Expression of the homeostatic factor DEL-1 is elevated in the neonatal period
To determine whether tissue expression of DEL-1 differs between adult and neonates, we analyzed different tissues from healthy adult and neonatal mice. We compared the DEL-1 mRNA expression in the lung, kidney, intestine, liver, heart, and brain of neonatal (4 days old) and adult (8-10wks) mice (Figure 1A, B). DEL-1 mRNA was highly expressed in murine neonatal brain and lung tissue, to a lesser degree in intestine and kidney, while very low expression of the DEL-1 transcript was observed in the heart and liver (Figure 1A). DEL-1 mRNA expression was higher in neonate mouse pups compared to adult mice in all tissues studied (lung, kidney, intestine) except for the brain (Figure 1B, C). As postnatal age advanced, DEL-1 mRNA expression was gradually reduced in tissues that were examined (lung and kidney) (Figure 1C). We also measured human DEL-1 protein in cord blood serum from newborns (gestational age 34 to 40wks), peripheral blood serum from children at the age of 4 years (Figure 1D). Median DEL-1 serum concentration was significantly higher in neonates compared to older children (Figure 1D).
The expression of ICAM-1 and the DEL-1 receptor, β2 integrin LFA-1 (CD11a/CD18), were also evaluated in neonatal tissues and neutrophils respectively (Supplemental Figure 1). Mean fluorescence intensity of CD11a protein in blood neutrophils did not differ between adult and neonate mice (Figure S1 B, C). There was no difference in ICAM-1 mRNA expression in various tissues from healthy adult and neonatal mice (4 days old) (Figure S1E).
DEL-1 expression is not suppressed upon sepsis in neonates
Earlier studies have shown that DEL-1 is suppressed upon acute inflammation in several animal models of disease and may resurge in the context of resolution of inflammation (15, 19, 25, 27-29). The regulation of DEL-1 expression during neonatal sepsis remains unknown. To address this, we subjected WT adult and neonate mice to the cecal slurry (CS) model of polymicrobial peritonitis, a gold standard model for neonatal sepsis studies (30).
Following intraperitoneal CS administration, mRNA expression of DEL-1 was determined in the lung of neonate pups and adult mice at different time points. DEL-1 mRNA was downregulated six hours after CS-induced sepsis in adults and returned to basal at 20 hours, while it was elevated six hours after CS-induced sepsis in neonate pups (Figure 2A). We then compared changes of DEL-1 expression in lung, kidney, and intestine six hours after CS-induced sepsis. DEL-1 was suppressed in the lung, intestine and kidney, while in neonates it was increased in the lung or remained unchanged in kidney and intestine (Figure 2A, B). A similar pattern was observed in human serum, where DEL-1 was suppressed in septic adults (during the first 24 hours of sepsis) compared to healthy controls, while such a decrease was not evident in neonates with sepsis during the same time interval (Figure 2C). Protein expression of the DEL-1 receptor, CD11a, was upregulated similarly in the lung of both neonatal and adult mice with sepsis (Figure S1 B, D). ICAM-1 mRNA expression was also upregulated in both neonate and adult septic mice, but to a lesser extent in neonates (Figure S1 F, G).
Neonates had increased bacterial load in peritoneal lavage fluid, lung, intestine and kidney tissues compared to adult septic mice (Figure 2D). DEL-1 regulates neutrophil recruitment to the site of inflammation. We, therefore, evaluated potential differences in neutrophil infiltration in the peritoneal cavity of neonatal and adult mice exposed to CS-induced sepsis. Septic neonate pups exhibited lower numbers of neutrophils in peritoneal lavage fluid and reduced MPO activity in the lung and kidney of septic neonatal pups compared to adults (Figure 2E). Thus, higher levels of DEL-1 in neonates, as compared to adults, are associated with reduced neutrophil recruitment and defective bacterial clearance.
