Innate Immune Deficiency of Extremely Premature Neonates Can Be Reversed by Interferon-γ

Background: Bacterial sepsis is a major threat in neonates born prematurely, and is associated with elevated morbidity and mortality. Little is known on the innate immune response to bacteria among extremely premature infants. Methodology/Principal Findings: We compared innate immune functions to bacteria commonly causing sepsis in 21 infants of less than 28 wks of gestational age, 24 infants born between 28 and 32 wks of gestational age, 25 term newborns and 20 healthy adults. Levels of surface expression of innate immune receptors (CD14, TLR2, TLR4, and MD-2) for Grampositive and Gram-negative bacteria were measured in cord blood leukocytes at the time of birth. The cytokine response to bacteria of those leukocytes as well as plasma-dependent opsonophagocytosis of bacteria by target leukocytes was also measured in the presence or absence of interferon-c. Leukocytes from extremely premature infants expressed very low levels of receptors important for bacterial recognition. Leukocyte inflammatory responses to bacteria and opsonophagocytic activity of plasma from premature infants were also severely impaired compared to term newborns or adults. These innate immune defects could be corrected when blood from premature infants was incubated ex vivo 12 hrs with interferon-c. Conclusion/Significance: Premature infants display markedly impaired innate immune functions, which likely account for their propensity to develop bacterial sepsis during the neonatal period. The fetal innate immune response progressivel

Innate immune functions of circulating immature neutrophils during sepsis

Le sepsis est associé à une réponse immunitaire innée et inflammatoire excessives en réaction à une infection bactérienne. Les phagocytes, en particulier les neutrophiles, sont des effecteurs essentiels de la réponse immune. Les neutrophiles représentent la population circulante prédominante. Lors d'une infection, une proportion significative de neutrophiles immatures est recrutée dans la circulation. Bien que les neutrophiles matures soient considérés comme étant des cellules essentielles de l'immunité innée, le rôle, les fonctions immunitaires et la capacité des neutrophiles immatures à initier une réponse immunitaire innée restent inconnus. Le but de cette thèse est d'explorer les fonctions immunitaires innées de ces neutrophiles immatures isolés chez des patients avec sepsis et de les comparer aux neutrophiles matures des patients avec sepsis et de donneurs sains. Nous démontrons dans ce travail de thèse que malgré leur immaturité, les neutrophiles immatures restent capables de médier les fonctions immunitaires innées essentielles lors d'infections sévères

Innate immune functions of immature neutrophils in patients with sepsis and severe systemic inflammatory response syndrome

A hallmark of sepsis and severe systemic inflammatory response syndrome (SIRS) is the massive recruitment of immature neutrophils from the bone marrow into the circulation (left shift, band forms). Their capacity to participate in innate defense against bacteria is ill defined. We aimed at comparing various innate immune functions of mature vs. immature neutrophils circulating during sepsis and SIRS

Increased FGF21 plasma levels in humans with sepsis and SIRS.

Fibroblast growth factor 21 (FGF21) is a key regulator in glucose and lipid metabolism and its plasma levels have been shown to be increased not only in humans in different situations such as type 2 diabetes, obesity, and nonalcoholic fatty liver disease but also in animal models of sepsis and pancreatitis. FGF21 is considered as a pharmacological candidate in conditions associated with insulin resistance. The aim of this study was to compare FGF21 plasma levels in patients with sepsis, in patients with systemic inflammatory response syndrome (SIRS), and in healthy controls. We measured FGF21 plasma concentrations in 22 patients with established sepsis, in 11 with SIRS, and in 12 healthy volunteers. Here, we show that FGF21 levels were significantly higher in plasma obtained from patients with sepsis and SIRS in comparison with healthy controls. Also, FGF21 levels were significantly higher in patients with sepsis than in those with noninfectious SIRS. FGF21 plasma levels measured at study entry correlated positively with the APACHE II score, but not with procalcitonin levels, nor with C-reactive protein, classical markers of sepsis. Plasma concentrations of FGF21 peaked near the onset of shock and rapidly decreased with clinical improvement. Taken together, these results indicate that circulating levels of FGF21 are increased in patients presenting with sepsis and SIRS, and suggest a role for FGF21 in inflammation. Further studies are needed to explore the potential role of FGF21 in sepsis as a potential therapeutic target

Toll-like receptor activation of human cells by synthetic triacylated lipid A-like molecules

Recognition of microbial molecules by mammalian host receptors is essential to mount an immune response. Hexaacylated LPS is the prototypic example of a bacterial molecule recognized by the receptor complex TLR4/MD-2 with its lipid A moiety, whereas bacterial lipopeptides are recognized by TLR2. Here we show that a series of synthetic triacylated lipid A-like molecules are weak Toll-like receptor (TLR) agonists (mainly TLR2 agonists) but very potent TLR4/MD-2 antagonists (submicromolar range). Not only do they block human cell responses to LPS but also to whole gram-negative bacteria, and they inhibit the phagocytosis of gram-negative bacteria. These compounds may represent promising immunomodulatory agents

Phagocytosis of bacteria by newborn and adult neutrophils, and plasma opsonic activity.

