Interleukin-34-NF-κB signaling aggravates myocardial ischemic/reperfusion injury by facilitating macrophage recruitment and polarization

Summary

Background

Macrophage infiltration and polarization are integral to the progression of heart failure and cardiac fibrosis after ischemia/reperfusion (IR). Interleukin 34 (IL-34) is an inflammatory regulator related to a series of autoimmune diseases. Whether IL-34 mediates inflammatory responses and contributes to cardiac remodeling and heart failure post-IR remains unclear.

Methods

IL-34 knock-out mice were used to determine the role of IL-34 on cardiac remodeling after IR surgery. Then, immunofluorescence, flow cytometry assays, and RNA-seq analysis were performed to explore the underlying mechanisms of IL-34-induced macrophage recruitment and polarization, and further heart failure after IR.

Findings

By re-analyzing single-cell RNA-seq and single-nucleus RNA-seq data of murine and human ischemic hearts, we showed that IL-34 expression was upregulated after IR. IL-34 knockout mitigated cardiac remodeling, cardiac dysfunction, and fibrosis after IR and vice versa. RNA-seq analysis revealed that IL-34 deletion correlated negatively with immune responses and chemotaxis after IR injury. Consistently, immunofluorescence and flow cytometry assays demonstrated that IL-34 deletion attenuated macrophage recruitment and CCR2+ macrophage polarization. Mechanistically, IL-34 deficiency repressed both the canonical and noncanonical NF-κB signaling pathway, leading to marked reduction of P-IKKβ and P-IκBα kinase levels; downregulation of NF-κB p65, RelB, and p52 expression, which drove the decline in chemokine CCL2 expression. Finally, IL-34 and CCL2 levels were increased in the serum of acute coronary syndrome patients, with a positive correlation between circulating IL-34 and CCL2 levels in clinical patients.

Interpretation

In conclusion, IL-34 sustains NF-κB pathway activation to elicit increased CCL2 expression, which contributes to macrophage recruitment and polarization, and subsequently exacerbates cardiac remodeling and heart failure post-IR. Strategies targeting IL-34-centered immunomodulation may provide new therapeutic approaches to prevent and reverse cardiac remodeling and heart failure in clinical MI patients after percutaneous coronary intervention.

Funding

This study was supported by the National Nature Science Foundation of China (81670352 and 81970327 to R T, 82000368 to Q F).

Research in context.

Evidence before this study

Macrophage activation and polarization plays indispensable roles in the pathology of heart failure after myocardial infarction (MI) or myocardial ischemia/reperfusion injury (IR). Therefore, identifying secreted regulators of macrophage recruitment and polarizations could provide promising therapeutic modifications for clinical intervention of MI. Interleukin 34 (IL-34) is widely reported as a secreted inflammatory regulator and participates in the progression of autoimmune diseases. However, whether IL-34 directly contributes to the onset and progression of cardiac remodeling and heart failure after IR, as well as its underlying mechanisms remain unknown.

Added value of this study

This study shows that IL-34 knockout attenuates infarct size, cardiac dysfunction, and fibrosis after IR and vice versa. Mechanistically, we reported that IL-34-null blocks the activation of NF-κB signaling, and subsequently decreases CCL2 expression, which prevents macrophage recruitment and polarization after IR injury. In line with pre-clinical findings, we observed that serum IL-34 levels were elevated in patients with acute coronary syndrome and positively correlated with CCL2 levels.

Implications of all the available evidence

In this study, evidence linking IL-34 with macrophage polarization, NF-κB signaling, and cardiac remodeling after IR is provided. This study broadens our understandings of the biological functions of IL-34 in macrophage activation and polarization, and provide as new therapeutic approaches to prevent and reverse cardiac remodeling and heart failure in clinical MI and reperfused MI after percutaneous coronary intervention.

Introduction

Heart failure is the terminal consequence of various cardiovascular diseases and remains one of the most common and costly public health issues worldwide.^1,2 Among them, coronary artery diseases, including myocardial infarction (MI) and ischemia/reperfusion injury (IR), account for nearly 50% of heart failure cases.³ Although myocardial reperfusion efficiently ameliorates the infarct size and adverse clinical outcomes in acute MI patients, the global incidence of heart failure and cardiac death remains high and a significant clinical challenge.^4,5 Therefore, identifying master regulators of cardiac ischemic injuries may offer potential therapeutic modifications to prevent and reverse the progression of heart failure after MI and IR.

Similar to MI, reperfusion after MI leads to extensive cardiomyocyte necrosis and orchestrated immune cells, fibroblasts, and endothelial cells responses to prevent cardiac injuries and promote tissue healing.⁶ The complex function of immune cells in the progression of MI and IR is gaining appreciation.⁷ Appropriate immune responses contribute to the clearance of necrotic cell debris and scar formation. However, continuous inflammatory activation exacerbates cardiac remodeling and heart failure.8, 9, 10 Therefore, coordinated immune cell recruitment and inflammatory responses are closely related to the development of heart failure after MI and IR.

Of note, macrophages constitute the largest immune cell subset in the heart, showing marked plasticity and flexibility under various pathological conditions.^11,12 Once cardiac ischemia has occurred, macrophages are recruited to the infarcted area and exhibit prominent inflammatory phenotypes with upregulated cytokines and chemokines (including IL-1, IL-6, IL-12, CCL2), reactive oxygen species (ROS), and reactive nitrogen species (RNS).^10,13 As the healing process advances, macrophages become polarized toward a reparative phenotype with the high expression of MRC1, CD206, IL-10, and ARG1, which are involved in limiting and suppressing the inflammatory response and tissue repair.¹⁴ Our laboratory focuses on the biphasic roles of macrophages, and have previously demonstrated that deletion of macrophage dectin-1, dectin-2, and Lgr4 receptors causes a decline in neutrophil recruitment and in macrophage polarization, and further ameliorates cardiac remodeling and cardiac dysfunction after MI or IR.15, 16, 17 However, ethical challenges and safety concerns about macrophage receptor deletion using CRISPR/CAS9 or via the adeno-associated viruses limit its full translation from the bench to the bedside. Therefore, identification of secreted regulators of macrophage recruitment and polarizations would hold promise for therapeutic strategies for clinical MI intervention.

Interleukin 34 (IL-34) is widely reported as an inflammatory regulator and is highly expressed in multiple tissues, including the brain, kidneys, and heart.¹⁸ IL-34 was first reported as a specific ligand of the colony-stimulating factor-1 receptor (CSF-1R) in 2008.¹⁹ It was proposed that IL-34 participates in the survival and proliferation of monocytes, macrophages, and dendritic cells under homeostatic and inflammatory conditions.²⁰ A series of publications revealed that cellular and circulating levels of IL-34 are upregulated in numerous autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, and inflammatory bowel diseases; suggesting a cross-link between IL-34 and immune responses.21, 22, 23 Moreover, we and other researchers have reported the relationship between serum IL-34 levels and the incidence and exacerbation of cardiovascular diseases, for example, 1) the serum IL-34 level is elevated in patients with coronary artery disease²⁴; 2) serum IL-34 level is increased in patients with ischemic cardiomyopathy and related to the severity of heart failure²⁵; and 3) the serum IL-34 level positively correlates with the incidence of cardiovascular death and re-hospitalization of heart failure patients.²⁶ Although these studies provide evidence for the link between IL-34 and immune responses and cardiac dysfunction, it remains unclear whether IL-34 contributes directly to the onset and progression of cardiac remodeling and heart failure after IR, and if so, whether IL-34 triggers cardiac damage after IR in an inflammation-dependent or -independent manner, and by what underlying mechanisms.

