NLR proteins are a diverse family of pattern recognition receptors that are essential mediators of inflammation and host defense in the gastrointestinal system. Recent studies have identified a sub-group of inflammasome forming NLRs that modulate the mucosal immune response during inflammatory bowel disease (IBD) and colitis associated tumorigenesis. To better elucidate the contribution of NLR family members in IBD and cancer, we conducted a retrospective analysis of gene expression metadata from human patients. These data revealed that NLRP1, an inflammasome forming NLR, was significantly dysregulated in IBD and colon cancer. To better characterize the function of NLRP1 in disease pathogenesis, we utilized Nlrp1b−/− mice in colitis and colitis associated cancer models. Here, we report that NLRP1 attenuates gastrointestinal inflammation and tumorigenesis. Nlrp1b−/− mice demonstrated significant increases in morbidity, inflammation and tumorigenesis compared to wild type animals. Similar to data previously reported for related inflammsome forming NLRs, the increased inflammation and tumor burden was correlated with attenuated levels of IL-1β and IL-18. Further mechanistic studies utilizing bone marrow reconstitution experiments revealed that the increased disease pathogenesis in the Nlrp1b−/− mice was associated with non-hematopoietic derived cells and suggests that NLRP1 functions in the colon epithelial cell compartment to attenuate tumorigenesis. Together, these data identify NLRP1 as an essential mediator of the host immune response during IBD and cancer. These findings are consistent with a model whereby multiple NLR inflammasomes attenuate disease pathobiology through modulating IL-1β and IL-18 levels in the colon.
IC. Caspase-11 attenuates gastrointestinal inflammation and experimental colitis pathogenesis.
Histiocytic sarcoma is an uncommon malignancy in both humans and veterinary species. Research exploring the pathogenesis of this disease is scarce; thus, diagnostic and therapeutic options for patients are limited. Recent publications have suggested a role for the NLR, NLRX1, in acting as a tumor suppressor. Based on these prior findings, we hypothesized that NLRX1 would function to inhibit tumorigenesis and thus the development of histiocytic sarcoma. To test this, we utilized Nlrx1−/− mice and a model of urethane-induced tumorigenesis. Nlrx1−/− mice exposed to urethane developed splenic histiocytic sarcoma that was associated with significant up-regulation of the NF-λB signaling pathway. Additionally, development of these tumors was also significantly associated with the increased regulation of genes associated with AKT signaling, cell death and autophagy. Together, these data show that NLRX1 suppresses tumorigenesis and reveals new genetic pathways involved in the pathobiology of histiocytic sarcoma.
Background Multi-walled carbon nanotubes (MWCNT) have been shown to elicit the release of inflammatory and pro-fibrotic mediators, as well as histopathological changes in lungs of exposed animals. Current standards for testing MWCNTs and other nanoparticles (NPs) rely on low-throughput in vivo studies to assess acute and chronic toxicity and potential hazard to humans. Several alternative testing approaches utilizing two-dimensional (2D) in vitro assays to screen engineered NPs have reported conflicting results between in vitro and in vivo assays. Compared to conventional 2D in vitro or in vivo animal model systems, three-dimensional (3D) in vitro platforms have been shown to more closely recapitulate human physiology, providing a relevant, more efficient strategy for evaluating acute toxicity and chronic outcomes in a tiered nanomaterial toxicity testing paradigm. Results As inhalation is an important route of nanomaterial exposure, human lung fibroblasts and epithelial cells were co-cultured with macrophages to form scaffold-free 3D lung microtissues. Microtissues were exposed to multi-walled carbon nanotubes, M120 carbon black nanoparticles or crocidolite asbestos fibers for 4 or 7 days, then collected for characterization of microtissue viability, tissue morphology, and expression of genes and selected proteins associated with inflammation and extracellular matrix remodeling. Our data demonstrate the utility of 3D microtissues in predicting chronic pulmonary endpoints following exposure to MWCNTs or asbestos fibers. These test nanomaterials were incorporated into 3D human lung microtissues as visualized using light microscopy. Differential expression of genes involved in acute inflammation and extracellular matrix remodeling was detected using PCR arrays and confirmed using qRT-PCR analysis and Luminex assays of selected genes and proteins. Conclusion 3D lung microtissues provide an alternative testing platform for assessing nanomaterial-induced cell-matrix alterations and delineation of toxicity pathways, moving towards a more predictive and physiologically relevant approach for in vitro NP toxicity testing. Electronic supplementary material The online version of this article (10.1186/s12989-019-0298-0) contains supplementary material, which is available to authorized users.
