Christine H. Wendt

Background: The systemic immune responses associated with persistent respiratory symptoms (PRS) after exposure to airborne environmental pollutants remain poorly understood. Objective: To identify immune disturbances associated with PRS, defined as persistent wheeze, cough, or breathlessness, we examined systemic immune responses and airway function in a cross-sectional cohort with detailed histories of airborne pollutant exposure. Methods: Never-smoking post-deployment Veterans with PRS (n=16) or without PRS (n=24) underwent chest computed tomography, pulmonary function testing, and oscillometry to assess structural and functional airway abnormalities. Peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3/CD28 antibodies, lipopolysaccharide, or β-glucan, and cytokine production was measured. Correlation analyses evaluated associations between cytokine responses and physiological measures of airway function. Results: Oscillometry, but not conventional pulmonary function testing or chest computed tomography, detected small-airway abnormalities in participants with PRS, including significantly greater frequency dependence of resistance and higher resonant frequency. Baseline PBMC cytokine concentrations were similar between groups. After stimulation, however, PBMCs from participants with PRS showed increased IL-17A production consistent with a type 17 (T17) response; innate stimulation also increased the type 2 (T2) cytokines IL-33 and IL-4. T2/T17 cytokine responses correlated positively with oscillometric measures of small-airway dysfunction. Conclusion: Individuals with PRS exhibited a stimulus-dependent systemic T2/T17 immune signature that was associated with early small-airway dysfunction. Clinical Implication: Stimulus-dependent systemic immune profiling, combined with oscillometry, may help identify early respiratory abnormalities in pollutant-exposed individuals whose conventional pulmonary tests remain normal. KEY MESSAGES: Oscillometry detected early small-airway dysfunction in pollutant-exposed individuals with persistent respiratory symptoms, whereas conventional pulmonary function tests and chest CT did not distinguish the groups. Immune stimulation revealed a mixed T2/T17 systemic signature in participants with persistent respiratory symptoms, including increased IL-17A and stimulus-dependent increases in IL-4 and IL-33. T2/T17 cytokine responses correlated with oscillometric abnormalities, linking systemic immune dysregulation to small-airway dysfunction and suggesting a potential approach for identifying early pollutant-associated respiratory disease. CAPSULE SUMMARY: A stimulus-dependent systemic T2/T17 immune signature correlated with oscillometric evidence of small-airway dysfunction, identifying a potential early respiratory phenotype in pollutant-exposed individuals with persistent respiratory symptoms.

Military personnel deployed to Iraq and Afghanistan were exposed to emissions from open-air burn pits, where plastics, metals, and medical waste were incinerated. These exposures have been linked to deployment-related respiratory diseases (DRRD) and may also impact neurological health via the lung–brain axis. To investigate molecular mechanisms, adult male rats were exposed to filtered air, naphthalene (a representative volatile organic compound), or a combination of naphthalene and carbon black (surrogate for particulate matter; CBN) via whole-body inhalation (six hours/day, three consecutive days). Lung, brain, and plasma samples were collected 24 h after the final exposure. Pro-inflammatory biomarkers were assessed using multiplex electrochemiluminescence and western blot. Differentially expressed genes (DEGs) were identified by RNA sequencing, and elastic net modeling was used to define exposure-predictive gene signatures. CBN exposure altered inflammatory biomarkers across tissues, with activation of nuclear factor kappa B (NF-κB) signaling. In the lung, gene set enrichment revealed activated pathways related to proliferation and inflammation, while epithelial–mesenchymal transition (EMT) and oxidative phosphorylation were suppressed. In the brain, EMT, inflammation, and senescence pathways were activated, while ribosomal function and oxidative metabolism were downregulated. Elastic net modeling identified a lung gene signature predictive of CBN exposure, including Kcnq3, Tgfbr1, and Tm4sf19. These findings demonstrate that inhalation of a surrogate burn pit mixture induces inflammatory and metabolic gene expression changes in both lung and brain tissues, supporting the utility of this animal model for understanding systemic effects of airborne military toxicants and for identifying potential biomarkers relevant to DRRD and Veteran health.

