Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1β during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca²⁺ pathway. The resulting increase in the intracellular calcium concentration triggers inflammasome assembly and Caspase-1 activation. We identified necrotic cells as one source for excess extracellular calcium triggering this activation. In vivo, increased calcium concentrations can amplify the inflammatory response in the mouse model of carrageenan-induced footpad swelling, and this effect was inhibited in GPRC6A^−/− mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation.

Extibacter muris is a newly described mouse gut bacterium which metabolizes cholic acid (CA) to deoxycholic acid (DCA) via 7α-dehydroxylation. Although bile acids influence metabolic and inflammatory responses, few in vivo models exist for studying their metabolism and impact on the host. Mice were colonized from birth with the simplified community Oligo-MM¹² with or without E. muris. As the metabolism of bile acids is known to affect lipid homeostasis, mice were fed either a low- or high-fat diet for eight weeks before sampling and analyses targeting the gut and liver. Multiple Oligo-MM¹² strains were capable of deconjugating primary bile acids in vitro. E. muris produced DCA from CA either as pure compound or in mouse bile. This production was inducible by CA in vitro. Ursodeoxycholic, chenodeoxycholic, and β-muricholic acid were not metabolized under the conditions tested. All gnotobiotic mice were stably colonized with E. muris, which showed higher relative abundances after HF diet feeding. The presence of E. muris had minor, diet-dependent effects on Oligo-MM¹² communities. The secondary bile acids DCA and surprisingly LCA and their taurine conjugates were detected exclusively in E. muris-colonized mice. E. muris colonization did not influence body weight, white adipose tissue mass, liver histopathology, hepatic aspartate aminotransferase, or blood levels of cholesterol, insulin, and paralytic peptide (PP). However, proteomics revealed shifts in hepatic pathways involved in amino acid, glucose, lipid, energy, and drug metabolism in E. muris-colonized mice. Liver fatty acid composition was substantially altered by dietary fat but not by E. muris.In summary, E. muris stably colonized the gut of mice harboring a simplified community and produced secondary bile acids, which affected proteomes in the liver. This new gnotobiotic mouse model can now be used to study the pathophysiological role of secondary bile acids in vivo.

Cryptococcosis, caused by Cryptococcus neoformans, has been demonstrated to be controlled by T helper (Th)1 cells while Th2 cells are associated with fungal growth and dissemination. Although cryptococcal immunoreactive protein antigens were previously identified, their association with Th1 or Th2 immune responses was not provided. In mice, Th1-dependent IFN-γ induces the production of IgG2a, whereas the Th2 cytokine IL-4 stimulates the expression of IgG1 rendering each isotype an indicator of the underlying Th cell response. Therefore, we performed an immunoproteomic study that distinguishes Th1- and Th2-associated antigens by their reactivity with Th1-dependent IgG2a or Th2-dependent IgG1 antibodies in sera from C. neoformans-infected wild-type mice. We additionally analysed sera from Th2-prone IL-12-deficient and Th1-prone IL-4Rα-deficient mice extending the results found in wild-type mice. In total, ten, four, and three protein antigens associated with IgG1, IgG2a, or both isotypes, respectively, were identified. Th2-associated antigens represent promising candidates for development of immunotherapy regimens, whereas Th1-associated antigens may serve as candidates for vaccine development. In conclusion, this study points to intrinsic immunomodulatory effects of fungal antigens on the process of Th cell differentiation based on the identification of cryptococcal protein antigens specifically associated with Th1 or Th2 responses throughout mice of different genotypes.

The obesity pandemic is presumed to be accelerated by endocrine disruptors such as phthalate-plasticizers, which interfere with adipose tissue function. With the restriction of the plasticizer di-(2-ethylhexyl)-phthalate (DEHP), the search for safe substitutes gained importance. Focusing on the master regulator of adipogenesis and adipose tissue functionality, the peroxisome proliferator-activated receptor gamma (PPARγ), we evaluated 20 alternative plasticizers as well as their metabolites for binding to and activation of PPARγ and assessed effects on adipocyte lipid accumulation. Among several compounds that showed interaction with PPARγ, the metabolites MINCH, MHINP, and OH-MPHP of the plasticizers DINCH, DINP, and DPHP exerted the highest adipogenic potential in human adipocytes. These metabolites and their parent plasticizers were further analyzed in human preadipocytes and mature adipocytes using cellular assays and global proteomics. In preadipocytes, the plasticizer metabolites significantly increased lipid accumulation, enhanced leptin and adipsin secretion, and upregulated adipogenesis-associated markers and pathways, in a similar pattern to the PPARγ agonist rosiglitazone. Proteomics of mature adipocytes revealed that both, the plasticizers and their metabolites, induced oxidative stress, disturbed lipid storage, impaired metabolic homeostasis, and led to proinflammatory and insulin resistance promoting adipokine secretion. In conclusion, the plasticizer metabolites enhanced preadipocyte differentiation, at least partly mediated by PPARγ activation and, together with their parent plasticizers, affected the functionality of mature adipocytes similar to reported effects of a high-fat diet. This highlights the need to further investigate the currently used plasticizer alternatives for potential associations with obesity and the metabolic syndrome.

Bisphenol A (BPA), which is used in a variety of consumer-related plastic products, was reported to cause adverse effects, including disruption of adipocyte differentiation, interference with obesity mechanisms, and impairment of insulin- and glucose homeostasis. Substitute compounds are increasingly emerging but are not sufficiently investigated.We aimed to investigate the mode of action of BPA and four of its substitutes during the differentiation of human preadipocytes to adipocytes and their molecular interaction with peroxisome proliferator-activated receptor γ (PPARγ), a pivotal regulator of adipogenesis.Binding and effective biological activation of PPARγ were investigated by surface plasmon resonance and reporter gene assay, respectively. Human preadipocytes were continuously exposed to BPA, BPS, BPB, BPF, BPAF, and the PPARγ-antagonist GW9662. After 12 days of differentiation, lipid production was quantified via Oil Red O staining, and global protein profiles were assessed using LC-MS/MS-based proteomics. All tested bisphenols bound to human PPARγ with similar efficacy as the natural ligand 15d-PGJ2in vitroand provoked an antagonistic effect on PPARγ in the reporter gene assay at non-cytotoxic concentrations. During the differentiation of human preadipocytes, all bisphenols decreased lipid production. Global proteomics displayed a down-regulation of adipogenesis and metabolic pathways, similar to GW9662. Interestingly, pro-inflammatory pathways were up-regulated, MCP1 release was increased, and adiponectin decreased. pAKT/AKT ratios revealed significantly reduced insulin sensitivity by BPA, BPB, and BPS upon insulin stimulation.Thus, our results show that not only BPA but also its substitutes disrupt crucial metabolic functions and insulin signaling in adipocytes under low, environmentally relevant concentrations. This effect, mediated through inhibition of PPARγ, may promote hypertrophy of adipose tissue and increase the risk of developing metabolic syndrome, including insulin resistance.