Yogesh Saini

Ground-level ozone (O₃), a criteria air pollutant, can cause significant adverse effects on lung health, including airway inflammation, compromised lung function, and increased susceptibility to lung infections. This study was designed to determine whether a history of acute lung injury from high-concentration O₃ modulates respiratory tract inflammatory responses to repetitive low-concentration O₃ exposures. Accordingly, we hypothesized that mice acutely exposed to a high concentration of O₃ would exhibit a modulated type-2 (Th2)-like inflammatory response to subsequent, repetitive O₃ exposures, even after full recovery from non-Th2 acute inflammation caused by the initial exposure. Ten-week-old C57BL6/J mice were exposed to either filtered air (FA) or 2 ppm O₃ for 3 h. After a three-week recovery period, the mice received daily exposures to FA or O₃ for 4 h per day over nine days. We evaluated immune cells and inflammatory mediators in cell-free bronchoalveolar lavage fluid (BALF), and examined mucous cell metaplasia (MCM) and epithelial cell injury in lung tissue sections. As expected, mice in the FA-FA group, which were not exposed to O₃, showed no overt signs of injury or inflammation. Mice in the O₃-FA group, which received acute exposure to 2 ppm O₃ and allowed to recover for 3 weeks, exhibited homeostatic immune cell composition within lung airspaces and no evidence of MCM, suggesting almost complete recovery from non-Th2 acute inflammation caused by the initial exposure. In contrast, mice in the FA-O₃ group, which received only repetitive exposures to O₃, exhibited elevated eosinophil counts and prominent MCM, suggesting Th2 inflammatory responses. These mice also displayed increased numbers of FIZZ1⁺ epithelial cells in the lungs. Compared with the FA-O₃ group, mice in the O₃-O₃ group, which received acute exposure to 2 ppm O₃, were allowed to recover for 3 weeks, and then repetitively exposed to 1 ppm O₃, exhibited attenuated Th2 inflammatory responses, as evidenced by diminished eosinophil and lymphocyte counts and attenuated MCM. Compared with the FA-O₃ group, bronchoalveolar lavage fluid (BALF) from the O₃-O₃ group showed lower levels of eotaxin, IL-1α, IL-1β, and IL-4, but higher levels of G-CSF, KC, IL-6, and IL-12, with a trend toward higher IL-10. Furthermore, lung sections in the O₃-O₃ group also displayed reduced numbers of mast cells and a trend toward fewer FIZZ1⁺ epithelial cells. These findings suggest that prior exposure to acute, high-concentration O₃ modulates inflammatory and remodeling responses induced by subsequent repeated exposures to low-concentration O₃. This study highlights the health impacts of pollution, particularly for populations experiencing intermittent high-level exposures to O₃.

Background: RNA-binding proteins, Zinc Finger Protein 36-Like 1 (ZFP36L1) and Zinc Finger Protein 36-Like 2 (ZFP36L2), post-transcriptionally regulate the expression of a large number of genes involved in various cellular processes. However, specific or redundant functions of ZFP36L1 and ZFP36L2 in liver homeostasis have never been explored. Here, we hypothesized that ZFP36L1 and ZFP36L2 are functionally redundant in the liver, and their combined deficiency would stabilize their direct mRNA targets, which would alter liver homeostasis. Methods: We generated combined liver-specific ZFP36L1-deficient and ZFP36L2-deficient mice (L1/L2 ^dKO ) and compared their liver homeostatic parameters with flox control (L1/L2 ^FLX ) mice. We performed detailed analyses of liver histology, serum biomarkers of liver injury, liver transcriptome, bile flow rate, and biliary total bile acid (TBA) excretion. Results: We demonstrated that the combined liver-specific deficiency of ZFP36L1 and ZFP36L2 in mice (L1/L2 ^dKO ) results in spontaneous cholestatic liver injury, which was characterized by elevated hepatic and serum levels of TBAs and liver injury biomarkers, including ALP, ALT, and AST, presence of bile infarcts followed by marked inflammation, cellular proliferation, and fibrosis. To determine the impact of deficiency of ZFP36L1 and ZFP36L2 on the liver transcriptome and their relevance to cholestatic liver injury, RNA sequencing of whole livers of L1/L2 ^dKO and L1/L2 ^FLX mice was performed, which showed significant perturbation of ZFP36L1/ZFP36L2 target genes associated with cholestasis in L1/L2 ^dKO mice. In addition, L1/L2 ^dKO mice exhibited reduced bile flow rate and decreased biliary TBA excretion. Subsequent analyses revealed impaired bile canalicular morphogenesis by postnatal day 7, as evidenced by F-actin and zonula occludens-1 staining patterns. Conclusions: Our findings demonstrate beneficial roles of ZFP36L1 and ZFP36L2 in maintaining liver homeostasis.

