Development of a Gut-On-A-Chip Model for High Throughput Disease Modeling and Drug Discovery.

A common bottleneck in any drug development process is finding sufficiently accurate models that capture key aspects of disease development and progression. Conventional drug screening models often rely on simple 2D culture systems that fail to recapitulate the complexity of the organ situation. In this study, we show the application of a robust high throughput 3D gut-on-a-chip model for investigating hallmarks of inflammatory bowel disease (IBD). Using the OrganoPlate platform, we subjected enterocyte-like cells to an immune-relevant inflammatory trigger in order to recapitulate key events of IBD and to further investigate the suitability of this model for compound discovery and target validation activities. The induction of inflammatory conditions caused a loss of barrier function of the intestinal epithelium and its activation by increased cytokine production, two events observed in IBD physiopathology. More importantly, anti-inflammatory compound exposure prevented the loss of barrier function and the increased cytokine release. Furthermore, knockdown of key inflammatory regulators RELA and MYD88 through on-chip adenoviral shRNA transduction alleviated IBD phenotype by decreasing cytokine production. In summary, we demonstrate the routine use of a gut-on-a-chip platform for disease-specific aspects modeling. The approach can be used for larger scale disease modeling, target validation and drug discovery purposes.

A novel Dutch mutation in UNC13D reveals an essential role of the C2B domain in munc13-4 function.

BACKGROUND: UNC13D, encoding the protein munc13-4, is essential in intracellular trafficking and exocytosis of lytic granules. Mutations in this gene are associated with familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a genetically heterogeneous, rare autosomal recessive immune disorder. How mutations affect function of munc13-4 is poorly understood. Since 2006 we genetically identified seven FHL patients with mutations in UNC13D. PROCEDURES: Here, we report for the first time a c.2695C>T (p.Arg899X) mutation in exon 28 of UNC13D in three young unrelated Dutch patients. The mutation causes a premature stop codon and encodes munc13-4(1-899), which lacks the C-terminal C2 domain. Genealogical research and haplotyping of the patient families demonstrated that a single ancestral founder introduced the mutation in the Netherlands. We then characterized the mutant protein phenotypically in cell biological and immunological assays. RESULTS: Munc13-4(1-899) was correctly targeted to CD63-positive secretory lysosomes, although its stability was reduced and dynamic turnover on the granule membrane became uncoupled from receptor signaling. In accord, and in contrast to wild-type munc13-4, ectopically expressed mutant failed to rescue degranulation in cells with silenced endogenous munc13-4. CONCLUSIONS: The functional and clinical data showed that this novel Dutch founder mutation leads to severe early onset of FHL3 due to misfolding and degradation of munc13-4(1-899).

The munc13-4-rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane.

Cytotoxic T lymphocytes (CTLs) kill target cells through the polarized release of lytic molecules from secretory lysosomes. Loss of munc13-4 function inhibits this process and causes familial hemophagocytic lymphohistiocytosis type 3 (FHL3). munc13-4 binds rab27a, but the necessity of the complex remains enigmatic, because studies in knockout models suggest separate functions. In the present study, we describe a noncanonical rab27a-binding motif in the N-terminus of munc13-4. Point mutants in this sequence have severely impaired rab27a binding, allowing dissection of rab27a requirements in munc13-4 function. The munc13-4-rab27a complex is not needed for secretory lysosome maturation, as shown by complementation in CTLs from FHL3 patients and in a mast cell line silenced for munc13-4. In contrast, fusion of secretory lysosomes with, and content release at the plasma membrane during degranulation, strictly required the munc13-4-rab27a complex. Total internal reflection fluorescence microscopy imaging revealed that the complex corrals motile secretory lysosomes beneath the plasma membrane during degranulation and controls their docking. The propensity to stall motility of secretory lysosomes is lost in cells expressing munc13-4 point mutants that do not bind rab27. In summary, these results uncovered a mechanism for tethering secretory lysosomes to the plasma membrane that is essential for degranulation in immune cells.