Pollen exposure induces allergic airway inflammation in sensitized subjects. The role of antigenic pollen proteins in the induction of allergic airway inflammation is well characterized, but the contribution of other constituents in pollen grains to this process is unknown. Here we show that pollen grains and their extracts contain intrinsic NADPH oxidases. The pollen NADPH oxidases rapidly increased the levels of ROS in lung epithelium as well as the amount of oxidized glutathione (GSSG) and 4-hydroxynonenal (4-HNE) in airway-lining fluid. These oxidases, as well as products of oxidative stress (such as GSSG and 4-HNE) generated by these enzymes, induced neutrophil recruitment to the airways independent of the adaptive immune response. Removal of pollen NADPH oxidase activity from the challenge material reduced antigen-induced allergic airway inflammation, the number of mucin-containing cells in airway epithelium, and antigen-specific IgE levels in sensitized mice. Furthermore, challenge with Amb a 1, the major antigen in ragweed pollen extract that does not possess NADPH oxidase activity, induced low-grade allergic airway inflammation. Addition of GSSG or 4-HNE to Amb a 1 challenge material boosted allergic airway inflammation. We propose that oxidative stress generated by pollen NADPH oxidases (signal 1) augments allergic airway inflammation induced by pollen antigen (signal 2).

The prevalence of allergies and asthma among the world’s population has been steadily increasing due to environmental factors. It has been described that exposure to ozone, diesel exhaust particles, or tobacco smoke exacerbates allergic inflammation in the lungs. These environmental oxidants increase the levels of cellular reactive oxygen species (ROS) and induce mitochondrial dysfunction in the airway epithelium. In this study, we investigated the involvement of preexisting mitochondrial dysfunction in the exacerbation of allergic airway inflammation. After cellular oxidative insult induced by ragweed pollen extract (RWE) exposure, we have identified nine oxidatively damaged mitochondrial respiratory chain-complex and associated proteins. Out of these, the ubiquinol-cytochrome c reductase core II protein (UQCRC2) was found to be implicated in mitochondrial ROS generation from respiratory complex III. Mitochondrial dysfunction induced by deficiency of UQCRC2 in airway epithelium of sensitized BALB/c mice prior the RWE challenge increased the Ag-induced accumulation of eosinophils, mucin levels in the airways, and bronchial hyperresponsiveness. Deficiency of UQCRC1, another oxidative damage-sensitive complex III protein, did not significantly alter cellular ROS levels or the intensity of RWE-induced airway inflammation. These observations suggest that preexisting mitochondrial dysfunction induced by oxidant environmental pollutants is responsible for the severe symptoms in allergic airway inflammation. These data also imply that mitochondrial defects could be risk factors and may be responsible for severe allergic disorders in atopic individuals.

A large percentage of redox-responsive gene promoters contain evolutionarily conserved guanine-rich clusters; guanines are the bases most susceptible to oxidative modification(s). Consequently, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most abundant base lesions in promoters and is primarily repaired via the 8-oxoguanine DNA glycosylase-1 (OOG1)-initiated base excision repair pathway. In view of a prompt cellular response to oxidative challenge, we hypothesized that the 8-oxoG lesion and the cognate repair protein OGG1 are utilized in transcriptional gene activation. Here, we document TNFα-induced enrichment of both 8-oxoG and OGG1 in promoters of pro-inflammatory genes, which precedes interaction of NF-κB with its DNA-binding motif. OGG1 bound to 8-oxoG upstream from the NF-κB motif increased its DNA occupancy by promoting an on-rate of both homodimeric and heterodimeric forms of NF-κB. OGG1 depletion decreased both NF-κB binding and gene expression, whereas Nei-like glycosylase-1 and -2 had a marginal effect. These results are the first to document a novel paradigm wherein the DNA repair protein OGG1 bound to its substrate is coupled to DNA occupancy of NF-κB and functions in epigenetic regulation of gene expression.

