The Cdh5-CreERT2 transgene causes conditional Shb gene deletion in hematopoietic cells with consequences for immune cell responses to tumors.

The tamoxifen-responsive conditional Cdh5-CreERT2 is commonly used for endothelial cell specific conditional deletion of loxP-flanked gene sequences. To address the role of endothelial cell Shb gene for B16F10 melanoma immune responses, tamoxifen-injected Cdh5-CreERT2/WT and Cdh5-CreERT2/Shbflox/flox mice received subcutaneous tumor cell injections. We observed a decrease of tumor myeloid cell Shb mRNA in the tamoxifen treated Cdh5-CreERT2/Shbflox/flox mice, which was not present when the mice had undergone a preceding bone marrow transplantation using wild type bone marrow. Differences in CD4+/FoxP3+ Tregs were similarly abolished by a preceding bone marrow transplantation. In ROSA26-mTmG mice, Cdh5-CreERT2 caused detectable floxing in certain bone marrow populations and in spleen cells. Floxing in bone marrow could be detected two months after tamoxifen treatment. In the spleen, however, floxing was undetectable two months after tamoxifen treatment, suggesting that Cdh5-CreERT2 is operating in a non-renewable population of hematopoietic cells in this organ. These data suggest that conditional gene deletion in hematopoietic cells is a potential confounder in experiments attempting to assess the role of endothelial specific effects. A cautious approach to achieve an endothelial-specific phenotype would be to adopt a strategy that includes a preceding bone marrow transplantation.

NHLRC2 variants identified in patients with fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA): characterisation of a novel cerebropulmonary disease.

A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction. In the affected children, neuropathology revealed increased angiomatosis-like leptomeningeal, cortical and superficial white matter vascularisation and congestion, vacuolar degeneration and myelin loss in white matter, as well as neuronal degeneration. Interstitial fibrosis and previously undescribed granuloma-like lesions were observed in the lungs. Hepatomegaly, steatosis and collagen accumulation were detected in the liver. A whole-exome sequencing of the two unrelated families with the affected children revealed the transmission of two heterozygous variants in the NHL repeat-containing protein 2 (NHLRC2); an amino acid substitution p.Asp148Tyr and a frameshift 2-bp deletion p.Arg201GlyfsTer6. NHLRC2 is highly conserved and expressed in multiple organs and its function is unknown. It contains a thioredoxin-like domain; however, an insulin turbidity assay on human recombinant NHLRC2 showed no thioredoxin activity. In patient-derived fibroblasts, NHLRC2 levels were low, and only p.Asp148Tyr was expressed. Therefore, the allele with the frameshift deletion is likely non-functional. Development of the Nhlrc2 null mouse strain stalled before the morula stage. Morpholino knockdown of nhlrc2 in zebrafish embryos affected the integrity of cells in the midbrain region. This is the first description of a fatal, early-onset disease; we have named it FINCA disease based on the combination of pathological features that include fibrosis, neurodegeneration, and cerebral angiomatosis.

Postnatal development and LPS responsiveness of pulmonary adenosine receptor expression and of adenosine-metabolizing enzymes in mice.

BACKGROUND: Adenosine levels are regulated by ecto-5'-nucleotidase/CD73 and adenosine deaminase (ADA). Adenosine regulates endothelial permeability and anti-inflammatory responses via adenosine receptors. Here, the adenosine receptors and purine-converting enzymes were studied during postnatal development and inflammation. METHODS: Newborn, 1-, 10-, 14-d-old and adult C57BL/6 mice were challenged intraperitoneally (i.p.) with lipopolysaccharide (LPS) for 6 h. The inflammatory response was evaluated by histochemistry. Expression levels of adenosine receptors (A1, A2A, A2B, and A3), CD73, and ADA were measured by quantitative reverse transcription polymerase chain reaction. A1 was studied by immunohistochemistry, and enzyme activities were analyzed by thin-layer chromatography. RESULTS: LPS caused respiratory distress in newborns within 24 h. LPS induced neutrophils at the basal stage and alveolar congestion. Low activity and expression of CD73 increased after birth. Expression of ADA after LPS increased 16-fold in adults and 2-fold in newborns (P < 0.05). A1 expression was high in newborns and increased after LPS (P < 0.05). A1 was localized to endothelial membranes. A2A decreased after LPS in newborns and increased in adults (P < 0.05). The expression of A3 increased in newborns and adults after LPS. CONCLUSION: Low pulmonary CD73 expression, LPS-induced suppression of A2A, LPS-induced increase of A1 expression, and severe respiratory distress were distinguishing responses in the newborns from those in the adults.

Surfactant protein D modulates levels of IL-10 and TNF-α in intrauterine compartments during lipopolysaccharide-induced preterm birth.

