2012 Annual Report
1a.Objectives (from AD-416):
This project proposes rodent models to provide molecular, genetic, and functional information to address the effects of nutrient requirements on mucosal immune responses to infectious pathogens, and pig models to provide physiologically relevant comparisons to human allergy and responses to probiotic bacteria. Common features are the use of targeted gene expression probes to elucidate innate and acquired immunity to both probiotic and pathogenic bacteria that activate Th1 responses, and allergies and worm infections that activate Th2 responses. The goal is to reveal interactions between dietary micronutrients and food components that modulate immune responses to food allergens, micro and macrobiotic organisms, and their products. Objective 1: To elucidate the role of vitamin A (VA) on the phenotype and function of alternatively activated macrophages and T regulatory cells, and identify macrophage-mediated modulation of localized nutrient delivery/partitioning in porcine models of allergy. Objective 2: To elucidate the mechanisms used by probiotic bacteria to improve respiratory and intestinal mucosal responses to allergens, and correlate intestinal microflora composition of pigs and humans with biomarkers of allergic and intestinal disease. Objective 3: To elucidate the mechanisms by which micronutrients affect gut physiology and immune competence in response to food-borne illness due to viruses, bacteria, and gastrointestinal parasites.
1b.Approach (from AD-416):
Studies will evaluate if pigs can be sensitized to peanut (PN) allergens by different routes of mucosal exposure without cholera toxin and orally challenge with over-the-counter unsalted dry-roasted PN; if Vitamin A (VA) via all-trans retinoic acid (ATRA) can exacerbate allergic disease via stimulation of Th2 dependent pathways at low doses of antigen; if alternatively activated macrophages (AAM) express retinal and retinol dehydrogenases leading to increased ATRA generation in vitro and in vivo; and if CD209 is a receptor for PN and parasite antigens that mediates functional polarization of AAM accompanied by generation of ATRA. Additional work will test if probiotic bacteria protect against allergy, and if changes in intestinal microflora in children affect the incidence of allergy and intestinal disorders such as chronic diarrhea. Finally, it will be determined if selenium (Se) deficiency impairs AAM function in a helminth-parasite infection model in mice, if chronic Se deficiency or genetic deficiencies in selenoprotein expression in immune cells or intestinal tissue alter immunity and pathology associated with Citrobacter rodentium; and if vitamin A status will alter gastrointestinal immunity to C. rodentium and Heligmosomoides polygyrus in mice.
Alveolar macrophages (AM) normally make up greater than 90% of the cells in the airspaces of the lung and are the first line of defense against airborne particles and pathogens that contribute to lung health and disease. Information on the biology and function of these cells comes largely from studies in mice with little comparable information available for pigs and humans. To address this, we studied basal gene expression in porcine AM by Illumina-based next generation sequencing technology. More than 200 new, full, or partial length porcine genes were cloned from this project including many key immune regulatory molecules and splice variants. Real time PCR results confirm the deep sequencing data with a correlation of 0.999. Correlations of gene expression among animal samples were greater than 0.90 including both biological and technical duplicates. Results from pathways and bio-function analysis were consistent with the biological roles of AM defined in the mouse.
DGIL scientists continue to enhance a database on genes and proteins related to those prominently studied in humans and mice. The database now contains 4,545 entries. New datafields were added to accommodate the unit’s new focus on epigenomics including descriptions of the promoter region of porcine genes and porcine micro RNAs. This will enhance the ability to look for regulatory regions that may be conserved among different species. As part of an international consortium, they annotated over 1,400 genes to provide the first comprehensive description of the portion of the pig genome involved in the immune response. These efforts, to be published in Nature, revealed an overwhelming similarity of pigs to humans, further validating their use as a biomedical model.
To establish safety and tolerability of probiotic interventions in humans fecal samples and whole blood have been collected from patients fed probiotic bacteria, Lactobacillus rhamnossus (LGG) or Bifidobacterium lactis. Measurement of probiotic colonization have been tracked by relative quantification of probiotic bacteria in fecal samples using specific single copy gene assays. The complete change in transcriptome of RNA from whole blood from probiotic treatment are being evaluated by Illumina-based transcriptomic analysis.
Mice that lack expression of selenoproteins in macrophages did not differ from normal mice infected with Citrobacter rodentium bacteria (Cr) or the parasitic nematode Heligmosomoides bakeri (Hb). This suggested that a selenium dependent defect in macrophages may not be responsible for the altered immune response to Hb as originally hypothesized. Similarly, glutathione peroxidase (GPX) isozyme 1 or 2 knockout (KO) mice had unaltered immune responses during primary and secondary Hb infection infections. In contrast, preliminary data indicated that GPX2KO mice had a higher colonic Cr burden than WT mice. Additional studies will be conducted with GPX1KO and GPX2KO to define the role of these selenoproteins in resistance to gastrointestinal bacterial infections.
Allergens from parasite worms mimic allergens that induce hypersensitivity. The study investigated the cross-reactivity between a major glutathione-S transferase allergen of cockroach (Bla g.
Probiotic worms alter the pig intestinal microbiota and improve function. The parasitic whipworm of pigs has been used therapeutically to treat humans with inflammatory bowel disease and other autoimmune diseases. The intestinal microbiota is thought to modulate intestinal disease and this concept was tested in a pig model. Major changes in the composition of bacterial populations in the intestine were detected by whole genome sequencing techniques following whipworm infection. There were significant differences in the bacterial populations that are associated with the worm burden. The host response in the intestine of pigs that were resistant to whipworm was enriched for protective gene expression patterns that were also associated with worm clearance and improved tissue healing and function. Other studies using mouse models have been used to determine the cellular populations in the intestine that are induced by worm infection to regulate healing of injured intestinal tissues. These findings should facilitate development of strategies for parasitic control in pigs and humans, and could optimize the successful application of helminth (worm) therapy to reduce inflammation during autoimmune diseases in humans. The application of helminth therapy is currently part of a multi-center NIH-funded trail of safety and efficacy. The work enhances the understanding of how the intestinal microbiota affects disease outcome.
