Obesity exacerbates influenza-induced respiratory disease via the arachidonic acid-p38 MAPK pathway.

Obesity is a risk factor for severe influenza, and asthma exacerbations caused by respiratory viral infections. We investigated mechanisms that increase the severity of airway disease related to influenza in obesity using cells derived from obese and lean individuals, and in vitro and in vivo models. Primary human nasal epithelial cells (pHNECs) derived from obese compared with lean individuals developed increased inflammation and injury in response to influenza A virus (IAV). Obese mice infected with influenza developed increased airway inflammation, lung injury and elastance, but had a decreased interferon response, compared with lean mice. Lung arachidonic acid (AA) levels increased in obese mice infected with IAV; arachidonic acid increased inflammatory cytokines and injury markers in response to IAV in human bronchial epithelial (HBE) cells. Obesity in mice, and AA in HBE cells, increased activation of p38 MAPK signaling following IAV infection; inhibiting this pathway attenuated inflammation, injury and tissue elastance responses, and improved survival. In summary, obesity increases disease severity in response to influenza infection through activation of the p38 MAPK pathway in response to altered arachidonic acid signaling.

Diet-induced obesity worsens allergen-induced type 2/type 17 inflammation in airways by enhancing DUOX1 activation.

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.

Mitoquinone mesylate attenuates pathological features of lean and obese allergic asthma in mice.

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.

Macrophage-intrinsic DUOX1 contributes to type 2 inflammation and mucus metaplasia during allergic airway disease.

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.

Oxidation-Dependent Activation of Src Kinase Mediates Epithelial IL-33 Production and Signaling during Acute Airway Allergen Challenge.

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 synthesis of OspD3 (ShET2) in Shigella flexneri is independent of OspC1.

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.

Defining the phylogenomics of Shigella species: a pathway to diagnostics.

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.

Characterization of intracellular growth regulator icgR by utilizing transcriptomics to identify mediators of pathogenesis in Shigella flexneri.

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 intracellular growth regulator. 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 ΔicgR 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.

Quantitative PCR for detection of Shigella improves ascertainment of Shigella burden in children with moderate-to-severe diarrhea in low-income countries.

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 <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the 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 <2.9 × 10(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.

Draft genome sequences of the diarrheagenic Escherichia coli collection.

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.

Vaccine-induced intestinal immunity to ricin toxin in the absence of secretory IgA.

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.