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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2025 Jun 18;195(9):1719–1735. doi: 10.1016/j.ajpath.2025.05.014

Macrophage–Kruppel-Like Transcription Factor 6 Signaling Promotes Experimental Atherogenesis

Hang Pong Ng ∗, Atif Zafar ∗, Rachel Diamond-Zaluski ∗, Gun-Dong Kim ∗, Kartik Bhat ∗, Owen Meadows ∗, Yashwant Pantra ∗, E Ricky Chan †, Jonathan D Smith ‡,§, Ganapati H Mahabeleshwar ∗,∗
PMCID: PMC12489348  PMID: 40553722

Abstract

A hallmark event in the development of atherosclerotic plaque is the accumulation of lipid-laden macrophages in the subendothelial layers of affected blood vessels. Macrophages are key players in all stages of atherogenesis, including plaque initiation, growth, and rupture, as well as healing of ruptured plaques. In this context, macrophages are the principal innate immune cells that modulate atherogenesis by engaging in various processes, such as inflammation, extracellular matrix degradation, phagocytosis, and efferocytosis. In the current study, Kruppel-like transcription factor 6 (KLF6) deficiency attenuated proinflammatory gene expression in macrophages and experimentally induced atherosclerotic plaque development. In vivo studies showed that myeloid-KLF6 deficiency on Apoe-null background significantly curtailed high-fat/high-cholesterol diet-induced atherosclerotic lesion formation and macrophage abundance in atherosclerotic plaques. Integrated transcriptomics and Gene Set Enrichment Analysis showed that KLF6 deficiency significantly curtailed a large number of tumor necrosis factor (TNF)–induced gene targets, TNF-induced interferon-γ response, interferon-α response, and inflammatory response signaling in macrophages. At the molecular level, KLF6 promoted interferon regulatory factor 1 (IRF1) signaling to enhance TNF-induced proinflammatory gene expression in macrophages. Collectively, study results show that KLF6 promoted proinflammatory gene expression in macrophages and enhanced experimentally induced atherosclerotic plaque formation in vivo.

Clinical and experimental studies support an important role for macrophages in the development and progression of atherosclerotic plaques.1 Atherosclerotic plaque development is initiated by the deposition of cholesterol-rich, apolipoprotein B–containing lipoproteins on the luminal surface of the vascular wall.2 Lipoproteins within the vascular wall acquire proinflammatory properties through a variety of modifications, such as oxidation, aggregation, enzymatic, and/or nonenzymatic cleavage. These proinflammatory lipoproteins not only activate resident macrophages but also recruit monocyte-derived macrophages into the subendothelial space. These macrophages actively phagocytose deposited lipoproteins and transform into lipid-loaded foam cells. A hallmark event in the development of atherosclerotic plaque is the accumulation of lipid-loaded foam cells in the subendothelial layers of affected blood vessels to form early atherosclerotic plaques or commonly referred to as fatty streaks. Macrophages modulate atherogenesis by engaging in several processes, such as inflammation, extracellular matrix degradation, phagocytosis, and efferocytosis.3,4 Macrophages that accumulate in mouse and human atherosclerotic plaques exist in a continuum of proinflammatory to anti-inflammatory states.5,6 Proinflammatory macrophages are mainly localized near rupture-prone shoulder regions, whereas anti-inflammatory macrophages can be found in the adventitia.7,8 Thus, proinflammatory macrophages are generally considered pro-atherogenic and contribute to plaque development, destabilization, and rupture. However, anti-inflammatory macrophages are efficient in lipid metabolism and cholesterol efflux, promote plaque regression, and are considered anti-atherogenic.9,10 Despite the acknowledged importance of macrophages in atherogenesis, the transcription factors that shape the macrophage functional phenotype determination within atherosclerotic plaques through regulation of gene expression, and their contribution to the pathogenesis of atherosclerosis, remain incompletely understood. The macrophage phenotypic transition is an explicitly robust biological response to local cues that involves alterations in broad gene expression profiles affecting a significant part of the cellular genome.11 Thus, transcription factors that govern broad innate-immune gene expression profiles play an essential role in shaping the macrophage contribution to atherosclerotic plaque development.

Kruppel-like transcription factor 6 (KLF6) modulates key biological processes, such as cellular development, proliferation, differentiation, cell death, metabolism, and signaling transduction.12 Alterations in the KLF6 expression or functions are associated with the pathogenesis of several human diseases, such as cancer, inflammatory bowel disease, hepatic fibrosis, and hepatic steatosis.13 Structurally, KLF6 protein contains an N-terminal acidic domain, a hydrophobic serine/threonine-rich central domain, and a C-terminal DNA-binding zinc finger domain.12 KLF6 interacts with other proteins, transcription factors, chromatin-modifying enzymes, coactivators, and corepressors through the N-terminal acidic domain.12 The highly conserved C-terminal C2H2-type zinc finger domain preferentially binds to GC-box or CACC motifs in promoter, regulatory, and enhancer regions of gene targets.12 KLF6 is highly expressed in resting human and murine macrophages.14 Proinflammatory cytokines elevate, and anti-inflammatory agents diminish, KLF6 expression in human and murine primary macrophages.14 Moreover, KLF6 promotes proinflammatory macrophage activation while repressing anti-inflammatory macrophage polarization.14 Macrophage-KLF6 deficiency is protective against experimentally induced cutaneous inflammation,15 ulcerative colitis,16 diet-induced obesity and type 2 diabetes,17 and endotoxin-induced systemic inflammatory response syndrome.18 Mechanistically, in macrophages, KLF6 boosts NF-κB and hypoxia-inducible factor-1α signaling while restraining peroxisome proliferator-activated receptor γ, B-cell lymphoma 6 (BCL6), and miR-223 functions.14, 15, 16, 17, 18 Whether macrophage-KLF6 deficiency impacts inflammatory or metabolic gene programs involved in atherogenesis has not been previously examined. Whether macrophage-KLF6 deficiency alters experimentally induced atherosclerotic plaque development has also not been investigated. The current study provided evidence that KLF6 deficiency alleviated key inflammatory signaling pathways in macrophages that are involved in the initiation and progression of atherosclerotic plaque development. Importantly, this study showed that macrophage-KLF6 deficient mice are highly protected from experimentally induced atherosclerotic plaque development in vivo.

Materials and Methods

Materials

Anti-KLF6 (sc-365633) and anti–β-actin (sc-69879) were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX). Anti–interferon regulatory factor 1 (IRF1) (8478S), phosphorylated STAT1 (9167S), STAT1 (14994S), and anti-F4/80 (70076S) antibodies were obtained from Cell Signaling (Danvers, MA). The horseradish peroxidase–conjugated goat anti-mouse (A16066) and horseradish peroxidase–conjugated goat anti-rabbit (A16096) antibodies were obtained from Thermo Fisher Scientific (Waltham, MA). The Lyz2cre (04781) and Apoe–/– (002052) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). The pCl-neo-KLF6 (number 49488) and MAC-N-IRF1 (number 167760) plasmids were obtained from Addgene (Watertown, MA), and IRF-luciferase reporter plasmid (LR-2020) was obtained from Signosis (Santa Clara, CA). The Lipofectamine3000 (L3000-008), bicinchoninic acid assay kit (23227), ECL Western Blotting Substrate (32106), random hexamers (N8080127), oligo-dT primer (18418020), Fast SYBR Green PCR Master Mix (4385612), and custom primers listed in this article were obtained from Thermo Fisher Scientific. The Sudan IV stain (198120), bovine serum albumin (A3608), cholesterol quantification assay kits (CS0005), and triglyceride quantification kits (MAK564) were obtained by MilliporeSigma (St. Louis, MO). The High Pure RNA Isolation Kits (11828665001), protease inhibitors (04693132001), and phosphatase inhibitors (04906837001) were obtained from Roche (Indianapolis, IN). The RAW264.7 cell line (TIB-71) was purchased from ATCC (Manassas, VA). The recombinant mouse macrophage colony-stimulating factor (416-ML), mouse tumor necrosis factor (TNF)-α protein (210-TA-100), and mouse interferon-γ (485-MI-100) were obtained from R&D Systems (Minneapolis, MN). The high-glucose Dulbecco's modified Eagle's medium (SH30249.02), phosphate-buffered saline (SH30028.03), and nitrocellulose membranes (10401197) were obtained from GE-Healthcare Life Sciences (Chicago, IL). The dual-Luciferase reporter assay system (E1960) was obtained from Promega (Madison, WI). The siKlf6 and siControl siRNAs were obtained from GE Dharmacon (Lafayette, CO). All other chemicals and reagents used were of analytical grade and were obtained from commercial sources.

