Short-term whole body cigarette smoke exposure induces regional differences in cellular response in the mouse larynx

Easwaran, Meena; Martinez, Joshua D.; Ramirez, Daniel J.; Gall, Phillip A.; Erickson‐DiRenzo, Elizabeth

doi:10.1016/j.toxrep.2021.04.007

Cited by 6 publications

(37 citation statements)

References 112 publications

(161 reference statements)

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“…Mainstream CS from Kentucky 3R4F reference cigarettes was generated using inExpose smoking equipment (SCIREQ, Montreal, QB, Canada), consistent with our previous studies [12][13][14] . Exposure conditions were per the Federal Trade Commission (FTC)/International Standard Organization (ISO) standards (ISO 1991).…”

Section: Cigarette Smoke Generationmentioning

confidence: 98%

“…HD mice were exposed to 4 h of CS per day in 1-1.5 h increments, with room air breaks. Earlier studies in the airways and speci cally in the larynx, have shown that 1-4 h/day of CSE is su cient to induce histopathological changes 12,13,77,78 . LD and HD CS exposures were performed for 5 days/week, M-F for a total of 4 weeks as per the subacute inhalation toxicity guidelines regulated by the Organization for Economic Co-operation and Development (OECD) 79 .…”

Section: Experimental Designmentioning

confidence: 99%

“…We did not perform these measurements in our study. Prior studies also indicate that mice lose body weight in response to successful in vivo CS inhalation 12,[74][75][76] . Correspondingly, we observed in this study that the HD mice exhibited a signi cant body weight reduction in comparison to the controls.…”

Section: It Is Currently Unknown Which Speci C Toxicants Present In C...mentioning

confidence: 99%

“…For histological analyses, larynges were xed in 4% paraformaldehyde overnight at 4 °C and transferred to 70% ethanol (n = 5 control, n = 6 each in both CSE groups). HistoWiz, Inc. (http://www.histowiz.com; Brooklyn, NY, USA) processed tissues for para n embedding and performed coronal section microtomy per established methods [12][13][14]80,81 . 5 µm coronal sections were used for immunohistochemical assessment of immune cell in ltrates.…”

Section: Cigarette Smoke Generationmentioning

confidence: 99%

“…The laryngeal mucosal surfaces (i.e., epithelium and lamina propria) are the rst exposed to harmful external stimuli such as CS. The larynx is lined by ciliated pseudostrati ed columnar respiratory epithelium except for the VF and epiglottic regions which are lined by strati ed squamous epithelium 12 .…”

Section: Introductionmentioning

confidence: 99%

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Modulation of mouse laryngeal inflammatory and immune cell responses by low and high doses of mainstream cigarette smoke

Easwaran

Martinez

Kim

et al. 2022

Preprint

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Cigarette smoking is a major risk factor for laryngeal diseases. Despite well-documented cigarette smoke (CS) induced laryngeal histopathological changes, the underlying immunopathological mechanisms remain largely unexplored. The goal of this study was to evaluate inflammatory and immune cell responses in a CS-exposed larynx. Specifically, we used a 4-week subacute whole-body CS inhalation mouse model to assess these responses in the laryngeal mucosa upon exposure to low (LD; 1 h/day) and high dose (HD; 4 h/day) CS. Laryngeal tissues were harvested and evaluated using a 254-plex NanoString inflammation panel and neutrophil/macrophage/T-cell immunohistochemistry (IHC). NanoString global and differential gene expression analysis revealed a unique expression profile only in the HD group, with 26 significant differentially expressed genes (DEGs). StringDB KEGG pathway enrichment analysis revealed the involvement of these DEGs with proinflammatory pathways including TNF and IL-17. Furthermore, inflammatory responses remained inhibited in conjunction with predicted activated states of anti-inflammatory regulators like PPARG and NFE2L2 upon Ingenuity Pathway Analysis. Neutrophil/macrophage/T-cell recruitment responses remained inhibited as corroborated by IHC outcomes. Overall, our key findings are consistent with HD exposures being anti-inflammatory and immunosuppressive. Furthermore, the identification of important regulatory genes, and enriched pathways may help improve clinical interventions for CS-induced laryngeal diseases.

