Lynn M. Schnapp

Sarcoidosis activates diverse transcriptional programs in bronchoalveolar lavage cells

BackgroundSarcoidosis is a multisystem immuno-inflammatory disorder of unknown etiology that most commonly involves the lungs. We hypothesized that an unbiased approach to identify pathways activated in bronchoalveolar lavage (BAL) cells can shed light on the pathogenesis of this complex disease.MethodsWe recruited 15 patients with various stages of sarcoidosis and 12 healthy controls. All subjects underwent bronchoscopy with lavage. For each subject, total RNA was extracted from BAL cells and hybridized to an Affymetrix U133A microarray. Rigorous statistical methods were applied to identify differential gene expression between subjects with sarcoidosis vs. controls. To better elucidate pathways differentially activated between these groups, we integrated network and gene set enrichment analyses of BAL cell transcriptional profiles.ResultsSarcoidosis patients were either non-smokers or former smokers, all had lung involvement and only two were on systemic prednisone. Healthy controls were all non-smokers. Comparison of BAL cell gene expression between sarcoidosis and healthy subjects revealed over 1500 differentially expressed genes. Several previously described immune mediators, such as interferon gamma, were upregulated in the sarcoidosis subjects. Using an integrative computational approach we constructed a modular network of over 80 gene sets that were highly enriched in patients with sarcoidosis. Many of these pathways mapped to inflammatory and immune-related processes including adaptive immunity, T-cell signaling, graft vs. host disease, interleukin 12, 23 and 17 signaling. Additionally, we uncovered a close association between the proteasome machinery and adaptive immunity, highlighting a potentially important and targetable relationship in the pathobiology of sarcoidosis.ConclusionsBAL cells in sarcoidosis are characterized by enrichment of distinct transcriptional programs involved in immunity and proteasomal processes. Our findings add to the growing evidence implicating alveolar resident immune effector cells in the pathogenesis of sarcoidosis and identify specific pathways whose activation may modulate disease progression.

Widespread activation of immunity and pro‐inflammatory programs in peripheral blood leukocytes of HIV‐infected patients with impaired lung gas exchange

HIV infection is associated with impaired lung gas transfer as indicated by a low diffusing capacity (DLCO), but the mechanisms are not well understood. We hypothesized that HIV‐associated gas exchange impairment is indicative of system‐wide perturbations that could be reflected by alterations in peripheral blood leukocyte (PBL) gene expression. Forty HIV‐infected (HIV+) and uninfected (HIV–) men with preserved versus low DLCO were enrolled. All subjects were current smokers and those with acute illness, lung diseases other than COPD or asthma were excluded. Total RNA was extracted from PBLs and hybridized to whole‐genome microarrays. Gene set enrichment analysis (GSEA) was performed between HIV+ versus HIV– subjects with preserved DLCO and those with low DLCO to identify differentially activated pathways. Using pathway‐based analyses, we found that in subjects with preserved DLCO, HIV infection is associated with activation of processes involved in immunity, cell cycle, and apoptosis. Applying a similar analysis to subjects with low DLCO, we identified a much broader repertoire of pro‐inflammatory and immune‐related pathways in HIV+ patients relative to HIV– subjects, with up‐regulation of multiple interleukin pathways, interferon signaling, and toll‐like receptor signaling. We confirmed elevated circulating levels of IL‐6 in HIV+ patients with low DLCO relative to the other groups. Our findings reveal that PBLs of subjects with HIV infection and low DLCO are distinguished by widespread enrichment of immuno‐inflammatory programs. Activation of these pathways may alter the biology of circulating leukocytes and play a role in the pathogenesis of HIV‐associated gas exchange impairment.

Soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1) Is Elevated in Bronchoalveolar Lavage Fluid of Patients with Acute Respiratory Distress Syndrome

Introduction Pulmonary vascular endothelial activation has been implicated in acute respiratory distress syndrome (ARDS), yet little is known about the presence and role of endothelial activation markers in the alveolar space in ARDS. We hypothesized that endothelial activation biomarkers would be differentially expressed in bronchoalveolar lavage fluid from patients with ARDS compared with healthy volunteers, and that biomarker concentrations would be associated with ARDS severity. Methods We performed a cross-sectional analysis of data from 26 intubated patients with ARDS undergoing evaluation for clinically suspected ventilator-associated pneumonia and five healthy volunteers. Patients underwent bronchoalveolar lavage a median of five days after intubation. Healthy volunteers also underwent bronchoalveolar lavage. Endothelial activation biomarkers (soluble vascular cell adhesion molecule-1 [sVCAM-1], soluble endothelial selectin [sESEL], angiopoietin-1 [Ang-1] and angiopoietin-2 [Ang-2]) were measured in bronchoalveolar lavage fluid. Clinically suspected ventilator-associated pneumonia was confirmed with microbiologic culture data. Results Patients with ARDS had significantly higher median sVCAM-1 concentrations in the bronchoalveolar lavage fluid compared with healthy volunteers (985 vs 119 pg/mL, p = 0.03). Additionally, there was a trend toward greater bronchoalveolar lavage fluid sVCAM-1 concentrations among patients with moderate/severe compared to mild ARDS (1395 vs 209 pg/mL, p = 0.06). We did not detect significant differences in bronchoalveolar lavage fluid levels of sESEL, Ang-1 or Ang-2 between patients with ARDS and healthy volunteers. Median bronchoalveolar lavage fluid biomarker levels did not differ between patients with and without microbiologically-confirmed ventilator-associated pneumonia. Conclusions sVCAM-1 concentrations were significantly higher in the bronchoalveolar lavage fluid of patients with ARDS compared to healthy controls, and tended to be higher in moderate/severe ARDS compared to mild ARDS. Our findings add to the growing evidence supporting the concept that endothelial activation plays an important mechanistic role in the pathogenesis of ARDS. Further studies are necessary to characterize the role and/or clinical significance of sVCAM-1 and other endothelial activation markers present in the alveolar space in ARDS.

Variability in Structure of University Pulmonary/Critical Care Fellowships and Retention of Fellows in Academic Medicine

INTRODUCTION Individual fellowship programs are challenged to find a format of training that not only meets the Accreditation Council for Graduate Medical Education requirements, but also grooms fellows to be trusted clinicians, and encourages them to enter academic careers. This study was undertaken as part of an internal effort to evaluate and revise the program structure of the pulmonary/critical care medicine fellowship at the Medical University of South Carolina. Our objectives were to characterize variation in the training structure and specifically research opportunities of university pulmonary/critical care medicine fellowship programs, and to identify factors associated with fellow retention in academic medicine and research. METHODS A 30-item survey was developed through rigorous internal review and was administered via email. Descriptive statistics, Cronbachs alpha, correlations, Wilcoxon sign-rank test, and ANOVA were carried out. RESULTS We had a response rate of 52%. Program directors reported that, within the past 5 years, 38% of their fellows remained in academic medicine and 20% remained in academics with significant research focus. We found a statistically significant association between obtaining a masters degree and remaining in academics (r = 0.559; P < 0.008). The survey also revealed statistically significant relationships between scholarly requirements (grant proposals, peer-reviewed original research projects) and the percent of fellows who graduated and remained in academics. CONCLUSIONS This survey offers some insights that may be useful to fellowship program directors. In particular, advanced education in research and maximizing scholarly activities might be associated with increased academic retention among fellowship trainees.

