Steven L. Brody

The epidemiology, pathogenesis and treatment of Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa is an important bacterial pathogen, particularly as a cause of infections in hospitalised patients, immunocompromised hosts and patients with cystic fibrosis. Surveillance of nosocomial P. aeruginosa infections has revealed trends of increasing antimicrobial resistance, including carbapenem resistance and multidrug resistance. Mechanisms of antimicrobial resistance include multidrug efflux pumps, β-lactamases and downregulation of outer membrane porins. Mechanisms of virulence include secreted toxins and the ability to form biofilms. The effective treatment of infections caused by P. aeruginosa includes prevention when possible, source control measures as necessary and prompt administration of appropriate antibacterial agents. Antibacterial de-escalation should be pursued in patients with an appropriate clinical response, especially when antibacterial susceptibilities are known. Multidrug-resistant P. aeruginosa may require treatment with less commonly used antibacterials (e.g. colistin), but newer anti-pseudomonal antibacterials are expected to be available in the near future.

Influenza Virus Receptor Specificity and Cell Tropism in Mouse and Human Airway Epithelial Cells

ABSTRACT Recent human infections caused by the highly pathogenic avian influenza virus H5N1 strains emphasize an urgent need for assessment of factors that allow viral transmission, replication, and intra-airway spread. Important determinants for virus infection are epithelial cell receptors identified as glycans terminated by an α2,3-linked sialic acid (SA) that preferentially bind avian strains and glycans terminated by an α2,6-linked SA that bind human strains. The mouse is often used as a model for study of influenza viruses, including recent avian strains; however, the selectivity for infection of specific respiratory cell populations is not well described, and any relationship between receptors in the mouse and human lungs is incompletely understood. Here, using in vitro human and mouse airway epithelial cell models and in vivo mouse infection, we found that the α2,3-linked SA receptor was expressed in ciliated airway and type II alveolar epithelial cells and was targeted for cell-specific infection in both species. The α2,6-linked SA receptor was not expressed in the mouse, a factor that may contribute to the inability of some human strains to efficiently infect the mouse lung. In human airway epithelial cells, α2,6-linked SA was expressed and functional in both ciliated and goblet cells, providing expanded cellular tropism. Differences in receptor and cell-specific expression in these species suggest that differentiated human airway epithelial cell cultures may be superior for evaluation of some human strains, while the mouse can provide a model for studying avian strains that preferentially bind only the α2,3-linked SA receptor.

Adenovirus‐Mediated in Vivo Gene Transfer

Adenovirus vectors are efficient vehicles for in vivo gene transfer to many different cell types. Recombinant adenovirus vectors containing exogenous genes for transfer are derived from adenovirus type 5 and are made replication deficient by the deletion of the E1 region. Based on the observation that many natural adenovirus infections are targeted to airway epithelial cells, a replication-deficient adenovirus vector was constructed containing the cystic fibrosis transmembrane conductance regulator cDNA for the potential therapy of the respiratory manifestations of cystic fibrosis. Using this vector, the normal human CFTR cDNA has been successfully transferred to airway epithelial cells of experimental animals via the trachea. This finding has led to the development of human gene therapy protocols for the evaluation of the safety and efficacy of adenovirus-mediated CFTR cDNA transfer to lungs of individuals with cystic fibrosis. In addition to the airways, adenovirus vectors have been demonstrated to mediate in vivo gene delivery to cells of the liver, blood vessels, brain, muscle, heart, peritoneum, and salivary glands. Adenovirus vectors containing marker genes have also been demonstrated to transfer genes to human tumor cells in nude mice. Such vectors may be useful for a variety of therapeutic applications for in vivo gene transfer for the therapy of cancer and other diseases.

Blocking airway mucous cell metaplasia by inhibiting EGFR antiapoptosis and IL-13 transdifferentiation signals

Epithelial hyperplasia and metaplasia are common features of inflammatory and neoplastic disease, but the basis for the altered epithelial phenotype is often uncertain. Here we show that long-term ciliated cell hyperplasia coincides with mucous (goblet) cell metaplasia after respiratory viral clearance in mouse airways. This chronic switch in epithelial behavior exhibits genetic susceptibility and depends on persistent activation of EGFR signaling to PI3K that prevents apoptosis of ciliated cells and on IL-13 signaling that promotes transdifferentiation of ciliated to goblet cells. Thus, EGFR blockade (using an irreversible EGFR kinase inhibitor designated EKB-569) prevents virus-induced increases in ciliated and goblet cells whereas IL-13 blockade (using s-IL-13Ralpha2-Fc) exacerbates ciliated cell hyperplasia but still inhibits goblet cell metaplasia. The distinct effects of EGFR and IL-13 inhibitors after viral reprogramming suggest that these combined therapeutic strategies may also correct epithelial architecture in the setting of airway inflammatory disorders characterized by a similar pattern of chronic EGFR activation, IL-13 expression, and ciliated-to-goblet cell metaplasia.

