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More than 50% of people with asthma in the United States are obese, and obesity often worsens symptoms of allergic asthma and impairs response to treatment. Based on previously established roles of the epithelial NADPH oxidase DUOX1 in allergic airway inflammation, we addressed the potential involvement of DUOX1 in altered allergic inflammation in the context of obesity. Intranasal house dust mite (HDM) allergen challenge of subjects with allergic asthma induced rapid secretion of IL-33, then IL-13, into the nasal lumen, responses that were significantly enhanced in obese asthmatic subjects (BMI >30). Induction of diet-induced obesity (DIO) in mice by high-fat diet (HFD) feeding similarly enhanced acute airway responses to intranasal HDM challenge, particularly with respect to secretion of IL-33 and type 2/type 3 cytokines, and this was associated with enhanced epithelial DUOX1 expression and was avoided in DUOX1-deficient mice. DIO also enhanced DUOX1-dependent features of chronic HDM-induced allergic inflammation. Although DUOX1 did not affect overall weight gain by HFD feeding, it contributed to glucose intolerance, suggesting a role in glucose metabolism. However, glucose intolerance induced by short-term HFD feeding, in the absence of adiposity, was not sufficient to alter HDM-induced acute airway responses. DIO was associated with enhanced presence of the adipokine leptin in the airways, and leptin enhanced DUOX1-dependent IL-13 and mucin production in airway epithelial cells. In conclusion, augmented inflammatory airway responses to HDM in obesity are associated with increases in airway epithelial DUOX1, and by increased airway epithelial leptin signaling.

Obesity is associated with severe, difficult-to-control asthma, and increased airway oxidative stress. Mitochondrial reactive oxygen species (mROS) are an important source of oxidative stress in asthma, leading us to hypothesize that targeting mROS in obese allergic asthma might be an effective treatment. Using a mouse model of house dust mite (HDM)-induced allergic airway disease in mice fed a low- (LFD) or high-fat diet (HFD), and the mitochondrial antioxidant MitoQuinone (MitoQ), we investigated the effects of obesity and ROS on HDM-induced airway inflammation, remodeling, and airway hyperresponsiveness (AHR). Obese allergic mice showed increased lung tissue eotaxin, airway tissue eosinophilia, and AHR compared with lean allergic mice. MitoQ reduced airway inflammation, remodeling, and hyperreactivity in both lean and obese allergic mice, and tissue eosinophilia in obese-allergic mice. Similar effects were observed with decyl triphosphonium (dTPP+), the hydrophobic cationic moiety of MitoQ lacking ubiquinone. HDM-induced oxidative sulfenylation of proteins was increased particularly in HFD mice. Although only MitoQ reduced sulfenylation of proteins involved in protein folding in the endoplasmic reticulum (ER), ER stress was attenuated by both MitoQ and dTPP+ suggesting the anti-allergic effects of MitoQ are mediated in part by effects of its hydrophobic dTPP+ moiety reducing ER stress. In summary, oxidative signaling is an important mediator of allergic airway disease. MitoQ, likely through reducing protein oxidation and affecting the UPR pathway, might be effective for the treatment of asthma and specific features of obese asthma.

The respiratory epithelium forms the first line of defense against inhaled pathogens and acts as an important source of innate cytokine responses to environmental insults. One critical mediator of these responses is the IL-1 family cytokine IL-33, which is rapidly secreted upon acute epithelial injury as an alarmin and induces type 2 immune responses. Our recent work highlighted the importance of the NADPH oxidase dual oxidase 1 (DUOX1) in acute airway epithelial IL-33 secretion by various airborne allergens associated with H2O2 production and reduction-oxidation–dependent activation of Src kinases and epidermal growth factor receptor (EGFR) signaling. In this study, we show that IL-33 secretion in response to acute airway challenge with house dust mite (HDM) allergen critically depends on the activation of Src by a DUOX1-dependent oxidative mechanism. Intriguingly, HDM-induced epithelial IL-33 secretion was dramatically attenuated by small interfering RNA– or Ab-based approaches to block IL-33 signaling through its receptor IL1RL1 (ST2), indicating that HDM-induced IL-33 secretion includes a positive feed-forward mechanism involving ST2-dependent IL-33 signaling. Moreover, activation of type 2 cytokine responses by direct airway IL-33 administration was associated with ST2-dependent activation of DUOX1-mediated H2O2 production and reduction-oxidation–based activation of Src and EGFR and was attenuated in Duox1−/− and Src+/− mice, indicating that IL-33–induced epithelial signaling and subsequent airway responses involve DUOX1/Src–dependent pathways. Collectively, our findings suggest an intricate relationship between DUOX1, Src, and IL-33 signaling in the activation of innate type 2 immune responses to allergens, involving DUOX1-dependent epithelial Src/EGFR activation in initial IL-33 secretion and in subsequent IL-33 signaling through ST2 activation.

