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Dichotomous roles of RIPK3 in regulating the IFN
response and NLRP3 inammasome in human
monocytes
Chao Yang,
1
Ruoxi Yuan,
1
Caroline Brauner,
1
Yong Du,
1,2
Marie Dominique Ah Kioon,
1
Franck J. Barrat,
1,3,2
Lionel B. Ivashkiv
1,3,4,
*
1
HSS Research Institute and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, 535 E 70th St, New York, NY 10021, United States
2
Department of Microbiology and Immunology, Weill Cornell Medicine, 1300 York Avenue, Box 62, New York, NY 10065, United States
3
Immunology and Microbial Pathogenesis Program, Weill Cornell Medicine, 1300 York Avenue, Box 65, New York, NY 10065, United States
4
Department of Medicine, Weill Cornell Medicine, 530 East 70th Street, M-522, New York, NY 10021, United States
*Corresponding author: Email: ivashkivl@hss.edu
Abstract
Regulation of the prole and magnitude of toll-like receptor (TLR) responses is important for effective host defense against infections
while minimizing inammatory toxicity. The chemokine CXCL4 regulates the TLR8 response to amplify inammatory gene and
inammasome activation while attenuating the interferon (IFN) response in primary monocytes. In this study, we describe an
unexpected role for the kinase RIPK3 in suppressing the CXCL4 + TLR8–induced IFN response and providing signal 2 to activate the
NLRP3 inammasome and interleukin (IL)-1 production in primary human monocytes. RIPK3 also amplies induction of inammatory
genes such as TNF,IL6, and IL1B while suppressing IL12B. Mechanistically, RIPK3 inhibits STAT1 activation and activates PI3K-Akt–
dependent and XBP1- and NRF2-mediated stress responses to regulate downstream genes in a dichotomous manner. These ndings
identify new functions for RIPK3 in modulating TLR responses and provide potential mechanisms by which RIPK3 plays roles in
inammatory diseases and suggest targeting RIPK3 and XBP1- and NRF2-mediated stress responses as therapeutic strategies to
suppress inammation while preserving the IFN response for host defense.
Keywords: CXCL4, NLRP3 inammasome, RIPK3, TLR8, gene regulation, inammation
1. Introduction
Toll-like receptors (TLRs) are a family of pattern recognition re-
ceptors and play key roles in host defense against pathogen infec-
tion by inducing production of type I interferon (IFN-I) and
inammatory cytokines such as tumor necrosis factor (TNF) and
interleukin (IL)-1β. However, imbalanced and excessive IFN-I
and inammatory cytokine expression could be detrimental to
the host during defense against pathogen infection, such as inu-
enza and COVID-19
1,2
; uncontrolled TLR responses to molecules
from host dying cells, such as HMGB1 and self-nucleic acids
(NAs), also cause inammation, which is related to inammatory
diseases and autoimmune disorders, such as rheumatoid arthritis
and systemic lupus erythematosus.
3–5
A moderate and balanced
IFN-I response and nuclear factor κB (NF-κB)–induced inamma-
tory response is critical for host defense against pathogen
infections and maintenance of homeostasis; however, the mech-
anisms that balance TLR responses are still not understood. One
example is the balance between IFN and IL-1, as previous work
has shown negative crossregulation between IFN-I and IL-1β
through the regulation of NLRP3 inammasome activation and
IL-1 expression and the induction of eicosanoids by IL-1β, which
is associated with Mycobacterium tuberculosis pathogenesis.
6–9
Endosomal TLRs, including TLR3, TLR7, TLR8, and TLR9, sense
NAs to induce IFN-I and cytokine production and are critical for
antiviral and anti-intracellular bacterial responses. TLR8 recog-
nizes single-stranded RNA (ssRNA) to induce IFN-I and cytokines.
CXCL4 is a cationic chemokine and has been implicated in various
inammatory diseases including atherosclerosis and systemic
sclerosis.
10–14
In our recent study,
10
we discovered that CXCL4 fa-
cilitates TLR8 ssRNA ligand ORN8L
15
internalization through
nanoparticle formation between CXCL4 and ORN8L; CXCL4 and
TLR8 signaling crosstalk synergistically activates cytokine and
chemokine genes involved in cytokine storm and inammatory
disease pathogenesis, and triggers a second signal-independent
NLRP3 inammasome activation and massive IL-1βproduction.
In contrast to this superactivation, CXCL4 modestly attenuates
TLR8-induced IFN responses, as determined by reduction of select
IFN-stimulated gene (ISG) expression after CXCL4 + TLR8 costi-
mulation relative to TLR8 alone. However, CXCL4 and TLR8 signal-
ing crosstalk strongly activates TBK1/IKKϵ-IRF3 axis to induce a
high amount of IFN-I expression compared with TLR8 alone.
This suggests that CXCL4 and TLR8 signaling activates a feedback
mechanism that negatively regulates IFN-I responses to set the
balance between IFN responses and NLRP3 inammasome activa-
tion and IL-1βproduction.
RIPK3 and RIPK1 are kinases that initiate apoptosis and necrop-
tosis. RIPK1 is a death domain (DD)–containing protein, which can
interact with DD of other proteins such as TNF receptor 1 and the
death receptor Fas; RIPK1 also harbors an amino-terminal kinase
domain and a bridging intermediate domain that contains an RIP
homotypic interaction motif (RHIM). Unlike RIPK1, RIPK3 lacks a
DD in the carboxy-terminal region but contains a RHIM protein:
protein interaction motif.
16
Although RIPK3 cannot directly inter-
act with or be activated by DD-containing death receptors, it can
bind proteins such as RIPK1, TRIF, and ZBP1
16
through RHIM do-
main–mediated interaction and become activated. RIPK3 can
Received: January 29, 2023. Revised: August 3, 2023. Accepted: August 7, 2023
© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail:
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Journal of Leukocyte Biology, 2023, 00, 1–15
https://doi.org/10.1093/jleuko/qiad095
Regular Article
also activate itself through autophosphorylation at serine 227,
which is generally achieved when RIPK3 interacts with other
proteins, like its target pseudokinase mixed lineage kinase
domain-like (MLKL).
17,18
RIPK3 is the key kinase that activates
MLKL to trigger a programed cell death termed necroptosis.
16
Increasing evidence suggests that RIPK3 not only is a pleiotropic
modulator of necroptosis, but also contributes to second signal-
independent NLRP3 inammasome activation and inammatory
responses and diseases.
16,19–24
RIPK3 is tightly controlled at base-
line by caspase-8; after activation of TNF receptor (TNFR) or TLR
signaling, RIPK3 can be activated by its upstream kinase RIPK1
or other kinases when caspase-8 activity is inhibited, which leads
to necroptosis and NLRP3 activation.
22,25
RIPK3 contributes to
lipopolysaccharide induction of inammatory genes such as Tnf
through activation of NF-κB in the absence of caspase-8 activity
in mouse bone marrow–derived macrophages.
26
RIPK3 also pro-
motes kidney brosis through the AKT-dependent activation of
adenosine triphosphate (ATP) citrate lyase, in which genetic or
chemical inhibition of RIPK3 suppressed AKT activation and ATP
citrate lyase in transforming growth factor β–experienced bro-
blasts.
27
RIPK3 plays a greater role in mouse models of inamma-
tion and tissue injury than its necroptotic executor client MLKL.
