An IL-10/DEL-1 axis supports granulopoiesis and survival from sepsis in early life

Abstract

The limited reserves of neutrophils are implicated in the susceptibility to infection in neonates, however the regulation of neutrophil kinetics in infections in early life remains poorly understood. Here we show that the developmental endothelial locus (DEL-1) is elevated in neonates and is critical for survival from neonatal polymicrobial sepsis, by supporting emergency granulopoiesis. Septic DEL-1 deficient neonate mice display low numbers of myeloid-biased multipotent and granulocyte-macrophage progenitors in the bone marrow, resulting in neutropenia, exaggerated bacteremia, and increased mortality; defects that are rescued by DEL-1 administration. A high IL-10/IL-17A ratio, observed in newborn sepsis, sustains tissue DEL-1 expression, as IL-10 upregulates while IL-17 downregulates DEL-1. Consistently, serum DEL-1 and blood neutrophils are elevated in septic adult and neonate patients with high serum IL-10/IL-17A ratio, and mortality is lower in septic patients with high serum DEL-1. Therefore, IL-10/DEL-1 axis supports emergency granulopoiesis, prevents neutropenia and promotes sepsis survival in early life.

Fig. 1. Tissue DEL-1 expression is elevated in neonatal age.

a DEL-1 relative mRNA expression in different tissues of healthy C57BL/6 neonate mice (4 days old) (expression of DEL-1 in the intestine was set as 1) (n = 5 animals per group in heart, liver and kidney and n = 6 animals per group for the rest of the groups). b DEL-1 relative mRNA expression in tissues from healthy C57BL/6 mice of adult (8–10weeks) or neonatal age (4 days old) (expression of DEL-1 in each tissue of adult mice was set as 1) (n = 5 animals per group in adult and neonatal kidney groups, n = 4 animals per group in adult and neonatal intestine groups, n = 8 animals per in adult and neonatal lung group and n = 4 animals per group on adult and neonatal brain tissue groups). DEL-1 relative mRNA expression in the c lung (n = 5 animals per group in adult and 1d, 14d and 21d neonate groups, n = 4 animal in 7d neonate group), d intestine (n = 5 animals per group in adult and 1d, 7d, and 14d neonate groups, n = 4 animal in 21d neonate group) and e kidney tissue (n = 5 animals per group in adult and 1d, 14d, and 21d neonate groups, n = 4 animal in 7d neonate group), from healthy C57BL/6 adult mice and healthy neonate mice of 1, 7, 14 or 21 days of postnatal age (the expression of DEL-1 in each adult tissue was set as 1). Statistical analysis by two-sided unpaired t test (b) and Kruskal Wallis with Dunn’s multiple comparison post-test (c–e). Mean ± SD (a, b) and median ± interquartile range (c–e) are depicted. Source data are provided as a Source Data file. Ad adults, Neo neonates, d days.

Fig. 2. DEL-1 expression in human neonates and adults.

a Median human DEL-1 protein concentration in cord blood serum from healthy newborns (gestational age 34–40 weeks old) and from healthy children at the age of 4 years old (n = 20 children and n = 20 newborns). b Median human DEL-1 protein serum concentration from healthy and septic adults and neonates (within <24 hours and >24 hours of sepsis onset) (n = 40 adult controls, n = 20 adults in <24 hours sepsis group, n = 20 adults in >24 hours sepsis group, n = 28 neonate controls, n = 15 neonates in <24 hours sepsis group and n = 9 neonates in >24 hours sepsis group). Statistical analysis by two-sided Mann-Whitney test (a) and Kruskal-Wallis test with Dunn’s multiple comparison post-test. Median ± interquartile range is depicted (a, b). Source data are provided as a Source Data file. Ctl control, h hours.

Fig. 3. Tissue DEL-1 expression is not suppressed upon sepsis in neonates and controls neonatal tissue neutrophil infiltration.

