Rho-Kinase Inhibition Attenuates Airway Inflammation In An Experimental Asthma Model In Sickle Cell Mice

Introduction Asthma in sickle cell disease is known to be associated with increased morbidity and an elevated rate of sickle cell complications, such as acute chest syndrome, stroke, vaso-occlusive episodes and early mortality. Experimental asthma increases eosinophil and collagen deposition in the lungs of SCD mice, induces profound increases in pulmonary inflammation, shifts in TH1 and TH2 cytokine production, and airway resistance. A small GTPase, Rho, and its target molecule, Rho-kinase (ROCK), play an important role in cell functions, including contractility, chemotaxis, adhesion, and migration, where the Rho/ROCK-mediated pathway facilitates infiltration of inflammatory cells both in vitro and in vivo. This study was designed to determine whether the Rho/Rho-kinase pathways are involved in eosinophil recruitment and inflammation. To investigate the role of Rho/ROCK in the pathogenesis of SCD asthma, we investigated the effect of fasudil, a specific inhibitor of Rho-kinase, on acute allergic inflammation in SCD mice model. Methods Berkeley SCD mouse bone marrow was transplanted into lethally-irradiated C57BL/6 animals to generate age- and gender-matched genetically identical cohorts of SCD mice. Female SCD mice were sensitized and challenged with ovalbumin (OVA). OVA-challenge mice were treated intraperitoneally with fasudil (10 mg/kg), 1 h before each OVA-challenge. At 48 h after OVA challenged total cell counts, differential cell counts, cytokines, and chemokine levels were measured by ELISA in bronchoalveolar lavage fluid (BALF), and the mRNA expressions of ROCK-1, ROCK-2, IL-6, MMP-8, MMP-9, MMP-12, TIMP-1, TIMP-2 were measured in lungs by RT-PCR. Results Intranasal challenge of OVA in SCD mice induced airway inflammation after 48h, characterized by increases in total leukocytes numbers (WBC) that were almost exclusively accounted for by eosinophils, when compared with non-sensitized mice (WBC: 12.8 ±1.5 vs 5.5 ± 0.68; eosinophils: 6.67 ± 1.2 vs 0.01 ± 0.01, p<0.05, respectively). When fasudil was administer to OVA-challenged SC mice, increased numbers of total cells and eosinophils in the BALF were significantly attenuated (WBC: 6.8 ± 0.6; eosinophils: 1.14 ± 0.33, p<0.05). The numbers of neutrophils were also reduced in animals treated with fasudil (0.84 ± 0.13 vs 2.32 ± 0.34, p<0.05). However, mononuclear cells were not affected by fasudil treatment. ROCK-1 and ROCK-2 mRNA expressions in mice of the asthmatic group (1.2 ± 0.18 and 0.84 ± 0.10, respectively) were similar than those of the non-sensitized group (1.1±0.05 and 0.97±0.04, respectively). When fasudil was administered, the expressions of ROCK-1 and ROCK-2 mRNA did not alter in the asthmatic group (0.96 ± 0.12 and 0.98 ± 0.15, respectively). MMP-2, MMP-8, MMP-9, MMP-12 and TIMP-1 gene expressions were not significantly different between SCD asthmatic mice and SCD non-sensitized mice. Increased IL-6 mRNA levels were detected in the lungs at 48h after OVA challenge (0.83 ± 0.09 vs 0.4 ± 0.1, p<0.05). Lung mRNA expressions of MMPs, TIMP-1 and cytokines were not significantly reduced in SCD mice treated with fasudil. OVA challenge in sensitized SCD mice induced IL-4, IL-5, IL-6, eotaxin, RANTES, MCP-1, TIMP-1 release into BALF (OVA-sensitized: 11.1 ± 3.2, 31.8 ± 7.7, 20.4 ± 4.0, 8.8 ± 2.2, 11.7 ± 1.8, 46.6 ± 7.1, 2239 ± 424.7, respectively vs OVA-nonsensitized:1.1 ± 0.6; 1.4 ± 0.6, 4.1 ± 2.0, 2.3 ± 1.5; 6.9 ± 1.9; 4.6 ± 0.97; 294.3 ± 45.6 pg/ml, p<0.05). Fasudil reduced levels of IL-5, L-6, RANTES, MCP-1, proMMP-9 and TIMP-1 in BALF of SCD mice after OVA challenges (-46.4 fold, -62.5 fold, -41.4 fold, -48.6 fold, -53.6 fold, p<0.05, respectively). Conclusion Administration of fasudil inhibits eosinophil recruitment in response to allergen challenge. The effects of this agent may be mediated by suppressing the production of chemokine and cytokines related to the pathophysiology of bronchial asthma, such as IL-5, RANTES and MCP-1. In addition, the Rho-kinase inhibitor reduced levels of proteins involved in the degraded the extracelular matrix, such as MMP-9, which directly or indirectly promoted the progression of pulmonary inflammatory responses. Our findings provide evidence that inhibition of the Rho/Rho-kinase pathway may be beneficial for pulmonary complications in sickle cell disease. Financial Support FAPESP/CNPq/INCTS Disclosures: No relevant conflicts of interest to declare.