Experimental Animal Studies

All animal procedures were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University (Cleveland, OH) and conformed to guidelines established by the American Association for Accreditation of Laboratory Animal Care. All mice were bred and maintained under pathogen-free conditions, fed standard laboratory chow (2916; ENVIGO, Indianapolis, IN), and kept on a 12-hour light/dark cycle. The control (Lyz2cre on C57BL/6 background) and myeloid-specific Klf6 deficient (Klf6fl/fl:Lyz2cre on C57BL/6 background) were generated by breeding male and female Klf6fl/fl:Lyz2cre mice. The Klf6fl/fl:Lyz2cre mice contained two Klf6 floxed and a Lyz2 Cre allele (C57BL/6 background). Mice with a Cre allele (Lyz2cre) were used as the control group (C57BL/6J background). For atherosclerosis studies, 8-week–old male and female Lyz2cre:Apoe−/− (C57BL/6) and Klf6fl/fl:Lyz2cre:Apoe−/− (C57BL/6) mice were fed a Western-style high-fat diet (HFD; TD 88137; Harlan Teklad, Indianapolis, IN: 21.2% fat, 0.2% cholesterol) for 20 weeks. These study mice received randomly numbered ear tags, and investigators were blinded to mice genotype information. At the end of the feeding period, total blood samples were collected for plasma cytokine analysis. These study mice were euthanized and perfused with saline. The aortic sinuses and ascending and descending aorta were collected. The entire aorta was stained with Sudan IV, and excessive adventitial tissues were removed. For en-face aortic lesion quantification, aortas were dissected by removing all branching vessels down to the femoral bifurcation and then sliced ventrally. The images of the aortas next to a ruler were digitally captured using a Canon (Melville, NY) EOS Rebel T6 DSLR camera. Lesion areas within the entire length of the aorta were quantified using ImageJ software version 1.54f (NIH, Bethesda, MD; https://imagej.nih.gov/ij). Results were expressed as absolute aortic surface plaque area as well as percentage lesion area relative to the total aortic surface area. Furthermore, aortic sinuses were cryosectioned using a Sakura (Torrance, CA) Tissue-Tek Cryo 2000 cryostat at a thickness of 10 μm. The serial sections were taken starting at the aortic valve plane and covering 450 μm in intervals of 50 μm. The serial sections were placed on Superfrost Plus microscope slides (Thermo Fisher Scientific), and the sections were stored at −20°C until use. These sections were stained with oil red O/hematoxylin staining for aortic root plaque quantification. Furthermore, aortic sinus cryosections were fixed with 10% buffered formalin. These sections were subjected to antigen retrieval steps with antigen unmasking solution. Samples were treated with 0.3% H2O2 for 30 minutes at room temperature, and the nonspecific binding was blocked with a blocking buffer. Samples were incubated with rabbit anti-F4/80 antibody overnight at 4°C. These tissue sections were subsequently incubated with biotin-conjugated goat anti-rabbit IgG for 30 minutes at room temperature. Samples were incubated in avidin-biotin complex (ABC) reagent, and the immunostaining was visualized using a diaminobenzidine reagent. Images were acquired using a microscope, and ImageJ software version 1.54f was used for quantification of aortic sinus plaque and macrophage area. These quantifications included at least seven sections for each mouse. The quantification of histologic data and statistical analyses were performed by two independent investigators (among H.P.N., A.Z., G.-D.K., E.R.C., and G.H.M.) who were blinded to sample genotypes and/or treatments. Furthermore, no experimental animals or data from the experiments were excluded from the analyses.

Cell Culture

RAW264.7 cells were cultured in Dulbecco's modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 10 μg/mL streptomycin, and 2 mmol/L glutamine in a humidified incubator (5% CO2 and 37°C). Bone marrow–derived macrophages (BMDMs) were generated by ex vivo differentiation of bone marrow cells. Briefly, bone marrow cells from 8-week–old Lyz2cre and Klf6fl/fl:Lyz2cre mice were harvested from the femur and tibia. The isolated bone marrow cells were cultured in Dulbecco's modified Eagle’s medium supplemented with 10 ng/mL recombinant mouse macrophage colony-stimulating factor for 7 days. The resulting BMDMs were harvested and used for indicated experiments. The primary macrophages from aortic plaques were obtained 20 weeks after the initiation of a control or Western-style diet (TD 88137; Harlan Teklad). Briefly, the entire aortic structure was collected, and periaortic adipose tissues were removed by microdissection. These aortas were washed with a copious amount of sterile, ice-cold phosphate-buffered saline to remove red blood cells. The aortic tissues were minced with scalpel blades and were incubated with 3 mg/mL collagenase-dispase mixture for 4 hours at room temperature with gentle shaking. The cellular suspension was collected using a tissue strainer. Anti-F4/80 microbeads (130-110-443; Miltenyi Biotec, Auburn, CA) were used to purify macrophages from these cellular suspensions. The total RNA and protein samples derived from aortic macrophages were used for quantitative RT-PCR and Western blot analyses.

RNA Extraction, Real-Time Quantitative PCR, and Western Blot Analysis

Total RNA was isolated from indicated samples using the High Pure RNA Isolation Kit. The total RNA samples were reverse transcribed using M-MuLV reverse transcriptase in the presence of random hexamers and oligo-dT primers. Real-time quantitative PCR was performed using Universal SYBR Green PCR Master Mix or TaqMan Universal Master Mix on Applied Biosystems (Foster City, CA) Step One Plus real-time PCR system in the presence of gene-specific primers. The list of primers used in this study is provided in Table 1.

Table 1.

List of Primers Used for Experiments

Target gene Forward primer Reverse primer
Klf6 5′-TCCCACTTGAAAGCACATCA-3′ 5′-ACTTCTTGCAAAACGCCACT-3′
36B4 5′-GCTCCAAGCAGATGCAGCA-3′ 5′-CCGGATGTGAGGCAGCAG-3′
Ptgs2 5′-TTCAACACACTCTATCACTGGC-3′ 5′-AGAAGCGTTTGCGGTACTCAT-3′
Cfb 5′-CAGACTCTCAGGCCCATCTG-3′ 5′-CATCCTTCACAGGGAGCAAC-3′
Cd69 5′-CCCTTGGGCTGTGTTAATAGTG-3′ 5′-AACTTCTCGTACAAGCCTGGG-3′
Gbp6 5′-GTTCCAGGAAGTAACAAAGGCT-3′ 5′-ATCCCTAGTCTATTCCCAGTGAC-3′
Vcam1 5′-ACGTCAGAACAACCGAATCC-3′ 5′-GTGGTGCTGTGACAATGACC-3′
Cxcl11 5′-GGCTTCCTTATGTTCAAACAGGG-3′ 5′-GCCGTTACTCGGGTAAATTACA-3′
Cxcl10 5′-CCAAGTGCTGCCGTCATTTTC-3′ 5′-GGCTCGCAGGGATGATTTCAA-3′
Gbp4 5′-GAGAGAGAGCAGCTCATCAAAG-3′ 5′-TTCCTCAGCTTGTGTCTTATATCC-3′
Ifi44 5′-AACTGACTGCTCGCAATAATGT-3′ 5′-GTAACACAGCAATGCCTCTTGT-3′
Il1a 5′-GCACCTTACACCTACCAGAGT-3′ 5′-AAACTTCTGCCTGACGAGCTT-3′
Mx1 5′-TTCAAGGATCACTCATACTTCAGC-3′ 5′-GGGAGGTGAGCTCCTCAGT-3′
Clec5a 5′-TCGGGGCTTATCGTAGTAGTG-3′ 5′-TGTAGGCATGGTACTTTCGTCAT-3′
Mmp14 5′-GGACTGAGATCAAGGCCAAT-3′ 5′-GCCCACCTTAGGGGTGTAAT-3′
Nlrp3 5′-CCCTTGGAGACACAGGACTC-3′ 5′-GGTGAGGCTGCAGTTGTCTA-3′
Lpar1 5′-AGCCATGAACGAACAACAGTG-3′ 5′-CATGATGAACACGCAAACAGTG-3′
Itgb8 5′-AGTGAACACAATAGATGTGGCTC-3′ 5′-TTCCTGATCCACCTGAAACAAAA-3′
Ccl5 5′-GCTGCTTTGCCTACCTCTCC-3′ 5′-TCGAGTGACAAACACGACTGC-3′
Nos2 5′-GTTCTCAGCCCAACAATACAAGA-3′ 5′-GTGGACGGGTCGATGTCAC-3′
Icam1 5′-CCCACGCTACCTCTGCTC-3′ 5′-GATGGATACCTGAGCATCACC-3′
Cd40 5′-TTGTTGACAGCGGTCCATCTA-3′ 5′-GCCATCGTGGAGGTACTGTTT-3′
Itgal 5′-CCCCAGACTTTTGCTACTGG-3′ 5′-CGTGTGTCCAGGTTGTAGCTC-3′
Irf1 5′-ATGCCAATCACTCGAATGCG-3′ 5′-TTGTATCGGCCTGTGTGAATG-3′
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The primary cells and cell lines were lysed after indicated treatment in ice-cold radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors. Protein concentration was measured by the bicinchoninic acid protein assay. An equal amount of protein samples were electrophoresed using 8% or 4% to 15% Mini-PROTEAN TGX precast gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membranes. These membranes were blocked with 5% nonfat dry milk or 5% bovine serum albumin in Tris-buffered saline with Tween 20 for 1 hour at room temperature. These blots were further incubated with primary antibodies diluted in 5% nonfat dry milk in Tris-buffered saline with Tween 20. After overnight incubation, primary antibodies were removed by washing with Tris-buffered saline with Tween 20. These blots were incubated for 1 hour at room temperature in horseradish peroxidase–conjugated secondary antibodies. Blots were visualized using enhanced chemiluminescence Western blotting substrate. The densitometry analyses were performed using ImageJ software version 1.54f. The primary antibodies were used at the following dilutions: KLF6 (1:1000), IRF1 (1:1000), STAT1 and phosphorylated STAT1 (1:2500), and β-actin (1:5000).

