Robert M. Tighe

Butylated hydroxytoluene (BHT) is a synthetic phenolic antioxidant utilized as a preservative in many products from foods to cosmetics. Population-based studies have detected BHT in the vast majority (>90%) of human specimens (serum, urine, fingernails) as well as home dust samples. Thus, BHT constitutes a ubiquitous environmental contaminant without obvious adverse health effects to humans. In contrast, exposure of mice to a single intraperitoneal dose of BHT triggers distal epithelial damage including loss of gas-exchanging alveolar type 1 epithelial cells, thus providing an invaluable tool to study alveolar repair and transient fibrosis. Presently, the molecular basis of BHT lung toxicity remains unknown. To address this, mouse lung single cell transcriptomic data were used to identify cytochrome P450 2B10 (CYP2B10) in AT1 cells as a BHT-activating enzyme. In cell culture systems, expression of CYP2B10 leads to marked BHT sensitization consistent with bioactivation of BHT into a toxic quinone methide. Targeted and proteome-wide experiments identify BHT-induced protein alkylation, DNA damage response, stress kinase activation and intrinsic apoptosis in a CYP2B10-dependent manner. Structure-activity relationship studies reveal the necessity of para-methyl and ortho-t-butyl groups necessary for BHT bioactivation. Our findings elucidate the molecular basis by which BHT is converted from an innocuous antioxidant into a highly toxic quinone methide and identify the first mammalian enzyme known to catalyze BHT bioactivation. Further, CYP2B10-catalyzed BHT bioactivation may provide a strategy for future targeted cell ablation technologies or environmental remediation of BHT.

Ozone (O ₃ )-driven pulmonary inflammation is partly regulated by damage associated molecular patterns (DAMPs) binding to scavenging receptors (SRs). However, how SRs and DAMPs regulate O ₃ -induced pulmonary inflammation remains incompletely understood. CD163 is a SR responsible for clearing cell free hemoglobin (CFH), a DAMP which accumulates during acute pulmonary injury and is associated with worsening respiratory outcomes. We hypothesized that increased CD163 is necessary for reducing CFH levels and resolving O ₃ -induced pulmonary injury. To test this hypothesis, we defined CD163 and CFH responses to O ₃ exposure in C57BL/6N (WT) and CD163 deficient ( Cd163 ^-/- ) mice, as well as in human bronchoalveolar lavage fluid (BALF). In WT mice, lung Cd163 expression was significantly increased by O ₃ during peak inflammation and declined 24 hours post exposure. Human exposure studies revealed a diversity of Cd163 expression and a reduction of CFH following O ₃ exposure, suggesting regulation of this pathway in humans. When compared to WT mice, Cd163 ^-/- mice had augmented O ₃ -induced pulmonary injury, inflammation, and oxidative stress. Further, the antioxidant EUK-134 did not reduce O ₃ -induced pulmonary oxidative stress in Cd163 ^-/- mice, suggesting a role for CD163 in the pulmonary response to oxidative insults. Furthermore, compared to WT controls, Cd163 ^-/- mice receiving an oropharyngeal aspiration of CFH had a significant increase in airspace inflammation. Combined, these findings suggest that CD163 mediated clearance of CFH is involved in resolving O ₃ -induced pulmonary injury, inflammation, and oxidative stress. New & Noteworthy: Ozone (O ₃ ) is known to induce damage associated molecular patterns (DAMPs) which drive lung inflammation. The scavenging receptor, CD163, binds and clears the DAMP cell free hemoglobin (CFH), which accumulates during sterile lung injury. Our findings indicate that O ₃ exposure alters CD163 expression in the lung and that mice lacking Cd163 expression have more lung inflammation. Our data indicate that CD163 serves a protective role in response to acute O ₃ exposure perhaps through CFH clearance.

