CXCR3 regulates CD4+ T cell cardiotropism in pressure overload-induced cardiac dysfunction

Abstract

Heart failure (HF) is associated in humans and mice with increased circulating levels of CXCL9 and CXCL10, chemokine ligands of the CXCR3 receptor, predominantly expressed on CD4+ Th1 cells. Chemokine engagement of receptors is required for T cell integrin activation and recruitment to sites of inflammation. Th1 cells drive adverse cardiac remodeling in pressure overload-induced cardiac dysfunction, and mice lacking the integrin ligand ICAM-1 show defective T cell recruitment to the heart. Here, we show that CXCR3+ T cells infiltrate the heart in humans and mice with pressure overload-induced cardiac dysfunction. Genetic deletion of CXCR3 disrupts CD4+ T cell heart infiltration and prevents adverse cardiac remodeling. We demonstrate that cardiac fibroblasts and cardiac myeloid cells that include resident and infiltrated macrophages are the source of CXCL9 and CXCL10, which mechanistically promote Th1 cell adhesion to ICAM-1 under shear conditions in a CXCR3-dependent manner. To our knowledge, our findings identify a previously unrecognized role for CXCR3 in Th1 cell recruitment into the heart in pressure overload-induced cardiac dysfunction.

Keywords: Cardiology; Heart failure; Inflammation; T cells.

Figure 1. CXCR3⁺ T cell infiltration in humans and mice with cardiac pressure overload.

(A and B) LV tissue sections from non-HF or nonischemic HF human subjects were stained for CXCR3 or isotype control by IHC (representative CXCR3⁺ leukocyte–shaped cells are indicated with red arrows) (A) and quantified in multiple fields of view using a 40× objective (B). (C and D) LV tissue sections were stained for DAPI (blue), CD3 (red), and CXCR3 (green) by immunofluorescence (C), and the number of cells showing colocalization were quantified in multiple fields of view per section using a 40× objective (D). Scale bar: 100 μm. n = 2 control, 3 HF. Error bars represent mean ± SEM (**P < 0.01; Mann-Whitney unpaired U test). (E and F) CD4⁺ T cells isolated from the LV tissue of mice 4 weeks after Sham or TAC surgery were analyzed (E) and quantified (F) for surface CXCR3 and LFA-1 expression within the CD4⁺ gate by flow cytometry. n = 3 Sham, 7 TAC. Error bars represent mean ± SEM (**P < 0.01; Mann-Whitney unpaired U test). (G–J) Chemokine and cytokine mRNA levels in the LV of WT mice at different time points after surgery were determined by qPCR for Cxcl9 (G), Cxcl10 (H), Ifng (I), and Tnfa (J). n = 4 Sham, 5 TAC 1 weeks (w); 5 Sham, 9 TAC 2w; 6 Sham, 8 TAC 4w. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni post hoc test).

Figure 2. CXCL9 and CXCL10 are produced by cardiac myeloid cells and fibroblasts in response to cardiac pressure overload.

REX3 mice were subjected to Sham and TAC surgery, and LV tissue sections were isolated for analysis of CXCL9 and CXCL10 expression by immunofluorescence. (A–C) The outline of cardiomyocytes was identified by wheat germ agglutinin (WGA) staining (A), leukocytes were identified with anti-CD45 (B), and endothelial cells identified with anti-CD31 (C) in Sham mice (top row) and TAC mice 4 weeks after surgery in nonfibrotic areas (middle row) and fibrotic areas (bottom row), also stained by WGA. Scale bars: 100 μm. n = 3 mice per group. (D–J) Cardiac endothelial cells (ECs), myeloid cells, T cells, and cardiac fibroblasts (CFBs) were identified by flow cytometry with the indicated gating strategy (D), to quantify the frequency of CXCL9- and CXCL10-producing cells 1 week after TAC surgery (E–G) and 4 weeks after TAC surgery (H–J). Statistical comparisons are indicated compared with ECs. n = 3 mice per group. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001; 1-way ANOVA with Bonferroni post hoc test).

Figure 3. CXCL9 and CXCL10 are produced by resident and recruited cardiac myeloid cells in response to cardiac pressure overload.

REX3 mice were subjected to TAC surgery, and LV tissue sections were isolated for analysis of CXCL9 and CXCL10 expression by flow cytometry, 4 weeks after TAC. (A and B) CD11b⁺Ly6G⁺ Neutrophils, CD11b⁺MerTK^–CCR2⁺ monocytes, CD11b⁺MerTK⁺CCR2^– resident macrophages, and CD11b⁺MerTK⁺CCR2⁺ recruited macrophages were identified by flow cytometry with the indicated gating strategy. (C–F) CXCL9 and CXCL10 expression was quantified as frequency (C and D) and absolute cell number per LV (E and F). Statistical comparisons are indicated compared with neutrophils. n = 3 mice per group. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001; 1-way ANOVA with Bonferroni post hoc test).

Figure 4. CXCR3⁺CD4⁺ T cells from mice and humans with cardiac pressure overload express high levels of LFA-1.

(A and B) Representative flow cytometry plots (A) and quantification (B) of CD4⁺ T cells isolated from the mLNs of WT mice at the indicated times after Sham and TAC surgery, indicating surface CXCR3 and LFA-1 expression within the CD4⁺ gate. Relative values to Sham 1 week (w) are indicated. n = 3 Sham, 3 TAC 1w; 4 Sham, 4 TAC 2w; 3 Sham, 4 TAC 4w. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001; 1-way ANOVA with Bonferroni post hoc test). (C–F) Histogram representation (C and E) and quantification (D and F) of LFA-1 expression on CXCR3⁺CD4⁺ and CXCR3^–CD4⁺ T cells in the mLNs of WT mice 4 weeks after TAC surgery (C and D) and in peripheral blood of nonischemic HF patients (E and F). n = 7 TAC mice, 20 nonischemic HF patients. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney unpaired U test).

