BackgroundHeparanase-1 (HPSE)-mediated degradation of endothelial glycocalyx heparan sulfate (HS) contributes to vascular endotheliopathy in sepsis, yet age-dependent differences in HPSE biology remain undefined. Thus, we sought to determine age-related differences in circulating HPSE activity during sepsis and its association with markers of endotheliopathy and organ dysfunction.MethodsHeparanase-1 enzymatic activity and HS disaccharide levels were measured in plasma from children (10 sepsis, 10 controls) and adults (16 sepsis, 15 controls) from prospective observational cohorts using liquid chromatography-tandem mass spectrometry. Associations with plasma angiopoietin-2 levels and change in serum albumin (markers of endotheliopathy) in addition to organ failure scores were assessed using Spearman correlations.ResultsHeparanase-1 activity and circulating HS were elevated in both sepsis cohorts compared to controls, with moderate-to-strong correlations between HPSE activity and HS levels. However, adults with sepsis demonstrated approximately 10-fold higher plasma HPSE activity than children (median 1,256 vs. 116, p < 0.001), despite children exhibiting greater endotheliopathy and higher organ failure scores. In both age groups, HPSE activity correlated with angiopoietin-2, serum albumin decline, and organ failure scores. Adult non-survivors had higher HPSE activity than survivors; no pediatric deaths occurred. Predominant neutrophilic/monocytic activation in adults versus greater platelet consumption in children may suggest developmental differences in cellular sources of circulating HPSE.ConclusionHeparanase-1-mediated glycocalyx degradation is a conserved feature of sepsis across the age spectrum, but the magnitude, cellular source, and clinical implications of circulating HPSE activity differ markedly by age, underscoring the need for age-stratified therapeutic approaches.

Smoking remains a major risk factor for the severity of bacterial pneumonias, although mechanisms driving this injury remain poorly understood. This manuscript provides a new insight on how the smoke-exposed lung and bacteria augment the release of enzymes capable of directly damaging pulmonary tissue, leading to worsening inflammation and tissue damage.

E-cigarette use has surged as an alternative to traditional smoking, but the pulmonary effects of e-cigarette vapor remain poorly understood. Previous work has shown that bacterial components like lipopolysaccharide (LPS) trigger the generation of pathogenic, neutrophil-derived extracellular vesicles (EVs) expressing destructive proteases, such as neutrophil elastase (NE). This study aimed to determine whether e-cigarette vapor modulates the EV signature linked to LPS exposure. Wild-type mice were pre-exposed to ambient air or e-cigarette vapor for 2 weeks. Mice then received intratracheal LPS treatment, and pulmonary inflammation and alveolar damage were assessed over time. Mice pre-exposed to e-cigarette vapor exhibited heightened inflammation and greater alveolar damage upon LPS treatment compared to air-exposed mice. The vapor-exposed, LPS-treated mice also had a more destructive EV signature with increased protease activity. When these EVs were transferred to naïve mice, they caused more severe lung damage compared to EVs from air-exposed LPS-treated mice. E-cigarette vapor appears to enhance lung injury and inflammation, highlighting the need for further investigation into the mechanisms by which vapor may increase lung susceptibility to damage. Immune Mechanisms of Human Disease (HUM)

BackgroundWhile e-cigarette use (vaping) has increased in the last decade, its effects on airway inflammation and extracellular vesicle (EV) biology remain unclear. This study examined how long-term and acute vapor exposures influence lung immune responses, neutrophilic inflammation, and EV-associated proteolytic activity.MethodsMice were exposed daily to vapor from commercial e-cigarettes or room air for up to 12 weeks. After exposure, we assessed immune cell recruitment, alveolar damage, and EV populations in the airways. To explore vapor-mediated effects on secondary lung injury, a lipopolysaccharide (LPS) challenge was administered after two weeks of vapor exposure. We then analyzed immune cell responses and isolated neutrophil-derived EVs (nEVs) for transfer into naïve mice to evaluate pathogenic potential.ResultsVapor exposure alone did not significantly alter immune cell infiltration, lung histology, or EV protease activity. However, mice pre-exposed to vapor and then challenged with LPS showed increased neutrophil infiltration, elevated neutrophil elastase activity in EVs, and greater alveolar damage. Furthermore, nEVs from these mice induced more severe emphysematous changes when transferred to unexposed mice.ConclusionsWhile e-cigarette vapor alone does not provoke marked airway inflammation or proteolytic EV release, it creates a primed immune state. This priming amplifies inflammatory and destructive responses to subsequent challenges. These findings suggest vaping may exacerbate lung damage when combined with infections or other environmental stressors, raising concerns about its role in worsening pulmonary disease.

