Epigenetic dysregulation, particularly DNA methylation variations, is implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Ten-eleven translocation (TET) proteins (TET1, TET2, and TET3) regulate DNA methylation and gene transcription. Impaired TET1 expression was previously associated with airway inflammation and asthma. Here we investigated TET gene associations with COPD severity. We found that reduced TET1 expression in peripheral blood mononuclear cells was associated with higher sputum and blood neutrophil counts, decreased lung function and increased disease severity in patients. These findings support a potential protective role and warrant further mechanistic investigations into the actions of TET1 in COPD.

Rationale While human surfactant protein D (hSP-D) has been of immunological interest for its antiviral capabilities over at least the past two decades, there do not currently exist experimentally derived structural images that include all of hSP-D's amino acids of importance, especially the N-Acetylglucosamine (NAG) binding site at Asparagine amino acid site number 90 (ASN90). Computational folding methods have improved capability to take given amino acid sequences and derive potential 3D conformations. In this study, we evaluated the computationally bound structures of three different, computationally folded SP-D structures with SARS-CoV-2 Spike Wild Type (WT) (PDB: 6VSB) and dimerized ACE2 (PDB: 6M18) to the computational bound structure of the X-Ray fragment of hSP-D (PDB: 3DBZ) that exists in RCSB with SARS-CoV-2 Spike WT and X-Ray fragment of porcine SP-D (PDB: 6BBE), which has numerous NAG sites, with SARS-CoV-2 Spike WT. Methods We used the computational folding tools I-TASSER, AlphaFold, and ESMFold with the amino acid sequence for hSP-D to find top 3D conformers. Then, we used a variation of the Quantum Approximate Optimization Algorithm (QAOA) implemented with a maximum-sized cut (MaxCut) of a graph, called QAOA-MaxCut, that utilizes only a single, initial Trotter-Suzuki decomposition step (SETS), dubbed “SETS-QAOA-MaxCut”, to prune the protein structures. Penultimately, we used ZDOCK to complete protein binding. Lastly, we used SAMSON's Biological Verification tool to identify top clashes and contacts for the top complexes for each of our structures, and to visualize the protein complexes. Results Figure 1 shows that the additional N-Acetylglucosamine sites on porcine SP-D appeared to have allowed porcine SP-D to produce a higher ZDOCK score than hSP-D, and all other bound SP-D structures outperform the X-Ray hSP-D structure without the ASN90 site for SARS-CoV-2 Spike WT binding. However, SP-D produced after using I-TASSER appears to bind best to ACE2. Conclusion No SP-D conformation bound to ACE2 or to SARS-CoV-2 Spike WT with a higher ZDOCK Score than SARS-CoV-2 Spike WT bound to ACE2. Clearly, computational folding methods can lead to dramatically different protein structure models than their X-ray or other experimentally-derived counterparts. This would make sense, considering that all three methods used here (AlphaFold, I-TASSER, ESMFold) use data from other, similar structures to be able to derive high confidence structures, but do not necessarily detail molecular conformations or even stoichiometry for effective protein structure determinations.

ABSTRACT Background The relationship between asthma and coccidioidomycosis has not been fully described. We have hypothesised that Coccidioides could trigger inflammatory airway responses, similar to other fungi. Objectives: To estimate the frequency of new‐onset asthma‐related symptoms after coccidioidomycosis and identify potentially associated factors. Patients/Methods We used a large health insurance claims database to identify patients with coccidiomycosis with and without an asthma diagnosis code or a short‐acting β 2 agonist prescription in the year after diagnosis. Results Thirteen per cent of 1657 patients with an asthma diagnosis code or a short‐acting β 2 agonist prescription (median 2.5 months later). Conclusions Increased healthcare provider awareness of asthma as a potential coccidioidomycosis complication could benefit patients, especially female patients and patients with severe pulmonary infection.