Here, we show that IL-34 deficiency significantly attenuates infarct size, cardiac dysfunction, and fibrosis after IR, and vice versa. IL-34 deletion prevents macrophage recruitment and the decrease in polarization of CCR2+ macrophages after IR. Mechanistically, the loss of IL-34 blocks the activation of NF-κB signaling, which subsequently decreases the expression of CCL2. In support of the regulatory role of the IL-34-NF-κB axis on the CCL2 level, we observed that serum IL-34 levels are elevated in patients with acute coronary syndrome and correlate positively with CCL2 levels.

Methods

MI and myocardial IR model

Eight-week-old wild-type (WT) male C57BL/6 mice were purchased from Shanghai JieSiJie Laboratory Animal Co., Ltd. (Shanghai, China). All mice used in the study were age-matched and exclusively male, because of female traits were more flexible than males due to the effects of ovulatory cycles and hormones. Mice were fed with a standard chow diet until sacrifice and were maintained under a 12:12 h light–dark cycle. All experimental procedures were approved by the Animal Care Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.

The MI model was constructed as previously described by permanent left anterior descending coronary artery ligation. Briefly, 8-week-old male IL-34 knockout and wild-type littermates were used in this study. The male mice were anesthetized with 1.5% isoflurane for 5 min, followed by tracheal intubation. Anesthesia and mechanical ventilation were continued during the surgery by mixing oxygen with 0.5% isoflurane. Mice were then placed on a 37 °C temperature-controlled plate and disinfected the operation area. The skin, connective tissue, and 3rd and 4th intercostal spatia were sequentially dissected to expose the heart. After removing the pericardium, the left anterior descending artery was ligated with an 8–0 silk suture. Then, the chest and skin were closed with 5–0 and 3–0 silk sutures, respectively. Sham surgery involved all procedures except artery ligation. The investigators carried out the animal experiments were not blinded to the groups.

The myocardial IR model was constructed with 45 min of ischemia followed by reperfusion. The ischemia procedures were similar to those used in the MI model. After 45 min of ligation, the mice were re-intubated, the heart were exposed, and the suture of the coronary artery was released to induce reperfusion injury. Cardiac reperfusion was visually confirmed by the rapid restoration of blood flow in the ischemic area.

IL-34 knock out mice

The IL-34 knock out mice were generated by Cyagen Biosciences with standard procedures. Briefly, the floxed IL-34 mice were generated by CRISPR/Cas9 mediated genome engineering. The LoxP was inserted in the exon 3 of Il34 genome (NCBI Reference Sequence: NM_001135100; Ensembl: ENSMUSG00000031750) with the homologous recombinases. The Il34 transcripts have 7 exons and ATG start codon appears on exon 2. Deletion of exon 3 would result in frameshift of this gene, further the loss function of IL-34. The homozygous IL-34 flox/flox mice were obtained by inter-cross the heterozygous mice. Then, the female IL-34 flox/flox mice were bred with a mouse line expressing Cre recombinase under the control of the EIIa promoter (EIIa-Cre) to obtain homozygous mice with the whole tissue deletion of Il34 (IL34-KO). Homozygous IL34-KO was identified by reverse transcription-polymerase chain reaction with followed primers:

F2: 5′-GCTGAGAGTCATGTCCACACTATT-3’;

R2: 5′-CCAGGATTTTCAGGAGACATGGTA-3’;

F1: 5′-CCTTAGGAAGGCCGTTGAACATA-3’.

Western blotting

To determine the protein expression of target genes, heart tissue followed differential treatment was lysed with SDS lysis buffer (50 mM Tris, pH 8.1, SDS, sodium pyrophosphate, β-glycerophosphate, sodium orthovanadate, sodium fluoride, EDTA, and leupeptin) containing phenylmethanesulfonyl fluoride and phosphatase inhibitor cocktail (Roche). To obtained the homogenized sample, tissue was cut into 1–3 mm³ pieces and sonicated for 15 min using a Qsonica Q700 sonicator (Qsonica LLC, Newtown, USA). After removing the insoluble materials with centrifugation (12,000 rpm, 20 min), the supernatant was collected and protein concentration was quantified by bicinchoninic acid assay (# CW2011S; CWBIO, Beijing, China). The protein samples were then denatured by mixing with 4 × laemmli sample buffer (#1610747; Bio-Rad, Melville, NY, USA) and boiled at 100 °C for 10 min. Next, 30 μg of cleared lysate was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After transferring, PVDF membranes were blocked with 5% nonfat dried milk or 5% bovine serum albumin (detecting phosphorylated proteins) at room temperature for 1 h and incubated with primary antibodies overnight at 4 °C. To probe the signaling of target genes, the PVDF membrane were wash three times with TBST buffer (20 mM Tris–HCl, pH 7.5, 0.5 M NaCl, 0.1% Tween 20), and incubated with the specific horseradish peroxidase-conjugated secondary antibodies (1:3000 in 5% milk) at room temperature for 1 h. Specific protein bands were detected using the ECL detection reagent (#180–5001, Tanon, Shanghai, China). The information on antibodies applied in this study referred to Reagent Validation.

Immunofluorescence

To probe the expression of IL-34, F4/80, LY6G, CCR2, and CCL2, we conducted the immunofluorescence using frozen section from indicated hearts. Briefly, mice were sacrificed with cervical dislocation and intraventricularly perfused with 10 mL PBS buffer and 4% paraformaldehyde buffer. Then heart was fixed with 4% paraformaldehyde at 4 °C for 2 h, cryoprotected with 20% sucrose in PBS buffer for 4 h and 30% sucrose buffer for 4 h. Heart were further embedded in optimum cutting temperature compound (OCT, Sakura Finetechnical, Tokyo, Japan) for frozen sectioning (7 μm). Frozen sections were washed with PBS buffer for three times, permeabilizated and blocked with PBSST (940 μL PBS, 10 μL Triton X-100, 50 μL donkey serum) for 30 min at room temperature. The sections were next stained with primary antibodies at 4 °C overnight. Negative control sections were incubated with IgG antibodies. After incubation, the sections were washed with PBS buffer, stained with secondary antibody (1:1000) at room temperature for 2 h and stained the nuclei with Hoechst 33,342 (1:1000) for 5 min. The information on antibodies applied in this study referred to Reagent Validation.

Bulk RNA sequencing analysis (RNA-seq)

WT or IL-34 knock out mice were subjected to MI or IR surgery, total RNA from indicated groups (n = 3 for each group) was extracted using TRIzol Reagent, reverse-transcribed, and amplified according to the standard procedures. Sequencing libraries were constructed using the BGISEQ-500 platform (BGI, Wuhan, China). RNA quality including the mRNA concentration, RIN value, 28S/18S, was examined with an Agilent Bioanalyzer 2100 (Agilent). Once passed, RNA was transcribed into cDNA using the SMART-Seq HT Kit (Takara Bio USA) and processed for libraries construction and sequencing.