a DEL-1 relative mRNA expression in lung tissue from C57BL/6 mice of adult (8–10 weeks) or neonatal age (4 days old) upon polymicrobial sepsis [6 and 12 hours after cecal slurry (CS) administration] (expression of DEL-1 in adult control lungs was set as 1) (n = 9 animals in adult 0 h, n = 6 animals in adult 6 h, n = 7 animals in adult 12 h, n = 10 animals in neonates 0 h, n = 7 animals in neonates 6 h and n = 5 animals in neonates 12 h group). b DEL-1 protein levels in lung lysates from C57BL/6 mice of adult (8–10 weeks) or neonatal age (4 days old) following 12 hours of CS-induced polymicrobial sepsis (n = 7 animals in adult control, n = 5 animals in adult sepsis and neonate control groups and n = 6 animals in neonatal sepsis group). c DEL-1 relative mRNA expression in small intestine from C57BL/6 mice of adult (8–10 weeks) or neonatal age (4 days old) following 6 hours of CS-induced polymicrobial sepsis (expression of DEL-1 in adult control intestines was set as 1) (n = 4 animals in adult control group, n = 6 animals in adult sepsis group, n = 5 animals in neonate control group and n = 6 animals in neonatal sepsis group). d Myeloperoxidase (MPO) activity in total lung lysates in wild type (WT) C57BL/6 adult and neonate mice 6 and 12 hours after CS-induced sepsis (n = 6 animals in adult 0 h, n = 6 animals in adult 6 h, n = 4 animals in adult 12 h, n = 6 animals in neonates 0 h, n = 7 animals in neonates 6 h and n = 5 animals in neonates 12 h group). e Neutrophil  numbers (CD11b⁺ Ly6G⁺) in the peritoneum in WT C57BL/6 adult and neonate mice 6 and 12 hours after CS-induced sepsis (n = 6 animals in adult 0 h, n = 6 animals in adult 6 h, n = 5 animals in adult 12 h, n = 8 animals in neonates 0 h, n = 6 animals in neonates 6 h and n = 5 animals in neonates 12 h group). f MPO activity in lung (n = 8 animals in WT +IgG-Fc control, n = 5 animals in Del1^−/− + IgG-Fc control, n = 5 animals in Del1^−/− + DEL-1-Fc control, n = 7 animals in WT + IgG-Fc sepsis, n = 6 animals in Del1^−/− + IgG-Fc sepsis and n = 5 animals in Del1^−/− + DEL-1-Fc sepsis group). and g total neutrophil count (CD11b⁺ Ly6G⁺) in peritoneal lavage in WT and Del1^−/− C57BL/6 neonate mice that received either DEL-1-Fc or IgG-Fc i.v. 15 min prior to CS-induced sepsis (6 hours) (n = 8 animals in WT +IgG-Fc control, n = 6 animals in Del1^−/− + IgG-Fc control, n = 5 animals in Del1^−/− + DEL-1-Fc control and n = 8 animals per group in WT + IgG-Fc sepsis, Del1^−/− + IgG-Fc sepsis and Del1^−/− + DEL-1-Fc sepsis groups). Mean ± SD (a–d) is depicted. Statistical analysis by one-way ANOVA with Bonferroni’s multiple comparison post-test (a–g), and two-sided unpaired t test (a and c, between adult mice indicated with #). Source data are provided as a Source Data file. Ctl control, h hours, U units, g grams.

Fig. 4. DEL-1 promotes neonatal sepsis survival via enhancement of circulating neutrophil pool.