RNA-Sequencing Analysis

Total RNA from primary macrophages was obtained using the High Pure RNA Isolation Kit. Quality control of total RNA samples was assessed using Qubit (Thermo Fisher Scientific) for quantification and Agilent 2100 BioAnalyzer (Santa Clara, CA) analysis to assess quality using a cutoff of RNA integrity number >7.0 to select specimens for further analysis. cDNA library for RNA sequencing was generated from 150 ng of total RNA using the Illumina (San Diego, CA) TruSeq Stranded Total RNA kit with Ribo Zero Gold for rRNA removal, according to the manufacturer's protocol. The resulting purified mRNA was used as input for the Illumina TruSeq kit in which libraries are tagged with unique adapter-indexes. Final libraries were validated on the Agilent 2100 BioAnalyzer, quantified via real-time quantitative PCR, and pooled at equimolar ratios. Pooled libraries were diluted, denatured, and loaded onto the Illumina NextSeq 550 System using a high output flowcell. The aligned reads were then analyzed with Cufflinks (version name: 2.2.1) to obtain gene-level expression data using the GENCODE gene annotation for M32 (version GRCm39) and reported as fragments per kilobase per million reads mapped. Differential expression analysis was also performed, and significantly differentially expressed genes were defined using an adjusted P < 0.05 (false discovery rate corrected). Gene expression tables for relevant pairwise comparisons were analyzed for Gene Set Enrichment Analysis19 using GenePattern (Broad Institute, Cambridge, MA; version v3.9.11-rc.5 b234). Pathway data sets that were examined included Hallmark and transcription factor target gene sets. A gene set was considered to be significantly enriched using a family-wise error rate cutoff <0.05. Heat maps were generated using ClustVis (latest release December 20, 2018).20 The sequencing data reported in this article have been deposited in Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE283475).

Transient Transfection and Luciferase Assay

Transfection of RAW264.7 cells and mice BMDMs was performed using Lipofectamine transfection reagents (Life Technologies, Carlsbad, CA), according to the manufacturer's instructions. Transfected cells were stimulated with 10 ng/mL TNF or phosphate-buffered saline and used for indicated experiments. RAW264.7 cells were transfected with IRF luciferase reporter plasmid or were cotransfected with pCl-neo-Klf6 plasmid or Klf6-specific siRNA using Lipofectamine transfection reagent. These cells were exposed to indicated cytokine. Luciferase reporter activity was measured and normalized according to the manufacturer's instructions. Results are presented as relative luciferase activity over the control group.

Quantification and Statistical Analysis

All phenotype data were analyzed by groups using a combination of letter and number codes that did not specify genotypes, sex, or diet. Thus, data acquisition was blinded. All data, unless indicated, are presented as the means ± SD. All data were normally distributed passing the Kolmogorov-Smirnov and/or D'Agostino and Pearson normality tests. Therefore, parametric statistics were used in this study. The statistical significance of differences between the two groups was analyzed using a t-test or two-way analysis of variance with Bonferroni multiple comparison tests. P < 0.05 was considered significant.

Results

KLF6 Deficiency Curtails Key Inflammatory Pathways Involved in Atherogenesis

TNF signaling promotes atherogenesis. TNF knockout or neutralization (infliximab) reduces,21, 22, 23 whereas recombinant TNF supplementation elevates, experimentally induced atherosclerotic plaque development in mice.24 Bone marrow transplantation studies show that TNF derived from bone marrow cells promotes atherogenesis.25 Whether KLF6 plays any role in regulating TNF-induced signaling pathways and inflammatory gene expression in macrophages is yet to be examined. Thus, this study was designed to analyze whether TNF treatment altered KLF6 expression in macrophages. Accordingly, Lyz2cre and Klf6fl/fl:Lyz2cre mice BMDMs were stimulated with TNF, and KLF6 protein levels were evaluated by Western blot analysis (Figure 1A). The analyses showed that TNF robustly elevated KLF6 expression in Lyz2cre mice BMDMs (Figure 1A). However, KLF6 protein was not detectable in unstimulated or TNF-induced Klf6fl/fl:Lyz2cre mice BMDMs (Figure 1A). The impact of KLF6 deficiency on TNF-induced gene expression in macrophages was analyzed next. Accordingly, Lyz2cre and Klf6fl/fl:Lyz2cre mice BMDMs were stimulated with TNF, and total RNA samples were subjected to gene expression profiling studies. TNF significantly elevated the expression of approximately 4785 gene targets in control macrophages (Figure 1B). However, KLF6 deficiency alone was sufficient to abrogate >50% of TNF-induced gene expression in macrophages (Figure 1B). Next, to identify the importance of KLF6 in promoting TNF-induced signaling pathways in macrophages, RNA-sequencing data were subjected to Gene Set Enrichment Analysis.19 KLF6 deficiency significantly decreased interferon (IFN)-γ response, IFN-α response, inflammatory response, allograft rejection, IL-6–Janus kinase (Jak)–STAT3 signaling, TNF-α signaling via NF-κB, and complement signaling following TNF challenge (Figure 1, C–G). Moreover, TNF stimulation significantly induced a large number of IFN-γ response gene targets in Lyz2cre mice BMDMs (Figure 1H). However, KLF6 deficiency significantly curtailed the induction of these gene targets (Itgb7, Pim1, Ptgs2, Cfb) following the TNF challenge (Figure 1H). Consistently, KLF6 deficiency significantly reduced TNF-induced IFN-α response (Ccrl2, Irf1, Cxcl11), inflammatory response (Rela, Irak2, Il1a), and allograft rejection (Cxcl9, Mmp9, Il6) target gene expression in macrophages (Figure 1, I–K). Collectively, the analyses reveal that KLF6 deficiency broadly attenuates TNF-induced signaling pathways and inflammatory target gene expression in macrophages.

Figure 1.

Figure 1

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KLF6 deficiency attenuates tumor necrosis factor (TNF)–induced gene programs in macrophages. A:Lyz2cre and Klf6fl/fl:Lyz2cre mice bone marrow–derived macrophages (BMDMs) were stimulated with 10 ng/mL TNF for 6 hours. The KLF6 protein levels were evaluated by Western blot analyses. B: Venn diagram of gene targets that are significantly [false discovery rate (FDR) <0.05] up-regulated in Lyz2cre mice BMDMs and significantly curtailed in Klf6fl/fl:Lyz2cre mice BMDMs following TNF exposure (10 ng/mL for 6 hours). C–G: Gene Set Enrichment Analysis of RNA-sequencing data (C) and enrichment plots (D–G) from TNF-treated Lyz2cre and Klf6fl/fl:Lyz2cre mice BMDMs using Hallmark gene sets. H–K: Heat map of interferon (IFN)-γ response (H), IFN-α response (I), inflammatory response (J), and allograft rejection (K) genes that are significantly (FDR <0.05) decreased in Klf6fl/fl:Lyz2cre mice BMDMs following TNF treatment are shown. Family-wise error rate P value is given. ∗∗P < 0.01, ∗∗∗P < 0.001. Jak, Janus kinase; KO, knockout; PBS, phosphate-buffered saline; WT wild type.

