Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype

Yuanyuan Ding ,Yu Sun ,Hongyan Wang ,Hongqin Zhao ,Ruihua Yin,Meng Zhang,*,Xudong Pan,*,Xiaoyan Zhu

Abstract Our previous study has demonstrated that lnc_000048 is upregulated in large-artery atherosclerotic stroke and promotes atherosclerosis in ApoE–/– mice.However,little is known about the role of lnc_000048 in classically activated macrophage (M1) polarization.In this study,we established THP-1-derived testing state macrophages (M0),M1 macrophages,and alternately activated macrophages (M2).Real-time fluorescence quantitative PCR was used to verify the expression of marker genes and the expression of lnc_000048 in macrophages.Flow cytometry was used to detect phenotypic proteins (CD11b,CD38,CD80).We generated cell lines with lentivirus-mediated upregulation or downregulation of lnc_000048.Flow cytometry,western blot,and real-time fluorescence quantitative PCR results showed that downregulation of lnc_000048 reduced M1 macrophage polarization and the inflammation response,while over-expression of lnc_000048 led to the opposite effect.Western blot results indicated that lnc_000048 enhanced the activation of the STAT1 pathway and mediated the M1 macrophage polarization.Moreover,catRAPID prediction,RNA-pull down,and mass spectrometry were used to identify and screen the protein kinase RNA-activated (PKR),then catRAPID and RPIseq were used to predict the binding ability of lnc_000048 to PKR.Immunofluorescence (IF)-RNA fluorescence in situ hybridization (FISH) double labeling was performed to verify the subcellular colocalization of lnc_000048 and PKR in the cytoplasm of M1 macrophage.We speculate that lnc_000048 may form stem-loop structure-specific binding and activate PKR by inducing its phosphorylation,leading to activation of STAT1 phosphorylation and thereby enhancing STAT1 pathway-mediated polarization of THP-1 macrophages to M1 and inflammatory factor expression.Taken together,these results reveal that the lnc_000048/PKR/STAT1 axis plays a crucial role in the polarization of M1 macrophages and may be a novel therapeutic target for atherosclerosis alleviation in stroke.

Key Words: atherosclerosis;inflammation;lnc_ 000048;lncRNA;macrophage;polarization;protein kinase RNA-activated (PKR);STAT1

Introduction

Atherosclerotic plaque formation is a chronic inflammatory reaction (Wolf and Ley,2019),and is the primary underlying pathology of ischemic stroke,the second leading cause of death worldwide (Banerjee and Chimowitz,2017;Roth et al.,2020).Atherosclerotic plaque formation results from the deposition of oxidized low-density lipoprotein in the arterial intima and the activation of macrophages (Libby et al.,2019).Macrophages,the most plastic type of immune cells,are polarized to specific phenotypes in response to environmental stimuli (Zhao et al.,2021).Macrophage polarization plays a crucial role in immune function and the inflammatory response (Ding et al.,2021).The number and phenotype of macrophages in atherosclerotic plaques influence atherosclerosis progression (Moore et al.,2013).Classically activated macrophages (M1) play a significant role in the progression of atherosclerosis and promote the formation of necrotic core and unstable plaque in the late stage of atherosclerosis,leading to thrombotic events (Barrett,2020).Inhibiting the polarization of the M1 phenotype and enhancing alternately activated macrophage (M2) polarization was shown to reduce inflammation and atherosclerosis (van Ingen et al.,2021).However,the molecular mechanisms that regulate macrophage polarization in atherosclerosis remain unclear.

Macrophage polarization is a tightly regulated process that involves a series of signaling pathways (Wang et al.,2021).Upon induction of IFN-γ,the JΑK1 kinase phosphorylates STAT1 protein to induce STAT1 activation and further dimerization (Ghafouri-Fard et al.,2021;Goldshmit et al.,2021),which plays a vital role in the polarization of macrophages to the M1 phenotype.The JAK-STAT1/2 pathway promotes M1 macrophage polarization (Li et al.,2018;Hu et al.,2020) and the p38/MΑPK pathway has also been reported to be associated with M1 macrophage polarization(Αbaricia et al.,2020).Αctivation of the PI3K/ΑKT signaling pathway promotes the transformation of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages(He et al.,2022;Wang et al.,2022).STAT6 is associated with M2 macrophage polarization (Li et al.,2018).Additionally,activating TGF-β1/Smad or Wnt/β-catenin pathways regulates the M2 macrophage polarization (Αbaricia et al.,2020;Zhang et al.,2020).Α previous study has shown that neuropeptide Y (NPY) can also affect the expression of macrophage surface markers and secretory profiles,thereby transforming macrophages into M2 phenotype.The NPY signaling pathway may also regulate macrophage balance and atherosclerosis(Profumo et al.,2022).These studies suggest that disruption of the expression or functions of proteins in these pathways will affect macrophage polarization.

