Bronchial Vascular Remodeling Is Attenuated by Anti-IL-17 in Asthmatic Responses Exacerbated by LPS

Abstract

Introduction: Although the major alterations associated with asthma are related to the airways, there is also evidence of the importance of peribronchial vascular inflammation and remodeling in its pathophysiology.

Objectives: To determine the effects of anti-IL-17 therapy on peribronchial vessels of an asthma model exacerbated by lipopolysaccharide.

Methods: We evaluated several factors, including lung function, inflammation, oxidative stress, vascular remodeling, and signaling pathways present in the peribronchial vessels of 66 male BALB/c mice exposed to ovalbumin and treated (or not) treated with anti-IL-17. Twenty-four hours before the end of the experimental protocol, groups of sensitized animals (OVA-LPS and OVA-LPS anti-IL-17) also received LPS.

Results: The OVA-LPS-anti-IL-17 group presented a decrease in several factors [airway resistance and elastance, bronchoalveolar lavage fluid (BALF) cell counts, inflammatory response, eosinophils, TSLP, IL-33, TARC, TNF-α, CD4+, CD8+, IL-4, IL-6, IL-10, IL-17, and VEGF positive cells/10⁴μm², peribronchovascular edema, and angiogenesis], including remodeling (MMP-9, MMP-12, TIMP-1 and TGF-β positive cells and volume fraction of collagen fibers I, collagen fibers III, collagen fibers V, decorin, lumican, actin, biglycan, fibronectin, and integrin), oxidative stress (iNOS positive cells and volume fraction of PGF2α), and signaling pathways (FoxP3), as well as dendritic cells, NF-kB, ROCK-1, ROCK-2, STAT-1, and phosphor-STAT1-positive cells compared to OVA-LPS (p < 0.05).

Conclusions: In this model of LPS-induced asthma exacerbation, IL-17 inhibition represents a promising therapeutic strategy, indicating the potential of bronchial vascular control of Th2 and Th17 responses and the activation of the remodeling and oxidative stress pathways, associated with the control of signaling pathways.

Keywords: LPS (lipopolysaccharide); anti-IL-17; asthma; vascular inflammation; vascular remodeling.

Figure 1

Timeline of the sensitization protocol. On days 1 and 14, the BALB/c mice received a solution of OVA intraperitoneally (i.p.). On days 22, 24, 26 and 28, the animals were submitted to inhalation for 30 min at 1% concentration. The control group received a saline solution i.p. and was exposed to 0.9% saline aerosol for 30 min as the inhalation challenge. Anti-IL-17 neutralizing antibody was administered i.p. 1 h prior to the intratracheal instillation of LPS. Twenty-four hours after the final antigen challenge, on day 29, the animals received LPS intratracheally. On the 29^th day, the mechanics of the respiratory system and bronchoalveolar lavage (Barlow et al., 2011; Starkhammar et al., 2012; Camargo et al., 2018).

Figure 2

Description of antibodies used for immunohistochemistry analysis.

Figure 3

Effects of anti-IL 17 treatment on vascular remodeling. The values represent the mean ± standard error (SE) of the collagen fibers I, III, V, MMP-9, actin, biglycan, integrin, MMP-12, decorin, lumican, fibronectin, and TIMP-1 for all experimental groups. The differences were considered significant when p < 0.05. *p < 0.05 vs. SAL group; **p < 0.05 vs. OVA and OVA–LPS groups. ^#p < 0.05 vs. OVA group.

Figure 4

Effects of anti-IL 17 treatment on TGF-β and neutrophils in BALF. Panel A shows the expression of positive cells for TGF-β in the pulmonary vessels and Panel B shows the number of neutrophil cells in the bronchoalveolar lavage fluid. The values represent the mean ± SE of TGF-β and neutrophils for all experimental groups. The differences were considered significant when p < 0.05. *p < 0.05 vs. SAL group; **p < 0.05 vs. OVA and OVA–LPS groups. ^#p < 0.05 vs. OVA group. Photomicrograph pulmonary vessels measuring TGF-β. The animals exposed to only ovalbumin and ovalbumin plus LPS (OVA and OVA–LPS groups) presented prominent increases in the numbers of positive cells, compared with the control animals (SAL group). Anti-IL-17 treatment in the OVA anti-IL-17 and OVA–LPS anti-IL-17 groups ameliorated all increases in these markers. The red arrows indicate the positive cells for TGF-β.