KLF6 Deficiency Dampens TNF-Induced Proinflammatory Gene Expression

KLF6 deficiency suppressed a large number of TNF-induced gene targets as well as signaling pathways in macrophages (Figure 1, B–K). Thus, it was postulated that KLF6 is required for TNF-induced proinflammatory gene expression in macrophages. To test this notion, Lyz2cre and Klf6fl/fl:Lyz2cre mice BMDMs were stimulated with TNF, and total RNA samples were evaluated for classic proinflammatory gene expression. TNF challenge significantly elevated proinflammatory genes involved in IFN-γ response signaling, including Ptgs2, Cfb, Cd69, Gbp6, and Vcam1 expression in Lyz2cre macrophages (Figure 2A). However, KLF6 knockdown significantly curtailed the induction of these proinflammatory gene targets following the TNF challenge (Figure 2A). Similarly, TNF exposure significantly heightened the expression of classic IFN-α response signaling gene targets, including Cxcl11, Cxcl10, Gbp4, Ifi44, and Mx1, in Lyz2cre macrophages (Figure 2B). Remarkably, KLF6 knockdown markedly dampened the induction of these proinflammatory gene targets following the TNF challenge (Figure 2B). TNF treatment markedly upregulated inflammatory response gene targets (Clec5a, Mmp14, Nlrp3, Lpar1, and Itgb8) expression in Lyz2cre macrophages (Figure 2C). TNF-induced expression of these proinflammatory gene targets was markedly reduced in KLF6-deficient macrophages (Figure 2C). TNF treatment also significantly up-regulated the expression of allograft rejection pathway gene targets, including Ccl5, Nos2, Icam1, Cd40, and Itgal, in Lyz2cre macrophages (Figure 2D). KLF6 knockdown significantly limited the up-regulation of these proinflammatory genes following TNF treatment (Figure 2D). Collectively, the analyses show that KLF6 deficiency significantly attenuates TNF-induced proinflammatory gene expression in macrophages.

Figure 2.

Figure 2

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KLF6 deficiency dampens tumor necrosis factor (TNF)–induced proinflammatory gene expression. A–D: Bone marrow–derived macrophages from Lyz2cre and Klf6fl/fl:Lyz2cre mice were stimulated with 10 ng/mL of TNF for 6 hours. Total RNA samples were evaluated for interferon (IFN)-γ response (Ptgs2, Cfb, Cd69, Gbp6, and Vcam1; A), IFN-α response (Cxcl11, Cxcl10, Gbp4, Ifi44, and Mx1; B), inflammatory response (Clec5a, Mmp14, Nlrp3, Lpar1, and Itgb8; C), and allograft rejection gene targets (Ccl5, Nos2, Icam1, Cd40, and Itgal; D), which were analyzed by quantitative RT-PCR. 36B4 was used as a housekeeping gene. Data were analyzed by analysis of variance followed by Bonferroni post-testing. Values are reported as means ± SD (A–D). n = 4 (A–D). ∗∗P < 0.01, ∗∗∗P < 0.001. Ccl5, C-C motif chemokine ligand 5; Cd40, CD40 antigen; Cd69, CD69 antigen; Cfb, complement factor B; Clec5a, C-type lectin domain family 5, member a; Cxcl10, C-X-C motif chemokine ligand 10; Cxcl11, chemokine (C-X-C motif) ligand 11; Gbp4, guanylate-binding protein 4; Gbp6, guanylate-binding protein 6; Icam1, intercellular adhesion molecule 1; Ifi44, interferon-induced protein 44; Itgal, integrin α L; Itgb8, integrin β 8; Lpar1, lysophosphatidic acid receptor 1; Mmp14, matrix metallopeptidase 14; Mx1, MX dynamin-like GTPase 1; Nlrp3, NLR family, pyrin domain-containing 3; Nos2, nitric oxide synthase 2-inducible; PBS, phosphate-buffered saline; Ptgs2, prostaglandin-endoperoxide synthase 2; Vcam1, vascular cell adhesion molecule 1.

Myeloid-KLF6 Deficiency Limits Atherosclerotic Plaque Development

TNF elevated KLF6 expression (Figure 1A), and KLF6 deficiency attenuated TNF-induced signaling pathways and proinflammatory gene expression in macrophages (Figures 1 and 2). Western-style HFD feeding significantly elevated KLF6 mRNA (Figure 3A) and protein levels (Figure 3, B and C) in aortic macrophages. However, whether macrophage-KLF6 plays any role in the initiation or progression of atherosclerotic plaques has not been investigated. KLF6 is highly expressed in human atherosclerotic plaques,26 and KLF6 expression is substantially elevated in human atherosclerotic plaque macrophages.27 Therefore, this study examined whether macrophage-KLF6 deficiency altered experimentally induced atherosclerotic plaque development in vivo. Accordingly, 8-week–old female and male control (Lyz2cre:Apoe−/−) and myeloid-KLF6-deficient (Klf6fl/fl:Lyz2cre:Apoe−/−) mice were fed Western-style HFD for 20 weeks (n = 12 per sex per group). At the end of the study period, mice were euthanized, and the extent of aortic surface atherosclerotic plaque development was assessed by staining en face preparation of aorta with Sudan IV dye. The quantification of the aortic surface lesion area shows that myeloid-KLF6 deficiency significantly diminished HFD-induced atherosclerotic plaque area in both female and male hosts when compared with the control group (Figure 3, D and E). To address any variations in total plaque area due to differences in total aortic surface area, the percentage lesion area of the entire aortic surface area was calculated. Myeloid-KLF6–deficient mice challenged with a HFD exhibited a significantly lower percentage of aortic lesion area in female (28.7% versus 11.7%) and male (27.9% versus 11.1%) hosts compared with their respective control groups (Figure 3F). It is plausible that attenuated blood cholesterol and triglyceride levels could contribute to diminished atherosclerotic plaque formation in myeloid-KLF6–deficient mice. Surprisingly, analyses of blood cholesterol and triglyceride levels revealed that myeloid-KLF6 deficiency did not significantly impact HFD-induced blood cholesterol and triglyceride levels (Figure 3, G and H). Collectively, these results show that myeloid-KLF6 deficiency substantially limits atherosclerotic plaque development in vivo.

Figure 3.

Figure 3

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Myeloid-KLF6 deficiency limits atherosclerotic plaque development. A–C: Aortic macrophages were obtained from Apoe-null mice fed on a control or Western-style high-fat diet (HFD) for 20 weeks. A and B: Expression of KLF6 mRNA (A) and protein (B) levels was analyzed by quantitative RT-PCR and western blot analyses, respectively. C: The KLF6 Western blot densitometry analysis was performed by using ImageJ software version 1.54f. D–F: Female and male Lyz2cre:Apoe−/− and Klf6fl/fl:Lyz2cre:Apoe−/− mice were fed on a Western-style high-fat diet for 20 weeks. Aortas were obtained at the end of the feeding period and stained for lipid-filled atherosclerotic plaques using Sudan IV stain. D: Representative en-face preparation of entire aortas stained with Sudan IV is shown. E and F: Quantification of atherosclerotic lesion area (E) and percentage lesion area (F) of the entire isolated en face aortic area was calculated. The dot plot represents the mean with SD. G and H: Blood samples were collected from mice fed on a control or Western-style high-fat diet at the end of the feeding period. The total plasma cholesterol (G) and triglyceride (H) levels were quantified. Data were analyzed by analysis of variance followed by Bonferroni post-testing. n = 3 to 5 (A and B); n = 12 (D–H). ∗P ≤ 0.05, ∗∗∗P ≤ 0.001. Scale bar = 1 cm (D). NS, not significant.

