Virpi Glumoff

Phosphatidylinositol 3-kinase is involved in Toll-like receptor 4-mediated cytokine expression in mouse macrophages.

Recent evidence suggests a role for phosphatidylinositol (PI) 3‐kinase in various inflammatory responses. In this study, the consequences of LPS‐induced PI 3‐kinase activation on cytokine and chemokine expression and the intracellular mechanisms of inflammatory activation were examined in mouse macrophages. LPS stimulation induced a complex formation between PI 3‐kinase and myeloid differentiation factor 88 (MyD88), which was followed by an induction of IL‐1β, tumor necrosis factor‐α (TNF‐α) and macrophage inflammatory protein (MIP)‐2. The induction of IL‐1β, but not of MIP‐2 or TNF‐α, was blocked by the PI 3‐kinase inhibitors LY294002 and wortmannin. The nuclear factor‐κB (NF‐κB) inhibitor pyrrolidinedithiocarbamate (PDTC) blocked the induction of IL‐1β and TNF‐α, but had no effect on MIP‐2 expression. Inhibition of PI 3‐kinase decreased the LPS‐induced transcriptional activity of NF‐κB, but it had no effect on the nuclear DNA binding activity of NF‐κB. These findings suggest that, while NF‐κB nuclear localization and DNA binding are necessary, they are not sufficient for transcriptional activation of the IL‐1β gene in the absence of PI 3‐kinase activity. Taken together, our results demonstrate that activation of Toll‐like receptor (TLR)‐4 results in PI 3‐kinase‐MyD88 complex formation, and that PI 3‐kinase activity selectively leads to cytokine induction downstream of TLR4.

Ontogeny of Toll-Like Receptors Tlr2 and Tlr4 in Mice

Toll-like receptors (Tlr) have recently been linked to the immunostimulatory function of microbial toxins in human and mice. Tlr signals activation of nuclear factor κB that leads to the production of a number of proinflammatory mediators. Tlr4 mediates the endotoxin-induced inflammatory response, whereas Tlr2 may be involved in the response to yeast and Gram-positive bacterial products. To better understand age-related changes in acute inflammatory response, we studied the ontogeny of Tlr2 and Tlr4 mRNA in murine fetal lung, liver, and placenta by quantitative reverse transcriptase-PCR. Different expression patterns were seen between the tissues and between the Tlr. This is in accordance with the evidence that there are differences in the receptors for different microbial toxins and that the response is organ specific. We additionally show that the expression of Tlr was dependent on the stage of differentiation. In the liver, the levels of Tlr2 and Tlr4 were high regardless of the age. In the lung, Tlr2 and Tlr4 expression levels were barely detectable in immature fetus (d 14–15). Tlr2 and Tlr4 were increased several-fold during prenatal development and further increased after birth. The present results support the finding of a deficient inflammatory response of the immature lung to microbial toxins.

Surfactant in respiratory distress syndrome and lung injury.

A deficiency in alveolar surfactant due to immaturity of alveolar type II epithelial cells causes respiratory distress syndrome (RDS). In contrast to animals, the fetal maturation of surfactant in human lungs takes place before term, exceptionally large quantities of surfactant accumulating in the amniotic fluid. The antenatal development of surfactant secretion is very variable but corresponds closely to the risk of RDS. The variation in SP-A and SP-B genes, race, sex and perinatal complications influence susceptibility to RDS. Surfactant therapy has improved the prognosis of RDS remarkably. Abnormalities in alveolar or airway surfactant characterize many lung and airway diseases. In the acute respiratory distress syndrome, deficiencies in surfactant components (phospholipids, SP-B, SP-A) are evident, and may be caused by pro-inflammatory cytokines (IL-1, TNF) that decrease surfactant components. The resultant atelectasis localizes the disease, possibly allowing healing (regeneration, increase in surfactant). In the immature fetus, cytokines accelerate the differentiation of surfactant, preventing RDS. After birth, however, persistent inflammation is associated with low SP-A and chronic lung disease. A future challenge is to understand how to inhibit or redirect the inflammatory response from tissue destruction and poor growth towards normal lung development and regeneration.

Surfactant protein A and D expression in the porcine Eustachian tube1

Surfactant proteins A and D are collectins which are considered to play an important role in the innate immunity of lungs. Our aim was to investigate whether surfactant protein A or D is expressed in the porcine Eustachian tube originating from the upper airways. Both surfactant proteins A and D were present in the epithelial cells of the Eustachian tube, as shown by strong immunostaining. Using RT‐PCR and Northern hybridization, these collectins were detected in the Eustachian tube. The present study is the first report demonstrating surfactant protein gene expression in the Eustachian tube. Surfactant proteins A and D may be important in the antibody‐independent protection of the middle ear.

Intra-amniotic lipopolysaccharide leads to fetal cardiac dysfunction. A mouse model for fetal inflammatory response.