The NADPH oxidase DUOX1 contributes to epithelial production of alarmins, including interleukin (IL)-33, in response to injurious triggers such as airborne protease allergens, and mediates development of mucus metaplasia and airway remodeling in chronic allergic airways diseases. DUOX1 is also expressed in non-epithelial lung cell types, including macrophages that play an important role in airway remodeling during chronic lung disease. We therefore conditionally deleted DUOX1 in either lung epithelial or monocyte/macrophage lineages to address its cell-specific actions in innate airway responses to acute airway challenge with house dust mite (HDM) allergen, and in chronic HDM-driven allergic airway inflammation. As expected, acute responses to airway challenge with HDM, as well as type 2 inflammation and related features of airway remodeling during chronic HDM-induced allergic inflammation, were largely driven by DUOX1 with the respiratory epithelium. However, in the context of chronic HDM-driven inflammation, DUOX1 deletion in macrophages also significantly impaired type 2 cytokine production and indices of mucus metaplasia. Further studies revealed a contribution of macrophage-intrinsic DUOX1 in macrophage recruitment upon chronic HDM challenge, as well as features of macrophage activation that impact on type 2 inflammation and remodeling.

Shigella flexneri is a Gram-negative pathogen that invades the colonic epithelium and causes millions of cases of watery diarrhea or bacillary dysentery predominately in children under the age of 5 years in developing countries. The effector Shigella enterotoxin 2 (ShET2), or OspD3, is encoded by the sen or ospD3 gene on the virulence plasmid. Previous literature has suggested that ospD3 is in an operon downstream of the ospC1 gene, and expression of both genes is controlled by a promoter upstream of ospC1. Since the intergenic region is 328 bases in length and contains several putative promoter regions, we hypothesized the genes are independently expressed. Here we provide data that ospD3 and ospC1 are not co-transcribed and that OspC1 is not required for OspD3/ShET2 function. Most importantly, we identified strong promoter activity in the intergenic region and demonstrate that OspD3/ShET2 can be expressed and secreted independently of OspC1. This work increases our understanding of the synthesis of a unique virulence factor and provides further insights into Shigella pathogenesis.

Shigellae cause significant diarrheal disease and mortality in humans, as there are approximately 163 million episodes of shigellosis and 1.1 million deaths annually. While significant strides have been made in the understanding of the pathogenesis, few studies on the genomic content of the Shigella species have been completed. The goal of this study was to characterize the genomic diversity of Shigella species through sequencing of 55 isolates representing members of each of the four Shigella species: S. flexneri , S. sonnei , S. boydii , and S. dysenteriae . Phylogeny inferred from 336 available Shigella and Escherichia coli genomes defined exclusive clades of Shigella ; conserved genomic markers that can identify each clade were then identified. PCR assays were developed for each clade-specific marker, which was combined with an amplicon for the conserved Shigella invasion antigen, IpaH3, into a multiplex PCR assay. This assay demonstrated high specificity, correctly identifying 218 of 221 presumptive Shigella isolates, and sensitivity, by not identifying any of 151 diverse E. coli isolates incorrectly as Shigella . This new phylogenomics-based PCR assay represents a valuable tool for rapid typing of uncharacterized Shigella isolates and provides a framework that can be utilized for the identification of novel genomic markers from genomic data.