Myeloid-KLF6 Deficiency Stifles Inflammatory Milieu in Atherosclerotic Plaques

KLF6 deficiency suppressed inducible proinflammatory gene expression (Figures 1 and 2) and attenuated atherosclerotic plaque development (Figure 3). The aortic root region is highly prone to the development of atherosclerotic plaques and exhibits hallmark features associated with disease progression. Thus, atheroprone regions of the aortic root were analyzed to determine whether myeloid-KLF6 deficiency alters atherosclerotic plaque structure and the inflammatory milieu in vivo. Accordingly, serial aortic root sections from female and male Lyz2cre:Apoe−/− and Klf6fl/fl:Lyz2cre: Apoe−/− mice fed on control or HFD (20 weeks) were evaluated for lipid-rich atherosclerotic plaque development by oil red O staining. Myeloid-KLF6 deficiency significantly diminished aortic root plaque formation on control as well as on the HFD diet (Figure 4, A and B). A hallmark event in atherosclerotic plaque development is the accumulation of inflammatory macrophages in the subendothelial layers of affected blood vessels.1 Thus, whether myeloid-KLF6 deficiency altered macrophage abundance in atherosclerotic plaques was assessed. Myeloid-KLF6 deficiency significantly attenuated macrophage abundance in aortic root atherosclerotic plaques on control as well as on HFD (Figure 4, C and D). Macrophages are known to accelerate atherosclerotic plaque development by producing inflammatory cytokines, chemokines, and matrix remodeling enzymes.1 Therefore, whether myeloid-KLF6 deficiency altered inflammatory gene expression in atherosclerotic plaque macrophages was evaluated. As shown in Figure 5A, myeloid-KLF6 deficiency significantly reduced the expression of gene targets involved in IFN-γ response (Ptgs2, Vcam1), IFN-α response (Ifi44, Mx1), inflammatory response (Nlrp3, Il1a), and allograft rejection (Icam1, Cd40) pathways in atherosclerotic plaque macrophages. Whether myeloid-KLF6 deficiency altered the systemic inflammatory milieu following the HFD challenge was assessed next. As shown in Figure 5B, blood levels of IL-1α, TNF, IL-6, IFN-γ, and chemokine (C-C motif) ligand 5 were significantly diminished in myeloid-KLF6–deficient mice (Klf6fl/fl:Lyz2cre:Apoe−/−) compared with those in the control mice group (Lyz2cre:Apoe−/−) on HFD. Collectively, these results show that myeloid-KLF6 deficiency significantly stifles aortic root atherosclerotic plaque development, macrophage abundance in atherosclerotic plaques, and proinflammatory gene expression in plaque macrophages, and dampens systemic proinflammatory milieu.

Figure 4.

Figure 4

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Impact of myeloid-KLF6 deficiency on aortic root plaque development. Female and male Lyz2cre:Apoe−/− and Klf6fl/fl:Lyz2cre:Apoe−/− mice were fed on a control or Western-style high-fat diet for 20 weeks. A and B: The aortic sinus serial sections were stained for lipid-filled atherosclerotic lesions with oil red O and counterstained with hematoxylin. A: Representative images of oil red O–stained aortic root sections are shown. B: The aortic root lesions area were quantified using ImageJ software version 1.54f. C and D: The aortic sinus serial sections were stained for macrophages using an anti-F4/80 antibody (C), and macrophage areas were quantified using ImageJ software version 1.54f (D). Data were analyzed by two-way analysis of variance followed by Bonferroni post-testing. n = 12 (A and C). ∗∗∗P < 0.001. Scale bar = 0.5 mm (A and C).

Figure 5.

Figure 5

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Myeloid-KLF6 deficiency curbs the aortic plaque inflammatory milieu. Eight-week–old Lyz2cre:Apoe−/− and Klf6fl/fl:Lyz2cre:Apoe−/− mice were fed on a control or Western-style high-fat diet (HFD) for 20 weeks. A: Aortic macrophages were obtained using anti-F4/80 microbeads. Total RNA samples derived from these macrophages were evaluated for expression of gene targets involved in interferon (IFN)-γ response (Ptgs2, Vcam1), IFN-α response (Ifi44, Mx1), inflammatory response (Nlrp3, Il1a), and allograft rejection (Icam1, Cd40) by quantitative RT-PCR. 36B4 was used as a housekeeping gene. B: Blood plasma samples from these mice were evaluated for IL-1α, tumor necrosis factor (TNF), IL-6, IFN-γ, and chemokine (C-C motif) ligand 5 (CCL5) levels using enzyme-linked immunosorbent assay kits. Data were analyzed by two-way analysis of variance. n = 5 (A and B). ∗∗P < 0.01, ∗∗∗P < 0.001.

KLF6 Deficiency Limits IRF1 Signaling in Macrophages

KLF6 deficiency abrogated a large number of TNF-induced gene targets (Figure 1B) as well as proinflammatory gene expression in macrophages (Figure 2). Concordantly, KLF6 deficiency reduced TNF-induced IFN-γ response, IFN-α response, inflammatory response, allograft rejection signaling pathways, and corresponding target gene expression in macrophages (Figure 1, C–K).

However, whether KLF6 harnessed specific transcription factors to promote TNF-induced proinflammatory gene expression in macrophages has yet to be examined. To address this issue, gene expression profiling data were used to perform transcription factor target analysis using the BROAD Gene Set Enrichment Analysis program.19 KLF6 deficiency significantly decreased the common IRF (STTTCRNTTT_IRF_Q6, IRF_Q6), ISRE_01, ICSBP_Q6, IRF2_01, and IRF7_01 target gene enrichment following TNF challenge (Figure 6, A–D). As expected, TNF treatment significantly elevated gene targets of common IRFs (STTTCRNTTT_IRF_Q6, IRF_Q6), IFN-stimulated response element targets (ISRE_01), and IRF1_01 targets in Lyz2cre mice BMDMs (Figure 6, E–H). Interestingly, TNF-induced expression of common IRFs, interferon-stimulated response element (ISRE), and canonical IRF1 gene targets was significantly reduced in KLF6-deficient macrophages (Figure 6, E–H). STAT1 promotes IRF1 expression,28 and STAT129 or IRF130 deficiency alleviates atherogenesis. Thus, this study examined whether KLF6 deficiency altered cytokine-induced expression and activation status of STAT1 in macrophages. KLF6 deficiency did not significantly alter IFN-γ–induced STAT1 phosphorylation or expression in macrophages (Figure 7A). TNF promotes IRF1 expression,31 and IRF1 is critical for TNF-driven interferon response.32 Therefore, it was hypothesized that KLF6 promotes TNF-induced interferon response by elevating IRF1 expression in macrophages. To test this notion, Lyz2cre and Klf6fl/fl:Lyz2cre mice BMDMs were stimulated with TNF, and total RNA samples were evaluated for Klf6 and Irf1 expression by quantitative RT-PCR analysis. The Lyz2cre and Klf6fl/fl:Lyz2cre mice BMDMs treated with IFN-γ were used as positive controls. Both TNF and IFN-γ treatment significantly elevated Klf6 expression in Lyz2cre mice BMDMs (Figure 7B). As anticipated, TNF or IFN-γ challenge did not alter Klf6 expression in Klf6fl/fl:Lyz2cre mice BMDMs (Figure 7B). Interestingly, both TNF and IFN-γ exposure robustly heightened Irf1 expression in Lyz2cre mice BMDMs (Figure 7C). Remarkably, KLF6 deficiency significantly curtailed TNF or IFN-γ–induced Irf1 mRNA expression in macrophages (Figure 7C). The next set of experiments was performed to determine whether these observations are recapitulated at the protein level. As shown in Figure 7D, KLF6 knockdown significantly curtailed TNF-induced IRF1 protein expression in macrophages. Moreover, KLF6 deficiency significantly attenuated HFD-induced IRF1 mRNA (Figure 7E) and protein (Figure 7F) expression in aortic macrophages. Taken together, the analyses show that KLF6 deficiency stifles TNF- or HFD-induced IRF1 expression as well as IRF1 target gene expression in macrophages.

Figure 6.

