Gregory Dolganov

Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages Insights into the Phagosomal Environment

Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2–deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon γ– and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and β-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of σE-dependent, sodium dodecyl sulfate–regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogens cell envelope.

Inhibition of Respiration by Nitric Oxide Induces a Mycobacterium tuberculosis Dormancy Program

An estimated two billion persons are latently infected with Mycobacterium tuberculosis. The host factors that initiate and maintain this latent state and the mechanisms by which M. tuberculosis survives within latent lesions are compelling but unanswered questions. One such host factor may be nitric oxide (NO), a product of activated macrophages that exhibits antimycobacterial properties. Evidence for the possible significance of NO comes from murine models of tuberculosis showing progressive infection in animals unable to produce the inducible isoform of NO synthase and in animals treated with a NO synthase inhibitor. Here, we show that O2 and low, nontoxic concentrations of NO competitively modulate the expression of a 48-gene regulon, which is expressed in vivo and prepares bacilli for survival during long periods of in vitro dormancy. NO was found to reversibly inhibit aerobic respiration and growth. A heme-containing enzyme, possibly the terminal oxidase in the respiratory pathway, likely senses and integrates NO and O2 levels and signals the regulon. These data lead to a model postulating that, within granulomas, inhibition of respiration by NO production and O2 limitation constrains M. tuberculosis replication rates in persons with latent tuberculosis.

Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma

Asthma is an increasingly common disease that remains poorly understood and difficult to manage. This disease is characterized by airway hyperreactivity (AHR, defined by exaggerated airflow obstruction in response to bronchoconstrictors), mucus overproduction and chronic eosinophilic inflammation. AHR and mucus overproduction are consistently linked to asthma symptoms and morbidity. Asthma is mediated by Th2 lymphocytes, which produce a limited repertoire of cytokines, including interleukin-4 (IL-4), IL-5, IL-9 and IL-13. Although each of these cytokines has been implicated in asthma, IL-13 is now thought to be especially critical. In animal models of allergic asthma, blockade of IL-13 markedly inhibits allergen-induced AHR, mucus production and eosinophilia. Furthermore, IL-13 delivery to the airway causes all of these effects. IL-13 is thus both necessary and sufficient for experimental models of asthma. However, the IL-13-responsive cells causing these effects have not been identified. Here we show that mice lacking signal transducer and activator of transcription 6 (STAT6) were protected from all pulmonary effects of IL-13. Reconstitution of STAT6 only in epithelial cells was sufficient for IL-13-induced AHR and mucus production in the absence of inflammation, fibrosis or other lung pathology. These results demonstrate the importance of direct effects of IL-13 on epithelial cells in causing two central features of asthma.

Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids

Airway inflammation and epithelial remodeling are two key features of asthma. IL-13 and other cytokines produced during T helper type 2 cell-driven allergic inflammation contribute to airway epithelial goblet cell metaplasia and may alter epithelial–mesenchymal signaling, leading to increased subepithelial fibrosis or hyperplasia of smooth muscle. The beneficial effects of corticosteroids in asthma could relate to their ability to directly or indirectly decrease epithelial cell activation by inflammatory cells and cytokines. To identify markers of epithelial cell dysfunction and the effects of corticosteroids on epithelial cells in asthma, we studied airway epithelial cells collected from asthmatic subjects enrolled in a randomized controlled trial of inhaled corticosteroids, from healthy subjects and from smokers (disease control). By using gene expression microarrays, we found that chloride channel, calcium-activated, family member 1 (CLCA1), periostin, and serine peptidase inhibitor, clade B (ovalbumin), member 2 (serpinB2) were up-regulated in asthma but not in smokers. Corticosteroid treatment down-regulated expression of these three genes and markedly up-regulated expression of FK506-binding protein 51 (FKBP51). Whereas high baseline expression of CLCA1, periostin, and serpinB2 was associated with a good clinical response to corticosteroids, high expression of FKBP51 was associated with a poor response. By using airway epithelial cells in culture, we found that IL-13 increased expression of CLCA1, periostin, and serpinB2, an effect that was suppressed by corticosteroids. Corticosteroids also induced expression of FKBP51. Taken together, our findings show that airway epithelial cells in asthma have a distinct activation profile and identify direct and cell-autonomous effects of corticosteroid treatment on airway epithelial cells that relate to treatment responses and can now be the focus of specific mechanistic studies.