This study addresses a critical knowledge gap regarding the response of alveolar macrophages to ozone, a potent inhaled toxicant. Specifically, we demonstrate that a single exposure to two experimentally relevant concentrations of ozone significantly alters functionally relevant gene expression in alveolar macrophages, as revealed by single-cell resolution analysis.

Electronic cigarette (ECIG) liquids are marketed with labeled nicotine concentrations and propylene glycol (PG) to vegetable glycerin (VG) ratios, yet quality control inconsistencies may alter vaping emissions. We quantified discrepancies between labeled and measured chemical content and evaluated how these differences affect emissions of particulate matter with an aerodynamic diameter of 2.5 µm or smaller (PM_2.5). Flavor-free liquids (n = 20) spanning nicotine labels of 0, 9, 18, and 48 mg/mL and PG content from 0% to 80% were purchased. Nuclear magnetic resonance spectroscopy measured nicotine, PG, and VG. Aerosols were generated using a standardized device in a controlled exposure chamber. PM_2.5 was measured using a pDR-1500 and SMPS/APS, with gravimetric correction factors calculated. Labeling inaccuracies were widespread: “nicotine-free” liquids contained 0.1 to 0.4 mg/mL nicotine, and labeled nicotine deviated by up to ±30%. PG/VG ratios were frequently incorrect; 70% of samples contained higher VG than labeled, including “100% VG” products with about 10% PG. Higher VG consistently increased PM_2.5 mass, while nicotine had a minimal effect. The pDR overestimated mass, whereas SMPS/APS underestimated due to volatilization losses. Overall, inaccurate ECIG liquid labeling can alter measured PM_2.5 emissions under controlled conditions.