Survival rates of wild type (WT) and Del1^−/− C57BL/6 neonate mice after cecal slurry (CS) induced sepsis of either a mild (n = 16 animals per group), b moderate (n = 16 animals per group), or c severe severity (n = 16 animals per group). Bacterial counts, expressed as colony forming units (cfu) in d blood (n = 13 animals in WT 6 h, n = 12 animals in WT 12 h, n = 14 animals in Del1^−/− 6 h and n = 9 animals in Del1^−/− mice 12 h group) (in the 0 h time point the value was set to 0), and e peritoneum (n = 13 animals in WT 6 h, n = 8 animals in WT 12 h, n = 13 animals in Del1^−/− 6 h and n = 8 animals in Del1^−/− 12 h) (in the 0 h time point the value was set to 0). Neutrophils (CD11b⁺ Ly6G⁺) in (f) peritoneal lavage (n = 7 animals in WT 0 h, n = 14 animals in WT 6 h and n = 12 animals in WT 12 h, n = 6 animals in Del1^−/− 0 h, n = 17 animals in Del1^−/− 6 h and n = 10 animals in Del1^−/− 12 h) and g blood (n = 4 animals in WT 0 h, n = 21 animals in WT 6 h, n = 12 animals in WT 12 h, n = 4 animals in Del1^−/− 0 h, n = 19 animals in Del1^−/− 6 h and n = 11 animals in Del1^−/− 12 h group) in WT and Del1^−/− C57BL/6 neonate mice 6 and 12 hours following CS-induced sepsis. h Survival rates of Del1^−/− C57BL/6 neonate mice treated with i.v. DEL-1-Fc or IgG-Fc 15 min prior to injection of CS (n = 25 animals per group). i Neutrophils (CD11b⁺Ly6G⁺) (n = 5 animals in IgG-Fc Del1^−/− 0 h, n = 17 animals in IgG-Fc Del1^−/− 6 h, n = 15 animals in IgG-Fc Del1^−/− 12 h, n = 5 animals in Del1^−/− 0 h, n = 8 animals in DEL-1-Fc in Del1^−/− 6 h and n = 10 animals in DEL-1-Fc Del1^−/− 12 h group) and j bacterial counts (cfu) in the blood of Del1^−/− neonate pups treated with either i.v. DEL-1-Fc or IgG-Fc 6 and 12 hours following CS-induced sepsis (n = 4 animals in IgG-Fc Del1^−/− 6 h, n = 8 animals in IgG-Fc Del1^−/− 12 h, n = 7 animals in DEL-1-Fc Del1^−/− 6 h and n = 9 animals in DEL-1-Fc Del1^−/− 12 h group) (in the 0 h time point the value was set to 0). Statistical analysis by Log-rank (Mantel-Cox) test in survival experiments (a–c, h). Mean ± SD (d, e, f, g, i, j) is depicted. Statistical analysis by one-way ANOVA with Bonferroni’s multiple comparison post-test (d–g, i, j) or by two-sided unpaired t test (d–g, i, j, indicated with *) to compare between WT and Del1^−/− or IgG-Fc Del1^−/− and DEL-1-Fc Del1^−/− groups  at a specific timepoint following CS-induced sepsis. Source data are provided as a Source Data file. Ctl control.

Fig. 5. DEL-1 expression correlates with enhanced circulating neutrophils and lower mortality in septic human subjects.

a Fold increase in neutrophil numbers within 24 hours in human neonates with sepsis with either low serum DEL-1 concentration (<700 pg/ml) or high serum DEL-1 concentration (>700 pg/ml) (in the group with low serum DEL-1, at day 0, the neutrophil numbers were set as 1) (n = 12 neonates per group). 28-day mortality rate (%) in (b) septic neonates (n = 12 neonates per group) and (c) adults (n = 25 adults in the group wtih low serum DEL-1 concentration and n = 15 adults in the group with high serum DEL-1 concentration) with either low DEL-1 or high DEL-1 concentration in the serum (the threshold for high/low DEL-1 concentration was determined to 125 pg/ml for adult patients and 700 pg/ml for neonates). Mean ± SD (a) and frequency (%) (b, c) are depicted. Statistical analysis with one-way ANOVA with Bonferroni multiple comparison post-test (a) and Chi-squared test (for 28-day mortality categorical data b, c). Source data are provided as a Source Data file.

Fig. 6. DEL-1 supports bone marrow neutrophil pool in neonatal sepsis.

a DEL-1 protein expression (pg/ml) per 10⁶ bone marrow cells from wild type (WT) C57BL/6 mice of adult and neonatal age at basal state and following 12 hours of cecal slurry (CS) -induced sepsis (n = 5 animals per group in adult control, neonate control, and adult sepsis group and n = 4 animals in neonate sepsis group). b DEL-1 mRNA relative expression in total bone marrow cells from WT C57BL/6 mice of adult and neonatal age at basal state and following 6 and 12 hours of CS-induced sepsis (expression of DEL-1 in adult control mice was set as 1) (n = 24 animals in adult control, n = 20 animals in neonate control, n = 7 animals in adult 6 h sepsis, n = 6 animals in neonate 6 h sepsis, n = 6 animals in adult 12 h sepsis and n = 9 animals in neonate 12 h sepsis group). c Total neutrophil numbers (CD11b⁺Ly6G⁺Ly6C⁻, normalized per femur and per mouse weight in grams) in bone marrow of WT adult, WT and Del1^−/− C57BL/6 neonate pups in steady state and following 6 and 12 hours of CS induced sepsis (n = 7 animals in WT adult control group, n = 5 animals per group in WT adult 6 h and 12 h sepsis, n = 16 animals in WT neonate control, n = 10 animals per group in WT neonate 6 h and 12 h sepsis, n = 7 animals in Del1^−/− control, n = 9 animals in Del1^−/− 6 h sepsis and n = 10 animals in Del1^−/− 12 h sepsis). d Total neutrophil numbers (CD11b⁺Ly6G⁺Ly6C⁻, normalized per femur and per mouse weight in grams) in bone marrow of WT neonates, and Del1^−/− neonate pups treated with i.v. DEL-1-Fc or IgG-Fc 15 min prior to injection of CS and following 12 hours of CS-induced sepsis (n = 5 animals per group). Mean ± SD is depicted (a–d). Statistical analysis by one-way ANOVA with Bonferroni’s and multiple comparison post-test (a, b, d) and two-sided unpaired t test between the indicated groups (c). Source data are provided as a Source Data file. Ctl control, Ad adults, Neo neonate, h hours, g grams.

