Background: A hallmark of mucosal inflammation is the accumulation of neutrophils (PMN) at sites of injury. Myeloperoxidase (MPO) is the primary antimicrobial enzyme in PMN. MPO generates hypochlorous acid via a reaction between hydrogen peroxide and chloride. Hypochlorous acid diffuses through the tissue and indiscriminately reacts with the phenol moiety in tyrosine to generate chlorinated tyrosine. It is unknown what impact tyrosine chlorination has on cellular function. The current study aims to identify the impact of PMN MPO on the development and resolution of colitis. Results: Previous studies have shown that therapeutic inhibition of MPO improves acute colitis in a murine model of DSS colitis. We expanded upon these studies and demonstrated that colonic inflammation in MPO KO mice resolved faster than WT mice, as shown by, a decrease in fecal lipocalin and tissue cytokines. Next, we examined the impact of MPO on the development of chronic colitis. MPO KO mice exhibited significantly less inflammation when compared to WT mice. MPO mice had longer colons, lower cytokine levels, and lower histological scores. To better understand the mechanism(s) involved in MPO associated tissue damage and inflammation we extended these studies to include in vitro models. Analysis of the extracellular loops of occludin reveals a high number of tyrosine residues, 11 in loop 1 and 7 in loop 2. Given the observation that MPO activation results in the indiscriminate chlorination of tyrosine we examined occludin for 3-chlorotyrosine. Following PMN transepithelial migration and exposure to activated MPO we observed 3-chlorotyrosine within occludin. Further, we then examined the impact of tyrosine chlorination within the binding domain of occludin. It has been reported that a short sequence within the second extracellular loop of occludin is required for tight junction function. Intestinal epithelial cells (IEC) were treated with a short peptide containing this sequence there are significant changes to tight junction length ratio, indicating an impairment of tight junction function. The occludin peptide was neutralized by the chlorination of the tyrosines within the sequence. This suggests that physiologically, the chlorination of tyrosines by PMN MPO disrupts occludin binding. This was further supported by a significant decrease in barrier function in IEC treated with activated MPO. Conclusions: The results of our study suggests the enzymatic action of MPO promotes chronic inflammation. Activated MPO not only damages tissue and inhibits wound healing, but as shown in our study, also disrupts IEC barrier function. Our work suggests that the barrier dysfunction is a result the chlorination of tyrosines within occludin. Taken together these studies highlight the need to further study the impact of MPO on the inflammatory microenvironment.

Prostaglandin (PG) E2 is a recognized inhibitor of granulocyte functions. However, most of the data supporting this was obtained when available pharmacological tools mainly targeted the EP2 receptor. Herein, we revisited the inhibitory effect of PGE2 on reactive oxygen species production, leukotriene biosynthesis and migration in human neutrophils. Our data confirm the inhibitory effect of PGE2 on these functions and unravel that the effect of PGE2 on human neutrophils is obtained by the combined action of EP2 and EP4 agonism. Accordingly, we also demonstrate that the inhibitory effect of PGE2 is fully prevented only by the combination of EP2 and EP4 receptor antagonists, underscoring the importance of targeting both receptors in the effect of PGE2. Conversely, we also show that the inhibition of reactive oxygen species production by human eosinophils only involves the EP4 receptor, despite the fact that they also express the EP2 receptor.

Neutrophils are massively recruited to combat infections in the lungs of people with cystic fibrosis (PwCF), yet they fail to eradicate pathogens and thus contribute to chronic lung inflammation and loss of lung function. In addition to the circulating normal-dense neutrophils (NDNs), high numbers of low-density neutrophil subtypes (LDNs) have been reported in chronic inflammatory diseases such as lupus [1]. LDNs have immunophenotype and function distinct from those of NDNs and are associated with disease severity, activity, and certain comorbidities such as vasculitis in lupus [2]. How LDNs contribute to the immune response in PwCF is unknown and is the objective of our project. Blood samples were collected from clinically stable adults PwCF with at least one copy of the F508del mutation to isolate NDNs and LDNs by negative selection. We used flow cytometry to determine the proportion, immunophenotype, and antimicrobial functions of NDNs and LDNs and searched for associations with clinical parameters of lung function decline. Sex and age frequency-matched healthy donors (HD) were used as controls. LDNs are composed of mature and immature cells as per the cell-surface expression of CD10 and CD16. The proportion of circulating LDNs in PwCF, specifically the proportion of mature LDNs (n=41; not on modulators) was significantly higher than in HD (n=34). Mature LDNs were associated with more frequent pulmonary exacerbations and with greater lung function decline over time [3]. Circulating mature and immature LDNs remain present in PwCF with highly-effective modulator treatment and display distinct antimicrobial functions including a reduced phagocytic capacity, reactive oxygen species production and degranulation in immature LDNs compared to mature LDNs and NDNs. Characterizing neutrophil heterogeneity and contribution to the immune response in PwCF could help to stratify patients into clinically more homogeneous subgroups, improving disease management and understanding of the CF pathogenesis. [1] Rahman S, et al. Ann Rheum Dis 2019, [2] Denny MF, et al. J Immunol 2010, [3] Murru A, et al. J Cyst Fibros 2023.