The obtained data were filtered using SOAPnuke to remove reads containing sequencing adapters. The resulting clean reads were saved in FASTQ format. Transcriptional reads were mapped to the mouse genome (GRCm38) using HISAT2 and STAR RNA-seq aligner, the transcripts of mapped genes were further counted. Normalized mRNA expression of transcriptome among samples (fragments per kilobase million (FPKM) values) were calculated accordingly. To identify differentially expressed genes (DEGs), the expression of all mapped genes was calculated and analyzed using DESeq2. The significant differences were then assessed using a cut-off false discovery rate value of 0.05. All RNA-seq analysis procedures, including RNA extraction, quantification, and cDNA library preparation, were performed by BGI Genomics, using standard and consistent procedures. For functional analysis, DEGs were subjected to KEGG (https://www.kegg.jp/) and GO (http://www.geneontology.org/) enrichment analysis using Phyper based on the hypergeometric test. Statistical differences between enriched terms and pathways were corrected using Bonferroni's test with a rigorous Q value threshold of ≤0.05. Gene set enrichment analysis (GSEA) based on the KEGG and GO pathway dataset downloaded from the official website was performed using the Dr. Tom BGI Genomics platform. DEGs of IL-34 knockout mice after IR and MI surgery were listed as Supplementary Table S2 and Supplementary Table S3, respectively.

In situ hybridization (FISH) assays

To probe the expression of IL-34 and CSPG4, we conducted the in-situ hybridization assays using frozen section from indicated hearts. Briefly, frozen sections were washed with PBS buffer for three times, and digested using proteinase K (20 μg/ml) at 37 °C for 5 min. Washed with sterilized water and PBS (5 min, 3 times). Sections were then prehybridized with hybridization buffer at 37 °C for 1 h and incubated with the IL34 and CSPG4 probe hybridization solution with concentration of 500 nM overnight at 40 °C. Then, the probe2 hybridization solution was added and incubated in a humidity chamber and hybridize overnight at 40 °C. For final signal hybridization, sections were probed with the signal probe hybridization solution overnight at 40 °C. Nucleus were stained with DAPI for 8 min in the dark, and then mounting with anti-fluorescence quenching sealing tablets for further imaging. The probe information of IL34 and CSPG4 are listed as followed:

IL34:

5′-CGTTTCCCAAAGCCACGTCAAGTAG-3′

5′-AGGTCACACTCCTTATCTTGGGTCA-3′

5′-GCAATCCTGTAGTTGATGGGGAAGT-3′

5′-ACTCTGAGTACCCCCTCATAAGGCA-3′

5′-CCAGCAATGTCTGAACCTCCTGTAG-3′

CSPG4:

5′-AACTGGAGCAGCAGGTCTACTCTGG -3′

5′-CAACACTCCGTCAACAGACAGCACA -3′

5′-AGCACAACCTTCATGAACATCGGAG -3′

5′-TCCACGTAGATAAAGTTGCCACGCT-3′

5′-AGTAGCATGGTACCTTGGCCTTTCT -3′

Patient characteristics

A total of 217 patients who underwent coronary angiography (CAG) at the cardiovascular center of Ruijin Hospital were involved in this study, their baseline characteristics have been summarized as Supplemental Table S4. Patients were excluded when: (1) they have less than 1 year of life expectancy; (2) patients with malignancy; (3) acute infection patients; and (4) patients with severe liver dysfunction or chronic kidney disease.

All enrolled patients have the CAG surgery, electrocardiography (ECG), and serum troponin I results. ACS patients were identified as followed: (1) patients with symptom of acute chest pain; (2) ECG showed ST-segment elevation, T-wave inversion, or pathological Q wave; (3) patients had increased serum troponin I level (>0.5 ng/mL); and (4) CAG surgery showed more than 50% stenosis of coronary artery. The patients of ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI) were enrolled into ACS groups. The control subjects had the symptom of pain chest, while the CAG showed intact coronary, or less than 50% coronary stenosis. Collected blood were rested in room temperature for 2 h, and the serum were obtained by centrifugation at 3000g for 15 min.

This study was carried out in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and the study protocol has been priorly approved by the Ruijin Hospital Ethics Committee on research on humans (The ethics committee reference number: 2016–019). All patients provided written informed consent.

Statistical analysis

Data are presented as means ± SEMs (for pre-clinical data) or means ± SDs (for clinical characteristics of patients). Data are graphically presented using GraphPad Prism (Version 7l GraphPad Inc., La Jolla, CA, USA) or Rstudio (version 1.4.1106) software and statistically analyzed using SPSS software (version 23; SPSS Inc., Chicago, IL, USA) or Rstudio (version 1.4.1106) software. Differences between two groups were compared using two-tailed unpaired Student's t-tests. Differences between more than two groups were compared using one-way analysis of variance, followed by Tukey's post-hoc test. Statistical significance was considered at ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Role of funders

The funders did not have any role in study design, data collection, data analyses, interpretation or writing of the report.

Results

Cardiac IL-34 is derived from pericyte and upregulated after IR

To determine the expression profile of IL-34 after IR, we constructed the IR model with 45 min of ischemia followed by time-series reperfusion at 1, 3, 7, and 28 days, and found that the mRNA and protein expression of IL-34 was significantly upregulated at days 1 and 3 and started to decline at days 7 after reperfusion (Fig. 1a–c). Previous studies have reported that IL-34 is distinctively expressed by skin keratinocytes,²⁷ neurons,²⁷ or renal tubular epithelial cells²⁸; however, the source of IL-34 in the heart remains unclear. Determining the source of IL-34 can help to interpret the function of IL-34 in cardiovascular diseases more precisely. Therefore, we investigated the expression pattern of IL-34 using our previous single-cell RNA sequencing (scRNA-seq) dataset of non-cardiomyocytes under homeostasis and at 3-day, 7-day after MI surgery.²⁹ Macrophages, neutrophils, endothelial cells, fibroblasts, and pericytes with differentially expressed marker genes were identified in this scRNA-seq.²⁹ We showed that IL-34 was highly expressed in Cspg4+ and Pdgfrb + pericytes (Fig. 1d). Then, we re-analyzed the single-nucleus RNA sequencing data (snRNA-seq) with human ischemic and normal heart samples, which included all cell types of human hearts, such as the cardiomyocytes, myeloid cells, endothelial cells, and pericytes.³⁰ We found that, IL-34 was highly expressed in CSPG4+ pericytes, moreover, the expression of IL-34 was markedly upregulated in the ischemic zone and border zone of ischemic hearts as compared to the control samples (Fig. 1e). To further verify the results of scRNA-seq and snRNA-seq data, we co-stained for IL-34 with PDGFRB and showed that the percentage of IL-34+ PDGFRB + pericytes were increased after IR (Fig. 1f and g). Taken together, these findings showed that IL-34 might be derived from pericytes, and that its expression is dramatically upregulated after IR.

Fig. 1.

IL-34 was upregulated after myocardial ischemic/reperfusion injury (IR). (a) Quantitative real-time polymerase chain reaction assay (RT-qPCR) determines the mRNA expression of Il34 in wild-type (WT) hearts with 45 min ischemia followed by different duration of reperfusion (n = 3 for each group. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.). The expression of Il34 is normalized to sham. (b) Representative immunoblotting images of IL-34 in sham, 1 day, 3 days, 7 days, 28 days after IR surgery. (c) Quantification of panel b (n = 6, 4, 4, 4, 4, respectively. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.). (d) Uniform manifold approximation and projection (UMAP) plot showing the scaled expression of Cspg4, Pdgfrb, and Il34 in non-cardiomyocyte scRNA-seq data. (e) UMAP plot showing the scaled expression of CSPG4 and IL34 in snRNA-seq data. The violin plot showing the scaled expression of IL34 in control, ischemic zone (IZ), border zone (BZ), remoted zone (RZ), and fibrotic zone (FZ) of snRNA-seq. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test. (f) Representative immunostaining images showing the co-localization and expression of IL-34 (green), Pdgfrb (red) at sham and 3 day after IR surgery. Scale bars: 20 μm. (g) Quantification of panel f (n = 4, 3, 3, respectively. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test).