RhoA-mediated apical actin enrichment is required for ciliogenesis and promoted by Foxj1

Programs that direct cellular differentiation are dependent on the strict temporal expression of regulatory factors that can be provided by Rho GTPases. Ciliogenesis is a complex sequence of events involving the generation and docking of basal bodies at the apical membrane, followed by ciliary axoneme generation. Although a cilia proteome has been assembled, programs that direct ciliated cell differentiation are not well established, particularly in mammalian systems. Using mouse primary culture airway epithelial cells, we identified a critical stage of ciliogenesis requiring the temporal establishment of an apical web-like structure of actin for basal body docking and subsequent axoneme growth. Apical web formation and basal body docking were prevented by interruption of actin remodeling and were dependent on RhoA activation. Additional evidence for this program was provided by analysis of Foxj1-null mice that failed to dock basal bodies and lacked apical actin. Foxj1 expression coincided with actin web formation, activated RhoA and RhoB, and persisted despite RhoA inhibition, suggesting that Foxj1 promoted RhoA during ciliogenesis. Apical ezrin localization was also dependent on Foxj1, actin remodeling, and RhoA, but was not critical for ciliogenesis. Thus, temporal Foxj1 and RhoA activity are essential regulatory events for cytoskeletal remodeling during mammalian ciliogenesis.

FoxJ1-dependent gene expression is required for differentiation of radial glia into ependymal cells and a subset of astrocytes in the postnatal brain

Neuronal specification occurs at the periventricular surface of the embryonic central nervous system. During early postnatal periods, radial glial cells in various ventricular zones of the brain differentiate into ependymal cells and astrocytes. However, mechanisms that drive this time- and cell-specific differentiation remain largely unknown. Here, we show that expression of the forkhead transcription factor FoxJ1 in mice is required for differentiation into ependymal cells and a small subset of FoxJ1+ astrocytes in the lateral ventricles, where these cells form a postnatal neural stem cell niche. Moreover, we show that a subset of FoxJ1+ cells harvested from the stem cell niche can self-renew and possess neurogenic potential. Using a transcriptome comparison of FoxJ1-null and wild-type microdissected tissue, we identified candidate genes regulated by FoxJ1 during early postnatal development. The list includes a significant number of microtubule-associated proteins, some of which form a protein complex that could regulate the transport of basal bodies to the ventricular surface of differentiating ependymal cells during FoxJ1-dependent ciliogenesis. Our results suggest that time- and cell-specific expression of FoxJ1 in the brain acts on an array of target genes to regulate the differentiation of ependymal cells and a small subset of astrocytes in the adult stem cell niche.

Long-term IL-33–producing epithelial progenitor cells in chronic obstructive lung disease

Chronic obstructive lung disease is characterized by persistent abnormalities in epithelial and immune cell function that are driven, at least in part, by infection. Analysis of parainfluenza virus infection in mice revealed an unexpected role for innate immune cells in IL-13-dependent chronic lung disease, but the upstream driver for the immune axis in this model and in humans with similar disease was undefined. We demonstrate here that lung levels of IL-33 are selectively increased in postviral mice with chronic obstructive lung disease and in humans with very severe chronic obstructive pulmonary disease (COPD). In the mouse model, IL-33/IL-33 receptor signaling was required for Il13 and mucin gene expression, and Il33 gene expression was localized to a virus-induced subset of airway serous cells and a constitutive subset of alveolar type 2 cells that are both linked conventionally to progenitor function. In humans with COPD, IL33 gene expression was also associated with IL13 and mucin gene expression, and IL33 induction was traceable to a subset of airway basal cells with increased capacities for pluripotency and ATP-regulated release of IL-33. Together, these findings provide a paradigm for the role of the innate immune system in chronic disease based on the influence of long-term epithelial progenitor cells programmed for excess IL-33 production.

