Additional file 5: Table E4. of RNA-sequencing analysis of lung primary fibroblast response to eosinophil-degranulation products predicts downstream effects on inflammation, tissue remodeling and lipid metabolism

Upstream Regulators of the genes listed in our dataset (target genes, Additional file 3: Table E2 and Additional file 4: Table E3) as predicted by IPA analysis. (PDF 141 kb

Additional file 9: Figure E3. of RNA-sequencing analysis of lung primary fibroblast response to eosinophil-degranulation products predicts downstream effects on inflammation, tissue remodeling and lipid metabolism

Global Molecular Network #2 generated by IPA downstream analysis. Interactions between the genes of our dataset #1 (300 genes) in relation with other neighboring genes present in a Global Molecular Network. Thirty-five total genes or group of genes are present in each network. This network characterized genes related to cellular movement, hematological system development and function and immune cell trafficking. It includes 15 genes and 4 groups from our dataset. (PDF 332 kb

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31 octobre 18791879/10/31 (A8,N2098)-1879/10/31.Appartient à l’ensemble documentaire : PoitouCh

Semaphorin-7A expression is inversely correlated with periostin expression in HLF.

<p>Expression of semaphorin-7A (sema7A), periostin, fibronectin and collagen-1 (COL1A1) were measured by real-time PCR in fibroblasts from non-fibrotic (●, n = 7) and IPF lung (■, n = 6). Semaphorin-7A expression was inversely associated with periostin expression level (<i>r</i>p = -0.608, <i>p</i> = 0.027, n = 13).</p

Endogenous Semaphorin-7A Impedes Human Lung Fibroblast Differentiation

<div><p>Semaphorin-7A is a glycosylphosphatidylinositol-anchored protein, initially characterized as an axon guidance protein. Semaphorin-7A also contributes to immune cell regulation and may be an essential pro-fibrotic factor when expressed by non-fibroblast cell types (exogenous). In mouse models, semaphorin-7A was shown to be important for TGF-ß1-induced pulmonary fibrosis characterized by myofibroblast accumulation and extracellular matrix deposition, but the cell-specific role of semaphorin-7A was not examined in fibroblasts. The purpose of this study is to determine semaphorin-7A expression by fibroblasts and to investigate the function of endogenously expressed semaphorin-7A in primary human lung fibroblasts (HLF).</p><p>Herein, we show that non-fibrotic HLF expressed high levels of cell surface semaphorin-7A with little dependence on the percentage of serum or recombinant TGF-ß1. Semaphorin-7A siRNA strongly decreased semaphorin-7A mRNA expression and reduced cell surface semaphorin-7A. Reduction of semaphorin-7A induced increased proliferation and migration of non-fibrotic HLF. Also, independent of the presence of TGF-ß1, the decline of semaphorin-7A by siRNA was associated with increased α-smooth muscle actin production and gene expression of periostin, fibronectin, laminin, and serum response factor (SRF), indicating differentiation into a myofibroblast. Conversely, overexpression of semaphorin-7A in the NIH3T3 fibroblast cell line reduced the production of pro-fibrotic markers. The inverse association between semaphorin-7A and pro-fibrotic fibroblast markers was further analyzed using HLF from idiopathic pulmonary fibrosis (IPF) (n = 6) and non-fibrotic (n = 7) lungs. Using these 13 fibroblast lines, we observed that semaphorin-7A and periostin expression were inversely correlated. In conclusion, our study indicates that endogenous semaphorin-7A in HLF plays a role in maintaining fibroblast homeostasis by preventing up-regulation of pro-fibrotic genes. Therefore, endogenous and exogenous semaphorin-7A may have opposite effects on the fibroblast phenotype.</p></div

Semaphorin-7A diminishes HLF proliferation and migration.

<p>HLF derived from non-fibrotic lungs were treated with either control or semaphorin-7A siRNA and cultured in 1% or 10% FBS for 48 h. A/ BrdU was incubated with cells for 6 h. Absorbance was measured using a spectrophotometric plate reader at dual wavelengths of 450–550 nm. Each condition was performed in quadruplicate (4 wells) and graphs show a mean ± SEM of 3 experiments. B/ Cells were resuspended in 0.1% FBS and migration on plastic or plexin-C1 (10 μg/ml) toward medium with 10% FBS was measured after 20 h. Graph shows an average ± SEM of 4 experiments, and * indicates statistical difference between control-siRNA and sema7A-siRNA-treated HLF.</p

Semaphorin-7A inhibits the expression of myofibroblast markers.