Airway pollutants, like the reactive oxygen species ozone, cause significant lung injury, which can lead to the development or exacerbation of lung diseases like asthma and chronic obstructive pulmonary disease (COPD), and drives worldwide morbidity and mortality. Altered epithelial function is a hallmark and trigger of ozone-induced lung injury, but its precise mechanisms are incompletely known. The extracellular matrix, and specifically its major component, hyaluronan (HA), plays a crucial role in cellular injury responses. We hypothesized that HA signaling mediates epithelial responses to ozone-induced injury. We exposed human and murine differentiated primary epithelia to ozone in vitro and evaluated epithelial integrity and transcriptomic responses. We used genetically deficient cells for cognate HA receptors cluster of differentiation 44 (CD44) and receptor for HA-mediated motility (RHAMM), and innate immune receptors toll-like receptor 4 (TLR4) and TLR5 to study signaling pathways, and evaluated high molecular weight HA (HMWHA) as a potential treatment for ozone-induced epithelial injury. In vitro ozone exposure caused significant reduction in epithelial integrity and very similar inflammatory changes in human and murine cells. CD44 deficiency led to decreased inflammation, while RHAMM deficiency exacerbated cell injury. HMWHA protected against ozone-induced epithelial injury, mediated by TLR4 and TLR5 but not CD44 or RHAMM. Our results identify novel contributions of HA signaling to ozone-induced epithelial injury and suggest that HMWHA protects epithelia via innate immune activation of TLR4 and TLR5.

Pneumonia is the leading cause of death from infectious disease worldwide. The diagnosis and treatment of patients with pneumonia lag behind other major conditions, relying on syndromic definitions that lack molecular resolution and ignore underlying endotypes. We sought to test the hypothesis that dynamic pathogen-specific host responses in the alveolar space represent distinct pneumonia endotypes linked to different clinical features and outcomes. We prospectively enrolled a cohort of 690 patients (including immunocompromised patients) with known or suspected pneumonia receiving mechanical ventilation in whom the etiology of pneumonia was determined by gold-standard analysis of distal lung fluid obtained by bronchoalveolar lavage (BAL) combined with clinical adjudication. From these patients, we analyzed 792 BAL fluid samples, including 310 serial samples, using flow cytometry (482 patients) and single-cell RNA-sequencing (170 patients; 263 samples, complemented by 9 healthy controls and 25 post-COVID-19 patients, yielding ~2.4 million single cells across 28 cell types), and extracted daily clinical data from the electronic health record (>15,000 patient-days). We used machine learning models to identify pathogen-specific host responses in the transcriptome of alveolar immune cells that were associated with changes in alveolar cell abundance and clinical features. Our results suggest that therapeutic strategies for pneumonia should be individualized to specific host-pathogen interactions.

Acute Respiratory Distress Syndrome (ARDS), a heterogeneous syndrome of hypoxic respiratory failure secondary to dysregulated pulmonary inflammation, is caused by diverse insults. This heterogeneity presents challenges for mechanistic and therapeutic research, as evidenced by conflicting results from trials of n−3 polyunsaturated fatty acid (PUFA) supplementation for ARDS. PUFAs and downstream oxylipins are important to pulmonary inflammation but are not well defined in ARDS. We hypothesized that differences in fatty acid metabolism, as measured by levels of n−3 and n−6 PUFAs and oxylipins, are associated with differences in ARDS outcomes, inflammation, and causes. To test this, PUFAs/oxylipins were measured by LC MS/MS in plasma samples from 90 patients with ARDS. Pro‐inflammatory cytokines and chemokines IL‐6 and IL‐8 were measured by ELISA. Multivariable linear regressions modeled the relationship between PUFAs/oxylipins, inflammation, and ARDS mortality, severity, and cause. Multiple n−3 and n−6 PUFA‐derived oxylipins were decreased in severe ARDS. We did not detect differences in PUFAs/oxylipins by mortality. PUFAs/oxylipins varied by cause of ARDS, especially between patients with sepsis and those with trauma. Furthermore, specific oxylipins were associated with IL‐6 and IL‐8. As we observed that oxylipins varied by disease severity and underlying cause, these metabolites may function as biomarkers and suggest dysregulated mechanisms of lung repair. Furthermore, while oxylipins are derived from PUFAs, differences in PUFAs did not directly correlate with changes in oxylipins, suggesting altered lipid metabolism as a mechanism. Further consideration of differences in lipid metabolism in ARDS could identify subgroups with differential responses to n−3 PUFA supplementation or other therapies.