Loss of IL-34 mitigates cardiac remodeling, cardiac dysfunction, and fibrosis after IR

To further determine the role of IL-34 in cardiac remodeling and heart failure after IR, IL-34 knockout mice were generated by targeted deletion of exon 3 of murine Il34 (Figure S1a–c). This knockout strategy led to an obvious loss of Il34 expression in murine hearts, which enabled the determination of IL-34 effects after IR and MI surgery (Figure S1d). Of note, IL-34 deletion did not lead to obvious abnormalities of murine organs (such as the hearts, livers, and brain) under homeostatic conditions (Figure S2a and b). Then, IL-34 knockout mice (IL34-KO) and their wild-type (WT) littermates were subjected to 45 min of myocardial ischemia followed by reperfusion for further analysis. Echocardiographic analysis revealed that IL-34 deletion improved cardiac function at 28-days post-IR surgery, as evidenced by increased left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), and decreased left ventricular end-systolic volume (LVESV), as compared with WT mice (Fig. 2a, Figure S3a, and Table S1). Consistent with attenuated cardiac function, the heart weight to tibia length and lung weight to tibia length ratios were decreased in the IL34-KO mice than WT mice at 28-days post-surgery (Figure S3b).

Fig. 2.

IL-34 deficiency alleviated IR-induced cardiac dysfunction and remodeling. (a) IL-34 deficient mice and their wild-type (WT) littermates are subjected to IR or sham surgery. Echocardiography determines cardiac function at 7 days and 28 days after IR or sham surgery (n = 14, 15, 10, 9, 8, 9, respectively. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.). (b) WT and IL-34 null mice are subjected to 45 min ischemia followed by 12 h and 24 h reperfusion, then their heart tissues are stained with Evans blue triphenyltetrazolium chloride staining. Scale bars: 1 mm. (c) Quantification of area at risk (AAR) to total area ratio, area of infarction (AOI) to AAR ratio of panel b (n = 4, 5, respectively. Data were analyzed by Student's t-tests.). (d) Masson's trichrome staining of WT and IL-34 null hearts 28 days after IR surgery. Scale bars: 1 mm. (e) Quantification of infarct size of panel d (n = 5, 5, respectively. Data were analyzed by Student's t-tests.). (f) Representative picrosirius red images of WT and IL-34 KO hearts 28 days after IR. Top: bright-field microscopy, scale bars: 1 mm. Bottom: polarized field showing enlarged picrosirius red images corresponding to the top field. Scale bars: 50 μm. (g) Quantification of panel f (n = 4, 4, respectively. Data were analyzed by Student's t-tests.). LVEF: left ventricular ejection fraction; LVFS: left ventricular fractional shortening; LVEDV: left ventricular end diastolic volume; LVESV: left ventricular end systolic volume.

Furthermore, we compared the histological differences between WT and IL-34-null hearts during the acute and chronic phases. The infarct size after IR injury was determined using Evans blue triphenyl tetrazolium chloride (TTC) staining and Masson staining. Compared to the WT littermates, IL-34 deletion led to a reduced infarct size at 12 h and 24 h post-IR surgery (Fig. 2b and c). By using the TUNEL assay, we determined the level of cardiomyocytes apoptosis after IR and revealed that a lack of IL-34 after reperfusion greatly reduced apoptosis of cardiac myocytes as demonstrated by the reduced number of TUNEL-positive myocytes by ∼50% compared to the WT littermates (Figure S3c). Consistently, infarct size and adverse cardiac remodeling were significantly attenuated in IL-34-KO mice compared to WT mice at 28 days after IR (Fig. 2d and e). Picrosirius red polarization staining showed differential collagen morphology in WT and IL34-KO hearts at 28 days after IR. Infarct scars from IL34-KO hearts contained thinner and greenish fibers, whereas thicker and orange collagen fibers were predominant in WT scars (Fig. 2f and g), indicating that IL-34 deletion improved cardiac remodeling after IR. Taken together, we showed that IL-34 deficiency mitigates IR-induced cardiac dysfunction and cardiac remodeling, highlighting the critical role of IL-34 in the pathogenesis of cardiac ischemic injuries.

IL-34 overexpression aggravates cardiac injuries and heart failure after IR

In the above experiments, we had revealed the protective role of IL-34 deficiency in cardiac remodeling and heart failure. To validate the effects of IL-34 on IR-induced heart failure, we overexpressed IL-34 in murine hearts by constructing an adeno-associated virus 9 (AAV9) with pericyte specific promoter region. Specifically, the 2.6 Kb (−600/+2000) of genomic sequence upstream of Pdgfrb, which was able to direct reporter gene expression as the same spatial and temporal pattern as the endogenous Pdgfrb, was cloned into an AAV9 package.³¹ We injected 2 × 10ˆ11 viral genome particles of AAV9-Pdgfrb IL-34 vector into 4-week-old mice via the tail vein, and performed the IR surgery at 4-week after gene transfer (Fig. 3a). At 4 weeks after vein injection, we showed that the cardiac IL-34 level was upregulated in AAV-IL34 hearts than the AAV-CTL mice (Fig. 3b). Meanwhile, we also performed in situ hybridization (FISH) assays of IL34 and CSPG4 and confirmed upregulation of IL34 in pericytes at 4-weeks after infection (Fig. 3c). Having confirming the efficiency of the AAV9 vector, the IL-34 overexpressed and the control mice were subjected to IR surgery. The echocardiographic analysis revealed that IL-34 overexpression remarkedly exacerbated cardiac dysfunction as compared to the empty control group, evidenced by decreased LVEF and LVFS at 7 and 28 days after IR surgery (Fig. 3d). Furthermore, the Evans-blue/TTC assay and masson assays were conducted at 1 day or 28 days after IR surgery to determine the degree of infarct size, and showed that IL-34 overexpression led to the increased infarct size and cardiac remodeling compared with control mice after IR surgery (Fig. 3e–g). Taken together, the loss-of-function and gain-of-function models consistently revealed that IL-34 is a key regulator of cardiac function and cardiac remodeling after IR.

Fig. 3.

