The NLRP3 inflammasome plays a critical role in host defense by facilitating caspase I activation and maturation of IL-1β and IL-18, whereas dysregulation of inflammasome activity results in autoinflammatory disease. Factors regulating human NLRP3 activity that contribute to the phenotypic heterogeneity of NLRP3-related diseases have largely been inferred from the study of Nlrp3 mutant mice. By generating a mouse line in which the NLRP3 locus is humanized by syntenic replacement, we show the functioning of the human NLRP3 proteins in vivo, demonstrating the ability of the human inflammasome to orchestrate immune reactions in response to innate stimuli. Humanized mice expressing disease-associated mutations develop normally but display acute sensitivity to endotoxin and develop progressive and debilitating arthritis characterized by granulocytic infiltrates, elevated cytokines, erosion of bones, and osteoporosis. This NLRP3-dependent arthritis model provides a platform for testing therapeutic reagents targeting the human inflammasome.
Neuronal active Caspase-6 (Casp6) is associated with Alzheimer disease (AD), cognitive impairment, and axonal degeneration. Caspase-1 (Casp1) can activate Casp6 but the expression and functionality of Casp1-activating inflammasomes has not been well-defined in human neurons. Here, we show that primary cultures of human CNS neurons expressed functional Nod-like receptor protein 1 (NLRP1), absent in melanoma 2, and ICE protease activating factor, but not the NLRP3, inflammasome receptor components. NLRP1 neutralizing antibodies in a cell-free system, and NLRP1 siRNAs in neurons hampered stress-induced Casp1 activation. NLRP1 and Casp1 siRNAs also abolished stress-induced Casp6 activation in neurons. The functionality of the NLRP1 inflammasome in serum-deprived neurons was also demonstrated by NLRP1 siRNA-mediated inhibition of speck formation of the apoptosis-associated speck-like protein containing a caspase recruitment domain conjugated to green fluorescent protein. These results indicated a novel stress-induced intraneuronal NLRP1/Casp1/Casp6 pathway. Lipopolysaccharide induced Casp1 and Casp6 activation in wild-type mice brain cortex, but not in that of Nlrp1(-/-) and Casp1(-/-) mice. NLRP1 immunopositive neurons were increased 25- to 30-fold in AD brains compared with non-AD brains. NLRP1 immunoreactivity in these neurons co-localized with Casp6 activity. Furthermore, the NLRP1/Casp1/Casp6 pathway increased amyloid beta peptide 42 ratio in serum-deprived neurons. Therefore, CNS human neurons express functional NLRP1 inflammasomes, which activate Casp1 and subsequently Casp6, thus revealing a fundamental mechanism linking intraneuronal inflammasome activation to Casp1-generated interleukin-1-β-mediated neuroinflammation and Casp6-mediated axonal degeneration.
UDP-Glucuronosyltransferases (UGTs) conjugate a glucuronyl group from glucuronic acid to a wide range of lipophilic substrates to form a hydrophilic glucuronide conjugate. The glucuronide generally has decreased bioactivity and increased water solubility to facilitate excretion. Glucuronidation represents an important detoxification pathway for both endogenous waste products and xenobiotics, including drugs and harmful industrial chemicals. Two clinically significant families of UGT enzymes are present in mammals: UGT1s and UGT2s. Although the two families are distinct in gene structure, studies using recombinant enzymes have shown considerable overlap in their ability to glucuronidate many substrates, often obscuring the relative importance of the two families in the clearance of particular substrates in vivo. To address this limitation, we have generated a mouse line, termed ΔUgt2, in which the entire Ugt2 gene family, extending over 609 kilobase pairs, is excised. This mouse line provides a means to determine the contributions of the two UGT families in vivo. We demonstrate the utility of these animals by defining for the first time the in vivo contributions of the UGT1 and UGT2 families to glucuronidation of the environmental estrogenic agent bisphenol A (BPA). The highest activity toward this chemical is reported for human and rodent UGT2 enzymes. Surprisingly, our studies using the ΔUgt2 mice demonstrate that, while both UGT1 and UGT2 isoforms can conjugate BPA, clearance is largely dependent on UGT1s.
