Macrophage RIPK3 triggers inflammation and cell death via the XBP1–Foxo1 axis in liver ischaemia–reperfusion injury

Background & Aims Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) is a central player in triggering necroptotic cell death. However, whether macrophage RIPK3 may regulate NOD1-dependent inflammation and calcineurin/transient receptor potential cation channel subfamily M member 7 (TRPM7)-induced hepatocyte death in oxidative stress-induced liver inflammatory injury remains elusive. Methods A mouse model of hepatic ischaemia–reperfusion (IR) injury, the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific RIPK3 knockout (RIPK3M-KO) and RIPK3-proficient (RIPK3FL/FL) mice. Results RIPK3M-KO diminished IR stress-induced liver damage with reduced serum alanine aminotransferase/aspartate aminotransferase levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the RIPK3FL/FL controls. IR stress activated RIPK3, inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α), x-box binding protein 1 (XBP1), nucleotide-binding oligomerisation domain-containing protein 1 (NOD1), NF-κB, forkhead box O1 (Foxo1), calcineurin A, and TRPM7 in ischaemic livers. Conversely, RIPK3M-KO depressed IRE1α, XBP1, NOD1, calcineurin A, and TRPM7 activation with reduced serum tumour necrosis factor α (TNF-α) levels. Moreover, Foxo1M-KO alleviated IR-induced liver injury with reduced NOD1 and TRPM7 expression. Interestingly, chromatin immunoprecipitation coupled with massively parallel sequencing revealed that macrophage Foxo1 colocalised with XBP1 and activated its target gene Zc3h15 (zinc finger CCCH domain-containing protein 15). Activating macrophage XBP1 enhanced Zc3h15, NOD1, and NF-κB activity. However, disruption of macrophage Zc3h15 inhibited NOD1 and hepatocyte calcineurin/TRPM7 activation, with reduced reactive oxygen species production and lactate dehydrogenase release after macrophage/hepatocyte coculture. Furthermore, adoptive transfer of Zc3h15-expressing macrophages in RIPK3M-KO mice augmented IR-triggered liver inflammation and cell death. Conclusions Macrophage RIPK3 activates the IRE1α–XBP1 pathway and Foxo1 signalling in IR-stress livers. The XBP1–Foxo1 interaction is essential for modulating target gene Zc3h15 function, which is crucial for the control of NOD1 and calcineurin-mediated TRPM7 activation. XBP1 functions as a transcriptional coactivator of Foxo1 in regulating NOD1-driven liver inflammation and calcineurin/TRPM7-induced cell death. Our findings underscore a novel role of macrophage RIPK3 in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases. Impact and implications Macrophage RIPK3 promotes NOD1-dependent inflammation and calcineurin/TRPM7-induced cell death cascade by triggering the XBP1–Foxo1 axis and its target gene Zc3h15, which is crucial for activating NOD1 and calcineurin/TRPM7 function, implying the potential therapeutic targets in stress-induced liver inflammatory injury.