Prematurity is the main cause of perinatal mortality and morbidity, and preterm birth is often associated with intrauterine inflammation. Surfactant protein D (SP-D) functions in lung homeostasis and has multiple roles in innate immunity. It is present in amniotic fluid and in gestational tissues. We propose that SP-D may regulate intrauterine inflammatory responses related to preterm labor. Our aim was to investigate the role of SP-D in lipopolysaccharide-induced preterm birth in mice that overexpress rat SP-D (rSP-D) under the human SP-C promoter. SP-D protein in amniotic fluid and in gestational tissues was detected by western analysis. TNF-α, IL-10, and IL-6 concentrations in serum and amniotic fluid and mRNA levels in gestational tissues were quantified using cytometric bead array and ribonuclease protection assay, respectively. Increased levels of SP-D protein were detected in the amniotic fluid and gestational tissues of rSP-D mice. Lipopolysaccharide given at 17 days post-coitum to rSP-D dams led to preterm birth of live-born offspring within 18 h. Preterm birth of live-born pups was induced with a lower dose of lipopolysaccharide compared to wild-type mice. In rSP-D mice, the lipopolysaccharide-induced levels of TNF-α and IL-10 in amniotic fluid and fetal serum and the expression of IL-10 in placenta and fetal membranes were significantly different from wild-type mice. We conclude that SP-D in fetal and gestational tissues modulates the levels of intrauterine inflammatory mediators involved in preterm birth and may contribute to inflammatory processes related to spontaneous preterm labor.

Transient defect in nitric oxide generation after rupture of fetal membranes and responsiveness to inhaled nitric oxide in very preterm infants with hypoxic respiratory failure.

OBJECTIVE: To study antenatal risk factors and inflammatory responses during hypoxic respiratory failure (HRF) in infants of very low gestational age (VLGA, ≤32.0 weeks). STUDY DESIGN: Of a cohort of 765 VLGA infants, 144 required mechanical ventilation. Airway specimens from these patients were prospectively studied. Infants who developed HRF (oxygenation index >25) with echocardiographic diagnosis of pulmonary hypertension were treated with inhaled nitric oxide (iNO). Three gestation comparison groups were formed on the basis of specific antenatal complications: prolonged preterm rupture of membranes (PPROM), spontaneous preterm birth, and preeclampsia. Chest radiographs were studied and airway specimens were analyzed for concentrations of tumor necrosis factor-α, interleukin (IL)-6, IL-8, IL-10, IL-12p70, IL-1ß, and nitrite + nitrate over 4 days. RESULTS: Seventeen (2.2% of all VLGA infants) developed HRF. In all 17 cases, PPROM complicated the antenatal course; these infants responded to iNO, regardless of infection or PPROM. The chest radiographs of HRF and non-HRF PPROM infants were similar. Airway proinflammatory cytokines and nitrite + nitrate levels were low in infants with HRF, but they increased during iNO treatment and remained elevated after discontinuation of iNO. Each of the 3 comparison groups had different and characteristic patterns of airway cytokines and nitrite + nitrate levels. CONCLUSIONS: Seven percent of VLGA infants with preterm rupture of membranes and 15% of those with PPROM developed HRF, characterized by pulmonary hypertension that acutely responds to iNO. These infants may have a transient deficiency in the inflammatory response, including a defect in nitric oxide generation in airspaces.

Genetic association of SP-C with duration of preterm premature rupture of fetal membranes and expression in gestational tissues.

BACKGROUND: Surfactant protein (SP) C has been shown to be expressed also outside pulmonary alveoli. Certain SP-C gene (SFTPC) polymorphisms associate with lung diseases and very preterm birth. AIMS: We investigated the association of SFTPC single nucleotide polymorphism (SNP) rs4715 with factors affecting spontaneous preterm birth and characterized the SP-C expression in human and mouse gestational tissues. METHODS: The SFTPC SNP rs4715 polymorphism was genotyped in a homogeneous northern European population of mothers and infants in spontaneous preterm birth and term controls. The expression and protein of SP-C in gestational tissues was analyzed. RESULTS: SFTPC SNP rs4715 did not associate with spontaneous preterm birth. However, fetuses with short interval (<72 hours) between preterm premature rupture of fetal membranes (PPROM) and preterm birth had significant over-representation of the minor allele A, whereas in fetuses with prolonged PPROM (>or=72 hours) the frequency was decreased. Maternal SFTPC did not associate with the duration of PPROM. SP-C mRNA and proprotein were detected in fetal membranes, placenta, and pregnant uterus. CONCLUSION: SFTPC SNP rs4715 associates with the duration of PPROM, and SP-C is expressed in gestational tissues. We propose that fetal SFTPC moderates the inflammatory activation within the fetal extra-embryonic compartment.

Blood cytokines during the perinatal period in very preterm infants: relationship of inflammatory response and bronchopulmonary dysplasia.

