Here, we describe a novel pathogenic entity, the activated PMN (polymorphonuclear leukocyte, i.e., neutrophil)-derived exosome. These CD63 + /CD66b + nanovesicles acquire surface-bound neutrophil elastase (NE) during PMN degranulation, NE being oriented in a configuration resistant to a1-antitrypsin (a1AT). These exosomes bind and degrade extracellular matrix (ECM) via the integrin Mac-1 and NE, respectively, causing the hallmarks of chronic obstructive pulmonary disease (COPD). Due to both ECM targeting and a1AT resistance, exosomal NE is far more potent than free NE. Importantly, such PMN-derived exosomes exist in clinical specimens from subjects with COPD but not healthy controls and are capable of transferring a COPD-like phenotype from humans to mice in an NE-driven manner. Similar findings were observed for another neutrophil-driven disease of ECM remodeling (bronchopulmonary dysplasia [BPD]). These findings reveal an unappreciated role for exosomes in the pathogenesis of disorders of ECM homeostasis such as COPD and BPD, providing a critical mechanism for proteolytic damage.

Premature infants are at high risk for developing bronchopulmonary dysplasia (BPD), characterized by chronic inflammation and inhibition of lung development, which we have recently identified as being modulated by microRNAs (miRNAs) and alterations in the airway microbiome. Exosomes and exosomal miRNAs may regulate cell differentiation and tissue and organ development. We discovered that tracheal aspirates from infants with severe BPD had increased numbers of, but smaller, exosomes compared with term controls. Similarly, bronchoalveolar lavage fluid from hyperoxia-exposed mice (an animal model of BPD) and supernatants from hyperoxia-exposed human bronchial epithelial cells (in vitro model of BPD) had increased exosomes compared with air controls. Next, in a prospective cohort study of tracheal aspirates obtained at birth from extremely preterm infants, utilizing independent discovery and validation cohorts, we identified unbiased exosomal miRNA signatures predictive of severe BPD. The strongest signal of reduced miR-876-3p in BPD-susceptible compared with BPD-resistant infants was confirmed in the animal model and in vitro models of BPD. In addition, based on our recent discovery of increased Proteobacteria in the airway microbiome being associated with BPD, we developed potentially novel in vivo and in vitro models for BPD combining Proteobacterial LPS and hyperoxia exposure. Addition of LPS led to a larger reduction in exosomal miR 876-3p in both hyperoxia and normoxia compared with hyperoxia alone, thus indicating a potential mechanism by which alterations in microbiota can suppress miR 876-3p. Gain of function of miR 876-3p improved the alveolar architecture in the in vivo BPD model, demonstrating a causal link between miR 876-3p and BPD. In summary, we provide evidence for the strong predictive biomarker potential of miR 876-3p in severe BPD. We also provide insights on the pathogenesis of neonatal lung disease, as modulated by hyperoxia and microbial product-induced changes in exosomal miRNA 876-3p, which could be targeted for future therapeutic development.

Cell-free heme (CFH) and hemoglobin (Hb) have emerged as distinct mediators of acute injury characterized by inflammation and microcirculatory dysfunction in hemolytic conditions and critical illness. Several reports have shown changes in Hb and CFH in specific pathophysiological settings. Using PBS, plasma from patients with sickle cell disease, acute respiratory distress syndrome (ARDS) patients and supernatants from red cells units, we found that commonly used assays and commercially available kits do not distinguish between CFH and Hb. Furthermore, they suffer from a variety of false-positive interferences and limitations (including from bilirubin) that lead to either over- or underestimation of CFH and/or Hb. Moreover, commonly used protocols to separate CFH and Hb based on molecular weight (MWt) are inefficient due to CFH hydrophobicity. In this study, we developed and validated a new approach based on absorbance spectrum deconvolution with least square fitting analyses that overcomes these limitations and simultaneously measures CFH and Hb in simple aqueous buffers, plasma or when associated with red cell derived microvesicles. We show how incorporating other plasma factors that absorb light over the visible wavelength range (specifically bilirubin), coupled with truncating the wavelength range analyzed, or addition of mild detergent significantly improves fits allowing measurement of oxyHb, CFH and metHb with >90% accuracy. When this approach was applied to samples from SCD patients, we observed that CFH levels are higher than previously reported and of similar magnitude to Hb.

dPneumococcal surface protein A (PspA) is a surface molecule on pneumococci that is required for full virulence in mouse models of infection. PspA has been reported to inhibit complement deposition on the pneumococcal surface. It has been assumed that this decreased complement deposition results in the inefficient phagocytosis of wild-type pneumococci. However, an effect of PspA on phagocytosis had not been shown. Our present studies demonstrated that a loss of PspA by capsular type 3 strains WU2 and A66.1 led to enhanced complement-dependent phagocytosis of the pneumococci by the mouse macrophage cell line J774A.1. This observation was made using human complement as well as mouse complement. Since this enhanced phagocytosis could be blocked by antibody to complement receptor CR3 on J774A.1, it was concluded that PspA's effect on phagocytosis was due to its effect on the amount of deposited complement, which in turn helped opsonize the pneumococci for phagocytosis. Since these studies included new independent mutants lacking PspA, the results provide solid confirmation of the previously reported effects of PspA on pneumococcal virulence and complement deposition. Finally, we showed that antibody to PspA, which is also known to enhance complement deposition, also enhances the phagocytosis of pneumococci in a largely complement-dependent manner.

Pneumococcal surface protein A (PspA) is a surface exposed, highly immunogenic protein of Streptococcus pneumoniae. Its N-terminal α-helical domain (αHD) elicits protective antibody in humans and animals that can protect mice from fatal infections with pneumococci and can be detected in vitro with opsonophagocytosis assays. The proline-rich domain (PRD) in the center of the PspA sequence can also elicit protection. This study revealed that although the sequence of PRD was diverse, PRD from different pneumococcal isolates contained many shared elements. The inferred amino acid sequences of 123 such PRDs, which were analyzed by assembly and alignment-free (AAF) approaches, formed three PRD groups. Of these sequences, 45 were classified as Group 1, 19 were classified as Group 2, and 59 were classified as Group 3. All Group 3 sequences contained a highly conserved 22-amino acid non-proline block (NPB). A significant polymorphism was observed, however, at a single amino acid position within NPB. Each of the three PRD groups had characteristic patterns of short amino acid repeats, with most of the repeats being found in more than one PRD group. One of these repeats, PKPEQP as well as the NPB were previously shown to elicit protective antibodies in mice. In this study, we found that sera from 12 healthy human adult volunteers contained antibodies to all three PRD groups. This suggested that a PspA-containing vaccine containing carefully selected PRDs and αHDs could redundantly cover the known diversity of PspA. Such an approach might reduce the chances of PspA variants escaping a PspA vaccine’s immunity.