WNT6/ACC2-induced storage of triacylglycerols in macrophages is exploited by Mycobacterium tuberculosis.

In view of emerging drug-resistant tuberculosis (TB), host-directed adjunct therapies are urgently needed to improve treatment outcomes with currently available anti-TB therapies. One approach is to interfere with the formation of lipid-laden "foamy" macrophages in the host, as they provide a nutrient-rich host cell environment for Mycobacterium tuberculosis (Mtb). Here, we provide evidence that Wnt family member 6 (WNT6), a ligand of the evolutionarily conserved Wingless/Integrase 1 (WNT) signaling pathway, promotes foam cell formation by regulating key lipid metabolic genes including acetyl-CoA carboxylase 2 (ACC2) during pulmonary TB. Using genetic and pharmacological approaches, we demonstrated that lack of functional WNT6 or ACC2 significantly reduced intracellular triacylglycerol (TAG) levels and Mtb survival in macrophages. Moreover, treatment of Mtb-infected mice with a combination of a pharmacological ACC2 inhibitor and the anti-TB drug isoniazid (INH) reduced lung TAG and cytokine levels, as well as lung weights, compared with treatment with INH alone. This combination also reduced Mtb bacterial numbers and the size of mononuclear cell infiltrates in livers of infected mice. In summary, our findings demonstrate that Mtb exploits WNT6/ACC2-induced storage of TAGs in macrophages to facilitate its intracellular survival, a finding that opens new perspectives for host-directed adjunctive treatment of pulmonary TB.

Innovative robust basophil activation test using a novel gating strategy reliably diagnosing allergy with full automation.

BACKGROUND: Allergy is one of the most common chronic diseases in Europe. Therefore, an increased need for specific and sensitive diagnostic tests that truly detect allergy exists. This study aimed at establishing a highly specific high-throughput and automated basophil activation test (BAT) that proves the existence of an allergy with utmost probability. METHODS: BAT from 1104 samples was analyzed; a novel gating strategy with three antibodies (FcεRIα, CD203c, CD63) was established and compared with our published protocol (12 antibodies). Based on the novel gating strategy, storage conditions, automated measurement, and analyses using R (1376 samples out of 1389) were optimized to set up a high-throughput BAT. RESULTS: No differences in sensitivity and specificity were found between the novel three antibody (FcεRIα, CD203c, CD63) and the 12 antibody gating strategy or between automated and manually analyzed samples (saving up to 90% of labor time). The time frame for basophil activation measurement after blood donation has been extended considerably (whole blood storage ≤7 days (RT) and 17 days (4°C) prior to BAT preparation and measurement). Respective storage conditions were optimized for samples after stimulation, staining, and preparation (≤7 days (RT) and 28 days (4°C)). These achievements were confirmed by a nationwide ring trial showing robustness and applicability of our BAT on a variety of flow cytometers. CONCLUSION: Our considerable optimizations overcame the hurdles that until now prevented the BAT from being used as high-throughput allergy diagnostic test in routine laboratories and shall allow for collaborative studies between clinics and research centers.

Extracellular ATP induces cytokine expression and apoptosis through P2X7 receptor in murine mast cells.

Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X(7), and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca(2+) influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-alpha. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X(7) receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X(7) receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.