24
In this study, we observed that CXCL4 and TLR8 costimulation
activated RIPK3 in human primary monocytes, and that RIPK3
promoted expression of inammatory genes such as TNF and ac-
tivated NLRP3 inammasome and IL-1βproduction while sup-
pressing IFN response and IL12B expression. Furthermore, we
demonstrated that RIPK3 activated XBP1-mediated endoplasmic
reticulum (ER) stress and NRF2-mediated oxidative stress re-
sponses in a PI3K-Akt–dependent manner in human monocytes
upon CXCL4 and TLR8 costimulation. XBP1- and NRF2-mediated
responses contributed to RIPK3-mediated regulation of inamma-
tory gene and ISG expression. In accord with a dampened IFN re-
sponse after CXCL4 and TLR8 costimulation, RIPK3 suppressed
STAT1 activation. In addition, RIPK3 played a role in the second
signal-independent NLRP3 inammasome activation and RIPK1
partnered with RIPK3 for NLRP3 inammasome activation and
IL-1βproduction when caspase-8 was inhibited. These ndings
support the important role of RIPK3 in inammatory conditions
by driving inammatory gene expression and NLRP3 inamma-
some activation, as has been suggested in nonalcoholic fatty liver
disease
21
and COVID-19.
28
Dampening IFN responses and IL12B
expression also suggests that RIPK3 may suppress T helper 1
and cytotoxic T lymphocyte immune responses, thereby dampen-
ing antipathogen
29,30
or antitumor immunity.
21,31,32
2. Materials and methods
2.1 Cell culture
Deidentied human buffy coats were purchased from the New York
Blood Center following a protocol approved by the Hospital for
Special Surgery Institutional Review Board. Peripheral blood mono-
nuclear cells were isolated with Lymphoprep (Accurate Chemical)
via density gradient centrifugation and monocytes were puried
from peripheral blood mononuclear cells with anti-CD14 magnetic
beads as recommended by the manufacturer (Miltenyi Biotec).
10
Monocytes were cultured overnight at 37 °C, 5% CO
2
in RPMI-1640
medium (Invitrogen) supplemented with 10% heat-inactivated de-
ned fetal bovine serum (FBS) (HyClone; Fisher Scientic), penicillin-
streptomycin (Invitrogen), L-glutamine (Invitrogen), and 20 ng/mL
human macrophage colony-stimulating factor (M-CSF). After at
least 12-h culture, the cells were treated as described in the gure
legends and were harvested and prepared for total RNA extraction,
protein extraction, and ow cytometry. Cells were stimulated with
CXCL4 (Sigma) and/or TLR8 ligand ORN8L (ChemGenes) as previous-
ly described
10
; testing of various lots of CXCL4 using the
Chromogenic LAL Endotoxin Assay Kit (Genscript) showed that
CXCL4 preparations contributed 0.01 to 0.06 EU/mL nal concentra-
tion of endotoxin in cultures, which does not contribute appreciably
to synergy with ORN8L.
10
2.2 RIPK3 overexpression
Monocytes (1 ×10
6
cells) were seeded in 24-well plates in complete
RPMI-1640 medium supplemented with 10% heat-inactivated de-
ned FBS, penicillin-streptomycin, L-glutamine, and 50 ng/mL hu-
man M-CSF for 5 d. The culture medium was replaced with fresh
medium on day 3 during the culture. On day 5 of culture, the old me-
dium was removed and replaced with 200 μL of RPMI-1640 medium
without penicillin-streptomycin with a nal concentration of 2%
heat-inactivated dened FBS and 50 ng/mL human M-CSF.
Enhanced green uorescent protein (GFP) or GFP-h-RIPK3 adeno-
viral particles (multiplicity of infection =100 plaque-forming
units/cell; Vector Biolabs) were added to the cells accordingly. The
cells were centrifuged at 1600 rpm for 30 min at room temperature
(RT). After culturing for 12 h, 300 μL of complete RPMI-1640 medium
supplemented with 10% heat-inactivated dened FBS, penicillin-
streptomycin, L-glutamine, and 20 ng/mL human M-CSF was added
to the cell culture. The cells were cultured for another day before
harvesting for experiments.
2.3 RNA sequencing
After RNA extraction, libraries for sequencing were prepared us-
ing the NEBNext Ultra II RNA Library Prep Kit for Illumina follow-
ing the manufacturer’s instructions (Illumina). Quality of all RNA
and library preparations was evaluated with BioAnalyzer 2100
(Agilent). Libraries were sequenced by the Genomics Resources
Core Facility at Weill Cornell Medicine using a NovaSeq, 50-bp
paired-end reads at a depth of ∼10 to 20 million reads per sample.
Paired-end reads were preprocessed using fastp (0.19.10; https://
github.com/OpenGene/fastp), which supports adapter trimming,
low-quality base trimming, and calculation of additional quality
control metrics. Trimmed reads were aligned to the human gen-
ome (hg38) genome using STAR (2.7.3a; https://github.com/
alexdobin/STAR). Low quality reads and multimapping align-
ments were ltered using SAMtools (1.9; https://github.com/
samtools/). Reads were counted within exons and summarized
at the gene level using featureCounts (v2.0.1; https://subread.
sourceforge.net) to produce a count matrix of reads per gene.
Differential gene expression analysis was performed in R
(4.1.0; R Foundation for Statistical Computing) using
edgeR (3.34.1; https://bioconductor.org/packages/release/bioc/
html/edgeR.html). Genes with low expression levels (<3 counts
per million in at least 1 group) were ltered from all downstream
analyses. The Benjamini-Hochberg false discovery rate procedure
was used to correct for multiple testing.
2.4 Ingenuity Pathway Analysis
Ingenuity Pathway Analysis was used to analyze differentially ex-
pressed genes. The Ingenuity Canonical Pathways were used to
predict activated or suppressed pathways based on the expression
pattern of genes regulated by CXCL4 and ORN8L in human pri-
mary monocytes. The Upstream Regulator analytic was used to
predict upstream regulators whose change in expression or func-
tion could explain the observed gene expression changes. The
2|Journal of Leukocyte Biology, 2023, Vol. 00, No. 0
overall activation/inhibition states of canonical pathways and
Upstream Regulators are predicted based on a z-score algorithm,
for which a negative or positive value represents the predicted in-
hibition or activation of the pathway and upstream regulator,
respectively.
2.5 Analysis of messenger RNA amounts
Total RNA was extracted from cultured human monocytes with
an RNeasy Mini Kit (Qiagen) and was used for complementary
DNA synthesis by reverse transcription with a RevertAid RT
Reverse Transcription Kit (Thermo Fisher Scientic; catalog num-
ber: K1691). Real-time polymerase chain reaction (PCR) was per-
formed with the Fast SYBR Green Master Mix and a 7500 Fast
Real-Time PCR System (Applied Biosystems). The primer sequen-
ces for the quantitative PCR (qPCR) reactions are listed in
Supplementary Table 1. Cycle threshold (CT) values of target
gene were normalized to GAPDH expression and are shown as per-
centage of GAPDH (100/2^
ΔCt
).
2.6 Flow cytometric analysis of cell death
Single-cell suspensions were stained with Fixable Viability Dye
eFluor 780 (eBioscience; cat. #65-0865-18) for 20 min at 4 °C.
Then, cells were washed with FACS buffer (phosphate-buffered
saline containing 2% calf serum and 1 mM EDTA) and were ana-
lyzed by ow cytometry. Dead cells were detected based on
eFluor 780 uorescence. Data were analyzed with FlowJo
software version 10 (TreeStar).
2.7 Flow cytometric analysis of phospho-NRF2
After treatment with CXCL4 and ORN8L in the presence/absence
of PI3K inhibitor Wortmanin for 3 h, cells were harvested and xed
with 4% paraformaldehyde for 15 min at RT. After washing with
FACS buffer, the cells were incubated in cold 90% methanol for
10 min and then incubated with anti-NRF2 antibody for 2 h at
RT. Cells were then incubated with PE-conjugated secondary anti-
body for 1 h at RT. Cells were analyzed using a BD FACSymphony
A5 Cell Analyzer to measure PE uorescence.