Overexpression of IL-34 aggravated cardiac dysfunction after IR. (a) Schematic graph showing the strategy of IL-34 overexpression. The adeno-associated virus 9 (AAV) contained a murine IL-34 transcript (AAV-IL34) and Pdgfrb promoter region is constructed and injected via tail vein at 4-week-old to induce the expression of IL-34 on pericytes. Mice are then subjected to IR surgery at 4-week after injection. (b) Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay determines the mRNA expression of Il34 in WT hearts transfecting with IL-34 AAV vector or the empty control (n = 4 for each group. Data were analyzed by Student's t-tests.). (c) Double fluorescent in situ hybridization for IL-34 (red) and CSPG4 (green) in AAV-CTL and AAV-IL34 hearts at 4-week after virus injection. Scale bars: 20 μm. (d) AAV-CTL and AAV-IL34 mice are subjected to IR surgery and echocardiography analyzes cardiac function at 7 days and 28 days after IR or sham surgery (n = 12, 11, 9, 9, 9, 9, respectively. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.). (e) AAV-CTL and AAV-IL34 mice are subjected to IR surgery, their hearts are collected for evans blue triphenyltetrazolium chloride staining 24 h after reperfusion. Scale bars: 1 mm. (f) The area at risk (AAR) to total area ratio, area of infarction (AOI) to AAR ratio of panel e is determined (n = 4, 5, respectively. Data were analyzed by Student's t-tests.). (g) Masson's trichrome staining of WT mice with or without IL-34 overexpression at 28 days after IR surgery (n = 5, 5, respectively. Data were analyzed by Student's t-tests.). Scale bars: 1 mm.

IL-34 deletion ameliorates macrophage recruitment and polarization after IR

Using IL-34 knockout and overexpression models, we demonstrated that IL-34 contributed to heart failure, and its deletion prevented cardiac dysfunction and cardiac remodeling after IR. Having confirmed the therapeutic benefits of IL-34 deletion in a murine IR model, we next performed bulk RNA-seq analysis of IL-34 knockout and WT hearts at 1-day post IR surgery (Figure S4a, Table S2). RNA-seq analysis showed reduced Il34 transcripts in IL34-KO hearts compared to those in WT hearts, which confirmed the efficiency of our knockout strategy (Figure S4b). Compared to the transcriptome of WT hearts, IL-34 deletion markedly mitigated cardiac inflammatory signaling after IR surgery. Specifically, using differentially downregulated genes in IL-34 knockout hearts of IR-1D RNA-seq data, Kyoto Encyclopedia of Genes and Genomes Pathway Analysis (KEGG) showed that cytokine–cytokine receptor interaction and IL-17 signaling pathways were significantly enriched in IL-34 deficient hearts (Fig. 4a). Of note, genes linked to immune activation, including Ccl2, Cxcl2, Ccl7, and Osm, were significantly downregulated in IL-34 deletion hearts after IR surgery (Figure S4c, Figure S5a). Gene set pathway enrichment analysis, which contributes to the understanding of the gene list expression at the genome-scale level, showed that IL-34 knockout was negatively correlated with immune responses compared with WT hearts (Fig. 4b).

Fig. 4.

IL-34 deletion attenuated inflammatory responses after IR. (a) IL-34 deficient mice and their wild-type (WT) littermates are subjected to IR surgery and their hearts are collected for RNA-seq analysis. Kyoto encyclopedia of genes and genomes analysis (KEGG) of downregulated differentially expressed genes showing inflammatory pathways are enriched in IL34-KO hearts. (b) Gene set enrichment analysis of IR-1-day RNA-seq data reveals IL-34 deletion is negatively correlated with immune responses compared to WT mice. (c) Representative immunostaining images of LY6G, F4/80, CCR2, and CD68 in WT and IL-34 deficient hearts at 1 days after IR surgery. Scale bars: 20 μm. (d) Quantification of panel c (n = 5, 4, respectively. Data were analyzed by Student's t-tests.). (e) Representative immunostaining images of CD68 and Ki67 in WT and IL-34 deficient hearts at 1 days after IR surgery. Scale bars: 20 μm. (f) Quantification of panel e (n = 5, 4, respectively. Data were analyzed by Student's t-tests.).

In addition to IR model, the study of murine MI model helps to improve the pathophysiological understandings of ischemic injury.^32,33 Therefore, to strengthen the mechanical understanding of IL-34 in ischemic responses, we performed the RNA-seq of MI models to confirm the results of a decreased inflammatory response in IL-34 deficient hearts. Consistently, IL-34 knockout downregulated expression of a series of inflammatory cytokines and chemokines expression after MI surgery (Figure S4d, Figure S5b, and Table S3). To further confirm the effects of IL-34 on the immune response, the numbers of neutrophils, macrophages were assessed by immunofluorescence assay and revealed that IL-34-KO mice and their WT littermates had comparable LY6G + neutrophils 1-day after IR. However, the number of macrophages (F4/80+) were markedly reduced in IL-34 deficient hearts 1 day after IR as compared with WT mice (Fig. 4c and d). Then, immunofluorescent co-staining CCR2 and CD68 was conducted in IR operated hearts, and showed IL-34 deletion led to decrease in CCR2+ CD68+ macrophages than their WT littermates (Fig. 4c and d). Defective macrophage proliferation might contribute to the decline in macrophage numbers and thereby attenuate IR-induced inflammatory responses. By co-staining Ki-67 and CD68 at 1-day after IR surgery, we showed that there were no differences in macrophage proliferation in IL34-KO and WT hearts (Fig. 4e and f), suggesting that macrophage recruitment and polarization, but not their proliferation, contributed to IL-34 induced immune responses after IR.

Subsequently, to verify the role of IL-34 on ischemic-induced immune responses, cardiac immune cells in IL-34 deletion and WT hearts were assessed using flow cytometry assays after different IR time courses (Figure S6). We showed that cardiac CD45+ leukocytes and neutrophils were unaffected in IL-34 defect and WT hearts in IR conditions (Fig. 5a–c). The number of monocytes were comparable in WT and IL-34-KO hearts at 12 h after IR (Fig. 5d), indicating that IL-34 had no effects on the initial recruitment of monocytes upon ischemia occurred. Notably, IR led to the influx of macrophages and CCR2+ macrophages in WT hearts, while IL-34 deficiency significantly mitigated macrophage numbers and polarization at 12 h and 24 h post-reperfusion (Fig. 5e–i), highlighting that IL-34 plays key roles in macrophage differentiation and polarization after IR.

Fig. 5.

IL-34 knock out inhibited IR-induced macrophages recruitment and polarization. (a) IL-34 deficient mice and their wild-type (WT) littermates are subjected to sham surgery or 45 min ischemia followed by different durations of reperfusion, including 12 h (12H), 24 h (24H), and 3 days (3D). Their hearts are collected for flow cytometry analysis. (b–e) The quantification of flow cytometry results to determine the numbers of leukocytes, neutrophils, monocytes, and macrophages at different time points after IR (n = 6, 6, 6, 6, 6, 6, 3, 3, respectively. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.). (f) Flow cytometry determining the numbers of CCR2+ macrophages, CCR2-macrophages, and the percentages of MHCII + CCR2-macrophages after IR. (g–i) Quantification of panel f (n = 6, 6, 6, 6, 6, 6, 3, 3, respectively. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.).

Having confirmed the inflammatory effects of IL-34 in IR injury, we further validated the result of attenuated cardiac immune responses in IL-34 deficient mice using MI models. Macrophages undergo polarization state changes over the first week of MI time course, accompanied by a inflammatory signature at day 1, the pro-phagocytotic signature at day 3, and the pro-reparative signature at day 7 after MI surgery.³⁴ Therefore, we determined the numbers of cardiac immune cells and polarized macrophages at 1, 3, and 7-day after MI surgery. We found that the number of cardiac neutrophils (CD45+ CD11B + LY6G+) and monocytes (CD45+ CD11B + LY6G- LY6C+) were similar in IL-34 KO hearts and WT hearts (Figure S7a–d and Figure S8a and b). However, the number of macrophages (CD45+ CD11B + LY6G- F4/80+ LY6Cint-low) and CCR2+ macrophages were remarkably reduced in IL-34 deficient hearts as compared to WT hearts at 1-day and 3-day after MI (Figure S8c–g). Conversely, no differences were found between WT and IL34-KO mice in terms of the number of CCR2-macrophages under sham and MI conditions (Figure S8f and g). These immunofluorescence and flow cytometry results indicated that IL-34-mediated cardiac damage is centered on macrophage recruitment and polarization.

