Extended Data Fig. 7: T3SS injection of Fpr1−/− neutrophils in vitro and in vivo.
a, b, Mouse plasma does not affect Fpr1−/− neutrophil resistance to T3SS injection. Y. pestis KIM D27(pMM83) translocation of YopM–Bla in mouse neutrophils, incubated in mouse plasma with (a) or without (b) prior heat inactivation (HI). One of three repeats is shown. Data are mean + s.e.m. (n = 3 biological replicates, shown as circles). One-way ANOVA with Bonferroni correction was used to identify significant differences. ***P < 0.001; **P < 0.01. c–e, The Fpr1−/− mutation does not abolish the influx of neutrophils into Y.-pestis-infected tissues. Age- and sex-matched C57BL/6J and Fpr1−/− mice (4 groups, n = 5 per group, 6–8 weeks old, 5 males and 5 females, 2 experimental replicates) were anaesthetized and infected by injection of 1,000 CFU Y. pestis CO92 into the inguinal region. Then, 4 h after the challenge, euthanized mice were necropsied, the dermis surrounding the injection site was removed and fixed in formalin for histopathological analysis. Consecutive thin-sectioned slides were stained with haematoxylin and eosin (H&E) or stained by immunohistochemistry with the neutrophil marker anti-Ly6G (α-Ly6G). c, Slides were analysed by a blinded investigator and assigned one of four pathology scores: 0, no neutrophil influx; 1, local infiltration; 2, moderate local infiltration; 3, widespread infiltration. d, e, Analysis of neutrophil influx was performed in male (n = 8) and in female (n = 10) mice (d) or in all mice (e; male and female, n = 18; 2 mice were excluded from the analysis owing to unclear histology). One-way ANOVA with Bonferroni correction was used to analyse differences. ns, not significant (d, e).
