Page 161 - Haematologica July

GHI ings (C and E) and mean fluorescence intensity over baseline (ΔMFI) (D, F, H and I) of P-selectin (alpha granules) (C, D and H) and LAMP-1 (lysosomal granules) (E, F and I) translocated to the surface of washed platelets measured by flow cytometry. N=8-11 (C-F), N=3 (H and I). (G) Mean release of PF4 (alpha granules) measured by ELISA. N=3-9. Color legend in (A) applies to all panels. Bar: mean; error bar: Standard Error of Mean. #P<0.05; †P<0.01; *P<0.001; compar- isons are with WT unless otherwise spec-
haematologica | 2018; 103(7)
Munc13-4 in hemostasis and airway inflammation
intermediate phenotype (Figure 6C). The proportion of vessels occluded at 30 min was 100% for Munc13-4+/+ mice, 50% for Munc13-4F/F mice and 0% for Munc13-4Δ/Δ mice.
Platelets contribute to allergic airway inflammation in a Munc13-4-dependent manner
We observed that Munc13-4−/− mice were partially pro- tected from developing AHR in a model of asthma. While looking for the cell responsible, we found that megakary- ocyte/platelet-specific Munc13-4 KO mice presented a similar phenotype. We then studied our Munc13-4 mutant mice under an OVA-dependent acute model of allergic asthma. As expected, all OVA-exposed animals had an ele- vated baseline Rrs (total respiratory system resistance) compared to the naïve controls (data not shown), and these
baseline values were used to normalize the dose-response curves (Figure 7A). Given that the results in mice express- ing Cre recombinase in their platelets (Munc13-4+/+ Cre+ mice) were indistinguishable from those of Munc13-4+/+ mice, we concluded that this effect was not a Cre-induced artifact. We found that, although there was no difference between Munc13-4+/+ and Munc13-4F/F mice, Munc13-4Δ/Δ mice completely mimicked the blunted AHR observed in Munc13-4−/− mice (Figure 7A and B). Analysis of metha- choline PC1000 (data not shown) confirmed the same differ- ences.
The protection observed in Munc13-4−/− and Munc13-4Δ/Δ mice was not related to changes in airway mucous metaplasia, as we observed no significant differ- ences of intracellular epithelial mucin content in periodic acid-fluorescent Schiff (PAFS)-stained sections (Figure 7C
AB
CD
EF
Figure 3. Deletion of Munc13-4 impairs dense and lysosomal granule release directly and alpha granule release indi- rectly in thrombin-stimulated platelets. Samples from wild-type mice for Munc-13-2 and -4 (WT), Munc13-4 +/−, −/−, F/F, Δ/Δ, Munc13-2 −/−, and Munc13-2 and -4 double knockout (DKO) mice were with thrombin (Thr) or thrombin and ADP (10 mM). Representative trac- ings (A) and mean release of ATP (dense granules) (B) measured by luminometry in whole blood. N=5-9. Representative trac-
ified.
1239