Effects of salinity stress on antioxidant status and inflammatory responses in females of a "Near Threatened" economically important fish species Notopterus chitala: a mechanistic approach.

In the present study, acute stress responses of adult female Notopterus chitala were scrutinized by antioxidant status and inflammation reaction in the gill and liver at five different salinity exposures (0, 3, 6, 9, 12 ppt). Oxidative defense was assessed by determining superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase, and glutathione reductase activities, while malondialdehyde (MDA), glutathione, and xanthine oxidase levels were determined as indicators of oxidative load. Pro-inflammatory cytokines (IL-1ß, IL-6, IL-10, and TNFα) and caspase 1 levels were also analyzed. Expression levels of transcription factors (NRF2 and NF-κB) and molecular chaperons (HSF, HSP70, and HSP90) were estimated to evaluate their relative contribution to overcome salinity stress. MDA showed a significant (P < 0.05) increase (gill, + 25.35-90.14%; liver, + 23.88-80.59%) with salinity; SOD (+ 13.72-45.09%) and CAT (+ 12.73-33.96%) exhibited a sharp increase until 9 ppt, followed by a decrease at the highest salinity (12 ppt) (gill, - 3.92%; liver, - 2.18%). Levels of cytokines were observed to increase (+ 52.8-127.42%) in a parallel pattern with increased salinity. HSP70 and HSP90 expressions were higher in gill tissues than those in liver tissues. NRF2 played pivotal role in reducing salinity-induced oxidative load in both the liver and gills. Serum cortisol and carbonic anhydrase were measured and noted to be significantly (P < 0.05) upregulated in salinity stressed groups. Gill Na+-K+-ATPase activity decreased significantly (P < 0.05) in fish exposed to 6, 9, and 12 ppt compared to control. Present study suggests that a hyperosmotic environment induces acute oxidative stress and inflammation, which in turn causes cellular death and impairs tissue functions in freshwater fish species such as Notopterus chitala.

In vivo assessment of molybdenum and cadmium co-induce nephrotoxicity via NLRP3/Caspase-1-mediated pyroptosis in ducks.

Excessive molybdenum (Mo) and cadmium (Cd) cause toxic effects on animals, but their joint effects on pyroptosis in kidney of ducks remain unclear. 160 healthy 7-day-old ducks were randomly divided into four groups which were fed with basal diet containing different dosages of Mo or/and Cd for 16 weeks. On the 4th, 8th, 12th and 16th weeks, kidney tissue and serum were collected. The results showed that Mo or/and Cd could significantly elevate their contents in kidney, disturb the homeostasis of trace elements, cause renal function impairment and histological abnormality, and oxidative stress as accompanied by increasing hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations and decreasing glutathione peroxidase (GSH-Px), catalase (CAT) and total-superoxide dismutase (T-SOD) activities. Simultaneously, Mo or/and Cd could markedly increase interleukin-1ß (IL-1ß), interleukin-18 (IL-18) contents and the expression levels of pyroptosis-related genes (NOD-like receptor protein-3 (NLRP3), Caspase-1, apoptosis-associated speck-like protein (ASC), NIMA-related kinase 7 (NEK7), Gasdermin A (GSDMA), Gasdermin E (GSDME), IL-1ß and IL-18) and proteins (NLRP3, Caspase-1 p20, ASC and Gasdermin D (GSDMD)). Moreover, the changes of above these indicators were more obvious in combined group. Taken together, the results illustrate that Mo and Cd might synergistically lead to oxidative stress and induce pyroptosis via NLRP3/Caspase-1 pathway, whose mechanism is somehow related to Mo and Cd accumulation in duck kidneys.

Digital signaling network drives the assembly of the AIM2-ASC inflammasome.

The AIM2-ASC inflammasome is a filamentous signaling platform essential for mounting host defense against cytoplasmic dsDNA arising not only from invading pathogens but also from damaged organelles. Currently, the design principles of its underlying signaling network remain poorly understood at the molecular level. We show here that longer dsDNA is more effective in inducing AIM2 assembly, its self-propagation, and downstream ASC polymerization. This observation is related to the increased probability of forming the base of AIM2 filaments, and indicates that the assembly discerns small dsDNA as noise at each signaling step. Filaments assembled by receptor AIM2, downstream ASC, and their joint complex all persist regardless of dsDNA, consequently generating sustained signal amplification and hysteresis. Furthermore, multiple positive feedback loops reinforce the assembly, as AIM2 and ASC filaments accelerate the assembly of nascent AIM2 with or without dsDNA. Together with a quantitative model of the assembly, our results indicate that an ultrasensitive digital circuit drives the assembly of the AIM2-ASC inflammasome.

OLT1177, a ß-sulfonyl nitrile compound, safe in humans, inhibits the NLRP3 inflammasome and reverses the metabolic cost of inflammation.