Long non-coding RNAs (lncRNAs) are noncoding transcripts of more than 200 nucleotides in length that play a role in various cellular functions and biological processes,such as the inflammatory immune response,differentiation(Qian et al.,2019;Josefs and Boon,2020;Gao et al.,2024)and intracellular communication regulation (Sherazi et al.,2023).LncRNΑ,as key regulatory molecules in all stages of atherosclerosis,can bind to proteins,DNA,and RNA to regulate endothelial cells,vascular smooth muscle cells,and macrophages in atherosclerosis,thereby affecting the growth and stability of atherosclerotic plaques (Josefs and Boon,2020;Ding et al.,2021;Ghafouri-Fard et al.,2021).Thus,lncRNΑs have been identified as a biomarker of atherosclerotic plaque progression and instability and potentially valuable therapeutic targets (Shapouri-Moghaddam et al.,2018;Josefs and Boon,2020).For example,the expression of lncRNACDKN2B-AS1reduces the inflammatory response of arterial plaque and promotes cholesterol efflux from macrophages (Li et al.,2019).Down-regulation of lncRNΑ-MALAT1can reduce macrophage pyroptosis in diabetic rats with atherosclerosis(Han et al.,2018).Additionally,down-regulation of lncRNACox2can reduce the percentage of M1 macrophages and increase the percentage of M2 macrophages (Wang et al.,2021).Research has shown that lncRNAs can regulate the polarization of resting state macrophages (M0) to M1 or M2 phenotype through various mechanisms,thus playing a pro-inflammatory or anti-inflammatory role (Ghafouri-Fard et al.,2021).A recent study analyzed the expression profile of lncRNAs in monocytes/macrophages after stroke and identified a stroke-induced up-regulated lncRNA_SNHG15which promotes the polarization of M2 macrophages and serves as a negative regulator of immunosuppressive inflammation induced by acute ischemic stroke (Sun et al.,2020).Another report has shown that down-regulation oflnc-MRGPRF-1inhibits the polarization of M1 macrophages through the TLR4-MyD88-MAPK signaling pathway,thus regulating the process of atherosclerosis (Hu et al.,2022).

Numerous studies have shown that lncRNAs exert biological functions by directly interacting with RNA-binding proteins(RBPs) (Ferrè et al.,2016;Ding et al.,2021).RBPs contain one or more RNΑ binding domains and influence the fate or function of the bound RNΑ (Hentze et al.,2018).For example,a study showed that lncRNAMAARSinteracts with HuR to induce the apoptosis of late plaque macrophages and reduce their foam formation by regulating p53,p27,caspase-9,and Bcl2 (Simion et al.,2020).SNHG12promotes RNA stability and XIAP transcription of non-small-cell lung cancer cells by binding to the RBP HUR (Tan et al.,2022).

Protein kinase RNA-activated (PKR) is an IFN-induced serine-threonine kinase encoded byEIF2AK2with two conserved double-stranded RNA (dsRNA) binding domains in its N-terminal region,and PKR is activated by autophosphorylation upon dsRNΑ binding or phosphorylation by upstream kinases (Saunders and Barber,2003;Thapa et al.,2013).Α previous study has shown that PKR is also regulated by the noncoding RNAs and related to innate immunity.For example,one study showed that an endogenous circular RNA,which forms a 16–26 bp imperfect RNA duplex,inhibits dsRNΑ-activated PKRs in the innate immune system (Liu et al.,2019).The non-coding RNA nc886 inhibits dsRNA-activated PKR;during T cell stimulation,nc886 participates in nuclear factor kappa-B (NF-κB) signaling and CREB phosphorylation and it also regulates PKR phosphorylation during activation of human monocyte-derived macrophages (Golec et al.,2019).PKR interacts with lncRNAIRENAat two different hairpin sites,which may recruit two PKR monomers to form the PKR2-IRENAcomplex and induce PKR phosphorylation to activate NF-κβ (Liu et al.,2021).However,current research on lncRNΑ regulation of PKR-mediated immune signaling pathways is limited.

In our previous study,we found thatlnc_000048was associated with large artery atherosclerotic stroke by sequencing peripheral blood and exosomal RNΑ from patients with large artery atherosclerotic stroke (Zhang et al.,2021),RNA-seq and gene expression data which can be accessed by the NCBIs Gene Expression Omnibus (GEO,http://www.ncbi.nlm.nih.gov/geo/) and the GEO Series accession number is GSE173719.In vitroandin vivoexperiments demonstrated thatlnc_000048promotes inflammation and collagen degradation in atherosclerotic plaques (Zhang et al.,2023).Whetherlnc_000048influences macrophage polarization has not been examined.

We hypothesized that atherosis-associatedlnc_000048may interact with PKR to enhance STAT1 pathway-mediated polarization of THP-1 macrophages to the M1 phenotype.In this study,we explored the underlying molecular mechanism oflnc_000048interaction with PKR and participation in M1 macrophage polarization.These results may serve as a critical reference for future studies of therapeutic targets for atherosclerosis-related stroke.

Methods

Cell culture and macrophage model construction

The THP-1 human leukemia monocytic cell line has been extensively used to study monocyte/macrophage functions,mechanisms,signaling pathways,and nutrient and drug transport (Chanput et al.,2014).The THP-1 cell line was purchased from Procell Life Science&Technology (Wuhan,China,Cat# CL-0233,RRID: CVCL_0006) and cultured in RPMI-1640 medium (Procell Life Science&Technology,Cat#PM150140) supplemented with 10% fetal bovine serum(FBS,Biological Industries,Israel,Cat# 04-010-1A),1%penicillin-streptomycin (Procell Life Science&Technology,Cat#PB180120) and 0.05 mM β-mercaptoethanol (Procell Life Science&Technology,Cat# PB180633) in an incubator at 37°C with 5% CO2.The medium was replaced with fresh medium every 2 to 3 days.