DEL-1 controls neonatal tissue neutrophil infiltration and is essential for neonate survival in sepsis
To evaluate the impact of DEL-1 on neonatal neutrophil recruitment, we subjected WT and Edil3−/− (hereafter designated Del1−/−) neonate mouse pups to CS-induced sepsis and evaluated neutrophil recruitment to the peritoneum and lung. Del1−/− septic neonate mice had elevated numbers of neutrophils in peritoneal lavage fluid (Figure 3A) and higher MPO activity in the lung (Figure 3B), compared to WT septic neonate mouse pups. In contrast, septic Del1−/− neonate mice that received i.v. recombinant DEL-1 protein fused with human IgG Fc (DEL-1-Fc), had reduced numbers of neutrophils in peritoneal lavage fluid and reduced MPO activity in the lung compared to septic Del1−/− neonates that received control IgG-Fc (Figure 3A, Β).
Next, we evaluated the effect of DEL-1 deficiency on survival from CS-induced sepsis in neonate pups. Del1−/− neonate pups exhibited reduced survival upon sepsis compared to WT ones when sepsis was mild or moderate, while at severe sepsis the difference was no longer evident (Figure 4A), suggesting that endogenous DEL-1 confers protection against sepsis. Consistent with this notion, DEL-1-Fc administration in the context of severe sepsis significantly improved the survival in Del1−/− septic neonatal pups (Figure 4B). Furthermore, we evaluated the 28-day mortality rate in humans, adult and neonate patients with sepsis that had either low or high serum DEL-1 concentration. Based on the median DEL-1 concentration of healthy sex- and age-matched humans that we included in this study (Figure 2D), the threshold for high/low DEL-1 concentration was determined to 125pg/ml for adult patients and 700pg/ml for neonates. The clinical characteristics, source of sepsis and outcome of the two groups of the neonates and adults are depicted in Supplemental Table 1 and 2 respectively. Neonates with sepsis in the high DEL-1 group appeared to exhibit lower 28-day mortality rate compared to the low DEL-1 group (8.3% vs. 25%, respectively) (Table S1). Furthermore, adult patients with sepsis in the high DEL-1 group exhibited lower 28-day mortality rate compared to the low DEL-1 adult group (13.3% vs 44%, respectively, p <0.05) (Table S2).
DEL-1 facilitates sustained neutrophil output in the blood circulation
To delineate the mechanism of DEL-1-mediated protection against neonatal sepsis, we evaluated cytokine responses, neutrophil infiltration and bacteria clearance in WT and Del1−/− neonate mice in the presence or absence of exogenous DEL-1 administration using DEL-1-Fc (26, 27). No difference was observed in serum TNFα and IL-10 protein concentration between WT and Del1−/− neonate pups or between Del1−/− neonate pups treated with either i.v DEL-1-Fc or control IgG 6 hours following CS-induced sepsis (Figure S2A, B). Administration of a broad-spectrum antibiotic (meropenem) significantly reduced the mortality rate of septic Del1−/− neonate mice 3-fold, while to a lesser degree in WT mice (Figure S2C), suggesting that defective bacterial clearance contributes to increased mortality in Del1−/− mice. Indeed, although at six hours following initiation of sepsis Del1−/− mice had less or similar bacterial burden in the peritoneum and blood, respectively, at 12 hours post-sepsis they exhibited higher bacterial load in the blood, as compared to WT neonate mice (Figure 5A). The increased bacterial load associated with DEL-1 deficiency was reversed by DEL-1-Fc administration (Figure 5C, left panel). The time course of neutrophil abundance in the peritoneum and blood followed the reverse pattern of that of the bacterial load. Specifically, at six hours following initiation of sepsis, Del1−/− mice had initially higher or similar neutrophil numbers in the peritoneum and blood respectively, but the number of neutrophils fell significantly at twelve hours post-sepsis and were significantly lower as compared to WT mice (Figure 5B), suggesting bone marrow exhaustion and a failure to replenish neutrophil numbers. Intriguingly, DEL-1-Fc administration prevented the decline in neutrophils numbers in DEL-1 deficient neonate pups (Figure 5C, right panel). Interestingly, neonate patients with sepsis that had high DEL-1 concentration on the first day of enrollment, exhibited a significant increase in the blood neutrophil count the following 24hours (2-fold) compared to the neonates of the low DEL-1 group (Figure 5D).
