| Abstract |
Neonatal sepsis is a major cause of morbidity and mortality among term and preterm infants
in both developed and developing countries worldwide. Several studies have identified E.
coli and Streptococcus agalactiae (GBS) as the leading causative agents of sepsis in this
vulnerable age group. Though these pathogens are commensals for healthy adults, in
neonates such infections have particularly devastating outcomes for both infant hosts and
their families. Despite antibiotic treatment there are no other available therapies for the
management of neonatal sepsis. A major contributor of neonatal susceptibility to infections
is the immature state of neonatal adaptive and innate immune responses. During this tender
period of life, adaptive immunity is still underdeveloped, whereas innate immune responses
are untrained and partly disarmed compared to those of adults. Since the molecular and
cellular mechanisms of neonatal innate immune defense against pathogens and sepsis have
not been clearly elucidated, their understanding is crucial to develop new targeted and
effective therapies for this detrimental condition.
DEL-1 is a molecule with profound immunomodulatory and anti-inflammatory properties
with fundamental role in inflammatory disease pathology. By utilizing wild type (WT) healthy
and septic mice of adult and neonatal age, we observed that DEL-1 expression was
systemically elevated in healthy neonates compared to adults and further increased under
septic conditions as opposed to septic adults. To investigate the role of DEL-1 in the context
of neonatal sepsis, we utilized WT and DEL-1-/- that we subjected to polymicrobial sepsis. We
found that DEL-1-/- mice displayed profound neutropenia, exaggerated bacteremia and
overall diminished survival in response to sepsis, defects that were rescued by the
exogenous administration of DEL-1. The basis of these defects relies on the diminished
numbers of myeloid-biased hematopoietic stem cells, granulocyte progenitors and
neutrophils in the bone marrow of septic DEL-1-/- mice, indicating the importance of DEL-1 as
an integral component of the neonatal bone marrow niche that exerts its protective effects
via supporting emergency granulopoiesis.
We also demonstrate that the sustained tissue production of DEL-1 in newborn sepsis was
attributed to the high IL-10/IL-17A ratio that neonates display. Specifically, we provide
evidence that as IL-10 upregulates DEL-1, as blockade of IL-10 signaling diminished both the
expression and the beneficial effects of DEL-1. Consistent with the mouse findings, DEL-1
and neutrophil numbers were higher in septic human adult and neonate patients with high
IL-10/IL-17A ratio. Furthermore, septic patients with high DEL-1 exhibited lower mortality
rates compared to patients with low DEL-1.
In conclusion, we highlight the role of a hitherto unappreciated IL-10–DEL-1 axis in the
maintenance of the bone marrow neutrophil pool, which is inadequate in infancy, by
supporting emergency granulopoiesis, thus preventing neutropenia and promoting sepsis
survival in early life.
Next, we studied the role of adult and neonatal macrophages in response to GBS infection.
GBS is a commensal for healthy adults but a leading cause of sepsis and meningitis among
neonates and young infants. To study the molecular mechanisms of macrophage immunity
against GBS we utilized thioglycolate elicited peritoneal macrophages from adult and
neonate mice that we stimulated with GBS. Unlike adult macrophages, GBS infection in neonatal macrophages was characterized by elevated intracellular bacterial load, enhanced
cytoplasmic escape and decreased targeting to lysosomes for degradation. Using confocal
microscopy analysis and a series of siRNA molecules we demonstrated that LC3 Associated
Phagocytosis (LAP) is the driver immune mechanism mediating GBS clearance in adult
macrophages. However, neonatal macrophages suffer from profound deficits in LAP
pathway as indicated by the diminished co-localization of GBS with LC3.
GBS infected neonatal macrophages acquired a unique metabolic and inflammatory profile,
baring both pro- and anti-inflammatory characteristics. RNA-seq analysis revealed that
neonatal macrophages produce exacerbated amounts of IL-6 in response to infections that
negatively impact LAP. In fact, the administration of IL-6 in murine adult macrophages
impeded their antibacterial activity. In contrast, the universal inhibition of IL-6 or the
selective inhibition of its trans signaling mode improved the bactericidal capacity of the GBS
infected macrophages in a LAP dependent manner. Similarly, the inhibition of the
downstream signaling mediators JAK1/2 and STAT-3 significantly decreased intracellular load
and cytoplasmic escape of GBS, while enhancing LC3 deposition on internalized bacteria.
In accordance with our findings on the murine model, the inhibition of IL-6R with
Tocilizumab, augments the capacity of human cord blood macrophages to eliminate GBS.
Finally, we demonstrate that the IL-6 targeting therapies can have potential in vivo and
prolong the survival of GBS infected murine pups. Hence, the administration of
immunomodulatory therapies targeting the IL-6/JAK1&2/STAT-3 axis may confer protection
from invasive GBS infection in newborns by effectively activating the intracellular defense
mechanisms of macrophages against invading pathogens.
The findings of the current study can potentially lead to the development of targeted
therapies that may improve neonatal sepsis prognosis and morbidity that remain too dismal.
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