Background We previously demonstrated that exposure to wildfire smoke is associated with increased IL-13 production from CD56bright NK cells. We also found that ozone (O3), a major component of wildfire smoke, diminished IFNy expression by lung-resident Natural Killer (NK) cells, but increased IL-33 expression in the lung. Human NK cells are classified into CD56bright(major producers of inflammatory cytokines) and CD56dim(a more cytotoxic subset) in the periphery. Whether environmental exposure effects are mediated via IL-33 on NK cell is not well understood. We hypothesize that IL-33 polarizes NK cells towards the NK2 phenotype. Methods Primary human NK cells were purchased from STEMCELL technologies and cultured in ImmunoCult™ NK Cell Base Medium. Media was supplemented with 200U/ml rhIL-2 alone and with cytokine cocktails shown to polarize to IFNγ-producing NK1 cells (10ng/mL rhIL-12 and 10 μg/ml anti-IL-4) or IL-13-producing NK2 cells (25ng/mL rhIL-4 and 10 μg/ml anti-IL-12) for 10 days ±10ng/ml of rhIL-33. Cells received fresh media containing respective cytokine cocktails at days 3 and 7, n=3 per condition. To activate NK cells for cytokine production, NK cells were incubated with 50ng/ml phorbol 12-myristate 13-acetate (PMA) and 0.5 µM Ionomycin for 5 hours. Surface markers for NK cell activating and inhibitory receptors, and intracellular cytokines IFNγ and IL-13 were measured by flow cytometry (FACS). A paired t-test and two-way ANOVA followed by Tukey correction for multiple post hoc comparisons were used to compare the effects of IL-33 and NK polarizing cytokines on NK cell markers and intracellular cytokine production. Results After 10 days, NK1 polarized cells cultured in the presence of IL-33 showed significantly increased IFNγ and NKp46 expression (p<0.0001 and p=0.0247, respectively), while there was decreased NKG2D (p<0.0001), CD69 (p<0.0001) and IL-13 (p<0.0001) expression compared to NK1 cells alone. NK2 polarized cells expressed significantly less IFNγ, similar to baseline expression by rhIL-2 controls, and was unchanged with IL-33. Notably, while the majority of cells were CD56dim NK cells on Day 0, CD56bright NK cells predominated after 10 days in culture, particularly in the NK2 polarized subset (Figure 1a). These cells show significant responsiveness to IL-33 in CD69, IL-13 and IFNγ expression which was markedly diminished (Figure 1b). Conclusions IL-33 significantly impacts CD56bright NK cell cytokine production and alters surface activation and inhibitory marker expression. We speculate that IL-33 is necessary and sufficient to mediate the effects of inhaled environmental exposures on NK cells.

Rationale: Emerging evidence supports that epigenetic dysregulation, specifically variations in DNA methylation, is implicated in chronic obstructive pulmonary disease (COPD). Genes related to oxidative stress, mucus production, and epithelial barrier integrity are under strong epigenetic control. Ten-eleven translocation (TET) proteins, known to catalyze the hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine, may play a significant role but their specific functions in COPD remain underexplored. We aimed to examine the relationship between TET gene expression and COPD clinical phenotypes, with a focus on inflammatory parameters. Methods: RNA-seq data from peripheral blood samples of former smoker COPD patients (n=17; 63-76 yrs old; 40% female) and healthy controls (n=15; 37-43 yrs old, 50% female) was evaluated. Correlations between TET1, TET2, and TET3 expression and immune cell counts, lung function, and COPD severity markers were assessed. Logistic regression and causal inference analyses were used to explore potential associations of TET1 with COPD. Results: TET1 expression was significantly downregulated in peripheral blood mononuclear cells of COPD patients. TET1 showed significant negative correlations with neutrophil counts in sputum and blood as well as with serum SP-D levels, a biomarker linked to COPD severity. Logistic regression indicated that higher TET1 expression was associated with reduced odds of COPD (OR=0.5201). In contrast, blood TET2 and TET3 were positively associated with neutrophil levels, and TET2 was inversely correlated with lung function (FEV1/FVC ratio). Causal inference analysis suggested that TET1's protective effect might be mediated through regulation of neutrophil levels. Conclusion: Our findings highlight a potential protective role for TET1 in COPD through the regulation of neutrophil counts, and serum SP-D, key indicators of airway inflammation severity. TET2 and TET3 on the other hand, were positively associated with inflammatory markers and negatively associated with lung function in COPD. This study suggests that members of the TET family may differentially regulate airway inflammation and may serve as novel epigenetic therapeutic targets in COPD.

