Zinc depletion regulates the processing and secretion of IL-1ß.

Sterile inflammation contributes to many common and serious human diseases. The pro-inflammatory cytokine interleukin-1ß (IL-1ß) drives sterile inflammatory responses and is thus a very attractive therapeutic target. Activation of IL-1ß in sterile diseases commonly requires an intracellular multi-protein complex called the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome. A number of disease-associated danger molecules are known to activate the NLRP3 inflammasome. We show here that depletion of zinc from macrophages, a paradigm for zinc deficiency, also activates the NLRP3 inflammasome and induces IL-1ß secretion. Our data suggest that zinc depletion damages the integrity of lysosomes and that this event is important for NLRP3 activation. These data provide new mechanistic insight to how zinc deficiency contributes to inflammation and further unravel the mechanisms of NLRP3 inflammasome activation.

Caspase-1: is IL-1 just the tip of the ICEberg?

Caspase-1, formerly known as interleukin (IL)-1-converting enzyme is best established as the protease responsible for the processing of the key pro-inflammatory cytokine IL-1ß from an inactive precursor to an active, secreted molecule. Thus, caspase-1 is regarded as a key mediator of inflammatory processes, and has become synonymous with inflammation. In addition to the processing of IL-1ß, caspase-1 also executes a rapid programme of cell death, termed pyroptosis, in macrophages in response to intracellular bacteria. Pyroptosis is also regarded as a host response to remove the niche of the bacteria and to hasten their demise. These processes are generally accepted as the main roles of caspase-1. However, there is also a wealth of literature supporting a direct role for caspase-1 in non-infectious cell death processes. This is true in mammals, but also in non-mammalian vertebrates where caspase-1-dependent processing of IL-1ß is absent because of the lack of appropriate caspase-1 cleavage sites. This literature is most prevalent in the brain where caspase-1 may directly regulate neuronal cell death in response to diverse insults. We attempt here to summarise the evidence for caspase-1 as a cell death enzyme and propose that, in addition to the processing of IL-1ß, caspase-1 has an important and a conserved role as a cell death protease.

Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness.

The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

Turbot TNFalpha gene: molecular characterization and biological activity of the recombinant protein.

The tumor necrosis factor (TNF) superfamily is composed by several proteins with similar structure and functions. One of the main representatives of this family is TNF-alpha (TNFalpha), a proinflammatory cytokine which is produced by different immune cells and presents a wide variety of activities. Using the RACE technique, we have cloned and sequenced the turbot TNF cDNA. The analysis of its sequence showed several conserved motifs characteristic of members of the TNFalpha family. A phylogenetic tree constructed with different TNFs of fish and mammals grouped our sequence within the fish TNFalpha cluster. Therefore, the turbot TNF here studied was identified as TNFalpha. The complete TNFalpha gene was obtained by gene walking, and, similarly to the other known fish TNFalpha genes, presented three introns and four exons. A PCR was designed to study the turbot TNFalpha expression in vivo using as stimulus the bacteria Vibrio pelagius strain Hq222 and virus VHSV. The expression of the cytokine happened early after injection, and it was dependent on the pathogen injected and organ analyzed. Virus induced a higher TNFalpha expression, but this response was shorter in time than that induced by bacteria. In addition, TNFalpha expression was in general higher in kidney than in liver, as expected since the former is the haematopoietic organ of fish. The turbot recombinant TNFalpha (rTNFalpha) was obtained by IPTG induction of bacteria transformed with the pET15b-TNFalpha construct, and it was purified in native conditions. The recombinant protein was approximately 20 kDa in size, and its biological activity was assessed in vitro. No effect of the rTNFalpha neither alone nor in combination with LPS was observed on the chemiluminescence activity of turbot macrophages at any time tested. However, NO production was enhanced by the recombinant protein alone or with LPS 72 h after the addition of the treatments. Finally, turbot rTNFalpha was able to recruit and activate inflammatory cells when injected in gilthead seabream, although to a lesser extent than gilthead seabream rTNFalpha.