We generated macrophages following a previous protocol with some modifications (Baxter et al.,2020).Briefly,THP-1 cells were differentiated into M0 macrophages by treatment with 25 ng/mL phorbol-12-myristate 13-acetate (PMΑ;MedChemExpress,Monmouth Junction,NJ,USA,Cat# HY-18739) for 24 hours.M1 macrophages were induced by treatment with 10 ng/mL lipopolysaccharide (LPS;Solarbio,Beijing,China,Cat# L8880) and 20 ng/mL interferon-γ (IFN-γ;PeproTech,Cranbury,NJ,USΑ,Cat# 300-02).M2 macrophages were induced by treatment with 20 ng/mL interleukin-4 (IL-4;PeproTech,Cat# 200-04) and 20 ng/mL interleukin-13 (IL-13)(PeproTech,Cat#200-13) were constructed for 48 hours.

Lentivirus transfection

The GV493 (shRNΑ silencing oflnc_000048) and GV367(overexpression oflnc_000048) lentivirus vectors were purchased from GeneChem Co.Ltd.(Shanghai,China).Logarithmically growing THP-1 cells were inoculated in 12-well plates at 1 × 105cells/mL,and 20 µl of infection enhancement solution was added to cells.According to cell multiplicity of infection (MOI) and the degree of virus drops,virus volume was calculated using the following formula: Virus volume (µL)=MOI × number of cells/virus titer (TU/mL).

THP-1 macrophage cells were divided into the following experimental groups: Sh-lnc_000048-M1 group (transfected with lnc_000048 shRNΑ lentiviral vector,MOI=30),Sh-NC-M1 group (transfected with control shRNA lentiviral vector,MOI=30),Oe-lnc_000048-M1 group,Oe-lnc_000048-M1+C16 group (transfected with lnc_000048 overexpression lentiviral vector,MOI=50),Oe-NC-M1 group,and Oe-NC-M1+C16 group (transfected with control lentiviral vector,MOI=50).Cells were incubated for 12 to 16 hours,and after 72 hours of infection,the cells were cultured in a medium containing 2.0µg/mL puromycin for 48 hours to screen the stable cell lines after infection following the manufacturer’s protocol (https://www.genechem.com.cn/).

Flow cytometry

M0 or M1 macrophages were harvested,washed twice with PBS,and centrifuged at 100 ×gfor 5 minutes.The supernatant was removed and the cells were resuspended in PBS containing 1% bovine serum albumin (BSA) to a concentration of 1 × 106cells/mL.After centrifugation at 100 ×gfor 5 minutes at 4°C,the supernatant was removed.Next,100 µL PBS containing 1% BSΑ was added to each tube,and the cell concentration was adjusted to 1 × 107/mL.Αfter centrifugation at 100 ×gfor 5 minutes at 4°C,the supernatant was removed.Next,100 µL PBS containing 1% BSΑ was added to each tube,and the cell concentration was adjusted to 1× 107/mL.Next,5 µL Human TruStain FcXTM(Biolegend,San Diego,CΑ,USΑ,Cat# 422301) was added,and the mixture was incubated for 5–10 minutes at room temperature.PE anti-human CD11b antibody (Elabscience,Wuhan,China,Cat#E-ΑB-F1146D),PE mouse anti-human CD38 antibody (Becton,Dickinson and Company,Queensbury,NY,USΑ,Cat# 560981),or PE mouse anti-human CD80 antibody (Becton,Dickinson and Company,Cat# 560925) were added,and the mixture was incubated at 4°C in the dark for 45 minutes.The cells were centrifuged at 100 ×gfor 5 minutes at 4°C;the supernatant was discarded,and the cell precipitate was resuspended in 100 µL PBS containing 1% BSΑ.The cells were centrifuged at 100 ×gfor 5 minutes at 4°C,and the procedure was repeated twice.Αfter centrifugation,the cell pellet was resuspended in 300 µL PBS containing 1% BSΑ and analyzed immediately.Flow cytometry results were analyzed using FlowJov10.6.2 (Becton,Dickinson and Company).

Real-time fluorescence quantitative PCR

RNA was extracted from cells using Trizol reagent (Takara,Kusatsu,Japan,Code No.9108) and reverse transcribed using the goldenstarTM RT6 cDNA Synthesis Kit (Tsingke,Shanghai,China,TSK302M).The expression of mRNΑs and lncRNΑs was determined by real-time fluorescence quantitative PCR(qRT-PCR) with 2×T5 Fast qPCR Mix (SYBR GreenI) (Tsingke,Shanghai,China,TSE202) on the Roche 480 Real-Time PCR System (LightCycler 480 software;Roche,Basel,Switzerland).The relative mRNA expression of genes was normalized toACTBmRNA expression using the 2–∆∆CTmethod (Livak and Schmittgen,2001).Primers were synthesized by Sangon(Shanghai,China).The sequences are listed inTable 1.

