Siva Kumar Solleti

Fibroblast Growth Factor Receptors Control Epithelial–Mesenchymal Interactions Necessary for Alveolar Elastogenesis

RATIONALE The mechanisms contributing to alveolar formation are poorly understood. A better understanding of these processes will improve efforts to ameliorate lung disease of the newborn and promote alveolar repair in the adult. Previous studies have identified impaired alveogenesis in mice bearing compound mutations of fibroblast growth factor (FGF) receptors (FGFRs) 3 and 4, indicating that these receptors cooperatively promote postnatal alveolar formation. OBJECTIVES To determine the molecular and cellular mechanisms of FGF-mediated alveolar formation. METHODS Compound FGFR3/FGFR4-deficient mice were assessed for temporal changes in lung growth, airspace morphometry, and genome-wide expression. Observed gene expression changes were validated using quantitative real-time RT-PCR, tissue biochemistry, histochemistry, and ELISA. Autocrine and paracrine regulatory mechanisms were investigated using isolated lung mesenchymal cells and type II pneumocytes. MEASUREMENTS AND MAIN RESULTS Quantitative analysis of airspace ontogeny confirmed a failure of secondary crest elongation in compound mutant mice. Genome-wide expression profiling identified molecular alterations in these mice involving aberrant expression of numerous extracellular matrix molecules. Biochemical and histochemical analysis confirmed changes in elastic fiber gene expression resulted in temporal increases in elastin deposition with the loss of typical spatial restriction. No abnormalities in elastic fiber gene expression were observed in isolated mesenchymal cells, indicating that abnormal elastogenesis in compound mutant mice is not cell autonomous. Increased expression of paracrine factors, including insulin-like growth factor-1, in freshly-isolated type II pneumocytes indicated that these cells contribute to the observed pathology. CONCLUSIONS Epithelial/mesenchymal signaling mechanisms appear to contribute to FGFR-dependent alveolar elastogenesis and proper airspace formation.

Genome-Wide Transcriptional Profiling Reveals Connective Tissue Mast Cell Accumulation in Bronchopulmonary Dysplasia

RATIONALE Bronchopulmonary dysplasia (BPD) is a major complication of premature birth. Risk factors for BPD are complex and include prenatal infection and O(2) toxicity. BPD pathology is equally complex and characterized by inflammation and dysmorphic airspaces and vasculature. Due to the limited availability of clinical samples, an understanding of the molecular pathogenesis of this disease and its causal mechanisms and associated biomarkers is limited. OBJECTIVES Apply genome-wide expression profiling to define pathways affected in BPD lungs. METHODS Lung tissue was obtained at autopsy from 11 BPD cases and 17 age-matched control subjects without BPD. RNA isolated from these tissue samples was interrogated using microarrays. Standard gene selection and pathway analysis methods were applied to the data set. Abnormal expression patterns were validated by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS We identified 159 genes differentially expressed in BPD tissues. Pathway analysis indicated previously appreciated (e.g., DNA damage regulation of cell cycle) as well as novel (e.g., B-cell development) biological functions were affected. Three of the five most highly induced genes were mast cell (MC)-specific markers. We confirmed an increased accumulation of connective tissue MC(TC) (chymase expressing) mast cells in BPD tissues. Increased expression of MC(TC) markers was also demonstrated in an animal model of BPD-like pathology. CONCLUSIONS We present a unique genome-wide expression data set from human BPD lung tissue. Our data provide information on gene expression patterns associated with BPD and facilitated the discovery that MC(TC) accumulation is a prominent feature of this disease. These observations have significant clinical and mechanistic implications.

The genome-wide transcriptional response to neonatal hyperoxia identifies Ahr as a key regulator.

Premature infants requiring supplemental oxygen are at increased risk for developing bronchopulmonary dysplasia (BPD). Rodent models involving neonatal exposure to excessive oxygen concentrations (hyperoxia) have helped to identify mechanisms of BPD-associated pathology. Genome-wide assessments of the effects of hyperoxia in neonatal mouse lungs could identify novel BPD-related genes and pathways. Newborn C57BL/6 mice were exposed to 100% oxygen for 10 days, and whole lung tissue RNA was used for high-throughput, sequencing-based transcriptomic analysis (RNA-Seq). Significance Analysis of Microarrays and Ingenuity Pathway Analysis were used to identify genes and pathways affected. Expression patterns for selected genes were validated by qPCR. Mechanistic relationships between genes were further tested in cultured mouse lung epithelial cells. We identified 300 genes significantly and substantially affected following acute neonatal hyperoxia. Canonical pathways dysregulated in hyperoxia lungs included nuclear factor (erythryoid-derived-2)-like 2-mediated oxidative stress signaling, p53 signaling, eNOS signaling, and aryl hydrocarbon receptor (Ahr) pathways. Cluster analysis identified Ccnd1, Cdkn1a, and Ahr as critical regulatory nodes in the response to hyperoxia, with Ahr serving as the major effector node. A mechanistic role for Ahr was assessed in lung epithelial cells, and we confirmed its ability to regulate the expression of multiple hyperoxia markers, including Cdkn1a, Pdgfrb, and A2m. We conclude that a global assessment of gene regulation in the acute neonatal hyperoxia model of BPD-like pathology has identified Ahr as one driver of gene dysregulation.