Introduction
Hepatic inflammation and injury initiated by ischaemia and reperfusion (IR) are the leading cause of hepatic dysfunction and failure following liver transplantation, resection, and haemorrhagic shock. 1 Endoplasmic reticulum (ER) and oxidative stress are the most important pathological mechanisms in IR injury (IRI), which causes inflammation and cell death through multiple pathways. 2][5] Nucleotide-binding oligomerisation domain-containing protein 1 (NOD1), a member of the NOD-like receptor family of cytosolic pattern recognition receptors, has been recognised as a crucial sensor of the innate immune system in response to invading pathogens and stress signals. 6,7Upon activation, NOD1 mediates distinct cellular responses.It initiates signal transduction mechanisms, including stimulation of NF-jB, mitogen-activated protein kinases (MAPKs), interferon regulatory factors, and programmed cell death.NOD1 stimulation recruited inflammatory cells and induced chemokine production. 8Activation of NOD1 increased macrophage accumulation and contributed to the progression of cardiovascular inflammation, 9 whereas disruption of NOD1 ameliorated vascular inflammation-mediated injury. 10oreover, cellular stress promoted NOD1-dependent inflammation by triggering NF-jB activation. 11ROS may induce NOD1 activation with pro-inflammatory gene expression in response to ER or oxidative stress. 6,12These results indicate that NOD1 signalling is critical in mediating innate immune activation during a stress-induced inflammatory response.
Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) contains a C-terminal domain unique from other RIP family members.The encoded protein is primarily localised to the cytoplasm. 13IPK3 can form a complex with tumour necrosis factor receptor 1 (TNFR1) to induce necroptosis by interaction with RIPK1 and mixed lineage kinase domain-like pseudokinase (MLKL). 14,15Indeed, the initiation of necroptosis is involved in the ligation of TNFR1, which binds to tumour necrosis factor (TNF). 15 Activation of RIPK3 phosphorylates the pseudokinase MLKL, which is translocated into the inner leaflet of the plasma membrane. 16Moreover, a transient disruption in membrane integrity results in an abrupt calcium influx.Increased mitochondrial Ca 2+ accumulation could result in cell death mediated by transient receptor potential cation channel subfamily M member 7 (TRPM7) under cell stress conditions. 17,18nterestingly, RIPK3 can also trigger inflammatory signalling pathways, especially in tissue injury and sterile inflammation. 19Activation of RIPK3 promoted inflammatory injury in non-alcoholic steatohepatitis, 20 whereas disruption of RIPK3 dampened inflammation and non-alcoholic steatohepatitis progression. 21Moreover, RIPK3 interacted with Toll-like receptor 4 (TLR4) to activate NF-jB and pro-inflammatory cytokine production in response to cell stress. 22Conversely, RIPK3 deletion prevented ER stress-induced cell death. 23Although the role of RIPK3-mediated necroptosis and inflammation has been characterised, we know very little about the mechanism of macrophage RIPK3 in regulating NOD1 function and TRPM7-induced cell death in IR stress-induced liver inflammatory injury.
Here, we identify a novel regulatory mechanism of macrophage RIPK3 on NOD1 function and the TRPM7-mediated cell death pathway in IR stress-induced liver inflammation.We demonstrate that macrophage RIPK3 modulates NOD1 and calcineurin-mediated TRPM7 activation by controlling the x-box binding protein 1 (XBP1)-forkhead box O1 (Foxo1) axis and its target gene Zc3h15 (zinc finger CCCH domain-containing protein 15), which is critical in triggering NOD1-driven inflammatory responses and calcineurin/TRPM7-induced hepatocyte death in IR-stressed livers.

Animals
The floxed RIPK3 (RIPK3 FL/FL ) mice (B6;129-RIPK3 tm1.1Fkmc /J) and the mice expressing Cre recombinase under the control of the lysozyme 2 (Lyz2) promoter (LysM-Cre) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA).The targeting vector is designed to insert a loxP site and a Flippase recognition target (FRT)-flanked neomycin resistance (neo) upstream of exon 10.An enhanced green fluorescent protein sequence, followed by a second loxP site, is inserted at the end of the coding region.Flp-mediated recombination removed the FRT-flanked neo cassette.This strain was maintained on a mixed 129 and C57BL/6 genetic background.To generate myeloid-specific RIPK3 knockout (RIPK3 M-KO ) mice, a homozygous loxP-flanked RIPK3 mouse was mated with a homozygous Lyz2-Cre mouse to create the F1 mice that were heterozygous for a loxP-flanked RIPK3 allele and heterozygous for the Lyz2-Cre.The F1 mice were then backcrossed to the homozygous loxPflanked RIPK3 mice, resulting in the generation of RIPK3 M-KO mice (25% of the offspring), which were homozygous for the loxPflanked RIPK3 allele and heterozygous for the Lyz2-Cre allele (Fig. S1).The myeloid-specific Foxo1 knockout (Foxo1 M-KO ) mice were generated as described. 1Mouse genotyping was performed using a standard protocol with primers described in the JAX Genotyping Protocol Database.Male mice at 6-8 weeks of age were used in all experiments.This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.Animal protocols were approved by the Institutional Animal Care and Use Committee of The University of California at Los Angeles.

Mouse liver IRI model
We used an established mouse model of warm hepatic ischaemia (90 min) followed by reperfusion (6 h). 3 Mice were injected with heparin (100 U/kg), and an atraumatic clip was used to interrupt the arterial/portal venous blood supply to the cephalad liver lobes.After 90 min of ischaemia, the clip was removed, and mice were sacrificed at 6 h of reperfusion.Some animals were injected via tail vein with Zc3h15-expressing bone marrow-derived macrophages (BMMs) or control cells (1 × 10 6 cells in 0.1 ml of PBS/mouse) 24 h before ischaemia.