OBJECTIVE Intrauterine infection is associated with increased lipopolysaccharide (LPS) and proinflammatory cytokines in amniotic fluid. We hypothesized that intra-amniotic LPS launches a fetal inflammatory response leading to cardiac dysfunction. METHODS A mouse model was established. At 15-16 days of gestation, 52 fetuses of nine dams received LPS and 46 fetuses of nine dams vehicle intra-amniotically. Five dams underwent a sham operation. Echocardiography was performed before and 6 h after the injection to obtain inflow and outflow blood velocity waveforms. Outflow mean velocity (V(mean)) and the proportions of isovolumetric relaxation (IRT%) and contraction (ICT%) times of the cardiac cycle were calculated. Pulsatility indices (PI) were calculated from the umbilical and intracranial arteries and the descending aorta. Pulsatility indices for veins (PIV) were obtained from ductus venosus. Toll-like receptor-4 (TLR4) and several other inflammatory mediators were determined using ELISA, immunohistochemistry, or ribonuclease protection assay. RESULTS In the LPS group, outflow V(mean) was significantly lower, and ICT% and IRT% longer than in the other groups. LPS increased PIs, except in the intracranial arteries, which showed a decrease in PIs. In ductus venosus, PIVs were increased after LPS. LPS increased interleukin (IL)-6 in amniotic fluid and induced the expression of proinflammatory cytokines in placenta and fetal membranes, but not in lung. In fetal myocardium, TLR4 was constitutional. LPS induced the expression of IL-1beta and tumor necrosis factor (TNF)-alpha mRNA in myocardium, whereas inducible nitric oxide synthase (NOS2) protein and nitrotyrosine remained undetectable. CONCLUSIONS As a response to endotoxin in amniotic fluid, fetal myocardium acutely generates cytokines and severe fetal cardiovascular compromise develops. These two may be linked through a mechanism that does not include NO.

Expression of toll-like receptor 4 and endotoxin responsiveness in mice during perinatal period.

Endotoxin [lipopolysaccharide (LPS)] from Gram-negative bacteria is found in amniotic fluid in intrauterine infections that associate with the risk for spontaneous premature birth, bronchopulmonary dysplasia (BPD), and respiratory distress syndrome. Toll-like receptor 4 (TLR4) is the signaling receptor for LPS. The aim was to investigate the primary inflammatory response in mice shortly after administration of LPS to the dam (14 and 17 d of pregnancy), to the newborn, or into the amniotic fluid. The expression levels of TLR4, IL-1, tumor necrosis factor-α, IL-6, IL-10, macrophage inflammatory protein-2, and IL-1 receptor 1 were studied with ribonuclease protection assay. In addition, TLR4 protein was analyzed with Western blotting. The fetal membranes expressed TLR4 mRNA and protein and showed an acute cytokine response to LPS when LPS was administrated into the amniotic fluid. There was distinct ontogeny in the responsiveness of fetal lung to LPS: on fetal day 14 (term 20 d), both the expression of TLR4 and the acute cytokine response were undetectable 5 h after LPS; they became detectable by fetal day 17. TLR4 and the cytokine response further increased after birth. In maternal lung, the TLR4 expression was strongest and up-regulated in parallel with the induction of the cytokines. We propose that TLR4 controls the magnitude of the LPS-induced cytokine response during the perinatal period.

Mechanism of Acute Fetal Cardiovascular Depression after Maternal Inflammatory Challenge in Mouse

Intra-amniotic lipopolysaccharide (LPS) causes an acute inflammatory response and cardiac dysfunction in fetal mice. We hypothesized that the placenta protects the fetus against maternally administered bacterial toxins, delaying the onset of a fetal inflammatory response and vascular compromise. At 14 to 15 days of gestation, DBA mice were randomized to receive LPS (2.4 mg/kg) or vehicle intraperitoneally. Doppler ultrasonography of fetal cardiovascular hemodynamics was performed before and 6 hours after maternal LPS. Six hours after the LPS, maternal serum concentrations of tumor necrosis factor-alpha and interleukin (IL)-6 (P < 0.05) were increased. Placenta showed severe maternal vascular dilatation and congestion. The expressions of tumor necrosis factor-alpha, IL-1alpha, and IL-6 (P < 0.05) were increased, and the expression of Toll-like receptor 4 was constitutive in placenta. The expression of Toll-like receptor 2 increased (P < 0.05) and was detected in labyrinthine macrophages. No inflammatory activation was found in fetal tissues, and amniotic fluid revealed no significant increase in cytokines. The ultrasonographic examination demonstrated increased fetal cardiac afterload after LPS, with 65% of the fetuses exhibiting atrioventricular valve regurgitation. In conclusion, maternal inflammatory insult activates placental labyrinthine macrophages leading to an acute increase in placental vascular resistance and fetal cardiac dysfunction without an inflammatory response in fetus.

Central nervous system-related symptoms and findings are common in acute Puumala hantavirus infection.