Shigella species Gram-negative bacteria which cause a diarrheal disease, known as shigellosis, by invading and destroying the colonic mucosa and inducing a robust inflammatory response. With no vaccine available, shigellosis annually kills over 600,000 children in developing countries. This study demonstrates the utility of combining high-throughput bioinformatic methods with in vitro and in vivo assays to provide new insights into pathogenesis. Comparisons of in vivo and in vitro gene expression identified genes associated with intracellular growth. Additional bioinformatics analyses identified genes that are present in S. flexneri isolates but not in the three other Shigella species. Comparison of these two analyses revealed nine genes that are differentially expressed during invasion and that are specific to S. flexneri . One gene, a DeoR family transcriptional regulator with decreased expression during invasion, was further characterized and is now designated icgR , for i ntra c ellular g rowth r egulator. Deletion of icgR caused no difference in growth in vitro but resulted in increased intracellular replication in HCT-8 cells. Further in vitro and in vivo studies using high-throughput sequencing of RNA transcripts (RNA-seq) of an isogenic Δ icgR mutant identified 34 genes that were upregulated under both growth conditions. This combined informatics and functional approach has allowed the characterization of a gene and pathway previously unknown in Shigella pathogenesis and provides a framework for further identification of novel virulence factors and regulatory pathways.

The RNA N-glycosidase ribosome inactivating proteins (RIPs) constitute a ubiquitous family of plant- and bacterium-derived toxins that includes the category B select agents ricin, abrin and shiga toxin. While these toxins are potent inducers of intestinal epithelial cell death and inflammation, very little is known about the mechanisms underlying mucosal immunity to these toxins. In the present study, we report that secretory IgA (SIgA) antibodies are not required for intestinal immunity to ricin, as evidenced by the fact that mice devoid of SIgA, due to a mutation in the polymeric immunoglobulin receptor, were impervious to the effects of intragastric toxin challenge following ricin toxoid immunization. Furthermore, parenteral administration of ricin-specific monoclonal IgGs, directed against either ricin's enzymatic subunit (RTA) or ricin's binding subunit (RTB), to wild type mice was as effective as monoclonal IgAs with comparable specificities in imparting intestinal immunity to ricin. These data are consistent with reports from others demonstrating that immunization of mice by routes known not to induce mucosal antibody responses (e.g., intramuscular and intradermal) is sufficient to elicit protection against both systemic and mucosal ricin challenges.

Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 10 4 ipaH copies per 100 ng of stool DNA for set 1 ( n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 10 4 ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella . Individuals with ≥2.9 × 10 4 ipaH copies have 5.6-times-higher odds of having diarrhea than those with 4 ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH , a 90% increase from the 172 cases detected by culture in both samples. Among a subset ( n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% ( n = 129) for culture to 17.6% ( n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 10 4 ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella .

Escherichia coli is a normal inhabitant of the healthy gut, but is also an important and widespread human and animal pathogen. E. coli has been associated with human infections including diarrhea, urinary tract infections, and meningitis. E. coli are designated into pathogenic variants (pathovars, also known as pathotypes) based largely on molecular markers associated with specific disease presentations. Many of these biomarkers are present on mobile genetic elements, which provide difficulties in accurate identification and surveillance of these pathogens. E. coli was one of the first bacteria to have the complete genome decoded and these organisms have been the focus of genomic studies for approximately the last 15 years. While early studies suggested the diversity of the species may be limited, the generation of multiple genomes rapidly identified significant genomic diversity. Whole genome sequence analysis has demonstrated significant genetic variability even within each pathovar. Furthermore, next generation sequencing has identified the distribution of genes across the E. coli species and provided insights into the evolution of the species. In the coming years, decreased sequencing costs are likely to positively affect the understanding of pathogenesis in this important human pathogen.

We report the draft genome sequences of the collection referred to as the Escherichia coli DECA collection, which was assembled to contain representative isolates of the 15 most common diarrheagenic clones in humans ( http://shigatox.net/new/ ). These genomes represent a valuable resource to the community of researchers who examine these enteric pathogens.