Statistical analysis
Data are expressed as mean ± SD and analysed using a permutation t test and Pearson correlation.Per comparison, two-sided p values less than 0.05 were considered statistically significant.Multiple group comparisons were made using one-way ANOVA followed by Bonferroni's post hoc test.When groups showed unequal variances, we applied Welch's ANOVA to make various group comparisons.All analyses were performed using SAS/STAT software, version 9.4.
For further details regarding the materials and methods used, please refer to the Supplementary CTAT Table and Supplementary information.

Results
Myeloid-specific RIPK3 deficiency alleviates IR-induced liver damage and diminishes macrophage/neutrophil infiltration and pro-inflammatory mediators in IR-stressed liver The myeloid-specific RIPK3-deficient (RIPK3 M-KO ) and RIPK3proficient (RIPK3 FL/FL ) mice were subjected to 90 min of warm ischaemia followed by 6 h of reperfusion.The primary hepatocytes and liver macrophages (Kupffer cells) were isolated from these ischaemic livers.Indeed, the RIPK3 expression was undetectable in liver macrophages but not in hepatocytes of RIPK3 M-KO mice (Fig. 1A).The liver damage was assessed using Suzuki's histological grading of liver IRI 24 (Fig. 1B).The RIPK3 FL/FL livers showed severe oedema, sinusoidal congestion, and extensive hepatocellular necrosis.In contrast, The RIPK3 M-KO livers displayed mild to moderate oedema, sinusoidal congestion, and mild necrosis (Fig. 1B).The liver function was measured by the serum alanine aminotransferase (sALT) and serum aspartate aminotransferase (sAST) levels.RIPK3 M- KO significantly decreased sALT and sAST levels at 6 h post liver reperfusion compared with the RIPK3 FL/FL controls (Fig. 1C).Moreover, RIPK3 M-KO markedly decreased accumulation of CD11b + macrophages (Fig. 1D) and Ly6G + neutrophils (Fig. 1E), with reduced mRNA levels of tumour necrosis factor a (TNF-ɑ), IL-1b, IL-6, C-X-C motif chemokine ligand 10 (CXCL-10), and monocyte chemoattractant protein 1 (MCP-1) in ischaemic livers and liver macrophages (Fig. 1F and Fig. S6).These results suggest that RIPK3 contributes to IR stress-induced liver inflammation and injury.

XBP1 interacts with Foxo1 and regulates NOD1 activation in macrophages
As IR stress activated the IRE1a-XBP1 pathway and Foxo1 signalling in ischaemic livers, we examined whether there is a crosstalk between the IRE1a-XBP1 pathway and Foxo1 signalling during an inflammatory response.Indeed, immunofluorescence staining showed increased nuclear XBP1s (Fig. 4A) and Foxo1 (Fig. 4B) expression in lipopolysaccharide (LPS)-stimulated BMMs.Interestingly, XBP1s and Foxo1 were colocalised in the nucleus (Fig. 4C).This result was further confirmed by Western blot assay, which showed that increased nuclear XBP1s and Foxo1 protein expression in macrophages after LPS stimulation (Fig. 4D).We next used a co-immunoprecipitation assay to detect the interaction between XBP1s and Foxo1 under inflammatory conditions.Strikingly, co-immunoprecipitation analysis revealed that XBP1s bound to endogenous Foxo1 in macrophages after LPS stimulation (Fig. 4E).Moreover, disruption of RIPK3 depressed NOD1 and P65 activation in LPS-stimulated RIPK3 M-KO macrophages (Fig. 4F).
Hence, these results suggest that the macrophage XBP1s-Foxo1 axis is crucial for the NOD1-driven inflammatory response in RIPK3-mediated immune regulation.

The XBP1-Foxo1 axis targets Zc3h15 and modulates NOD1driven inflammatory response in macrophages
To explore the potential mechanism of the XBP1-Foxo1 axis in the modulation of NOD1 function in macrophages, we performed Foxo1 chromatin immunoprecipitation (ChIP) coupled to massively parallel sequencing (ChIP-seq).Indeed, Foxo1 ChIP-seq peaks were identified within the Zc3h15 gene.One was located in the promoter region, and the others were within the intron or exon (Fig. 5A).To validate the ChIP-seq peak in the Zc3h15 promoter region, ChIP-PCR was performed using Foxo1 and XBP1s antibodies in macrophages.The primer was designed to detect the Foxo1 DNA-binding site in the Zc3h15 promoter by PCR analysis.
Collectively, these data indicate the critical roles of the XBP1-Foxo1 axis and its target gene Zc3h15 in the modulation of NOD1driven inflammatory responses.