5)and the glutathione-S transferase of a human filarial parasite Wuchereria bancrofti (WbGST) and a mouse gastrointestinal parasite Heligmosomoides bakeri (HbGST). The molecular and structural similarities between Bla g 5 and WbGST were analyzed in silico by linear epitope mapping. The levels of IgE, IgG, and IgG4 antibodies were measured in filarial-infected and filarial uninfected subjects. Mice infected with H. bakeri were skin-tested for cross-reactive allergic responses and showed a immediate-type skin reactivity to Bla g 5. These studies demonstrated that worm parasites can enhance allergic reactions to common environmental allergens, and that conflicting reports of worm-induced reductions in allergic reactions may be related to the immune modulating capacity of persistent worm infection.
The pattern of gene expression exhibited by macrophages exposed to ATRA led us to test the hypothesis that VA may be involved in responses to hypoxia and glucose deprivation. A glial cell line used to model stroke exhibits increased reactive oxygen species/reactive nitrogen species (ROS/RNS), depolarization of the inner mitochondrial membrane potential increased cell swelling and increased intracellular calcium when deprived of oxygen and glucose. All-trans retinoic acid prevented all of these hallmark features of stroke. These studies indicate that VA has the potential to reduce brain edema and associated neural damage in ischemic injury.
Diarrhea is a leading cause of mortality and morbidity in children less than five years of age in impoverished regions of the world. We compared the fecal microbiota of healthy children to children with clinical diarrhea in a population from a tropical highland in Colombia, South America. Our results indicated that the composition of the fecal microbiota was affected by host demographic factors: age, health status, location, and gender. The relative abundance of Bifidobacterium species was inversely correlated with incidence of diarrhea, particularly rotavirus, while certain Lactobacillus species were directly correlated with clinical diarrhea regardless of location. Delivery of Bifidobacterium species or a diet rich in bifidogenic components that promote growth of Bifidobacterium species could provide a prophylactic effect to ameliorate the impact of diarrhea in children at risk.
Selenium deficient mice failed to expel the gastrointestinal nematode parasite Heligmosomoides bakeri and had reduced expression of multiple Th2-related genes including resistin-like-beta. Resistin-like-beta is known to play a key role in H. bakeri expulsion by interfering with worm feeding and reducing metabolic ATP levels in the worm. Selenium deficient mice had lower levels of resistin-like-beta protein in the intestinal mucosa and worms from these mice had higher ATP levels than those from selenium adequate mice. These results indicated that resistin-like-beta is modulated by selenium levels and contributes to protective immunity to worm infection. Similar parasitic worms that infect humans are a significant world-wide public health problem.
VA deficiency dramatically alters host resistance to a natural mouse pathogen that mimics many aspects of food-borne E. coli infections in humans. However, we have demonstrated the timing of VA repletion to deficient mice with respect to disease onset may actually exacerbate infections. Repletion of VA via supplementation is very common in underdeveloped areas of the world. Although overall mortality is decreased by such repletion, several studies have shown age-dependent effects and repletion of some groups with VA actually increases disease morbidity. Our studies suggest that both positive and negative effects could result from VA repletion if coincident with existing infections.
Wu, S., Li, R.W., Li, W., Urban Jr, J.F., Dawson, H.D., Beshah, E. 2012. Worm burden-dependent disruption of the porcine colon microbiota by Trichuris suis infection. PLoS One. 7(4):e35470.
Li, R.W., Wu, S., Li, W., Hill, D.E., Urban, Jr., J.F., Navarro, K., Couch, R.D. 2012. Alterations in the Porcine Colon Microbiota Induced by the Gastrointestinal Nematode Trichuris suis. Infection and Immunity. 80(6):2150-2157.
Chen, F., Liu, Z., Rozo, C., Wu, W., Bowdridge, S., Wynn, T., Urban Jr, J.F., Gauge, W. 2012. An essential role for TH2-type response in limiting acute tissue damage during experimental helminth infection.. Nature Medicine. 18(2):260-6.
Santiago, H.C., Leevan, E., Bennuru, S., Ribeiro-Gomes, F., Mueller, E., Wilson, M., Wynn, T., Garboczi, D., Urban Jr, J.F., Nutman, T.B. 2012. Molecular mimicry between cockroach and helminth glutathione S-transferases promotes cross-reactivity and cross-sensitization. Journal of Allergy Clinical Immunology. 130:248-256.
Hang, L., Setiawan, T., Blum, A., Urban Jr, J.F., Stoyanoff, K., Reinecker, H.C., Weinstock, J. 2010. Helogmosomoides polygyrus infection can inhibit colitis through direct interaction with innate immunity. Journal of Immunology. 185(6):3184-9.
Kvist, P.H., Iburg, T., Dawson, H.D., Jensen, H.E. 2010. Effect of subcutaneous glucose sensor implantation on skin mRNA expression in pigs. Diabetes Technology & Theraputics. 10:791-799.
Bhagwat, A.A., Kannan, P., Leow, Y., Dharne, M., Smith, A.D. 2012. Role of anionic charges of osmoregulated periplasmic glucans of Salmonella enterica Serovar Typhimurium SL1344 in mice virulence. Archives Of Microbiology. 194(6):541-548.