2.8 Western blotting
Cells were lysed with 50 µl of cold lysis buffer comprising 50 mM
Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (vol/vol) Triton
X-100, 2 mM Na
3
VO
4
, 1 ×phosSTOP EASYPACK, 1 mM Pefabloc,
and 1×EDTA-free complete protease inhibitor cocktail (Roche),
and incubated for at least 10 min on ice. Then, cell debris was pel-
leted using Eppendorf Centrifuge 5424 R at 21,130 gat 4 °C for
5 min. The protein fraction in the supernatant was mixed with
4×Laemmli Sample buffer (Bio-Rad; cat. #1610747) supplemented
with 10% 2-mercroptoehanol (BME) (Sigma-Aldrich). Samples for
Western blotting were subjected to electrophoresis on Bis-Tris
gels (Invitrogen). Proteins were transferred to polyvinylidene di-
uoride membrane was as previously reported
10
Membranes
were blocked in 5% (w/v) bovine serum albumin in Tris-buffered
saline (20 mM Tris, 50 mM NaCl, pH 8.0) with 0.1% (v/v) Tween
20 (TBST) at RT for at least 1 h with shaking. Membranes were
then incubated with target primary antibodies at 4 °C overnight
with shaking and then washed 3 times in TBST for 10 min, then in-
cubated with anti-mouse or anti-rabbit immunoglobulin G sec-
ondary antibodies conjugated to horseradish peroxidase (GE
Healthcare; cat: NA9310V and NA9340V) diluted in TBST at
room temperature for 1 h with shaking. Next, membranes were
washed 3 times in TBST at RT for with shaking. Antibody binding
was detected using enhanced chemiluminescent substrates for
horseradish peroxidase (ECL Western blotting reagents
[PerkinElmer; cat: NEL105001EA]) or SuperSignal West Femto
Maximum Sensitivity Substrate (Thermo Fisher Scientic; cat:
34,095) according to the manufacturer’s instructions, and visual-
ized using premium autoradiography lm (Thomas Scientic; cat:
E3018). For membranes that required probing twice or more using
different primary antibodies, Restore PLUS Western blotting strip-
ping buffer (Thermo Fisher Scientic) was applied on the blots
with shaking for 15 to 20 min at RT following rst-time develop-
ment. To detect multiple proteins on the same experimental lter,
membranes were cut horizontally based on the molecular mass
markers and molecular size of the target proteins. Antibodies
used are identied in Supplementary Table 1.
2.9 Cytokine detection by enzyme-linked
immunosorbent assay
Levels of IL-1βwere measured in supernatants of cells using
enzyme-linked immunosorbent assay kits (R&D Systems) accord-
ing to the manufacturer’s instructions.
2.10 Statistical analysis
GraphPad Prism Version 8 for Windows (GraphPad Software) was
used for all statistical analysis. Information about the specic
tests used and the number of independent experiments are pro-
vided in gure legends. Two-way analysis of variance with Sidak
correction for multiple comparisons was used for grouped data.
Otherwise, 1-way analysis of variance with the Greenhouse–
Geisser correction and Tukey’s post hoc test for multiple compar-
isons was performed. For paired data, when the data did not pass
the normal distribution by Ftest the Wilcoxon signed rank test
was performed; otherwise, a paired ttest was used.
3. Results
3.1 RIPK3 suppresses ISG induction after
CXCL4 + TLR8 costimulation
In our recent study, we found that CXCL4 and TLR8 costimulation
synergistically activates inammatory gene expression and ma-
ture IL-1 production at least in part via NF-κB, AP-1, and synergis-
tic activation of TBK1-IRF5 signaling.
10
As RIPK3 can promote
NF-κB activity and inammasome activation, we tested the role
of RIPK3 in CXCL4 + TLR8–induced monocyte activation.
23,26,33
Here, we found that treatment of human primary monocytes
with the combination of CXCL4 and TLR8 ligand ORN8L activated
RIPK3, as determined by increased phosphorylation at serine 227
compared with baseline (Fig. 1A and Supplementary Fig. 1B).
Both CXCL4 and ORN8L contributed to this increase in a time-
course experiment (Fig. 1A). CXCL4 + TLR8 stimulation induces
production of TNF,
10
which can potentially activate RIPK3 in an
autocrine manner. We found that blockade of TNF signaling using
anti-TNF neutralizing antibodies did not affect CXCL4 + TLR8–in-
duced RIPK3 phosphorylation, suggesting that CXCL4 and TLR8
signaling–induced RIPK3 activation is TNF independent
(Supplementary Fig. 1B). Strikingly, inhibition of RIPK3 using its
specic inhibitor GSK872
34,35
had a dichotomous effect on
CXCL4 + TLR8–induced gene expression: induction of the NF-κB
target gene TNF was nearly abolished, whereas induction of the
ISG CXCL10 was increased (Fig. 1B); induction of IL12B, which is
strongly potentiated by IFNs,
36
was also increased, whereas IL6
and IL1B induction were less affected (Supplementary Fig. 1A).
Inhibition of RIPK3 did not affect NF-κB activation, as deter-
mined by p65 phosphorylation (Supplementary Fig. 1C). To
Yang et al. |3
Fig. 1. RIPK3 regulates CXCL4 and TLR8–induced inammatory and IFN-stimulated genes. Human blood monocytes were isolated with CD14
+
magnetic beads and rested overnight with M-CSF (20 ng/mL). (A) Immunoblot of phospho-RIPK3 and total RIPK3 using whole cell lysates of cells
stimulated with CXCL4 (10 μg/mL) and TLR8 ligand ORN8L (20 μg/mL) in the presence/absence of GSK872 (10 µM) for the indicated times. (B) mRNA was
measured by qPCR and normalized relative to GAPDH mRNA after 6 h of CXCL4 and TLR8 costimulation of human blood monocytes with/without RIPK3
inhibitor GSK872. (C) Immunoblot of RIPK3 and phospho-p65 with whole cell lysates of control enhanced GFP (eGFP)– or RIPK3-overexpressing cells
stimulated with CXCL4 and ORN8L for 6 h. (D) mRNA of inammatory genes was measured by qPCR and normalized relative to GAPDH mRNA in cells
from panel C. (E) mRNA of CXCL10 and IFIT1 was measured by qPCR and normalized relative to GAPDH mRNA in cells from eGFP- or
RIPK3-overexpressing cells stimulated with CXCL4 and ORN8L with/without IFN-β(100 ng/mL) for 3 h. (F, G) Immunoblot of RIPK3 and phospho-RIPK3
using whole cell lysates of cells stimulated with CXCL4 and ORN8L in the presence/absence of caspase-8 inhibitor zVAD (50 µM), RIPK1 inhibitor
necrostatin-1 s (30 µM), and GSK872 (F) or of HSP90 inhibitor luminespib (1 µM) (G) for 6 h. (H) mRNA was measured by qPCR and normalized relative to
GAPDH mRNA in cells stimulated with CXCL4 and ORN8L with/without luminespib for 6 h. Data are depicted as mean ±SEM of 5 to 11 healthy donors (B,
D, E, and H) or 1 representative blot of at least 3 independent experiments (A, C, F, and G). ***P≤0.001; **P≤0.01; *P≤0.05 by Wilcoxon matched signed
rank test (B, D), paired ttest (D, H), and 2-way analysis of variance with Sidak correction for multiple comparisons (E). OE =overexpressed.
