Age-dependent differences in efferocytosis determine the outcome of opsonophagocytic protection from invasive pathogens

Abstract

In early life, susceptibility to invasive infection skews toward a small subset of microbes, whereas other pathogens associated with diseases later in life, including Streptococcus pneumoniae (Spn), are uncommon among neonates. To delineate mechanisms behind age-dependent susceptibility, we compared age-specific mouse models of invasive Spn infection. We show enhanced CD11b-dependent opsonophagocytosis by neonatal neutrophils improved protection against Spn during early life. The augmented function of neonatal neutrophils was mediated by higher CD11b surface expression at the population level due to dampened efferocytosis, which also resulted in more CD11b^hi "aged" neutrophils in peripheral blood. Dampened efferocytosis during early life could be attributed to the lack of CD169⁺ macrophages in neonates and reduced systemic expressions of multiple efferocytic mediators, including MerTK. On experimentally impairing efferocytosis later in life, CD11b^hi neutrophils increased and protection against Spn improved. Our findings reveal how age-dependent differences in efferocytosis determine infection outcome through the modulation of CD11b-driven opsonophagocytosis and immunity.

Keywords: CD11b; complement; early-life immunity; efferocytosis; invasive infection; macrophages; myeloid development; neutrophils; opsonophagocytosis; streptococci.

Figure 1:. Neonates are resistant to systemic infection by Streptococcus pneumoniae, a ‘non-neonatal pathogen’.

[A] Survival of WT neonatal (NNT, solid circle black line, n = 26), juvenile (JUV, half solid circle grey spotted line, n = 25) and adult mice (AD, open circle grey line, n = 18) following intraperitoneal (IP) challenge with P2406, an Spn strain with invasive potential in humans adapted in mice, at 5 x 10¹ – 1 x 10² colony forming units (CFU) per gram of body weight. [B] Recoverable live Spn in blood of NNT and AD to assess systemic dissemination at two early time points, 24 hours (n = 16 for NNT, n = 11 for AD) and 32 hours (n = 19 for NNT, n = 13 for AD) post-infection, following IP challenge with Spn at 5 x 10¹ – 1 x 10² CFU per gram of body weight. [C] Similar as described in [B], but for spleen at 24 hours (n = 16 for NNT, n = 11 for AD) and 32 hours post-infection (n = 17 for NNT, n = 11 for AD); BW, body weight. [D] Recoverable live Spn in blood of NNT (n = 10) and JUV (n = 13) to assess systemic dissemination at 32 hours post-infection, following IP challenge with Spn at 5 x 10¹ – 1 x 10² CFU per gram of neonatal body weight. [E] Similar as described in [D] but for spleen (n = 10 for NNT, n = 13 for JUV). [F] Blood neutrophil counts from mice treated with an IgG isotype control clone 2A3 (n = 6) or neutrophil-specific depletion antibody clone 1A8 (n = 6) following IP challenge with Spn at 5 x 10¹ – 1 x 10² CFU per gram body weight at 32 hours post-infection. [G] Recoverable live bacteria in spleen from mice described in [F]. [H] Blood monocyte counts from mice that were either wild-type, Ccr2^+/+ (n = 15), or CCR2-deficient, Ccr2^−/− (n = 9), following IP challenge with Spn at 5 x 10¹ – 1 x 10² CFU per gram body weight at 32 hours post-infection. [I] Recoverable live bacteria in spleen from mice described in [H]. Each data point represents an individual mouse, and data are representative of experiments repeated at least twice. Data with error bars are presented as mean ± SEM. N.S., not significant.

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⁺ Ly6C^mid) 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.

Figure 3:. Circulating neutrophils in neonatal mice bias towards an ‘aged’ phenotype.

[A] MFI of CD62L on peripheral blood neutrophils from NNT (n = 10) versus AD mice (n = 7). [B] Frequency of CD62L^lo ‘aged’ neutrophils from peripheral blood of NNT (n = 10) versus AD mice (n = 7) as proportion of all hematopoietic cells [Live, CD45⁺ cells] (left) and of all neutrophils [Ly6G⁺ cells] (right); FMO, fluorescence-minus-one control. [C-H] MFI analysis of known markers between CD62L^lo ‘aged’ (red dots) and CD62L^hi ‘non-aged’ (blue dots) neutrophils in neonatal blood (n = 10). Each data point represents a biological replicate (refer to STAR Methods for number of mice pooled), and data are representative of experiments repeated three times. Data with error bars are presented as mean ± SEM. N.S., not significant. See also Figure S2.

