Group 2 innate lymphoid cells contribute to IL-33-mediated alleviation of cardiac fibrosis

Abstract

Rationale: The major cause of heart failure is myocardium death consequent to detrimental cardiac remodeling and fibrosis following myocardial infarction. The cardiac protective cytokine interleukin (IL)-33, which signals by ST2 receptor binding, is associated with group 2 innate lymphoid cell (ILC2) activation and regulates tissue homeostasis and repair following tissue injury in various tissues. However, the distribution and role of IL-33-responsive ILC2s in cardiac fibrosis remain unclear. In this study, we elucidated the roles of IL-33-responsive cardiac-resident ILC2s and IL-33-mediated immunomodulatory functions in cardiac fibrosis. Methods: We examined the distribution of cardiac ILC2s by using flow cytometry. The roles of IL-33-mediated ILC2 expansion in cardiac fibrosis was evaluated in the mouse model of catecholamine-induced cardiac fibrosis. ILC-deficient Rag2^‒/‒IL2Rγc^‒/‒ mice were implemented to determine the contribution of endogenous ILC in the progression of cardiac fibrosis. Histopathological assessments, speckle tracking echocardiography, and transcriptome profile analysis were performed to determine the effects of IL-33-mediated cardiac protective functions. Results: We identified the resident cardiac ILC2s, which share similar cell surface marker and transcriptional factor expression characteristics as peripheral blood and lung tissue ILC2s. IL-33 treatment induced ILC2 expansion via ST2. In vivo, ILC-deficient Rag2^‒/‒IL2Rγc^‒/‒ mice developed exacerbated cardiac fibrosis following catecholamine-induced stress cardiac injury. IL-33 treatment expanded cardiac ILC2s and revealed protective effects against cardiac tissue damage with reduced cardiomyocyte death, immune cell infiltration, tissue fibrosis, and improved myocardial function. Transcriptome analysis revealed that IL-33 attenuated extracellular matrix synthesis- and fibroblast activation-associated gene expressions. IL13-knockout or epidermal growth factor receptor (EGFR) inhibition abolished IL-33-mediated cardiac protective function, confirming IL-13 and EGFR signaling as crucial for IL-33-mediated cardioprotective responses. Moreover, ILC2-produced BMP-7 served as a novel anti-fibrotic factor to inhibit TGF-β1-induced cardiac fibroblast activation. Conclusion: Our findings indicate the presence of IL-33-responsive ILC2s in cardiac tissue and that IL-33-mediated ILC2 expansion affords optimal cardioprotective function via ILC2-derived factors. IL-33-mediated immunomodulation is thus a promising strategy to promote tissue repair and alleviate cardiac fibrosis following acute cardiac injury.

Keywords: ILC2; cardiac fibrosis; fibroblast activation; interleukin-33; myocardial injury.

Figure 1

Identification and characterization of the cardiac resident ILC2 population. Wild-type C57BL/6J mice were intraperitoneally injected with Saline or IL-33 (2 μg/mouse for 5 consecutive days). The heart tissues were collected for flow cytometry analyses of ILC2 population surface markers and transcription factors. (A) Representative plots and gating strategy for ILC (CD45⁺Lin^-Thy1.2⁺) and ILC2 population (CD45⁺Lin^-Thy1.2⁺ST2⁺) identification in mouse heart cells. (B) Representative plots for characterization of GATA3 expression in mouse cardiac ILC2s. Lineage markers (CD3, CD19, FcεRI, CD11c, CD11b, F4/80, and CD49b) were used in the flow cytometry. (C) Total number of CD45⁺ leukocytes, ILC, ST2⁺ ILC2, and (D) GATA3⁺ ILC2 in the heart (n = 5 per group). (E) Anti-CD45 antibody were i.v. administered into saline- or IL-33-injected mice 3 minutes before hearts were harvested. Intravascular (CD45 i.v.⁺; blue arrows) and interstitial (CD45 i.v. ^-; red arrows) ILC (Lin^-Thy1.2⁺) and ILC2 (Lin^-Thy1.2⁺ST2⁺) were determined by flow cytometry. (F) Proportion of intravascular (IV⁺, blue) and interstitial (IV^-, red) CD45⁺ leukocytes (left), Lin^-Thy1.2⁺ ILC (middle), and Lin^-Thy1.2⁺ST2⁺ ILC2 (right) in mouse hearts. The percentage are indicated inside the bars; the numbers below the bars represent the total number of each population in the heart (x 10³). Each panel shows the representative data from two independent experiments (n = 5 per group). (G) Representative plots and gating strategy for ILC2 population identification in hearts from saline or IL-33-treated ST2^‒/‒, IL33^‒/‒, and IL33^-/+ST2^-/+ heterozygote littermates. Dashed lines indicate the isotype control, and solid red lines indicate the ST2 signal intensity. Data are representative of three independent experiments. (H) Total cell number of leukocytes, ILC, and ILC2s in the cardiac tissues (n = 3 in each group). (I) The heart tissues were harvested and the mRNA were isolated for qRT-PCR analysis of gene expression (n = 3 in each group). *P < 0.05 by one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test. All values are means ± SD. Each dot indicates a biological replicate.

