MODELING INFLAMMATORY BOWEL DISEASE PROGRESSION IN HUMAN ORGAN-CHIPS

Background: Abdominal inflammatory pain is a common and persistent symptom in inflammatory bowel disease (IBD, up to 60% of patients experience chronic abdominal pain regardless of disease activity). Pain can arise from different mechanisms and persists due to changes in afferents sensory neurons activating threshold. A key role in this process is played by a protease activated receptor 2 (PAR2), a G-Protein-Coupled Receptors (GPCRs), which upon activation on the plasma membrane internalize in endosomes where it continues to signal. AIM: To test the hypothesis that blocking PAR2 endosomal signaling can reduce inflammation and pain in preclinical rodent models of IBD. Methods: T-84 and HEK293 cells were used to assess PAR2 internalization, activation and signaling blockage in intracellular compartments. DSS cand TNBS models of colitis were used to characterize colonocytes PAR2 trafficking from plasma membrane to endosome and to study the role of endosomal PAR2 persistent signaling in inflammatory pain in the colon of C57BL/6 and PAR2-GFP mice. Immunofluorescence was used to localize the receptor on the plasma membrane, and following DSS and TNBS, the trafficking of the receptor to endosome. Nanotechnology delivering cargo (PAMAM_G3-Az3451, a par2 antagonist) was used to specifically target the receptor in the endosome and inhibit endosomal signaling to study the effect of persistent PAR2 endosomal signaling in preclinical rodents models of IBD.
Results: In vitro: in T-84 and HEK293 cells, upon stimulation with PAR2 agonist, the receptor internalize in endosomes and other intracellular compartments, PAMAM_G3-AZ3451 inhibit receptor to signal from endosome, while the "free" AZ3451 drug inhibit plasma membrane signal but failed to inhibit endosomal PAR2 signal. In vivo: PAR2 is activated and internalized into endosome in a DSS and TNBS preclinical models of IBD. PAMAM_G3-AZ3451 nanoparticles, injected by enema into the colonic lumen are able to inhibit sustained PAR2 endosomal signaling, abdominal pain (measured using abdominal Von Frey filament stimulation) and to reduce proinflammatory cytokine levels (measured using qRT-PCR), free drug instead has little or no effect. Summary and Conclusion: in IBD, PAR2 receptor is activated and internalized to endosome, where it continues to signal persistent inflammation and pain. inhibition of endosomal signaling using smart delivery system, blocked PAR2 endosomal signaling. Inflammation and pain were greatly reduced. Drugs should target the receptor not only on the plasma membrane, where the signal is quickly desensitized, but also in intracellular compartments where the signal is sustained. Many GPCRs FDA approved drugs to treat pain, fail to cure chronic pain. A better understanding of GPCRs endosomal signaling is may needed to improve management of chronic pain and to discover new therapies.

Background: The circuitous network of anatomical, cellular, and molecular connections between the central and enteric nervous systems, gastrointestinal (GI) tract, and hepatobiliary (HB) system, is known as the gut-liver-brain axis, or simply the gut-brain axis (GBA). GBA dysregulation and the resulting neuroimmunoendocrine imbalance lead to symptoms such as abdominal pain, diarrhea, swallowing problems, and itching, which are associated with disorders including inflammatory bowel disease (IBD) and certain fibrotic conditions. These symptoms are also consistent with increased mast cell activity. Mast Cells (MCs) are emerging as a key component of the GBA. Triggered by external stressors, MCs can amplify and advance inflammatory and fibrotic responses through the release and activation of proteases, which in turn stimulate the production of inflammatory cytokines, alter epithelial barrier integrity, promote infiltration of immune cells, and activate fibrotic factors. The complexity of the GBA and its impact on GI disorders provide a clear rationale to deploy AI to develop precision therapeutics. Methods: We utilized a proprietary AI/ML platform that uses natural language processing (NLP) algorithms to identify therapeutic targets downstream of MC activation, as well as candidate GI and HB indications for a MC modulator. This platform was trained on hundreds of thousands of sentences and extracts biological entities, including cell types, genes, and diseases, and scores both directional and inferred relationships between entities from the literature to build testable hypotheses. Results: Our platform identified high-scoring associations between MC activation and downstream inflammatory/fibrotic targets including TNF, IL6, IL1β, TGFβ, and chymase. We selected chymase for indication prioritization based on its preferential expression by MCs and NLP scores. By combining the NLP scores for MC activation to GI and HB indications with unmet medical need analysis, we identified IBD and certain fibrotic conditions as candidate indications. Using a chymase inhibitor, we validated our in-silico results in vivo in mouse models. In both cases we observed significant symptom amelioration and reduction in markers of inflammation for certain fibrotic conditions, suggesting chymase inhibition as an attractive therapeutic avenue for these chronic conditions. Conclusions: Using our AI platform, we identified chymase as a candidate therapeutic target for selected GBA-related disorders. Our preliminary preclinical data support a significant role for chymase in IBD.