Whether IL-34 affected the number of circulating monocytes and neutrophils and furthered their infiltration into ischemic hearts remained undetermined. Therefore, circulating leukocytes (CD45+), neutrophils (CD45+ CD11B + LY6G+), monocytes (CD45+ CD11B + CD115+) and LY6C + monocytes were assessed after MI and IR surgery (Figure S9). A similar increase in the number of blood leukocytes, neutrophils, and LY6C + monocytes was observed in WT and IL34-KO mice in both the MI (Figure S9a and b) and IR models (Figure S9c and d). However, IL-34 deficiency decreased the number of CD115+ monocytes compared to that in WT mice at 3-days after MI and 24 h after IR surgery (Figure S9a–d). Together, these findings suggest that IL-34 plays a central role in macrophage recruitment and polarization, which triggers cardiac remodeling and heart failure after ischemic injuries.

Loss of IL-34 abolished IR-induced NF-κB signaling activation

Next, we explored the underlying mechanisms responsible for the attenuated macrophage recruitment and polarization achieved by IL-34 deletion. First, we determined whether macrophage colony-stimulating factor 1 (Csf1) would compensate for the absence of IL-34 under ischemic conditions, and revealed that the mRNA expression of Csf1 did not increase in IL-34 deficient hearts as compared to their WT littermate (Figure S10a). Consistently, the CSF1 protein level was comparable among IL34-KO and WT hearts (Figure S10b), suggesting that CSF1 does not compensate for the absence of IL-34 and that IL-34 driven ischemic responses in a CSF1 independent manner.

Next, Gene ontology (GO) analysis of RNA-seq data revealed that immune pathways and NF-kappaB (NF-κB) activity were significantly enriched in IL-34 deficient hearts (Fig. 6a). Consistently, the gene set enrichment analysis showed that, IL-34 deletion resulted in downregulation of NF-κB transcription factor activity in both MI and IR models (Fig. 6b). To confirm the reduced activity of NF-κB signaling in IL34-KO hearts, we collected the target gene list of NF-κB signaling (https://www.bu.edu/nf-kb/gene-resources/target-genes/) and evaluated their transcriptional expression on our RNA-seq data. We observed that IL-34 deletion resulted in the broad downregulation of NF-κB target genes in both MI and IR models (Figure S10c and d). Together, these data suggested that IL-34 might provoke macrophage inflammatory responses via NF-κB signaling.

Fig. 6.

Loss of IL-34 mitigated the canonical and non-canonical NF-κB signaling. (a) Gene ontology analysis of differentially expressed genes between IL34-KO and WT hearts after MI surgery. Top enriched categories are exhibited. (b) Gene set enrichment analysis of RNA-seq data reveals IL-34 deletion negatively correlates with NF-κB signaling in both MI and IR models. (c) Representative immunoblotting images of NF-κB p65, NF-κB1, P-IKKα/β, P-IκBα in WT and IL34-KO hearts after sham or IR surgery. (d) Quantification of panel c (n = 4 for each group. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.). (e) Representative immunoblotting images of NF-κB2, RelB, c-Rel in WT and IL34-KO hearts after sham or IR surgery. (f) Quantification of panel e (n = 4 for each group. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.). (g) Bone marrow derived macrophages are polarized to a proinflammatory M1 phenotype (LPS and interferon-γ treated), and stimulated with PBS or IL-34 recombinant proteins for 24 h; their cytoplasmic and nuclear fraction are isolated for western blotting assay. (h) Quantification of panel g (n = 4 for each group. Data were analyzed by Student's t-tests.). (i) Representative immunofluorescence images of NF-κB p65 (green) in bone marrow derived macrophages with or without IL-34 treatment. Scale bars: 20 μm.

NF-κB signaling is the master regulator of macrophage inflammatory responses in cardiac MI and IR injuries.^35,36 We further determined the differences in NF-κB signaling between WT and IL-34 deletion mice. IR-induced significant upregulation of NF-κB p65 protein expression 1 and 3 days after IR surgery; however, IL-34 deletion significantly repressed p65 protein levels after IR surgery (Fig. 6c and d). It is known that NF-κB p65 is sequestered by the inhibitors of NF-κB (IκB). When phosphorylated by the specific IκB kinase (IKK) complex, P-IκB protein is degraded and releases the p65 element for nuclear translocation and transactivation,³⁷ which represents the canonical pathway of NF-κB activation. Consistent with downregulated p65 expression, the phosphorylated levels of IκBα and IKKα/β were markedly decreased in IL-34 null hearts compared to WT littermates at 1 day after IR (Fig. 6c and d). In addition to the canonical pathway, NF-κB signaling can be activated by a non-canonical pathway.³⁶ We discovered that IL-34 deletion significantly decreased the protein levels of p52 and RelB 1 and 3 days after IR compared to WT mice (Fig. 6e and f), indicating that IL-34 deletion repressed the non-canonical NF-κB pathway after IR. Moreover, bone marrow derived macrophages (BMDMs) were treated with IL-34 recombinant protein or the vehicle, the western blotting with nuclear/cytoplasmic extract and immunofluorescence assays revealed that IL-34 treatment increased the nuclear translocation of NF-κB p65 (Fig. 6g–i). On the other hand, anti–IL-34 (neutralizing) Ab blockaded the effects of IL-34 and led to a decline in NF-κB and RelB protein levels (Figure S10e), supporting that IL-34 contributes to the activation of NF-κB signaling. Taken together, our RNA-seq results, in vivo, and in vitro experiments confirmed that IL-34 is a key regulator of NF-κB activation and macrophage inflammatory responses after IR injury.

IL-34 deficiency downregulates the expression of CCL2 via NF-κB pathways

We further aimed to explore the functional consequence of IL-34 mediated NF-κB activation, whether specific genes are mandatorily governed by the IL-34-NF-κB axis, and whether increased IL-34 can be mirrored in clinical acute coronary syndrome (ACS) patients. To address these issues, we investigated the transcriptional expression of NF-κB target genes in the RNA-seq data to determine which genes were differentially expressed in IL-34 knockout hearts during ischemic injuries. Interestingly, the Venn diagram showed that 4 NF-κB target genes were consistently downregulated in IL-34-KO hearts in both MI and IR models, including the Gzma, Prf1, Ccl2, and Ebi3 (Fig. 7a). Among these differentially expressed genes (DEGs), Ccl2 exhibited the most marked abundance and differential expression (Fig. 7b). Therefore, we focused on the effect of IL-34 on transcriptional regulation of Ccl2. In line with the RNA-seq results, ischemia induced the expression of Ccl2 mRNA expression in WT hearts; however, IL-34 deletion reversed IR-induced Ccl2 upregulation at 1 day after surgery (Fig. 7c). Immunofluorescence and western blotting assays showed that the protein level of CCL2 was markedly decreased in IL-34 deficient hearts as compared to that of WT hearts at 1 day after IR (Fig. 7d and e). Moreover, supplement with IL-34 recombinant proteins significantly upregulated the mRNA expression of Ccl2 in BMDMs; while neutralizing IL-34 with antibody or inhibiting the activity of NF-κB with its specific inhibitor, QNZ,^38,39 downregulated the protein level of NF-κB p65, moreover, efficiently prevented the upregulation of Ccl2 after IL-34 treatment (Fig. 7f–h). Therefore, these pre-clinical findings provide evidences for the link between the IL-34-NF-κB-CCL2 axis and cardiac dysfunction after ischemic injury.