Figure 2

Deficiency of endogenous ILCs develops exacerbated cardiac fibrosis following injury. (A) BALB/cByJ, Rag2^-/-, and Rag2^-/-IL2Rγc ^-/- mice were subcutaneously administered saline or isoproterenol (ISO, 30 mg/kg per day for 3 days). The cardiac tissues were collected for histological analysis on day 4 after the last injection. Representative images of Picrosirius red staining for fibrotic area are shown. Scale bar = 50 µm. (B) Quantification of fibrosis area to assess susceptibility to isoproterenol (ISO)-induced cardiac fibrosis. Data are pooled from two independent experiments and expressed as the mean ± SD (n = 5-9 per group). (C) Picrosirius red staining for fibrotic area. (D) Quantification of fibrosis area. *P < 0.05, **P < 0.01 by one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test. All values are means ± SD. Each dot indicates a biological replicate.

Figure 3

IL-33 expands cardiac ILC2 cells and reduces ISO-induced cardiac fibrosis. (A) BALB/cByJ mice were subcutaneously administered saline or ISO followed by saline or IL-33 on days 3, 5, and 7. The heart tissues were harvested on day 10 after the last ISO injection. Flow cytometry analysis for the CD45⁺Lin^-Thy1.2⁺ ILC2 cells in the cardiac tissues on day 10 after the last ISO injection. (B) Frequency of ILC2 among CD45⁺ cells. (C) Number of ILC2s per heart. (D) Picrosirius red staining and quantification of cardiac fibrosis area. Data are pooled from two independent experiments and expressed as the means ± SD (n = 5-10 per group). Scale bar = 100 µm. *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001; ns, not significant by one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test. All values are means ± SD. Each dot indicates a biological replicate. (E) Gating strategy for each leukocyte population and the comparison of each condition. (F) ST2⁺ cell gating for the gated leukocyte population. (G) Proportion of ST2-positive leukocytes. (H) Mean fluorescence intensity (MFI) of ST2 on the surface of ILC (Lineage^- Thy1.2⁺), DC (CD11c⁺), macrophages (F4/80⁺), B cells (CD19⁺), and eosinophils (CD11b⁺ SiglecF⁺).

Figure 4

IL-33 treatment prevents ISO-induced progressive cardiac function impairment. (A) BALB/cByJ mice were subcutaneously administered isoproterenol (ISO) (60 mg/kg) for three days followed by intraperitoneal Saline or IL-33 (0.5 μg/mouse) treatment on days 3, 5, and 7. Cardiac functions were evaluated by M-mode echocardiography (on day 10), pulse wave Doppler (on day 10), and speckle-tracking echocardiography (STE, on day -3 before ISO challenge as baseline, day 3 after the last ISO injection before IL-33 or saline administration, and day 10 post-ISO challenge) (n = 6-10 per group) before the mice were euthanized (SAC). (B) Heart weight to tibia length ratio of the mice between groups. (C) Analysis of the ejection fraction (EF) and fraction shortening (FS) by M-mode echocardiography. (D) Pulse wave Doppler analysis of the ratio of peak velocity of early to late filling of mitral inflow (E/A) (E) Schematic overview of anatomical segments in the parasternal long-axis view. Anterior (AA) and posterior (AP) apex; anterior (BA) and posterior (BP) base; anterior (MA) and posterior (MP) mid. (F) Quantification of the global longitudinal strain (GLS) and (G) global radial strain (GRS) of the mice. (H) Quantification of the segmental longitudinal peak strain upon ISO and IL-33 treatment. (I) Quantitative analysis of segmental radial peak strain. *P < 0.05 by one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test. All values are means ± SD. Each dot indicates a biological replicate. *P < 0.05, **P<0.01 by one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test.

Figure 5

IL-33 treatment reverses isoproterenol (ISO)-induced differentially regulated gene expression as shown by transcriptome profiling. BALB/cByJ mice were subcutaneously administered saline or ISO (60 mg/kg per day for 3 days) followed by saline or IL-33 (0.5 μg/mouse, intraperitoneal injection) on day 3, 5, 7. The heart tissues were harvested on day 10 after the last injection for mRNA isolation and microarray analysis. The mRNAs from three hearts were pooled as one sample for transcriptome analysis by Affymatrix microarray. (A) Principle component analysis (PCA) for the gene expression profile between groups. (B) Number of differentially expressed genes (DEGs) between (ISO vs Saline) and (ISO vs ISO+IL-33) groups. (C) Functional annotation clusters identified by gene ontology of DEGs between (ISO vs Saline) and (ISO vs ISO+IL-33) groups. (D) Representative cluster DEGs for extracellular matrix (ECM), ECM remodeling enzymes, inflammatory cytokines and chemokines, and cardiac injury-related genes. Fold increase in the ISO and ISO+IL-33 group represents fold change in gene expression of ISO vs Saline and ISO+IL-33 vs ISO group, respectively. (E) qRT-PCR validation of the ECM-associated gene expression (Postn, Tnc, Timp1, Col1a2, Col3a1, and Lox) in the cardiac tissues. *P < 0.05 by one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test. (n = 8-10 per group). (F-G) Immunohistochemical staining for periostin (Postn) expression and quantification of positive-stained areas in the heart tissue sections. Scale bar = 100 µm. **P < 0.01, ****P < 0.0001 by one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test. All values are means ± SD. Each dot indicates a biological replicate.