Nanomaterials interact with lipids and proteins forming a corona that determines the cellular response.

Ozone (O3) is an air pollutant that induces pulmonary inflammation and injury, leading to increased susceptibility and exacerbation of chronic lung diseases. Furthermore, ambient O3 concentrations are expected to rise with increasing global temperatures. Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid found primarily in oily fish that reduces inflammation and enhances the resolution of inflammation. This is attributed partially to DHA-derived oxylipins termed specialized proresolving mediators that have antiinflammatory and/or proresolving properties. However, whether dietary DHA protects the lungs from O3-induced inflammation and injury is unclear. We hypothesized that dietary DHA supplementation increases pulmonary specialized proresolving mediators and thereby decreases O3-induced pulmonary inflammation. To test this, C57BL/6J mice were fed a control diet or a DHA-enriched diet (2% kcal from DHA) for 6 weeks, exposed to filtered air or 1 ppm O3 for 3 hours (comparable with an O3 action day for humans), and necropsied 24 or 48 hours after exposure. DHA supplementation reduced airspace neutrophilia, decreased cytokine production, and promoted transcriptomic signatures for leukocyte chemotaxis and fatty acid oxidation. Furthermore, dietary DHA increased pulmonary DHA and its oxylipins while decreasing proinflammatory omega-6 polyunsaturated fatty acids and their oxylipins. Oropharyngeal aspiration of DHA oxylipins monohydroxylated 14-hydroxy-DHA and maresin 1 decreased O3-induced airspace neutrophilia and reduced bone marrow–derived macrophage production of neutrophil chemokines Cxcl1 and Cxcl2. These findings reveal that dietary DHA protects the lungs from O3 exposure by driving monohydroxylated 14-hydroxy-DHA and maresin 1 production, which reduces neutrophil-recruiting chemokine production by macrophages. This pathway highlights a potential therapeutic dietary approach for mitigating air pollution–induced pulmonary inflammation.

Deployed military personnel often develop respiratory symptoms and disorders due to exposure to particulate matter such as dust, blast materials, and burn pit emissions. A range of deployment-related respiratory diseases have been reported, including toxic lung injury, eosinophilic pneumonia, asthma, chronic obstructive pulmonary disease, bronchiolitis, and interstitial lung disease. The Sergeant First Class Heath Robinson Honoring Our Promise to Address Comprehensive Toxics Act of 222, which was enacted in 2022, expanded coverage of medical care for veterans and improved awareness of deployment-related respiratory diseases. This law added 23 diagnoses presumed to be connected to deployment but has failed to address issues related to the diagnosis of deployment-related respiratory disorders. Diagnosing some of the respiratory disorders associated with deployment can be challenging, as symptoms are often nonspecific. Veterans who present with respiratory symptoms should undergo a comprehensive assessment, including a detailed medical and exposure history, pulmonary function tests, imaging, and serologic screening for autoimmune disorders. A decision on whether a surgical lung biopsy should be performed should be made on a case-by-case basis on the basis of multidisciplinary review and an informed discussion with the patient. The clinical care team should discuss pharmacological and nonpharmacological treatment options with the patient and direct them to reliable sources of information. Long-term follow-up is essential to monitor for worsening of pulmonary function or symptoms. Further research is needed to characterize associations between deployment-related exposures and respiratory health outcomes and to inform better means of assessment and treatment of military veterans.