Rationale: Inhaled environmental pollutants may cause systemic chronic oxidative stress but the underlying mechanisms are poorly understood. At UC Davis, a novel exposure facility adjacent to a major freeway tunnel (Caldecott Tunnel) system was developed to provide a platform for real-world, near-roadway experimental traffic-related air pollution (TRAP) exposure studies. We aimed to investigate oxidative stress-induced changes in splenocytes collected from rat models exposed to TRAP. These animals were housed in the Caldecott Tunnel Exposure Facility from 1 month of age and were studied 32 weeks later. Control animals were exposed to filtered air in the same facility. We hypothesized that chronic inhalational TRAP exposure alters splenocyte function. Methods: Frozen splenocytes form filtered air and TRAP-exposed age-and sex-matched Fischer 344 rats (36 weeks of age) were thawed up and cultured in RPMI with L-glutamine, 10% FBS, and 1% penicillin-streptomycin, with or without stimulation by phorbol myristate acetate (PMA) and ionomycin. Cells were plated in 96-well plates at varying concentrations (0.1, 0.75, 2.0 and 4 million/mL). Freshly isolated splenocytes from filtered air and TRAP-exposed rats were similarly plated, stimulated PMA/ionomycin and exposed to 200 µM tert-butyl hydroperoxide (TBHP) for 3 hours to induce oxidative stress. Protein content was measured by the BCA assay. Viability was assessed using a Trypan blue exclusion assay at 24 and 48h. Alamar blue assay was performed to evaluate metabolic activity. Results: The viability of frozen splenocytes measured at 48h significantly varied between the culture conditions with the lowest (4.3%) seen in the PMA/ionomycin and TBHP stimulated TRAP-exposed cells and the highest (67%) observed in the unstimulated cells from the filtered air-exposed control animals. Cell counts did not differ significantly between control or TRAP-exposed freshly isolated/cultured splenocytes that were also 90%< viable. However, alamar blue assay in these cells revealed that splenocytes from TRAP-exposed rats had significantly lower metabolic activity than those from control rats (p<0.05, n=4) with metabolic activity of the TRAP-exposed, PMA/ionomycin stimulated cells treated with TBHP displaying the lowest levels. Decreased metabolic activity corresponded with increased total protein concentrations in these samples. Conclusions: TRAP-exposed rat splenocytes are more susceptible to TBHP-induced oxidative stress effects and display significantly altered cellular metabolism compared with filtered air-exposed cells. Our results suggest that chronic inhalational exposure to air pollution impairs immune cell metabolism.

