Viral modulation of type II interferon increases T cell adhesion and virus spread.

During primary varicella zoster virus (VZV) infection, infected lymphocytes drive primary viremia, causing systemic dissemination throughout the host, including the skin. This results in cytokine expression, including interferons (IFNs), which partly limit infection. VZV also spreads from skin keratinocytes to lymphocytes prior to secondary viremia. It is not clear how VZV achieves this while evading the cytokine response. Here, we show that VZV glycoprotein C (gC) binds IFN-γ and modifies its activity, increasing the expression of a subset of IFN-stimulated genes (ISGs), including intercellular adhesion molecule 1 (ICAM1), chemokines and immunomodulatory genes. The higher ICAM1 protein level at the plasma membrane of keratinocytes facilitates lymphocyte function-associated antigen 1-dependent T cell adhesion and expression of gC during infection increases VZV spread to peripheral blood mononuclear cells. This constitutes the discovery of a strategy to modulate IFN-γ activity, upregulating a subset of ISGs, promoting enhanced lymphocyte adhesion and virus spread.

Analysis of CFTR mRNA and Protein in Peripheral Blood Mononuclear Cells via Quantitative Real-Time PCR and Western Blot.

The Cystic Fibrosis Conductance Transmembrane Regulator gene encodes for the CFTR ion channel, which is responsible for the transport of chloride and bicarbonate across the plasma membrane. Mutations in the gene result in impaired ion transport, subsequently leading to perturbed secretion in all exocrine glands and, therefore, the multi-organ disease cystic fibrosis (CF). In recent years, several studies have reported on CFTR expression in immune cells as demonstrated by immunofluorescence, flow cytometry, and immunoblotting. However, these data are mainly restricted to single-cell populations and show significant variation depending on the methodology used. Here, we investigated CFTR transcription and protein expression using standardized protocols in a comprehensive panel of immune cells. Methods: We applied a high-resolution Western blot protocol using a combination of highly specific monoclonal CFTR antibodies that have been optimized for the detection of CFTR in epithelial cells and healthy primary immune cell subpopulations sorted by flow cytometry and used immortalized cell lines as controls. The specificity of CFTR protein detection was controlled by peptide competition and enzymatic Peptide-N-Glycosidase-F (PNGase) digest. CFTR transcripts were analyzed using quantitative real-time PCR and normalized to the level of epithelial T84 cells as a reference. Results: CFTR mRNA expression could be shown for primary CD4+ T cells, NK cells, as well as differentiated THP-1 and Jurkat T cells. In contrast, we failed to detect CFTR transcripts for CD14+ monocytes and undifferentiated THP-1 cells, as well as for B cells and CD8+ T cells. Prominent immunoreactive bands were detectable by immunoblotting with the combination of four CFTR antibodies targeting different epitopes of the CFTR protein. However, in biosamples of non-epithelial origin, these CFTR-like protein bands could be unmasked as false positives through peptide competition or PNGase digest, meaning that the observed mRNA transcripts were not necessarily translated into CFTR proteins, which could be detected via immunoblotting. Our results confirm that mRNA expression in immune cells is many times lower than in that cells of epithelial origin. The immunoreactive signals in immune cells turned out to be false positives, and may be provoked by the presence of a high-affinity protein with a similar epitope. Non-specific binding (e.g., Fab-interaction with glycosyl branches) might also contribute to false positive signals. Our findings highlight the necessity of accurate controls, such as CFTR-negative cells, as well as peptide competition and glycolytic digest in order to identify genuine CFTR protein by immunoblotting. Our data suggest, furthermore, that CFTR protein expression data from techniques such as histology, for which the absence of a molecular weight or other independent control prevents the unmasking of false positive immunoreactive signals, must be interpreted carefully as well.

Pulmonary Alveolar Proteinosis and new therapeutic concepts.

Pulmonary alveolar proteinosis (PAP) is an umbrella term used to refer to a pulmonary syndrome which is characterized by excessive accumulation of surfactant in the lungs of affected individuals. In general, PAP is a rare lung disease affecting children and adults, although its prevalence and incidence is variable among different countries. Even though PAP is a rare disease, it is a prime example on how modern medicine can lead to new therapeutic concepts, changing ways and techniques of (genetic) diagnosis which ultimately led into personalized treatments, all dedicated to improve the function of the impaired lung and thus life expectancy and quality of life in PAP patients. In fact, new technologies, such as new sequencing technologies, gene therapy approaches, new kind and sources of stem cells and completely new insights into the ontogeny of immune cells such as macrophages have increased our understanding in the onset and progression of PAP, which have paved the way for novel therapeutic concepts for PAP and beyond. As of today, classical monocyte-derived macrophages are known as important immune mediator and immune sentinels within the innate immunity. Furthermore, macrophages (known as tissue resident macrophages (TRMs)) can also be found in various tissues, introducing e. g. alveolar macrophages in the broncho-alveolar space as crucial cellular determinants in the onset of PAP and other lung disorders. Given recent insights into the onset of alveolar macrophages and knowledge about factors which impede their function, has led to the development of new therapies, which are applied in the context of PAP, with promising implications also for other diseases in which macrophages play an important role. Thus, we here summarize the latest insights into the various forms of PAP and introduce new pre-clinical work which is currently conducted in the framework of PAP, introducing new therapies for children and adults who still suffer from this severe, potentially life-threatening disease.

Co-chaperones DNAJA1 and DNAJB6 are critical for regulation of polyglutamine aggregation.

Huntington's disease (HD) is caused by CAG repeat expansion in the huntingtin gene. The expanded polyglutamine (polyQ) repeat of the encoded protein leads to protein misfolding and aggregation, resulting in increased neuronal cell death. DNAJ co-chaperones play a crucial role in transferring misfolded/unfolded proteins to HSP70 chaperones, which play an essential role for protein folding. Here, we investigated the effect of knock out (KO) of three individual DNAJ genes in HEK293 cells expressing polyglutamine74exon1 huntingtin (polyQ74htt). Flourescence microscopy analysis revealed that KO of DNAJB6 resulted in a 5-fold increase in polyQ74htt aggregation and that DNAJA1 KO resulted in a 4-fold decrease of polyQ74htt aggregation. KO of DNAJB1 did not change the propensity of polyQ74htt to aggregate in cells. These findings where confirmed both by fluorescence microscopy analysis and filter trap assay (FTA). DNAJB6 KO cells displayed an increased rate of cell death as assessed by trypan blue exclusion and propidium iodide (PI) uptake assays. These results demonstrate that the DNAJ proteins DNAJA1 and DNAJB6 can modulate polyQ aggregation in opposite manners, and thus that fine-tuning the cellular levels of DNAJ proteins is critical for suppression of polyQ aggregation and cell survival.