Among the insidious DNA base lesions, 8-oxo-7,8-dihydroguanine (8-oxoG) is one of the most abundant, a lesion that arises through the attack by reactive oxygen species on guanine, especially when located in cis-regulatory elements. 8-oxoG is repaired by the 8-oxoguanine glycosylase 1 (OGG1)–initiated DNA base excision repair pathway. In this study, we investigated whether 8-oxoG repair by OGG1 in promoter regions is compatible with a prompt gene expression and a host innate immune response. For this purpose, we used a mouse model of airway inflammation, supplemented with cell cultures, chromatin immunoprecipitation, small interfering RNA knockdown, real-time PCR, and comet and reporter transcription assays. Our data show that exposure of cells to TNF-α altered cellular redox, increased the 8-oxoG level in DNA, recruited OGG1 to promoter sequences, and transiently inhibited base excision repair of 8-oxoG. Promoter-associated OGG1 then enhanced NF-κB/RelA binding to cis-elements and facilitated recruitment of specificity protein 1, transcription initiation factor II-D, and p-RNA polymerase II, resulting in the rapid expression of chemokines/cytokines and inflammatory cell accumulation in mouse airways. Small interfering RNA depletion of OGG1 or prevention of guanine oxidation significantly decreased TNF-α–induced inflammatory responses. Taken together, these results show that nonproductive binding of OGG1 to 8-oxoG in promoter sequences could be an epigenetic mechanism to modulate gene expression for a prompt innate immune response.

Pollen is known to induce allergic asthma in atopic individuals, although only a few inhaled pollen grains penetrate into the lower respiratory tract. We sought to provide evidence that subpollen particles (SPPs) of respirable size, possessing both antigenic and redox properties, are released from weed pollen grains and to test their role in allergic airway inflammation. The release of SPPs was analyzed by means of microscopic imaging and flow cytometry. The redox properties of SPPs and the SPP-mediated oxidative effect on epithelial cells were determined by using redox-sensitive probes and specific inhibitors. Western blotting and amino acid sequence analysis were used to examine the protein components of the SPP. The allergenic properties of the SPP were determined in a murine model of experimental asthma. Ragweed pollen grains released 0.5 to 4.5 microm of SPPs on hydration. These contained Amb a 1, along with other allergenic proteins of ragweed pollen, and possessed nicotinamide adenine dinucleotide (reduced) or nicotinamide adenine dinucleotide phosphate (reduced) [NAD(P)H] oxidase activity. The SPPs significantly increased the levels of reactive oxygen species (ROS) in cultured cells and induced allergic airway inflammation in the experimental animals. Pretreatment of the SPPs with NAD(P)H oxidase inhibitors attenuated their capacity to increase ROS levels in the airway epithelial cells and subsequent airway inflammation. The allergenic potency of SPPs released from ragweed pollen grains is mediated in tandem by ROS generated by intrinsic NAD(P)H oxidases and antigenic proteins. Severe clinical symptoms associated with seasonal asthma might be explained by immune responses to inhaled SPPs carrying allergenic proteins and ROS-producing NAD(P)H oxidases.

8-Oxo-7,8-dihydroguanine (8-oxoG), arguably the most abundant base lesion induced in mammalian genomes by reactive oxygen species, is repaired via the base excision repair pathway that is initiated with the excision of 8-oxoG by OGG1. Here we show that OGG1 binds the 8-oxoG base with high affinity and that the complex then interacts with canonical Ras family GTPases to catalyze replacement of GDP with GTP, thus serving as a guanine nuclear exchange factor. OGG1-mediated activation of Ras leads to phosphorylation of the mitogen-activated kinases MEK1,2/ERK1,2 and increasing downstream gene expression. These studies document for the first time that in addition to its role in repairing oxidized purines, OGG1 has an independent guanine nuclear exchange factor activity when bound to 8-oxoG.