Fig. 7. DEL-1 supports multipotent progenitors upon sepsis in neonates.

a Long-term hematopoietic stem cells (LT-HSCs, CD48⁻CD150⁺LSK) [n = 4 animals per group in control wild type (WT) and Del1^−/− groups, and n = 5 animals per group in septic WT and Del1^−/− groups] b short-term HSCs (ST-HSCs, CD48⁻CD150⁻LSK) (n = 4 animals per group in control WT and Del1^−/− groups, and n = 5 animals per group in septic WT and Del1^−/− groups) c multipotent progenitors (MPPs, CD48⁺CD150⁻LSK) and (n = 4 animals per group in control WT and Del1^−/− groups, and n = 5 animals per group in septic WT and Del1^−/− groups) d myeloid-biased MPPs (MPP3s, Flt3⁻CD48⁺CD150⁻LSK) (n = 4 animals per group in control WT and Del1^−/− control groups and n = 5 animals per group in septic WT and Del1^−/− groups) and e representative flow cytometry plots of MPP and MPP3 cells in the bone marrow of WT and Del1^−/− C57BL/6 neonate mice at steady state (gray background) and at 12 hours of cecal slurryinduced polymicrobial sepsis. Mean ± SD is depicted (a–d). Statistical analysis by unpaired two-sided t test between WT and Del1^−/− control groups (gray background, a–d) and between WT and Del1^−/− septic groups (a–d). Source data are provided as a Source Data file. LSK (Lin⁻Sca-1⁺cKit⁺), Ctl control, h hours.

Fig. 8. DEL-1 prevents the decline of granulocyte progenitors upon sepsis in neonates.

a Representative flow cytometry plots of granulocyte-macrophage progenitors (GMPs) (Lin⁻c-Kit⁺ Sca-1⁻CD16/32⁺CD34⁺) in wild-type (WT) and Del1^−/− C57BL/6 neonate pups at 12 hours of cecal slurry (CS)-induced sepsis b percentage of GMPs in WT and Del1^−/− C57BL/6 neonate pups at basal state and following 12 and 24 hours of CS-induced sepsis (n = 9 animals in control WT, n = 11 animals in Del1^−/− control, n = 7 animals in WT 12 h sepsis, n = 6 animals in Del1^−/− 12 h sepsis, n = 6 animals in WT 24 h sepsis and n = 8 animals in Del1^−/− 12 h sepsis groups). c percentage of GMPs in WT neonate mice and Del1^−/− C57BL/6 neonate mice treated with i.v. DEL-1-Fc or IgG-Fc 15 min prior to injection of CS and following 12 hours of CS-induced sepsis (n = 5 animals per group). Mean ± SD is depicted (b, c). Statistical analysis by one-way ANOVA with Bonferroni’s multiple comparison post-test (c) and unpaired two-sided t test between WT and Del1^−/− control groups of the same time point (b, as indicated). Source data are provided as a Source Data file. Ctl control, h hours.

Fig. 9. IL-10 promotes DEL-1 expression in neonatal sepsis.

