Prenatal Maternal Lipopolysaccharide and Mild Newborn Hyperoxia Increase Intrapulmonary Airway but Not Vessel Reactivity in a Mouse Model.

Maternal infection is a risk for preterm delivery. Preterm newborns often require supplemental oxygen to treat neonatal respiratory distress. Newborn hyperoxia exposure is associated with airway and vascular hyperreactivity, while the complications of maternal infection are variable. In a mouse model of prenatal maternal intraperitoneal lipopolysaccharide (LPS, embryonic day 18) with subsequent newborn hyperoxia (40% oxygen × 7 days) precision-cut living lung slices were used to measure intrapulmonary airway and vascular reactivity at 21 days of age. Hyperoxia increased airway reactivity to methacholine compared to room air controls. Prenatal maternal LPS did not alter airway reactivity in room air. Combined maternal LPS and hyperoxia exposures increased airway reactivity vs. controls, although maximal responses were diminished compared to hyperoxia alone. Vessel reactivity to serotonin did not significantly differ in hyperoxia or room air; however, prenatal maternal LPS appeared to attenuate vessel reactivity in room air. Following room air recovery, LPS with hyperoxia lungs displayed upregulated inflammatory and fibrosis genes compared to room air saline controls (TNFαR1, iNOS, and TGFß). In this model, mild newborn hyperoxia increases airway but not vessel reactivity. Prenatal maternal LPS did not further increase hyperoxic airway reactivity. However, inflammatory genes remain upregulated weeks after recovery from maternal LPS and newborn hyperoxia exposures.

Scavenging roles of chemokine receptors: chemokine receptor deficiency is associated with increased levels of ligand in circulation and tissues.

In vitro studies have implicated chemokine receptors in consumption and clearance of specific ligands. We studied the role that various signaling chemokine receptors play during ligand homeostasis in vivo. We examined the levels of ligands in serum and CNS tissue in mice lacking chemokine receptors. Compared with receptor-sufficient controls, Cx3cr1(-/-) mice exhibited augmented levels of CX3CL1 both in serum and brain, and circulating levels of CXCL1 and CXCL2 were increased in Cxcr2(-/-) mice. CCR2-deficient mice showed significantly increased amounts of circulating CCL2 compared with wild-type mice. Cxcr3(-/-) mice revealed increased levels of circulating and brain CXCL10 after experimental autoimmune encephalomyelitis (EAE) induction. CCR2-deficient peripheral blood and resident peritoneal cells exhibited reduced binding capacity and biologic responses to the CCR1 ligand CCL3, suggesting that elevated levels of CCR2 ligands had down-regulated CCR1. The results indicate that signaling chemokine receptors clear chemokines from circulation and tissues. These homeostatic functions of signaling chemokine receptors need to be integrated into safety and efficacy calculations when considering therapeutic receptor blockade.

Control of microglial neurotoxicity by the fractalkine receptor.

Microglia, the resident inflammatory cells of the CNS, are the only CNS cells that express the fractalkine receptor (CX3CR1). Using three different in vivo models, we show that CX3CR1 deficiency dysregulates microglial responses, resulting in neurotoxicity. Following peripheral lipopolysaccharide injections, Cx3cr1-/- mice showed cell-autonomous microglial neurotoxicity. In a toxic model of Parkinson disease and a transgenic model of amyotrophic lateral sclerosis, Cx3cr1-/- mice showed more extensive neuronal cell loss than Cx3cr1+ littermate controls. Augmenting CX3CR1 signaling may protect against microglial neurotoxicity, whereas CNS penetration by pharmaceutical CX3CR1 antagonists could increase neuronal vulnerability.

Chronic expression of monocyte chemoattractant protein-1 in the central nervous system causes delayed encephalopathy and impaired microglial function in mice.

Increased central nervous system (CNS) levels of monocyte chemoattractant protein 1 [CC chemokine ligand 2 (CCL2) in the systematic nomenclature] have been reported in chronic neurological diseases such as human immunodeficiency virus type 1-associated dementia, amyotrophic lateral sclerosis, and multiple sclerosis. However, a pathogenic role for CCL2 has not been confirmed, and there is no established model for the effects of chronic CCL2 expression on resident and recruited CNS cells. We report that aged (>6 months) transgenic (tg) mice expressing CCL2 under the control of the human glial fibrillary acidic protein promoter (huGFAP-CCL2hi tg+ mice) manifested encephalopathy with mild perivascular leukocyte infiltration, impaired blood brain barrier function, and increased CD45-immunoreactive microglia, which had morphologic features of activation. huGFAP-CCL2hi tg+ mice lacking CC chemokine receptor 2 (CCR2) were normal, showing that chemokine action via CCR2 was required. Studies of cortical slice preparations using video confocal microscopy showed that microglia in the CNS of huGFAP-CCL2hi tg+ mice were defective in expressing amoeboid morphology. Treatment with mutant CCL2 peptides, a receptor antagonist and an obligate monomer, also suppressed morphological transformation in this assay, indicating a critical role for CCL2 in microglial activation and suggesting that chronic CCL2 exposure desensitized CCR2 on microglia, which in the CNS of huGFAP-CCL2hi tg+ mice, did not up-regulate cell-surface expression of major histocompatibility complex class II, CD11b, CD11c, or CD40, in contrast to recruited perivascular macrophages that expressed enhanced levels of these markers. These results indicate that huGFAP-CCL2hi tg+ mice provide a useful model to study how chronic CNS expression of CCL2 alters microglial function and CNS physiology.

Isolation of murine microglial cells for RNA analysis or flow cytometry.

There is increasing interest in the isolation of adult microglia to study their functions at a morphological and molecular level during normal and neuroinflammatory conditions. Microglia have important roles in brain homeostasis, and in disease states they exert neuroprotective or neurodegenerative functions. To assay expression profiles or functions of microglia, we have developed a method to isolate microglial cells and infiltrating leukocytes from adult mouse brain. This protocol uses a digestion cocktail containing collagenase and dispase, and it involves separation over discontinuous percoll gradients. Isolated cells can be used for RNA analysis, including RNase protection analysis (RPA), quantitative RT-PCR, high-density microarray, proteomic or flow cytometric characterization of cell surface markers or adoptive transfer. Cell isolation can be completed in less than 4 h.