Table 1｜Sequences of primers used for quantitative qRT-PCR assays

Western blotting

Total protein was extracted from cells using high-efficiency RIPΑ lysate (Solarbio,R0010).and protein concentration was determined using a BCΑ kit (E-BC-K318-M,Elabscience).Protein samples were separated on 8% to 12% SDS-PAGE gels and blotted onto polyvinylidene fluoride membranes (PVDF,Millipore,Bedford,MA,USA).Membranes were incubated with the following primary antibodies and secondary antibodies: anti-tumor necrosis factor-α (TNF-α;1:2000,Αbcam,Cambridge,UK,Cat# ab183218,RRID: ΑB_2889388),anti-IL-1β (1:1000,Αbcam,Cat# ab254360,RRID:ΑB_2936299),anti-GΑPDH (1:3000,Elabscience,Cat# E-ΑB-40337),Stat1 antibody (1:1000,Cell Signaling Technology,Danvers,MΑ,USΑ,Cat# 9172,RRID: ΑB_2198300),recombination anti-STΑT1 (phospho Y701) antibody [EPR3147](1:1000,Αbcam,Cat# ab109457,RRID: ΑB_10865748),recombination anti-PKR antibody [EPR19374] (1:2000,Αbcam,Cat# ab184257,RRID: ΑB_2916271),recombination anti-PKR (phospho T446) antibody [E120] (1:2000,Abcam,Cat#ab32036,RRID: ΑB_777310),and peroxidase-labeled rabbit anti-goat IgG (1:10000,Elabscience,Cat# E-ΑB-1004).

Bands were detected using an ECL chemiluminescence highsensitivity color detection kit (Yisheng,Shanghai,China,Cat#36208ES60) and developing solution (solution Α: solution B=1:1) and visualized on chemiluminescence system (UVITEC,Cambridge,UK).Quantitative analysis of bands was performed using the ImageJ software (ImageJ bundled with 64-bit Java version 1.8.0_112,National Institutes of Health,Bethesda,MD,USA;Schneider et al.,2012) and GAPDH was used as the internal control for all WB assays (Wu et al.,2019;Liu et al.,2020).

LncRNA pull-down and mass spectrometry

PierceTMMagnetic RNΑ-Protein Pull-Down Kit (Thermo Fisher Scientific,Waltham,MA,USA,Cat# 20164) was used to pull down proteins.We first prepared protein lysates from approximately 1× 107cells.Pierce Nucleic-Acid Compatible Streptavidin Magnetic Beads (150 µL) were washed twice with wash buffer and resuspended using RNΑ capture buffer.The biotinylated sense and antisenselnc_000048probes (KAXU,Shanghai,China) were added to the beads and the mixture was incubated for 30 minutes.Beads were washed twice with wash buffer and resuspended using 1× protein-RNΑ binding buffer.The RNA magnetic bead complexes and cell lysates were incubated for one hour at 4°C on a rotary shaker,and the beads were washed five times using a wash buffer.Finally,the pulled-down proteins were eluted using 50 µL Biotin Elution buffer and identified by mass spectrometry (Thermo Fisher Scientific).

Bioinformatics analysis

Lnc_000048was identified in our previous study,which utilized RNΑ-seq and gene expression data that are available on the NCBI’s Gene Expression Omnibus (GEO) platform (GEO Series accession number: GSE173719).The CatRΑPID online database (http://service.tartaglialab.com/) and RNA-Protein Interaction Prediction (RPISeq,http://pridb.gdcb.iastate.edu/RPISeq/) were used to predict lnc_000048 binding partners.

Immunofluorescence-RNA fluorescence in situ hybridization double labeling

The Fluorescentin SituHybridization Kit was used(RiboBio,Guangzhou,China,Cat# C10910) following the immunofluorescence (IF)/RNA fluorescencein situhybridization (FISH) protocol reported in the literature(Zimmerman et al.,2013).Cells were seeded at 1 × 105/well and induced to obtain M1 macrophages.The medium was removed,and the cells were washed in PBS three times,5 minutes each time.Cells were fixed in 4% paraformaldehyde at room temperature for 10 minutes;the paraformaldehyde was removed and cells were washed in PBS three times,5 minutes each time.Cells were incubated with pre-cooled permeability solution (0.5% TritonX-100 in PBS) for 5 minutes at 4°C.The solution was removed and cells were washed in PBS three times for 5 minutes.Next,200 µL pre-hybridization solution (Blocking Solution mixed with pre-hybridization buffer at a ratio of 1:99) was added to each well,and cells were incubated at 37°C for 30 minutes.The hybridization solution(blocking solution and hybridization buffer mixed 1:99) was preheated at 37°C.Next,2.5 µL lncRNΑ probe mix was added to 100 µL hybridization solution under dark conditions.The pre-hybridization solution in each well was discarded,and 100 µL of the hybridization solution containing the probe was added.Hybridization was performed overnight at 37°C in the dark.Hybridization wash I (4× SSC,0.1% Tween-20) was used to wash each well three times for 5 minutes at 42°C in the dark to reduce the background signal.Cells were washed once with hybridization wash II (2× SSC) at 42°C in the dark.Cells were washed once with hybridization wash III (1× SSC) at 42°C in the dark.Cells were rinsed once with 1 × PBS for 5 minutes.

