Blood myeloid cells differentiate to lung resident cells and respond to pathogen stimuli in a 3D human tissue-engineered lung model.

Introduction: Respiratory infections remain a leading global health concern. Models that recapitulate the cellular complexity of the lower airway of humans will provide important information about how the immune response reflects the interactions between diverse cell types during infection. We developed a 3D human tissue-engineered lung model (3D-HTLM) composed of primary human pulmonary epithelial and endothelial cells with added blood myeloid cells that allows assessment of the innate immune response to respiratory infection. Methods: The 3D-HTLM consists of small airway epithelial cells grown at air-liquid interface layered on fibroblasts within a collagen matrix atop a permeable membrane with pulmonary microvascular endothelial cells layered underneath. After the epithelial and endothelial layers had reached confluency, an enriched blood monocyte population, containing mostly CD14+ monocytes (Mo) with minor subsets of CD1c+ classical dendritic cells (cDC2s), monocyte-derived dendritic cells (Mo-DCs), and CD16+ non-classical monocytes, was added to the endothelial side of the model. Results: Immunofluorescence imaging showed the myeloid cells migrate through and reside within each layer of the model. The myeloid cell subsets adapted to the lung environment in the 3D-HTLM, with increased proportions of the recovered cells expressing lung tissue resident markers CD206, CD169, and CD163 compared with blood myeloid cells, including a population with features of alveolar macrophages. Myeloid subsets recovered from the 3D-HTLM displayed increased expression of HLA-DR and the co-stimulatory markers CD86, CD40, and PDL1. Upon stimulation of the 3D-HTLM with the toll-like receptor 4 (TLR4) agonist bacterial lipopolysaccharide (LPS), the CD31+ endothelial cells increased expression of ICAM-1 and the production of IL-10 and TNFα was dependent on the presence of myeloid cells. Challenge with respiratory syncytial virus (RSV) led to increased expression of macrophage activation and antiviral pathway genes by cells in the 3D-HTLM. Discussion: The 3D-HTLM provides a lower airway environment that promotes differentiation of blood myeloid cells into lung tissue resident cells and enables the study of respiratory infection in a physiological cellular context.

A Synthetic Hydrogel, VitroGel® ORGANOID-3, Improves Immune Cell-Epithelial Interactions in a Tissue Chip Co-Culture Model of Human Gastric Organoids and Dendritic Cells.

Immunosurveillance of the gastrointestinal epithelium by mononuclear phagocytes (MNPs) is essential for maintaining gut health. However, studying the complex interplay between the human gastrointestinal epithelium and MNPs such as dendritic cells (DCs) is difficult, since traditional cell culture systems lack complexity, and animal models may not adequately represent human tissues. Microphysiological systems, or tissue chips, are an attractive alternative for these investigations, because they model functional features of specific tissues or organs using microscale culture platforms that recreate physiological tissue microenvironments. However, successful integration of multiple of tissue types on a tissue chip platform to reproduce physiological cell-cell interactions remains a challenge. We previously developed a tissue chip system, the gut organoid flow chip (GOFlowChip), for long term culture of 3-D pluripotent stem cell-derived human intestinal organoids. Here, we optimized the GOFlowChip platform to build a complex microphysiological immune-cell-epithelial cell co-culture model in order to study DC-epithelial interactions in human stomach. We first tested different tubing materials and chip configurations to optimize DC loading onto the GOFlowChip and demonstrated that DC culture on the GOFlowChip for up to 20 h did not impact DC activation status or viability. However, Transwell chemotaxis assays and live confocal imaging revealed that Matrigel, the extracellular matrix (ECM) material commonly used for organoid culture, prevented DC migration towards the organoids and the establishment of direct MNP-epithelial contacts. Therefore, we next evaluated DC chemotaxis through alternative ECM materials including Matrigel-collagen mixtures and synthetic hydrogels. A polysaccharide-based synthetic hydrogel, VitroGel®-ORGANOID-3 (V-ORG-3), enabled significantly increased DC chemotaxis through the matrix, supported organoid survival and growth, and did not significantly alter DC activation or viability. On the GOFlowChip, DCs that were flowed into the chip migrated rapidly through the V-ORG matrix and reached organoids embedded deep within the chip, with increased interactions between DCs and gastric organoids. The successful integration of DCs and V-ORG-3 embedded gastric organoids into the GOFlowChip platform now permits real-time imaging of MNP-epithelial interactions and other investigations of the complex interplay between gastrointestinal MNPs and epithelial cells in their response to pathogens, candidate drugs and mucosal vaccines.

p38 MAPK signaling mediates retinoic acid-induced CD103 expression in human dendritic cells.