Caspase-6 is highly activated in neuropil threads, neurofibrillary tangles, and neuritic plaques of Alzheimer Disease (AD) brains. Caspase-6 activity restricted to the entorhinal cortex and hippocampal CA1 of aged non-AD individuals, correlates with lower cognitive performance. Expression of active Caspase-6 in mouse brain CA1 is sufficient to cause age-dependent spatial and episodic memory deficits. Caspase-1 activates Caspase-6 in primary cultures of human neurons. Here, we investigated which inflammasome may be responsible for Caspase-1-mediated Caspase-6 activation in human neurons and in AD brains. qRT-PCR and western blots identified the levels of specific inflammasome receptor components in primary cultures of fetal human neurons. Neurons were challenged by serum deprivation or purinergic P 2 X 7 receptor agonist, benzylated ATP to activate the inflammasomes. Caspase-1 and Caspase-6 activities were determined by fluorogenic assays, interleukin-1-beta and tubulin cleaved by Caspase-6 production. A cell free system treated with blocking antibodies identified specific functional inflammasomes. Null mice, injected with the lipopolysaccharide inflammatory agent, LPS, confirmed inflammasome-mediated activation of Caspase-1 and Caspase-6 in vivo. AD inflammasome mRNA and protein were assessed by RT-PCR and immunohistochemistry. Caspase-1 activation appeared before Caspase-6 activation in challenged primary cultures of human neurons. These neurons expressed the Nod-like receptor protein 1 (NLRP1), absent in melanoma 2 (AIM2), and ICE protease activating factor (IPAF-1) receptor components of the inflammasomes, but not NLRP3. Antibodies to NLRP1, but not to AIM2 and IPAF-1, blocked Caspase-1 and Caspase-6 activation in neuronal S100 sub-cellular fractions. LPS-mediated activation of Caspase-1 and Caspase-6 in wild-type mice brains was repressed in NLRP1 -/- and Casp1 -/- mice. NLRP1 mRNA levels and NLRP1-immunopositive neurons were increased in AD brains. We conclude that the NLRP1 inflammasome can regulate Caspase-1-mediated activation of Caspase-6 in human neurons, in mice brains, and in AD. The presence of functional inflammasomes in neurons indicates a mechanism by which neuronal production of interleukin-1-beta could initiate the inflammatory microglial response in AD brains. Our data suggest that the NLRP1 could represent a novel upstream target to prevent both the inflammatory response and Caspase-6-mediated axonal degeneration in AD.
Representative members of surface water microbiota were obtained from three unrelated municipal sites in Oklahoma by direct plating under selection by the hydrophobic biocide triclosan. Multiple methods were employed to determine if intrinsic triclosan resistance reflected resistance to hydrophobic molecules by virtue of outer membrane impermeability. While all but one organism isolated in the absence of triclosan were able to initiate growth on MacConkey agar, only one was able to initiate significant growth with triclosan present. In contrast, all bacteria selected with triclosan were identified as Pseudomonas spp. using 16S RNA gene sequencing and exhibited growth comparable to Pseudomonas aeruginosa controls in the presence of hydrophobic antibacterial agents to include triclosan. Two representative bacteria isolated in the absence of triclosan allowed for greater outer membrane association with the fluorescent hydrophobic probe 1-N-phenylnapthylamine than did two triclosan-resistant isolates. Compound 48/80 disruption of outer membrane impermeability properties for hydrophobic substances either partially or fully sensitized nine of twelve intrinsically resistant isolates to triclosan. These data suggest that outer membrane exclusion underlies intrinsic resistance to triclosan in some, but not all Pseudomonas spp. isolated by selection from municipal surface waters and implicates the involvement of concomitant triclosan resistance mechanisms.