XBP1 is required for the Foxo1-targeted Zc3h15 activation and NOD1 function in macrophages
To elucidate the mechanistic role of the XBP1 in RIPK3-mediated immune regulation, we used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated XBP1 KO or activation approach.Immunofluorescence staining revealed that Foxo1-targeted Zc3h15 expression was reduced in LPS-stimulated RIPK3 FL/FL macrophages after transfection with CRISPR/Cas9-mediated XBP1 KO vector (Fig. 6A).CRISPR/Cas9-mediated XBP1 KO inhibited the protein expression of NOD1 and p-P65 in RIPK3 FL/FL macrophages after LPS stimulation (Fig. 6B).However, activation of XBP1 augme-nted Zc3h15 expression in LPS-stimulated RIPK3 M-KO macrophages, evidenced by immunofluorescence staining, which showed that CRISPR/ Cas9-mediated XBP1 activation increased Zc3h15 expression (Fig. 6C), with enhanced NOD1 and P-65 activation (Fig. 6D) in RIPK3-deficient macrophages after LPS stimulation.Interestingly, the Zc3h15 expression was significantly increased in Foxo1 FL/FL macrophages but not in Foxo1 M-KO macrophages after LPS stimulation (Fig. 6E and F), suggesting that the XBP1 is a key coactivator in mediating Foxo1-targeted Zc3h15 activation in RIPK3mediated immune and inflammatory response.Zc3h15 is crucial to regulate NOD1-driven inflammatory response and calcineurin/TRPM7-induced cell death in response to oxidative stress To further test the functional role of Zc3h15 in regulating macrophage NOD1 function and hepatocyte calcineurin/TRPM7 activation under cell stress conditions, we used a macrophage/ hepatocyte coculture system.BMMs from the RIPK3 FL/FL mice were transfected with CRISPR/Cas9-mediated Zc3h15 KO or control vector followed by LPS stimulation and then cocultured with primary hepatocytes supplemented with H 2 O 2 .Indeed, immunofluorescence staining showed that disruption of Zc3h15 reduced NOD1 expression in LPS-stimulated RIPK3 FL/FL macrophages compared with the control vector-treated cells (Fig. 7A).Unlike in control cells, Zc3h15 deletion diminished the protein expression of NOD1 and p-P65 (Fig. 7B), with reduced mRNA levels coding for TNF-a, IL-1b, IL-6, CXCL-2, and CXCL-10 (Fig. 7C) in Zc3h15-deficient macrophages after LPS stimulation.Interestingly, increased Zc3h15 release was observed in the supernatant after coculture of LPS-stimulated RIPK3 FL/FL BMMs with H 2 O 2stressed hepatocytes (Fig. S4A).The coculture of LPS-stimulated BMMs with H 2 O 2 -stressed hepatocytes markedly increased the release of TRPM7 compared with those in hepatocytes exposed to H 2 O 2 alone or exposed to H 2 O 2 plus cocultured BMM without LPS stimulation (Fig. S4C), suggesting that LPS-stimulated macrophages with Zc3h15 release are critical in mediating TRPM7 activation in H 2 O 2 -stressed hepatocytes after coculture.Strikingly, LPS-stimulated Zc3h15-deficient macrophages displayed reduced hepatocyte calcineurin A and TRPM7 expression in hepatocytes with or without H 2 O 2 treatment after coculture (Fig. 7D and Fig. S7A).This result was confirmed by immunofluorescence staining, which showed that disruption of macrophage Zc3h15 reduced hepatocyte TRPM7 expression after coculture (Fig. 7E).Moreover, CRISPR/Cas9-mediated Zc3h15 KO decreased TNF-a release from coculture supernatant (Fig. 7F).Notably, unlike in the control groups, LPS-stimulated Zc3h15-deficient macrophage showed reduced ROS production (Fig. 7G) and LDH release (Fig. 7H) in H 2 O 2 -stressed hepatocyte after coculture.Collectively, these results indicate that macrophage Zc3h15 is a critical regulator in activating NOD1-driven inflammatory response and Calcineurin/TRPM7-induced cell death in response to oxidative stress.