Fig. 7.

IL-34-NF-κB signaling drove the expression of CCL2. (a) Combining the RNA-seq data to identify the commonly differentially expressed NF-κB target genes under MI and IR surgery. (b) RNA-seq data showing the differential expression of NF-κB target genes (Ccl2, Gzmb, Prf1, Ebi3) between IL34-KO and WT hearts (n = 3 for each group). (c) Reverse-transcription-quantitative polymerase chain reaction (RT-qPCR) determines the mRNA expression of Ccl2 in WT and IL34-KO hearts after sham or IR surgery (n = 4, 4, 5, 5, respectively. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.). (d) Representative immunofluorescence images of CCL2 (green), CD68 (red) and quantification in WT and IL34-KO hearts 1 day after IR surgery (n = 5 for each group. Data were analyzed by Student's t-tests.). Scale bars: 20 μm. (e) Representative immunoblotting images and quantification of CCL2 in WT and IL34-KO hearts after sham or IR surgery (n = 3 for each group. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.). (f) Bone marrow derived macrophages (BMDMs) are treatment with IL-34 recombinant proteins with or without Anti-IL-34 neutralizing antibody, the expression of Ccl2 is determined by RT-qPCR (n = 8 for each group. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.). (g) BMDMs are treatment with IL-34 recombinant proteins with or without NF-κB inhibitor (QNZ) for 24 h. Western blot determining the protein level of p65 (n = 4 for each group. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.). (h) BMDMs are treatment with IL-34 recombinant proteins with or without NF-κB inhibitor (QNZ) for 24 h. RT-qPCR determining the mRNA expression of Ccl2 (n = 3 for each group. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test.). (i) The serum of normal and acute coronary syndrome (ACS) patients (n = 101, 116, respectively) are collected for enzyme-linked immunosorbent assay to analyze the levels of IL-34 and CCL2. Data were analyzed by Student's t-tests. (j) The correlation among serum IL-34 and CCL2 is evaluated using Pearson correlation coefficient (n = 217, linear regression analysis).

Next, to support the relationship of IL-34 with clinical ischemic events in humans, we collected serum from ACS patients and control patients, and determined the serum levels of IL-34 and CCL2 by enzyme-linked immunosorbent assay. A total of 217 patients were included in the final analysis (ACS: 116 patients, controls: 101 patients). Their basic characteristics were summarized in Table S4. We found that serum levels of IL-34 and CCL2 were higher in ACS patients than in normal subjects (Fig. 7i). Moreover, serum IL-34 levels were positively correlated with CCL2 levels (R = 0.28, P = 3.2E-05, Fig. 7j). These data were consistent with our and other previous studies, which revealed that IL-34 was increased in patients with ischemic cardiomyopathy and was positively correlation with the adverse cardiovascular events of heart failure patients.^24,26 Therefore, we demonstrated the underlying mechanisms of IL-34 on cardiac dysfunction after ischemic events, and highlighted that the IL-34-CCL2 pathway was the key regulator of immune response after IR injury.

Taken together, our results indicate that IL-34 is elevated after myocardial IR injury, and that IL-34 triggers the activation of both canonical and non-canonical NF-κB pathways, with upregulated P-IκBα, P-IKKα/β, p52, RelB, and p65 subunits. After translocation to the nucleus, these NF-κB transcriptional factors foster the expression of CCL2, ultimately leading to macrophage recruitment and polarization, which exacerbates inflammatory responses and tissue injury after IR and finally the progression of cardiac remodeling and heart failure (Fig. 8).

Fig. 8.

Summary of IL-34 on cardiac IR injury. The graphical model for IL-34-NF-κB axis mediated macrophage recruitment and polarization, further cardiac remodeling after IR. Once IR occurred, pericytes-derived IL-34 is markedly upregulated to stimulate macrophages recruitment and polarization through activation of both the canonical and non-canonical NF-κB pathways, increasing cellular p65, p52, and RelB levels, which leads to the upregulation of CCL2 expression, furthermore, the aggravation of cardiac remodeling and heart failure.

Discussion

Here, we revealed that the IL-34-NF-κB axis contributes to macrophage recruitment and polarization by upregulating CCL2 expression, which triggers cardiac remodeling and heart failure after IR. Moreover, serum IL-34 levels positively correlated with CCL2 levels in patients with coronary arteriography. Thus, this study revealed that IL-34 is a critical regulator of macrophage polarization and cardiac remodeling after IR. Identifying the functional characteristics of IL-34 provides mechanistic insight relevant to the treatment of patients with MI following percutaneous coronary intervention (PCI).

Upregulated IL-34 constitutes the central driver of cardiac dysfunction after IR

IL-34 is widely recognized as a functional regulator of macrophage survival and proliferation via binding to CSF-1R.²⁷ IL-34 is upregulated in several inflammatory diseases (such as Crohn's disease and ulcerative colitis), autoimmune diseases (such as systemic lupus erythematosus and nephritis), metabolic diseases (diabetes and obesity), and tumors.^21,40, 41, 42 We previously reported that the serum level of IL-34 is increased in patients with ischemic cardiomyopathy²⁵ and positively correlates with the prognosis of heart failure patients,²⁶ suggesting a functional link between IL-34 and the progression of heart failure. Consistent with these studies, we revealed that the mRNA and protein expression of IL-34 increased significantly at 1 and 3 days after IR surgery. Hence, one may wonder: what is the source of cardiac IL-34 and why is it upregulated in IR and other inflammatory diseases. By leveraging scRNA sequencing, snRNA sequencing, and immunofluorescence assays, we demonstrated that cardiac IL-34 might derive from pericytes, and the amount of IL-34+ PDGFRB + pericytes increases markedly after IR. Furthermore, previous studies have confirmed that inflammatory cytokines, including tumor necrosis factor (TNF) and IL-1β, and microRNAs regulate IL-34 expression. Stimulation of synovial fibroblasts with TNF upregulates IL-34 expression in a time-dependent manner.⁴³ Bostrom et al. demonstrated that the expression of IL-34 is enhanced by TNF and IL-1β through cellular NF-κΒ and c-Jun N-terminal kinase pathways.⁴⁴ In addition, miRNAs, including miR-31–5p and miR-28–5p, directly modulate IL-34 mRNA and protein levels through post-transcriptional modification.^45,46 These studies partially explain the upregulation of IL-34 after myocardial ischemic injury. IR results in cardiomyocyte apoptosis and necrosis, accompanied by the expression and release of TNF and IL-1β; on the other hand, necrotic debris and damage-associated molecular patterns from dying cardiomyocytes activate macrophages, and upregulate TNF and IL-1β expression. Therefore, increased TNF and IL-1β after MI and IR might stimulate pericytes via their corresponding receptors to induce the expression of IL-34 in a paracrine manner.