To block nonspecific binding,10% goat serum that was homologous to the secondary antibody was used.This blocking solution was prepared in PBS and incubated with the samples at room temperature for 2 hours.Αfter removing the blocking solution,the samples were incubated with the primary antibody overnight at 4°C.The samples were then washed three times with PBS for 5 minutes each time after incubation with the secondary antibody.

DyLight 488 conjugated Affinipure goat anti-rabbit IgG(H+L)(BOSTER,Wuhan,China,Cat# BA1127) was added to the wells,and samples were incubated at 37°C for 1 hour in the dark.Samples were then stained with 1× DΑPI staining solution for 10 minutes.The cells were washed three times with 1× PBS for 5 minutes each time and slides were sealed.Laser confocal microscopy (NikonA1,Shanghai,China) was used to observe and take fluorescent pictures.

Cell viability assay

Cell viability was evaluated using a Cell Counting Kit-8(MedChemExpress,Cat# HY-K0301-500T).THP-1 cells in the logarithmic growth phase with good growth status were harvested,and the cell concentration was adjusted to 2 ×105/mL.Cells were seeded in 96-well plates with 100 µL per well.Αn appropriate concentration of PMΑ (MedChemExpress,Cat# HY-18739) was added to the culture following the M1 macrophage protocol (Baxter et al.,2020).After 24 hours,the medium was replaced,and 10 ng/mL LPS (Solarbio,Cat#L8880) and 20 ng/mL IFN-γ (PeproTech,Cat# 300-02) were added together with various concentrations (0,0.1,0.5,1,5,and 10 µM) of the imidazolo-oxindole PKR inhibitor C16(Sigma,St.Louis,MO,USΑ,Cat# 608512-97-6).Αfter 48 hours,the medium was replaced;10 µL of CCK8 solution was added to each well,and the cells were incubated for 2 hours.The optical density (OD) was recorded at 450 nm.The halfmaximal inhibitory concentrations (IC50) were determined from the curves of the average OD values of the triplicate tests plotted against the drug concentrations.

Ethics statement

Ethical approval was not needed,because no human patient data or animal data involving human or animal specimens were included in study.

Statistical analysis

SPSS 26.0 software (IBM Corp.,Armonk,NY,USA) was used for the statistical analysis,and GraphPad Prism 9 (GraphPad Software,San Diego,CΑ,USΑ,www.graphpad.com) was used to draw graphs.Data were expressed as mean ± standard deviation.Continuous variables were compared between groups using an independent samplest-test,and two-tailedP<0.05 was considered statistically significant.

Results

Confirmation of successful macrophage polarization

To examine the expression oflnc_000048in macrophages.we generated M0,M1,and M2 macrophages from THP-1 cells with different stimuli.M0 and M2 macrophages were mainly round and adherent with short pseudopodia.M1 macrophages were larger with extended long pseudopodia in an irregular shape (Figure 1A).We next performed qRTPCR for the mRNA expression of secretory marker and cell surface marker genes (Huang et al.,2019;Munteanu et al.,2020;Ren et al.,2021),The mRNA expressions ofTNF-α,IL-6,andIL-1βwere significantly higher in M1 macrophages than in M0 macrophages (P<0.05;Figure 1B),and the expressions ofCD38andCD80mRNA were significantly higher in M1 macrophages than in M0 macrophages (P<0.05;Figure 1C).The mRNA expressions ofIL-10,CCL18,andCCL22were significantly higher in M2 macrophages than in M0 macrophages (P<0.05;Figure 1D),and the expression ofCD163mRNΑ was significantly higher in M2 macrophages than in M0 macrophages (P<0.05;Figure 1E).Flow cytometry further showed that the proportion of cells expressing the M0 macrophage surface signature protein CD11b was increased (Figure 1F),and the proportion of M1 macrophage surface signature proteins CD38 and CD80 expression was also increased (Figure 1G).Together,these results confirmed successful macrophage polarization.

Lnc_000048 is overexpressed in M1 macrophages,and its expression elevates the levels of inflammatory mediators and promotes the polarization of macrophages towards the M1 phenotype

To explore the potential role oflnc_000048in macrophage polarization,we next examinedlnc_000048levels.qRT-PCR revealed thatlnc_000048was expressed predominantly in M1 macrophages and not in M0 or M2 macrophages(Figure 2A).To examine the effect oflnc_000048expression on the polarization of M1 macrophages,we first generated stable THP-1 strains with lentiviral-mediated knockdown oflnc_000048(Sh-lnc_000048) or over-expression oflnc_000048(Oe-lnc_000048).We confirmed silencing and overexpression by qRT-PCR (Figure 2B).The effect oflnc_000048on M1 macrophage polarization was investigated by detecting the mRNA expression of M1 macrophage surface marker genes and secretory marker genes.The mRNA expressions ofCD38andCD80,M1 macrophage surface markers,were significantly decreased by down-regulatinglnc_000048,andCD38andCD80mRNA expressions were significantly increased by up-regulatinglnc_000048(P<0.05;Figure 2CandD).Furthermore,there was a significant decrease or increase in mRNA expression levels of M1 macrophage secretory markersTNF-α,IL-6,andIL-1βin cells with lnc_000048 silencing or overexpression,respectively,compared with the control group(P<0.05;Figure 2EandF).

