An Inflamed Human Alveolar Model for Testing the Efficiency of Anti-inflammatory Drugs in vitro

Abstract

A large number of prevalent lung diseases is associated with tissue inflammation. Clinically, corticosteroid therapies are applied systemically or via inhalation for the treatment of lung inflammation, and a number of novel therapies are being developed that require preclinical testing. In alveoli, macrophages and dendritic cells play a key role in initiating and diminishing pro-inflammatory reactions and, in particular, macrophage plasticity (M1 and M2 phenotypes shifts) has been reported to play a significant role in these reactions. Thus far, no studies with in vitro lung epithelial models have tested the comparison between systemic and direct pulmonary drug delivery. Therefore, the aim of this study was to develop an inflamed human alveolar epithelium model and to test the resolution of LPS-induced inflammation in vitro with a corticosteroid, methylprednisolone (MP). A specific focus of the study was the macrophage phenotype shifts in response to these stimuli. First, human monocyte-derived macrophages were examined for phenotype shifts upon exposure to lipopolysaccharide (LPS), followed by treatment with MP. A multicellular human alveolar model, composed of macrophages, dendritic cells, and epithelial cells, was then employed for the development of inflamed models. The models were used to test the anti-inflammatory potency of MP by monitoring the secretion of pro-inflammatory mediators (interleukin [IL]-8, tumor necrosis factorα [TNF-α], and IL-1β) through four different approaches, mimicking clinical scenarios of inflammation and treatment. In macrophage monocultures, LPS stimulation shifted the phenotype towards M1, as demonstrated by increased release of IL-8 and TNF-α and altered expression of phenotype-associated surface markers (CD86, CD206). MP treatment of inflamed macrophages reversed the phenotype towards M2. In multicellular models, increased pro-inflammatory reactions after LPS exposure were observed, as demonstrated by protein secretion and gene expression measurements. In all scenarios, among the tested mediators the most pronounced anti-inflammatory effect of MP was observed for IL-8. Our findings demonstrate that our inflamed multicellular human lung model is a promising tool for the evaluation of anti-inflammatory potency of drug candidates in vitro. With the presented setup, our model allows a meaningful comparison of the systemic vs. inhalation administration routes for the evaluation of the efficacy of a drug in vitro.

A High‐Throughput Distal Lung Air–Blood Barrier Model Enabled By Density‐Driven Underside Epithelium Seeding

“…[ 25 ] Some models also include cocultured or transmigrating immune cells that further enhance physiologic relevance. [ 26–30 ] However, these coculture models are almost exclusively in 6‐, 12‐, or 24‐well plates and in low‐throughput microfluidic devices that limit their utility. [ 31,32 ]…”

Modulating the Barrier Function of Human Alveolar Epithelial (hAELVi) Cell Monolayers as a Model of Inflammation

“…81 Drasler et al investigated the anti-inflammatory effect of a reduced secretion of IL-8, after the methylprednisolone treatment of a multicellular human alveolar model consisting of macrophage, dendritic and epithelial cells. 25 In June 2020, dexamethasone was reported to reduce by one-third the 28-day mortality of Covid-19 patients with severe disease progression that were undergoing intensive mechanical ventilation. 82 Future studies should determine whether improved barrier properties in our inflammation model, as characterised by a higher TEER (up to 5000 Ω · cm 2 ; see Figure 5a), could be mediated and stabilised by treatment with corticosteroids such as dexamethasone or methylprednisolone.…”

“…primary cells, A549, Calu-3, BEAS-2B or CFTE29o- cells), macrophages and dendritic cells are suitable for investigating pulmonary fibrosis or the efficacy of anti-inflammatory drugs in vitro . 25 , 26 These sophisticated cell culture models can be used as part of lung-on-a-chip systems, 26 with a more physiological reconstitution of the lung being feasible, but they still lack standardised end-user formats. 27…”

From preclinical to clinical models of acute respiratory distress syndrome

“…In vitro monocultures can also be used to study important cellular functions, such as wound healing after a scratch assay [64] or transepithelial fluid transport by alveolar epithelial cells (often called "alveolar fluid clearance" in vivo), using transwell experiments [65,66]. For example, monolayer cultures of commercialized, immortalized or primary isolated alveolar epithelial type I (AT I) or type II (AT II) cells have been used to mimic the alveolar epithelium and its barrier function [67]). Non-sterile inflammation was first studied in 2D monoculture or classical culture models exposed to LPS in vitro, mimicking the clinical infection with Gramnegative bacteria [68][69][70][71][72].…”

“…Unlike 2D cultures, co-culture or multicellular systems can model complex interactions between different cell types in a more relevant environment, such as a model of alveolar-capillary barrier using epithelial and endothelial cells [81,82]. For example, a 3D multicellular model composed of an alveolar epithelial cell layer cultured in interaction with alveolar macrophages on one side and monocyte-derived dendritic cells on the other has been recently described [67,81,83]. Ex vivo organoid cultures have also been proposed to better model the multiple features of ARDS.…”