Military personnel deployed to Iraq and Afghanistan were exposed to emissions from open-air burn pits, where plastics, metals, and medical waste were incinerated. These exposures have been linked to deployment-related respiratory diseases (DRRD) and may also impact neurological health via the lung–brain axis. To investigate molecular mechanisms, adult male rats were exposed to filtered air, naphthalene (a representative volatile organic compound), or a combination of carbon black (surrogate for particulate matter) and naphthalene (CBN) via whole-body inhalation (six hours/day, three consecutive days). Lung, brain, and plasma samples were collected 24 hours after final exposure. Proinflammatory biomarkers were assessed using multiplex electrochemiluminescence and western blotting. Differentially expressed genes (DEGs) were identified by RNA sequencing, and elastic net modeling was used to define exposure-predictive gene signatures. CBN exposure altered inflammatory biomarkers across tissues, with activation of nuclear factor kappa B (NF-κB) signaling. In the lung, gene set enrichment revealed activated pathways related to proliferation and inflammation, while epithelial-mesenchymal transition (EMT) and oxidative phosphorylation were suppressed. In the brain, EMT, inflammation, and senescence pathways were activated, while ribosomal function and oxidative metabolism were downregulated. Elastic net modeling identified a lung gene signature predictive of CBN exposure, including Kcnq3, Tgfbr1, and Tm4sf19. These findings demonstrate that inhalation of a surrogate burn pit mixture induces inflammatory and metabolic gene expression changes in both lung and brain tissues, supporting the utility of this animal model for understanding systemic effects of airborne military toxicants and for identifying potential biomarkers relevant to DRRD and Veteran health.

Rationale: Obstructive lung disease is increasingly common among persons living with HIV (PLWH). There are currently no validated biomarkers that identify individuals at risk of developing obstructive lung disease (OLD), and specific mechanisms contributing to HIV-associated OLD remain elusive, independent of smoking. We sought to identify biomarkers and biological pathways associated with OLD using a broad proteomic approach.Methods: We performed tandem mass tagging and mass spectrometry (MS) analysis on bronchoalveolar lavage fluid samples from persons living with HIV with OLD (n=26) and without OLD (n=26). We combined untargeted MS with a targeted SomaScan aptamer-based approach. We used Pearson correlation tests to identify associations between each protein and lung function (forced expiratory volume in 1 s (FEV₁) % pred). We adjusted for multiple comparisons using a false discovery rate adjustment. Significant proteins were entered into a pathway over-representation analysis. Protein-driven endotypes were constructed using K-means clustering.Measurements and main results: We identified over 3800 proteins by MS and identified 254 proteins that correlated with FEV₁% pred when we combined the MS and SomaScan proteomes when adjusting for smoking status. Pathway analysis revealed cell adhesion molecules as significant.Conclusions: Protein expression differs in the lung of PLWH and decreased lung function (FEV₁% pred). Pathway analysis reveals cell adhesion molecules having potentially important roles in this process.

Background: Obstructive lung disease (OLD) is increasingly prevalent among persons living with HIV (PLWH). However, the role of proteases in HIV-associated OLD remains unclear. Methods: We combined proteomics and peptidomics to comprehensively characterize protease activities. We combined mass spectrometry (MS) analysis on bronchoalveolar lavage fluid (BALF) peptides and proteins from PLWH with OLD (n = 25) and without OLD (n = 26) with a targeted Somascan aptamer-based proteomic approach to quantify individual proteases and assess their correlation with lung function. Endogenous peptidomics mapped peptides to native proteins to identify substrates of protease activity. Using the MEROPS database, we identified candidate proteases linked to peptide generation based on binding site affinities which were assessed via z-scores. We used t-tests to compare average forced expiratory volume in 1 s per predicted value (FEV1pp) between samples with and without detection of each cleaved protein and adjusted for multiple comparisons by controlling the false discovery rate (FDR). Findings: We identified 101 proteases, of which 95 had functional network associations and 22 correlated with FEV1pp. These included cathepsins, metalloproteinases (MMP), caspases and neutrophil elastase. We discovered 31 proteins subject to proteolytic cleavage that associate with FEV1pp, with the top pathways involved in small ubiquitin-like modifier mediated modification (SUMOylation). Proteases linked to protein cleavage included neutrophil elastase, granzyme, and cathepsin D. Interpretations: In HIV-associated OLD, a significant number of proteases are up-regulated, many of which are involved in protein degradation. These proteases degrade proteins involved in cell cycle and protein stability, thereby disrupting critical biological functions.