Discussion
This study is the first to document the key role of macrophage RIPK3 signalling in regulating NOD1-driven inflammatory response and calcineurin/TRPM7-induced cell death in IR stressinduced liver injury.There are several principal findings: (i) IR stress activates macrophage IRE1a-XBP1 pathway and Foxo1 signalling in ischaemic livers; (ii) macrophage RIPK3 promotes NOD1 activation and calcineurin/TRPM7-induced hepatocyte death by triggering the XBP1-Foxo1 axis; (iii) the XBP1-Foxo1 interaction is essential for modulating its target gene Zc3h15 function; (iv) XBP1 functions as a transcriptional coactivator of Foxo1 in regulating NOD1 and hepatocyte calcineurin/TRPM7 activation; and (v) Zc3h15 is crucial for NOD1-driven inflammation and calcineurin/TRPM7-induced cell death cascade.Our results highlight the importance of the macrophage RIPK3mediated XBP1-Foxo1-Zc3h15 signalling as a critical regulator of the NOD1 and calcineurin/TRPM7 function in IR-triggered liver inflammation.
Necroptosis is a regulated inflammatory type of cell death by activating RIPK3 and its downstream necroptosis executioner MLKL. 27RIPK3 also functions as a signalling adaptor for the inflammatory response.Loss of membrane integrity results in releasing intracellular immunogenic contents, which induces an inflammatory response, 28 suggesting that necroptosis is a key driver for RIPK3-induced inflammation.Our current study revealed that IR stress induced RIPK3 activation and promoted Foxo1 signalling, with enhanced NOD1 and NF-jB activity in ischaemic livers.Moreover, NOD1 has been linked to ER stress-induced innate immune and inflammatory responses.Indeed, IR stress activated IRE1a, an ER stress sensor.Activation of IRE1a promotes the splicing of the mRNA coding for XBP1, thereby increasing protein XBP1s, a transcription factor that regulates gene expression that is involved in immune function. 29Notably, macrophage RIPK3 deficiency inactivated the IRE1a-XBP1 pathway and depressed NOD1 activation, indicating the distinct ability of macrophage RIPK3 in controlling the IRE1a-XBP1 pathway in ER stress-mediated immune response and inflammatory cascades during liver IRI.
Foxo1 signalling pathway regulates multiple transcriptional targets involved in various cellular processes, including cell survival, stress response, apoptosis, metabolism, and inflammation. 30Increasing Foxo1 activity activated TLR4 or NLR family pyrin domain containing 3 (NLRP3)-driven inflammatory response and tissue injury. 1,31Disruption of Foxo1 signalling reduced susceptibility to cell death induced by oxidative stress. 32nder cell stress conditions, Foxo1 is regulated by JNK, 33 which induces Foxo1 intracellular localisation from the cytoplasm to the nucleus and enhances Foxo1 transcription activity. 33onsistent with this result, we found that IR stress activated JNK, which in turn stimulated Foxo1 nuclear localisation, leading to enhanced Foxo1 transcription activity.Disruption of macrophage Foxo1 reduced IR-induced liver damage with depressed NOD1 and NF-jB activation.Moreover, our in vivo findings showed that RIPK3 activated the IRE1a-XBP1 pathway and augmented XBP1s nuclear translocation in response to IR stress.Thus, we speculate that nuclear localisation of endogenous Foxo1 and XBP1 may be essential for modulating NOD1 activation in IRstressed livers.
induced inflammatory response.Our in vitro study provided further evidence revealing that macrophage XBP1s and Foxo1 colocalised in the nucleus and increased nuclear expression of XBP1s and Foxo1 in response to LPS stimulation.Notably, XBP1s interacted with Foxo1 via direct binding.The ChIP and ChIP-sequencing data further revealed that XBP1s was colocalised with Foxo1 on the promoter of Zc3h15, suggesting that Zc3h15 is a target gene of Foxo1 regulated by the XBP1-Foxo1 complex.Indeed, disruption of XBP1s diminished Zc3h15, whereas activation of XBP1s increased Zc3h15 expression.Furthermore, activation of XBP1s augmented Zc3h15 induction in Foxo1 FL/FL macrophages but not in Foxo1 M-KO macrophages under inflammatory conditions, indicating that XBP1s acts as a transcriptional coactivator of Foxo1 in macrophagemediated inflammatory response.
It is interesting to note that Zc3h15 is critical in activating NOD1-driven inflammatory response in IR-stressed livers.Indeed, Zc3h15 is a highly conserved eukaryotic protein associated with cell growth, transcription, and immune response. 38c3h15 can regulate NF-jB signalling and MAPK activity by interacting with tumour necrosis factor receptor-associated factor 2 (TRAF2). 38TRAF2 is an essential adaptor protein that participates in pro-inflammatory TLR signalling in macrophages. 39merging evidence suggests that several CCCH zinc finger proteins, such as Zc3h15, also function as RNA-binding proteins to induce cytokine production, immune cell activation, and immune homoeostasis by modulating mRNA degradation, phosphorylation, and translation. 40These results suggest that Zc3h15 modulates innate immune response via multiple mechanisms.In line with these findings, we found that RIPK3 FL/FL macrophages displayed increased expression of Zc3h15 and NOD1 under inflammatory conditions.However, disruption of macrophage Zc3h15 inhibited NOD1 and NF-jB activation, with reduced proinflammatory mediators.Furthermore, in vivo adoptive transfer of Zc3h15-expressing macrophages exacerbated IR-induced liver damage and augmented NOD1 activation and ROS production in IR-stressed livers.Thus, our findings reveal a novel role of Zc3h15 in controlling NOD1-driven inflammatory response in RIPK3mediated immune and inflammatory regulation.
Another striking finding was that macrophage RIPK3mediated Zc3h15 could be involved in triggering IR-induced cell death pathways.Indeed, IR stress induces TNF-a and ROS production.ROS generation contributes to TNF-a-induced cell death by inducing mitochondrial membrane permeabilisation. 41s an ion channel and functional kinase, TRPM7 is regulated by calcineurin, 42 a calcium and calmodulin-dependent serine/threonine protein phosphatase. 43Activation of calcineurin increases inward Ca 2+ permeation mediated by TRPM7, leading to increased ROS production under cell stress conditions. 18Moreover, an influx of Ca 2+ into the cytosol also leads to mitochondrial accumulation of Ca 2+ in response to cell stress. 44A Ca 2+ overload links the process of necrosis and apoptosis, which is crucial for the mitochondrial permeability transition.Increased mitochondrial Ca 2+ and ROS generation act synergistically to produce the mitochondrial permeability transition, leading to the structural and functional collapse of mitochondria and cell death. 45In line with these findings, we found that IR stress activated calcineurin and TRPM7, whereas disruption of RIPK3 inhibited calcineurin and TRPM7 activation in IR-stressed livers, suggesting that calcineurin-mediated TRPM7 activation is crucial in RIPK3dependent necroptosis during liver IRI.Our in vitro coculture system provided further evidence showing that macrophage Zc3h15 deficiency diminished TNF-a release and ROS production and inhibited calcineurin and TRPM7 in hepatocytes after coculture.Moreover, in vivo adoptive transfer of Zc3h15expressing macrophages exacerbated IR-induced liver damage with enhanced calcineurin/TRPM7 activity and ROS generation.Thus, our in vitro and in vivo findings reveal the essential role of the macrophage RIPK3-mediated Zc3h15 in modulating the calcineurin/TRPM7-induced cell death cascade in IR stress-induced liver inflammatory injury.