4|Journal of Leukocyte Biology, 2023, Vol. 00, No. 0
further test the function of RIPK3 in regulation of the gene expres-
sion in CXCL4 + TLR8 costimulation, we took a complementary
gain-of-function approach by overexpressing RIPK3 using adeno-
viral transduction of primary human macrophages and found
that, in line with a previous report that RIPK3 contributes to
lipopolysaccharide-induced NF-κB activation and inammatory
gene expression in mouse bone marrow–derived macrophages
when caspase-8 is inhibited,
26
forced expression of RIPK3 in-
creased the amounts of NF-κB p65 phosphorylation under condi-
tions of CXCL4 + TLR8 costimulation (Fig. 1C) and increased
expression of NF-κB target inammatory genes IL1B and IL6 with
a trend toward increased TNF expression that was not signicant
but did not affect IL12B expression (Fig. 1D and Supplementary
Fig. 1D). The results showing the effects of GSK872 and RIPK3 over-
expression on p65 activation (Fig. 1C and Supplementary Fig. 1C)
are consistent with a redundant role for endogenous RIPK3 in
p65 activation in the context of primary human monocytes costi-
mulated with CXCL4 +TLR8, but that the role of RIPK3 in this sig-
naling pathway can be revealed by supraphysiological RIPK3
expression. The lack of IL12B superinduction after RIPK3 overex-
pression is consistent with the RIPK3-mediated suppressive path-
way targeting IL12B but not TNF shown in Fig. 1B, which would
counterbalance the positive effects of RIPK3-mediated NF-κB acti-
vation on IL12B expression. Strikingly, forced RIPK3 expression
strongly suppressed induction of the ISGs CXCL10 and IFIT1
(Fig. 1E). Thus, both loss- and gain-of-function experiments con-
cordantly show that RIPK3 attenuates induction of ISGs by
CXCL4 + TLR8 costimulation, suggesting a feedback inhibition
mechanism that may selectively target the IFN response.
Next, we investigated how RIPK3 was activated after CXCL4
and TLR8 costimulation. The pan caspase inhibitor zVAD, which
inhibits caspase-8, or necrostatin-1s, which inhibits RIPK1, min-
imally affected RIPK3 activation; in contrast, inhibition of RIPK3
using its inhibitor GSK872 completely removed its phosphoryl-
ation (Fig. 1A, F). These results suggest that in our system RIPK3
is not predominantly activated by the RIPK1/Caspase-8 pathway
and instead RIPK3 activation might depend on autophosphoryla-
tion. RIPK3 is a client of chaperone protein HSP90, and chemical
inhibitors of HSP90 disrupt the HSP90-RIPK3 complex and prevent
RIPK3 phosphorylation on serine 227 and activation.
37–39
Strikingly, inhibition of RIPK3 using HSP90 inhibitor luminespib,
which is under clinical development as a cancer therapy, sup-
pressed RIPK3 phosphorylation (Fig. 1G) and phenocopied the ef-
fects of the RIPK3 inhibitor GSK872 on gene expression (Fig. 1H):
superinduction of CXCL10,IRF1, and IL12B with concomitant sup-
pression of TNF and IL1B expression. This result opens HSP90 in-
hibition as a novel approach to modulating the balance between
TLR-induced inammatory and IFN responses.
3.2 RIPK3 attenuates CXCL4 + TLR8–induced
STAT1 activation
To investigate mechanisms by which RIPK3 attenuates ISG induc-
tion, we examined regulation of IRF1 and STAT1, 2 key compo-
nents of the IFN-Jak-STAT-IRF pathway that activates ISG
expression. As expected based upon a previously described
IFN-β-mediated autocrine loop,
40
CXCL4 + TLR8 costimulation ac-
tivated STAT1 by inducing tyrosine 701 phosphorylation (Fig. 2A).
STAT1 activation was increased when RIPK3 was inhibited
(Fig. 2A), as was induction of its target gene IRF1 at both messenger
RNA (mRNA) and protein levels (Fig. 2B, C). In a complementary
gain-of-function experiment, RIPK3 strongly suppressed CXCL4
+ TLR8–induced STAT1 activation and IRF1 induction (Fig. 2D,
compare lane 4 with lane 3; densitometry showed >90% decreases
in band intensity [Supplementary Fig. 1E]). Thus, RIPK3 suppresses
CXCL4 + TLR8–induced STAT1 activation; however, RIPK3 did not
signicantly affect induction of IFNB (Fig. 2E). Moreover, adding ex-
ogenous IFN-βonly partially rescued STAT1 phosphorylation and
IRF1 protein expression (Fig. 2D, compare lane 6 with lane 5) and
only partially rescued RIPK3-mediated suppression of ISGs in
CXCL4 + TLR8 costimulation (Fig. 1E, columns 5 and 6).
Additional experiments supported that RIPK3 does not suppress
ISG induction by exogenous IFN-β(Fig. 2F). Thus, overall the data
suggest that RIPK3 contributes to suppression of macrophage
IFN responses. The suppression of IFN-induced gene expression
under conditions of CXCL4 + TLR8 costimulation, but not when ex-
ogenous IFN was added alone, suggests induction of a cofactor(s)
that cooperates with RIPK3 to regulate IFN-mediated STAT activa-
tion, which we next investigated using a transcriptomic approach.
3.3 Genome-wide regulation of CXCL4 + TLR8–
induced gene expression by RIPK3
We had previously observed gasdermin D (GSDMD) activation in
CXCL4 + TLR8–stimulated monocytes,
10
which can lead to cell
death via pyroptosis (see the following) in addition to IL-1βrelease.
RIPK3 can also induce necroptosis via activation of MLKL. To at-
tenuate secondary effects of cell death on gene induction and to fo-
cus on the effects of upstream signaling pathways, we followed a
standard approach in the eld of suppressing cell death and inhib-
ited MLKL and GSDMD activity using necrosulfonamide (NSA)
(5μM),
41
and used the selective RIPK3 inhibitor GSK872 to identify
RIPK3 effects on gene expression. Inhibition of GSDMD had min-
imal effects on CXCL4 + TLR8–induced expression of inammatory
NF-κB targets with a trend toward increased expression of TNF
(Supplementary Fig. 2A). In contrast, induction of the ISGs
CXCL10 and IRF1, and of IL12B, was strongly suppressed
(Supplementary Fig. 2A), and this suppression was reversed by
RIPK3 inhibition (Supplementary Fig. 2B). These results suggest
that inhibition of MLKL and GSDMD (and attendant cell death)
more fully reveals the regulatory function of RIPK3 in gene expres-
sion, possibly related to prolonged signaling when cell viability is
maintained, and supports using this system for studying
RIPK3-mediated gene regulation. Thus, we performed transcrip-
tomic analysis using RNA sequencing in primary human mono-
cytes stimulated with CXCL4 + TLR8 in the presence of NSA and
GSK872 (Supplementary Fig. 3A). Principal component analysis
showed that GSK872 effectively reversed the effects of NSA
(Supplementary Fig. 3A), further supporting the notion that inhib-
ition of MLKL and GSDMD highlights the effects RIPK3 on gene ex-
pression. Signicantly differentially expressed genes (n =6,750;
false discovery rate <0.05, fold change >2) were clustered into 4
groups based on pattern of expression (Fig. 3A). In this study, we
mainly focused on the genes that were upregulated by CXCL4
and TLR8 costimulation (clusters 1 and 4). Cluster 4 contained
genes negatively regulated by RIPK3 and contained various ISGs
(Fig. 3A, compare column 4 with column 3); pathway analysis
showed most signicant enrichment of interferon signaling path-
ways in cluster 4 (Fig. 3B). Accordingly, the most signicantly en-
riched transcription factors (TFs) associated with these genes
were IRFs and STAT1 (Fig. 3C). Increased expression of a represen-
tative panel of ISGs when RIPK3 was inhibited is displayed in a heat
map in Fig. 3D. These results show that RIPK3 broadly suppresses
the CXCL4 + TLR8–induced IFN response and suggest that RIPK3
activity may explain why ISG expression was suppressed in
Yang et al. |5
CXCL4 + TLR8 costimulation relative to TLR8 alone at late time
points in our recent study.
