Bacterial Indole as a Multifunctional Regulator of Klebsiella oxytoca Complex Enterotoxicity.

Gastrointestinal microbes respond to biochemical metabolites that coordinate their behaviors. Here, we demonstrate that bacterial indole functions as a multifactorial mitigator of Klebsiella grimontii and Klebsiella oxytoca pathogenicity. These closely related microbes produce the enterotoxins tilimycin and tilivalline; cytotoxin-producing strains are the causative agent of antibiotic-associated hemorrhagic colitis and have been associated with necrotizing enterocolitis of premature infants. We demonstrate that carbohydrates induce cytotoxin synthesis while concurrently repressing indole biosynthesis. Conversely, indole represses cytotoxin production. In both cases, the alterations stemmed from differential transcription of npsA and npsB, key genes involved in tilimycin biosynthesis. Indole also enhances conversion of tilimycin to tilivalline, an indole analog with reduced cytotoxicity. In this context, we established that tilivalline, but not tilimycin, is a strong agonist of pregnane X receptor (PXR), a master regulator of xenobiotic detoxification and intestinal inflammation. Tilivalline binding upregulated PXR-responsive detoxifying genes and inhibited tubulin-directed toxicity. Bacterial indole, therefore, acts in a multifunctional manner to mitigate cytotoxicity by Klebsiella spp.: suppression of toxin production, enhanced conversion of tilimycin to tilivalline, and activation of PXR. IMPORTANCE The human gut harbors a complex community of microbes, including several species and strains that could be commensals or pathogens depending on context. The specific environmental conditions under which a resident microbe changes its relationship with a host and adopts pathogenic behaviors, in many cases, remain poorly understood. Here, we describe a novel communication network involving the regulation of K. grimontii and K. oxytoca enterotoxicity. Bacterial indole was identified as a central modulator of these colitogenic microbes by suppressing bacterial toxin (tilimycin) synthesis and converting tilimycin to tilivalline while simultaneously activating a host receptor, PXR, as a means of mitigating tissue cytotoxicity. On the other hand, fermentable carbohydrates were found to inhibit indole biosynthesis and enhance toxin production. This integrated network involving microbial, host, and metabolic factors provides a contextual framework to better understand K. oxytoca complex pathogenicity.

Cytotoxin-producing Klebsiella oxytoca in the preterm gut and its association with necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating intestinal inflammatory disease of premature infants associated with gut bacterial dysbiosis. Using 16S rRNA-based methods, our laboratory identified an unclassified Enterobacteriaceae sequence (NEC_unk_OTU) with high abundance in NEC fecal samples. We aimed to identify this bacterium and determine its potential role in the disease. NCBI database searches for the 16S sequence, selective culture systems, biotyping and polymerase chain reaction were employed to refine classification of NEC_unk_OTU and identify toxin-encoding genes from the index NEC case. Bacterial cytotoxin production was confirmed by mass spectrometry and apoptosis assays. Additional fecal samples from 9 NEC and 5 non-NEC controls were analyzed using similar methods and multi-locus sequence typing (MLST) was performed to investigate clonal relationships and define sequence types of the isolates. NEC_unk_OTU was identified as Klebsiella oxytoca, a pathobiont known to cause antibiotic-associated hemorrhagic colitis, but not previously linked to NEC. Including the index case, cytotoxin-producing strains of K. oxytoca were isolated from 6 of 10 subjects with NEC; in these, the K. oxytoca 16S sequence predominated the fecal microbiota. Cytotoxin-producing strains of K. oxytoca also were isolated from 4 of 5 controls; in these, however, the abundance of the corresponding 16S sequence was very low. MLST analysis of the toxin-positive isolates demonstrated no clonal relationships and similar genetic clustering between cases and controls. These results suggest cytotoxin-producing strains of K. oxytoca colonize a substantial proportion of premature infants. Some, perhaps many, cases of NEC may be precipitated by outgrowth of this opportunistic pathogen.

Long-term glycemic control with hepatic insulin gene therapy in streptozotocin-diabetic mice.

