Decreased expression of the host author self-archiving of the accepted manuscript version of this article is solely
long-noncoding RNA-GM facilitates viral escape by inhibiting the kinase activity governed by the terms of such publishing agreement and applicable law.
TBK1 via S-glutathionylation. Immunity. 2020;53:1168–81.

Cell Death & Differentiation (2023) 30:1279 – 1292