Rationale: Approximately half of asthma patients present with severe neutrophilic inflammation, for which biomarkers are limited and therapeutic response is poor. IL-17A involvement has been identified as a major pathogenic player, and can promote glucocorticoid resistance. Group 2 innate lymphoid cells (ILC2s) are elevated in circulation blood and bronchoalveolar lavage (BAL) of asthmatics. These cells are prominent source of T2 cytokines. ILC produce a variety of cytokines, and while canonical ILC2s do not secrete IL-17A, plasticity of ILC cytokine secretion profile has been reported. The mechanisms and significance of ILC2s and their role in pathogenic cytokine production is not well understood. To understand the role of ILC2s, Ozone (O3) exposure was utilized to model ILC2-dependent airway hyperreactivity in response to oxidative stress. Methods: Methacholine (MCh) responsiveness, bronchoalveolar lavage (BAL), and circulating immune cells were characterized in a cohort of rhesus macaques with established airway hyperreactivity, housed at the California National Primate Research Center. Macaques were exposed to 0.3 ppm O3 for 6.5 hours, with lung function measured 8 hours and necropsy 42 hours post-O3 respectively. Baseline measurements were taken one week prior to O3 exposure. Techniques included methacholine challenge, flow cytometry, rtPCR, and Luminex®. Half of the cohort (n=6) received anti-oxidant treatment before O3 exposure, controlled for in post-O3 classification. All procedures were conducted with approval of the University of California, Davis Institutional Animal Cares and Use Committee. Results: In rhesus macaques, O3 exposure induced increased populations of neutrophils, eosinophils, and ILC2s, but not ILC3s, in the BAL, compared to baseline. Expression of Il17a in ILC2s was observed and correlated significantly with canonical ILC2 markers Gata3 and Il13, and Il22. Il17 and Il22 are typically produced by ILC3s, suggesting ILC2 plasticity. ILC2 Il17a expression was used to rank the monkeys into high (inflammatory ILC2s iILC2s), and low natural ILC2s (nILC2s) expressors. ILC2 Il17a was significantly correlated with reduced lung function (EC150) in the iILC2 group when compared with nILC2 macaques (p<0.05). Conclusions: The presence of iILC2s in macaques with reduced lung function following O3 exposure indicates that production of IL-17 by ILC2s specifically may be clinically significant in asthma patients and are a potential therapeutic target in cases of glucocorticoid resistance. Moreover, IL17+ILC2s hold potential as a biomarker in asthma, particularly in low T-2 asthma that currently lacks reliable biomarkers.

Background: Wildfires are increasingly common with wildfire smoke affecting millions globally, yet its impact on immune responses is poorly understood. Natural Killer (NK) cells play a role in mediating air pollutant effects and regulating vaccine immunity. Objective: This real-world study, conducted on participants in the Pfizer BNT162b2 COVID-19 vaccine trial, studied the effects of wildfire smoke exposure on long-term vaccine effects. Methods: We collected blood samples from 52 healthy, non-smoking participants (ages 26-83) before and 1 month after placebo or vaccine injections during heavy wildfire smoke events in Sacramento. The study included 28 vaccinated (Group 1) and 24 placebo-injected (Group 2) individuals, the latter vaccinated several months later, outside wildfire season. Blood samples from both Group 1 and 2 were also investigated 6 months after the second dose of vaccine. We analyzed intracellular cytokines, B and NK cell markers by flow cytometry, and serum immunoglobulin levels against common coronaviruses using multiplex assays. Results: A robust S-RBD-specific IgG response observed 1 month post booster, declined variably 6 months later. Wildfire smoke acutely increased IL-13 expression by CD56bright NK cells. IL-13+CD56bright NK cells at the time of vaccination negatively correlated with anti-S-RBD IgG (r=-0.41, p<0.05) one month later. Total IgG levels on the other hand, positively correlated with the air quality index (AQI) measured during vaccination (r=0.96, p<0.01). Similarly to age (but not sex, BMI or race/ethnicity), the two-week AQI averages during vaccination showed a significant negative correlation with anti-S-RBD IgG levels 6 months later (r=-0.41, p<0.05). Conclusion: Wildfire smoke may lead to inappropriate immunoglobulin production and diminished vaccine immunity. Our novel findings highlight a previously unrecognized pathway involving NK-cell derived IL-13 and non-specific B-cell activation and underscore the significance of environmental exposures in shaping immunity.

Wildfires are increasing in intensity, duration, and frequency with smoke plums affecting the lives of millions over large geographic areas. The immune modulatory effects of wildfire smoke are unclear. We previously showed that a major wildfire smoke component, ozone, inhibited dendritic cell lymph node homing (an essential process in vaccine immunity) by suppressing NK cell-derived IFN-γ expression in mice.

