Adela Cota-Gomez

Muc5b is required for airway defence

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b−/− mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

Widespread colonization of the lung by Tropheryma whipplei in HIV infection

RATIONALEnLung infections caused by opportunistic or virulent pathogens are a principal cause of morbidity and mortality in HIV infection. It is unknown whether HIV infection leads to changes in basal lung microflora, which may contribute to chronic pulmonary complications that increasingly are being recognized in individuals infected with HIV.nnnOBJECTIVESnTo determine whether the immunodeficiency associated with HIV infection resulted in alteration of the lung microbiota.nnnMETHODSnWe used 16S ribosomal RNA targeted pyrosequencing and shotgun metagenomic sequencing to analyze bacterial gene sequences in bronchoalveolar lavage (BAL) and mouths of 82 HIV-positive and 77 HIV-negative subjects.nnnMEASUREMENTS AND MAIN RESULTSnSequences representing Tropheryma whipplei, the etiologic agent of Whipples disease, were significantly more frequent in BAL of HIV-positive compared with HIV-negative individuals. T. whipplei dominated the community (>50% of sequence reads) in 11 HIV-positive subjects, but only 1 HIV-negative individual (13.4 versus 1.3%; P = 0.0018). In 30 HIV-positive individuals sampled longitudinally, antiretroviral therapy resulted in a significantly reduced relative abundance of T. whipplei in the lung. Shotgun metagenomic sequencing was performed on eight BAL samples dominated by T. whipplei 16S ribosomal RNA. Whole genome assembly of pooled reads showed that uncultured lung-derived T. whipplei had similar gene content to two isolates obtained from subjects with Whipples disease.nnnCONCLUSIONSnAsymptomatic subjects with HIV infection have unexpected colonization of the lung by T. whipplei, which is reduced by effective antiretroviral therapy and merits further study for a potential pathogenic role in chronic pulmonary complications of HIV infection.

A novel class of microbial phosphocholine‐specific phospholipases C

In this report we describe the 1500‐fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17u2003117.2u2003Da (PlcR2) and 78u2003417u2003Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1u2003:u20031. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline‐containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the con‐clusion that the members of this PC‐PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.

The Human Immunodeficiency Virus-1 Tat Protein Activates Human Umbilical Vein Endothelial Cell E-selectin Expression via an NF-κB-dependent Mechanism

Human immunodeficiency virus infection is associated with inflammation and endothelial cell activation that cannot be ascribed to direct infection by the virus or to the presence of opportunistic infections. Factors related to the virus itself, to the host and/or to environmental exposures probably account for these observations. The HIV protein Tat, a viral regulator required for efficient transcription of the viral genome in host cells is secreted from infected cells and taken up by uninfected by-stander cells. Tat can also act as a general transcriptional activator of key inflammatory molecules. We have examined whether Tat contributes to this endothelial cell activation by activating NF-κB. Human endothelial cells exposed to Tat in the culture medium activated E-selectin expression with delayed kinetics compared with tumor necrosis factor (TNF). Tat-mediated E-selectin up-regulation required the basic domain of Tat and was inhibited by a Tat antibody. Transfection of human E-selectin promoter-luciferase reporter constructs into Tat-bearing cells or into endothelial cells co-transfected with a Tat expression vector resulted in induction of luciferase expression. Either Tat or TNF activated p65 translocation and binding to an oligonucleotide containing the E-selectin κB site 3 sequence. Tat-mediated p65 translocation was also delayed compared with TNF. Neither agent induced new synthesis of p65. A super-repressor adenovirus (AdIκBαSR) that constitutively sequesters IκB in the cytoplasm as well as cycloheximide or actinomycin D inhibited Tat- or TNF-mediated κB translocation and E-selectin up-regulation.

HIV-1 Tat increases oxidant burden in the lungs of transgenic mice

Chronic human immunodeficiency virus infection is associated with higher incidence of pulmonary complications including hypertension, vasculopathy, lymphocytic alveolitis, and interstitial pneumonitis not attributed to either opportunistic infections or presence of the virus. The Tat (transactivator of transcription) protein, a required transactivator for expression of full-length viral genes, is pleiotropic and influences expression of cellular inflammatory genes. Tat-dependent transactivation of cellular genes requires specific mediators, including NF-κB, widely recognized as sensitive to changes in cellular oxidant burden. We hypothesized that overproduction of Tat leads to increased oxidant burden and to alterations in basal inflammatory status as measured by NF-κB activation. We engineered transgenic mouse lines that express Tat (86-amino-acid isoform) in the lung under the control of the surfactant protein C promoter. Tat-transgenic mice exhibit increased pulmonary cellular infiltration, increased nitrotyrosine and carbonyl protein modifications, and increased levels of NF-κB, MnSOD, and thioredoxin-interacting protein. These data indicate that Tat increases oxidant burden and resets the threshold for inflammation, which may increase susceptibility to secondary injuries.

The human immunodeficiency virus-1 Tat protein activates endothelial cell E-selectin expression via an NF-κ-dependent mechanism

Human immunodeficiency virus infection is associated with inflammation and endothelial cell activation that cannot be ascribed to direct infection by the virus or to the presence of opportunistic infections. Factors related to the virus itself, to the host and/or to environmental exposures probably account for these observations. The HIV protein Tat, a viral regulator required for efficient transcription of the viral genome in host cells is secreted from infected cells and taken up by uninfected by-stander cells. Tat can also act as a general transcriptional activator of key inflammatory molecules. We have examined whether Tat contributes to this endothelial cell activation by activating NF-κB. Human endothelial cells exposed to Tat in the culture medium activated E-selectin expression with delayed kinetics compared with tumor necrosis factor (TNF). Tat-mediated E-selectin up-regulation required the basic domain of Tat and was inhibited by a Tat antibody. Transfection of human E-selectin promoter-luciferase reporter constructs into Tat-bearing cells or into endothelial cells co-transfected with a Tat expression vector resulted in induction of luciferase expression. Either Tat or TNF activated p65 translocation and binding to an oligonucleotide containing the E-selectin κB site 3 sequence. Tat-mediated p65 translocation was also delayed compared with TNF. Neither agent induced new synthesis of p65. A super-repressor adenovirus (AdIκBαSR) that constitutively sequesters IκB in the cytoplasm as well as cycloheximide or actinomycin D inhibited Tat- or TNF-mediated κB translocation and E-selectin up-regulation.