Figure 6

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KLF6 deficiency limits interferon regulatory factor (IRF) signaling in macrophages. A–H:Lyz2cre and Klf6fl/fl:Lyz2cre mice bone marrow–derived macrophages (BMDMs) were stimulated with 10 ng/mL tumor necrosis factor (TNF) for 6 hours. Total RNA samples from this experiment were subjected to RNA-sequencing (RNAseq) analyses. The RNAseq data were subjected to Gene Set Enrichment Analysis–based transcription factor target (TFT) analyses. A–D: A list of significantly [family-wise error rate (FWER) P <0.05] affected transcription factors (A) and corresponding gene enrichment plots (B–D) are shown. E–H: Heat map of STTTCRNTTT_IRF_Q6 (E), IRF_Q6 (F), ISRE_01 (G), and IRF1_01 (H) gene targets that are significantly (false discovery rate <0.05) attenuated in Klf6fl/fl:Lyz2cre mice BMDMs following TNF treatment are shown. FWER P values are given. n = 3 (A–H). ∗∗P < 0.01, ∗∗∗P < 0.001. KO, knockout; PBS, phosphate-buffered saline; WT, wild type.

Figure 7.

Figure 7

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KLF6 promotes proinflammatory gene expression in macrophages through IRF1. A:Lyz2cre and Klf6fl/fl:Lyz2cre mice bone marrow–derived macrophages (BMDMs) were stimulated with 10 ng/mL interferon (IFN)-γ for 1 hour. Total protein samples were analyzed for phosphorylated STAT1 (pSTAT1) and total STAT1 expression by Western blot analyses. The pSTAT1 densitometry analysis was performed by using ImageJ software version 1.54f. B and C:Lyz2cre and Klf6fl/fl:Lyz2cre mice BMDMs were stimulated with 10 ng/mL IFN-γ or tumor necrosis factor (TNF) for 6 hours. Total RNA samples were analyzed for expression of Klf6 (B) and Irf1 (C) by quantitative RT-PCR. D:Lyz2cre and Klf6fl/fl:Lyz2cre mice BMDMs were challenged with 10 ng/mL TNF for 6 hours. Total protein samples were analyzed for IRF1 expression by Western blot analysis. IRF1 densitometry analysis was performed by using ImageJ software version 1.54f. β-Actin was used as a housekeeping gene. E and F: Aortic macrophages were obtained from Lyz2cre:Apoe−/− and Klf6fl/fl:Lyz2cre:Apoe−/− mice fed on a control or Western-style high-fat diet (HFD) for 20 weeks. Total RNA (n = 5) and protein (n = 3) samples derived from these macrophages were evaluated for expression of IRF1 mRNA and protein levels by quantitative RT-PCR and Western blot, respectively. G and H: RAW264.7 cells were transfected with an IRF-luciferase reporter construct in the presence of pCIneo-Klf6 plasmid (G) or Klf6-specific siRNA (H). These cells were stimulated with 10 ng/mL TNF for 18 hours, and cell lysates were analyzed for luciferase activity. I: RAW264.7 cells were cotransfected with a combination of Klf6-specific siRNA or Mac_N_IRF1 and stimulated with 10 ng/mL TNF for 6 hours. Total RNA from these experiments was evaluated for expression of Vcam1, Ifi44, Nlrp3, and Icam1 by quantitative RT-PCR. A–G: Data were analyzed by analysis of variance followed by Bonferroni post-testing. All values are reported as means ± SD (A–E and G–I). n = 3 (A, D, G, and H); n = 4 (B, C, and I). ∗∗∗P < 0.001. NS, not significant; PBS, phosphate-buffered saline.

IRF1 Overexpression Rescues Attenuated KLF6 Targets in Macrophages

Given the newly ascribed role of KLF6 in promoting IRF1 and its target gene expression, this study examined whether altering KLF6 levels affected TNF-induced IRF1 transcriptional activity in macrophages. Accordingly, RAW264.7 cells were cotransfected with IRF1-driven luciferase reporter plasmid in the presence of pCI-neo-KLF6 plasmid (Figure 7G) or Klf6-specific siRNA (Figure 7H). These cells were stimulated with TNF, and luciferase activities were recorded (Figure 7, G and H). Overexpression of KLF6 increased, whereas deficiency of KLF6 decreased, TNF-induced IRF1 luciferase reporter activity in macrophages (Figure 7, G and H). Overall, KLF6 promoted TNF-induced IRF1 expression (Figure 7, C and D), IRF1 transcriptional activity (Figure 7, G and H), and IRF1 target gene expression (Figure 6) in macrophages. On the basis of these observations, it was hypothesized that KLF6 may use IRF1 to promote cytokine-induced proinflammatory gene expression in macrophages. To test this hypothesis, whether IRF1 overexpression rescues diminished KLF6 target genes in KLF6-deficient macrophages was examined. Accordingly, RAW264.7 macrophages were cotransfected with Mac_N_IRF1 plasmid in the presence of siControl or Klf6-specific siRNA. These cells were stimulated with TNF, and total RNA samples were evaluated for KLF6 target gene expression by quantitative RT-PCR (Figure 7I). As shown in Figure 7I, TNF challenge significantly elevated KLF6 gene targets, such as Vcam1, Ifi44, Nlrp3, and Icam1 expression in macrophages. As predicted, KLF6 deficiency significantly abrogated TNF-induced KLF6 target gene (Vcam1, Ifi44, Nlrp3, and Icam1) expression in macrophages (Figure 7I). Interestingly, IRF1 overexpression fully reversed attenuated KLF6 gene target (Vcam1, Ifi44, Nlrp3, and Icam1) expression following TNF treatment (Figure 7I). Collectively, the analyses show that KLF6 uses IRF1 signaling to promote TNF-induced proinflammatory gene expression in macrophages.

Discussion

The principal finding of this study is that KLF6 promoted broad proinflammatory gene expression programs in macrophages and accelerated experimentally induced atherosclerotic plaque development. The main observations are as follows: i) Myeloid-KLF6 deficiency suppressed experimentally induced atherosclerotic plaque development. ii) Myeloid-KLF6 deficiency attenuated inflammatory milieu in atherosclerotic plaques. iii) KLF6 deficiency alone was sufficient to abrogate a large number of TNF-induced gene expression in macrophages. iv) KLF6 deficiency suppressed key inflammatory pathways involved in atherogenesis. v) KLF6 deficiency limited IRF1 signaling and IRF1 target gene expression in macrophages. vi) IRF1 overexpression rescued attenuated KLF6 gene targets in macrophages. Collectively, this study offers the first evidence that macrophage-KLF6 deficiency limited experimentally induced atherosclerotic plaque development by attenuating key inflammatory signaling pathways and proinflammatory target gene expression (Figure 8).

Figure 8.

Figure 8

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Macrophage-KLF6 signaling promotes atherogenesis. KLF6 promotes proinflammatory cytokine-induced IRF1 signaling, inflammatory gene expression, macrophage inflammatory response, and atherogenesis.

The importance of macrophages in the initiation of inflammation and inflammatory disease pathogenesis has been extensively studied. However, because of their phenotypic plasticity, functional adaptability to local environment, and tissue-specific functions, full-scale contributions to atherosclerotic plaque development are not fully explored. Atherosclerosis is a lipid-driven inflammatory vascular disease characterized by the accumulation of lipid-laden macrophages in subendothelial space. Specifically, lipid build-up in the intimal extracellular space is handled by macrophages via various lipid handling processes, such as autophagy, storage, and efflux. In such microenvironment, native and modified lipoprotein particles trigger inflammatory responses that help to recruit additional inflammatory cells as well as contribute to disease pathogenesis. This study showed that macrophage-KLF6 deficiency significantly curtailed lipid accumulation and macrophage abundance in the aortic root area of mice on control or following HFD challenge. However, the limitation of this study is that the use of Apoe–/– mice background to induce atherosclerosis in mice may not fully recapitulate the pathophysiological mechanisms operative in human disease pathogenesis. Similarly, use of a single concentration of cytokines to induce inflammatory gene expression may not capture the full-spectrum of the action of these cytokines in inflammatory gene expression in macrophages. Macrophages are known to secrete a large number of proinflammatory molecules, prothrombotic mediators, extracellular matrix-degrading proteases, cytokines, and chemokines that influence atherosclerotic plaque cellular composition, growth, and stability. The current study corroborated a previous study showing that macrophage-KLF6 deficiency significantly limits inducible IL-1β and TNF expression in macrophages.14 Consistently, KLF6 deficiency also reduced intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 expression in aortic plaque macrophages. Many of the cytokines (Il1a, Il6, Il12, Il15, and Il18), chemokines (Ccl2, Ccl3, Ccl4, Ccl5, Cxcl1, Cxcl2, and Cxcl10), and cell-surface receptors (Cd44, Itgal, Itga4, and Tlr4) known to promote atherogenesis were also attenuated in KLF6-deficient macrophages. Consistent with previous observations, results from this study showed that KLF6 deficiency substantially reduced IFN-α/γ response, inflammatory response, allograft rejection, IL-6–STAT3, and TNF-induced NF-κB signaling in macrophages. Furthermore, macrophage-KLF6 deficiency attenuated inflammatory gene expression in atherosclerotic plaque macrophages and circulating blood cytokine levels following the HFD challenge. Collectively, this study showed that KLF6 promoted pathogenic proinflammatory gene expression that promoted atherogenesis.

