Differential Expression of NLRP3 among Hematopoietic Cells

Department of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland.
The Journal of Immunology (Impact Factor: 4.92). 02/2011; 186(4):2529-34. DOI: 10.4049/jimmunol.1002720
Source: PubMed


Although the importance of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in health and disease is well appreciated, a precise characterization of NLRP3 expression is yet undetermined. To this purpose, we generated a knock-in mouse in which the Nlrp3 coding sequence was substituted for the GFP (enhanced GFP [egfp]) gene. In this way, the expression of eGFP is driven by the endogenous regulatory elements of the Nlrp3 gene. In this study, we show that eGFP expression indeed mirrors that of NLRP3. Interestingly, splenic neutrophils, macrophages, and, in particular, monocytes and conventional dendritic cells showed robust eGFP fluorescence, whereas lymphoid subsets, eosinophils, and plasmacytoid dendritic cells showed negligible eGFP levels. NLRP3 expression was highly inducible in macrophages, both by MyD88- and Trif-dependent pathways. In vivo, when mice were challenged with diverse inflammatory stimuli, differences in both the number of eGFP-expressing cells and fluorescence intensity were observed in the draining lymph node. Thus, NLRP3 levels at the site of adaptive response initiation are controlled by recruitment of NLRP3-expressing cells and by NLRP3 induction.

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Available from: Kate Schroder, Apr 22, 2015
    • "To examine whether the loss of pro-caspase-1 was caused by this autocleavage, the effect of ziram was studied in mouse bone-marrow macrophages (Fig. 3) because in J774A.1 cells no cleavage of pro-caspase- 1 was detected in response to various stimulations that are known to cause autocleavage in primary bone-marrow macrophages. In bone-marrow macrophages, ATP alone did not affect either pro- caspase-1, NLRP3 or ASC, whereas LPS treatment increased the NLRP3 level probably because LPS increased the expression of NLRP3 (Bauernfeind et al., 2009; Guarda et al., 2011). As observed in macrophage-like cell lines, ziram caused the loss of both pro- caspase-1 and NLRP3 in bone-marrow macrophages, while not affecting ASC and b-actin. "
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    ABSTRACT: The NLRP3 inflammasome, composed of caspase-1, NLRP3 and ASC, plays a critical role in the clearance of microbial pathogens. Here, we found that the treatment of mouse macrophages with the zinc-containing dithiocarbamate ziram, a widely used fungicide in agriculture, caused a decrease in pro-caspase-1 and NLRP3 levels while not affecting ASC level. Ziram did not affect levels of pro-caspase-1 and NLRP3 mRNA, and no cleavage products of pro-caspase-1 including p10 subunit, which is an autocleavage product of pro-caspase-1, were detected, indicating that the decrease was associated with degradation of these proteins. The decrease was inhibited by SH-type antioxidants, N-acetyl cysteine, dithiothreitol and 2-mercaptoethanol, or a metal chelator EDTA but not by inhibitors of proteasome, lysosomes, autophagy and matrix metalloproteases. Thiram, a comparator for ziram that does not contain zinc, showed a weaker decrease in protein levels. Furthermore, the zinc-containing dithiocarbamate, zinc diethyldithiocarbamate, efficiently decreased the levels of pro-caspase-1 and NLRP3, whereas dithiocarbamates, dimethyldithiocarbamate and diethyldithiocarbamate without zinc, were less active. The organic zinc compound [3,4-Toluenedithiolato(2-)]zinc hydrate did not induce a decrease in protein levels. Ziram also inhibited IL-1β production by macrophages in response to lipopolysaccharide and bacterial clearance during Salmonella infection of macrophage cells. These results indicate that ziram causes degradation of pro-caspase-1 and NLRP3 in a zinc- and oxidative property-dependent manner and suggest that exposure to ziram may compromise the clearance of microbial pathogens. Copyright © 2015. Published by Elsevier Ireland Ltd.
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    • "In contrast, human dental pulp fibroblasts (HDPFs), the main cells that constitute the dental pulp, produce pro-inflammatory factors (which are involved in the innate immune response) when stimulated by caries-related bacterial virulence factors. TLR, nucleotidebinding oligomerization domain containing 1 (NOD1) and NOD2 expression has been detected in HDPFs (Guarda et al. 2011). Taken together, these findings suggest that odontoblasts and HDPFs play a role in the innate immune response and thereby protect against bacterial infection in the dental pulp. "
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    ABSTRACT: The NLRP3/caspase-1 inflammasome pathway plays an important role in cellular immune defence against bacterial infection; however, its function in human dental pulp tissue and human dental pulp fibroblasts remains poorly understood. We demonstrate that NLRP3 protein expression occurs to a greater extent in pulp tissue with irreversible pulpitis than in normal pulp tissue and in tissue with reversible pulpitis. Caspase-1 is present in its active (cleaved) form only in pulp tissue with irreversible pulpitis. NLRP3 and caspase-1 are expressed in the odontoblast layers in normal human dental pulp tissue, whereas in inflamed pulp tissue, the odontoblast layers are disrupted and dental pulp cells are positive for NLRP3 and caspase-1. Additionally, we investigate the role of the NLRP3/caspase-1 inflammasome pathway in human dental pulp fibroblasts and show that ATP activates the P2X7 receptor on the cell membrane triggering K(+) efflux and inducing the gradual recruitment of the membrane pore pannexin-1. Extracellular lipopolysaccharide is able to penetrate the cytosol and activate NLRP3. Furthermore, the low intracellular K(+) concentration in the cytosol triggers reactive oxygen species generation, which also induces the NLRP3 inflammasome. Thus, the NLRP3/caspase-1 pathway has a biological role in the innate immune response mounted by human dental pulp fibroblasts.
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    • "NLRP3, a member of the NLRP protein family, is expressed in response to inflammatory stimuli mainly within the hematopoietic compartment in both lymphocytic and myelogenic lineages. To a lesser extent, it can also be expressed by other cell types of the non-hematopoietic compartment, such as skin, keratinocyte, and osteoblasts (47). NLRP3 consists of a carboxy-terminal LRR domain, a central NOD domain, and an amino-terminal PYD, mainly interacting with apoptosis-associated speck-like protein containing a CARD (ASC) (8) (Figure 1). "
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    ABSTRACT: Nucleotide-binding and oligomerization domain NOD-like receptors (NLRs) are highly conserved cytosolic pattern recognition receptors that play, in combination with toll-like receptors, a critical role in innate immunity and inflammation. These proteins are characterized by a central oligomerization domain termed nucleotide-binding domain, and a protein interaction domain containing leucine-rich repeats. Some NLRs, including NOD1 and NOD2, sense the cytosolic presence of conserved bacterial molecular signatures and drive the activation of mitogen-activated protein kinase and the transcription factor NF-κB. A different set of NLRs induces caspase-1 activation through the assembly of large protein complexes known as inflammasomes. Activation of NLR proteins results in secretion of pro-inflammatory cytokines and subsequent inflammatory responses. The critical role of NLRs in innate immunity is underscored by the fact that polymorphisms within their genes are implicated in the development of several immune-mediated diseases, including inflammatory bowel disease. Over the past few years, the role of NLRs in intestinal homeostasis has been highlighted, however the mechanism by which dysfunction in these proteins leads to aberrant inflammation is still the focus of much investigation. The purpose of this review is to systematically evaluate the function of NLRs in mucosal innate immunity and understand how genetic or functional alterations in these components can lead to the disruption of intestinal homeostasis, and the subsequent development of chronic inflammation.
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