[Solidworks 2019 SP1 Activator SSQ 64 Bit
Eliora Shopbell
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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.
Airway inflammation is associated with a number of pulmonary diseases, including asthma (Louis et al., 2000; Angelis et al., 2014). In particular, eosinophilic airway inflammation is found in up to 80% of people with asthma and 40% with chronic obstructive pulmonary disease, indicated by an increased eosinophil count in their sputum (Pavord, 2013). Eosinophils are immune cells that play an essential role in tissue inflammation and remodeling (Lee et al., 2010). These cells can be activated to undergo degranulation and cytolysis, releasing cytotoxic granule proteins and a variety of proinflammatory soluble mediators that impact surrounding cells, including fibroblasts (Acharya and Ackerman, 2014; Spencer et al., 2014; Esnault et al., 2020). Fibroblasts are mesenchymal cells often found in connective tissues that provide structural support in various organs (Plikus et al., 2021). They play a key role in the immune response and wound healing via the uptake and secretion of various inflammatory signals (Desjardins-Park et al., 2018; Davidson et al., 2021).
In asthma, chronic airway inflammation is accompanied by the activation and differentiation of lung fibroblasts, which eventually leads to enhanced subepithelial thickness, fibrosis, and irreversible airway obstruction (Al-Muhsen et al., 2011; Torr et al., 2015; Mostao-Guidolin et al., 2019). Eosinophils release cytokines that promote fibroblast activation, including transforming growth factor β (TGF-β) and interleukin-1 β (IL-1β) (Hinz et al., 2007; Esnault et al., 2012; Mia et al., 2014; McBrien and Menzies-Gow, 2017). Previous work has shown that eosinophil-mediated signaling could lead to either pro-inflammatory or pro-fibrotic fibroblast phenotypes (Phipps et al., 2002; Bernau et al., 2018). However, these studies did not employ ex vivo coculture of primary human eosinophils with primary human lung fibroblasts (HLFs), which may exhibit important differential signaling processes compared to these previous studies. Thus, coculture methods of these primary cells provide a unique advantage in the investigation of the potential mechanisms of airway inflammation via fibroblast activation.
We previously reported an ex vivo eosinophil degranulation model, where primary human blood eosinophils activated with interleukin-3 (IL-3) can robustly degranulate and lyse on well plates coated with heat aggregated immunoglobulin G (HA-IgG) (Esnault et al., 2017b). To study the impact of the degranulation products and cellular content from eosinophils on HLFs, we previously isolated supernatants from eosinophils in that model, and exposed them to primary lung fibroblasts, including HLFs and human bronchial fibroblasts (HBFs) (Esnault et al., 2017a; Bernau et al., 2018, Bernau et al., 2021). In these previous studies, we observed a proinflammatory fibroblast phenotype after the treatment with eosinophil supernatants (Esnault et al., 2017a; Bernau et al., 2018, Bernau et al., 2021). However, these studies were performed using eosinophil-conditioned media collected from these cells after they had degranulated and lysed, which only allowed one-way signaling from eosinophils to fibroblasts. This approach lacked signaling from fibroblasts to eosinophils, as well as the real-time assessment of cell-cell soluble factor bidirectional signaling.
Microfluidic coculture offers several advantages for studying cell-cell signaling over conventional approaches, such as conditioned media or Transwell inserts (Bhatia and Ingber, 2014; Li et al., 2016). Besides its ability to realize bidirectional communication in coculture, the versatile configurations of microfluidic coculture platforms provide more accurate recapitulation of the microenvironment (Young and Beebe, 2010); the flexibility in designs also enables different cell seeding ratios, culture chamber dimensions and numbers with a potential for triculture. Additionally, microfluidic devices operate with less volume, which minimizes the use of expensive reagents and conserves precious primary samples isolated from patients (Sackmann et al., 2014). Moreover, the emerging open microfluidic technology provides easy accessibility and can be operated with a pipette in a standard biological laboratory without specialized equipment (Casavant et al., 2013; Berry et al., 2017; Humayun et al., 2018; lvarez-Garca et al., 2018; Berthier et al., 2019; Lee et al., 2019). In light of this, we recently developed a novel open microfluidic coculture device using the common cell culture material polystyrene, where two culture chambers are separated by a half wall, the connection of which can be temporally controlled to allow soluble factor signaling via a liquid bridge (Zhang et al., 2020) (Figure 1). The current study builds on our prior work using this device to study cell signaling in the kidney (Zhang et al., 2020); and extends its application to include immune cell-fibroblast interactions, thus, establishing a new biological model. Further, in this work we expand the readouts possible with this device beyond fluorescence microscopy and gene expression reported in Zhang et al. (2020), demonstrating the ability to conduct phase contrast microscopy to study eosinophil morphology changes upon degranulation/cytolysis and monitor HLFs in real time (facilitated by both culture chambers being in the same focal plane). Further, the open nature of our microfluidic device (in contrast to conventional closed channels) allows for easy sampling and measurement of soluble factors using multiplexed immunoassays.
In this study, we adapt the open microfluidic coculture platform to establish a novel coculture model of degranulating eosinophils and primary HLFs to investigate the mechanisms of airway inflammation. We validate the model by characterizing eosinophil degranulation [release of eosinophil-derived neurotoxin (EDN)], HLF proinflammatory gene expression (interleukin 6 (IL6), C-X-C motif chemokine ligand 8 (CXCL8), and intercellular adhesion molecule 1 (ICAM1)), as well as soluble factors released in the culture media [interleukin-6 (IL-6) and interleukin-8 (IL-8)] that were previously found in the eosinophil-conditioned media studies (Esnault et al., 2017a; Bernau et al., 2018, Bernau et al., 2021). Additionally, we present a new analyte [granulocyte-macrophage colony-stimulating factor (GM-CSF)] found in our coculture system which has not been characterized by our group before. In the future, we will continue using this validated model to discover novel bidirectional signals and the consequences on the biology of both cell types that are conferred in cocultures. Although the present study uses eosinophils derived from participants with allergy and asthma, in the future, we envision that this coculture model system could be more broadly applied to other immune cells and organs (e.g., liver, breast, prostate, gastrointestinal tract). Taken together, we demonstrate that our open microfluidic coculture device can be effectively used to dissect soluble factor signaling mechanisms between immune cells and fibroblasts.
The open microfluidic coculture device (Figure 1) was designed with Solidworks (Solidworks, Waltham, MA). An engineering drawing of the device with dimensions and original design files are included in Supplementary Figure S1 and the Supplemental Materials of the previous work (Zhang et al., 2020). The coculture devices were fabricated using a DATRONneo (Datron Dynamics, Milford, NH) Computer Numerical Control (CNC) mill. Device top and bottom pieces were milled from 2-mm and 1.2-mm thick polystyrene (PS) sheets (Goodfellow United States, Coraopolis, PA), respectively. After milling, the pieces were cleaned thoroughly with pressurized air to remove most of the plastic particles. The top and bottom pieces were then further cleaned via sonication and solvent bonded together to form a complete device chip using previously established protocols (Young et al., 2013; Zhang et al., 2020) with a few modifications to improve the bonding efficiency as described in the following paragraphs.
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