Activation of the NLRP3 inflammasome induces maturation of IL-1ß and IL-18, both validated targets for treating acute and chronic inflammatory diseases. Here, we demonstrate that OLT1177, an orally active ß-sulfonyl nitrile molecule, inhibits activation of the NLRP3 inflammasome. In vitro, nanomolar concentrations of OLT1177 reduced IL-1ß and IL-18 release following canonical and noncanonical NLRP3 inflammasome activation. The molecule showed no effect on the NLRC4 and AIM2 inflammasomes, suggesting specificity for NLRP3. In LPS-stimulated human blood-derived macrophages, OLT1177 decreased IL-1ß levels by 60% and IL-18 by 70% at concentrations 100-fold lower in vitro than plasma concentrations safely reached in humans. OLT1177 also reduced IL-1ß release and caspase-1 activity in freshly obtained human blood neutrophils. In monocytes isolated from patients with cryopyrin-associated periodic syndrome (CAPS), OLT1177 inhibited LPS-induced IL-1ß release by 84% and 36%. Immunoprecipitation and FRET analysis demonstrated that OLT1177 prevented NLRP3-ASC, as well as NLRP3-caspase-1 interaction, thus inhibiting NLRP3 inflammasome oligomerization. In a cell-free assay, OLT1177 reduced ATPase activity of recombinant NLRP3, suggesting direct targeting of NLRP3. Mechanistically, OLT1177 did not affect potassium efflux, gene expression, or synthesis of the IL-1ß precursor. Steady-state levels of phosphorylated NF-κB and IkB kinase were significantly lowered in spleen cells from OLT1177-treated mice. We observed reduced IL-1ß content in tissue homogenates, limited oxidative stress, and increased muscle oxidative metabolism in OLT1177-treated mice challenged with LPS. Healthy humans receiving 1,000 mg of OLT1177 daily for 8 d exhibited neither adverse effects nor biochemical or hematological changes.

Assessment of Inflammasome Formation by Flow Cytometry.

Inflammasomes are large protein complexes formed in response to cellular stresses that are platforms for recruitment and activation of caspase 1. Central to most inflammasome functions is the adapter molecule ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) that links the inflammasome initiator protein to the recruited caspases. ASC is normally diffuse within the cell but within minutes of inflammasome activation relocates to a dense speck in the cytosol. The dramatic redistribution of ASC can be monitored by flow cytometry using parameters of fluorescence peak height and width when immunostained or tagged with a fluorescent protein. This can be used to define cells with active inflammasomes within populations of primary macrophages and monocytes, allowing quantification of responses and flow-sorting of responding cells. Protein structural requirements for ASC speck formation and recruitment of caspases to ASC specks can be assessed by expressing components in HEK293 cells. This provides rapid quantification of responding cell number and correlation with the expression level of inflammasome components within single cells. © 2016 by John Wiley & Sons, Inc.

Assessment of Inflammasome Activation by Cytokine and Danger Signal Detection.

The evaluation of the inflammasome activation usually addresses the presence of extracellular IL-1ß and IL-18 or the secretion of danger signal proteins such as HMGB-1 through their quantification using an enzyme-linked immunosorbent assay (ELISA). The ELISA is a routine laboratory technique that uses antibodies and colorimetric changes to identify a substance of interest. ELISA uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid or wet sample. Using 96 well plates, the ELISA technique enables to quantify the concentration of a single cytokine in multiple samples. However, a limitation of IL-1ß and IL-18 ELISA is the absence of discrimination between active and non-active form of the proteins, parameter critical, for example, to distinguish the biologically relevant IL-1ß from its poorly active form pro-IL-1ß. This issue can be solved using western blots or immunoblots (IB), a common analytical procedure to detect the presence of different proteins in biological samples. Using denaturating conditions, IB allows the visualization of different sizes of the proteins of choice and is a commonly used technique in the inflammasome field to evaluate, for instance, the maturation of pro-IL-1ß, pro-IL-18, and pro-caspase-1 into mature IL-1ß, mature IL-18, and mature caspase-1, respectively. Moreover inflammasome activation may lead to the release of inflammasome particles outside the cell through caspase-1- or caspase-11-dependent cell death mechanism termed pyroptosis. In this case, NLR, ASC, and caspase-1 components are detectable outside the cell using IB analysis. ELISA and IB can be performed on cell culture supernatant or cell extract and on ex vivo samples from organ homogenates or biological fluids such as serum and plasma or bronchoalveolar lavages.

Assessment of inflammasome activation in primary human immune cells.

Inflammasomes are central regulators of inflammation, responsible for cleavage of the inactive pro--inflammatory cytokines IL-1ß and IL-18 into their biologically active counterparts. Several regulatory stages within the pathways responsible for the production of these cytokines have been identified. In this chapter, methods are described for assessing the degree of activity of these regulatory stages, which include mRNA transcription, caspase-1 activation, and secretion of the bioactive proteins.