Elevated levels of circulating cell-free DNA and neutrophil proteins are associated with neonatal sepsis and necrotizing enterocolitis in immature mice, pigs and infants

Abstract

Preterm infants are highly susceptible to late-onset sepsis (LOS) and necrotizing enterocolitis (NEC), but disease pathogenesis and specific diagnostic markers are lacking. Circulating cell-free DNA (cfDNA) and immune cell-derived proteins are involved in multiple immune diseases in adults but have not been investigated in preterm neonates. We explored the relation of circulating neutrophil-associated proteins and cfDNA to LOS and/or NEC. Using a clinically relevant preterm pig model of spontaneous LOS and NEC development, we investigated neutrophil-associated proteins and cfDNA in plasma, together with cytokines in gut tissues. The changes in cfDNA levels were further studied in preterm pigs and neonatal mice with induced sepsis, and in preterm infants with or without LOS and/or NEC. Fifteen of 114 preterm pigs spontaneously developed both LOS and NEC, and they showed increased intestinal levels of IL-6 and IL-1b and plasma levels of cfDNA, neutrophil-associated proteins, and proteins involved in platelet-neutrophil interaction during systemic inflammation. The abundance of neutrophil-associated proteins highly correlated with cfDNA levels. Further, Staphylococcus epidermidis challenge of neonatal mice and preterm pigs increased plasma cfDNA levels and bacterial accumulation in the spleen. In infants, plasma cfDNA levels were elevated at LOS diagnosis and 1-6 d before NEC. In conclusion, elevated levels of plasma cfDNA and neutrophil proteins are associated with LOS and NEC diagnosis.

Proteomic and Post‐Translational Modification Profiling of Exosome‐Mimetic Nanovesicles Compared to Exosomes

“…Cell lysates (20 µg) were lysed in 0.5% sodium deoxycholate in 100 m m triethylammonium and incubation at 95 °C for 5 min. Samples were reduced with 12.5 m m tris(2‐carboxyethyl)phosphine hydrochloride (Sigma‐Aldrich, C4706) at 28 °C for 4 h with gentle rotation, alkylated with 12.5 m m chloroacetamide (Sigma‐Aldrich) for 30 min at 37 °C, and digested with 2 µg bovine sequencing grade trypsin (Promega, V5111) at 37 °C for 18 h. For cell lysate samples, sodium deoxycholate was removed following digestion as previously described . Subsequently, peptides were purified and extracted using reverse‐phase C18 StageTips (Sep‐Park cartridges, Waters, MA) in 85% v/v acetonitrile (ACN) in 0.5% v/v formic acid (FA).…”

Neonatal gut and immune maturation is determined more by postnatal age than by postconceptional age in moderately preterm pigs

“…Preterm pigs delivered at this age mimic the multiple clinical signs of prematurity in moderately preterm infants, including the immaturity of the gut, immune system, and multiple other organs (16,36,42,43,46). Similar to preterm infants, preterm newborn pigs are leukopenic, poorly responsive to bacterial and viral antigens (36), and spontaneously develop NEC and systemic infections when feeding regime and bacterial colonization are suboptimal (37,52). After one month of life, preterm pigs remain to show deficiencies in some gut functions (5,16), systemic immune responses (36), and neurodevelopment (2), but it remains unclear if these differences are explained by reduced PCA alone.…”

Markers of NETosis Do Not Predict Neonatal Early Onset Sepsis: A Pilot Study

“…Maternal age [infection 33.17 (28.43-36.99) vs. controls 31.63 (29. [25][26][27][28][29][30][31][32][33][34][35].48) years, p = 0.61] was also comparable between both groups. Only one child in the infection, but none in the control group, received steroids to promote fetal lung maturation (p = 0.31).…”

“…Recent studies evaluating neonatal, and not umbilical cord blood, surrogate markers of NET formation showed an association with sepsis (25,26): elevated circulating cfDNA levels in neonatal plasma have been associated with late-onset sepsis, as well as necrotizing enterocolitis, diagnosis (25). Moreover, neutrophils capable of releasing NETs have also been described as potential sepsis biomarkers in neonates (11,26).…”