10
Genes in cluster 1 were positively regulated by RIPK3 and in-
cluded TNF, in accordance with the reverse-transcription qPCR
data (Fig. 1B). Notably, Ingenuity Pathway Analysis of cluster 1
genes showed most signicant enrichment of NRF2-mediated oxi-
dative stress response (OSR) and ER stress pathways and accord-
ingly showed most signicant enrichment of XBP1 and NFE2L2
(encoding NRF2) TFs as upstream regulators of these genes
(Fig. 3E, F). The regulation of known XBP1 and NRF2 target genes
is shown in Supplementary Fig. 3B. Out of the XBP1 and NRF2 tar-
get genes, 19 genes were regulated by both XBP1 and NRF2
(Fig. 3G); these genes mainly function in ER stress response
(Fig. 3H), and a subset were dependent on RIPK3, such as ATF4
and MCFD2 (Supplementary Fig. 3C). In addition to stress path-
ways that regulate inammatory gene expression,
42
RIPK3 pro-
moted expression of genes in the PI3K-Akt pathway (Fig. 3E),
which has previously been implicated in context-dependent posi-
tive or negative regulation of TLR responses
43
and also regulates
stress responses.
44–47
These ndings led us to test whether
RIPK3 regulates PI3K-Akt signaling and NRF2-mediated OSR and
ER stress pathways even in the absence of MLKL and GSDMD in-
hibition, and whether these pathways contribute to downstream
regulation of gene expression and suppression of ISGs.
3.4 PI3K-Akt pathway activity is dependent on
RIPK3 and regulates ISGs and inammatory genes
In accord with previously described activation of PI3K-Akt signal-
ing by TLRs,
48
treatment of monocytes with CXCL4 and the TLR8
ligand ORN8L increased Akt phosphorylation at serine 473 in
most blood donors (Fig. 4A and Supplementary Fig. 4A).
Inhibition of RIPK3 strongly diminished Akt phosphorylation in
all 5 donors tested (Fig. 4A,Supplementary Fig. 4A, and data not
shown). We next used inhibitors of PI3K, which is upstream of
Akt, to test the role of PI3K-Akt signaling in CXCL4 + TLR8–induced
gene activation. The PI3K inhibitor Wortmanin signicantly in-
creased CXCL10 and IRF1 induction and slightly suppressed TNF
expression in CXCL4 and TLR8 costimulated human monocytes
(Fig. 4B). This result was corroborated with inhibitors targeting in-
dividual PI3K catalytic subunits: α,β,δ, and γor combined inhib-
ition of the 4 subunits (Fig. 4C and Supplementary Fig. 4B, C).
Strikingly, PI3K inhibition phenocopied RIPK3 inhibition: in-
creased induction of ISGs and IL12B with suppression of TNF in-
duction. Together, the results show that Akt activity is
dependent on RIPK3 and support a role for PI3K-Akt signaling in
mediating effects of RIPK3 on gene expression.
3.5 RIPK3 regulates XBP1 and NRF2 activation to
regulate ISG and inammatory gene expression
Emerging evidence shows that PI3K-Akt pathways activate both
XBP1- and NRF2-mediated responses in regulating ER stress and
the effector function and survival of human natural killer (NK)
cells in regulating antioxidant function and growth factor
gene expression and neuroinammation and in maintaining
memory CD4 T cells regulated by Akt-GSK3β–mediated
NRF2 nuclear translocation and in preventing ferroptosis,
respectively.
44–47,49–51
Given the role of stress pathways in inam-
matory responses
42
and the RNA sequencing data that RIPK3
Fig. 2. RIPK3 regulates the CXCL4 + TLR8–induced IFN response. (A) Immunoblot of STAT1 and phospho-STAT1 with whole cell lysates of cells
stimulated for 3 h. (B, C) mRNA level (B) and protein level (C) of IRF1 measured in cells stimulated under the indicated conditions for 6 h. (D) Immunoblot
of STAT1, phospho-STAT1, IRF1, and RIPK3 using whole cell lysates of eGFP- or RIPK3-overexpressing cells stimulated with CXCL4 and ORN8L with/
without IFN-β(100 ng/mL) for 3 h. (E) mRNA of IFNB1 was measured by qPCR and normalized relative to GAPDH mRNA in cells stimulated with CXCL4
and ORN8L in the presence/absence of GSK872 for 3 h. (F) mRNA of CXCL10 and IFIT1 was measured by qPCR and normalized relative to GAPDH mRNA
in cells from eGFP- or RIPK3-overexpressing cells stimulated with IFN-β(100 ng/mL) for 3 h. Data are depicted as mean ±SEM of 3 to 6 healthy donors (B,
E, and F) or 1 representative blot from at least 3 independent experiments (A, C, and D). **P≤0.01 by 1-way analysis of variance with Tukey’s post hoc
test for multiple comparisons (B and E). ns =no signicance; OE =overexpressed.
6|Journal of Leukocyte Biology, 2023, Vol. 00, No. 0
positively regulates ER stress and NRF2-mediated OSR (Fig. 3), we
hypothesized that RIPK3 regulates ER stress and NRF2 pathways,
which in turn affect ISG and inammatory gene expression in hu-
man monocytes during CXCL4 and TLR8 costimulation. CXCL4 +
TLR8 costimulation indeed activated ER stress, as determined by
the upregulation of ER chaperone BIP and ER stress–related TF
CHOP, and the increased ratio of spliced XBP1 (s-XBP1)/XBP1 (Fig. 5A,
B). CXCL4 + TLR8 costimulation also signicantly increased NRF2 ac-
tivation, determined by phosphorylation at serine 40, relative to
stimulation with either factor alone (Fig. 5C). Inhibition of RIPK3 using
GSK872 signicantly reduced BIP,CHOP, and the ratio of s-XBP1/XBP1
mRNA and NRF2 phosphorylation (Fig. 5A—C). Furthermore, inhib-
ition of PI3K signicantly reduced XBP1 activation, BIP expression,
and NRF2 phosphorylation after CXCL4 and TLR8 costimulation of
human monocytes (Fig. 5D, E). These ndings indicate that RIPK3 ac-
tivates a stress response mediated by XBP1 and NRF2 in a PI3K-Akt–
dependent manner in CXCL4 + TLR8–costimulated monocytes.
Next, we tested the role of XBP1 and NRF2 in regulation of
downstream ISG and inammatory gene expression. Inhibition
of XBP1 using its inhibitor 4µ8C, but not inhibition of other ER
stress pathways, signicantly reduced TNF expression without
substantially affecting expression of other inammatory genes
or ISGs (Fig. 5F and Supplementary Fig. 5A). Inhibition of NRF2 us-
ing ML385 increased IRF1 and reduced TNF expression (Fig. 5G and
Supplementary Fig. 5B). Similar to the effect of RIPK3 in regulation
of the gene expression pattern, inhibition of both XBP1 and NRF2
together signicantly increased ISG CXCL10 and IRF1 expression
while reducing inammatory TNF and IL1βexpression (Fig. 5H).
These data suggest that XBP1 and NRF2 contribute at least in
part to RIPK3-mediated regulation of gene expression in CXCL4
and TLR8 costimulation of human monocytes.
