a b s t r a c tThe aggregation by antigen of the IgE bound to its high affinity receptor on mast cells initiates a complex series of biochemical events that result in the release of inflammatory mediators. The essential role of the protein tyrosine kinase Syk has been appreciated for some time, and newer results have defined the mechanism of its activation. The use of siRNA has defined the relative contribution of Syk, Fyn and Gab2 to signaling and has made possible a screening study to identify previously unrecognized molecules that are involved in these pathways.Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

The pathophysiology of sickle cell disease (SCD) is driven by chronic inflammation. We show that elevated cell-free DNA (cfDNA) in SCD patients is not just a prognostic biomarker but contributes to the pathological inflammation. Within the elevated cfDNA, SCD patients had significantly higher proportion of mitochondrial (cf-mtDNA) to nuclear cfDNA (cf-nDNA) compared to healthy controls. Additionally, mtDNA in patient samples showed significant disproportionately increased hypomethylation compared to healthy controls, that was further increased in crises when compared to steady-state. Using flow cytometry, structured illumination microscopy and electron microscopy, we showed that circulating SCD red blood cells abnormally retained their mitochondria, and thus likely to be the source of the elevated cf-mtDNA in SCD patients. Patient plasma containing high levels of cf-mtDNA triggered formation of neutrophil extracellular traps (NETs) that was substantially reduced by inhibition of TANK-binding kinase 1 (TBK1) implicating activation of the cGAS-STING pathway. cf-mtDNA is an erythrocytic-DAMP, highlighting an underappreciated role of mitochondria in sickle pathology. Clinicaltrials.gov identifiers: #NCT00081523, #NCT03049475, #NCT00047996

The clustering of the high affinity receptor for IgE (Fc⑀RI) 2 by multivalent antigen on mast cells results in degranulation, with the release of inflammatory mediators such as leukotrienes and cytokines (1-3). In this signaling pathway phosphorylation of the immunoreceptor tyrosine-based activation motif of the ␤ and ␥ subunits of Fc⑀RI recruits and activates Syk, which is critical for the downstream propagation of signals (4 -9). Sykmediated phosphorylation and/or activation of phospholipase C␥, LAT, SLP-76, and PI3K are important for calcium mobilization and degranulation (7, 10 -12). The increase in intracellular calcium concentration leads to activation of molecules including MAPKs Erk, p38, and JNK, the transcription factors NFB and NFAT (13-19), all of which play important roles in the control of IgE-mediated cytokine synthesis.After Fc⑀RI aggregation the Src family kinase Lyn is thought to be the enzyme that phosphorylates the tyrosine residues of the immunoreceptor tyrosine-based activation motif of the ␤ and ␥ subunits of the receptor (3,20,21). However, bone marrow-derived mast cells (BMMC) from Lyn Ϫ/Ϫ mice have normal or enhanced Fc⑀RI-induced degranulation, suggesting that other tyrosine kinases could substitute for Lyn (22)(23)(24)(25)(26). Such a kinase could be Fyn, which is activated following IgE-Fc⑀RI stimulation and its tyrosine phosphorylation is increased in Lyn negative cells (22,27). In BMMC from Fyn deficient mice, there is decreased PI3K activation as reflected by Akt tyrosine phosphorylation, with reduced activation of JNK, p38 MAPK, and of the transcription factor NFB; these changes parallel decreased degranulation and release of leukotrienes (27, 28).Studies of Fyn-deficient BMMC suggest an interaction between Fyn and the adaptor protein Gab2 (27). Gab2 is tyrosine phosphorylated after Fc⑀RI stimulation and has potential binding sites for signaling molecules such as Grb2, SHP2, p85 subunit of PI3K, and Shc (27,29,30) . Mast cells from Gab2Ϫ/Ϫ mice show decreased Kit-mediated signal transduction resulting in decreased proliferation in vitro and lower number of mast cells in various tissues in vivo (31, 32). Bone-marrow derived mast cells from Gab2 Ϫ/Ϫ mice have decreased activation of PI3K, reduced tyrosine phosphorylation of phospholipase C␥1, Akt, and JNK resulting in reduced Fc⑀RI-induced degranulation and cytokine generation (33). In Fyn-deficient cells there is decreased tyrosine phosphorylation of Gab2 while in Syk Ϫ/Ϫ cells there is complete absence of Gab2 phosphorylation (27, 32). Gab2 might regulate mast cells degranulation by interfering with the granule translocation to the plasma membrane, through a calcium-independent mechanism that also involves Fyn and the small GTPase RhoA (34). These studies with Fyn and Gab2 deficient bone marrow mast cells suggest a role for these proteins in signaling from Fc⑀RI.In the present experiments, we used transient transfection with small interference RNA (siRNA) targeting Syk, Fyn, or Gab2 to investigate the relative roles of these mo...