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Section: Cigarette Smoke Generationmentioning

confidence: 98%

Section: Experimental Designmentioning

confidence: 99%

Section: It Is Currently Unknown Which Speci C Toxicants Present In C...mentioning

confidence: 99%

Section: Cigarette Smoke Generationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Modulation of mouse laryngeal inflammatory and immune cell responses by low and high doses of mainstream cigarette smoke

Easwaran

Martinez

Kim

et al. 2022

Preprint

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Method for Collecting Single Epithelial Cells from the Mouse Larynx

Khan,

Easwaran,

Martinez

et al. 2023

The Laryngoscope

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ObjectiveThe larynx is lined by specialized epithelial cell populations. Studying molecular changes occurring in individual epithelial cell types requires a reliable method for removing these cells from the larynx. Our objective was to develop a method to harvest individual epithelial cells from the mouse larynx while minimizing contamination from non‐laryngeal sites and non‐epithelial laryngeal cells.MethodsMice were euthanized, and the larynx was carefully exposed and separated from non‐laryngeal sites. A small dental brush was inserted into the laryngeal inlet and rotated to obtain epithelial cells. Cells were transferred to collection media, counted, and cytospin preparations stained for laryngeal epithelial (i.e., Pan‐Keratin, EpCAM, NGFR, p63, K5, β‐tubulin, MUC5AC) and non‐epithelial (i.e., vimentin) cell markers. Histopathology was completed on brushed laryngeal tissue sections to evaluate the depth of cell collection. Preliminary Single‐cell RNA sequencing (scRNA‐seq) was performed to confirm this method can capture diverse laryngeal cell types.ResultsWe collected 6000–8000 cells from a single larynx and 35000–40000 cells from combining brushings from three tissues. Histopathology demonstrated brushing removed the epithelial layer of the larynx and some underlying tissue. Immunofluorescence staining demonstrated the phenotype of harvested cells was primarily epithelial. Preliminary scRNA‐seq was successfully conducted and displayed nine unique cell clusters.ConclusionWe developed a reliable method of harvesting individual epithelial cells from the mouse larynx. This method will be useful for collection of laryngeal cells for a variety of downstream cellular and molecular assays, including scRNA‐seq, protein analyses, and cell‐culture‐based experiments, following laryngeal injury.Level of EvidenceN/A Laryngoscope, 2023

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Effects of Short‐term Electronic(e)‐Cigarette Aerosol Exposure in the Mouse Larynx

Easwaran,

Maria,

Martinez

et al. 2023

The Laryngoscope

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ObjectivesThe effects of electronic cigarettes (e‐cigarettes) on the larynx are relatively unknown. This study examined the short‐term effects of e‐cigarette inhalation on cellular and inflammatory responses within the mouse laryngeal glottic and subglottic regions after exposure to pod‐based devices (JUUL).MethodsMale C57BL6/J mice (8–9 weeks) were assigned to control (n = 9), JUUL flavors Mint (JMi; n = 10) or Mango (JMa; n = 10). JUUL mice were exposed to 2 h/day for 1, 5, and 10 days using the inExpose inhalation system. Control mice were in room air. Vocal fold (VF) epithelial thickness, cell proliferation, subglandular area and composition, inflammatory cell infiltration, and surface topography were evaluated in the harvested larynges. Mouse body weight and urinary nicotine biomarkers were also measured. Chemical analysis of JUUL aerosols was conducted using selective ion flow tube mass spectrometry.ResultsJUUL‐exposed mice had reduced body weight after day 5. Urinary nicotine biomarker levels indicated successful JUUL exposure and metabolism. Quantitative analysis of JUUL aerosol indicated that chemical constituents differ between JMi and JMa flavors. VF epithelial thickness, cellular proliferation, glandular area, and surface topography remained unchanged after JUUL exposures. Acidic mucus content increased after 1 day of JMi exposure. VF macrophage and T‐cell levels slightly increased after 10 days of JMi exposures.ConclusionsShort‐term e‐cigarette exposures cause minimal flavor‐ and region‐specific cellular and inflammatory changes in the mouse larynx. This work provides a foundation for long‐term studies to determine if these responses are altered with multiple e‐cigarette components and concentrations.Level of EvidenceN/A Laryngoscope, 2023

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Short-term whole body cigarette smoke exposure induces regional differences in cellular response in the mouse larynx

Abstract: Graphical abstract

Cited by 6 publications

References 112 publications

Modulation of mouse laryngeal inflammatory and immune cell responses by low and high doses of mainstream cigarette smoke

Modulation of mouse laryngeal inflammatory and immune cell responses by low and high doses of mainstream cigarette smoke

Method for Collecting Single Epithelial Cells from the Mouse Larynx

Effects of Short‐term Electronic(e)‐Cigarette Aerosol Exposure in the Mouse Larynx

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