DEL-1 supports emergency granulopoiesis in the bone marrow of neonates
It has been recently shown that DEL-1, derived from endothelial and mesenchymal stromal cells in the bone marrow, promotes myelopoiesis under both steady-state and hematopoietic stress conditions (24). Since Del1−/− mice failed to sustain circulating neutrophils in the blood early upon sepsis, we evaluated the neutrophil pool and DEL-1 expression in the bone marrow in healthy and septic WT and Del1−/− mice of neonatal age and compared them to adult WT mice. WT neonate mice had smaller pool of neutrophils in the bone marrow both under steady-state and septic conditions compared to WT adult mice (Figure 6A, B). In comparison to WT neonates, Del1−/− neonates had similar numbers of neutrophils in the bone marrow at steady-state conditions but significantly lower during sepsis (Figure 6A, B). DEL-1 expression in the bone marrow was not significantly altered upon sepsis in adult mice but was increased 12 hours after sepsis in septic neonate mice compared to healthy ones (Figure 6C), ostensibly to support emergency granulopoiesis. Indeed, compared to WT neonates, Del1−/− neonates had similar numbers of granulocyte-macrophage progenitors (GMPs) (Lin–cKit+Sca1–CD16/32+CD34+) in the bone marrow at steady-state conditions but the percentage and the absolute number of GMPs upon sepsis were significantly decreased in Del1−/− neonatal mice (Figure 6D, E). The expression of DEL-1 receptor, avβ3 integrin (comprising the av integrin CD51 and the β3 integrin CD61) in the bone marrow, did not differ among neonates and adult mice, in either normal or septic conditions (assessed by flow cytometry analysis in bone marrow total cells, supplemental figure 3).
IL-10 expression in neonates promote DEL-1 upregulation in sepsis
DEL-1 is negatively regulated by inflammatory cytokines such as TNFα and IL-17A (26, 31, 32). To identify the key cytokines that control DEL-1 expression in septic neonates, we analyzed serum expression of inflammatory (TNFα, IL-17A, IL-6) and the anti-inflammatory cytokine IL-10 in septic neonatal and adult mice. Induction of TNFα in neonates was similar to adults, induction of IL-17A was less in neonates, while IL-6 and especially IL-10 were higher in septic neonates compared to adult (Figure 7A). Unlike TNFα and IL-17A that are known to downregulate DEL-1 (26), the effect of IL-6 and IL-10 on DEL-1 expression in endothelial cells has not been previously examined. To this end, we treated human endothelial cells (human umbilical vein endothelial cells, HUVECs and Ea.hy926 endothelial cell line) with human recombinant IL-6 or IL-10. IL-6 did not affect DEL-1 expression, while IL-10 promoted a two-fold upregulation of DEL-1 mRNA expression (Figure 7B). Stimulation with IL-10 reversed the suppressive effect of IL-17A on DEL-1 expression (Figure 7C), indicating opposing actions on DEL-1 expression by IL-10 and IL-17A.
To determine whether differences in DEL-1 expression observed in different tissues during neonatal sepsis reflected respective changes in IL-17A and IL-10, we measured IL-17A and IL-10 mRNA expression in tissue extracts from neonates six hours following exposure to CS-induced sepsis. We found that in neonatal lung, intestine and kidney, where DEL-1 was not suppressed in CS sepsis (Figure 2A, B), a higher IL-10 to IL-17A ratio was observed (Figure 7D). Consistent with these findings in mouse tissues, the median human DEL-1 protein concentration was higher in septic adults and septic neonates that exhibited high (>2) serum IL-10 to IL-17A ratio, compared to those with low (<2) IL-10 to IL-17A ratio (Figure 7E), further supporting that the IL-10/IL-17A balance regulates DEL-1 levels in sepsis. Moreover, we showed that IL-10 induced DEL-1 expression in human mesenchymal stromal cells (Figure 7F), suggesting that this cytokine might control DEL-1 expression in the human bone marrow.
To determine whether IL-10 regulates DEL-1 expression in the bone marrow during sepsis, we treated neonatal septic mice with an IL-10 receptor blocking antibody (anti-IL-10R). Administration of anti-IL-10R led to suppression of DEL-1 in the bone marrow of WT neonates (Figure 7G) and reduction of the neutrophil pool in the bone marrow and blood (Figure 7H). Importantly, treatment with anti-IL-10R resulted in higher bacterial load in blood (Figure 7I) and reduced survival in septic neonate mice (Figure 7J). Together, these findings indicate that a novel IL-10–DEL-1 axis promotes neutrophil production and host survival under septic stress conditions in neonates