Introduction and Rationale: Acute human exposures to common indoor concentrations of 1,000-2,500 ppm of CO2 cause significant cognitive deficits in measures of executive function. Rodent inhalation studies at these concentrations leads to PMN activation, oxidative burst, and systemic vascular inflammation, and vascular leak in brain, potentially providing a mechanism for the observed declines in executive function. We sought to test if mechanistic findings in rodents are observed in human subjects. Methods: Twelve college students completed standardized tests of executive function (Strategic Management System, SMS) for 2 h while breathing in a chamber , one week apart, either filtered ambient air (CO2=600 ppm) or air with CO2 at 2,500 ppm, in a blinded, randomized within subject cross-over design. Four hours after exposure, isolated PMNs were examined for markers of activation. Results: CO2 exposure impaired SMS performance, and increased baseline O2 consumption from 13 ± 2.9 to 21 ± 3.3 pmole/min. For oxidative burst, neutrophils were treated with PMA, and basal O2 consumption was measured. There was a significant increase in O2 consumption; however, relative to air, CO2 exposure delayed the time to peak from 56 ± 3.5 to 98 ± 9.2 minutes. Additionally, CO2 reduced the total oxidative burst as determined by the area under the curve (11 ± 0.9 vs. 8 ± 1.3 nmoles). All changes were statistically significant. Discussion: CO2 exposure increased non-mitochondrial O2 consumption without external stimulation. This was observed in all subjects and is indicative of increased NADPH oxidase function. Most importantly, CO2 abrogated the PMA-mediated oxidative burst presumably as the cells were already stimulated. These data support an inflammatory mechanism for the loss of executive function following CO2.

Exposure to air pollutants like ozone contributes to the pathogenesis and severity of inflammatory diseases including asthma, emphysema, and ARDS. We previously demonstrated that lung injury following of ozone is due, not only to its direct effects on the lung, but indirectly to the actions of inflammatory mediators released by activated macrophages; moreover, macrophage activation is controlled, at least in part, by epigenetic regulators particularly, noncoding microRNAs (miRNA)s. Extracellular vesicles (EVs) are cell-derived particles that facilitate cell-cell communication by delivering cargo, including miRNAs, from donor to recipient cells. We found that miRNA cargo released from lung cell EVs after acute ozone exposure (0.8 ppm, 3 h) regulates macrophage-mediated inflammatory responses. Herein, we analyzed the EV-cargo miRNA profiles isolated from bronchoalveolar lavage fluid (BAL) after chronic ozone exposure. Mice (C57Bl6/J, 11-12 wk) were exposed to air or ozone (1.5 ppm, 2 h), 2x/wk for 6 wk. BAL was collected 24 h after the last exposure, EVs isolated and analyzed by flow cytometry to assess their origin. Ozone exposure resulted in histo-pathologic changes in the lung consistent with inflammation and chronic disease. In both control and ozone treated mice, BAL EVs originated mainly from CD45+ macrophages, with a smaller % from CD326+ epithelial cells and CD31+ endothelial cells. Ozone had no effect on the origin of the EVs, or absolute numbers of EVs that originated from these cells. Next, we assessed the effects of ozone on EV-cargo miRNA and their pre-miRNAs precursors by RNA sequencing. Among the 1,935 miRNAs detected in BAL EVs after ozone exposure, 8 miRNAs were found to be significantly upregulated (more than 2.85-fold change) relative to EVs from mice exposed to air control. We also identified 3 pre-miRNAs which were significantly altered (1.5 - 2 fold) after ozone exposure. All of these miRNAs and pre-miRNAs are newly identified, and their function and regulatory activity are unknown. These findings are important as they suggest novel pathways mediating macrophage activation, which can be targeted to reduce lung injury and chronic disease.