In this study, we used the global IL-34 knockout and pericyte specific overexpression models to determine the role of IL-34 on cardiac remodeling after IR, we confirmed that IL-34 deficiency attenuated IR-induced cardiac dysfunction, cardiac remodeling, and fibrosis, while overexpression of IL-34 dramatically aggravates heart failure after IR.

IL-34 fosters macrophage recruitment and polarization to aggravate cardiac damage

We further explored the mechanisms underlying IL-34-mediated cardiac damage. By analyzing RNA-seq of WT and IL-34 knockout hearts 1 day after MI and IR surgery, we showed that IL-34 deletion mitigates cardiac inflammatory responses. Consistent with the RNA-seq results, immunofluorescence, and flow cytometry assays verified that IL-34 null mitigated macrophage recruitment and polarization after IR. Macrophages exhibit significant heterogeneity and plasticity during myocardial ischemic repair as early inflammatory macrophages and late reparative macrophages.¹³ Persistent macrophage recruitment and activation constitute the inflammatory environment, thereby leading to the expansion of infarct size, ventricular wall thinning, and cardiac dysfunction.^47,48 Conversely, accelerating polarization of macrophages to reparative macrophages can alleviates inflammatory responses and improve cardiac remodeling after MI.⁴⁹ In this regard, targeting macrophages, particularly macrophage phenotypic polarization, confers therapeutic benefits for the treatment of MI patients undergoing PCI.

Here, we revealed that IL-34 is a key regulator of macrophage polarization during MI and IR. In line with our results, a series of publications have demonstrated that IL-34 helps to maintain macrophage function in multiple organs: in the skin, IL-34 deficiency leads to reduced epidermal Langerhans cells in neonatal and in adult mice and impairs the survival of Langerhans cells in response to epidermal damage.^27,50 In the kidney, IL-34 deletion inhibits the recruitment and proliferation of macrophages and neutrophils in renal tissue during acute kidney injury.^28,51 In addition to the maintenance of tissue macrophages, IL-34 also exhibited dual property on macrophage polarization during diseases. For example, in Japanese flounder, IL-34 treatment in peripheral blood leukocytes induced an inflammatory response with production of cytokines and ROS via JAK/STAT signaling pathways.⁵² On the other hand, Foucher et al. revealed that IL-34 induces the differentiation of monocytes into M2 macrophages.⁵³ One caveat concerning the distinct actions of IL-34 in macrophage polarization related to differing cells (BMDMs, peripheral blood mononuclear cells, THP-1), organs (hearts, kidney, brain, skin, etc.), or experimental injury protocols. Taken together, in this study, we underpinned and broadened the biological functions of IL-34 in terms of macrophage activation and polarization in cardiovascular diseases, providing pre-clinical evidences for targeting macrophage function after IR.

Although we have revealed the deleterious roles of IL-34 on macrophage polarization and cardiac function, previous studies have indicated that CSF1 treatment after MI and IR attenuated ischemia and left ventricular dysfunction by upregulating the levels of transforming growth factor-β1, collagen I and III, and vascular endothelial growth factor (VEGF) production.^54,55 Whether CSF1 compensate for the loss of IL-34? Why are they acted distinct after ischemic conditions remain unclear. By performing RNA-seq and immunofluorescent assays, we showed that Csf1 expression was comparable among IL34-KO and WT hearts after IR and MI surgery, suggesting that CSF-1 does not compensate for the absence of IL-34 under IR conditions. Regarding the differential effects of IL-34 and CSF1, previous study had demonstrated that IL-34 and CSF-1 exhibited clearly different effects on growth and development. Csf1 deficiency mice (Csf1^op/op) have reduced osteoclasts and tissue macrophages, decreased body weight, and are infertile.⁵⁶ On the contrary, IL-34 deficient mice have relatively normal phenotypes, including similar circulating and heart macrophages, normal body weight, heart function, and fertility as compared to WT mice, except for reduced microglia and Langerhans cells.²⁷ These data collectively confirmed that the actions of IL-34 and CSF1 was distinct on macrophage fate, development, and disease progression. One caveat regrading this discrepancy would be attributed to the differing binding receptors of CSF1 and IL-34. Although these two ligands can interact with CSF-1R, IL-34 has a lower affinity and correspondingly lower activity than CSF1, moreover, researches have revealed that IL-34 could specifically interact with PTPRZ,²⁸ SDC1,⁵⁷ and TREM2,⁵⁸ which may elicit differential signaling cascades and consequences. Collectively, our data revealed that IL-34 exhibited a nonredundant, CSF1-independent role in the progression of cardiac remodeling after IR.

IL-34 mediates NF-κB activation to elicit inflammatory response

We further reveal that NF-κB signaling is responsible for IL-34 mediated macrophage polarization under IR injury. The NF-κB pathway plays a critical role in inflammatory responses, proliferation, differentiation, and cell survival of macrophages, which consists of five transcription factors: p65, RelB, c-Rel, p105/p50, and p100/p52.³⁷ Once activated, these transcriptional factors can form homo- and heterodimeric complexes for nuclear translocation and transactivation. Here, we found that IL-34 deletion markedly mitigated the activity of canonical NF-κB signaling, as evidenced by the downregulated protein levels of P-IKKα/β, P-IκBα, and p65. In addition, IL-34 deficiency inhibited non-canonical NF-κB signaling by decreasing RelB and p52 protein levels. Therefore, we confirmed that IL-34 directly modulated the macrophage polarization by sustaining the activation of NF-κB signaling. Previous studies have also reported a connection between IL-34 and NF-κB signaling. IL-34 administration on fibroblasts significantly upregulated the expression of IL-6, IL-8, CCL2, and IL-1β via the NF-κB, Akt, or p38 pathways.⁵⁹

In this study, we provided evidence linking IL-34 with macrophage polarization, NF-κB signaling, and cardiac remodeling after IR. However, some limitations of this study should be addressed in future studies. First, we did not depict the direct mechanisms for IL-34 mediated NF-κB activation, and why the protein levels of p65, RelB, and p50 were reduced after IL-34 deletion, is it attributed to reduced degradation via ubiquitination or de novo transcriptional inhibition? Secondly, we only used global IL-34 knockout mice to determine the effects of IL-34 on cardiac dysfunction after IR, further experiments with pericyte specific IL-34 knockout mice should be used. Then, whether IL-34 directly binds to CSF-1R to elicit downstream effects after IR or other receptors, such as the PTPRZ, SDC1, and TREM2, may integrate remains unclear. Finally, we included only male mice in all pre-clinical experiments, further studies that include female mice should be conducted to survey any sexual dimorphism of different immune responses in IL-34 defect mice.

In conclusion, our study demonstrates the inflammatory role of IL-34 in macrophage polarization, cardiac remodeling, and heart failure after IR. Targeting IL-34 may represent a new avenue for relieving cardiac inflammation and preventing cardiac dysfunction in clinical MI and MI with PCI patients.

Contributors

RT, LZ, and QF designed the study, LZ and XZ supervised the study. LZ, XZ, and QY performed most experiments and analysis. LZ, RT, and QF involved in data statistical analysis. XZ and QY help collect clinical samples. LZ and RT verified the data, wrote the manuscript, and made revisions. All authors read and approved the final version of the manuscript.

Data sharing statement

All relevant data are available in the figures and supplementary materials. Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon reasonable request.

Declaration of interests

The authors declared that they have no conflict of interest.