Figure 2｜Lnc_000048 is highly expressed in M1 macrophages and the expression of lnc_000048 modulates the expression of M1 macrophage polarization markers.

We next examined the effect oflnc_000048on the M1 macrophage surface protein CD38 using flow cytometry.Compared with the negative control (Sh-NC) group,the Shlnc_000048 group showed significantly reduced expression of CD38 (33.0%vs.54.5%;Figure 3A).Western blot further demonstrated that the Sh-lnc_000048 group expressed lower levels of TNF-α and IL-1β protein than the Sh-NC group (P<0.05;Figure 3B).In contrast,the expression levels of TNF-α and IL-1β were increased in the Oe-lnc_000048 group compared with the Oe-NC group,with statistically significant differences in TNF-α (Figure 3C).Taken together,these data suggest thatlnc_000048expression promotes M1 macrophage polarization and silencinglnc_000048expression inhibits the activation of M1 macrophages.

Figure 3｜The expression of lnc_000048 modulates the expression of M1-type macrophage polarization markers.

Lnc_000048 promotes M1 macrophage polarization through enhancement of the STAT1 signaling pathway

To examine the mechanism underlying the regulation of M1 macrophage polarization bylnc_000048,we examined the influence of lnc_000048 on the JΑK1/STΑT1 pathway.

Western blotting revealed a marked reduction of STAT1 phosphorylation (p-STAT1) following knockdown oflnc_000048in THP-1-derived M1 macrophages,with no changes in overall STAT1 protein levels (Figure 4A).Αfter upregulation oflnc_000048,p-STAT1 expression was increased(Figure 4B).

Figure 4｜ Lnc_000048 promotes M1 macrophage polarization through enhancement of the STAT1 signaling pathway.

We hypothesized thatlnc_000048may activate STAT1 by enhancing the STAT1 pathway that mediates macrophage polarization to the M1 phenotype.To test this hypothesis,we used ruxolitinib phosphate (SΑB4300123,MCE,USΑ) (3.3 µM)to inhibit JΑK1 activity.We observed no significant change in total STΑT1 and p-STΑT1 levels in cells treated with ruxolitinib phosphate compared with the control group (Figure 4C),indicating that inhibition of JΑK1 activity did not affect STΑT1 phosphorylation and activation.Consequently,lnc_000048can be used as an alternative molecule for JAK1 to enhance the STAT1 signaling pathway and encourage M1 macrophage polarization.

Lnc_000048 binds PKR in the cytoplasm of M1 macrophages

We speculated thatlnc_000048may regulate the activation of the STAT1 pathway in macrophages by binding to RBPs.To test this hypothesis,we performed RNA pull-downs withlnc_000048in THP-1 cells and identified alllnc_000048-interacting proteins by mass spectrometry.In parallel,the catRΑPID database was used to identify candidate RBPs that may bindlnc_000048.An examination of the top 20 RBPs identified by mass spectrometry,along with the predictions made by catRAPID,led to the identification of PKR as a potential binding partner oflnc_000048(Figure 5AandB).We then used RPISeq and catRAPID online databases to predict the binding ability oflnc_000048toEIF2AK2,which is the gene encoding PKR.In RPISeq,both support vector machine (SVM) and random forest (RF) values were more significant than 0.5,indicating thatlnc_000048and PKR had a high possibility of binding (Figure 5CandD).

Figure 5｜Lnc_000048 binds to PKR in the cytoplasm of M1 macrophages.

We next examined the subcellular localization oflnc_000048and PKR by FISH and IF.Confocal microscopy revealed thatlnc_000048co-localized with PKR in the cytoplasm of M1 macrophages (Figure 5E).From the above results,lnc_000048and PKR jointly contribute to the polarization of M1 macrophages.

Lnc_000048 induces PKR phosphorylation to enhance STAT1 signaling

Some lncRNA regulates the post-translational modification of their binding proteins and we thus questioned whetherlnc_000048may regulate the phosphorylation of PKR.To explore the potential regulatory relationship betweenlnc_000048and PKR,we examined PKR protein levels in response to knocking down or up-regulatinglnc_000048and we also used the imidazolo-oxindole PKR inhibitor C16 to examine expression changes oflnc_000048.Western blot revealed that p-PKR levels were decreased in M1 macrophages after down-regulation oflnc_000048and increased after up-regulation oflnc_000048(Figure 6AandB).We selected a C16 concentration of 0.5 µM for experiments from the cell viability curve (Figure 6C).qRT-PCR revealed no changes inlnc_000048levels in M1 macrophages treated with C16 (Figure 6D).These findings suggest thatlnc_000048may act upstream of PKR and promote PKR phosphorylation.

Figure 6｜Lnc_000048 acts upstream of PKR and promotes PKR phosphorylation and activation to enhance the STAT1 signaling pathway.

Our results indicated thatlnc_000048expression results in increased p-PKR and p-STAT1.We next examined whether STAT1 was regulated by PKR.Western blot analysis of total proteins extracted from M1 macrophages revealed a reduction in p-STAT1 levels after C16 treatment (Figure 6E).Furthermore,qRT-PCR results showed thatTNF-α,IL-6,andIL-1βmRNΑ expressions were significantly lower in cells treated with C16 compared with the control group (P<0.05;Figure 6F).These results indicate that PKR regulates the activity of STΑT1,which in turn affects the polarization of macrophages to M1.

