A Novel Macrophage Containing Organotypic Small Intestinal Tissue for Modeling Gut Inflammation

Kevin Causey, Zachary Stevens, Hailey Scott, Timothy Landry, Yulia Kaluzhny, Anna Langerveld, Elisabeth Lehigh, Stephanie Wheeler, Alex Armento, and Seyoum Ayehunie

As gatekeepers of intestinal immunehomeostasis, macrophages play a critical role in inflammation in the gut. In this study, were constructed a new macrophage-containing primary cell-based full-thickness small intestinal(SMI+M) tissue model and characterized it for:1) macrophage incorporation (immunohistochemistry, IHC), 2) barrier properties(TEER), and 3)functionality by measuring inflammatory responses following exposure to ligands for TLR4 (lipopolysaccharide;LPS),NOD1(C12-iE-DAP)andNOD2(L18-MDP) either individually or synergistically. For identification of inflammatory responses, we utilized AffymetrixGeneChiparrays. Structurally, the SMI+M tissues have 3D polarity and their morphology and physiological barrier properties mimic those of native in vivo tissues. IHC of SMI+M tissues showed CD14 (macrophagemarker) cells in the underlying fibroblast/collagen layer. Using gene upregulation of >1.9 fold as a cut-off following ligand stimulation, tissues without macrophages (SMI-M) showed fewer upregulated genes (1400genes) compared to SMI+M (4400genes). Furthermore, when gene upregulation levels by ligand-induced SMI+M were compared to stimulated SMI-M, even higher differences in upregulated genes (>5200genes) were noted. In SMI+M, upregulated genes include chemokines, chemokine receptors, FC receptors, co-stimulatory molecules, interferons, and HLA’s which are characteristic of immune cells. When we looked at 15 upregulated genes (>6-fold) in SMI+M, the synergistic effect of ligands in inducing inflammatory gene upregulation was much more pronounced compared to stimulated SMI-M tissues. Furthermore, increased cytokine release of IL-6, IL-8, and TNF-α into the culture medium from ligand exposed SMI+M was also confirmed by BioPlex ELISA. In summary, our results demonstrate that the 3D SMI+M tissue model can serve as an in vitro tool to study the complex cellular interactions manifested during inflammation in the gut microenvironment.


EpiIntestinal, SMI-200-FT, Macrophages, TLR4, lipopolysaccharide; LPS, NOD1, C12-iE-DAP, NOD2, L18-MDP, gene analysis, chemokines, chemokine receptors, FC receptors, co-stimulatory molecules, interferons, HLA, IL-6, IL-8, TNF-a, caspase 5, CCL20, , CCL3, CCL3L3, CCL3L1, CEACAM7, CFB complement factor B, CXCL1, CXCL3, CXCL8, IL23A, lipopolysaccharide binding protein

Materials Tested


Request a copy of this paper, click here.