<p>(A) Phagocytosis of fluorescent <i>E.coli</i>, <i>S.aureus</i>, and <i>S.epidermidis</i> by neutrophils from one representative subject from each group (ELBW, extremely low birth weight premature infants born before 28 wks of gestational age; VLBW, very low birth weight premature infants born between 28–32 wks of gestational age; TN, term newborns; and CA, control adults). Phagocytosis was measured by flow cytometry after 10 min (red lines) and 20 min (green lines) incubation with bacteria opsonised with autologous plasma. Neutrophils pre-incubated with the phagocytosis blocker cytochalasin D are presented with filled curves (negative control). (B) The opsonic capacity of patient's plasma using neutrophil-like human HL-60 cells is tested. Fluorescent <i>E.coli</i>, <i>S.aureus</i>, and <i>S.epidermidis</i> were opsonised with autologous plasma and incubated 40 min with DMSO-differentiated HL-60 cells. Intracellular fluorescence (phagocytosis of bacteria) was measured by flow cytometry and expressed as mean phagocytic index (± SEM) of the four groups (ELBW, extremely low birth weight premature infants born before 28 wks of gestational age N = 21; VLBW, very low birth weight premature infants born between 28–32 wks of gestational age, N = 24; TN, term newborns, N = 25; CA, control adults, N = 20). On each line, a reference group (*) is compared to other groups and respective P value expressed using Mann-Whitney <i>U</i> test.</p

LPS responsiveness of leukocytes and opsonic capacity of plasma from premature infants with and without the addition of interferon-γ.

<p>(A) Whole blood from 7 extremely premature infants (born before 28 wks of gestational age) was treated <i>ex vivo</i> or not for 12 hrs with interferon (IFN)-γ. LPS was then added for an additional 12 hrs and IL-6, TNF-α, and IL-10 were measured (mean ± SEM) in conditioned plasma. (B) The plasma from whole blood obtained in seven extremely premature infants (born before 28 wks of gestational age) treated <i>ex vivo</i> or not with for 12 hrs with IFN-γ was incubated with fluorescent <i>E.coli</i>, <i>S.aureus</i> and <i>S.epidermidis</i> (opsonisation). Opsonised bacteria were then incubated with neutrophil-like HL-60 cells for 40 min; intracellular fluorescence was measured by flow cytometry, and expressed as mean phagocytic index (± SEM). Statistical significance was tested with Wilcoxon matched-pairs signed rank test.</p

TLR2, TLR4, CD14, and MD-2 surface expression of blood leukocytes and plasma soluble MD-2 activity from premature infants, term newborns, and control adults.

<p>(A) Representative flow cytometry plot showing forward and side-scatter characteristics gating used to identify neutrophils (R1), and monocytes (R2). (B) Surface expression of TLR4, MD-2, CD14 and TLR2 of phagocytes (neutrophils and monocytes) from extremely low birth weight infants (ELBW, born before 28 wks of gestational age, N = 18), very low birth weight infants (VLBW, born between 28–32 wks of gestational age, N = 17), term newborn (TN, N = 25), and control adult (CA, N = 20). In addition the expression of the Fcγ receptor CD16 on neutrophils and the major histocompatibility HLA-DR on monocytes are shown. Receptor expression was measured by flow cytometry, and expressed as mean fluorescence index (MFI, geo mean receptor/geo mean IgG control). Errors bars are means ± SEM, On each line, a reference group (*) is compared to other groups and respective P value expressed using Mann-Whitney <i>U</i> test. (C) Plasma soluble MD-2 activity was measured as the capacity of plasma to support TLR4-HEK293 cell activation after a 30 ng/mL LPS challenge <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032863#pone.0032863-Pugin3" target="_blank">[50]</a>. Human recombinant soluble MD-2 (1 µg/mL) was used as a positive control. (ELBW, extremely low birth weight premature infants born before 28 wks of gestational age N = 20; VLBW, very low birth weight premature infants born between 28–32 wks of gestational age, N = 20; TN, term newborns, N = 20; CA, control adults, N = 20). Errors bars are means ± SEM.</p

Baseline Characteristics and outcome of newborns.

<p>Continuous data are expressed as median and interquartile range [IQR] and categorical data as number (%).</p><p>ELBW, extremely low birth weight born before 28 wks of gestational age; VLBW, very low birth weight born between 28 and 32 wks of gestational age, WBC, white blood cell; IVH, intraventricular haemorrhage. †, WBC count was performed in term newborn if there was an infectious risk at delivery (WBC count performed in ELBW, N = 13; VLBW, N = 24; and in term newborns, N = 8). Four premature infants (ELBW, N = 3; VLBW, N = 1) did not receive pulmonary maturation with antenatal corticosteroid therapy.</p

Cooperation between PU.1 and CAAT/Enhancer-binding Protein β Is Necessary to Induce the Expression of the MD-2 Gene*

Myeloid differentiation factor 2 (MD-2) binds Gram-negative bacterial lipopolysaccharide with high affinity and is essential for Toll-like receptor 4-dependent signal transduction. MD-2 has recently been recognized as a type II acute phase protein. Plasma concentrations of the soluble form of MD-2 increase markedly during the course of severe infections. Its production is regulated in hepatocytes and myeloid cells by interleukin-6 (IL-6) but not IL-1β. In the present work we show that two transcription factors (TF), PU.1 and CAAT/enhancer-binding protein β (C/EBPβ), participate in the activation of the human MD-2 gene in hepatocytic cells after stimulation with IL-6. PU.1 TF and proximal PU.1 binding sites in the MD-2 promoter were shown to be critical for the basal activity of the promoter as well as for IL-6-induced soluble MD-2 production. Deletions of proximal portions of the MD-2 promoter containing PU.1 and/or NF-IL-6 consensus binding sites as well as site-directed mutagenesis of these binding sites abrogated IL-6-dependent MD-2 gene activation. We show that the cooperation between C/EBPβ and PU.1 is critical for the transcriptional activation of the MD-2 gene by IL-6. PU.1 was essentially known as a TF involved in the differentiation of myeloid precursor cells and the expression of surface receptors of the innate immunity. Herein, we show that it also participates in the regulation of an acute phase protein, MD-2, in nonmyeloid cells cooperatively with C/EBPβ, a classical IL-6-inducible TF