High mobility group box 1 (HMGB1), a highly conserved nuclear protein, is released into the extracellular milieu serving as a modulator of inflammatory responses. HMGB1 levels are elevated in the airspaces of CF patients and mice with cystic fibrosis (CF)--like lung disease. To reduce extracellular release of HMGB1 into the lung airspaces, we previously introduced airway epithelial cell-specific HMGB1 deficiency in Scnn1b-Tg+ (Tg+) mice, a model of human CF-like lung disease. While the deletion of airway epithelial cell-specific HMGB1 did not reduce bronchoalveolar lavage fluid (BALF) HMGB1 levels, the intracellular HMGB1 deficiency resulted in exaggerated inflammatory responses. In this study, because HMGB1 protein levels were found significantly elevated in Tg+ BALF cells, we hypothesized that myeloid cell-derived HMGB1 deficiency will reduce extracellular HMGB1 levels in the lung airspaces of Tg+ mice, which will modulate pathological features of lung disease in Tg+ mice. Accordingly, we introduced myeloid cell-specific HMGB1 deficiency in Tg+ mice. The deletion of HMGB1 in myeloid cells did not result in any obvious alterations in the lungs of WT mice. The BALF levels of HMGB1 were comparable between myeloid cell-specific HMGB1-deficient Tg+ and HMGB1-sufficient Tg+ mice. As expected, as compared with the HMGB1-sufficient WT, HMGB1-sufficient Tg+ mice displayed increased total cell counts consisting of neutrophils, eosinophils, and lymphocytes. As compared with the HMGB1-sufficient Tg+ mice, the myeloid cell-specific HMGB1-deficient Tg+ mice exhibited significantly increased BALF neutrophil and eosinophil counts, which was associated with increased BALF levels of granulocyte-specific chemoattractants, i.e., KC/CXCL1, MCP-1/CCL2, MIP-1α/CCL3, and MIP-1β/CCL4, Th2 lymphocyte-specific chemoattractants, i.e., TARC/CCL17 and MDC/CCL22, and monocyte-specific chemoattractants, i.e., MCP-3/CCL7 and MCP-5/CCL12. As compared with the HMGB1-sufficient Tg+ mice, the alveolar macrophages from HMGB1-deficient Tg+ mice were significantly enlarged, suggesting their higher activation status. Myeloid cell-specific HMGB1 deletion also resulted in significantly improved clearance of spontaneous bacterial infection in Tg+ mice. Furthermore, myeloid cell-specific HMGB1 deletion significantly worsened pathological manifestations in Tg+ mice, i.e., exaggerated airway mucus obstruction, increased peribronchiolar infiltration of inflammatory cells, increased alveolar space enlargement, and increased lymphoid hyperplasia. Collectively, although myeloid cells are not the source of extracellular HMGB1 found in the inflamed airspaces of Tg+ mice, the myeloid cell-specific HMGB1 deficiency modulates key pathological features of mucoinflammatory lung disease in these mice.

Tristetraprolin (TTP) is an anti-inflammatory protein that mediates messenger RNA (mRNA) decay of certain transcripts, especially those encoding proinflammatory cytokines. TTP modulates various pathological outcomes in diverse inflammatory diseases; however, its role in ozone (O3)-induced acute lung injury (ALI) has never been tested. Here, we hypothesized that the loss of TTP would exacerbate O3-induced ALI and that the systemic overexpression of TTP would mitigate O3-induced ALI. Accordingly, TTP-knockout (TTPKO), airway epithelial cell–specific TTP-deficient (EpiKO), myeloid cell–specific TTP-deficient (MyeKO), and systemic TTP-overexpressing (TTPΔARE) adult male and female mice, along with their respective littermate control TTP-sufficient mice, were exposed to either O3 (3 ppm) or filtered air for 3 h. The endpoints, including bronchoalveolar lavage fluid cellularity, cytokine levels, and histopathological changes, were assessed 21 to 24 h after O3 or filtered air exposure. As compared with the O3-exposed TTP-sufficient mice, the O3-exposed TTPKO and O3-exposed cell-specific TTP-deficient mice exhibited a significant worsening of ALI outcomes (i.e., neutrophil infiltration, cytokine/chemokine production, and lung pathology). The severity of these outcomes was comparatively milder in O3-exposed EpiKO and O3-exposed MyeKO mice than in O3-exposed TTPKO mice. Conversely, the O3-exposed TTPΔARE mice were protected against O3-induced ALI, as indicated by relatively reduced levels of inflammatory cytokines/chemokines, reduced neutrophil infiltration, and mitigated lung pathology. Collectively, our data suggest that TTP is a critical regulator of inflammation in O3-induced ALI. These findings indicate that enhancing TTP expression could be a potential therapeutic strategy for simultaneously targeting multiple inflammatory cytokines in O3-induced ALI and possibly other inflammatory diseases.