BACKGROUND: Insulin self-administration is burdensome and can produce dangerous hypoglycemia. Insulin gene therapy may improve and simplify the treatment of diabetes mellitus. In rats, metabolically responsive hepatic insulin gene therapy (HIGT) delivered by adenovirus normalizes random blood sugars but with a limited duration. To prolong glycemic control, we delivered a metabolically regulated insulin transgene by adeno-associated virus (AAV). METHODS: We administered increasing doses of self-complementary (SC), pseudotyped AAV8 expressing the (GlRE)3 BP1-2xfur insulin transgene to streptozotocin-diabetic CD-1 mice, and monitored blood sugar and body weight. We also compared responses to intraperitoneal glucose and chow withdrawal, assessed for viral genomes in liver by Southern blotting, and measured hepatic glycogen. RESULTS: Glucose lowering required the combination of SC genomes and AAV capsid pseudotyping. HIGT controlled glycemia in diabetic mice (DM) for > 1 year. However, glycemic responses were variable. Approximately 30% of mice succumbed to hypoglycemia, and approximately 30% of mice again became hyperglycemic. During an intraperitoneal glucose tolerance test, blood sugars declined to normal within 180 min in HIGT-treated DM compared to 90 min in control mice. Hypoglycemia was common among HIGT-treated mice during a 24-h fast. However, HIGT mice lost less weight than either diabetic or nondiabetic controls as a result of increased water intake. HIGT treatment reduced the hepatic glycogen content of fed mice. CONCLUSIONS: Our studies demonstrate the possibility for long-term glycemic correction following AAV-mediated HIGT in mice. However, the dose-response relationship is irregular, and metabolic responsiveness may be less than that observed in rats.

Transmission of murine cytomegalovirus in breast milk: a model of natural infection in neonates.

Vertical transmission of viruses in breast milk can expose neonates to infectious pathogens at a time when the capacity of their immune system to control infections is limited. We developed a mouse model to study the outcomes of acquisition of murine cytomegalovirus (MCMV) when neonates are breastfed by mothers with acute or latent infection. Breast milk leukocytes collected from lactating mice were examined for the presence of MCMV IE-1 mRNA by reverse transcription-PCR (RT-PCR) with Southern analysis. As determined by this criterion, breast milk leukocytes from both acute and latent mothers were positive for MCMV. This mimics the outcome seen in humans with latent cytomegalovirus infection, where reactivation of virus occurs specifically in the lactating mammary gland. Interestingly, intraperitoneal injection of breast milk collected from mothers with latent infection was sufficient to transfer MCMV to neonatal mice, demonstrating that breast milk was a source of virus. Furthermore, we found that MCMV was transmitted from infected mothers to breastfed neonates, with MCMV IE-1 mRNA or infectious virus present in multiple organs, including the brain. In fact, 1 day of nursing was sufficient to transmit MCMV from latent mothers to breastfed neonatal mice. Together, these data validate this mouse model of vertical transmission of MCMV from mothers with acute or latent MCMV infection to breastfed neonates. Its relevance to human disease should prove useful in future studies designed to elucidate the immunological and pathological ramifications of neonatal infection acquired via this natural route.

Airway epithelial indoleamine 2,3-dioxygenase inhibits CD4+ T cells during Aspergillus fumigatus antigen exposure.

Indoleamine 2,3-dioxygenase (IDO) suppresses the functions of CD4(+) T cells through its ability to metabolize the essential amino acid tryptophan. Although the activity of IDO is required for the immunosuppression of allergic airway disease by the Toll-Like-Receptor 9 (TLR9) agonist, oligonucleotides comprised of cytosine and guanine nucleotides linked by phosphodiester bonds (CpG) DNA, it is unclear whether IDO expression by resident lung epithelial cells is sufficient to elicit these effects. Therefore, we created a transgenic mouse inducibly overexpressing IDO within nonciliated airway epithelial cells. Upon inhalation of formalin-fixed Aspergillus fumigatus hyphal antigens, the overexpression of IDO from airway epithelial cells of these mice reduced the number of CD4(+) T cells within the inflamed lung and impaired the capacity of antigen-specific splenic CD4(+) effector T cells to secrete the cytokines IL-4, IL-5, IL-13, and IFN-γ. Despite these effects, allergic airway disease pathology was largely unaffected in mice expressing IDO in airway epithelium. In support of the concept that dendritic cells are the major cell type contributing to the IDO-inducing effects of CpG DNA, mice expressing TLR9 only in the airway epithelium did not augment IDO expression subsequent to the administration of CpG DNA. Furthermore, the systemic depletion of CD11c(+) cells rendered mice incapable of CpG DNA-induced IDO expression. Our results demonstrate that an overexpression of IDO within the airway epithelium represents a novel mechanism by which the number of CD4(+) T cells recruited to the lung and their capacity to produce cytokines can be diminished in a model of allergic airway disease, and these results also highlight the critical role of dendritic cells in the antiasthmatic effects of IDO induction by CpG DNA.

