Exocytic machineries differentially control mediator release from allergen-triggered RBL-2H3 cells.

BACKGROUND: Mast cells utilize SNAREs (soluble-N-ethyl-maleimide sensitive factor attachment protein receptors) and SM (Sec1/Munc18) proteins to secrete/exocytose a variety of proinflammatory mediators. However, whether a common SNARE-SM machinery is responsible remains unclear. METHODS: Four vesicle/granule-anchored SNAREs (VAMP2, VAMP3, VAMP7, and VAMP8) and two Munc18 homologs (Munc18a and Munc18b) were systematically knocked down or knocked out in RBL-2H3 mast cells and antigen-induced release of ß-hexosaminidase, histamine, serotonin, and TNF was examined. Phenotypes were validated by rescue experiments. Immunofluorescence studies were performed to determine the subcellular distribution of key players. RESULTS: The reduction of VAMP8 expression inhibited the exocytosis of ß-hexosaminidase, histamine, and serotonin but not TNF. Unexpectedly, however, confocal microscopy revealed substantial co-localization between VAMP8 and TNF, and between TNF and serotonin. Meanwhile, the depletion of other VAMPs, including knockout of VAMP3, had no impact on the release of any of the mediators examined. On the other hand, TNF exocytosis was diminished specifically in stable Munc18bknockdown cells, in a fashion that was rescued by exogenous, RNAi-resistant Munc18b. In line with this, TNF was co-localized with Munc18b (47%) to a much greater extent than with Munc18a (13%). CONCLUSION: Distinct exocytic pathways exist in mast cells for the release of different mediators.

PKC-dependent phosphorylation of Munc18a at Ser313 in activated RBL-2H3 cells.

Protein Kinase C (PKC) regulates the release of pro-inflammatory compounds from IgE/antigen-activated mast cells by unknown mechanisms. In this study, we show for the first time that PKC inhibitor Ro-03-0432, which inhibits RBL-2H3 exocytosis/degranulation in a concentration-dependent fashion, prevents the phosphorylation of membrane fusion factor Munc18a at Ser 313. Our study provides fresh evidence that PKC-dependent protein phosphorylation may contribute to the intricate regulation of mast cell degranulation by directly targeting the fusion factors.

Author Correction: PKC-dependent phosphorylation of Munc18a at Ser313 in activated RBL-2H3 cells.

Acknowledgements This work was supported by the University of Southern Mississippi Development Grant DE01475, the National Institute of Allergy and Infectious Diseases Grant 1R15AI133430-01, and by the Mississippi INBRE, which was funded by an Institutional Development Award (IDeA) from NIGMS under Grant no. P20GM103476.

Munc18a clusters SNARE-bearing liposomes prior to trans-SNARE zippering.

Sec1-Munc18 (SM) proteins co-operate with SNAREs {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein] receptors} to mediate membrane fusion in eukaryotic cells. Studies of Munc18a/Munc18-1/Stxbp1 in neurotransmission suggest that SM proteins accelerate fusion kinetics primarily by activating the partially zippered trans-SNARE complex. However, accumulating evidence has argued for additional roles for SM proteins in earlier steps in the fusion cascade. Here, we investigate the function of Munc18a in reconstituted exocytic reactions mediated by neuronal and non-neuronal SNAREs. We show that Munc18a plays a direct role in promoting proteoliposome clustering, underlying vesicle docking during exocytosis. In the three different fusion reactions examined, Munc18a-dependent clustering requires an intact N-terminal peptide (N-peptide) motif in syntaxin that mediates the binary interaction between syntaxin and Munc18a. Importantly, clustering is preserved under inhibitory conditions that abolish both trans-SNARE complex formation and lipid mixing, indicating that Munc18a promotes membrane clustering in a step that is independent of trans-SNARE zippering and activation.

TNFR1 links TNF exocytosis to TNF production in allergen-activated RBL-2H3 cells.

We previously reported that the maximal production of Tumor Necrosis Factor (TNF or TNFα) in antigen-activated RBL-2H3 cells (a tumor analog of mucosal mast cells) requires Munc13-4, a regulator of exocytic fusion. In this study, we investigated the involvement of various fusion catalysts in TNF production. We observed a strong correlation between the total TNF level and TNF exocytosis in RBL-2H3 cells. RT-qPCR shows that TNFR1 (TNF receptor 1) is the sole TNFR expressed in these cells, and that its transcription is upregulated upon allergen-mediated activation. Importantly, the addition of soluble TNFR1 inhibits antigen-elicited TNF production in a dosage-dependent fashion. Likewise, TNF production is diminished in the presence of TACE (TNFα Converting Enzyme) inhibitor KP-457, which prevents the generation of soluble TNF (sTNF). Together, these findings indicate that sTNF and TNFR1 function as autocrine agent and receptor respectively at the mast cell surface to boost TNF proliferation during allergic inflammation.

TNF Production in Activated RBL-2H3 Cells Requires Munc13-4.

Mast cell activation triggers intricate signaling pathways that promote the expression and/or release of a wide range of mediators including tumor necrosis factor (TNF; also known as TNFα). In this study, we investigated the connection between TNF secretion and TNF production, exploiting RBL-2H3 cells (a tumor analog of mucosal mast cells) that are depleted of Munc13-4, a crucial component of the mast cell exocytic machinery. We showed that antigen/IgE elicited robust TNF production in RBL-2H3 cells, but not in Munc13-4 knockout cells. The production defect was corrected when Munc13-4 was reintroduced into the knockout cell line, suggesting that the phenotype was not caused by any secondary effect derived from the knockout approach. Furthermore, pre-incubation of RBL-2H3 cells with R-7050, an antagonist of TNF receptor-dependent signaling, was shown to block TNF production without inhibiting TNF release. These observations provide fresh evidence for a robust feed-back loop to boost TNF production in activated mast cells.

Correction: Surveillance of Influenza A Virus and Its Subtypes in Migratory Wild Birds of Nepal.

[This corrects the article DOI: 10.1371/journal.pone.0133035.].

Surveillance of Influenza A Virus and Its Subtypes in Migratory Wild Birds of Nepal.

Nepal boarders India and China and all three countries lie within the Central Asian Flyway for migratory birds. Novel influenza A H7N9 caused human fatalities in China in 2013. Subclinical infections of influenza A H7N9 in birds and the potential for virus dispersal by migratory birds prompted this study to assess avian H7N9 viral intrusion into Nepal. Surveillance of influenza A virus in migratory birds was implemented in early 2014 with assistance from the Food and Agricultural Organization (FAO). Of 1811 environmental fecal samples collected from seven wetland migratory bird roosting areas, influenza A H9N2 was found in one sample from a ruddy shelduck in Koshi Tappu Wildlife Reserve located in southern Nepal. Avian H7N9 and other highly pathogenic avian influenza viruses were not detected. This study provides baseline data on the status of avian influenza virus in migratory bird populations in Nepal.