Figure 2.
Platelet depletion recapitulates the reduced organ pathology and increased survival observed in Nbeal2−/−mice. (A) Representative flow cytometry plots of platelets in mouse whole blood when platelets are present (left, platelet intact mice) or absent (right, megakaryocyte/platelet–depleted mice). Platelets in whole blood of platelet-depleted anti-CD41–treated C57BL/6J mice (α-CD41) and diphtheria toxin–treated (−) MK mice normalized to their respective platelet intact control groups as quantified by flow cytometry (n = 29-37 mice/group). C57BL/6J mice were intraperitoneally injected with 100 μg of either an anti-CD41 or isotype control antibody on day 1 after PbA infection, and platelets were measured on day 3 after PbA infection. PF4-Cre–negative iDTR ((+) MK) (megakaryocyte and platelet sufficient) and (−) MK (megakaryocyte and platelet deficient) mice were intraperitoneally injected with 20 ng diphtheria toxin on days −7, −4, and −1 prior to PbA infection, and platelets were measured on day 0 of PbA infection. (B) Frequency of infected RBCs in peripheral blood of C57BL/6J mice given either an isotype control or anti-CD41 (α-CD41) antibody and diphtheria toxin–treated (+) MK and (−) MK mice at day 6 after infection with PbA as determined by staining and counting of thin blood smears (n = 14-20 mice/group). (C-E) Bioluminescence quantification of sequestered PbA schizonts expressing luciferase under the AMA-1 promoter in spleens (C), lungs (D), and brains (E) of PbA-infected mice in designated groups (n = 8-9 mice/group). Values are normalized to naïve control mice from each respective group (n = 4 mice/group). (F-G) Lung permeability (F) and brain permeability (G) in platelet-intact and depleted mice as described in panel A injected IV with 200 μL of 1% Evans blue dye at day 6 after infection with PbA (n = 7-11 mice/group). Representative images and quantification of dye extracted from whole organs are shown. Optical density values are normalized to naive control mice from each respective group (n = 4-5 mice/group). (H) Survival curves of PbA-infected C57BL/6J mice given an anti-CD41 (α-CD41, red line) or isotype control (green line) antibody on day 1 after infection (n = 15-21 mice/group). (I) Survival curves of PbA-infected (−) MK (blue line) and (+) MK (green line) mice given diphtheria toxin on days −7, −4, and −1 prior to infection (n = 11-13 mice/group). The gray shaded regions represent the typical timeframe of death from ECM. Bar graphs in panels A-B and F-G represent the mean ± standard error of the mean. Boxes in panels C-E represent the median ± the 25th and 75th percentiles with minimum/maximum whiskers. Statistical analyses were performed using the Mann-Whitney U test for isotype/α-CD41 and (+) MK/(−) MK groups, respectively (as separated by gray dotted line) (A-G) and log-rank Mantel-Cox test (H-I). Only statistically significant (P < .05) values are shown. Figures represent combined data from 2 (C-G,I) or ≥3 (A-B,H) independent experiments. FSC, forward scatter.

Platelet depletion recapitulates the reduced organ pathology and increased survival observed in Nbeal2−/−mice. (A) Representative flow cytometry plots of platelets in mouse whole blood when platelets are present (left, platelet intact mice) or absent (right, megakaryocyte/platelet–depleted mice). Platelets in whole blood of platelet-depleted anti-CD41–treated C57BL/6J mice (α-CD41) and diphtheria toxin–treated (−) MK mice normalized to their respective platelet intact control groups as quantified by flow cytometry (n = 29-37 mice/group). C57BL/6J mice were intraperitoneally injected with 100 μg of either an anti-CD41 or isotype control antibody on day 1 after PbA infection, and platelets were measured on day 3 after PbA infection. PF4-Cre–negative iDTR ((+) MK) (megakaryocyte and platelet sufficient) and (−) MK (megakaryocyte and platelet deficient) mice were intraperitoneally injected with 20 ng diphtheria toxin on days −7, −4, and −1 prior to PbA infection, and platelets were measured on day 0 of PbA infection. (B) Frequency of infected RBCs in peripheral blood of C57BL/6J mice given either an isotype control or anti-CD41 (α-CD41) antibody and diphtheria toxin–treated (+) MK and (−) MK mice at day 6 after infection with PbA as determined by staining and counting of thin blood smears (n = 14-20 mice/group). (C-E) Bioluminescence quantification of sequestered PbA schizonts expressing luciferase under the AMA-1 promoter in spleens (C), lungs (D), and brains (E) of PbA-infected mice in designated groups (n = 8-9 mice/group). Values are normalized to naïve control mice from each respective group (n = 4 mice/group). (F-G) Lung permeability (F) and brain permeability (G) in platelet-intact and depleted mice as described in panel A injected IV with 200 μL of 1% Evans blue dye at day 6 after infection with PbA (n = 7-11 mice/group). Representative images and quantification of dye extracted from whole organs are shown. Optical density values are normalized to naive control mice from each respective group (n = 4-5 mice/group). (H) Survival curves of PbA-infected C57BL/6J mice given an anti-CD41 (α-CD41, red line) or isotype control (green line) antibody on day 1 after infection (n = 15-21 mice/group). (I) Survival curves of PbA-infected (−) MK (blue line) and (+) MK (green line) mice given diphtheria toxin on days −7, −4, and −1 prior to infection (n = 11-13 mice/group). The gray shaded regions represent the typical timeframe of death from ECM. Bar graphs in panels A-B and F-G represent the mean ± standard error of the mean. Boxes in panels C-E represent the median ± the 25th and 75th percentiles with minimum/maximum whiskers. Statistical analyses were performed using the Mann-Whitney U test for isotype/α-CD41 and (+) MK/(−) MK groups, respectively (as separated by gray dotted line) (A-G) and log-rank Mantel-Cox test (H-I). Only statistically significant (P < .05) values are shown. Figures represent combined data from 2 (C-G,I) or ≥3 (A-B,H) independent experiments. FSC, forward scatter.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal