Necrotizing enterocolitis (NEC) is an inflammatory bowel disease of preterm human newborns with yet unresolved etiology. An established neonatal murine model for NEC employs oral administration of lipopolysaccharides (LPS) combined with hypoxia/hypothermia. In adult mice, feeding dextran sodium sulfate (DSS) represents a well-established model for experimental inflammatory bowel disease. Here we investigated the effect of DSS administration on the neonatal murine intestine in comparison with the established NEC model. 3-day-old C57BL/6J mice were either fed formula containing DSS or LPS. LPS treated animals were additionally stressed by hypoxia/hypothermia twice daily. After 72 h, mice were euthanized, their intestinal tissue harvested and analyzed by histology, qRT-PCR and flow cytometry. For comparison, adult C57BL/6J mice were fed with DSS for 8 days and examined likewise. Untreated, age matched animals served as controls. Adult mice treated with DSS exhibited colonic inflammation with significantly increased Cxcl2 mRNA expression. In contrast, tissue inflammation in neonatal mice treated with DSS or LPS plus hypoxia/hypothermia was present in colon and small intestine as well. Comparative analysis of neonatal mice revealed a significantly increased lesion size and intestinal Cxcl2 mRNA expression after DSS exposure. Whereas LPS administration mainly induced local neutrophil recruitment, DSS treated animals displayed increased monocytes/macrophages infiltration. Our study demonstrates the potential of DSS to induce NEC-like lesions accompanied by a significant humoral and cellular immune response in the small and large intestine of neonatal mice. The new model therefore represents a good alternative to LPS plus hypoxia/hypothermia administration requiring no additional physical stress.

The high susceptibility of newborn infants to sepsis is as cribed to animmaturity of the neonatal immune system, but the molecular mechanisms remain unclear. Newborn monocytes massively release the alarmins S100A8/S100A9. In adults, these are major regulators of immunosuppressive myeloid‐derived suppressor cells (MDSCs). We investigated whether S100A8/S100A9 cause an expansion of monocytic MDSCs (Mo‐MDSCs) in neonates, thereby contributing to an immunocompromised state. Mo‐MDSCs have been assigned to CD14 ⁺ /human leukocyte antigen (HLA)‐DR ^−/low /CD33 ⁺ monocytes in humans and to CD11b ⁺ /Gr‐1 ^int /Ly6G ⁻ /Ly6C ^hi cells in mice. We found monocytes with these phenotypes significantly expanded in their respective newborns. Functionally, however, they did not prove immunosuppressive but rather responded inflammatorily to microbial stimulation. Their expansion did not correlate with high S100A8/S100A9 levels in cord blood. Murine studies revealed an excessive expansion of CD11b ⁺ /Gr‐1 ^int /Ly6G ⁻ /Ly6C ^hi monocytes in S100A9 ^−/− neonates compared to wild‐type neonates. This strong baseline expansion was associated with hyperinflammatory responses during endotoxemia and fatal septic courses. Treating S100A9 ^−/− neonates directly after birth with S100A8/S100A9 alarmins prevented excessive expansion of this inflammatory monocyte population and death from septic shock. Our data suggest that a specific population of inflammatory monocytes promotes fatal courses of sepsis in neonates if its expansion is not regulated by S100A8/S100A9 alarmins.—Heinemann, A. S., Pirr, S., Fehlhaber, B., Mellinger, L., Burgmann, J., Busse, M., Ginzel, M., Friesenhagen, J., von Köckritz‐Blickwede, M., Ulas, T., von Kaisenberg, C. S., Roth, J., Vogl, T., Viemann, D. In neonates S100A8/S100A9 alarmins prevent the expansion of a specific inflammatory monocyte population promoting septic shock. FASEB J. 31, 1153–1164 (2017). www.fasebj.org

The development of the mammalian gut was first described more than a century ago. Since then, it has been believed that a series of highly orchestrated developmental processes occur before the intestine achieves its final formation. The key steps include the formation of the umbilicus, the so-called “physiological herniation” of the midgut into the umbilical cord, an intestinal “rotation”, and the “return of the gut” into the abdominal cavity. However, this sequence of events is predominantly based on histological sections of dissected embryos, a 2D technique with methodological limitations. For a better understanding of spatial relationships in the embryo, we utilized microcomputed tomography (µCT), a nondestructive 3D imaging method. Here, we show the detailed processes and mechanisms of intestinal development in rat embryos, including the development of the umbilicus, the formation of loops inside the umbilical coelom, and the subsequent shift of these loops into the abdominal cavity. Our 3D datasets of developing intestines will substantially advance the understanding of normal mammalian midgut embryology and offer new possibilities to reveal unknown mechanisms in the pathogenesis of congenital disorders.

Aim Neonate and preterm patients are threatened by exaggerated inflammation of the gut. This study tests the hypothesis that the neonatal gut is prone to inflammation, by comparing the inflammatory reaction of neonatal and adult murine intestine to ischemia and reperfusion. Methods Neonatal (4 days, n = 36) and adult (4 weeks, n = 12) C57BL/6J mice were randomly divided between ischemia-reperfusion (IR) and untreated controls (Con). In IR animal's intestinal ischemia was induced by clamping the superior mesenteric artery (30 minutes) followed by reperfusion (4 hours). After the experiment, RNA was extracted from the small intestines and the expression of the chemokines CXCL1/KC and CXCL2/MIP-2 were determined by quantitative real-time reverse transcription-polymerase chain reaction. Flow cytometry was used to analyze neutrophil influx (Live+ Ly-6G + ) in isolated cell populations. Results We observed a strong increase in all measured proinflammatory endpoints after IR in both adult and neonatal mice. However, the inflammatory reaction was significantly stronger in neonatal murine intestines, with a significantly higher increase in CXCL1/KC expression and neutrophil accumulation as compared with adults (p < 0.05). Conclusion The intestines of neonatal mice reacted with an increased inflammatory response to the ischemic insult. This increased susceptibility could help to explain the exaggerated inflammation seen in diseases such as necrotizing enterocolitis.

Newborns and especially preterm infants are much more susceptible to infections than adults. Due to immature adaptive immunity, especially innate immune cells play an important role in a newborn’s infection defense. Neonatal neutrophils exhibit profound differences in their functionality compared to neutrophils of adults. In particular, neonates possess a relevant population of suppressive neutrophils, which not only inhibit but also specifically modulate the function of T-cells. In this study, we investigated whether neonatal neutrophils are already involved in T-cell development in the thymus. For this purpose, we used a newly developed model of antibody-mediated immune cell depletion in which we administered a depleting antibody to pregnant and then lactating dams. Using this method, we were able to sufficiently deplete Ly6G-positive neutrophils in offspring. We demonstrated that the depletion of neutrophils in newborn mice resulted in altered peripheral T-cell homeostasis with a decreased CD4+/CD8+ T-cell ratio and decreased expression of CD62L. Neutrophil depletion even affected T-cell development in the thymus, with increased double positive thymocytes and a decreased CD4+/CD8+ single positive thymocyte ratio. Altogether, we demonstrated a previously unknown mechanism mediating neutrophils’ immunomodulatory effects in newborns.