3.6 RIPK3 and RIPK1 regulate NLRP3
inammasome activation in CXCL4 and TLR8
costimulation
As RIPK3, together with RIPK1 and caspase-8, has been shown to
contribute to noncanonical NLRP3 inammasome activation (i.e.
independent of second activation signals such as ATP and nigeri-
cin),
19,25,52,53
we wished to test whether RIPK3 or RIPK1 contrib-
uted to CXCL4 + TLR8–induced NLRP3 inammasome activation,
which does not require a second activation signal such as ATP
Fig. 3. Transcriptomic analysis of RIPK3 function in the CXCL4 and TLR8 response. (A) K-means clustering (K =4) of differentially expressed genes
induced more than 2-fold with false discovery rate <0.05 in indicated conditions (n =4 healthy donors). (B, C, E, and F) Analysis of signaling pathways
and upstream transcription factors by Ingenuity Pathway Analysis for the genes in clusters 1 and 4 dened in panel A. (D) Heatmap of representative
ISGs regulated by RIPK3 in cluster 4. (G) Venn diagram showing the overlap of XBP1 and NRF2 target genes from panel F. (H) Top signicant signaling
pathways of the 19 overlapping genes in panel G analyzed with the Enrichr program. SLE =systemic lupus erythematosus.
Yang et al. |7
or nigericin.
10
RIPK3 can be activated by recruitment to RHIM do-
main–containing signaling adaptors or in a cytoplasmic complex
with RIPK1 and caspase-8, in which RIPK1 promotes RIPK3 activa-
tion and caspase-8 restrains this activation by proteolytic cleav-
age. Thus, inhibition of caspase-8 potentiates RIPK3 activation
by RIPK1 in the RIPK1-RIPK3-Caspase-8 complex.
16
CXCL4 + TLR8–induced inammasome activation, as assessed by
cleavage of GSDMD, caspase-1, or IL-1βamounts in culture superna-
tants, was decreased upon inhibition of RIPK3 using GSK-872 alone
(in the absence of stabilization of the RIPK1-RIPK3-caspase-8 com-
plex) (Fig. 6A, lane 6; Fig. 6B, column 7; Fig. 6C, lane 8). In contrast
to RIPK3, inhibition of RIPK1 using necrostatin-1s alone had minimal
effect on IL-1 production (Fig. 6B). However, when caspases were inhib-
ited using pan caspase inhibitor zVAD to activate the RIPK1/RIPK3/
caspase-8 axis, a signicant inhibitory effect of necrostatin-1s on
IL-1 production was observed (Fig. 6B, column 5). Overall, the results
implicate RIPK3 in inammasome activation in CXCL4 + TLR8–stimu-
lated human monocytes; in this system, RIPK3 activity did not require
inhibition of caspase-8, which is consistent with the results presented
previously in Fig. 1. Our results also suggest that, in contrast to RIPK3,
activation of RIPK1 by CXCL4 + TLR8 requires inhibition of caspase-8.
Interestingly, addition of exogenous ATP as a second signal in-
creased IL-1βproduction and caspase-1 cleavage in CXCL4 +
TLR8–stimulated monocytes under conditions of RIPK3 inhibition
(Fig. 6D, E), which is consistent with the notion that RIPK3 signal-
ing contributes to the second activation signal. In contrast to in-
ammasome activation, CXCL4 + TLR8–induced CXCL10 and TNF
expression that was sensitive to RIPK3 inhibition was not affected
by RIPK1 and/or caspase inhibition (Fig. 6F). Similar to CXCL10 and
TNF expression, RIPK1 or caspase inhibition did not substantially
or consistently affect the induction of NLRP3 and IL-1βprecursor
(Fig. 6C and data not shown), which occur at the level of gene activa-
tion; the role of RIPK1 in this system merits further study.
Activation of RIPK3 in a RIPK1/RIPK3/caspase-8 complex typic-
ally occurs downstream of TNF family receptors, requires inhib-
ition of caspase-8 in in vitro cell culture systems, and activates
MLKL, which not only induces necroptotic cell death, but also
can contribute to inammasome activation.
16,54,55
We conrmed
this model in our system using TNF stimulation of primary human
monocytes and found a large increase in cell death after caspase-8
inhibition that was sensitive to MLKL inhibition using low-dose
NSA (0.5 μM) (Supplementary Fig. 6A). In contrast to TNF,
CXCL4 + TLR8–induced cell death occurred in the absence of
caspase-8 inhibition and was not increased by caspase-8 inhib-
ition (Supplementary Fig. 6B, C), which is consistent with an alter-
native activation pathway. However, under conditions of
caspase-8 inhibition, cell death was decreased by inhibition of
RIPK1 and RIPK3 (Supplementary Fig. 6B, C). Furthermore, when
caspase-8 was inhibited, cell death was decreased in a dose-
dependent way by NSA, which inhibits MLKL at a low concentra-
tion (0.5 μM) but begins to inhibit GSDMD, which induces pyrop-
totic cell death, at higher concentrations (5 μM) (Supplementary
Fig. 6C). Overall, these ndings are consistent with both pyroptosis
and necroptosis contributing to CXCL4 + TLR8–induced cell death,
at least under conditions of caspase inhibition, although this re-
quires further investigation. Distinct types of cell death can occur
Fig. 4. PI3K-Akt signaling is dependent on RIPK3 and regulates ISGs and inammatory genes. (A) Immunoblot of phospho-AKT and AKT using whole
cell lysates of cells stimulated (CXCL4 +ORN8L) in the presence/absence of GSK872 for 3 h. A representative blot for RIPK3 inhibition from 5
independent experiments; see also related data in Fig. S4A. (B, C) mRNA was measured by qPCR and normalized relative to GAPDH mRNA in CXCL4- and
ORN8L-stimulated cells with/without PI3K inhibitor Wortmannin (Wort) (B) and combined inhibitors of PI3Kα/β/δ/γ(C) for 6 h. Data are depicted as
mean ±SEM of 6 (B) and 7 (C) healthy donors. ***P≤0.001; *P≤0.05 by paired ttest (C) or by Wilcoxon matched signed rank test (D). DMSO, dimethyl
sulfoxide.
8|Journal of Leukocyte Biology, 2023, Vol. 00, No. 0
concomitantly and can be difcult to distinguish, which is in ac-
cordance with emerging ideas about panoptosis.
56
4. Discussion
CXCL4 and endosomal TLR signaling crosstalk augments TLR re-
sponses, which may play important roles in multiple inammatory
and autoimmune disorders.
3,10,11,13,57–60
Recently, we identied
signaling and epigenomic mechanisms by which CXCL4 boosts
TLR8-mediated inammatory gene induction in human mono-
cytes.
10
The major advances of the current study are to identify a
role for RIPK3 in CXCL4 + TLR8–mediated responses, and to dene
activation of a RIPK3-Akt-stress pathway as a mechanism by which
CXCL4 negatively regulates aspects of the TLR8 response, most not-
ably ISG and IL12B induction. Moreover, RIPK3 plays a dichotomous
role in regulating CXCL4 and TLR8 responses, as it also contributes
Fig. 5. XBP1 and NRF2 activation contributes to RIPK3-AKT mediated regulation of gene expression. (A, B, and D) BIP,CHOP, and s-XBP1/XBP1 mRNA was
measured by qPCR and normalized relative to GAPDH mRNA (n =6 for panels A and B, n =8 for panel D). (C, E) Representative ow plot and bar graph
showing phospho-NRF2 in the indicated conditions. (F–H) Gene expression was evaluated by qPCR and normalized relative to GAPDH mRNA in the
indicated conditions at 6 h (at least 6 healthy donors). Data are depicted as mean ±SEM. The representative ow plots are from at least 3 independent
experiments (C and E). **P≤0.01; *P≤0.05 by 1-way analysis of variance with Tukey’s post hoc test for multiple comparisons (A, B, D, and F) or by
Wilcoxon matched signed rank test (G and H).
