An Integrated Pharmacological, Structural, and Genetic Analysis of Extracellular Versus Intracellular ROS Production in Neutrophils.

The neutrophil NADPH oxidase produces both intracellular and extracellular reactive oxygen species (ROS). Although oxidase activity is essential for microbial killing, and ROS can act as signaling molecules in the inflammatory process, excessive extracellular ROS directly contributes to inflammatory tissue damage, as well as to cancer progression and immune dysregulation in the tumor microenvironment. How specific signaling pathways contribute to ROS localization is unclear. Here we used a systems pharmacology approach to identify the specific Class I PI3-K isoform p110ß, and PLD1, but not PLD2, as critical regulators of extracellular, but not intracellular ROS production in primary neutrophils. Combined crystallographic and molecular dynamics analysis of the PX domain of the oxidase component p47phox, which binds the lipid products of PI 3-K and PLD, was used to clarify the membrane-binding mechanism and guide the design of mutant mice whose p47phox is unable to bind 3-phosphorylated inositol phospholipids. Neutrophils from these K43A mutant animals were specifically deficient in extracellular, but not intracellular, ROS production, and showed increased dependency on signaling through the remaining PLD1 arm. These findings identify the PX domain of p47phox as a critical integrator of PLD1 and p110ß signaling for extracellular ROS production, and as a potential therapeutic target for modulating tissue damage and extracellular signaling during inflammation.

Follicular Dendritic Cells Modulate Germinal Center B Cell Diversity through FcγRIIB.

Follicular dendritic cells (FDCs), a rare and enigmatic stromal cell type in the B cell follicles of secondary lymphoid organs, store and present antigen to B cells. While essential for germinal center (GC) responses, their exact role during GC B cell selection remains unknown. FDCs upregulate the inhibitory IgG Fc receptor FcγRIIB during GC formation. We show that the stromal deficiency of FcγRIIB does not affect GC B cell frequencies compared to wild-type mice. However, in the absence of FcγRIIB on FDCs, GCs show aberrant B cell selection during autoreactive and selective foreign antigen responses. These GCs are more diverse as measured by the AidCreERT2 -confetti system and show the persistence of IgM+ clones with decreased numbers of IgH mutations. Our results show that FDCs can modulate GC B cell diversity by the upregulation of FcγRIIB. Permissive clonal selection and subsequent increased GC diversity may affect epitope spreading during autoimmunity and foreign responses.

Kinetics and Role of Plasma Matrix Metalloproteinase-9 Expression in Acute Lung Injury and the Acute Respiratory Distress Syndrome.

Primed neutrophils that are capable of releasing matrix metalloproteinases (MMPs) into the circulation are thought to play a significant role in the pathophysiology of acute respiratory distress syndrome (ARDS). We hypothesized that direct measurement of plasma MMP-9 activity may be a predictor of incipient tissue damage and subsequent lung injury, which was investigated in both an animal model of ARDS and a small cohort of 38 critically ill human patients. In a mouse model of ARDS involving instillation of intratracheal lipopolysaccharide (LPS) to induce lung inflammation, we measured neutrophil-mediated inflammation, along with MMP-9 activity in the airways and lung tissue and MMP-9 expression in the plasma. Neutrophil recruitment, inflammation, and MMP-9 activity in the airways and lung tissue increased throughout the 72 h after LPS instillation, whereas plasma MMP-9 expression was greatest at 12 to 24 h after LPS instillation. The results suggest that the peak in plasma MMP-9 activity may precede the peak of neutrophil inflammation in the airways and lung tissue in the setting of ARDS. Based on this animal study, a retrospective observational cohort study involving 38 patients admitted to a surgical intensive care unit at a tertiary care university hospital with acute respiratory failure requiring intubation and mechanical ventilation was conducted. Plasma samples were collected daily, and MMP-9 activity was compared with lung function as determined by the PaO2/FiO2 ratio. In patients who developed ARDS, a notable increase in plasma MMP-9 activity on a particular day correlated with a decrease in the PaO2/FiO2 ratio on the following day (r = -0.503, P < 0.006). Taken together, these results suggest that plasma MMP-9 activity changes, as a surrogate for primed neutrophils may have predictive value for the development of ARDS in a selected subset of critically ill patients.

Danger-associated molecular patterns and danger signals in idiopathic pulmonary fibrosis.

The chronic debilitating lung disease, idiopathic pulmonary fibrosis (IPF), is characterized by a progressive decline in lung function, with a median mortality rate of 2-3 years after diagnosis. IPF is a disease of unknown cause and progression, and multiple pathways have been demonstrated to be activated in the lungs of these patients. A recent genome-wide association study of more than 1,000 patients with IPF identified genes linked to host defense, cell-cell adhesion, and DNA repair being altered due to fibrosis (Fingerlin, et al. Nat Genet 2013;45:613-620). Further emerging data suggest that the respiratory system may not be a truly sterile environment, and it exhibits an altered microbiome during fibrotic disease (Molyneaux and Maher. Eur Respir Rev 2013;22:376-381). These altered host defense mechanisms might explain the increased susceptibility of patients with IPF to microbial- and viral-induced exacerbations. Moreover, chronic epithelial injury and apoptosis are key features in IPF, which might be mediated, in part, by both pathogen-associated (PA) and danger-associated molecular patterns (MPs). Emerging data indicate that both PAMPs and danger-associated MPs contribute to apoptosis, but not necessarily in a manner that allows for the removal of dying cells, without further exacerbating inflammation. In contrast, both types of MPs drive cellular necrosis, leading to an exacerbation of lung injury and/or infection as the debris promotes a proinflammatory response. Thus, this Review focuses on the impact of MPs resulting from infection-driven apoptosis and necrosis during chronic fibrotic lung disease.

P-Rex1, a PtdIns(3,4,5)P3- and Gbetagamma-regulated guanine-nucleotide exchange factor for Rac.

Rac, a member of the Rho family of monomeric GTPases, is an integrator of intracellular signaling in a wide range of cellular processes. We have purified a PtdIns(3,4,5)P3-sensitive activator of Rac from neutrophil cytosol. It is an abundant, 185 kDa guanine-nucleotide exchange factor (GEF), which we cloned and named P-Rex1. The recombinant enzyme has Rac-GEF activity that is directly, substantially, and synergistically activated by PtdIns(3,4,5)P3 and Gbetagammas both in vitro and in vivo. P-Rex1 antisense oligonucleotides reduced endogenous P-Rex1 expression and C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line. P-Rex1 appears to be a coincidence detector in PtdIns(3,4,5)P3 and Gbetagamma signaling pathways that is particularly adapted to function downstream of heterotrimeric G proteins in neutrophils.