Figure 4:. Impaired efferocytosis in early life increases ‘aged’ neutrophil numbers in blood.

[A] Neutrophil counts in peripheral blood from NNT (6d old, n = 7; 9d old, n = 10), JUV (n = 6) and AD (n = 11) mice. [B-F] Transcriptional analysis of whole spleen from naïve NNT versus AD for [B] Cd169, [C] Lxra, [D] Mertk, [E] Abca1 and [F] Gas6. Expression was calculated as fold change in NNT relative to AD (n = 5 for both groups). [G] Confocal images of naive NNT (n = 5) and AD (n = 4) lungs immunostained for CD169 (blue), MerTK (red), F4/80 (yellow), Ly6G (green) and CD3 (cyan). Scale bar is at 50μm. Quantification of densitometry values are adjacent to the images. Each data point represents an individual mouse, 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, S3-S4.

Figure 5:. Impairing efferocytosis later in life protects against Spn in a CD11b-effector activity dependent manner.

[A] Frequency of CD62L^lo ‘aged’ neutrophils from peripheral blood of WT versus CD169-DTR adult mice per all Live, CD45⁺ cells (n = 9 for both groups). [B] Absolute counts of CD62L^lo ‘aged’ neutrophils in peripheral blood of WT versus CD169-DTR adult mice (n = 9 for both groups). [C] MFI of CD11b surface expression on CD62L^lo ‘aged’ and CD62L^hi ‘non-aged’ neutrophils in blood of WT versus CD169-DTR adults (n = 9 for both groups). [D] MFI of CD11b surface expression on neutrophils in bone marrow of WT (n = 11) versus CD169-DTR adults (n = 8). [E] Recoverable live Spn in blood (left) or spleen (right) of WT and CD169-DTR adults to assess systemic dissemination at 32 hpi (n = 9 for both groups) following IP Spn challenge. [F] Survival of WT (open circle grey line, n = 12), CD169-DTR (open circle pink line, n = 13) and both groups of mice treated with 25μg of cobra venom factor (WT, open circle dotted grey line, n = 9; CD169-DTR, open circle dotted pink line, n = 9) following IP Spn challenge. [G] Frequency of CD62L^lo ‘aged’ neutrophils from peripheral blood of Mertk^fl/fl (n = 6) versus Lyz2^Cre/+ Mertk^fl/fl (n = 9) adult mice per all Live, CD45⁺ cells. ^[H] Recoverable life Spn in blood (left) or spleen (right) of Mertk^fl/fl (n = 4) versus Lyz2^Cre/+ Mertk^fl/fl (n = 9) adult mice to assess systemic dissemination at 32hpi following IP Spn challenge. Each data point represents an individual mouse, 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 S5-S6.

Figure 6:. Dependence on CD11b-driven immunity predicts streptococcal infection outcome in early life.

[A] Survival of WT NNT (solid circle black line, n = 17), Itgam^−/− NNT (solid circle green line, n = 10) and both groups of mice treated with 25μg of cobra venom factor (WT NNT, solid circle dotted black line, n = 12; Itgam^−/− NNT, solid circle dotted green line, n = 11) and WT AD mice (open circle grey line, n = 11) following IP challenge with GAS5, a Streptococccus pyogenes strain with necrotizing fasciitis potential in humans, at 5 – 50 CFU per gram of body weight. [B] Survival of WT NNT (solid circle black line, n = 13), WT AD (open circle grey line, n = 7) and Itgam^−/− (open circle muted green line, n = 8) mice following IP challenge with GBS5, a Streptococcus agalactiae strain at 3 x 10⁵ CFU per gram of body weight. Each data point represents an individual mouse, and data are representative of experiments repeated at least twice. N.S., not significant.