Expression of TNF and its receptors (TNFR1 and TNFR2) are elevated in aortic plaques,33 and TNF is one of the most potent cytokines that promotes atherogenesis. The HFD challenge of TNF knockout mice on Apoe–/– background results in a significant reduction in inflammatory gene expression as well as atherosclerotic plaque development.21,22 Furthermore, bone marrow transplantation studies using TNF knockout mice indicate that TNF derived from bone marrow cells is the key driver of atherogenesis in experimental studies.25 Herein, TNF induced KLF6 expression in macrophages. KLF6 deficiency attenuated >50% of TNF-induced target gene expression in macrophages. In addition, KLF6 deficiency substantially curtailed TNF-induced signaling pathways and inflammatory gene expression in macrophages. Administration of recombinant TNF elevates atherosclerotic plaque development through NF-κB signaling.24 Consistent with this observation, KLF6 deficiency diminished TNF-induced NF-κB signaling and its target gene expression in macrophages in the current study. Exogenous administration of IFN-γ significantly elevates atherosclerotic plaque formation in Apoe–/– mice.34 Similarly, IFN-γ deficiency significantly alleviates experimentally induced atherosclerosis plaque development.35 IFN-γ boosts IRF1 expression to exert a broad proinflammatory gene expression profile in immune cells.36 Interestingly, IRF1 deficiency significantly hampered experimentally induced atherosclerotic plaques in vivo.30 TNF promotes IRF1 expression to sustain elevated levels of cytokines and chemokine expression in macrophages.31 Moreover, TNF receptor deficiency also significantly restrains TNF-induced IRF1 expression in macrophages.31 Herein, KLF6 deficiency significantly dampened TNF-induced IFN-γ response, IFN-α response, and inflammatory response in macrophages. Thus, it served as a critical mediator of TNF-induced interferon signaling in macrophages. Furthermore, transcription factor target analysis showed that KLF6 deficiency significantly diminished the TNF-induced IRF gene targets as well as ISREs regulated gene targets in macrophages. IRF1 is a critical mediator of TNF-driven interferon response in rheumatoid arthritis.32,37 Moreover, in endothelial cells, TNF induces IRF1 expression to promote monocyte recruitment to the site of inflammation.38 Outcomes from the current study show that KLF6 deficiency significantly reduced TNF-induced interferon response, IRF target gene expression, and a large number of inflammatory gene targets involved in atherogenesis. Furthermore, macrophage-KLF6 deficiency significantly decreased macrophage abundance in aortic root plaques and HFD-induced atherosclerotic plaque development in vivo.

In summary, this study highlighted the importance of KLF6 signaling in governing TNF-induced inflammatory gene expression in macrophages. The in vivo data showed that macrophage-KLF6 deficiency significantly limited macrophage abundance in aortic plaques and experimentally induced atherosclerotic plaque development. KLF6 deficiency decreased TNF-induced IFN-γ response, IFN-α response, and inflammatory response in macrophages. More importantly, KLF6 deficiency substantially reduced TNF-induced expression of IRF gene targets and ISRE-regulated genes in macrophages. At the molecular level, KLF6 promoted TNF-induced IRF1 expression and IRF1-dependent proinflammatory gene expression in macrophages. Furthermore, overexpression of IRF1 rescued the attenuated proinflammatory gene expression observed in KLF6-deficient macrophages.

Disclosure Statement

None declared.

Acknowledgments

Author Contributions

G.H.M. conceived the study; G.H.M., H.P.N., A.Z., R.D.-Z., G.-D.K., K.B., Y.P., and O.M. performed experiments; G.H.M., H.P.N., A.Z., G.-D.K., and E.R.C. analyzed the data; J.D.S. supervised the atherosclerosis studies; and G.H.M. wrote the manuscript which was edited and approved by all authors.

Footnotes

Supported by NIH grant HL126626 (G.H.M.).