<p>Non-fibrotic HLF were treated with control-siRNA (C), sema7A-siRNA (S) or plexin C1-siRNA (P) for 24 h before starvation in BSA for 24 h. Then, the cells were either kept in BSA or activated with TGF-ß (1 ng/ml) for 20 h. Real-time PCR was used to measure the level of expression of the indicated genes. For each gene, the first 2 graphs show the difference between C, S or P treatment, with C fixed at 1. The third graph displays the difference between TGF-ß and BSA after treatment with the control-siRNA. Graphs are an average of 3 experiments. <i>P</i> values from ANOVA analyses are shown. # indicates a statistical difference between TGF-ß and BSA.</p

Semaphorin-7A reduces serum-induced ERK and p38 phosphorylation in HLF.

<p><b>HLF derived from non-fibrotic lungs</b> were treated with control-siRNA (Ctrl.) or semaphorin7A-siRNA (sema7A) for 24 h before starvation in BSA for 24 h. A/ Then cells were activated with 1% or 10% FBS for 15 min. ERK phosphorylation (P-ERK) was analyzed by western-blot. The graph shows ratios of phospho-ERK versus total ERK1, and is an average of 3 experiments. * indicates that the increase of phospho-ERK after semaphorin-7A (Sema7A)-siRNA-treatment is statistically different from phospho-ERK after control-siRNA (Ctrl) treatment. B/ Cells were activated with 1% FBS for 30 min and a representative blot of 2 experiments shows phosphorylation of p38 (P-p38). C/ Cells were treated with the indicated siRNA and cultured for 48 h in 1% FBS along with a phospho-ERK inhibitor (U0126) or its inactive analog (U0124) during the last 24 h. Proliferation was determined by adding BrdU in the last 6 h of the culture. One representative experiment of 2 using 2 different HLF lines is shown. Average ±SEM of 4 wells is shown.</p

Semaphorin-7A blocks α-SMA and periostin production in HLF.

<p>HLF derived from non-fibrotic lungs were treated with the indicated siRNAs for 24 h. HLF were subsequently starved in BSA for 24 h before either adding TGF-ß (1 ng/ml) or left in BSA for 20 h. The amounts of α-SMA and periostin in the cell lysates were determined by western-blot. A/ Graph shown is an average of 3 experiments, and * indicates that control (Ctrl) siRNA-treated HLF produce significantly less α-SMA than sema7A-siRNA-treated HLF. B/ Amount of intracellular periostin is representative of 2 experiments.</p

DataSheet1_An open microfluidic coculture model of fibroblasts and eosinophils to investigate mechanisms of airway inflammation.PDF

Interactions between fibroblasts and immune cells play an important role in tissue inflammation. Previous studies have found that eosinophils activated with interleukin-3 (IL-3) degranulate on aggregated immunoglobulin G (IgG) and release mediators that activate fibroblasts in the lung. However, these studies were done with eosinophil-conditioned media that have the capacity to investigate only one-way signaling from eosinophils to fibroblasts. Here, we demonstrate a coculture model of primary normal human lung fibroblasts (HLFs) and human blood eosinophils from patients with allergy and asthma using an open microfluidic coculture device. In our device, the two types of cells can communicate via two-way soluble factor signaling in the shared media while being physically separated by a half wall. Initially, we assessed the level of eosinophil degranulation by their release of eosinophil-derived neurotoxin (EDN). Next, we analyzed the inflammation-associated genes and soluble factors using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and multiplex immunoassays, respectively. Our results suggest an induction of a proinflammatory fibroblast phenotype of HLFs following the coculture with degranulating eosinophils, validating our previous findings. Additionally, we present a new result that indicate potential impacts of activated HLFs back on eosinophils. This open microfluidic coculture platform provides unique opportunities to investigate the intercellular signaling between the two cell types and their roles in airway inflammation and remodeling.</p