Abnormal tensional cellular homeostasis is now considered a hallmark of cancer. Despite this, the origin of this abnormality remains unclear. In this work, we investigated the role of tissue transglutaminase (TG2), a protein associated with poor prognosis and increased metastatic potential, and its relationship to the EGF receptor in the regulation of the mechanical state of tumor cells. Remarkably, we observed a TG2-mediated modulation of focal adhesion composition as well as stiffness-induced FAK activation using either a pharmacological or shRNA-based approach, linked with a distinctive increase in cell contractility. Additionally, the increased contractility could be reproduced in non-malignant cells upon TG2 expression. Moreover, the increased cell contractility mediated by TG2 was largely due to the loss of EGFR-mediated inhibition of cell contractility. These findings establish intracellular TG2 as a regulator of cellular tensional homeostasis and suggest the existence of signaling switches that control the contribution of growth factor receptors in determining a cell's mechanical state.
Trans-epithelial Electrical Resistance (TEER) can be used as a measure of cell monolayer confluence, health, and integrity. An EndOhm chamber connected to an Epithelial Voltohmmeter (EVOM) may be used to take TEER measurements that are more reproducible than the chopstick electrode. This method details how to take TEER readings using an EndOhm chamber and EVOM2 manufactured by World Precision Instruments. Read the manufacturer’s manual carefully as the conditions described here were determined to meet the manufacturer’s specifications. Disclaimer: The contents of this article have been reviewed by the US Environmental Protection Agency and approved for publication and do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.NOTE: There is a consolidated PDF version of this protocol in the supplementary files section below.
Members of the NLR family of pattern recognition receptors are essential mediators of the host immune response. Recently, our lab and others identified a novel sub-group of NLRs that function as negative regulators of inflammation. One of the members of this sub-group, NLRX1, is a potent regulator of interferon, NF-κB and autophagy signaling. Each of these signaling pathways has been linked to diverse types of cancer. Thus, we hypothesized that NLRX1 attenuates tumorigenesis through the negative regulation of overzealous innate immune system signaling. To evaluate this hypothesis, we utilized a urethane based model of tumorigenesis and explored disease progression in Nlrx1-/- mice. Mice lacking NLRX1 exhibited dramatically increased morbidity and mortality and were found to develop a rare neoplasia, diagnosed as histiocytic sarcoma. Of the already rare histiocytic disorders in humans, histiocytic sarcoma is exceedingly uncommon and the rarity of this disease has resulted in a paucity of data pertaining to diagnostic and therapeutic options. Mechanistically, our data indicates that NLRX1 attenuates tumorigenesis through the negative regulation of AKT and NF-κB signaling. These pathways were found to be associated with macrophage hyperproliferation in the absence of NLRX1. Our data extends the function of NLRX1 beyond its currently characterized role in host-pathogen defense and reveals a significant contribution for this NLR in cancer pathobiology.
NLR proteins are a diverse family of pattern recognition receptors that modulate immune system homeostasis in the gastrointestinal system. Recent studies have identified a sub-group of inflammasome forming NLRs that modulate the mucosal immune response during inflammatory bowel disease (IBD) and colitis associated tumorigenesis. To better elucidate the contribution of NLR family members in IBD and cancer, we conducted a retrospective analysis of gene expression metadata from human patients. These data revealed that NLRP1, an inflammasome forming NLR, was significantly dysregulated in IBD and colon cancer. To better characterize the function of NLRP1 in disease pathogenesis, we utilized Nlrp1b-/- mice in experimental colitis and colitis associated cancer models. Nlrp1b-/- mice demonstrated significant increases in inflammation and tumorigenesis compared to wild type mice. The increased inflammation and tumor burden was correlated with attenuated levels of IL-1β and IL-18. Further mechanistic studies revealed the increased disease pathogenesis in the Nlrp1b-/- mice was associated with non-hematopoietic derived cells and suggests that NLRP1 functions in the colon epithelial cells to attenuate tumorigenesis. Together, these data identify NLRP1 as an essential mediator of the host immune response during IBD and cancer. These findings are consistent with a model whereby multiple NLR inflammasomes attenuate disease pathobiology through modulating IL-1β and IL-18 levels in the colon.
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