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

Abstract

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).

Keywords: IL-34; Inflammation; Macrophage; Myocardial infarction; Myocardial ischemia-reperfusion; NF-κB.

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).

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.

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.

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.).

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.).

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.

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).

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.