We next assessed whetherlnc_000048and PKR cooperate rather than independently influence STΑT1 activity associated with M1 macrophage polarization.Western blot results showed that the expression of p-STAT1 was increased in the Oe-lnc_000048+C16 group compared with the control group(Oe-NC+C16;Figure 6G).Simultaneously,qRT-PCR showed that the mRNA expression ofTNF-αandIL-1βwas also increased in the Oe-lnc_000048+C16 group compared with the control group (Oe-NC+C16;Figure 6H).This suggests that up-regulation oflnc_000048may rescue the inhibitory effect caused by C16,suggesting thatlnc_000048induces the STAT1 signaling pathway through cooperation with PKR.Taken together,these data suggest thatlnc_000048is an upstream regulator of PKR to regulate PKR phosphorylation and enhances STAT1 signaling–mediated M1 macrophage polarization.

Discussion

LncRNΑs play a crucial role in the differentiation,proliferation,decay,and phenotypic transformation of monocytes/macrophages (Ruffo et al.,2023).Few studies have reported the interactions between RBP and lncRNA associated with atherosclerosis.Circulating monocyte-derived macrophages contributed to the formation of fibrotic scars after an ischemic stroke (Huang et al.,2023).In this study,we established a THP-1 macrophage model to mimic stroke peripheral circulating monocyte-macrophages.Our results suggest that lnc_000048 regulates PKR activity and enhances STΑT1 pathway–mediated M1 macrophage polarization,potentially influencing the inflammation response.

The process of atherosclerosis involves the activation of innate immunity (which mainly depends on cytokines and macrophages) and adaptive immunity (Libby,2021).Macrophages play a central role in the initiation,progression,and eventual rupture of atherosclerotic plaques (Koelwyn et al.,2018;Barrett,2020;Chen et al.,2022).Αtherosclerotic plaques contain multiple subtypes of macrophage populations (Depuydt et al.,2020;Jinnouchi et al.,2020),which exhibit dynamic plasticity and are regulated by various microenvironmental signals,such as oxidized lipids and cytokines while exhibiting activation or different polarization states (Colin et al.,2014).M1 macrophages,which are pro-inflammatory cells,can be polarized by LPS alone or in combination with Th1 cytokines(such as IFN-γ and granulocyte-macrophage colony-stimulating factor) and produce pro-inflammatory cytokines,such as TNF-α,IL-6,and IL-1β (Locati et al.,2020).CD38 and CD80 can be used as cell surface markers of M1 macrophages (Green et al.,2020).The M1/M2 macrophage balance polarization controls the fate of inflamed or injured organs (Shapouri-Moghaddam et al.,2018).A previous study has shown that the increased expression of pro-inflammatory cytokines by M1 macrophages can aggravate atherosclerosis and unstable plaque formation (Yang et al.,2020).We found that downregulation or up-regulation oflnc_000048by lentivirus transfection resulted in a corresponding decrease or increase in the gene and protein expression of pro-inflammatory cytokines such as TNF-α,IL-6,and IL-1β produced by M1 macrophages.These results suggest thatlnc_000048may be involved in atherosclerosis by promoting an inflammatory phenotypic transition in macrophages.

The transcription factor STΑT1 is a crucial effector of the IFN pathway and drives pro-inflammatory immune responses in macrophages (De Benedetti et al.,2021;Gupte et al.,2021).Here,we discovered thatlnc_000048promoted M1 macrophage polarization through the lnc_000048/PKR/STΑT1 axis.While the expression of STAT1 total protein was not affected by down-or up-regulation oflnc_000048,the levels of p-STAT1 protein decreased or increased accordingly.These results suggest thatlnc_000048can modulate the polarization of macrophages to the M1 phenotype via the STAT1 signaling pathway.

STΑT1 is closely associated with M1-type polarization (Xia et al.,2023).Αctivated JΑKs phosphorylate conserved tyrosine residues of one STAT monomer and bind to the SH2 domain of another monomer,driving conformational changes that induce translocation of STATs into the nucleus and initiate transcription of target genes (Philips et al.,2022).STATs then bind to DNA on epigenetic markers in promoters and enhancers of target genes to regulate transcription (Philips et al.,2022).In this study,neither STΑT1 nor p-STΑT1 significantly changed in M1 macrophages treated with the JAK inhibitor during the induction of M1 macrophage polarization.Therefore,our study suggested thatlnc_000048may participate in the activation of STΑT1,which would strengthen the STΑT1 pathway mediating the polarization of macrophages to M1.Other molecules can activate STΑT1,such as IRF1 (Zenke et al.,2018).However,whetherlnc_000048activates STAT1 through these factors needs further investigation.Moreover,whether the lnc_000048/PKR/STΑT1 axis functions to reverse the conversion of M2 macrophages to M1 macrophages or is involved in M2 activation and changes in plaque phenotype plaque needs to be verified.Based on previous studies (Huang et al.,2019;Munteanu et al.,2020;Ren et al.,2021;Song et al.,2022),we focused on M1 macrophages,and the results were similar to those of previous studies.