Protein expression of (a) TNF (n = 6 animals in adult control, n = 5 animals in adult sepsis, n = 8 animals in neonate control and n = 8 animals in neonate sepsis group), b IL-17A (n = 7 animals in adult control, n = 9 animals in adult sepsis, n = 5 animas in neonate control and n = 7 animals in neonate sepsis group), c IL-6 (n = 7 animals in adult control, n = 6 animals in adult sepsis, n = 5 animals in neonate control and n = 18 animals in neonate sepsis group) and d IL-10 (n = 6 in adult control, n = 8 in adult sepsis, n = 7 in neonate control and n = 13 in neonate sepsis group) protein expression in serum of C57BL/6 neonate and adult mice 6 hours after exposure to cecal slurry (CS)–induced sepsis. e DEL-1 mRNA relative expression in HUVECs at several time points upon stimulation with human recombinant IL-10 or IL-6 protein (expression of DEL-1 in control group was set as 1) (n = 5 biological replicates in control, n = 4 biological replicates in IL-10 4 h, n = 6 biological replicates in IL-10 24 h, n = 8 biological replicates in IL-6 4 h group and n = 7 biological replicates in IL-6 24 h group). f DEL-1 mRNA relative expression in Ea.hy926 endothelial cells upon stimulation with human recombinant IL-17, IL-10 or both, for 4 hours (expression of DEL-1 in control group was set as 1) (n = 10 biological replicates in control, n = 10 biological replicates in IL-10, n = 8 biological replicates in IL-17 and n = 12 biological replicates in IL-10 and IL-17 group). g Tissue (lung, kidney and intestine) IL-10 to IL-17A mRNA ratio in C57BL/6 neonate and adult mice exposed to CS sepsis (6 hours) (n = 5 animals per group in adult and neonatal lung groups, n = 4 animals in adult and n = 6 animals in neonatal kidney groups, n = 4 animals in adult and n = 6 animals in neonatal intestine groups and n = 4 animals per group on adult and neonatal brain tissue groups). h Median human DEL-1 protein in the serum of septic adults and neonates that exhibited either low (<2) or high (≥2) serum IL-10 to IL-17 ratio (n = 17 adults in ≤2 group, n = 24 adults in ≥2 group, n = 16 neonates in ≤2 group, and n = 8 neonates ≥2 group). i DEL-1 mRNA relative expression in human mesenchymal stromal cells upon 4 hours of IL-10 stimulation (expression of DEL-1 in control group was set as 1) (n = 4 biological replicates per group). j DEL-1 mRNA expression in murine bone marrow of wild type (WT) neonate mice that received either i.v anti-IL-10R (anti-IL-10 receptor) or IgG (expression of DEL-1 in control group was set as 1) and subjected to CS-induced sepsis (12 hours) (n = 6 animals in control, n = 8 animals in septic IgG group and n = 6 animals in septic a-IL-10R group). k Total neutrophils in bone marrow of WT neonate mice (left panel) and Del1^−/− neonate mice (right panel) that received either i.v. anti-IL-10R or IgG and subjected to CS-induced sepsis (12 hours) (n = 5 animals in IgG WT, n = 4 animals in a-IL-10R WT n = 4 animals in IgG Del1^−/− group and n = 4 animals in septic a-IL-10R Del1^−/− group), l Bacterial colony forming units (cfu) in the blood of WT neonate septic mice that received i.v. either anti-IL-10R or IgG (n = 5 animals in septic IgG group and n = 5 in septic anti-IL-10R group) and m, survival in WT neonate septic mice that received i.v. either anti-IL-10R or IgG and subjected to CS-induced sepsis (n = 21 animals in septic IgG group and n = 16 animals in septic a-IL-10R group). Mean ± SD (a–g, i–k), median ± interquartile range (h) and frequency % (m) are depicted. Statistical analysis by one-way ANOVA with Bonferroni’s multiple comparison post-test (a–e, j), two-sided Mann-Whitney test (h) unpaired two-sided t test (f, g, i, k, l) and Fisher’s exact test (m). Source data are provided as a Source Data file. Ctl control, Ad adults, Neo neonates, h hours.

Fig. 10. Mechanisms of IL-10/DEL-1 mediated protection in sepsis in early life.

Schematic illustration of the main findings of this study. Upon neonatal sepsis, a distinct cytokine response is developed, characterized by significant expression of interleukin (IL)-10 and reduced expression of IL-17 compared to adult sepsis. High IL-10 to IL-17 ratio augments DEL-1 expression in sepsis in early life. DEL-1 subsequently promotes emergency granulopoiesis in septic neonate mice, via support of myeloid-biased (MPPs) and granulocyte macrophage progenitors (GMPs) in the bone marrow. Emergency granulopoiesis further supports neutrophil production in the bone marrow and sustains the output of neutrophils in the peripheral circulation, leading to control of bacteremia and survival from sepsis in early life. Created with BioRender.com.