Background: Population growth and climate change have led to more frequent and larger wildfires, increasing the exposure of individuals to wildfire smoke. Notably, asthma exacerbations and allergic airway sensitization are prominent outcomes of such exposure. Summary: Key research questions relate to determining the precise impact on individuals with asthma, including the severity, duration, and long-term consequences of exacerbations. Identifying specific risk factors contributing to vulnerability, such as age, genetics, comorbidities, or environmental factors, is crucial. Additionally, reliable biomarkers for predicting severe exacerbations need exploration. Understanding the long-term health effects of repeated wildfire smoke exposures in individuals with asthma and addressing healthcare disparities are important research areas. Key Messages: This review discusses the need for comprehensive research efforts to better grasp wildfire smoke-induced respiratory health, particularly in vulnerable populations such as farmworkers, firefighters, pregnant women, children, the elderly, and marginalized communities. Effective mitigation would require addressing the current limitations we face by supporting research aimed at a better understanding of wildfire smoke-induced airway disease.

Rationale:Pulmonary innate immune cells play a central role in the initiation and perpetuation of chronic obstructive pulmonary disease (COPD), however the precise mechanisms that orchestrate the development and severity of COPD are poorly understood. Objectives:We hypothesized that the recently described family of innate lymphoid cells (ILCs) play an important role in COPD. Methods:Subjects with COPD and healthy controls were clinically evaluated, and their sputum samples were assessed by flow cytometry. A mouse model of spontaneous COPD [genetically deficient in surfactant protein-D (SP-D -/- )] and ozone (O 3 ) exposure were used to examine the mechanism by which lack of functional SP-D may skew ILC2s to produce IL-17A in combination with IL-5 and IL-13, leading to a mixed inflammatory profile and more severe disease. Measurements and Main Results:COPD was characterized by poor spirometry, sputum inflammation, and the emergence of sputum GATA3 + ILCs (ILC2s), but not T-bet + ILCs (ILC1s) nor RORγt + ILCs (ILC3s). COPD subjects with elevated sputum ILC2s (the ILC2 high group) had worse spirometry and sputum neutrophilia and eosinophilia than healthy and ILC2 low subjects. This was associated with the presence of dual-positive IL-5 + IL-17A + and IL-13 + IL-17A + ILCs and nonfunctional SP-D in the sputum in ILC2 high subjects. SP-D -/- mice showed spontaneous airway neutrophilia. Lack of SP-D in the mouse lung licensed ILC2s to produce IL-17A, which was dose-dependently inhibited by recombinant SP-D. SP-D -/- mice showed enhanced susceptibility to O 3 -induced airway neutrophilia, which was associated with the emergence of inflammatory IL-13 + IL-17A + ILCs. Conclusions:We report that the presence of sputum ILC2s predicts the severity of COPD, and unravel a novel pathway of IL-17A plasticity in lung ILC2s, prevented by the immunomodulatory protein SP-D.

OBJECTIVES: To analyze the temporal trend in enrollment rates in a COVID-19 platform trial during the first three waves of the pandemic in the United States. DESIGN: Secondary analysis of data from the I-SPY COVID randomized controlled trial (RCT). SETTING: Thirty-one hospitals throughout the United States. PATIENTS: Patients who were approached, either directly or via a legally authorized representative, for consent and enrollment into the I-SPY COVID RCT. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Among 1,338 patients approached for the I-SPY COVID trial from July 30, 2020, to February 17, 2022, the number of patients who enrolled ( n = 1,063) versus declined participation ( n = 275) was used to calculate monthly enrollment rates. Overall, demographic and baseline clinical characteristics were similar between those who enrolled versus declined. Enrollment rates fluctuated over the course of the COVID-19 pandemic, but there were no significant trends over time (Mann-Kendall test, p = 0.21). Enrollment rates were also comparable between vaccinated and unvaccinated patients. In multivariable logistic regression analysis, age, sex, region of residence, COVID-19 severity of illness, and vaccination status were not significantly associated with the decision to decline consent. CONCLUSIONS: In this secondary analysis of the I-SPY COVID clinical trial, there was no significant association between the enrollment rate and time period or vaccination status among all eligible patients approached for clinical trial participation. Additional studies are needed to better understand whether the COVID-19 pandemic has altered clinical trial participation and to develop strategies for encouraging participation in future COVID-19 and critical care clinical trials.