Figure 6

IL-33 treatment upregulates ILC2-associated factors. (A) ELISA analysis of cytokine levels in conditioned medium from ILC2 cells cultured in Ctrl (IL-2+IL-7) and IL-33 (IL-2+IL-7+IL-33) media. Each dot indicates a biological replicate. *P < 0.05 by nonparametric Mann-Whitney test. (B) qRT-PCR analysis of ILC2-associated gene expression in cardiac tissues. *P < 0.05 by one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test. Each dot indicates a biological replicate. Data are pooled from two independent experiments and expressed as the mean ± SD (n = 5-10 per group). (C) Serum cytokine levels of mice treated with Saline, IL-33, ISO, or ISO+IL-33. *P < 0.05 by one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test. Data pooled from three independent experiments and expressed as the mean ± SD (n ≥ 3 per group). Each dot indicates a biological replicate.

Figure 7

IL-13 is required for IL-33-mediated anti-fibrotic responses through the activation of alternatively activated macrophages (M2ϕ). (A) qRT-PCR validation of M2ϕ gene expression (Arg1 and YM1) in cardiac tissues. Data are pooled from two independent experiments and expressed as the means ± SD (n = 5-10 per group). *P < 0.05 by one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test. (B) IL13^-/- mice were subcutaneously administered with isoproterenol (ISO) (60 mg/kg) for three days then intraperitoneally administered with Saline or IL-33 (0.5 μg/mouse) on days 3, 5, and 7. The cardiac tissues were collected for Picrosirius red staining of the fibrotic area. Scale bar = 100 µm. (C) Quantification of the fibrosis area in the heart sections. ns, not significant. Data are expressed as the means ± SD. ns, not significant. (D) qRT-PCR analyses of M2ϕ gene expression (Arg1 and Ym1) in the cardiac tissues (n=3 in each group). (E) Intracellular flow cytometry analysis for CD45⁺F4/80⁺ macrophages and M2ϕ marker RELMα in the heart of wild-type and IL13^-/- mice. (F) Number of RELMα⁺ macrophages. (G) Mean-fluorescence intensity (MFI) of RELMα in macrophages (n =5 per group). ***P<0.001, ****P<0.0001 by one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test. All values are means ± SD. Each dot indicates a biological replicate.

Figure 8

ILC2-derived factors inhibit TGF-β1-induced cardiac fibroblast activation. (A) Primary mouse cardiac fibroblasts were cultured with Saline IL-33 (30 ng/mL) or ILC2-conditioned media (ILC2-CM, 1:100 dilution) for 24 h in the presence or absence of TGF-β1. Cell morphology of the mouse cardiac fibroblasts was analyzed by immunostaining for vimentin (red). Cell nuclei were stained by DAPI (blue). (B) Transcriptome analysis for the gene expressions associated with ECM and ECM remodeling. Fold increase in the TGF-β1 and TGF-β1+ILC2-CM group represents fold change in gene expression of TGF-β1 vs Saline and TGF-β1+ILC2-CM vs TGF-β1 group, respectively. (C) mRNA expression of Postn and Tnc was analyzed by qRT-PCR. (D) qRT-PCR analysis on TGF-β1-induced Postn and Tnc in the presence or absence of BMP-7 neutralization antibody (1 μg/mL). *P < 0.05, **P < 0.01, ns, not significant by one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test. All values are means ± SD. Each dot indicates a biological replicate. (E) Working model for IL-33-mediated ILC2s expansion for protection against cardiac fibrosis. IL-33 treatment expands and activates cardiac ILC2s to secrete soluble factors including IL-13, IL-5, Areg, IL-10, G-CSF, and BMP-7. IL-13 is crucial for IL-33-mediated cardiac protective function by regulating M2 macrophage (M2ϕ) polarization. ILC2-derived BMP-7 alleviates TGF-β1-induced cardiac fibroblast activation. Taken together, in combination with the increased M2 polarization, reduced ECM production, reduced CF activation, and reduced cell death within the myocardium, IL-33 alleviates cardiac fibrosis via the expansion of ILC2 and the effector functions of ILC2-derived paracrine factors.