Isolation of primary hepatocytes, Kupffer cells, and bone marrow-derived
macrophages.Primary hepatocytes, Kupffer cells, and BMMs from the RIPK3 FL/FL , RIPK3 M-KO , or wild-type (WT) mice were isolated as described [2].In brief, livers were perfused in situ with warmed (37°C) HBSS solution, followed by a collagenase buffer (collagenase type IV, Sigma-Aldrich).The Perfused livers were dissected and teased through 70-μm nylon mesh cell strainers (BD Biosciences, San Jose, CA).The nonparenchymal cells (NPCs) were separated from hepatocytes by centrifuging at 50 × g 2min three times.The NPCs were then suspended in HBSS and layered onto a 50%/25% two-step Percoll gradient (Sigma) in a 50-ml conical centrifuge tube and centrifuged at 1800 × g at 4°C for 15min.The Kupffer cells in the middle layer were collected and plated to cell culture dishes in DMEM with 10% FBS, 10mM HEPES, 2mM GlutaMax, 100 U/ml penicillin, and 100 μg/ml streptomycin for 15min at 37°C.Murine bone-derived macrophages (BMMs) were generated as described [2].In brief, bone marrow cells were removed from the femurs and tibias of the RIPK3 FL/FL , RIPK3 M-KO , or WT mice and cultured in DMEM supplemented with 10% FCS and 15% L929-conditioned medium for seven days.