Air pollution is a significant environmental risk to health, leading to many diseases. People with chronic respiratory diseases and children are especially vulnerable to detrimental respiratory effects [1, 2]. Up to 45% of the US population, predominately poor, urban populations and racial minorities, resides in areas of high trafficrelated air pollution (TRAP) exposure [3]. Exposure to TRAP, including PM2.5 (particulate matter with particles<2.5 μm in diameter), has been consistently associated with childhood asthma exacerbations and incidence, persistent lung function changes, higher risk of asthma and other lung diseases, and worse respiratory infection outcomes such as COVID severity [4]. Similar health effects have been observed for other gaseous air pollutants such as oxides of nitrogen, volatile organic compounds, and ozone. Although progress has been made over the past decade in reducing exposure to air pollutants, TRAP and other sources of air pollutants (e.g., repeated wildfire smoke exposure due to climate change) still pose substantial risks to public health and the environment. Although the cellular toxicity of air pollutants varies, the major affected pathways are similar. These pollutants typically induce lung inflammation and alter lung function. Cytokine production leading to immune and inflammatory cell infiltration (e.g., neutrophils) and differentiation is activated similarly by PM2.5 (from various sources) and ozone. Exposure to these pollutants elicits an elevated Th17 response, a prominent contributor to asthma susceptibility and difficult-to-treat disease [5, 6]. A significant contributor to this mechanism is the release of epithelial alarmins such as IL-33 [7, 8]. Oxidative stress impairs epithelial integrity and dysregulates innate immunity, leading to increased severity of airway inflammation and loss of protection against respiratory infection. Oxidative stress is strongly linked with chronic asthma, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis [9]. Although therapeutic strategies to target oxidative stress in a recent nonhuman primate model of Th2-low asthma showed some promising effects on ozone-induced airway inflammation [10], other approaches using nutritional, pharmacologic, and environmental interventions have had mixed results [11]. Indeed, to target oxidative stress for the development of effective disease prevention and treatment options, especially for those highly exposed and vulnerable, a

We assessed the humoral immune responses to a COVID-19 vaccine in a well-controlled rhesus macaque model compared to humans immunized with two mRNA vaccines over several months post-second dose. The plasma IgG levels against seven coronaviruses (including SARS-CoV-2) and antibody subtypes (IgG 1-4 and IgM) against SARS-CoV-2 were evaluated using multiplex assays. The neutralization capacity of plasma antibodies against the original SAR-CoV-2 isolate and nine variants was evaluated in vaccinated humans and non-human primates. Immunization of macaques and humans with SARS-CoV-2 vaccines induced a robust neutralizing antibody response. In non-SIV-infected adult macaques immunized with an adenoviral vector expressing S-RBD (n = 7) or N protein (n = 3), elevated levels of IgG and neutralizing antibodies were detected 2 weeks post-second dose. Immune responses to the S-RBD vaccine in SIV-infected adult macaques (n = 2) were similar to the non-SIV-infected animals. Adult humans immunized with Pfizer (n = 35) or Moderna (n = 18) vaccines developed IgG and neutralizing antibodies at 4 weeks post-second dose. In both vaccine groups, IgG 1 was the predominant subtype, followed by IgG 3. The IgG levels, including total and IgG 1,2,3 elicited by the Moderna vaccine, were significantly higher than the corresponding levels elicited by the Pfizer vaccine at 4 weeks post-second dose. A significant correlation was observed between the plasma total IgG antibody levels and neutralization titers in both macaques and humans. Furthermore, broad-spectrum neutralization antibodies against several variants of SARS-CoV-2 were detected in the plasma of both macaques and humans after two vaccinations.