NO2 inhalation induces maturation of pulmonary CD11c+ cells that promote antigenspecific CD4+ T cell polarization.

BACKGROUND: Nitrogen dioxide (NO2) is an air pollutant associated with poor respiratory health, asthma exacerbation, and an increased likelihood of inhalational allergies. NO2 is also produced endogenously in the lung during acute inflammatory responses. NO2 can function as an adjuvant, allowing for allergic sensitization to an innocuous inhaled antigen and the generation of an antigen-specific Th2 immune response manifesting in an allergic asthma phenotype. As CD11c+ antigen presenting cells are considered critical for naïve T cell activation, we investigated the role of CD11c+ cells in NO2-promoted allergic sensitization. METHODS: We systemically depleted CD11c+ cells from transgenic mice expressing a simian diphtheria toxin (DT) receptor under of control of the CD11c promoter by administration of DT. Mice were then exposed to 15 ppm NO2 followed by aerosolized ovalbumin to promote allergic sensitization to ovalbumin and were studied after subsequent inhaled ovalbumin challenges for manifestation of allergic airway disease. In addition, pulmonary CD11c+ cells from wildtype mice were studied after exposure to NO2 and ovalbumin for cellular phenotype by flow cytometry and in vitro cytokine production. RESULTS: Transient depletion of CD11c+ cells during sensitization attenuated airway eosinophilia during allergen challenge and reduced Th2 and Th17 cytokine production. Lung CD11c+ cells from wildtype mice exhibited a significant increase in MHCII, CD40, and OX40L expression 2 hours following NO2 exposure. By 48 hours, CD11c+MHCII+ DCs within the mediastinal lymph node (MLN) expressed maturation markers, including CD80, CD86, and OX40L. CD11c+CD11b- and CD11c+CD11b+ pulmonary cells exposed to NO2 in vivo increased uptake of antigen 2 hours post exposure, with increased ova-Alexa 647+ CD11c+MHCII+ DCs present in MLN from NO2-exposed mice by 48 hours. Co-cultures of ova-specific CD4+ T cells from naïve mice and CD11c+ pulmonary cells from NO2-exposed mice produced IL-1, IL-12p70, and IL-6 in vitro and augmented antigen-induced IL-5 production. CONCLUSIONS: CD11c+ cells are critical for NO2-promoted allergic sensitization. NO2 exposure causes pulmonary CD11c+ cells to acquire a phenotype capable of increased antigen uptake, migration to the draining lymph node, expression of MHCII and co-stimulatory molecules required to activate naïve T cells, and secretion of polarizing cytokines to shape a Th2/Th17 response.

Nitrogen dioxide promotes allergic sensitization to inhaled antigen.

Allergen sensitization and allergic airway disease are likely to come about through the inhalation of Ag with immunostimulatory molecules. However, environmental pollutants, including nitrogen dioxide (NO2), may promote adaptive immune responses to innocuous Ags that are not by themselves immunostimulatory. We tested in C57BL/6 mice whether exposure to NO2, followed by inhalation of the innocuous protein Ag, OVA, would result in allergen sensitization and the subsequent development of allergic airway disease. Following challenge with aerosolized OVA alone, mice previously exposed via inhalation to NO2 and OVA developed eosinophilic inflammation and mucus cell metaplasia in the lungs, as well as OVA-specific IgE and IgG1, and Th2-type cytokine responses. One hour of exposure to 10 parts per million NO2 increased bronchoalveolar lavage fluid levels of total protein, lactate dehydrogenase activity, and heat shock protein 70; promoted the activation of NF-kappaB by airway epithelial cells; and stimulated the subsequent allergic response to Ag challenge. Furthermore, features of allergic airway disease were not induced in allergen-challenged TLR2-/- and MyD88-/- mice exposed to NO2 and aerosolized OVA during sensitization. These findings offer a mechanism whereby allergen sensitization and asthma may result under conditions of high ambient or endogenous NO2 levels.

The tick salivary protein, Salp15, inhibits the development of experimental asthma.