Ground-level ozone (O ₃ ), a criteria air pollutant, can cause significant adverse effects on lung health, including airway inflammation, compromised lung function, and increased susceptibility to lung infections. This study was conceived to determine whether a history of high-concentration O ₃ -induced acute lung injury responsiveness of respiratory tract to low-concentration repetitive O ₃ exposures. Accordingly, we hypothesized that prior acute O ₃ exposure would modulate the lung’s Th2 responses to subsequent repetitive O ₃ exposures. We exposed 8-10-week-old C57BL6/J mice to either filtered air (FA) or 2 ppm O ₃ for three hours, followed by a three-week recovery period, after which the mice received daily exposures to FA or 1 ppm O ₃ for four hours over 9 days. We evaluated immune cell recruitment, inflammatory mediators in cell-free bronchoalveolar lavage fluid (BALF) and examined mucous cell metaplasia and epithelial cell injury in lung tissue sections. As expected, FA-FA (no O ₃ exposure) mice did not exhibit any signs of injury or inflammation. The O ₃ -FA (acute O ₃ exposure only) groups exhibited baseline immune cell populations and no evidence of MCM suggesting almost complete recovery from acute lung injury. In contrast, FA-O ₃ group (repetitive O ₃ exposure only) demonstrated loss of body weight, marked immune cell recruitment, and prominent MCM. These mice also displayed elevated BALF levels of eotaxin, IL-1α, IL-1β, and IL-4, along with increased number of mast cells and FIZZ1 ⁺ epithelial cells in the lungs. Compared with FA-O ₃ group, the O ₃ -O ₃ group (both acute and repetitive O ₃ ) exhibited attenuated responses, as evidenced by diminished eosinophil and lymphocyte counts, and attenuated MCM. BALF analysis revealed lower levels of eotaxin, IL-1α, IL-1β, and IL-4, but elevated levels of G-CSF, KC, IL-6, IL-10, and IL-12 in the O ₃ -O ₃ group. Furthermore, these mice displayed reduced numbers of mast cells and FIZZ1 ⁺ epithelial cells. These findings suggest that prior exposure to acute high-concentration O ₃ modulates inflammatory and remodeling responses induced by subsequent repeated low-concentration O ₃ exposures. The findings from this study highlight the health impacts of O ₃ pollution, particularly in populations experiencing intermittent high-level exposures.

Introduction: Ambient exposure to ozone (O₃), one of the six criteria pollutants, is associated with the exacerbation of respiratory symptoms in individuals with underlying lung diseases. Using Scnn1b-Tg+ (Tg+) mice, a widely used model of muco-inflammatory lung disease, we have demonstrated that O₃ exposure during the early stages of postnatal lung development leads to exacerbated muco-inflammatory outcomes. However, it remains unclear whether O₃ affects the developed lungs differently than the underdeveloped lungs of Tg+ mice.Methods: We exposed 3-week-old wild-type (WT) and Tg+ mice to either filtered air (FA) or 0.8 ppm O₃ for 3 weeks and examined the lung phenotypes 12-16 h post-last exposure.Results: As compared to FA-exposed WT mice, O₃-exposed WT mice showed increased bronchoalveolar lavage fluid (BALF) proteins, increased immune cells, increased inflammation, alveolar space enlargement, and tissue consolidation. As compared to FA-exposed WT mice, the FA-exposed Tg+ mice showed increased immune cells, elevated levels of inflammatory mediators, e.g., IL-5, G-CSF, MIP-2, KC, MIP-1α, MIP-1β, IP-10, TNF-α, and IL-17, increased inflammation, alveolar space enlargement, tissue consolidation. As compared to FA-exposed Scnn1b-Tg+ mice, O₃-exposed Tg+ mice had increased total protein, total dsDNA, and phagocytosed lipid contents, in addition to exaggerated granulocytic recruitment, peripheral and bronchiolar inflammation, alveolar space enlargement, and tissue consolidation.Discussion: Together, our data using Tg+ mice with developed lungs exhibited several findings consistent with previous findings observed in Tg+ neonates. Interestingly, however, as opposed to the previous report in O₃-exposed neonatal Tg+ mice, where the hallmark features of Tg+ airway disease, i.e., mucus obstruction and expression of major gel-forming mucins (MUC5B and MUC5AC) were found exacerbated by O₃ exposure, the FA- and O₃-exposed Tg+ mice with developed lungs exhibited comparable responses. These differential responses suggest that the stage of lung development is an important factor in the modulation of epithelial remodeling following O₃ exposure.