Figure 5

Effects of anti-IL-17 treatment on resistance (Rrs) and elastance (Ers). Dose/response curves of methacholine dose/3, 30, and 300 mg/ml. Data are presented as mean ± sem. The differences were considered significant when p < 0.05. *p < 0.05 vs. baseline. Values of the logarithm of the area under the curve (AUC) of resistance (Rrs) and elastance of the respiratory system (Ers) were considered a significant difference when p < 0.05. *p < 0.05 vs. SAL group; **p < 0.05 vs. OVA and OVA–LPS groups. #p < 0.05 vs. OVA group.

Figure 6

Effects of anti-IL-17 treatment on angiogenesis, peribronchovascular edema, and vascular endothelial growth factor (VEGF). The values represent the mean ± standard error (SE) of the angiogenesis, peribronchovascular edema, and vascular endothelial growth factor (VEGF) for all experimental groups. The differences were considered significant when p < 0.05. *p < 0.05 vs. SAL group; **p < 0.05 vs. OVA and OVA–LPS groups. ^#p < 0.05 vs. OVA group.

Figure 7

Effects of anti-IL 17 treatment on vascular inflammation. The values represent the mean ± SE of CD4, CD8, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, eosinophils, TNF-α, and TARC for all experimental groups. The differences were considered significant when p < 0.05. *p < 0.05 vs. SAL group; **p < 0.05 vs. OVA and OVA–LPS groups. ^#p < 0.05 vs. OVA group.

Figure 8

Effects of anti-IL 17 treatment on TSLP and vascular IL-33. The values represent the mean ± SE of TSLP and IL-33 for all experimental groups. The differences were considered significant when p < 0.05. *p < 0.05 vs. SAL group; **p < 0.05 vs. OVA and OVA–LPS groups. Photomicrograph pulmonary vessels measuring IL-33. The animals exposed to only ovalbumin and ovalbumin plus LPS (OVA and OVA–LPS groups) presented prominent increases in the numbers of positive cells, compared with the control animals (SAL group). Anti-IL-17 treatment in the OVA anti-IL-17 and OVA–LPS anti-IL-17 groups attenuated all increases in these markers. The IL-33 positive cells are identified by the red circle.

Figure 9

Effects of anti-IL 17 treatment on antigen-presenting cells (A), FOxP3 (B), signaling pathways (C–G), and oxidative stress (H–J). The values represent the mean ± SE of dendritic cells, FoxP3, ROCK-1, ROCK-2, Phosphor-STAT1, STAT1, NF-k, iNOS, NOex, and isoprostane for all experimental groups. The differences were considered significant when p < 0.05. *p < 0.05 vs. SAL group; **p < 0.05 vs. OVA and OVA–LPS groups. ^#p < 0.05 vs. OVA group.

Figure 10

Qualitative analysis of vascular inflammation and remodeling. Photomicrographs showing the presence of inflammation around the pulmonary vessels. The slides were stained for IL-17 expressing positive cells and collagen fiber I content. The experimental groups include SAL, OVA, OVA-anti-IL-17, OVA–LPS, and OVA–LPS-anti-IL-17. The red arrows indicate the positive cells for IL-17 and collagen fiber I content.

Figure 11

Qualitative analysis of vascular signaling pathways. Photomicrographs showing the presence of inflammation around the pulmonary vessels. The slides were stained for NF-kB and STAT-1-expressing positive cells. The experimental groups include SAL, OVA, OVA-anti-IL-17, OVA–LPS, and OVA–LPS-anti-IL-17. The red arrows indicate the positive cells for NF-kB and STAT-1.

Figure 12

Qualitative analysis of vascular stress oxidative. Photomicrographs showing the presence of inflammation around the pulmonary vessels. The slides were stained for iNOS expressing positive cells and isoprostane content. The experimental groups include SAL, OVA, OVA-anti-IL-17, OVA–LPS, and OVA–LPS-anti-IL-17. The red arrows indicate the positive cells for iNOS and isoprostane content.