Yang et al. |9
to increased expression of inammatory genes such as TNF and
IL1B, and NLRP3 inammasome activation. Negative regulation of
IFN responses by RIPK3 targets STAT1 activation and involves
PI3K-Akt activity that promotes XBP1-mediated ER stress and
NRF2-mediated oxidative responses. The PI3K-Akt, XBP1, and
NRF2 pathways also had a dichotomous role, as they promoted
the expression of inammatory genes while attenuating the IFN re-
sponse. These results implicate a RIPK3-mediated signaling axis in
regulating the balance between inammatory gene, ISG, and in-
ammasome activation, which is important for mounting effective
host defense while limiting concomitant inammatory pathology.
Potentiation of TLR8-induced inammatory responses by
CXCL4 has been attributed to a chaperone function that delivers
increased amounts of NA ligands to endolysosomal locations to
increase TLR8 activation, through synergistic activation of TBK1
signaling that drives inammatory outcomes via IRF5-mediated
gene induction and inammasome-mediated IL-1βproduction,
and by genome-wide regulation of chromatin accessibility,
10
but
signaling pathways in regulation of CXCL4 and TLR8–induced
IFN response and other inammatory gene induction such as
TNF and IL1βas well as NLRP3 inammasome activation have
not been very well dened. RIPK3 plays critical role in a programed
cell death, namely necroptosis, which contributes to effective
host defense against intracellular pathogen infections while un-
controlled necroptosis also causes tissue damage and host injury.
In addition to driving necroptosis, RIPK3 is upregulated in many
conditions,
27,61
and mounting evidence shows that RIPK3 plays
roles in the activation signal–independent NLRP3 inammasome
activation, inammatory diseases, and tumorigenesis.
21,25,32
Instead of increasing RIPK3 expression, CXCL4 and TLR8 signaling
induced RIPK3 activation in human primary monocytes, which
can occur independently of RIPK1. In contrast to synergistic
Fig. 6. CASP8-RIPK1-RIPK3 axis regulates NLRP3 inammasome. (A) Immunoblot of GSDMD with whole cell lysates in the indicated conditions at 6 h.
(B, D) IL-1βprotein concentrations in culture supernatants were measured by enzyme-linked immunosorbent assay after inhibition of CASP8, RIPK1,
and/or RIPK3 (B) or after additionally adding ATP (5 mM) (D) (n =4–6 healthy donors). (C) Immunoblot of NLRP3, IL-1β, and cleaved CASP1 using whole
cell lysates in the indicated conditions at 6 h. The inhibitors targeting caspases, RIPK1, and RIPK3 are zVAD (50 μM), necrostatin-1 s (30 μM), and GSK872
(10 μM), respectively. (E) Immunoblot of cleaved CASP1 using whole cell lysates in the indicated conditions. (F) CXCL10,TNF, and IL12B mRNA was
measured by qPCR and normalized relative to GAPDH mRNA (n =5). Data are depicted as 1 of at least 3 representative blots (A, C, and E) and as mean ±
SEM for the rest. ****P≤0.0001; **P≤0.01; *P≤0.05 by 1-way analysis of variance with Tukey’s post hoc test for multiple comparisons (B, D, and F).
10 |Journal of Leukocyte Biology, 2023, Vol. 00, No. 0
activation of TBK1 and IRF5 by CXCL4 + TLR8 costimulation,
10
synergistic activation of P-RIPK3 by costimulation relative to acti-
vation by single agent treatments was not observed. This suggests
that RIPK3 activity plays more of a regulatory role, being required
for full activation of inammatory genes and the inammasome,
while modulating and attenuating expression of other genes such
as ISGs. Further, we show that the activation of RIPK3 by CXCL4 +
TLR8 costimulation relies on HSP90 and autophosphorylation. It
has been reported that HSP90 activates RIPK3 through an inter-
action that stabilizes RIPK3
38,39
; however, at the 6-h time point
where we conducted the experiments, blockade of HSP90 activity
did not affect RIPK3 protein amounts. Thus, we speculate that
HSP90 may act as a platform for RIPK3 conjugation and autophos-
phorylation. Additionally, the specic kinase inhibitor of RIPK3,
GSK872, impeded CXCL4 and TLR8 signaling-induced RIPK3 phos-
phorylation, suggesting that the activation of RIPK3 we observed
could be caused by RIPK3 autophosphorylation. We speculate
that RIPK3 activation contributes to CXCL4 and TLR8–mediated
synergistic effects. Inhibiting the kinase function of RIPK3 skewed
the CXCL4 and TLR8–induced gene expression pattern: reducing
the expression of inammatory gene TNF while increasing ISG
CXCL10 and T cell–activating cytokine IL-12 subunit IL12B expres-
sion. In line with a previous study that RIPK3 activates NF-κB and
increases its target inammatory gene expression,
26
ectopic ex-
pression of RIPK3 increased CXCL4 + TLR8–induced NF-κB acti-
vation and its target inammatory genes TNF,IL6, and IL1B
but not IL12B. This suggests that there might exist a
RIPK3-mediated suppressive pathway to balance NF-κB activa-
tion to attenuate IL12B expression in RIPK3 overexpressing cells.
The role of RIPK3 in regulation of STAT1 activation and ISG ex-
pression in CXCL4 + TLR8 costimulation is also recapitulated in
RIPK3-overexpressing cells. Moreover, administration of
exogenous IFN-βonly partially rescued STAT1 activation and
ISG expression in CXCL4 + TLR8–stimulated cells. However, in
the cells that did not experience CXCL4 + TLR8 costimulation,
overexpression RIPK3 cannot suppress exogenous IFN-βre-
sponse, suggesting that CXCL4 + TLR8 costimulation may induce
a cofactor(s) that cooperates with RIPK3 to regulate
IFN-mediated STAT activation, and that RIPK3 acquires the abil-
ity to regulate IFN response only in certain situations, which
will be interesting to explore in future work.
Crosstalk between the RIPK and JAK-STAT pathways has been
previously described, e.g. Yu et al.,
62
which shows that a
RIPK1-RIPK3 complex enhances IFN-γ-induced STAT1 activation
and downstream gene expression in intestinal epithelial cells.
This enhancement of IFN-γsignaling occurred via a proximal sig-
naling mechanism, namely interaction of RIPK1-RIPK3 with JAK1,
which increased STAT1 activation. In contrast, in our system us-
ing human monocytes, RIPK3 suppresses autocrine IFN-I re-
sponses by downstream activation of the PI3K-Akt and NRF2
pathways. Interaction between these 2 signaling pathways is like-
ly to be complex and deserves future study.
One strength of our study is the use of freshly isolated primary
human monocytes ex vivo, which closely reects physiological
regulation of primary cells. We previously found that as mono-
cytes differentiate into macrophages during culture ex vivo, regu-
lation of inammasome activation changes as cells lose the ability
to produce CXCL4 + TLR8–induced mature IL-1βwithout addition
of a second signal such as ATP.
10
Interestingly, the regulation of
CXCL4 + TLR8 responses by RIPK3 also wanes during culture.
This leads to a technical limitation of our system for studying
RIPK3 function, as it precludes the ability to use genetic ap-
proaches such as short interfering RNA to study the RIPK3 func-
tions described in this study.
Fig. 7. RIPK3 regulates the prole and balance of the CXCL4-costimulated TLR8 response. RIPK3 provides signal 2 for inammasome activation and
regulates stress pathways to amplify induction of inammatory genes while suppressing STAT1 activation and IFN responses. The gure was created
with BioRender.com.
Yang et al. |11
The PI3K-Akt pathway regulates macrophage survival, migra-
tion, and proliferation and also participates in cell metabolism
and regulation of inammatory responses to TLR signals.
63,64
The PI3K-Akt pathway restricts macrophage overreaction to TLR
signals,
64
but it can enhance IFN and ISG expression for host de-
fense against intracellular pathogen infection.