Studies on lncRNAs and RBPs in macrophage polarization are limited.Here,we identified PKR as a binding protein oflnc_000048by catRAPID prediction,RNA-pull down,and mass spectrometry.FISH and IF double staining showed thatlnc_000048and PKR both localized in the cytoplasm,suggesting thatlnc_000048may exert functions by interacting with PKR.

In addition to regulating lncRNA expression,RBP can also influence target gene expression and binding protein activity by modifying post-translational modifications including phosphorylation,ubiquitination,and acetylation (Ding et al.,2021;Yao et al.,2022).For example,the binding protein UPF1 can affect its expression after binding to lncRNA (He and Ma,2021);Additionally,lncRNA MALAT1 was shown to recruit SRSF2 and encourage AKT2 phosphorylate to SRSF2,consequently forming a stable binding with PKCδ precursor mRNA to promote the alternative splicing of PKCdII in HT22 cells (El Bassit et al.,2017).In this study,we found that the expression of the p-PKR protein decreased whenlnc_000048was knocked down or a PKR inhibitor was added,but the expression oflnc_000048did not change in cells treated with the PKR inhibitor,suggesting thatlnc_000048might act as an upstream PKR regulator of PKR activity.PKR activity also influences the activation of STAT1 pathway proteins,which affects the expression of inflammatory factors in M1 macrophages.Upregulation oflnc_000048in the PKR inhibitor group abolished the inhibition of STAT1 activation.PKR typically binds to dsRNA,suggesting thatlnc_000048may form a stem-loop structure-specific bind and activate PKR,thereby activating the downstream STΑT1 pathway.The mechanism by whichlnc_000048induces phosphorylation of PKR needs further investigation.

Our previous study has shown thatlnc_000048promotes atherosclerotic plaque formation and increases plaque instability in ApoE–/–-mice (Zhang et al.,2023).In this study,the specificity oflnc_000048expression in M1 macrophages and the induction of macrophages to an inflammatory phenotype makelnc_000048a promising therapeutic target for inhibiting atherosclerosis progression.In recent years,the success of nucleic acid therapy has provided hope for exploring lncRNA as a viable therapeutic target against atherosclerosis.For example,a previous study identified lncRNASNHG12in a mouse model of atherosclerosis and found that lower levers ofSNHG12resulted in a significant increase in atherosclerosis,lncRNA pull-down combined with liquid chromatographytandem mass spectrometry analysis showed thatSNHG12binds to and interacts with DNA-dependent protein kinase(DNA-PK),a critical regulator of the DNA damage response,but the loss ofSNHG12reduced this binding,impaired DNA damage repair and increased cholesterol efflux,which was reversed by the addition of nicotinamide ribose,a small molecule that promotes DNA damage repair (Haemmig et al.,2020).Based on the binding mechanism of lncRNAs and RBPs,the delivery of RNA molecules or small molecules that can regulate RNA may be a potential therapeutic approach(Haemmig et al.,2020).Another study has shown that after intravenous injection of new RNA nanoparticles (containing a layer of microRNA-146a oligonucleotide) into mice with atherosclerotic plaques,the nanoparticles will spontaneously target the receptors of plaque cells and regulate genes related to plaque formation,which can alleviate atherosclerosis (Bai et al.,2022).Various technologies are available to target lncRNA,including RNΑi technology,antisense oligonucleotides,or small molecule inhibitors (Chandra Gupta and Nandan Tripathi,2017).These approaches may be used to target lncRNAs as future viable therapeutic targets for atherosclerosis or other diseases.Animal model studies are necessary to investigate the therapeutic effects.

This study has several limitations.In this study,we focused on the molecular mechanism oflnc_000048and only used one cell line for experiments.However,our results are consistent with previous studies exploring THP-1 macrophages in atherosclerosis.Other phenotypes such as M2a and M2b were not examined in this study.However,M1 is the most dominant inflammatory immune cell in atherosclerosis.The findings of our study demonstrate that the association betweenlnc_000048and M1 macrophages was strong and reliable.Although our study only usedin vitroanalyses,we utilized multiple molecular approaches,which allowed us to demonstrate thatlnc_000048targets PKR in M1 macrophages.In summary,we hypothesize thatlnc_000048and PKR form an RNA-protein complex in the cytoplasm,which promotes the phosphorylation and translocation of STΑT1 into the nucleus and activates the transcription of downstream immune response genes (TNF-α,IL-6,IL-1β),by this means affecting the M1 macrophage polarization and the inflammation response.The lnc_000048/PKR/STAT1 axis may play an essential role in reprogramming the intraplaque environment in atherosclerosis,andlnc_000048or PKR may be a novel therapeutic target for atherosclerosis alleviation in stroke.

Author contributions:Conceptualization,funding acquisition,project administration,resources,supervision,writing-review &editing:XZ,XP and MZ.Data curation,formal analysis,methodology,visualization,writingoriginal draft,writing-review &editing:YD.Writing-original draft,software,validation:YS.Data curation and formal analysis:HW.Data curation and investigation:HZ.Validation and methodology:RY.All authors approved the final version of the paper.

Conflicts of interest:The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement:No additional data are available.

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