Interferon-β decreases LPS-induced neutrophil recruitment to cardiac fibroblasts

Abstract

Introduction: Cardiac fibroblasts (CF) are crucial cells in damaged heart tissues, expressing TLR4, IFN-receptor and responding to lipopolysaccharide (LPS) and interferon-β (IFN-β) respectively. While CF interact with immune cells; however, their relationship with neutrophils remains understudied. Additionally, theimpact of LPS and IFN-β on CF-neutrophil interaction is poorly understood. Methods: Isolated CF from adult rats were treated with LPS, with or without IFN-β. This study examined IL-8 secretion, ICAM-1 and VCAM-1 expression, and neutrophil recruitment, as well as their effects on MMPs activity. Results: LPS triggered increased IL-8 expression and secretion, along with elevated ICAM-1 and VCAM-1 expression, all of which were blocked by TAK-242. Pre-treatment with IFN-β countered these LPS effects. LPS treated CF showed higher neutrophil recruitment (migration and adhesion) compared to unstimulated CF, an effect prevented by IFN-β. Ruxolitinib blocked these IFN-β anti-inflammatory effects, implicating JAK signaling. Analysis of culture medium zymograms from CF alone, and CF-neutrophils interaction, revealed that MMP2 was mainly originated from CF, while MMP9 could come from neutrophils. LPS and IFN-β boosted MMP2 secretion by CF. MMP9 activity in CF was low, and LPS or IFN-β had no significant impact. Pre-treating CF with LPS, IFN-β, or both before co-culture with neutrophils increased MMP2. Neutrophil co-culture increased MMP9 activity, with IFN-β pre-treatment reducing MMP9 compared to unstimulated CF. Conclusion: In CF, LPS induces the secretion of IL-8 favoring neutrophils recruitment and these effects were blocked by IFN-. The results highlight that CF-neutrophil interaction appears to influence the extracellular matrix through MMPs activity modulation.

Keywords: Interleukin-8; TLR4; cardiac fibroblast; metalloprotease; neutrophils.

FIGURE 1

LPS induces the expression and secretion of IL-8 in cardiac fibroblasts (CF). (A) CF were stimulated with LPS (1 μg/ml) for 4, 8, and 24 h mRNA of IL-8 was analyzed by RT-qPCR. (B) CF were stimulated with LPS (1 μg/ml) at 8, 24 or 48 h. Secreted IL-8 levels were quantified using an ELISA kit. (C) CF were pre-stimulated with the TLR4 inhibitor TAK-242 (4 uM) and subsequently stimulated with LPS (1 μg/ml) for 8 h. IL-8 mRNA was determined with an RT-qPCR. (D) IL-8 secretion via TLR4 in CF. CF were pre-stimulated with the TLR4 inhibitor TAK-242 (4 uM) and subsequently stimulated with LPS (1 μg/ml) for 24 h. IL-8 secretion was determined with an ELISA kit. VEH: DMSO (vehicle for TAK-242). ***p < 0.001; **p < 0.01; *p < 0.05 vs. control; #p < 0.05 vs. LPS (-). Results are expressed as mean ± SEM. (n = 3).

FIGURE 2

LPS induces ICAM-1 and VCAM-1 expression in cardiac fibroblasts (CF) thorough TLR4. (A, B) CF were stimulated with LPS (1 μg/ml) for 4, 8, 24 and 48 h, and then the expression of ICAM-1 (A) and VCAM-1 (B) was analyzed by WB. A representative image of the experiment is shown in the upper panel and its quantification, in the lower panel. (C, D) CF were stimulated with LPS (1 μg/ml) for 4, 8, and 24, and then the mRNA expression of ICAM-1 (C) and VCAM-1 (D) was analyzed by RT-qPCR. (E, F) CF were pre-stimulated with the TLR4 inhibitor TAK-242 (4 uM) for 1 h and subsequently stimulated with LPS (1 μg/ml) for 24 h for the subsequent analysis of ICAM-1 (C) and VCAM-1 (D) protein expression by WB. A representative image of the experiment is shown in the upper panel and its quantification, in the lower panel Proteins were analyzed by Western Blot. GAPDH was used as a loading control. (G) and (H) CF were pre-stimulated with the TLR4 inhibitor TAK-242 (4 uM) for 1 h and subsequently stimulated with LPS (1 μg/ml) for 8 h for the subsequent analysis of ICAM-1 (C) and VCAM-1 (D) mRNA expression by RT-qPCR. ***p < 0.001; **p < 0.01; *p < 0.05 vs. control; #p < 0.05 vs. LPS. Results are expressed as mean ± SEM. (n = 3).