Abstract Background. Puumala hantavirus (PUUV) causes a hemorrhagic fever with renal syndrome (HFRS) also called nephropathia epidemica (NE). Recent case reports and retrospective studies suggest that NE may damage the pituitary gland. Based on these observations, our goal was to explore the nature of this complication prospectively. Methods. A total of 58 hospitalized patients with acute NE volunteered to participate. Central nervous system (CNS) symptoms were recorded, cerebrospinal fluid (CSF) samples were collected, human leukocyte antigen (HLA) haplotype was analyzed, brain magnetic resonance imaging (MRI) was acquired, and electroencephalography (EEG) was recorded. Patients with abnormal pituitary MRI finding were examined by an endocrinologist. Results. Most patients experienced CNS symptoms, and half of the CSF samples were positive for PUUV IgM, elevated protein level, or leukocyte count. CSF of patients negative for DR15(2)-DQ6 haplotype was less frequently affected. MRI revealed pituitary hemorrhage in two patients; these two patients suffered sudden loss of vision associated with headache, and they both developed hypopituitarism. Only one patient required long-term hormonal replacement therapy. Conclusion. CNS-related symptoms and inflammation in the CSF are common in acute NE. Genetic properties of the host may predispose to CNS involvement. It does seem that pituitary injury and subsequent hormonal insufficiency may complicate the recovery.

Damaging heterozygous mutations in NFKB1 lead to diverse immunologic phenotypes

Background The nuclear factor &kgr; light‐chain enhancer of activated B cells (NF‐&kgr;B) signaling pathway is a key regulator of immune responses. Accordingly, mutations in several NF‐&kgr;B pathway genes cause immunodeficiency. Objective We sought to identify the cause of disease in 3 unrelated Finnish kindreds with variable symptoms of immunodeficiency and autoinflammation. Methods We applied genetic linkage analysis and next‐generation sequencing and functional analyses of NFKB1 and its mutated alleles. Results In all affected subjects we detected novel heterozygous variants in NFKB1, encoding for p50/p105. Symptoms in variant carriers differed depending on the mutation. Patients harboring a p.I553M variant presented with antibody deficiency, infection susceptibility, and multiorgan autoimmunity. Patients with a p.H67R substitution had antibody deficiency and experienced autoinflammatory episodes, including aphthae, gastrointestinal disease, febrile attacks, and small‐vessel vasculitis characteristic of Behçet disease. Patients with a p.R157X stop‐gain experienced hyperinflammatory responses to surgery and showed enhanced inflammasome activation. In functional analyses the p.R157X variant caused proteasome‐dependent degradation of both the truncated and wild‐type proteins, leading to a dramatic loss of p50/p105. The p.H67R variant reduced nuclear entry of p50 and showed decreased transcriptional activity in luciferase reporter assays. The p.I553M mutation in turn showed no change in p50 function but exhibited reduced p105 phosphorylation and stability. Affinity purification mass spectrometry also demonstrated that both missense variants led to altered protein‐protein interactions. Conclusion Our findings broaden the scope of phenotypes caused by mutations in NFKB1 and suggest that a subset of autoinflammatory diseases, such as Behçet disease, can be caused by rare monogenic variants in genes of the NF‐&kgr;B pathway. Graphical abstract Figure. No Caption available.

Nasal Middle Meatal Specimen Bacteriology as a Predictor of the Course of Acute Respiratory Infection in Children

Aims: To test our hypothesis that children with potentially pathogenic bacteria (Streptococcus pneumoniae, Haemophilus influenzae or Moraxella catarrhalis) in the nasal middle meatus might have more prolonged symptoms of acute respiratory infection than children without such bacteria, we conducted a prospective cohort study of such children. Materials and Methods: We recruited prospectively child volunteers between 6 and 13 years of age with acute respiratory infections present for fewer than 10 days. Nasal middle meatal bacterial culture was taken with a rigid endoscope at enrollment and again after 3 weeks and evaluated for presence or absence of 3 potential pathogens: S. pneumoniae, H. influenzae and M. catarrhalis. The subsequent persistence of acute symptoms (nasal discharge: clear/colored, nasal obstruction and cough) was determined by means of a diary. Viral etiology was studied with polymerase chain reaction methods. Results: The 82 children had had symptoms for an average of 4 days (range, 1–10) at entry, and viruses were detected in 54% (39 of 72). The endoscopic procedure and bacteriologic sampling succeeded in all cases. Thirty-eight children (46%) had at least 1 of the 3 pathogens in the middle meatus specimen. The children with nasal pathogens present at entry had a significantly longer mean duration of symptoms than those with nonpathogenic bacteria (difference, 3.6 days; 95% confidence interval, 0.7–6.5; P = 0.025). The effect remained significant after adjustment for age, sex, allergic symptoms and the presence of virus (adjusted relative hazard of delayed recovery, 2.0; 95% confidence interval, 1.1–3.6). Conclusions: We found that the use of endoscopic swab culture sampling from the nasal middle meatus is well-tolerated by children older than 6 years of age and that it can be useful in selected situations to determine pathogenic bacteria in the culture of these specimens.