Loss of TLR2 worsens spontaneous colitis in MDR1A deficiency through commensally induced pyroptosis.

Variants of the multidrug resistance gene (MDR1/ABCB1) have been associated with increased susceptibility to severe ulcerative colitis (UC). In this study, we investigated the role of TLR/IL-1R signaling pathways including the common adaptor MyD88 in the pathogenesis of chronic colonic inflammation in MDR1A deficiency. Double- or triple-null mice lacking TLR2, MD-2, MyD88, and MDR1A were generated in the FVB/N background. Deletion of TLR2 in MDR1A deficiency resulted in fulminant pancolitis with early expansion of CD11b(+) myeloid cells and rapid shift toward TH1-dominant immune responses in the lamina propria. Colitis exacerbation in TLR2/MDR1A double-knockout mice required the unaltered commensal microbiota and the LPS coreceptor MD-2. Blockade of IL-1ß activity by treatment with IL-1R antagonist (IL-1Ra; Anakinra) inhibited colitis acceleration in TLR2/MDR1A double deficiency; intestinal CD11b(+)Ly6C(+)-derived IL-1ß production and inflammation entirely depended on MyD88. TLR2/MDR1A double-knockout CD11b(+) myeloid cells expressed MD-2/TLR4 and hyperresponded to nonpathogenic Escherichia coli or LPS with reactive oxygen species production and caspase-1 activation, leading to excessive cell death and release of proinflammatory IL-1ß, consistent with pyroptosis. Inhibition of reactive oxygen species-mediated lysosome degradation suppressed LPS hyperresponsiveness. Finally, active UC in patients carrying the TLR2-R753Q and MDR1-C3435T polymorphisms was associated with increased nuclear expression of caspase-1 protein and cell death in areas of acute inflammation, compared with active UC patients without these variants. In conclusion, we show that the combined defect of two UC susceptibility genes, MDR1A and TLR2, sets the stage for spontaneous and uncontrolled colitis progression through MD-2 and IL-1R signaling via MyD88, and we identify commensally induced pyroptosis as a potential innate immune effector in severe UC pathogenesis.

Toll-like receptor 4 variant D299G induces features of neoplastic progression in Caco-2 intestinal cells and is associated with advanced human colon cancer.

BACKGROUND & AIMS: The Toll-like receptor (TLR) 4 mediates homeostasis of the intestinal epithelial cell (IEC) barrier. We investigated the effects of TLR4-D299G on IEC functions. METHODS: We engineered IECs (Caco-2) to stably overexpress hemagglutinin-tagged wild-type TLR4, TLR4-D299G, or TLR4-T399I. We performed gene expression profiling using DNA microarray analysis. Findings were confirmed by real-time, quantitative, reverse-transcriptase polymerase chain reaction, immunoblot, enzyme-linked immunosorbent assay, confocal immunofluorescence, and functional analyses. Tumorigenicity was tested using the CD1 nu/nu mice xenograft model. Human colon cancer specimens (N = 214) were genotyped and assessed for disease stage. RESULTS: Caco-2 cells that expressed TLR4-D299G underwent the epithelial-mesenchymal transition and morphologic changes associated with tumor progression, whereas cells that expressed wild-type TLR4 or TLR4-T399I did not. Caco-2 cells that expressed TLR4-D299G had significant increases in expression levels of genes and proteins associated with inflammation and/or tumorigenesis compared with cells that expressed other forms of TLR4. The invasive activity of TLR4-D299G Caco-2 cells required Wnt-dependent activation of STAT3. In mice, intestinal xenograft tumors grew from Caco-2 cells that expressed TLR4-D299G, but not cells that expressed other forms of TLR4; tumor growth was blocked by a specific inhibitor of STAT3. Human colon adenocarcinomas from patients with TLR4-D299G were more frequently of an advanced stage (International Union Against Cancer [UICC] ≥III, 70% vs 46%; P = .0142) with metastasis (UICC IV, 42% vs 19%; P = .0065) than those with wild-type TLR4. Expression of STAT3 messenger RNA was higher among colonic adenocarcinomas with TLR4-D299G than those with wild-type TLR4. CONCLUSIONS: TLR4-D299G induces features of neoplastic progression in intestinal epithelial Caco-2 cells and associates with aggressive colon cancer in humans, implying a novel link between aberrant innate immunity and colonic cancerogenesis.

TLR2 mediates gap junctional intercellular communication through connexin-43 in intestinal epithelial barrier injury.

Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1alpha-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.

Visualization of degenerating axons in a dysmyelinating mouse mutant with axonal loss.

Mice homozygously deficient for the myelin component P0 show loss of axons in peripheral nerves. In order to investigate the morphological characteristics of degenerating axons, we crossbred the myelin mutants with a transgenic mouse line expressing yellow fluorescent protein (YFP) in a small proportion of neurons. Peripheral nerves of the double mutants were prepared into small fiber bundles and investigated by fluorescence microscopy. We could identify the tips of degenerating axon as bulb-like structures. Additionally, by electron microscopy, these structures were characterized as axoplasmic extensions containing numerous membraneous compartments. By immunoelectron microscopy, the degenerating end bulbs were in contact with ensheathing Schwann cells that contained YFP-immunoreactivity possibly reflecting phagocytosis of axon material by these cells. Immunohistochemistry using antibodies against macrophages revealed that YFP-positive bulbs, but also other axonal swellings, were often associated with macrophages supporting our previous findings that myelin-related axonal loss is partially mediated by these cells.