Figure 5. CD4⁺ T cell recruitment to the LV, but not CD4⁺ T cell activation, is impaired in Cxcr3^–/– mice in response to cardiac pressure overload.

CD4⁺ T cells were isolated from the mLN of WT and Cxcr3^–/– mice, 4 weeks after Sham and TAC surgeries. (A and B) CD62L^loCD44^hi effector CD4⁺ T cells were identified by flow cytometry (A) and quantified (B). (C and D) CD4⁺ T cells recruited to the LV were identified by IHC (C) and quantified (D) per LV section. n = 4 Sham, 4 TAC WT; 3 Sham, 5 TAC Cxcr3^–/–. Scale bars: 100 μm. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni post hoc test). (E–H) The indicated gating strategy shown for WT TAC (E) was used to quantify the total cell number per LV of CD4⁺ T cells (F), CD11b⁺ myeloid cells (G), and CD11b⁺MerTK⁺CCR2⁺ recruited macrophages (H) by flow cytometry in WT and Cxcr3^–/– mice, 4 weeks after Sham and TAC surgeries. n = 3 Sham, 3 TAC WT; 6 TAC Cxcr3^–/– mice. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni post hoc test). (I–K) mRNA levels in the LV of WT and Cxcr3^–/– mice at 4 weeks after surgery was determined by qPCR for Cxcl9 (I), Cxcl10 (J), and Ifng (K). n = 9 Sham, 10 TAC WT; 3 Sham, 5 TAC Cxcr3^–/–. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni post hoc test).

Figure 6. Cxcr3^–/– mice are protected from adverse cardiac remodeling induced by cardiac pressure overload.

LV tissue sections were isolated from WT and Cxcr3^–/– mice, 4 weeks after Sham and TAC surgeries. IHC was used to determine perivascular fibrosis (A) (quantified in C) as well as interstitial fibrosis (B) (quantified in D) by Picrosirius red staining. Scale bars: 100 μm. (E and F) Mean cardiomyocyte area was quantified by wheat germ agglutinin (WGA) IHC of LV tissue sections (E) (as shown in F). n = 4 Sham, 5 TAC WT; 3 Sham, 5 TAC Cxcr3^–/– mice. Scale bars: 50 μm. Error bars represent mean ± SEM (*P < 0.05, ***P < 0.001; 1-way ANOVA with Bonferroni post hoc test). (G) The relative LV mRNA expression of the α and β myosin heavy chain isoforms were determined by qPCR to assess pathological cardiomyocyte hypertrophy. n = 3 Sham, 3 TAC WT; 3 Sham, 5 TAC Cxcr3^–/– mice. Error bars represent mean ± SEM (*P < 0.05; 1-way ANOVA with Bonferroni post hoc test).

Figure 7. Cardiac function is preserved in Cxcr3^–/– mice subjected to cardiac pressure overload induced by TAC.

(A–C) Transthoracic short axis M mode images of the mid LV were acquired by echocardiography (A) of WT and Cxcr3^–/– mice, 4 weeks after Sham and TAC surgeries to quantify fractional shortening (B) and ejection fraction (C). n = 5 Sham, 7 TAC WT; 5 Sham, 7 TAC Cxcr3^–/– mice. Error bars represent mean ± SEM (***P < 0.001; 1-way ANOVA with Bonferroni post hoc test). (D–F) Intraventricular hemodynamic measurements were acquired by a pressure volume transducer catheter to quantify maximum LV pressure (D), as well as dP/dt max (E) and dP/dt min (F) as parameters of cardiac contractility and relaxation, respectively. n = 3 Sham, 3 TAC WT; 3 Sham, 4 TAC Cxcr3^–/–. Error bars represent mean ± SEM (*P < 0.05; 1-way ANOVA with Bonferroni post hoc test).

Figure 8. CXCL9 and CXCL10 induce LFA-1–dependent T cell adhesion to ICAM-1 through CXCR3.

Naive WT and Cxcr3^–/– T cells were differentiated in vitro to Th1 cells and treated with either PMA (50 ng/ml), CXCL9 (100 ng/ml), or CXCL10 (100 ng/ml) for 5 minutes at 37°C to induce integrin LFA-1 activation, prior to perfusion over immobilized ICAM-1–coated coverslips at 1 dyne/cm² in a parallel plate flow chamber. (A and B) Representative images (A) and quantification (B) of real-time videos of WT Th1 cell adhesion to ICAM-1 after treatment with anti–LFA-1 function-blocking antibody or IgG isotype control (20 μg/ml 30 minutes at 37°C). Scale bars: 100 μm. Statistical comparisons are indicated compared with untreated cells. n = 5 independent experiments, analysis of 6 different fields of view per experiment (*P < 0.05, ***P < 0.001; 1-way ANOVA with Bonferroni post hoc test). (C and D) Representative images (C) and quantification (D) of real-time videos of Cxcr3^–/– Th1 cells. Scale bars: 100 μm. Statistical comparisons are indicated compared with untreated cells. n = 3 independent experiments, analysis of 6 different fields of view per experiment. Error bars represent mean ± SEM (***P < 0.001; 1-way ANOVA with Bonferroni post hoc test).