The Ca2+-dependent pathway contributes to changes in the subcellular localization and extracellular release of interleukin-33.

Interleukin-33 (IL-33) is a member of the IL-1 cytokine family and plays critical roles in facilitating type-2 immune responses. IL-33 is localized in the nucleus and released to the extracellular milieu during cell death, although the precise mechanisms underlying IL-33 mobilization remain unclear. Here, we found that nigericin, a toxin derived from Streptomyces hygroscopicus, promoted IL-33 translocation from the nucleus to the cytosol before extracellular release. This translocation was inhibited by chelating Ca2+ with EGTA or membrane protection by glycine treatment. Ca2+ ionophore A23187 stimulation caused IL-33 translocation to the cytoplasm but was not sufficient for extracellular release. However, IL-33 release was induced by detergent treatment, which indicates that membrane rupture is required for IL-33 release. The pore-forming pyroptosis executor gasdermin D was cleaved following nigericin stimulation, and overexpression of the cleaved gasdermin D-N-terminal fragment that forms the membrane pore sufficiently induced IL-33 release, which was blocked by EGTA and glycine. Together, these findings suggest that Ca2+-dependent signals and gasdermin D pore formation are required for robust IL-33 production.

Non-canonical Inflammasome-Mediated IL-1ß Production by Primary Endometrial Epithelial and Stromal Fibroblast Cells Is NLRP3 and Caspase-4 Dependent.

Inflammation of the post-partum uterus is a normal physiological event, crucial for tissue involution and repair. However, in the bovine, some cows fail to resolve this inflammation, resulting in endometritis, which compromises fertility. Earlier work has identified upregulated expression of the potent inflammatory cytokine IL-1ß early post-partum, in cows which subsequently develop endometritis. As a result, targeting IL-1ß expression holds potential as a novel treatment for this disease, yet the regulatory mechanisms contributing to IL-1ß expression in the bovine endometrium remain unknown. To investigate this, endometrial tissue samples were obtained 7 and 21 days post-partum (DPP) from cows that were diagnosed with endometritis at 21 DPP and cows that experienced a physiological level of inflammation throughout involution. IL-1ß was measured by qPCR, ELISA, and immunohistochemistry. Seven DPP, endometrial IL-1ß protein levels were significantly higher in animals that proceeded to develop endometritis at 21 DPP. IL-1ß production could be detected in luminal and glandular epithelium, in underlying stromal fibroblasts as well as infiltrating immune cells. To investigate the mechanisms regulating IL-1ß expression, primary endometrial epithelial cells, stromal fibroblasts and PBMCs were stimulated with LPS and the inflammasome activator nigericin. Stromal fibroblast cells were particularly potent producers of IL-1ß. Basolateral LPS stimulation of polarized epithelial cells induced IL1B mRNA and a previously undescribed IL-1ß protein isoform, with preferential protein secretion into the apical compartment. Key inflammasome components [nod-like receptor protein 3 (NLRP3), nima-related kinase-7 (NEK7), apoptosis speck like protein containing a CARD (ASC), and gasdermin-D] were expressed by endometrial cells following stimulation. Endometrial cell stimulation in the presence of NLRP3 receptor (MCC950) and pan-caspase (Z-VAD-FMK) inhibitors blocked IL-1ß production, demonstrating its dependence on the NLRP3 inflammasome and on caspase activity. Furthermore, caspase-4 specific siRNA prevented IL-1ß production, confirming that inflammasome activation in endometrial cells is caspase-4 but not caspase-1 dependent, as shown in other species. Identifying the tissue- and species-specificity of inflammasome assembly and activation has critical relevance for our understanding of inflammation and suggests new therapeutic targets to enhance the resolution of inflammatory pathologies including endometritis in cattle.

Human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase.

BACKGROUND: The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. OBJECTIVE: We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. METHODS: After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. RESULTS: Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1ß processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1ß release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1ß processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. CONCLUSION: Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK.

Inflammatory immune response by lipopolysaccharide-responsive nucleotide binding oligomerization domain (NOD)-like receptors in the Japanese pufferfish (Takifugu rubripes).

Some of NOD-like receptors (NLRs), the cytosolic pattern recognition receptors form a multi-protein complex, inflammasome consisting of one or more NLRs, the adaptor protein ASC and inflammatory caspase to generate mature inflammatory cytokines, interleukin (IL)-1ß and IL-18. However, inflammasome-mediated inflammatory cascade involving any NLR member is unknown in a lower vertebrate like fish. Also, inflammatory cytokine induction pathway in response to a specific ligand, namely bacterial lipopolysaccharide (LPS) has not yet been clarified. Therefore, 13 predicted NLR sequences of the Japanese pufferfish, Fugu (Takifugu rubripes) were retrieved in silico and categorized as NLR-C1∼13. Expression analysis of these genes in Fugu head kidney (HK) cells stimulated with a heat-killed Lactobacillus paracasei spp. paracasei (Lpp), LPS, nigericin and a combination of nigericin + LPS showed consistent up-regulations of NLR-C1, 5, 7, 10 and 12 genes in both Lpp and LPS stimulations and NLR-C9 gene in LPS stimulation only. However, nigericin and nigericin + LPS caused an increased expression of NLR-C10 and 12 in HK cells and leukocytes. Fugu treated with Lpp and LPS (in vivo), and infected with Vibrio harveyi had an elevated expression of NLR-C10 and 12. Increased transcription of caspase-1, ASC, IL-1ß and IL-18 was recorded in nigericin-stimulated HK cells and leukocytes. Results suggested activation of probable inflammasome-mediated inflammatory cytokine response in Fugu. Moreover, LPS may be a key ligand that induces some of the Fugu NLR-Cs (NLR-C9, 10 and 12). Further characterization and functional analysis of Fugu NLR-C10 and 12 for ligand sensing, and processing of pro-inflammatory cytokine, IL-1ß would elucidate the inflammasome evolution in fish.

[Development of monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic assay for lasalocid and semduramicin].

Monoclonal antibodies (MAbs) against lasalocid and semduramicin were prepared using keyhole limpet hemocyanin conjugates for the immunization of mice. With these MAbs, we developed quantitative enzyme-linked immunosorbent assay (ELISA) methods for lasalocid and semduramicin. The ELISAs were quantitative in the ranges of 0.1-50 ng/mL for lasalocid and 0.05-12.5 ng/mL for semduramicin, and showed 50% inhibition concentrations of 1.2 ng/mL for lasalocid and 0.5 ng/mL for semduramicin. The coefficient of variations (CV%) of lasalocid were 0.3-4.4% for intra-assay and 0.5-5.1% for inter-assay and those of semduramicin were 0.1-4.6% for intra-assay and 0.3-5.2% for inter-assay. The detection limits for lasalocid and semduramicin were 10 ng/g and 5 ng/g in chicken liver and muscle, respectively. Based on the immunochromatographic method, rapid test kits for lasalocid and semduramicin were also developed. With these kits, the detection limits of lasalocid were 50 ng/mL for standard solution and 125 ng/g for chicken muscle, and those of semduramicin were 10 ng/mL for standard solution and 100 ng/g for chicken muscle.