IL-33 induces granzyme C expression in murine mast cells via an MSK1/2-CREB dependent pathway

Granzymes comprise a group of proteases involved in the killing of infected or cancerous cells by the immune system. Although best studied in T cells and natural killer (NK) cells, they are also expressed in some innate immune cells. Granzymes B and C are encoded in the mouse chymase locus that also encodes a number of mast cell-specific proteases. In line with this, mast cells can express granzyme B, although how this is regulated and their ability to express other granzymes is less well studied. We therefore examined how IL-33, a cytokine able to activate mast cells but not induce degranulation, regulated granzyme B and C levels in mast cells. Granzyme C, but not B, mRNA was strongly up-regulated in bone marrow-derived mast cells following IL-33 stimulation and there was a corresponding increase in granzyme C protein. These increases in both granzyme C mRNA and protein were blocked by a combination of the p38α/β MAPK inhibitor VX745 and the MEK1/2 inhibitor PD184352, which blocks the activation of ERK1/2. ERK1/2 and p38α activate the downstream kinases, mitogen and stress-activated kinases (MSK) 1 and 2, and IL-33 stimulated the phosphorylation of MSK1 and its substrate CREB in an ERK1/2 and p38-dependent manner. The promoter for granzyme C contains a potential CREB-binding site. Bone marrow-derived mast cells from either MSK1/2 double knockout or CREB Ser133Ala knockin mice were unable to up-regulate granzyme C. Together these results indicate that IL-33-induced granzyme C expression in mast cells is regulated by an MSK1/2-CREB-dependent pathway

DIA label-free proteomic analysis of murine bone-marrow-derived macrophages.

Here, we describe an optimized protocol to analyze murine bone-marrow-derived macrophages using label-free data-independent acquisition (DIA) proteomics. We provide a complete step-by-step protocol describing sample preparation utilizing the S-Trap approach for on-column digestion and peptide purification. We then detail mass spectrometry data acquisition and approaches for data analysis. Single-shot DIA protocols achieve comparable proteomic depth with data-dependent MS approaches without the need for fractionation. This allows for better scaling for large sample numbers with high inter-experimental reproducibility. For complete details on the use and execution of this protocol, please refer to Ryan et al. (2022)