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Mast cell activation in the skin contributes to inflammation, neurogenic inflammation and
hyperalgesia in sickle mice (Vincent et al., Blood 2013). However, the underlying cause
of mast cell activation and challenges in inhibiting mast cell activation remain unclear.
Hemolysis in sickle cell disease leads to excess free heme, which contributes to
endothelial activation and neutrophil extracellular trap (NET) formation. We therefore
hypothesized that free heme activates mast cells and extracellular trap formation in a
sickle microenvironment. We examined hemin-induced activation in mast cells from the
skin of HbAA-BERK control and HbSS-BERK sickle mice. Mast cells in culture were
incubated with vehicle, or I ng/ml TNF-α for 4 hours, or 40 μM hemin for 2 hours, or with
hemin for 2 hours after 2 hours of priming with TNF-α to create an inflammatory sickle
microenvironment. Cells were stained with cell impermeable dye SYTOX orange and cell
permeable dye SYTO13 to visualize the extracellular DNA fibers. Incubation with TNF-α
or hemin did not show significant eruption of DNA from the cell body. However, sickle
mast cells incubated with both TNF-α and hemin showed distinct DNA containing fibers
exploding from the cell body with web like formation similar to NETs. The length of
majority of individual fibers was more than 50 μm, indicative of TRAP formation. This
response was blunted in mast cells from control mice. Thus, mast cells in a sickle
microenvironment are primed and activated, and further priming with cytokines such as
TNF-α leads to hemin-induced mast cell extracellular trap (MET) formation. Sickle mast
cells express significantly higher TLR4 and FcεRI as compared to control mice (Vincent
et al., Blood 2013). Therefore, we examined if silencing of FcεRI and/or inhibition of
TLR4 attenuated TNF-α primed/hemin-induced MET formation. TAK242 at a dose of 1
μM but not at 0.5 μM inhibited TNF-α primed/hemin-induced MET formation, but
silencing of FcεRI had no effect. Since cannabinoids, imatinib, palmitoylethanolamide
(PEA) and cromolyn are known mast cell inhibitors, we examined their effect on TNFα/hemin-induced METs. Imatinib (30 – 100 μM), a known mast cell inhibitor, showed no
significant effect, while cromolyn (100 μM) led to a modest decrease in MET formation.
Cannabinoid CP 55,940 and PEA at a relatively low dose of 30 μM completely blocked
MET formation. Functionally, imatinib, PEA and CP 55,940 reduced the release of TNFα from mast cells incubated with TNF-α/hemin for 24 hours. Hemin in the presence of
TNF-α stimulated sustained activation of NLRP3 inflammasome signaling. Thus, mast
cells in a sickle microenvironment are activated due to a rich inflammatory cytokine
milieu, via activation of the inflammasome signaling upon stimulation with hemin. The
unique phenomenon of MET formation is orchestrated by a combined action of
inflammatory milieu and hemin, imparting resistance to common mast cell inhibitors such
as cromolyn and imatinib.