Mast cells modulate early responses to Mycobacterium bovis Bacillus Calmette-Guerin by phagocytosis and formation of extracellular traps.

The early interactions between the vaccine Mycobacterium bovis Bacillus Calmette Guerin (BCG) and host peripheral innate immune cells like Mast cells (MCs) may pave the way for generating appropriate protective innate and adaptive immune responses. Mice on administration of BCG by intratracheal instillation showed a massive increase in MC numbers in the infected lung. In vitro co-culture of BCG and rodent Rat Basophilic Leukaemia (RBL-2H3) MCs led to significant killing of BCG. RBL-2H3 MCs were able to phagocytose BCG, take up BCG-derived antigens by macropinocytosis, generate Reactive Oxygen Species (ROS) and degranulate. Further, a few MCs died and released MC extracellular traps (MCETs) having DNA, histones and tryptase to trap BCG. This study highlights the multi-pronged effector responses of MCs on encountering BCG. These responses or their evasion may lead to success or failure of BCG vaccine to provide long term immunity to infections.

The cysteine-rich domain of synaptosomal-associated protein of 23 kDa (SNAP-23) regulates its membrane association and regulated exocytosis from mast cells.

Synaptosomal-associated protein of 23 kDa (SNAP-23) plays an important role during regulated exocytosis of various inflammatory mediators, stored in secretory granules, from mast cells in response to physiological triggers. It is however synthesized as a soluble protein, and the mechanisms by which free SNAP-23 gets peripherally associated with membrane for the regulation of exocytosis, are poorly defined. SNAP-23 contains a hydrophobic domain with five closely spaced cysteines which get palmitoylated, and we show that SNAP-23 cysteine mutants show differential membrane association when transfected in rat basophilic leukemia (RBL) mast cells. SNAP-23 Cys- mutant, devoid of all five cysteines, and SNAP-23 P119A (proline to alanine) mutant, that likely interferes with palmitoylation of SNAP-23 by palmitoyl transferases are completely cytosolic. Mutating specific cysteines (Cys; C) to leucine or phenylalanine (L or F; retains hydrophobicity but lacks palmitoylation) partially decreases the membrane association of SNAP-23 which is further hampered by alanine (A; has lesser hydrophobicity, and lacks palmitoylation) mutation at C79, C80 or C83 position. Cloning a transmembrane domain MDR31-145 from multidrug resistance protein into SNAP-23 Cys- mutant is able to partially restore its membrane association. Regulated exocytosis studies using co-transfected human growth hormone (hGH) secretion reporter plasmid revealed that overexpression of SNAP-23 Cys- and P119A mutants significantly inhibits the overall extent of exocytosis from RBL mast cells, whereas expression of SNAP-23 Cys--MDR31-145 fusion protein is able to restore exocytosis. These results establish that the cysteine-rich domain of SNAP-23 regulates its membrane association and thereby also regulates exocytosis from mast cells.

Phosphorylation of SNAP-23 regulates its dynamic membrane association during mast cell exocytosis.

Upon allergen challenge, mast cells (MCs) respond by releasing pre-stored mediators from their secretory granules by the transient mechanism of porosome-mediated cell secretion. The target SNARE SNAP-23 has been shown to be important for MC exocytosis, and our previous studies revealed the presence of one basal (Thr102) and two induced (Ser95 and Ser120) phosphorylation sites in its linker region. To study the role of SNAP-23 phosphorylation in the regulation of exocytosis, green fluorescence protein-tagged wild-type SNAP-23 (GFP-SNAP-23) and its phosphorylation mutants were transfected into rat basophilic leukemia (RBL-2H3) MCs. Studies on GFP-SNAP-23 transfected MCs revealed some dynamic changes in SNAP-23 membrane association. SNAP-23 was associated with plasma membrane in resting MCs, however, on activation a portion of it translocated to cytosol and internal membranes. These internal locations were secretory granule membranes. This dynamic change in the membrane association of SNAP-23 in MCs may be important for mediating internal granule-granule fusions in compound exocytosis. Further studies with SNAP-23 phosphorylation mutants revealed an important role for the phosphorylation at Thr102 in its initial membrane association, and of induced phosphorylation at Ser95 and Ser120 in its internal membrane association, during MC exocytosis.