OBJECTIVE: To evaluate the influence of chorioamnionitis (CA) on plasma cytokines and the cytokine-associated risk of bronchopulmonary dysplasia (BPD) during the perinatal period. STUDY DESIGN: Eleven cytokines from 128 very low gestational age infants were analyzed from cord blood and from plasma at ages 1 day and 7 days after birth. The diagnosis of CA was based on histology of the placenta, fetal membranes, and umbilical cord. Neonatal risk factors were recorded. RESULTS: In the 48 infants born with CA, high concentrations of inflammatory cytokines in cord blood decreased during the first postnatal day. Inflammatory cytokines in cord blood was associated with the severity of CA. At 1 day after birth, the concentration of interleukin (IL)-8 predicted the risk of BPD. For the 75 infants born without CA, cytokine concentrations increased after birth. For the 128 infants born with or without CA, at 1 day after birth, the concentrations of IL-8, granulocyte colony-stimulating factor, and anti-inflammatory IL-10 were associated with the risk of BPD, after adjustment for the duration of gestation and severity of respiratory distress during the first day. CONCLUSIONS: In infants exposed to CA, insufficient inhibition of high fetal inflammatory cytokine response shortly after birth may increase the risk of BPD.

Bacterial ligand of TLR2 signals Stat activation via induction of IRF1/2 and interferon-alpha production.

Both type I interferons (IFNs) and interferon regulatory factors (IRFs) are well characterized in viral infections, whereas they are far less studied in bacterially activated toll-like receptor (TLR) pathways. Here, we studied the involvement of IRF1 and IRF2 in TLR2-mediated responses. In mouse macrophages, IRF2 was activated by lipoteichoic acid (LTA) of Staphylococcus aureus, resulting in up-regulation of IRF1 and rapid secretion of IFN-alpha. In addition, LTA-induced activation of Signal transducers and activators of transcription 1 (Stat1) and Stat3 via IRF2. The secretion of IFN-alpha was reduced in IRF2-silenced macrophages, resulting in a disappearance of tyrosine-phosphorylated Stat3 and a reduction of pro-inflammatory responses, despite induction of Mal adapter protein. These results provide a mechanistic insight into the pro-inflammatory responses against S. aureus LTA in mouse macrophages. IRFs can be intersecting factors of viral and bacterial responses in activated TLR signalling pathways.

Maternal endotoxin-induced preterm birth in mice: fetal responses in toll-like receptors, collectins, and cytokines.

Major cause of prematurity is spontaneous preterm birth (PTB) associated with intrauterine inflammation. Our aim was to establish a model of endotoxin Lipopolysaccharide-induced PTB of live-born pups and to study early immune activation in fetal and maternal compartments. Expression of several proteins that bind microbes (Toll-like receptors TLR4, TLR2; surfactant proteins SP-A, SP-D) was analyzed. At 16 or 17 d of gestation, C57BL/6 dams received a single dose of intraperitoneal LPS, leading to PTB within 17 h. Cytokine levels increased in maternal serum, followed by a modest increase in fetal serum and in amniotic fluid. In uterus, placenta, and fetal membranes, LPS mostly increased the expressions of TLR, SPs, and cytokines. The number of TLR2-positive macrophages increased in labyrinthine placenta. In fetal lung, intestine, liver, and brain there were modest changes in cytokine expressions. In fetal lung, SP and TLR mRNAs decreased and TLR2-positive macrophages redistributed around vessels. LPS-induced fetal deaths associated with early age (16 d gestation) rather than with proinflammatory activation. Here we propose that maternal LPS response leads to PTB and acute decrease of immune proteins in epithelial lining of fetal lung. Instead, acceleration of lung maturity has been previously observed in intraamniotic inflammation.

Crosstalk between Jagged1 and GDNF/Ret/GFRalpha1 signalling regulates ureteric budding and branching.

Glial-Cell-Line-Derived Neurotrophic Factor (GDNF) is the major mesenchyme-derived regulator of ureteric budding and branching during nephrogenesis. The ligand activates on the ureteric bud epithelium a receptor complex composed of Ret and GFRalpha1. The upstream regulators of the GDNF receptors are poorly known. A Notch ligand, Jagged1 (Jag1), co-localises with GDNF and its receptors during early kidney morphogenesis. In this study we utilized both in vitro and in vivo models to study the possible regulatory relationship of Ret and Notch pathways. Urogenital blocks were exposed to exogenous GDNF, which promotes supernumerary ureteric budding from the Wolffian duct. GDNF-induced ectopic buds expressed Jag1, which suggests that GDNF can, directly or indirectly, up-regulate Jag1 through Ret/GFRalpha1 signalling. We then studied the role of Jag1 in nephrogenesis by transgenic mice constitutively expressing human Jag1 in Wolffian duct and its derivatives under HoxB7 promoter. Jag1 transgenic mice showed a spectrum of renal defects ranging from aplasia to hypoplasia. Ret and GFRalpha1 are normally downregulated in the Wolffian duct, but they were persistently expressed in the entire transgenic duct. Simultaneously, GDNF expression remained unexpectedly low in the metanephric mesenchyme. In vitro, exogenous GDNF restored the budding and branching defects in transgenic urogenital blocks. Renal differentiation apparently failed because of perturbed stimulation of primary ureteric budding and subsequent branching. Thus, the data provide evidence for a novel crosstalk between Notch and Ret/GFRalpha1 signalling during early nephrogenesis.

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-alpha, 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.