Activation of Th2 CD4(+) T cells is necessary and sufficient to elicit allergic airway disease, a mouse model with many features of human allergic asthma. Effectively controlling the activities of these cells could be a panacea for asthma therapy. Blood-feeding parasites have devised remarkable strategies to effectively evade the immune response. For example, ticks such as Ixodes scapularis, which must remain on the host for up to 7 days to feed to repletion, secrete immunosuppressive proteins. Included among these proteins is the 15-kDa salivary protein Salp15, which inhibits T cell activation and IL-2 production. Our objective for these studies was to evaluate the T cell inhibitory properties of Salp15 in a mouse model of allergic asthma. BALB/cJ mice were Ag sensitized by i.p. injection of OVA in aluminum hydroxide, with or without 50 mug of Salp15, on days 0 and 7. All mice were challenged with aerosolized OVA on days 14-16 and were studied on day 18. Compared with control mice sensitized with Ag, mice sensitized with Ag and Salp15 displayed significantly reduced airway hyperresponsiveness, eosinophilia, Ag-specific IgG1 and IgE, mucus cell metaplasia, and Th2 cytokine secretion in vivo and by CD4(+) T cells restimulated with Ag in vitro. Our results demonstrate that Salp15 can effectively prevent the generation of a Th2 immune response and the development of experimental asthma. These studies, and those of others, support the notion that a lack of ectoparasitism may contribute to the increasing prevalence of allergic asthma.

Host cells reduce glucose uptake and glycogen deposition in response to hepatic insulin gene therapy.

BACKGROUND: Hepatic insulin gene therapy (HIGT) restores weight gain and near-normal glycemia in rodent models of insulin-deficient diabetes mellitus. However, the effect of transgenic insulin on endogenous genes and recipient cell function is relatively unexplored. To investigate hepatocellular effects of transgenic insulin expression, we evaluated intermediary glucose metabolism in primary cultured hepatocytes treated with HIGT. METHODS: Rat hepatocytes were transduced with adenovirus expressing a glucose-responsive human insulin transgene and cultured in high-glucose and high-insulin conditions. We determined glycogen content in cell cultures and intact liver directly. Glycogenolysis was compared using glucose production of cultured cells. Glucose uptake, oxidative, and glycolytic processing were determined by radiotracer analysis or direct end-product assessment. Quantitative real-time reverse transcriptase polymerase chain reaction was used to determine expression of glucose transporter 2 (GLUT2) and glucokinase genes. GLUT2 protein abundance was determined by Western blot analysis. RESULTS: HIGT-treated hepatocytes contained significantly less glycogen than either untreated hepatocytes or those treated with an empty virus. Glucose release owing to glycogenolysis remained normal. However, HIGT treatment significantly impaired glucose uptake and processing. Metabolic synthetic processes were not generally inhibited, as indicated by enhanced beta-hydroxybutyrate secretion. While preserving cell viability, HIGT treatment diminished expression of both glucokinase and GLUT2. In HIGT-treated streptozocin-treated diabetic rats, total liver glycogen was intermediate between diabetic animals and normal controls. CONCLUSIONS: These results suggest gene-specific effects in recipient hepatocytes following HIGT treatment and underscore the need for expanded studies examining host cell responses to the transfer of metabolically active transgenes.

Glucose-responsive hepatic insulin gene therapy of spontaneously diabetic BB/Wor rats.

Hepatic insulin gene therapy (HIGT) ameliorates hyperglycemia in multiple rodent models of diabetes mellitus, with variable degrees of glucose control. We demonstrate here that adenoviral delivery of a glucose-regulated transgene into rat hepatocytes produces near-normal glycemia in spontaneously diabetic BB/Wor rats without administration of exogenous insulin. We compared growth, glycemia, counterregulatory hormones, and lipids in HIGT-treated diabetic rats to nondiabetic rats and diabetic rats treated with either insulin injections or sustained-release insulin pellets. HIGT-treated rats achieved near-normal blood glucose levels within 1 week and maintained glycemic control for up to 3 months. Rats treated with sustained release insulin implants had similar blood sugars, but more hypoglycemia and gained more weight than HIGT-treated rats. HIGT-treated rats normalized blood glucose within 2 hr after a glucose load, and tolerated a 24-hr fast without hypoglycemia. HIGT treatment suppressed ketogenesis similarly to peripheral insulin. However, glucagon levels and free fatty acids were increased in HIGT-treated rats compared to either nondiabetic controls or rats treated with exogenous insulin. In addition to extending successful application of HIGT to a rat model of autoimmune diabetes, these findings emphasize the relative contribution of hepatic insulin effect in the metabolic stabilization of diabetes mellitus.