Loss of integrin |[alpha]|v|[beta]|6-mediated TGF-|[beta]| activation causes Mmp12-dependent emphysema

Integrins are heterodimeric cell-surface proteins that regulate cell growth, migration and survival. We have shown previously that the epithelial-restricted integrin αvβ6 has another critical function; that is, it binds and activates latent transforming growth factor-β (TGF-β). Through a global analysis of pulmonary gene expression in the lungs of mice lacking this integrin (Itgb6 null mice) we have identified a marked induction of macrophage metalloelastase (Mmp12)—a metalloproteinase that preferentially degrades elastin and has been implicated in the chronic lung disease emphysema. Here we report that Itgb6-null mice develop age-related emphysema that is completely abrogated either by transgenic expression of versions of the β6 integrin subunit that support TGF-β activation, or by the loss of Mmp12. Furthermore, we show that the effects of Itgb6 deletion are overcome by simultaneous transgenic expression of active TGF-β1. We have uncovered a pathway in which the loss of integrin-mediated activation of latent TGF-β causes age-dependent pulmonary emphysema through alterations of macrophage Mmp12 expression. Furthermore, we show that a functional alteration in the TGF-β activation pathway affects susceptibility to this disease.

A multistage tuberculosis vaccine that confers efficient protection before and after exposure

All tuberculosis vaccines currently in clinical trials are designed as prophylactic vaccines based on early expressed antigens. We have developed a multistage vaccination strategy in which the early antigens Ag85B and 6-kDa early secretory antigenic target (ESAT-6) are combined with the latency-associated protein Rv2660c (H56 vaccine). In CB6F1 mice we show that Rv2660c is stably expressed in late stages of infection despite an overall reduced transcription. The H56 vaccine promotes a T cell response against all protein components that is characterized by a high proportion of polyfunctional CD4+ T cells. In three different pre‐exposure mouse models, H56 confers protective immunity characterized by a more efficient containment of late-stage infection than the Ag85B-ESAT6 vaccine (H1) and BCG. In two mouse models of latent tuberculosis, we show that H56 vaccination after exposure is able to control reactivation and significantly lower the bacterial load compared to adjuvant control mice.

Human Rad50 is physically associated with human Mre11: identification of a conserved multiprotein complex implicated in recombinational DNA repair.

In this report, we describe the identification and molecular characterization of a human RAD50 homolog, hRAD50. hRAD50 was included in a collection of cDNAs which were isolated by a direct cDNA selection strategy focused on the chromosomal interval spanning 5q23 to 5q31. Alterations of the 5q23-q31 interval are frequently observed in myelodysplasia and myeloid leukemia. This strategy was thus undertaken to create a detailed genetic map of that region. Saccharomyces cerevisiae RAD50 (ScRAD50) is one of three yeast RAD52 epistasis group members (ScRAD50, ScMRE11, and ScXRS2) in which mutations eliminate meiotic recombination but confer a hyperrecombinational phenotype in mitotic cells. The yeast Rad50, Mre11, and Xrs2 proteins appear to act in a multiprotein complex, consistent with the observation that the corresponding mutants confer essentially identical phenotypes. In this report, we demonstrate that the human Rad50 and Mre11 proteins are stably associated in a protein complex which may include three other proteins. hRAD50 is expressed in all tissues examined, but mRNA levels are significantly higher in the testis. Other human RAD52 epistasis group homologs exhibit this expression pattern, suggesting the involvement of human RAD52 epistasis group proteins in meiotic recombination. Human RAD52 epistasis group proteins are highly conserved and act in protein complexes that are analogous to those of their yeast counterparts. These findings indicate that the function of the RAD52 epistasis group is conserved in human cells.