Rationale:Streptococcus pneumoniae is a major causative pathogen of community acquired pneumonia. Smokers have a higher risk of acquiring and developing S.pneumoniae lung infection and they have worse outcomes compared to non-smokers.While cigarette smoking is a strong risk factor in pneumococcal pneumonia, there remains much to be learned about the molecular basis for susceptibility to pneumonia. To investigate whether cigarette smoke promoted colonization of the upper-respiratory tract results in increased inflammation and bacterial translocation into the lung, we developed a 2-hit murine model combining a murine model of S.pneumoniae infection with a model of cigarette smoke (CS) exposure. We have used this model to investigate the synergistic interaction of bacteria and smoke in augmenting tissue damage.Methods:C57BL/6 mice (n=5-6/group) were exposed to CS generated from the whole body “InExpose” smoking system (SCIREQ) for two weeks. Then the mice were split into two groups, one was inoculated intranasally with 2x106 CFU Streptococcal pneumoniae EF3030 serotype 19F and the other group was treated with intranasal saline (PBS). Control groups exposed to air were also included. All mice were sacrificed at 48-hours post-infection and tissue was collected. Ac-PGP levels were measured with mass spectrometry. MMP-9, PE and acrolein levels were measured in BALF using a commercial ELISA kit. The lung homogenate was immunoprecipitated using anti-acrolein antibody and immunoblotted with anti-PE, anti-MMP-9, and anti-LTA4H. Statistical analysis was performed using one way ANOVA with Tukey's multiple comparison post-test.Results:We found that animals with CS exposure had significantly higher levels of Ac-PGP compared to control mice and animals with CS and pneumococcus inoculation had the highest levels of Ac-PGP. Coinciding with elevated Ac-PGP levels, the smoke-exposed mice with S.pneumonaie infection also had increased inflammatory cells in the airways and increased levels of PGP-generating enzymes PE and MMP-9. We detected higher acrolein levels in the BALF of smoke exposed mice with infection than in control mice. Using immunoprecipitation with anti-acrolein antibodies we detected an increase in total acroleinated proteins (acroleinated PE, MMP-9 and LTA4H) in lung lysates in the smoke and infection group.Conclusions:The findings from these experiments provide evidence for a discrete mechanism by which protease activity is altered, leading to persistence of PGP in smoking-induced bacterial infection. Acrolein is a key reactive aldehyde generated with smoke and bacterial infection and this can enhance PGP persistence in the lung. This PGP persistence leads to ongoing PMN inflammation and tissue injury.

RATIONALE Mounting evidence demonstrates a role for extracellular vesicles (EVs) in driving lung disorders, such as COPD. While cigarette smoke (CS) is the primary risk factor for COPD, a link between CS and the EVs that could lead to COPD is unknown. OBJECTIVE To ascertain whether exposure to CS elicits a proteolytic EV signature capable of driving disease pathogenesis. METHODS Protease expression and enzymatic activity was measured in EVs harvested from the BAL fluid of smoke-exposed mice and otherwise healthy human smokers. Pathogenicity of EVs was examined using pathological tissue scoring following EV transfer into naïve recipient mice. MEASUREMENTS & MAIN RESULTS Our analyses reveal a unique EV profile defined by neutrophil and macrophage-derived EVs. These EVs are characterized by abundant surface expression of neutrophil elastase (NE) and matrix metalloproteinase (MMP-12), respectively. CS-induced mouse or human-derived airway EVs had a robust capacity to elicit rapid lung damage in naïve recipient mice, with an additive effect of NE and MMP-12-expressing EVs. CONCLUSIONS These studies demonstrate the capacity of CS to drive the generation of unique EV populations containing NE and MMP-12. The coordinated action of these EVs is completely sufficient to drive emphysematous disease, and their presence could operate as a prognostic indicator for COPD development. Furthermore, given the robust capacity of these EVs to elicit emphysema in naïve mice, they provide a novel model to facilitate pre-clinical COPD research. Indeed, the development of this model has led to the discovery of a previously unrecognized CS-induced protective mechanism against EV-mediated damage.

Leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme, with dual activities critical in defining the scale of tissue inflammation and pathology. LTA4H classically operates intracellularly, primarily within myeloid cells, to generate pro-inflammatory leukotriene B4. However, LTA4H also operates extracellularly to degrade the bioactive collagen fragment proline-glycine-proline to limit neutrophilic inflammation and pathological tissue remodeling. While the dichotomous functions of LTA4H are dictated by location, the cellular source of extracellular enzyme remains unknown. We demonstrate that airway extracellular LTA4H concentrations are governed by the level of pulmonary vascular permeability and influx of an abundant repository of blood-borne enzyme. In turn, blood LTA4H originates from liver hepatocytes, being released constitutively but further upregulated during an acute phase response. These findings have implications for our understanding of how inflammation and repair are regulated and how perturbations to the LTA4H axis may manifest in pathologies of chronic diseases.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of prematurity. Exposure to noxious stimuli such as hyperoxia, volutrauma, and infection in infancy can have long-reaching impacts on lung health and predispose towards the development of conditions such as chronic obstructive pulmonary disease (COPD) in adulthood. BPD and COPD are both marked by lung tissue degradation, neutrophil influx, and decreased lung function. Both diseases also express a change in microbial signature characterized by firmicute depletion. However, the relationship between pulmonary bacteria and the mechanisms of downstream disease development has yet to be elucidated. We hypothesized that murine models of BPD would show heightened acetylated proline-glycine-proline (Ac-PGP) pathway and neutrophil activity, and through gain- and loss-of-function studies we show that Ac-PGP plays a critical role in driving BPD development. We further test a inhaled live biotherapeutic (LBP) using active Lactobacillus strains in in vitro and in vivo models of BPD and COPD. The Lactobacillus-based LBP is effective in improving lung structure and function, mitigating neutrophil influx, and reducing a broad swath of pro-inflammatory markers in these models of chronic pulmonary disease via the MMP-9/PGP (matrix metalloproteinase/proline-glycine-proline) pathway. Inhaled LBPs show promise in addressing common pathways of disease progression that in the future can be targeted in a variety of chronic lung diseases.

The enzyme leukotriene A4 hydrolase (LTA4H) is classically known for its epoxide hydrolase activity that converts leukotriene A4 (LTA4) to the neutrophil chemoattractant LTB4 [1]. In 2010, our group published a study in Science which demonstrated that during an influenza model of acute airway inflammation, LTA4H was released from cells to degrade proline-glycine-proline (PGP), a non-canonical CXCR1 and −2 agonist of polymorphonuclear neutrophil (PMN) recruitment and activation [2], thereby attenuating PMN inflammation [3]. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of Interest: Jonathan Grigg reports lecture honoraria from GSK; outside the submitted work. Conflict of interest: None to disclose

Leukotriene A4 4 hydrolase (LTA4H) 4 H) is a bifunctional enzyme, with dual activities critical in defining the scale of tissue inflammation and pathology. LTA4H 4 H classically operates intracellularly, primarily within myeloid cells, to generate pro-inflammatory leukotriene B4. 4 . However, LTA4H 4 H also operates extracellularly to degrade the bioactive collagen fragment proline-glycine-proline to limit neutrophilic inflammation and pathological tissue remodeling. While the dichotomous functions of LTA4H 4 H are dictated by location, the cellular source of extra- cellular enzyme remains unknown. We demonstrate that airway extracellular LTA4H 4 H concentrations are governed by the level of pulmonary vascular permeability and influx of an abundant repository of blood-borne enzyme. In turn, blood LTA4H 4 H originates from liver hepatocytes, being released constitutively but further up- regulated during an acute phase response. These findings have implications for our understanding of how inflammation and repair are regulated and how perturbations to the LTA4H 4 H axis may manifest in pathologies of chronic diseases.