Background: Insulin resistance is a common feature of type 2 diabetes (T2D), obesity, and non-alcoholic fatty liver disease (NAFLD) and is defined as a blunted response to insulin, resulting in impaired insulin signaling. Recent reports indicate that diverse signaling pathways utilize the properties of biomolecular condensates to accomplish their tasks. Biomolecular condensates are cellular compartments wherein proteins and nucleic acids concentrate without being physically delimitated by a membrane. The previous evidence that diverse signaling pathways involve the formation of biomolecular condensates and that chronic signaling can alter the material properties of signaling condensates led us to consider a condensate model for Insulin receptor (IR) signaling and its dysfunction in insulin resistance.

Methods: To study the behavior of IR within condensates, endogenous IR fused to a fluorescent protein was visualized by super-resolution microscopy and single-molecule localization microscopy in engineered liver cell lines. Results were validated in primary human hepatocyte spheroids, primary human adipocytes, and human liver tissue.

Results: We have found that IR is incorporated into liquid-like condensates at the plasma membrane, in the cytoplasm and in the nucleus of human hepatocytes and adipocytes. Insulin stimulation promotes further incorporation of IR into these dynamic condensates in insulin-sensitive cells but not in insulin-resistant cells, where IR accumulation, activity, and dynamic behavior in condensates are reduced. Treatment of insulin-resistant cells with metformin, a first-line drug used to treat type 2 diabetes, can rescue IR accumulation, activity and the dynamic behavior of these condensates. This rescue is associated with metformin’s role in reducing reactive oxygen species that interfere with normal condensate dynamics. These results indicate that changes in the physico-mechanical features of IR condensates contribute to insulin resistance and have implications for improved therapeutic approaches.

Conclusions: Our results show that IR is incorporated into biomolecular condensates during the response to insulin stimulation and that changes in the physico-mechanical features of IR condensates contribute to insulin resistance. These results have implications for development of novel therapeutics for T2D, NAFLD, and other diseases that involve condensate dysregulation. For example, the condensate assays described here might be leveraged to develop new therapeutics that improve clinical outcomes for patients. Such therapeutics might also provide benefits to patients with other diseases where condensate dysregulation is also thought to play a role.

Patients with inflammatory bowel disease (IBD) have regions of their intestine that experience altered barrier function and increased inflammation leading to active flares of abdominal pain, cramping, and bleeding alternating with symptom-free periods, and some of these lesions develop into cancers over time. As this disease emerges in local regions of the intestine, the tissue microenvironment, and particularly stromal-epithelial interactions have been implicated as potential contributors to IBD as well as cancer formation. To gain additional insight into this disease, we leveraged human organ-on-a-chip (Organ Chip) microfluidic culture technology to engineer human colon chips lined by intestinal epithelial cells isolated from healthy or IBD patient-derived colon organoids interfaced with stromal fibroblasts isolated from the same surgical resections. We have shown that healthy human Colon Chips reconstitute normal tissue morphology, exhibit physiological tissue barrier integrity, and secrete a mucus layer with a thickness and microstructure similar to that observed in vivo. In contrast, the IBD Colon Chip exhibited multiple features reminiscent of inflamed regions of the IBD colon, including increased barrier permeability, inflammatory cytokine secretion, and stromal collagen production with decreased mucus production. (Figure 1). The transcriptomic analysis also revealed elevated expression of genes involved in both inflammatory and wound-healing responses. Interestingly, when we generated heterotypic tissue recombinants on-chip, we found that combining the IBD stroma with a healthy epithelium results in an IBD-like phenotype, including increased epithelial expression of acute and chronic inflammation-related genes in the epithelium and compromised barrier function. Moreover, these features normally exhibited by IBD epithelium when in contact with IBD stroma, could be partially reversed (normalized) by replacing the stroma with fibroblasts isolated from healthy normal colon. As peristalsis can be suppressed in IBD patients due to inflammation and fibrosis, we also carried out studies to explore the effects of culturing the cells in the presence or absence of cyclic mechanical distortion. We also found peristalsis-like mechanical strain upregulated acute and chronic inflammatory cytokines and genes, which proposes peristalsis in IBD may be influential on its progression. Finally, when we exposed homotypic healthy and IBD Colon Chips to the known carcinogen ENU, we found that the IBD chips were more sensitive to this mutagen as they displayed increased disruption of cell-cell junctions and β-catenin localization in the cytosol as well as increased gene reduplication compared to health Colon Chips. These data show that Colon Chip can be used to study the role of stromal-epithelial interactions in IBD pathogenesis and early cancer progression.