Whole-Exome Capture and Sequencing Identifies HEATR2 Mutation as a Cause of Primary Ciliary Dyskinesia

Motile cilia are essential components of the mucociliary escalator and are central to respiratory-tract host defenses. Abnormalities in these evolutionarily conserved organelles cause primary ciliary dyskinesia (PCD). Despite recent strides characterizing the ciliome and sensory ciliopathies through exploration of the phenotype-genotype associations in model organisms, the genetic bases of most cases of PCD remain elusive. We identified nine related subjects with PCD from geographically dispersed Amish communities and performed exome sequencing of two affected individuals and their unaffected parents. A single autosomal-recessive nonsynonymous missense mutation was identified in HEATR2, an uncharacterized gene that belongs to a family not previously associated with ciliary assembly or function. Airway epithelial cells isolated from PCD-affected individuals had markedly reduced HEATR2 levels, absent dynein arms, and loss of ciliary beating. MicroRNA-mediated silencing of the orthologous gene in Chlamydomonas reinhardtii resulted in absent outer dynein arms, reduced flagellar beat frequency, and decreased cell velocity. These findings were recapitulated by small hairpin RNA-mediated knockdown of HEATR2 in airway epithelial cells from unaffected donors. Moreover, immunohistochemistry studies in human airway epithelial cells showed that HEATR2 was localized to the cytoplasm and not in cilia, which suggests a role in either dynein arm transport or assembly. The identification of HEATR2 contributes to the growing number of genes associated with PCD identified in both individuals and model organisms and shows that exome sequencing in family studies facilitates the discovery of novel disease-causing gene mutations.

Pseudomonas aeruginosa Acquires Biofilm-Like Properties within Airway Epithelial Cells

ABSTRACT Pseudomonas aeruginosa can notably cause both acute and chronic infection. While several virulence factors are implicated in the acute phase of infection, advances in understanding bacterial pathogenesis suggest that chronic P. aeruginosa infection is related to biofilm formation. However, the relationship between these two forms of disease is not well understood. Accumulating evidence indicates that, during acute infection, P. aeruginosa enters epithelial cells, a process viewed as either a host-mediated defense response or a pathogenic mechanism to avoid host-mediated killing. We investigated the possibility that epithelial cell entry during early P. aeruginosa-epithelial cell contact favors bacterial survival and is linked to chronic infection. Using electron microscopy and confocal microscopy to analyze primary culture airway epithelial cells infected with P. aeruginosa, we found that epithelial cells developed pod-like clusters of intracellular bacteria with regional variation in protein expression. Extracellular gentamicin added to the medium after acute infection led to the persistence of intracellular P. aeruginosa for at least 3 days. Importantly, compared to bacterial culture under planktonic conditions, the intracellular bacteria were insensitive to growth inhibition or killing by antibiotics that were capable of intraepithelial cell penetration. These findings suggest that P. aeruginosa can use airway epithelial cells as a sanctuary for persistence and develop a reversible antibiotic resistance phenotype characteristic of biofilm physiology that can contribute to development of chronic infection.

Foxj1 is required for apical localization of ezrin in airway epithelial cells

Establishment and maintenance of epithelial cell polarity depend on cytoskeletal organization and protein trafficking to polarized cortical membranes. ERM (ezrin, radixin, moesin) family members link polarized proteins with cytoskeletal actin. Although ERMs are often considered to be functionally similar, we found that, in airway epithelial cells, apical localization of ERMs depend on cell differentiation and is independently regulated. Moesin was present in the apical membrane of all undifferentiated epithelial cells. However, in differentiated cells, ezrin and moesin were selectively localized to apical membranes of ciliated airway cells and were absent from secretory cells. To identify regulatory proteins required for selective ERM trafficking, we evaluated airway epithelial cells lacking Foxj1, an F-box factor that directs programs required for cilia formation at the apical membrane. Interestingly, Foxj1 expression was also required for localization of apical ezrin, but not moesin. Additionally, membrane-cytoskeletal and threonine-phosphorylated ezrin were decreased in Foxj1-null cells, consistent with absent apical ezrin. Although apical moesin expression was present in null cells, it could not compensate for ezrin because ERM-associated EBP50 and the β2 adrenergic receptor failed to localize apically in the absence of Foxj1. These findings indicate that Foxj1 regulates ERM proteins differentially to selectively direct the apical localization of ezrin for the organization of multi-protein complexes in apical membranes of airway epithelial cells.