ABSTRACT
The therapeutic application of siRNA shows promise as an alternative approach to small-molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the difficulty in delivering these large anionic molecules in vivo. In this study, we have investigated whether siRNA-mediated knockdown of p38 MAP kinase mRNA in mouse lung is influenced by conjugation to the nonviral delivery vector cholesterol and the cell penetrating peptides (CPP) TAT(48-60) and penetratin. Initial studies in the mouse fibroblast L929 cell line showed that siRNA conjugated to cholesterol, TAT(48-60), and penetratin, but not siRNA alone, achieved a limited reduction of p38 MAP kinase mRNA expression. Intratracheal administration of siRNA resulted in localization within macrophages and scattered epithelial cells and produced a 30-45% knockdown of p38 MAP kinase mRNA at 6 h. As with increasing doses of siRNA, conjugation to cholesterol improved upon the duration but not the magnitude of mRNA knockdown, while penetratin and TAT(48-60) had no effect. Importantly, administration of the penetratin or TAT(48-60) peptides alone caused significant reduction in p38 MAP kinase mRNA expression, while the penetratin-siRNA conjugate activated the innate immune response. Overall, these studies suggest that conjugation to cholesterol may extend but not increase siRNA-mediated p38 MAP kinase mRNA knockdown in the lung. Furthermore, the use of CPP may be limited due to as yet uncharacterized effects upon gene expression and a potential for immune activation.
Subject(s)
Carrier Proteins/pharmacology , Drug Delivery Systems , Gene Expression Regulation/drug effects , Gene Products, tat/chemistry , Immunity, Innate/drug effects , Lung/physiology , RNA, Small Interfering/pharmacology , Animals , Base Sequence , Cell Line , Cell-Penetrating Peptides , Cholesterol/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Regulation/physiology , Immunity, Innate/physiology , Macrophages/metabolism , Mice , Molecular Sequence Data , RNA, Messenger/metabolism , RNA, Small Interfering/chemistry , Time Factors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Respiratory infections are the third leading cause of death worldwide. Complications arise directly as a consequence of pathogen replication or indirectly due to aberrant or excessive immune responses. In the following report, we evaluate the efficacy, in a murine model, of nasally delivered DNA encoding TGF-beta1 to suppress immunopathology in response to a variety of infectious agents. A single nasal administration suppressed lymphocyte responses to Cryptococcus neoformans, influenza virus and respiratory syncytial virus. The suppression did not depend on the phenotype of the responding T cell, since both Th1 and Th2 responses were affected. During Th2-inducing infection, pulmonary eosinophilic responses were significantly suppressed. In all cases, however, suppressed immunity correlated with increased susceptibility to infection. We conclude that nasal TGF-beta treatment could be used to prevent pulmonary, pathogen-driven eosinophilic disease, although anti-pathogen strategies will need to be administered concordantly.
Subject(s)
Pulmonary Eosinophilia/drug therapy , Transforming Growth Factor beta/pharmacology , Animals , Cryptococcosis/drug therapy , Cryptococcosis/immunology , Female , Gene Expression , Inflammation/drug therapy , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/immunology , Plasmids , Pulmonary Eosinophilia/immunology , Pulmonary Eosinophilia/prevention & control , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Virus Infections/immunology , T-Lymphocytes, Helper-Inducer/drug effects , T-Lymphocytes, Helper-Inducer/physiology , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/physiologyABSTRACT
In a healthy individual, the lung contains few lymphoid cells. However, amplified immune responses, as exemplified during lung infection, can cause extensive tissue damage. We have previously demonstrated that one lung infection modulates the immunopathological outcome to a subsequent unrelated pathogen. Mimicking heterologous immunity may provide a means of enhancing both innate and acquired immunity. We now show that prior lung administration of a modified heat-labile toxin from Escherichia coli (LTK63) enhances immunity to respiratory syncytial virus, influenza virus, and the fungus Cryptococcus neoformans. Treatment with LTK63 decreased lung inflammation and tissue damage and improved the ability to resolve the infection. APCs expressing the activation markers MHC class II, CD80, and CD40 increased in number in the lung. LTK63 treatment increased the pathogen-specific IgA response in the nasal mucosa and simultaneously decreased inflammatory cytokine production (IFN-gamma and TNF-alpha) after infection. The number of activated CD8(+)CD44(+) T cells and the respiratory syncytial virus- or influenza-specific CD8-proliferative responses increased, although the total inflammatory infiltrate was reduced. LTK63 treatment matured lung APCs (LTK63 prevented efficient presentation of whole OVA to DO11.10 cells, whereas OVA peptide presentation was unaffected), enhanced immunity in both a Th1 and Th2 environment, was long lasting, and was not pathogen or host strain specific; the protective effects were partially independent of T and B cells. Innate imprinting by toxin-based immunotherapeutics may provide generic protection against infectious disease in the lung, without the need for coadministered pathogen-specific Ag.