Bronchoscopy for research purposes is a valuable tool to understand lung-specific biology in human participants. Despite published reports and active research protocols using this procedure in critically ill patients, no recent document encapsulates the important safety considerations and downstream applications of this procedure in this setting. The objectives were to identify safe practices for patient selection and protection of hospital staff, provide recommendations for sample procurement to standardize studies, and give guidance on sample preparation for novel research technologies. Seventeen international experts in the management of critically ill patients, bronchoscopy in clinical and research settings, and experience in patient-oriented clinical or translational research convened for a workshop. Review of relevant literature, expert presentations, and discussion generated the findings presented herein. The committee concludes that research bronchoscopy with bronchoalveolar lavage in critically ill patients on mechanical ventilation is valuable and safe in appropriately selected patients. This report includes recommendations on standardization of this procedure and prioritizes the reporting of sample management to produce more reproducible results between laboratories. This document serves as a resource to the community of researchers who endeavor to include bronchoscopy as part of their research protocols and highlights key considerations for the inclusion and safety of research participants.

Background: HIV is a risk factor for obstructive lung disease (OLD), independent of smoking. We used mass spectrometry (MS) approaches to identify metabolomic biomarkers that inform mechanistic pathogenesis of OLD in persons with HIV (PWH). Methods: We obtained bronchoalveolar lavage fluid (BALF) samples from 52 PWH, in case:control (+OLD/−OLD) pairs matched on age, smoking status, and antiretroviral treatment. Four hundred nine metabolites from 8 families were measured on BALF and plasma samples using a MS-based Biocrates platform. After filtering metabolites with a high proportion of missing values and values below the level of detection, we performed univariate testing using paired t tests followed by false discovery rate corrections. We used distance-weighted discrimination (DWD) to test for an overall difference in the metabolite profile between cases and controls. Results: After filtering, there were 252 BALF metabolites for analysis from 8 metabolite families. DWD testing found that collectively, BALF metabolites differentiated cases from controls, whereas plasma metabolites did not. In BALF samples, we identified 3 metabolites that correlated with OLD at the false discovery rate of 10%; all were in the phosphatidylcholine family. We identified additional BALF metabolites when analyzing lung function as a continuous variable, and these included acylcarnitines, triglycerides, and a cholesterol ester. Conclusions: Collectively, BALF metabolites differentiate PWH with and without OLD. These included several BALF lipid metabolites. These findings were limited to BALF and were not found in plasma from the same individuals. Phosphatidylcholine, the most common lipid component of surfactant, was the predominant lipid metabolite differentially expressed.

HIV is an independent risk factor for lung disease, including chronic obstructive pulmonary disease (COPD) and emphysema. Angiotensin receptor blockers may be beneficial in COPD and emphysema through pathways that have been implicated in HIV-related lung disease. We performed a randomized comparison of the effects of losartan versus placebo on the plasma concentrations of the pneumoproteins, surfactant protein D (SPD) and club cell secretory protein (CCSP), in people living with HIV (PLWH). A total of 108 PLWH were included (52 assigned to losartan and 56 assigned to placebo). We found no difference in the change from baseline in log ₂ concentrations of CCSP or SPD over 1 year of follow-up. For SPD, we found a strong interaction by CD4+ counts, where those with CD4+ counts >350 cells/mm ³ treated with losartan had more reduction (improvement) in SPD concentration than those treated with placebo ( p value for interaction <.001). In conclusion, we did not find a beneficial effect of losartan on pneumoprotein concentrations in PLWH, but PLWH with higher CD4+ counts may have improvement in SPD when treated with losartan.