References

  • 1.Hopkins P.N. Molecular biology of atherosclerosis. Physiol Rev. 2013;93:1317–1542. doi: 10.1152/physrev.00004.2012. [DOI] [PubMed] [Google Scholar]
  • 2.Moore K.J., Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell. 2011;145:341–355. doi: 10.1016/j.cell.2011.04.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bäck M., Yurdagul A., Jr., Tabas I., Öörni K., Kovanen P.T. Inflammation and its resolution in atherosclerosis: mediators and therapeutic opportunities. Nat Rev Cardiol. 2019;16:389–406. doi: 10.1038/s41569-019-0169-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Susser L.I., Rayner K.J. Through the layers: how macrophages drive atherosclerosis across the vessel wall. J Clin Invest. 2022;132 doi: 10.1172/JCI157011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Ley K., Miller Y.I., Hedrick C.C. Monocyte and macrophage dynamics during atherogenesis. Arterioscler Thromb Vasc Biol. 2011;31:1506–1516. doi: 10.1161/ATVBAHA.110.221127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Quintar A.A., Hedrick C.C., Ley K. Monocyte phenotypes: when local education counts. J Exp Med. 2015;212:432. doi: 10.1084/jem.2124insight1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Stoger J.L., Gijbels M.J., van der Velden S., Manca M., van der Loos C.M., Biessen E.A., Daemen M.J., Lutgens E., de Winther M.P. Distribution of macrophage polarization markers in human atherosclerosis. Atherosclerosis. 2012;225:461–468. doi: 10.1016/j.atherosclerosis.2012.09.013. [DOI] [PubMed] [Google Scholar]
  • 8.Chinetti-Gbaguidi G., Baron M., Bouhlel M.A., Vanhoutte J., Copin C., Sebti Y., Derudas B., Mayi T., Bories G., Tailleux A., Haulon S., Zawadzki C., Jude B., Staels B. Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPARgamma and LXRalpha pathways. Circ Res. 2011;108:985–995. doi: 10.1161/CIRCRESAHA.110.233775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Johnson J.L., Newby A.C. Macrophage heterogeneity in atherosclerotic plaques. Curr Opin Lipidol. 2009;20:370–378. doi: 10.1097/MOL.0b013e3283309848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Tabas I., Bornfeldt K.E. Intracellular and intercellular aspects of macrophage immunometabolism in atherosclerosis. Circ Res. 2020;126:1209–1227. doi: 10.1161/CIRCRESAHA.119.315939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Glass C.K., Natoli G. Molecular control of activation and priming in macrophages. Nat Immunol. 2016;17:26–33. doi: 10.1038/ni.3306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Syafruddin S.E., Mohtar M.A., Wan M., Nazarie W.F., Low T.Y. Two sides of the same coin: the roles of KLF6 in physiology and pathophysiology. Biomolecules. 2020;10:1378. doi: 10.3390/biom10101378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.McConnell B.B., Yang V.W. Mammalian Krüppel-like factors in health and diseases. Physiol Rev. 2010;90:1337–1381. doi: 10.1152/physrev.00058.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Date D., Das R., Narla G., Simon D.I., Jain M.K., Mahabeleshwar G.H. Kruppel-like transcription factor 6 regulates inflammatory macrophage polarization. J Biol Chem. 2014;289:10318–10329. doi: 10.1074/jbc.M113.526749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Kim G.D., Das R., Goduni L., McClellan S., Hazlett L.D., Mahabeleshwar G.H. Kruppel-like factor 6 promotes macrophage-mediated inflammation by suppressing B cell leukemia/lymphoma 6 expression. J Biol Chem. 2016;291:21271–21282. doi: 10.1074/jbc.M116.738617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Goodman W.A., Omenetti S., Date D., Di Martino L., De Salvo C., Kim G.D., Chowdhry S., Bamias G., Cominelli F., Pizarro T.T., Mahabeleshwar G.H. KLF6 contributes to myeloid cell plasticity in the pathogenesis of intestinal inflammation. Mucosal Immunol. 2016;9:1250–1262. doi: 10.1038/mi.2016.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Kim G.D., Ng H.P., Patel N., Mahabeleshwar G.H. Kruppel-like factor 6 and miR-223 signaling axis regulates macrophage-mediated inflammation. FASEB J. 2019;33:10902–10915. doi: 10.1096/fj.201900867RR. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Kim G.D., Ng H.P., Chan E.R., Mahabeleshwar G.H. Kruppel-like factor 6 promotes macrophage inflammatory and hypoxia response. FASEB J. 2020;34:3209–3223. doi: 10.1096/fj.201902221R. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Subramanian A., Tamayo P., Mootha V.K., Mukherjee S., Ebert B.L., Gillette M.A., Paulovich A., Pomeroy S.L., Golub T.R., Lander E.S., Mesirov J.P. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545–15550. doi: 10.1073/pnas.0506580102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Metsalu T., Vilo J. ClustVis: a web tool for visualizing clustering of multivariate data using principal component analysis and heatmap. Nucleic Acids Res. 2015;43:W566–W570. doi: 10.1093/nar/gkv468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Ohta H., Wada H., Niwa T., Kirii H., Iwamoto N., Fujii H., Saito K., Sekikawa K., Seishima M. Disruption of tumor necrosis factor-alpha gene diminishes the development of atherosclerosis in ApoE-deficient mice. Atherosclerosis. 2005;180:11–17. doi: 10.1016/j.atherosclerosis.2004.11.016. [DOI] [PubMed] [Google Scholar]
  • 22.Xiao N., Yin M., Zhang L., Qu X., Du H., Sun X., Mao L., Ren G., Zhang C., Geng Y., An L., Pan J. Tumor necrosis factor-alpha deficiency retards early fatty-streak lesion by influencing the expression of inflammatory factors in apoE-null mice. Mol Genet Metab. 2009;96:239–244. doi: 10.1016/j.ymgme.2008.11.166. [DOI] [PubMed] [Google Scholar]
  • 23.Tuleta I., França C.N., Wenzel D., Fleischmann B., Nickenig G., Werner N., Skowasch D. Hypoxia-induced endothelial dysfunction in apolipoprotein E-deficient mice; effects of infliximab and L-glutathione. Atherosclerosis. 2014;236:400–410. doi: 10.1016/j.atherosclerosis.2014.08.021. [DOI] [PubMed] [Google Scholar]
  • 24.Zhang Y., Yang X., Bian F., Wu P., Xing S., Xu G., Li W., Chi J., Ouyang C., Zheng T., Wu D., Zhang Y., Li Y., Jin S. TNF-[alpha] promotes early atherosclerosis by increasing transcytosis of LDL across endothelial cells: crosstalk between NF-[kappa]B and PPAR-[gamma] J Mol Cell Cardiol. 2014;72:85–94. doi: 10.1016/j.yjmcc.2014.02.012. [DOI] [PubMed] [Google Scholar]
  • 25.Brånén L., Hovgaard L., Nitulescu M., Bengtsson E., Nilsson J., Jovinge S. Inhibition of tumor necrosis factor-alpha reduces atherosclerosis in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol. 2004;24:2137–2142. doi: 10.1161/01.ATV.0000143933.20616.1b. [DOI] [PubMed] [Google Scholar]
  • 26.Dhaouadi N., Li J.Y., Feugier P., Gustin M.P., Dab H., Kacem K., Bricca G., Cerutti C. Computational identification of potential transcriptional regulators of TGF-[beta]1 in human atherosclerotic arteries. Genomics. 2014;103:357–370. doi: 10.1016/j.ygeno.2014.05.001. [DOI] [PubMed] [Google Scholar]
  • 27.Patino W.D., Kang J.G., Matoba S., Mian O.Y., Gochuico B.R., Hwang P.M. Atherosclerotic plaque macrophage transcriptional regulators are expressed in blood and modulated by tristetraprolin. Circ Res. 2006;98:1282–1289. doi: 10.1161/01.RES.0000222284.48288.28. [DOI] [PubMed] [Google Scholar]
  • 28.Forero A., Ozarkar S., Li H., Lee C.H., Hemann E.A., Nadjsombati M.S., Hendricks M.R., So L., Green R., Roy C.N., Sarkar S.N., von Moltke J., Anderson S.K., Gale M., Jr., Savan R. Differential activation of the transcription factor IRF1 underlies the distinct immune responses elicited by type I and type III interferons. Immunity. 2019;51:451–464.e6. doi: 10.1016/j.immuni.2019.07.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Agrawal S., Febbraio M., Podrez E., Cathcart M.K., Stark G.R., Chisolm G.M. Signal transducer and activator of transcription 1 is required for optimal foam cell formation and atherosclerotic lesion development. Circulation. 2007;115:2939–2947. doi: 10.1161/CIRCULATIONAHA.107.696922. [DOI] [PubMed] [Google Scholar]
  • 30.Du M., Wang X., Mao X., Yang L., Huang K., Zhang F., Wang Y., Luo X., Wang C., Peng J., Liang M., Huang D., Huang K. Absence of interferon regulatory factor 1 protects against atherosclerosis in apolipoprotein E-deficient mice. Theranostics. 2019;9:4688–4703. doi: 10.7150/thno.36862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Yarilina A., Park-Min K.H., Antoniv T., Hu X., Ivashkiv L.B. TNF activates an IRF1-dependent autocrine loop leading to sustained expression of chemokines and STAT1-dependent type I interferon-response genes. Nat Immunol. 2008;9:378–387. doi: 10.1038/ni1576. [DOI] [PubMed] [Google Scholar]
  • 32.Bonelli M., Dalwigk K., Platzer A., Olmos Calvo I., Hayer S., Niederreiter B., Holinka J., Sevelda F., Pap T., Steiner G., Superti-Furga G., Smolen J.S., Kiener H.P., Karonitsch T. IRF1 is critical for the TNF-driven interferon response in rheumatoid fibroblast-like synoviocytes : JAKinibs suppress the interferon response in RA-FLSs. Exp Mol Med. 2019;51:1–11. doi: 10.1038/s12276-019-0267-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Niemann-Jönsson A., Söderberg I., Lindholm M.W., Jovinge S., Nilsson J., Fredrikson G.N. Medial expression of TNF-[alpha] and TNF receptors precedes the development of atherosclerotic lesions in apolipoprotein E/LDL receptor double knockout mice. Int J Biomed Sci. 2007;3:116–122. [PMC free article] [PubMed] [Google Scholar]
  • 34.Whitman S.C., Ravisankar P., Elam H., Daugherty A. Exogenous interferon-gamma enhances atherosclerosis in apolipoprotein E-/- mice. Am J Pathol. 2000;157:1819–1824. doi: 10.1016/s0002-9440(10)64820-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Whitman S.C., Ravisankar P., Daugherty A. IFN-gamma deficiency exerts gender-specific effects on atherogenesis in apolipoprotein E-/- mice. J Interferon Cytokine Res. 2002;22:661–670. doi: 10.1089/10799900260100141. [DOI] [PubMed] [Google Scholar]
  • 36.Negishi H., Fujita Y., Yanai H., Sakaguchi S., Ouyang X., Shinohara M., Takayanagi H., Ohba Y., Taniguchi T., Honda K. Evidence for licensing of IFN-gamma-induced IFN regulatory factor 1 transcription factor by MyD88 in Toll-like receptor-dependent gene induction program. Proc Natl Acad Sci U S A. 2006;103:15136–15141. doi: 10.1073/pnas.0607181103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Marotte H., Tsou P.S., Rabquer B.J., Pinney A.J., Fedorova T., Lalwani N., Koch A.E. Blocking of interferon regulatory factor 1 reduces tumor necrosis factor [alpha]-induced interleukin-18 bioactivity in rheumatoid arthritis synovial fibroblasts by induction of interleukin-18 binding protein a: role of the nuclear interferon regulatory factor 1-NF-[kappa]B-c-jun complex. Arthritis Rheum. 2011;63:3253–3262. doi: 10.1002/art.30583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Venkatesh D., Ernandez T., Rosetti F., Batal I., Cullere X., Luscinskas F.W., Zhang Y., Stavrakis G., García-Cardeña G., Horwitz B.H., Mayadas T.N. Endothelial TNF receptor 2 induces IRF1 transcription factor-dependent interferon-[beta] autocrine signaling to promote monocyte recruitment. Immunity. 2013;38:1025–1037. doi: 10.1016/j.immuni.2013.01.012. [DOI] [PMC free article] [PubMed] [Google Scholar]

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