65,66
However, in
the current study, instead of suppressing CXCL4 + TLR8–induced
inammatory gene expression, surprisingly, the PI3K-Akt path-
way plays a more complex role in a dichotomous way, similar to
RIPK3, to regulate TNF,IL12B, and ISG expression. Consistent
with previous studies that RIPK3 can activate the Akt pathway
in inammatory conditions,
27
RIPK3 is also important for
PI3K-Akt pathway activity in CXCL4 and TLR8 costimulation in
human monocytes. PI3K-Akt activation induced by TLR4
67
regu-
lates inammatory responses to overcome excessive inamma-
tion and tissue damage.
43,64
However, the unexpected activation
of PI3K-Akt pathway by RIPK3 in the context of costimulation of
TLR8 signaling with CXCL4 might execute different functions
such as balancing IFN response while contributing to inamma-
tory gene expression and promoting tissue brosis.
27
Our transcriptomic analysis and functional experiments indicate
that RIPK3 activates ER stress responses and NRF2 oxidative re-
sponse after CXCL4 and TLR8 costimulation of human monocytes.
Stress pathway activation may reect hyperactivation of monocytes
during CXCL4 and TLR8 costimulation, as adequate ER stress re-
sponses and NRF2 oxidative response are protective mechanisms
to maintain cellular homeostasis and cell survival.
68,69
However, un-
controlled or excessive activation of ER stress and NRF2 oxidative re-
sponses also contribute to inammation, contribute to cell death,
and suppress antitumoral innate and adaptive immunity.
42,68,70
In
the current study, XBP1-mediated ER stress response coordinated
with NRF2 oxidative response to contribute to RIPK3-mediated in-
creases in inammatory gene expression, while suppressing IFN re-
sponse. IFN response is not only important for antipathogen
response, but also critical for antitumor immunity through activat-
ing tumor microenvironment immune cells.
71
Thus, we speculate
that RIPK3-activated XBP1 and NRF2 might play pleiotropic roles
in inammatory diseases and host defense. How the activation of
ER stress response and NRF2 is initiated in disease context (such
as inammatory diseases, tumorigenesis, and infections) is still
not well dened. The PI3K-Akt pathway has been shown play critical
roles in activating ER stress and NRF2 in multiple conditions, such as
inammation and cell survival,
44–46,51
which is recapitulated in
CXCL4 and TLR8 signaling crosstalk in human primary monocytes
in this study. The study reveals that RIPK3 can initiate signaling
by a PI3K-Akt-XBP1/NRF2 axis to modulate cellular responses.
In general, NLRP3 inammasome activation requires 2 signals:
a priming signal to upregulate the expression of inammasome
components NLRP3, caspase-1, and pro-IL-1β, and a second signal
to promote the inammasome complex assembly and activation
of caspase-1, thereby cleaving pro-IL-1βto mature IL-1βand
GSDMD to active N-terminal GSDMD, which induces plasma
membrane pores that allow IL-1βsecretion and can lead to pyrop-
totic cell death.
25,52,72,73
In line with many studies,
22,25,52,73
we
demonstrate that RIPK3 and the caspase-8–RIPK1/3 axis contrib-
ute to an alternative NLRP3 inammasome activation without af-
fecting inammasome component expression and the canonical
second step of NLRP3 inammasome activation by exogenous
ATP. RIPK3 seems to play a key role in the NLRP3 inammasome
activation because inhibition of RIPK3 signicantly affected
NLRP3 inammasome activation, while the involvement of
RIPK1 only occurred when caspases were inhibited, which is in ac-
cordance with the established model that caspase-8 regulates
RIPK1 kinase activation.
16
Our study suggests that a distinct path-
way from the RIPK1/RIPK3/Caspase-8 complex activates RIPK3,
and RIPK3 plays a key role in CXCL4 + TLR8–induced NLRP3 in-
ammasome activation and balance of IFN response and inam-
matory gene expression.
23,27,74,75
Of the 3 RIPK3-regulated downstream outcomes of CXCL4 +
TLR8 signaling assessed in the study—gene expression, inamma-
some activation, and cell death—gene expression was entirely un-
affected by RIPK1 inhibition, and inammasome activation and
cell death were modestly or minimally affected by RIPK1 inhib-
ition under standard culture conditions, in the absence of a
caspase-8 inhibitor. This argues for a predominant RIPK3 activa-
tion mechanism by CXCL4 + TLR8 that occurs independently of
RIPK1 and the RIPK1/RIPK3/caspase-8 complex (i.e. activated by
DD-containing receptors). Activation of RIPK3 independently of
RIPK1 typically occurs via recruitment to RHIM domain–contain-
ing signaling molecules such as TRIF and ZBP1
16
; this activation
mechanism can also involve interaction with HSP90 and RIPK3 au-
tophosphorylation.
37–39
Our study supports a role for HSP90 in
RIPK3 activation after CXCL4 + TLR8 costimulation, and future
work will address interaction of RIPK3 with RHIM-containing or
other signaling molecules activated by receptors engaged by
CXCL4-ssRNA complexes.
10
In cell culture systems, activation of
RIPK3 by the RIPK1/RIPK3/caspase-8 complex typically occurs
downstream of DD-containing receptors of the TNFR family and
requires pharmacological inhibition of caspase-8, which prevents
inactivating cleavage of these kinases. Interestingly, even when
caspases were inhibited using the standard approach of adding
zVAD in our system, RIPK1 played no role in regulation of gene ex-
pression; however, these conditions revealed a role for RIPK1 in in-
ammasome activation and cell death. It is possible that CXCL4 +
TLR8 activates this caspase-8 and RIPK1–mediated pathway indir-
ectly via induction of DD-containing receptors and or ligands.
In summary, RIPK3 modulates the CXCL4 and TLR8 response in
human monocytes by suppressing STAT1 activation and activating
NLRP3 and a PI3K-Akt-XBP1/NRF2 pathway that augments expres-
sion of select inammatory genes while suppressing the IFN re-
sponse (Fig. 7). These ndings provide potential mechanisms by
which RIPK3 calibrates TLR responses in inammatory diseases
and tumorigenesis, and suggest that RIPK3-PI3K-Akt-XBP1/NRF2
axis–mediated stress responses can be targeted for suppression of
inammation while preserving the IFN response for host defense
against pathogen infections.
Acknowledgments
The authors thank the Weill Cornell Medicine Genomic Resources
Core Facility for next-generation sequencing, the Weill Cornell
Medicine–HSS Flow Cytometry Core Facility for ow cytometry
support, and David Oliver (HSS Genomics Center) for advice and
discussions.
Author contributions
C.Y. conceptualized, designed, and performed most of the experi-
ments; performed bioinformatic analysis; prepared the gures; and
wrote the manuscript. R.Y. and C.B. contributed to experiments
and Y.D. and M.D.A.K. contributed experimental expertise and pro-
vided advice. F.J.B. contributed expertise and intellectual input and
edited the manuscript. L.B.I. oversaw the study and edited the manu-
script. All authors reviewed and provided input on the manuscript.
12 |Journal of Leukocyte Biology, 2023, Vol. 00, No. 0
Supplementary material
Supplementary materials are available at Journal of Leukocyte
Biology online.
Funding
This work was supported by grants from the National Institutes of
Health (L.B.I), from the National Institutes of Health
1R01AI132447 (F.J.B.), the Scleroderma Research Foundation
(F.J.B.), and the Scleroderma Foundation (F.J.B.). The David
Z. Rosensweig Genomics Center at HSS is supported by the Tow
Foundation.
Conict of interest statement. F.J.B. is a founder of IpiNovyx, a
startup biotechnology company. The other authors declare no
relevant conicts of interest.
Data availability
The datasets that support the ndings of this study and were gen-
erated by the authors as part of this study have been deposited in
the Gene Expression Omnibus database with the accession code
GEO: GSE201909. Otherwise, the data are either contained within
the manuscript or available from the authors on request.
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