Retinoic acid (RA) is an active derivative of vitamin A and a key regulator of immune cell function. In dendritic cells (DCs), RA drives the expression of CD103 (integrin αE ), a functionally relevant DC subset marker. In this study, we analyzed the cell type specificity and the molecular mechanisms involved in RA-induced CD103 expression. We show that RA treatment caused a significant up-regulation of CD103 in differentiated monocyte-derived DCs and blood DCs, but not in differentiated monocyte-derived macrophages or T cells. DC treatment with an RA receptor α (RARα) agonist led to an increase in CD103 expression similar to that in RA treatment, whereas RARA gene silencing with small interfering RNA blocked RA-induced up-regulation of CD103, pointing to a major role of RARα in the regulation of CD103 expression. To elucidate RA-induced signaling downstream of RARα, we used Western blot analysis of RA-treated DCs and showed a significant increase of p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, DCs cultured with RA and a p38 MAPK inhibitor had a significantly reduced expression of CD103 compared with DCs cultured with RA only, indicating that p38 MAPK is involved in CD103 regulation. In summary, these findings suggest that the RA-induced expression of CD103 is specific to DCs, is mediated primarily through RARα and involves p38 MAPK signaling.

CD103 (αE Integrin) Undergoes Endosomal Trafficking in Human Dendritic Cells, but Does Not Mediate Epithelial Adhesion.

Dendritic cell (DC) expression of CD103, the α subunit of αEß7 integrin, is thought to enable DC interactions with E-cadherin-expressing gastrointestinal epithelia for improved mucosal immunosurveillance. In the stomach, efficient DC surveillance of the epithelial barrier is crucial for the induction of immune responses to H. pylori, the causative agent of peptic ulcers and gastric cancer. However, gastric DCs express only low levels of surface CD103, as we previously showed. We here tested the hypothesis that intracellular pools of CD103 in human gastric DCs can be redistributed to the cell surface for engagement of epithelial cell-expressed E-cadherin to promote DC-epithelial cell adhesion. In support of our hypothesis, immunofluorescence analysis of tissue sections showed that CD103+ gastric DCs were preferentially localized within the gastric epithelial layer. Flow cytometry and imaging cytometry revealed that human gastric DCs expressed intracellular CD103, corroborating our previous findings in monocyte-derived DCs (MoDCs). Using confocal microscopy, we show that CD103 was present in endosomal compartments, where CD103 partially co-localized with clathrin, early endosome antigen-1 and Rab11, suggesting that CD103 undergoes endosomal trafficking similar to ß1 integrins. Dynamic expression of CD103 on human MoDCs was confirmed by internalization assay. To analyze whether DC-expressed CD103 promotes adhesion to E-cadherin, we performed adhesion and spreading assays on E-cadherin-coated glass slides. In MoDCs generated in the presence of retinoic acid, which express increased CD103, intracellular CD103 significantly redistributed toward the E-cadherin-coated glass surface. However, DCs spreading and adhesion did not differ between E-cadherin-coated slides and slides coated with serum alone. In adhesion assays using E-cadherin-positive HT-29 cells, DC binding was significantly improved by addition of Mn2+ and decreased in the presence of EGTA, consistent with the dependence of integrin-based interactions on divalent cations. However, retinoic acid failed to increase DC adhesion, and a CD103 neutralizing antibody was unable to inhibit DC binding to the E-cadherin positive cells. In contrast, a blocking antibody to DC-expressed E-cadherin significantly reduced DC binding to the epithelium. Overall, these data indicate that CD103 engages in DC-epithelial cell interactions upon contact with epithelial E-cadherin, but is not a major driver of DC adhesion to gastrointestinal epithelia.

Differential regulation of CD103 (αE integrin) expression in human dendritic cells by retinoic acid and Toll-like receptor ligands.

CD103 (αE integrin) is an important dendritic cell (DC) marker that characterizes functionally distinct DC subsets in mice and humans. However, the mechanism by which CD103 expression is regulated in human DCs and the role of CD103 for DC function are not very well understood. Here, we show that retinoic acid (RA) treatment of human monocyte-derived DCs (MoDCs) increased the ability of the DCs to synthesize RA and induced MoDC expression of CD103 and ß7 at the mRNA and protein level. In contrast, RA was unable to induce the expression of CD103 in primary human DCs isolated from the gastric mucosa. Inhibition of TGF-ß signaling in MoDCs down-regulated RA-induced CD103 expression, indicating that TGF-ß-dependent pathways contribute to the induction of CD103. Conversely, when RA-treated MoDCs were stimulated with live Helicobacter pylori, commensal bacteria, LPS, or a TLR2 agonist, the RA-induced up-regulation of CD103 and ß7 integrin expression was completely abrogated. To determine whether CD103 expression impacts DC priming of CD4+ T cells, we next investigated the ability of CD103+ and CD103─ DCs to induce mucosal homing and T cell proliferation. Surprisingly, RA treatment of DCs enhanced both α4ß7 expression and proliferation in cocultured T cells, but no difference was seen between RA-treated CD103+ and CD103─ DCs. In summary, our data demonstrate that RA, bacterial products, and the tissue environment all contribute to the regulation of CD103 on human DCs and that DC induction of mucosal homing in T cells is RA dependent but not CD103 dependent.