Transcriptional Survey of Alveolar Macrophages in a Murine Model of Chronic Granulomatous Inflammation Reveals Common Themes with Human Sarcoidosis

Mohan A, Malur A, McPeek M, Barna BP, Schnapp LM, Thomassen MJ, Gharib SA. Transcriptional survey of alveolar macrophages in a murine model of chronic granulomatous inflammation reveals common themes with human sarcoidosis. Am J Physiol Lung Cell Mol Physiol 314: L617-L625, 2018. First published December 6, 2017; doi: 10.1152/ajplung.00289.2017 . To advance our understanding of the pathobiology of sarcoidosis, we developed a multiwall carbon nanotube (MWCNT)-based murine model that shows marked histological and inflammatory signal similarities to this disease. In this study, we compared the alveolar macrophage transcriptional signatures of our animal model with human sarcoidosis to identify overlapping molecular programs. Whole genome microarrays were used to assess gene expression of alveolar macrophages in six MWCNT-exposed and six control animals. The results were compared with the transcriptional profiles of alveolar immune cells in 15 sarcoidosis patients and 12 healthy humans. Rigorous statistical methods were used to identify differentially expressed genes. To better elucidate activated pathways, integrated network and gene set enrichment analysis (GSEA) was performed. We identified over 1,000 differentially expressed between control and MWCNT mice. Gene ontology functional analysis showed overrepresentation of processes primarily involved in immunity and inflammation in MCWNT mice. Applying GSEA to both mouse and human samples revealed upregulation of 92 gene sets in MWCNT mice and 142 gene sets in sarcoidosis patients. Commonly activated pathways in both MWCNT mice and sarcoidosis included adaptive immunity, T-cell signaling, IL-12/IL-17 signaling, and oxidative phosphorylation. Differences in gene set enrichment between MWCNT mice and sarcoidosis patients were also observed. We applied network analysis to differentially expressed genes common between the MWCNT model and sarcoidosis to identify key drivers of disease. In conclusion, an integrated network and transcriptomics approach revealed substantial functional similarities between a murine model and human sarcoidosis particularly with respect to activation of immune-specific pathways.

Fli-1 Governs Pericyte Dysfunction in a Murine Model of Sepsis

Background Pericytes are vascular mural cells and are embedded in the basement membrane of the microvasculature. Recent studies suggest a role for pericytes in lipopolysaccharide (LPS)-induced microvascular dysfunction and mortality, but the mechanisms of pericyte loss in sepsis are largely unknown. Methods By using a cecal ligation and puncture (CLP)-induced murine model of sepsis, we observed that CLP led to lung and renal pericyte loss and reduced lung pericyte density and pericyte/endothelial cell (EC) coverage. Results Up-regulated Friend leukemia virus integration 1 (Fli-1) messenger ribonucleic acid (RNA) and protein levels were found in lung pericytes from CLP mice in vivo and in LPS-stimulated lung pericytes in vitro. Knockout of Fli-1 in Foxd1-derived pericytes prevented CLP-induced pericyte loss, vascular leak, and improved survival. Disrupted Fli-1 expression by small interfering RNA inhibited LPS-induced inflammatory cytokines and chemokines in cultured lung pericytes. Furthermore, CLP-induced pericyte pyroptosis was mitigated in pericyte Fli-1 knockout mice. Conclusions Our findings suggest that Fli-1 is a potential therapeutic target in sepsis.

Enteric Species F Human Adenoviruses use Laminin-Binding Integrins as Co-Receptors for Infection of Ht-29 Cells

The enteric species F human adenovirus types 40 and 41 (HAdV-40 and -41) are the third most common cause of infantile gastroenteritis in the world. Knowledge about HAdV-40 and -41 cellular infection is assumed to be fundamentally different from that of other HAdVs since HAdV-40 and -41 penton bases lack the αV-integrin-interacting RGD motif. This motif is used by other HAdVs mainly for internalization and endosomal escape. We hypothesised that the penton bases of HAdV-40 and -41 interact with integrins independently of the RGD motif. HAdV-41 transduction of a library of rodent cells expressing specific human integrin subunits pointed to the use of laminin-binding α2-, α3- and α6-containing integrins as well as other integrins as candidate co-receptors. Specific laminins prevented internalisation and infection, and recombinant, soluble HAdV-41 penton base proteins prevented infection of human intestinal HT-29 cells. Surface plasmon resonance analysis demonstrated that HAdV-40 and -41 penton base proteins bind to α6-containing integrins with an affinity similar to that of previously characterised penton base:integrin interactions. With these results, we propose that laminin-binding integrins are co-receptors for HAdV-40 and -41.