Rationale: Combined pulmonary fibrosis and emphysema (CPFE) is a unique phenotype with important prognosis and management implications in patients with idiopathic pulmonary fibrosis (CPFE-IPF) and other forms of fibrotic interstitial lung disease (CPFE-fILD). However, the epidemiology of CPFE is not well characterized, creating a barrier to clinical research needed to advance our understanding and management. Objectives: To investigate the incidence, prevalence, and long-term outcomes of CPFE among a regional cohort of veterans. Methods: We retrospectively reviewed records for veterans in the Veterans Affairs Mid-Atlantic Health Care Network (includes North Carolina and Virginia) with International Classification of Disease, Ninth Revision, codes for pulmonary fibrosis between January 1, 2008, and December 31, 2015. We stratified pulmonary fibrosis into IPF and fILD using diagnostic codes and chart review. We reviewed computed tomography reports and classified cases as having CPFE according to documented emphysema; a thoracic radiologist overread a subset of scans for validation. We calculated annual incidence and prevalence of CPFE and compared characteristics between veterans with CPFE and veterans with fibrosis without emphysema using chi-square tests, Mann-Whitney U tests, and paired t tests. We used Kaplan-Meier and Cox models to determine overall survival from diagnosis. Results: We identified 2,414 veterans with fILD. Among 1,880 veterans with IPF, 734 (39.0%) had CPFE-IPF; among 534 veterans with fILD, 194 (36.3%) had CPFE-fILD. Agreement between computed tomography reports and thoracic radiologist review was high (kappa = 0.78). Annual CPFE prevalence ranged from 71 to 100 per 100,000 veterans, and incidence ranged from 16 to 39 per 100,000 veterans. CPFE was associated with male sex, lower body mass index, greater tobacco history, higher forced vital capacity, reduced forced expiratory volume in 1 second/forced vital capacity ratio, reduced diffusing capacity of the lung for carbon monoxide, and increased oxygen use. CPFE was associated with increased mortality in unadjusted models. However, after adjustment for age, sex, and body mass index, CPFE was not associated with survival for CPFE-IPF versus IPF without emphysema (hazard ratio, 1.13; 95% confidence interval, 0.96–1.33) as well as CPFE-fILD versus fILD without emphysema (hazard ratio, 1.16, 95% confidence interval, 0.82–1.63). Conclusions: CPFE has a high incidence and prevalence among veterans with IPF and fILD and has a distinct phenotype with diagnostic and therapeutic implications. Further studies investigating diagnosis, treatment considerations, and long-term impacts in CPFE are merited.

Interstitial lung disease (ILD) is present in over 60% of patients with systemic sclerosis (SSc) and is the leading cause of SSc-related deaths. Profibrotic monocyte-derived alveolar macrophages (MoAM) play a causal role in the pathogenesis of pulmonary fibrosis in animal models where their persistence in the niche requires signaling through Colony Stimulating Factor 1 Receptor (CSF1R). We hypothesized that the presence and proportion of MoAM in bronchoalveolar lavage (BAL) fluid from patients with SSc-ILD may be a biomarker of ILD severity. To test this hypothesis, we analyzed BAL fluid from 9 prospectively enrolled patients with SSc-ILD and 13 healthy controls using flow cytometry and single-cell RNA sequencing. Patients with SSc-ILD had more MoAM and interstitial macrophages in BAL fluid than healthy controls, and their abundance was associated with lung fibrosis severity. We identified changes in the MoAM transcriptome as a function of treatment with mycophenolate, an effective therapy for SSc-ILD. In SSc-ILD lung explants, spatial transcriptomics identified an expanded population of interstitial macrophages spilling into the alveolar space. Our findings suggest that the proportion of profibrotic MoAM and interstitial macrophages in BAL fluid may serve as a biomarker of SSc-ILD and credential them as possible targets for therapy.