Figure 2: Increased CD11b expression in early life enhances neutrophil function and drives systemic immunity against Spn.
[A] Flow cytometry analysis of CD11b surface expression via relative median fluorescence intensity (MFI) on bone marrow neutrophils (gated on Singlets+ CD45+ CD11b+ Ly6G+ Ly6Cmid) from mice at different ages [3 days old (3d), n = 4; 6 days old (6d), n = 5; 9 days old (9d), n = 5; JUV, n = 5; AD, n= 10].
[B] RT-PCR analysis of CD11b (Itgam) mRNA transcript in NNT (n = 15) versus AD (n = 11) bone marrow neutrophils isolated via density centrifugation.
[C] MFI of CD11b on peripheral blood neutrophils from NNT (n = 10) versus AD mice (n = 7).
[D] CD11b marker expression of neutrophils from mass cytometry experiment comparing newborns aged 0 – 5 days (n = 75) and 31 – 81 days after birth (n = 31), mean expression from binned days used for coloring.
[E] Opsonophagocytic uptake of mouse sera opsonized GFP-fluorescent Spn (P2531) by neutrophils from bone marrow suspensions of NNT versus AD mice at multiplicity of infection (MOI) of 25 (n = 8 for both groups).
[F] MFI of GFP on GFP+ neutrophils from [E] (n = 8 for both groups).
[G] Recoverable live bacteria in blood (left) and spleen (right) of WT (Itgam+/+) and CD11b-deficient (Itgam−/−) NNT mice challenged IP with Spn at 32 hours post-infection (n = 8 for Itgam+/+ n = 10 for Itgam−/−).
[H] MFI of CD11b on neutrophils from Itgam+/+ NNT (n = 3), CD11b-heterozygous (Itgam+/−) NNT (n = 6), and Itgam+/+ AD (n = 6). Itgam−/− AD is included as negative control (n = 2).
[I] Survival of Itgam+/+ NNT (solid circle black line; n = 20), Itgam+/− NNT (solid circle orange line; n = 17), Itgam−/− NNT (solid circle green line; n = 17) and Itgam−/− AD (open circle muted green line; n = 12) mice challenged IP with Spn at 5 x 101 – 1 x 102 CFU/g body weight.
[J] Neutrophil counts in peripheral blood from Itgam+/+ (n = 10), Itgam+/− (n = 10) and Itgam−/− (n = 9) NNT mice.
[K] Survival of Itgam+/+(solid circle dotted black line, n = 9) and Itgam−/− (solid circle dotted green line, n = 9) NNT mice treated with 25μg cobra venom factor at 24 hours prior infection, followed by IP challenge with Spn at 5 x 101 – 1 x 102 CFU/g body weight.
[L] Survival of WT NNT (solid circle black line; n = 18) versus AD mice (open circle grey line; n = 20), and both groups of mice treated with 25μg cobra venom factor (WT NNT, solid circle dotted black line, n = 9; WT AD, open circle dotted grey line, n = 11) following IP challenge with complement sensitive capsule switch mutant, P2453, at 5 x 101 – 1 x 102 CFU/g body weight.
[M] Recoverable live bacteria in blood of Itgam−/− neonate recipients receiving either Itgam+/+ neonatal neutrophils (solid circle, n = 9), Itgam+/− neonatal neutrophils (solid orange circles, n = 8) or Itgam−/− neonatal neutrophils (solid green circle, n = 8) relative to that of recipients receiving Itgam+/+ adult neutrophils (open circle; n = 9, n = 9, n = 7, respectively) at 20 hours post infection with Spn via IP. Each data point represents a biological replicate (refer to STAR Methods for number of mice pooled), and data are representative of experiments repeated at least twice. Data with error bars are presented as mean ± SEM. N.S., not significant. See also Figure S1.