Airway epithelial cell PPARγ modulates cigarette smoke-induced chemokine expression and emphysema susceptibility in mice.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent, chronic inflammatory lung disease with limited existing therapeutic options. While modulation of peroxisome proliferator-activating receptor (PPAR)-γ activity can modify inflammatory responses in several models of lung injury, the relevance of the PPARG pathway in COPD pathogenesis has not been previously explored. Mice lacking Pparg specifically in airway epithelial cells displayed increased susceptibility to chronic cigarette smoke (CS)-induced emphysema, with excessive macrophage accumulation associated with increased expression of chemokines, Ccl5, Cxcl10, and Cxcl15. Conversely, treatment of mice with a pharmacological PPARγ activator attenuated Cxcl10 and Cxcl15 expression and macrophage accumulation in response to CS. In vitro, CS increased lung epithelial cell chemokine expression in a PPARγ activation-dependent fashion. The ability of PPARγ to regulate CS-induced chemokine expression in vitro was not specifically associated with peroxisome proliferator response element (PPRE)-mediated transactivation activity but was correlated with PPARγ-mediated transrepression of NF-κB activity. Pharmacological or genetic activation of PPARγ activity abrogated CS-dependent induction of NF-κB activity. Regulation of NF-κB activity involved direct PPARγ-NF-κB interaction and PPARγ-mediated effects on IKK activation, IκBα degradation, and nuclear translocation of p65. Our data indicate that PPARG represents a disease-relevant pathophysiological and pharmacological target in COPD. Its activation state likely contributes to NF-κB-dependent, CS-induced chemokine-mediated regulation of inflammatory cell accumulation.

MicroRNA expression profiling defines the impact of electronic cigarettes on human airway epithelial cells.

While all forms of tobacco exposure have negative health effects, the significance of exposure to electronic cigarettes (eCig) is not fully understood. Here, we studied the global effects of eCig on the micro RNA (miRNA) transcriptome in human lung epithelial cells. Primary human bronchial epithelial (NHBE) cells differentiated at air-liquid interface were exposed to eCig liquid. Exposure of NHBE to any eCig liquid resulted in the induction of oxidative stress-response genes including GCLM, GCLC, GPX2, NQO1 and HO-1. Vaporization of, and/or the presence of nicotine in, eCig liquid was associated with a greater response. We identified 578 miRNAs dysregulated by eCig exposure in NHBE, and 125 miRNA affected by vaporization of eCig liquid. Nicotine containing eCig vapor displayed the most profound effects upon miRNA expression. We selected 8 miRNAs (29A, 140, 126, 374A, 26A-2, 147B, 941 and 589) for further study. We validated increased expression of multiple miRNAs, including miR126, following eCig exposure. We also found significant reduction in the expression of two miR126 target genes, MYC and MRGPRX3, following exposure. These data demonstrated that eCig exposure has profound effects upon gene expression in human lung epithelial cells, some of which are epigenetically programmed at the level of miRNA regulation.

Serpine2 deficiency results in lung lymphocyte accumulation and bronchus-associated lymphoid tissue formation

Serine proteinase inhibitor, clade E, member 2 (SERPINE2), is a cell‐ and extracellular matrix‐associated inhibitor of thrombin. Although SERPINE2 is a candidate susceptibility gene for chronic obstructive pulmonary disease, the physiologic role of this protease inhibitor in lung development and homeostasis is unknown. We observed spontaneous monocytic‐cell infiltration in the lungs of Serpine2‐deficient (SE2‐/‐) mice, beginning at or before the time of lung maturity, which resulted in lesions that resembled bronchus‐associated lymphoid tissue (BALT). The initiation of lymphocyte accumulation in the lungs of SE2‐/‐ mice involved the excessive expression of chemokines, cytokines, and adhesion molecules that are essential for BALT induction, organization, and maintenance. BALT‐like lesion formation in the lungs of SE2‐/‐ mice was also associated with a significant increase in the activation of thrombin, a recognized target of SE2, and excess stimulation of NF‐κB, a major regulator of chemokine expression and inflammation. Finally, systemic delivery of thrombin rapidly stimulated lung chemokine expression in vivo. These data uncover a novel mechanism whereby loss of serine protease inhibition leads to lung lymphocyte accumulation.—Solleti, S. K., Srisuma, S., Bhattacharya, S., Rangel‐Moreno, J., Bijli, K. M., Randall, T. D., Rahman, A., Mariani, T. J. Serpine2 deficiency results in lung lymphocyte accumulation and bronchus‐associated lymphoid tissue formation. FASEB J. 30, 2615‐2626 (2016). www.fasebj.org