Acute Lung Inflammation in Sickle Mice Is Mediated by Increase of CXC Chemokines and Matrix Metalloproteinases
Authors: Franco-Penteado Carla Fernanda , Lanaro Carolina , Albuquerque Dulcinéia M , Gimenes Ana Paula , Passos Luiz Augusto C , Corat Marcus A.F. , Costa Fernando F. 
Published: April 2020

Abstract
Studies in vitro, and in vivo using animal models show that leukocytes play a key role in vasoocclusion and clinical research suggests that high leukocyte counts correlate with mortality, stroke and acute chest syndrome in sickle cell disease (SCD). Lungs are particularly vulnerable to vaso-occlusive events because of their anatomic features in SCD. Transgenic mice expressing exclusively human sickle hemoglobin (SS) are well-established models for the study of vascular inflammation. Previous studies have shown that systemic LPS challenge causes exaggerated inflammation, including increased serum and broncoalveolar lavage (BAL) TNF-α and IL-1 cytokines and sVCAM-1 in sickle mice. The aim of this study was to examine the contribution of acute airway inflammation in SCD using SS mice and the role of chemokines and matrix metalloproteinases (MMPs) in this process. Acute lung inflammation and injury were induced by intranasal administration of lipopolysaccharide (LPS, 50 μl of 250 μg/ml) in control (C57BL/6) and SS mice. The vehicle mice group received a similar volume of sterile PBS. BAL was performed 4 h after LPS challenge. qRT-PCR analysis was used to examine gene expression and ELISA protein production. The intranasal administration of LPS to mice triggered a huge influx of leukocytes (neutrophils, NS) in BAL of control and SS mice compared with the respective vehicle groups, but this influx was greater in SS mice, when compared with control mice (1.4 ± 0.06 vs 0.66 ± 0.12 WBCx106/BAL); p=0.0006, 1.06 ± 0.1 vs 0.40 ±0.12 NSx106/BAL; p=0.004, respectively). At baseline levels, KC and MIP-2 chemokines (functional homologues of human IL-8 in mice) are higher in BAL fluid of SS mice compared to control mice (186.6 ± 14.1 pg/ml vs 14.1 ± 5.8 pg/ml; 41.2 ± 7.9 pg/ml vs 11.4 ± 7.3 pg/ml, p=, respectively). Corresponding with influx of NS, lung lavage levels of KC and MIP-2 were significantly higher in SS BALF compared to control mice (2491 ± 454 pg/ml vs 798.1 vs 98.2 pg/ml; 1726 ± 307 pg/ml vs 887.3 ± 149.5 pg/ml, respectively). Enhanced levels of TNF-α were also observed at baseline and after LPS instillation compared to those of the control mice (20.8 ± 8.8 pg/ml vs 2.5 ± 1.6 pg/ml; 4250 ± 636 pg/ml vs 1585 ± 263 pg/ml, respectively). Instillation of LPS markedly increased KC, TNF-α, MMP-8, MMP-9 and TIMP-1 mRNA levels in the lungs of control and SS mice compared to animals that received PBS instead of LPS (Control, KC: 0.19 ± 0.047 vs 0.01 ± 0.005; TNF-α: 0.30 ± 0.07 vs 0.01 ± 0.002; MMP-8: 0.2 ± 0.06 vs 0.016 ± 0.004; MMP-9: 0.22 ± 0.03 vs 0.08 ± 0.01; TIMP-1: 0.32 ± 0.06 vs 0.09 ± 0.03); (SS, KC: 0.42 ± 0.1 vs 0.039 ± 0.02; TNF-α: 0.23 ± 0.025 vs 0.02 ± 0.007; MMP-8: 0.42 ± 0.06 vs 0.06 ± 0.03; MMP-9: 0.49 ± 0.11 vs 0.11 ± 0.05; TIMP-1: 0.49 ± 0.11 vs 0.09 ± 0.03). However, the LPS-induced KC, MMP-8 and MMP-9 expression was significantly higher in SS mice lung compared than that of the control group (p&lt;0.05). Lung MMP-2, MMP-12 and TIMP-2 gene expressions were similar in the PBS and LPS groups and were not significantly different between SS and control mice. Our results indicate that chemokines and MMPs are critically involved in the recruitment of neutrophils to the lung following LPS challenge, and suggest that these inflammatory mediators may play a role in the development of pulmonary diseases in SCD. The findings from this study provide further support to the claim that a proinflammatory state is present in SCD and have important implications for the pathophysiology of lung injury in SCD.