High-Throughput Human Gut Immune Co-Culture Model for Evaluating Inflammatory Bowel Disease Anti-Inflammatory Therapies

Abstract

Current treatments for inflammatory bowel disease (IBD) are often ineffective long-term, as many patients ultimately become unresponsive to anti-inflammatory drugs. The need for improved therapeutics is urgent. Animal models utilized for drug development are limited by interspecies variability and poor translatability. However, most in vitro models lack the sophistication to model the key interplay of the immune system with the intestinal epithelium in line with the known role of the immune system in the etiology of the disease. To address this gap, we developed a primary intestinal epithelial cell co-culture system to incorporate elements of innate immune signaling. This system models immune-epithelial interactions using RepliGut(R) - Planar Transverse Colon cultured on a Transwell system with THP-1 derived macrophages in a receiver compartment of a 96-well plate. Epithelial barrier integrity and cell viability were maintained in co-culture with unstimulated macrophages. However, similar to the pathology associated with IBD, epithelial integrity was compromised in co-culture with LPS + IFN-{gamma} pre-stimulated macrophages as evidenced by declining TEER and cell viability and increased inflammatory cytokine release. Cotreatment with anti-inflammatory IBD therapeutics adalimumab or tofacitinib mitigated these effects, demonstrating the models ability to replicate key inflammatory responses and prevention. Reproducibility and scalability of the model system further position the model for high-throughput screening of anti-inflammatory drugs, improving drug discovery, and accelerating the translation of new IBD therapies into clinical practice. HighlightsO_LICo-culture model: RepliGut(R) - Planar Transverse Colon with THP-1 derived macrophages C_LIO_LIHigh throughput and human-relevant model C_LIO_LI"Healthy" co-culture resembling healthy intestine C_LIO_LI"Inflamed" co-culture mimicking IBD innate inflammatory signaling C_LIO_LIPotential to screen anti-inflammatory drugs relevant to IBD C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/654072v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@101a675org.highwire.dtl.DTLVardef@157605borg.highwire.dtl.DTLVardef@1770c97org.highwire.dtl.DTLVardef@dbc9c0_HPS_FORMAT_FIGEXP M_FIG Gut-immune co-culture model simulating healthy and inflamed intestine. The immune co-culture model consists of mature differentiated primary human transverse colon epithelial cells cultured on a 96-well Transwell(R) plate with macrophage differentiated THP-1 cells (THP-1m) cultured in the receiver plate. In this configuration, the THP-1m are located basally to the epithelial cells, allowing for apical treatment in the transwell and basal treatment in the receiver plate. In the unstimulated state, intestinal cells and immune cells maintain a stable co-culture. Upon stimulation with LPS and IFN-y, both cell types initiate an inflammatory response that results in release of cytokines, loss of intestinal barrier integrity, and cytotoxicity. C_FIG