Flow cytometry analysis. 1X10 5 primary hepatocytes or liver macrophages (Kupffer cells)
were washed with staining medium (phosphate-buffered saline containing 3% fetal bovine serum), and then incubated with fluorescence-conjugated antibodies.We used the Alexa Fluor 488conjugated anti-ASGR1 polyclonal antibody (ThermoFisher Scientific) to detect hepatocytes, and Alexa Fluor 488 mouse IgG1k isotype control (BD Biosciences) was used.The BD Horizon PE-CF594 rat anti-mouse F4/80 (BD Biosciences) was used for detecting macrophages, and the PEconjugated mouse IgG1k isotype control (BD Biosciences) was also used.The fluorescencelabeled cells were run through a flow cytometer (LSRFortessaX-20, BD Biosciences).All data were analyzed with FlowJo software (Tree Star, Inc.) Co-culture of macrophages and primary hepatocytes.Primary hepatocytes were cultured in 6-well plates at a concentration of 4x10 5 cells per well.After 24h, the 0.4μm-pore size transwell inserts (Corning) containing 1x10 6 BMMs were placed into the 6-well plate with the initially seeded hepatocytes.The co-cultures were incubated for 12h with or without adding H2O2 (200 µM) in the lower chamber.
LDH activity assay.BMMs (1x10 6 ) were cultured with primary hepatocytes (4x10 5 /well) for 12h with or without adding H2O2 (200 µM) in the lower chamber.The activity of lactate dehydrogenase (LDH) in the cell culture medium from the lower chamber was measured with a commercial LDH activity assay kit (Stanbio Laboratory, Boerne, TX) according to manufacturer's instructions.

Immunoprecipitation analysis.
BMMs after LPS stimulation were lysed in NP-40 lysis buffer (50mM Tris pH7.4, 10 mM EDTA, 150 mM NaCl, 1% NP-40, ThermoFisher Scientific) containing protease inhibitors.The lysates were incubated with XBP1s (Cell Signaling Technology), Foxo1 (Cell Signaling Technology), or control IgG and protein A/G beads at 4 o C overnight.After immunoprecipitation, the immunocomplexes were washed with lysis buffer three times and analyzed by standard immunoblot procedures.Chromatin immunoprecipitation (ChIP).The ChIP analysis was carried out using ChIPAssay Kit (Abcam).Briefly, BMMs were treated with 1% formaldehyde for 10 min to cross-link proteins and chromatin.The reaction was stopped by adding 0.125M glycine for 5 min.Cells were washed with ice-cold PBS and then resuspended with ChIP lysis buffer for 10 min.Cell lysates were centrifuged to pellet the nuclei.The cell nuclei were resuspended in nuclei lysis buffer and then subjected to sonication for 15 min.Purified chromatin was analyzed on a 1.5 % agarose gel to analyze DNA fragment size.The sheared chromatin was immunoprecipitated with XBP1s (Cell Signaling Technology) or Foxo1 antibody (Cell Signaling Technology) overnight.As a control, the normal IgG was used as a replacement for XBP1s or Foxo1 antibody.The antibody/chromatin samples were mixed with protein A sepharose beads.Protein-DNA complexes were washed and eluted, followed by a cross-link reversal step, and the resulting DNA was purified.For sequential ChIP, sheared chromatin was first immunoprecipitated with XBP1s antibody, followed by elution

Fig. S4 .
Fig. S4.ELISA analysis of Zc3h15, RIPK3, TRPM7, and LDH assay.Bone marrow-derived macrophages (BMMs) and primary hepatocytes were isolated from the RIPK3 FL/FL mice.(A) ELISA analysis of Zc3h15 in cell supernatant after BMM/hepatocyte co-culture.(B-C) ELISA analysis of RIPK3 and TRPM7 in cell supernatant of hepatocytes, H2O2-treated hepatocytes alone, H2O2-treated hepatocytes plus co-cultured BMM with or without LPS stimulation.(D) LDH assay was performed in the cell culture medium of hepatocytes, H2O2-treated hepatocytes alone, H2O2-treated hepatocytes plus co-cultured BMM with or without LPS stimulation.The data represent the mean±SD.Statistical analysis was performed using a Permutation t-test.*p<0.05,**p<0.01,***p<0.001.

Table S1 :
Primers used in qRT-PCR studies.