Inhibition of the IRE-1α/XBP-1 pathway prevents chronic GVHD and preserves the GVL effect in mice

Hematopoietic stem cell transplantation (HCT) is a curative procedure for hematological malignancies, but chronic graft-versus-host disease (cGVHD) remains a major complication after allogeneic HCT. Because donor B cells are essential for cGVHD development and B cells are sensitive to endoplasmic reticulum (ER) stress, we hypothesized that the IRE-1α/XBP-1 pathway is required for B-cell activation and function and for the development of cGVHD. To test this hypothesis, we used conditional knock-out mice deficient of XBP-1 specifically in B cells. Recipients transplanted with donor grafts containing XBP-1-deficient B cells displayed reduced cGVHD compared with controls. Reduction of cGVHD correlated with impaired B-cell functions, including reduced production of anti-double-stranded DNA immunoglobulin G antibodies, CD86, Fas, and GL7 surface expression, and impaired T-cell responses, including reduced interferon-γ production and follicular helper T cells. In a bronchiolitis obliterans cGVHD model, recipients of transplants containing XBP-1-deficient B cells demonstrated improved pulmonary function correlated with reduced donor splenic follicular helper T cells and increased B cells compared with those of wild-type control donor grafts. We then tested if XBP-1 blockade via an IRE-1α inhibitor, B-I09, would attenuate cGVHD and preserve the graft-versus-leukemia (GVL) effect. In a cutaneous cGVHD model, we found that prophylactic administration of B-I09 reduced clinical features of cGVHD, which correlated with reductions in donor T-cell and dendritic cell skin infiltrates. Inhibition of the IRE-1α/XBP-1 pathway also preserved the GVL effect against chronic myelogenous leukemia mediated by allogeneic splenocytes. Collectively, the ER stress response mediated by the IRE-1α/XBP-1 axis is required for cGVHD development but dispensable for GVL activity.

Leadership Primer for Current and Aspiring Pulmonary, Critical Care, and Sleep Medicine Academic Division Chiefs

An academic medical career traditionally revolves around patient care, teaching, and scholarly projects. Thus, when an opportunity for a leadership role arises, such as division chief, the new leader is often unprepared with little or no formal leadership training. In this focused review, academic leaders of the Association of Pulmonary, Critical Care, and Sleep Division Directors describe several leadership concepts adapted from the business sector and apply their years of experience to aid new division chiefs with their first day on the job. The first 90 days are highlighted to include achieving early wins; performing a division-wide Strengths, Weaknesses, Opportunities, Threats analysis; establishing division rapport; redefining the division infrastructure; avoiding conflicts; and managing the relationship with the department chair. The five levels of leadership applicable to academic medicine are discussed: position, permission, production, people, and pinnacle. Finally, emotional intelligence and behavior styles crucial to leadership success are reviewed.

Addressing Gender Inequality in Our Disciplines: Report from the Association of Pulmonary, Critical Care and Sleep Division Chiefs

&NA; Despite the increasing proportion of women in U.S. medical schools, there are relatively few women in leadership positions, and a number of recent publications have highlighted many factors that could contribute to gender inequity and inequality in medicine. The Association of Pulmonary, Critical Care, and Sleep Division Directors, an organization of Division Directors from across the United States, convened a workshop to review data and obtain input from leaders on the state of gender equity in our field. The workshop identified a number of factors that could contribute to gender inequality and inequity: gender climate (including implicit and perceived biases); disproportionate family responsibilities; lack of women in leadership positions; poor retention of women; and lack of gender equality in compensation. The panel members developed a roadmap of concrete recommendations for societies, leaders, and individuals that should promote gender equity to achieve gender equality and improve retention of women in the field of pulmonary, critical care, and sleep medicine.