Rationale: Mounting evidence demonstrates a role for extracellular vesicles (EVs) in driving lung disorders, such as chronic obstructive pulmonary disease (COPD). Although cigarette smoke (CS) is the primary risk factor for COPD, a link between CS and the EVs that could lead to COPD is unknown. Objective: To ascertain whether exposure to CS elicits a proteolytic EV signature capable of driving disease pathogenesis. Methods: Protease expression and enzymatic activity were measured in EVs harvested from the BAL fluid of smoke-exposed mice and otherwise healthy human smokers. Pathogenicity of EVs was examined using pathological tissue scoring after EV transfer into naive recipient mice. Measurements and Main Results: The analyses revealed a unique EV profile defined by neutrophil- and macrophage-derived EVs. These EVs are characterized by abundant surface expression of neutrophil elastase (NE) and matrix metalloproteinase 12 (MMP12), respectively. CS-induced mouse or human-derived airway EVs had a robust capacity to elicit rapid lung damage in naive recipient mice, with an additive effect of NE- and MMP12-expressing EVs. Conclusions: These studies demonstrate the capacity of CS to drive the generation of unique EV populations containing NE and MMP12. The coordinated action of these EVs is completely sufficient to drive emphysematous disease, and their presence could operate as a prognostic indicator for COPD development. Furthermore, given the robust capacity of these EVs to elicit emphysema in naive mice, they provide a novel model to facilitate preclinical COPD research. Indeed, the development of this model has led to the discovery of a previously unrecognized CS-induced protective mechanism against EV-mediated damage.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of prematurity. Exposure to noxious stimuli such as hyperoxia, volutrauma, and infection in infancy can have long-reaching impacts on lung health and predispose towards the development of conditions such as chronic obstructive pulmonary disease (COPD) in adulthood. BPD and COPD are both marked by lung tissue degradation, neutrophil influx, and decreased lung function. Both diseases also express a change in microbial signature dominated by Proteobacteria abundance and Lactobacillus scarcity. However, the relationship between pulmonary microbial dysbiosis and the mechanisms of downstream disease development has yet to be elucidated. We hypothesized that a double-hit hyperoxia and LPS murine model of BPD would show heightened Ac-PGP pathway and neutrophil activity. Through gain- and loss-of-function studies in the same model we showed that Ac-PGP plays a critical role in driving BPD development. We tested a novel inhaled live biotherapeutic using active Lactobacillus strains to counteract lung dysbiosis in in vitro and in vivo models of BPD and COPD. The Lactobacillus LBP is effective in improving lung structure and function, reducing neutrophil influx, and reducing a broad swath of pro-inflammatory markers in these models of chronic pulmonary disease. Live inhaled microbiome-based therapeutics show promise in addressing common pathways of disease progression that in the future can be targeted in a variety of chronic lung diseases.

Protamine sulfate facilitates the removal of neutrophil elastase (NE) from the surface of extracellular vesicles from activated neutrophils. This “free” NE is no longer protected from inhibition by its endogenous anti-protease, α-1-anti-trypsin. This function of protamine sulfate highlights it as a potential therapeutic strategy for COPD, which may attenuate the disease process.

Introduction: Patients with chronic obstructive pulmonary disease (COPD) often develop cardiovascular disorders such as right ventricular hypertrophy (RVH) and pulmonary hypertension (PH). We hypothesized that the neutrophil chemoattractant proline-glycine-proline (PGP), generated by the stepwise proteolytic cleavage of collagen by matrix metalloproteinases and prolyl endopeptidase (PE), would be involved in ongoing PH pathogenesis. Methods: Mice were administered with acetylated PGP (Ac-PGP) (250 µg/dose) intratracheally for 6 and 10 weeks or exposed to cigarette smoke for 6 weeks to evaluate lung inflammation and RVH. To further elucidate the impact of PGP on pulmonary vascular remodeling, the PE inhibitor benzyloxycarbony-proline-prolinal (ZPP) was intratracheally administered in a 6-week smoking model. Results: In mice treated with Ac-PGP at 6 weeks and 10 weeks, there was increased right ventricular systolic pressures (RVSP) when compared to the control group. The average ratio of RV/(LV+S) also showed significant increase with Ac-PGP administration. Ac-PGP levels in the blood was significantly increased after 6 weeks of smoke exposure. Treating the mice with ZPP intratracheally prior to smoke exposure significantly decreased inflammation in the lungs and prevented mice from developing PH. Furthermore, nitrite and nitrate levels in blood were elevated after smoke exposure and the nitrite/nitrate ratio returned to baseline levels after ZPP treatment, suggesting reduced oxidative stress. Conclusion: These results demonstrate that Ac-PGP induced the development of PH and that targeting of PGP peptides in a smoke model affects the development of RVH and PH.

Chronic obstructive pulmonary disease (COPD) is a debilitating chronic disease and the third-leading cause of mortality worldwide. It is characterized by airway neutrophilia, promoting tissue injury through release of toxic mediators and proteases. Recently, it has been shown that neutrophil-derived extracellular vesicles (EVs) from lungs of patients with COPD can cause a neutrophil elastase–dependent (NE-dependent) COPD-like disease upon transfer to mouse airways. However, in vivo preclinical models elucidating the impact of EVs on disease are lacking, delaying opportunities for therapeutic testing. Here, we developed an in vivo preclinical mouse model of lung EV–induced COPD. EVs from in vivo LPS-activated mouse neutrophils induced COPD-like disease in naive recipients through an α-1 antitrypsin–resistant, NE-dependent mechanism. Together, these results show a key pathogenic and mechanistic role for neutrophil-derived EVs in a mouse model of COPD. Broadly, the in vivo model described herein could be leveraged to develop targeted therapies for severe lung disease.