Protective Effects Of Recombinant β-defensin-2 Against Acute Lung Injury In Rats

Background and objective Defensins are cysteine-rich cationicantimicrobial polypeptides with three to four disulfide bridges found in plants, insects and mammals. In humans, there are two families of defensins termed α-defensins and β-defensins, which are classified based on the organization of three intermolecular disulfide bonds. The group of α-defensins includes human neutrophil peptides 1-3 (HNP1-3), which are major components of the azurophil granules of neutrophils, and a fourth, HNP4, which is much less abundant in the same location. Two other human defensins, human defensin 5 and human defensin 6, are principally located in intestinal Paneth cells and are thought to be involved in local host defense. Human β-defensins (hBDs) include over 30 subtypes, of which hBD1-4 are well characterized. They are found in epithelial cells of a diverse range of tissues or mononuclear cells. In addition to their antimicrobial activity as part of the initial defense against invading micro-organisms, defensins show chemotactic activity for monocytes, immature dendritic cells and memory T cells, thereby linking innate immunity and adaptive immunity.β-Defensins are epithelium-derived. Human β-defensin 2 (hBD-2) is an inducible antimicrobial peptide in pulmonary epithelium, the expression of which can be induced by microbe infection and proinflammatory factors. Recent studies showed elevated hBD-2 levels in the bronchoaveolar lavage fluid (BALF) of infected individuals, whereas in severe septic patients the inducible expression ofhBD-2 was impaired. Furthermore, inactivated hBD-2 in the respiratory tract is associated with the incidence of Pseudomonas Aeruginosa infections in cystic fibrosis patients. HBD2 may exert an antimicrobial effect in the pulmonary tissues by either directly killing micro-organisms or by modulating the inflammatory process.However, the role of hBD-2 in ALI /ARDS remains unclear.Acute lung injury (ALI) and acute respiratory dysfunction syndrome (ARDS), are common clinical disorders characterized by acute respiratory dysfunction resulting from pulmonary inflammation, non-cardiogenic pulmonary edema and disruption of endothelium and epithelium. ALI can result from various pathophysiological processes including sepsis, pneumonia and trauma, with sepsis being the leading cause of ALI. ALI/ARDS show an annual incidence of 75 per 100,000, with a mortality rate of 30% - 50%. With the improvement of supportive care, such as mechanical ventilation and better nutrition, the mortality rate of ALI/ARDS decreased over the past decade, however, mortality remains high, especially in sepsis-induced ALI/ARDS. Ineffective antibiotic therapy coupled with the failure of most anti-inflammatory treatments in clinic trials has made it necessary to search for new therapeutic interventions to treat ALI/ARDS and improve its outcome.In order to investigate the role of recombinant β-defensin-2 in ALI, gene transfer can be used to achieve over-expression of BD2. The aim of this study was to identify possible vectors and maintain the biological activity of BD2 production, as well as a high level of expression. The adenovirus vector is one of the most popular systems in use that can infect mammalian cells, transfer and express genes efficiently. Up to now, there are no reports on transferring gene of P-defensin-2 by adenovirus vector in mammalian cells.In this study, we investigated the role of rat P-defensin-2 (rBD2), a homologue of hBD-2, by means of gene transfer using a recombinant adenovirus vector in ALI/ARDS models produced either by pulmonary infection withPseudomonas Aeruginosa or cecal ligation and puncture (CLP). Our study was performed in three stages, as lined out below.Part I Construction and expression of a recombinant adenovirus expression vector of p-Defensin-2Objective To explore the possibility of a stable expression of P-defensin-2 in eukaryocyte cells by adenovirus vector.Methods The rat p-defensin-2 (rBD2) gene was cloned at the downstream of the CMV promoter at the adenoviral shuttle plasmid pShuttle-CMV. Then pShuttle-CMV-rBD2 was transformed into E. coli BJ5183-AD-1 containing pAdEasy-1, and recombination occurred between the plasmids to forming pAdEasy-rBD2. After confirmation by endonuclease, linear pAdEasy-rBD2 was transformed into 292 cells to obtain a packaged adenoviral expression vector, which was used to infect COS-7 cells and to establish a respiratory adenovirus infection rat model. In vivo and in vitro expression activity of the recombinant adenovirus was detected by Western blot and immunohistochemistry.Results The inserted cDNA into pShuttle-CMV-rBD2 was consisted with rat β-defensin-2 gene. The observed cellular pathological effect of infected cells, electron microscopy observation and PCR confirmed that the recombinant adenovirus vector had been reconstructed successfully. The concentration of the adenovirus was 10~9 PFU/ml. Western blot and immunohistochemistry analysis showed that the recombinant vector could express rat p-defensin-2 efficiently both in vivo and in vitro.Conclusion The recombinant adenovirus vector expressed P-defensin-2 in eukaryocyte cells.Part II Protective effects of recombinant β-defensin-2 againstacute lung injury in rats induced by Pseudomonas AeruginosainfectionObjective To investigate the preventive function of P-defensin-2 in acute lung injury induced by Pseudomonas Aeruginosa infection in rats.Methods 12 Sprague-Dawley male rats weighting 250-300g were randomly divided into a Ad-rBD2 group (n=6) and a Ad-LacZ group (n=6), in which 10~7PFU adenovirus, Ad-rBD2 or Ad-LacZ, was titrated into the trachea via intubation. Fourty-eight hours later, 200μ16×10~8 CFU/mL Pseudomonas Aeruginosa ATCC27853 was titrated into the trachea in both groups. After 24 hours, all the rats were killed, the amounts of Pseudomonas Aeruginosa and white blood cells (WBC) in bronchial alveolar lavage fluid (BALF) were counted, and the pathological alterations of the lung tissue were examined.Results Compared to the Ad-LacZ group, the amount of WBC and Pseudomonas Aeruginosa were significantly less (P<0.05) and the damage of lung tissue decreased in the Ad-rBD2 group.Conclusion The recombinant rat β-defensin-2 may protect against acute lung injury induced by Pseudomonas Aeruginosa infection as a result of the antibacterial activities in lung tissue.Part III Protective effects of recombinant β-defensin-2 against sepsis-induced acute lung injury in ratsObjective To explore the effect of recombinant β-defensin-2 on sepsis induced acute lung injury in rats.Methods Recombinant adenovirus Ad-rBD2 (10~7PFU) or control adenovirus Ad-LacZ (10~7PFU) was administered intratracheally to Sprague-Dawley rats (n=84,42 rats in each group) 48h before sepsis-inducedacute lung injury caused by cecal ligation and puncture (CLP).The rats were sacrificed at 0, 12, 24, 36 and 72h after CLP (n=6 per time point in each group). Histological changes, expression of ICAM-1 and apoptosis level in lung tissue, as well as the one-week survival rate were determined. Quantitative bacterial analysis of intra-abdominal lavage fluid was also performed.Results The pathological damage of lung tissue, the expression level of ICAM-1 and the apoptosis of lung tissue were reduced in the Ad-rBD2 group(P<0.05), and survival was significantly improved, compared with controls (P＜0.05). The CFU of intra-abdominal bacteria was comparable to that in the control rats (P＜0.05).Conclusion The expression of recombinant BD2 protects against sepsis-induced acute lung injury and improves the outcome, based on its inflammation-modulating activities.