Experimental Study On The Therapeutics Of IκBα Mutant, A Novel Inhibitor Of NF-κB, For Asthma

Bronchial asthma (abbreviated asthma) is, per se, a chronic airway inflammatory disease involving in a wide range of inflammatory cells, mediators and cytokine network with multiple susceptible genes for atopy. As the most impotant effector cells, eosinophils (EOS) play a critical role in asthma characteristic of airway eosinophilia. Dendritic cells (DCs) function as the most potent antigen-presenting cells identified with distinct ability to stimulate the proliferation of naive T cells. It is now evident that asthma is a CD4⁺ T cell mediated disorder, in which DCs induce type 2 T helper (Th2) cells-dependent sensitization leading to eosinophilic airway inflammation.The nuclear factor κ B (NF- κ B), a p50 (NF- κ B1)/p65 (RelA) heterodimer, regulates the maximal transcription of genes associated with immune and inflammatory response. Several lines of evidence suggest a central role of NF- κ B in the pathogenesis of asthma: (1) activated NF- κ B has been identified in both macrophages of induced sputum and bronchial biopsy specimens from asthmatics; (2) agents such as allergens, ozone, and viral infections, which are associated with exacerbation of asthma, stimulate activation of NF- κ B; and (3) glucocorticoids, the most effective treatment for asthma, are potent inhibitors of NF- κ B (I κ B). In addition, some anti-inflammatory agents, eg, antioxidants, arsenic trioxide (As₂O₃), selenite, gliotoxin, asprin, sulfasalazine, curcumin and erythromycin also belong to potent I κ B. Both pharmacologic and gene transfer strategies have been used to target blockade of NF- κ B activation: (1) overexpression of I κ B or dominant-negative mutants of I κ B kinase(IKK); (2) double-stranded decoy oligonucleotides that bind NF- k B; and (3) antisense oligonucleotides that decrease p65 synthesis.Although myriad stimuli activate NF- k B via diverse initial transmembrane pathways of signal transduction, it is the phosphorylation of I κ B α, the most important member of I κ B family, at serine (Ser) 32/36 residues of N-terminal domain and subsequent degradation that leads to the common signaling pathway for NF- k B αctivation. Clearly, as far as the inflammatory cascade of asthma is concerned, NF- k B repression in airways through suppression of I κ B α degradation or augmentation of I κ B α synthesis can downregulate transcription of a series of target genes, such as cytokines, enzymes, adhesion molecules, chemokines, etc, which is more effective than blockade of single downstream inflammatory or immune gene. Therefore, I κ B α becomes a pivotal candidate to selectively inhibit NF- k B αctivation for regulation of NF- k B-dependent genes.In the present study, four experiments were further undertaken on the basis of our previous findings: (1) investigated the effect of As₂O₃ on NF- k B repression and I κ B α expression in a murine model of asthma to elucidate the possible therapeutic mechanism of As₂O₃ for broad anti-inflammation, and to explore the role of I κ B α in asthma therapy; (2) optimized the novel 801 bp I κ B α mutant (I κ BaM) gene derived from human placenta tissue by deleting the N-terminal phosphorylation sites of Ser 32/36 with site-directed mutagenesis, followed by construction and identification of the replication-deficient recombinant adenovirus AdI κ BaM, containing cytomegalovirus (CMV) promoter, I κ B α M cDNA and Poly A signals; (3) investigated the effect of adenoviral gene transfer of novel I κ B α M on apoptosis, phenotype and function of DCs derived from human peripheral blood mononuclear cells (PBMC) in vitro to explore the potential strategy for DC-based immunotherapy of asthma; and (4) investigated the inhibitory effect and mechanism of adenoviral gene transfer of novel I κ B α M on allergen-induced pulmonary EOS infiltration in a murine asthma model to provide a potential approach for I κ B α M transgene-based immunotherapy ofasthma in the future.Part I Inhibition of nuclear factor κ B activation through induction of I κ B a expression by arsenic tri...