The Mycobacterium tuberculosis regulatory network and hypoxia

We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub.

In vitro susceptibility to rhinovirus infection is greater for bronchial than for nasal airway epithelial cells in human subjects

BACKGROUND Human rhinoviruses (HRVs) characteristically cause upper respiratory tract infection, but they also infect the lower airways, causing acute bronchitis and exacerbating asthma. OBJECTIVE Our purpose was to study ex vivo the differences in the response to HRV infection of nasal and bronchial epithelial cultures from the same healthy and asthmatic individuals using conditions favoring development of fully differentiated, pseudostratified mucociliary epithelium. METHODS Cells from the inferior turbinates and bronchial tree of 5 healthy and 6 asthmatic individuals were cultured at an air-liquid interface. Cultures were infected with HRV-16, and after 48 hours, the degree of infection was measured. RESULTS Baseline median transepithelial resistance was lower in human bronchial epithelial (HBE) cell cultures than in human nasal epithelial (HNE) cell cultures (195 Omega.cm2 [95% CI, 164-252] vs 366 Omega.cm2 [95% CI, 234-408], respectively; P < .01). Virus replicated more easily in HBE cells than in HNE cells based on virus shedding in apical wash (log tissue culture infective dose of 50%/0.1 mL = 2.0 [95% CI, 1.0-2.5] vs 0.5 [95% CI, 0.5-1.5], P < .01) and on a 20- to 30-fold greater viral load and number of infected cells in HBE cell cultures than in HNE cell cultures. The increases in expression of RANTES and double-stranded RNA-dependent protein kinase were greater in HBE cell cultures than in HNE cell cultures, as were the concentrations of IL-8, IL-1alpha, RANTES, and IP-10 in basolateral medium. However, no significant differences between asthmatic and healthy subjects (including IFN-beta1 expression) were found. CONCLUSIONS Differentiated nasal epithelial cells might have mechanisms of increased resistance to rhinovirus infection compared with bronchial epithelial cells. We could not confirm previous reports of increased susceptibility to HRV infection in epithelial cells from asthmatic subjects.

Integrin αvβ8-Mediated Activation of Transforming Growth Factor-β by Perivascular Astrocytes : An Angiogenic Control Switch

Brain hemorrhage is a severe complication of both neoplastic and nonneoplastic brain disease. Mice deficient in the αvβ8 integrin display defective brain vessel formation resulting in hemorrhage and perinatal death, but the mechanism of brain hemorrhage is unknown. Because the αvβ8 integrin is expressed by astrocytes and not expressed by endothelium, paracrine interactions between astrocytes and endothelial cells could contribute to the maintenance of brain vessel integrity. We have investigated the mechanisms underlying astrocytic-endothelial paracrine signaling and have found that integrin-mediated activation of transforming growth factor (TGF)-β by astrocytes influences endothelial cell function. Thus, we identified the integrin αvβ8 in human perivascular glial cell processes surrounding developing blood vessels. Human astrocytic αvβ8 was a major cell surface receptor for latent TGF-β, and αvβ8-dependent activation of TGF-β was the major mechanism of TGF-β activation in primary cultures of astrocytes or freshly dissociated fetal brain cells. This activation of TGF-β was sufficient to inhibit endothelial migration in fibrin gels and to alter expression of genes affecting proteolytic and angiogenic pathways. Taken together, our data suggest that astrocytic αvβ8 acts as a central regulator of brain vessel homeostasis through regulation of TGF-β activation and expression of TGF-β-responsive genes that promote vessel differentiation and stabilization, most notably plasminogen activator inhibitor-1 and thrombospondin-1.