FIGURE 3

Effect of the IFN/JAK pathway on IL-8 secretion and ICAM and VCAM expression in cardiac fibroblasts (CF). The CF were pre-stimulated with ruxolitinib 30 min before treatment with IFN-β; then CF were pre-stimulated with IFN-β (500 IU) for 1 h and subsequently stimulated with LPS (1 μg/ml) for 24 h. (A) The expression of ICAM-1 and VCAM-1 protein levels were analyzed by Western Blot. GAPDH was used as a loading control. A representative image of the experiment is shown in the upper panel and its quantification, in (C). (B) IL-8 secretion was analyzed by ELISA from cell culture media. **p < 0.01 vs. control; #p < 0.01 vs. LPS; &p < 0.05 vs. LPS + IFN-β. Results are expressed as mean ± SEM. (n = 3).

FIGURE 4

The IFN-β/JAK signaling pathway prevents LPS-induced IL-8-dependent neutrophil migration. Neutrophils were previously labeled with Calcein^(R), a fluorescent probe, and seeded in a Transwell plate. The migration was analyzed at 3 h. (A) Migration of neutrophils in conditioned culture medium secreted by CF treated with/without LPS (1 μg/ml) for 24 h. The culture medium was preincubated for 30 min with the anti-IL-8 blocking antibody. (B) Migration of neutrophils in conditioned culture medium secreted by CF treated with ruxolitinib for 30 min before treatment with IFN-β. Then the cells were stimulated with IFN-β (500 IU/mL) for 1 h, and later stimulated with/without LPS (1 μg/ml) for 24 h. The number of fluorescent neutrophils that migrated to the lower compartment was measured by a spectrofluorometer. The percentage of migration was expressed based on the measured fluorescence vs. the fluorescence emitted by the total aggregated neutrophils. Results represent the mean ± SEM of four independent experiments. **p < 0.01 vs. control; #p < 0.01; ##p < 0.01 vs. LPS and andp < 0.05 vs. LPS + IFN-β. Results are expressed as mean ± SEM. (n = 3).

FIGURE 5

The IFN-β/JAK signaling pathway prevents LPS-induced neutrophil adhesion. Representative diagrams of adhesion of neutrophils to CF pre-stimulated with IFN-β (500 IU/mL) for 1 h and/or LPS (LPS (1 μg/ml) for 24 h in the absence of ruxolitinib ((A): cardiac fibroblast; (B) neutrophils and (C) other leukocytes and cell death). CF with adherent leukocytes were trypsinized for their removal from the plate and then were incubated with the anti RP-1(PE) antibody, specific for neutrophils, for 30 min. Adhesion levels were determined by flow cytometry. The graph shows adhesion levels of neutrophils to a CF monolayer treated as observed in the graph. The percentage of neutrophil adhesion was calculated as follows: 100 x (n° cells RP-1⁺ adhered/n° total cells RP-1⁺). **p < 0.01 vs. control; ##p < 0.01 vs. LPS; &&p < 0.01 vs. LPS + IFN-β. Results are expressed as mean ± SEM (n = 3).

FIGURE 6

Activity of MMP2 and MMP9 in cardiac fibroblasts and CF-neutrophils co-cultures. (A) CF were pre-stimulated with ruxolitinib for 30 min before IFN-β treatment. CF were pre-stimulated with IFN-beta (500 IU/mL) for 1 h and subsequently stimulated with LPS (1 μg/ml) for 24 h. In treated CF alone and in pre-treated CF co-cultured with the MMPs activity was measured as indicated in materials and methods. A representative image of the experiment is shown in the upper panel and its quantification appears in the lower panel. (A) MMP2 activity in CF alone (white bars) and in a CF-Neutrophils co-culture (black bars). *p < 0.05; **p < 0.01 vs. non-treated CF; #p < 0.05 Ruxolitinib + IFN-β vs. Ruxolitinib + IFN-β + LPS; ##p < 0.01 IFN-β vs. Ruxolitinib + IFN-β; &p < 0.05 CF vs. CF + neutrophils. (B) MMP9 activity in CF alone (white bars) and in a co-culture of CF-Neutrophils (black bars).). *p < 0.05; ***p < 0.001 CF vs. treated CF-neutrophils; #p < 0.05 no treated CF vs. IFN-β and Ruxolitinib + IFN-β. Results are expressed as mean ± SEM. (n = 3).