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Nicotinic alpha 7 receptor expression and modulation of the lung epithelial response to lipopolysaccharide

View Article: PubMed Central - PubMed

ABSTRACT

Nicotine modulates multiple inflammatory responses in the lung through the nicotinic acetylcholine receptor subtype alpha7 (α7). Previously we reported that α7 modulates both the hematopoietic and epithelium responses in the lung to the bacterial inflammogen, lipopolysaccharide (LPS). Here we apply immunohistochemistry, flow cytometry and RNA-Seq analysis of isolated distal lung epithelium to further define α7-expression and function in this tissue. Mouse lines were used that co-express a bicistronic tau-green fluorescent protein (tGFP) as a reporter of α7 (α7G) expression and that harbor an α7 with a specific point mutation (α7E260A:G) that selectively uncouples it from cell calcium-signaling mechanisms. The tGFP reporter reveals strong cell-specific α7-expression by alveolar macrophages (AM), Club cells and ATII cells. Ciliated cells do not express detectible tGFP, but their numbers decrease by one-third in the α7E260A:G lung compared to controls. Transcriptional comparisons (RNA-Seq) between α7G and α7E260A:G enriched lung epithelium 24 hours after challenge with either intra-nasal (i.n.) saline or LPS reveals a robust α7-genotype impact on both the stasis and inflammatory response of this tissue. Overall the α7E260A:G lung epithelium exhibits reduced inflammatory cytokine/chemokine expression to i.n. LPS. Transcripts specific to Club cells (e.g., CC10, secretoglobins and Muc5b) or to ATII cells (e.g., surfactant proteins) were constitutively decreased in in the α7E260A:G lung, but they were strongly induced in response to i.n. LPS. Protein analysis applying immunohistochemistry and ELISA also revealed α7-associated differences suggested by RNA-Seq including altered mucin protein 5b (Muc5b) accumulation in the α7E260A:G bronchia, that in some cases appeared to form airway plugs, and a substantial increase in extracellular matrix deposits around α7E260A:G airway bronchia linings that was not seen in controls. Our results show that α7 is an important modulator of normal gene expression stasis and the response to an inhaled inflammogen in the distal lung epithelium. Further, when normal α7 signaling is disrupted, changes in lung gene expression resemble those associated with long-term lung pathologies seen in humans who use inhaled nicotine products.

No MeSH data available.


Related in: MedlinePlus

Isolation of CD45- lung epithelium and characterization of RNA transcript expression.A) BALF was collected from groups of 3–5 mice, pooled, and blocked with anti-CD16/CD32 antibodies (FcRγ) to prevent non-specific binding. These cells were then stained with monoclonal antibodies directed against mouse CD45 (marker of bone marrow derived cells) and CD31 (endothelial cell marker) and analyzed using flow cytometry. In the BALF 97% or more of the cells were CD45+. B) The lung tissue was removed, trachea and proximal structures removed by dissection, and the remaining tissue (interstitium) was digested. These single cell suspensions Fc-blocked and stained as above and described in the Methods. Stained samples were analyzed using flow cytometry. More than 94% of the cells were CD45- and greater than 90% were epCam positive defining epithelial cells. C) Cells from CD45- fractions were then collected for each α7-genotype, RNA prepared and RNA-Seq performed (Methods and text). The results show a log(2) plot comparison of transcript expression between the α7g male-female or α7E260A:G male female analyses. Linear regression (R2) of the results demonstrates high correspondence between the respective genders of each genotype. D) Comparisons of RNA-Seq results (Log(2) plots) between CD45- distal lung cell fractions from α7G or α7E260A:G genotypes both following exposure to intranasal saline (control) or i.n. lipopolysaccharide (i.n.LPS). E) Examples of RNA-Seq results for two genes (interleukin1-β (IL-1β) and chemokine Cxcl10) overlaid with the UCSC RefSeq. The read coverage graphs taken from the UCSC browser reflect the results of raw counts (total) per sample. Because the library sizes for each sample were similar, differences in magnitude do not account for shifts in the reported relative transcript values (not shown). The quantitation (shown adjacent to the plot for each sample) after different treatments is also compared with the results of qPCR analysis for these gene transcripts. Note the overall agreement between these measures that was typical for these results. Further, neither the IL-1β or Cxcl10 gene transcript in the α7E260A:G lung CD45- fraction was responsive relative to the substantial increase in transcription in the control following LPS exposure. F) Plots comparing the result of RNA samples analyzed by RNA-Seq versus the Affymetrix based array platform. Overall good agreement between these methods was achieved although the expected compression of the dynamic range in Affymetrix samples is evident.
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pone.0175367.g004: Isolation of CD45- lung epithelium and characterization of RNA transcript expression.A) BALF was collected from groups of 3–5 mice, pooled, and blocked with anti-CD16/CD32 antibodies (FcRγ) to prevent non-specific binding. These cells were then stained with monoclonal antibodies directed against mouse CD45 (marker of bone marrow derived cells) and CD31 (endothelial cell marker) and analyzed using flow cytometry. In the BALF 97% or more of the cells were CD45+. B) The lung tissue was removed, trachea and proximal structures removed by dissection, and the remaining tissue (interstitium) was digested. These single cell suspensions Fc-blocked and stained as above and described in the Methods. Stained samples were analyzed using flow cytometry. More than 94% of the cells were CD45- and greater than 90% were epCam positive defining epithelial cells. C) Cells from CD45- fractions were then collected for each α7-genotype, RNA prepared and RNA-Seq performed (Methods and text). The results show a log(2) plot comparison of transcript expression between the α7g male-female or α7E260A:G male female analyses. Linear regression (R2) of the results demonstrates high correspondence between the respective genders of each genotype. D) Comparisons of RNA-Seq results (Log(2) plots) between CD45- distal lung cell fractions from α7G or α7E260A:G genotypes both following exposure to intranasal saline (control) or i.n. lipopolysaccharide (i.n.LPS). E) Examples of RNA-Seq results for two genes (interleukin1-β (IL-1β) and chemokine Cxcl10) overlaid with the UCSC RefSeq. The read coverage graphs taken from the UCSC browser reflect the results of raw counts (total) per sample. Because the library sizes for each sample were similar, differences in magnitude do not account for shifts in the reported relative transcript values (not shown). The quantitation (shown adjacent to the plot for each sample) after different treatments is also compared with the results of qPCR analysis for these gene transcripts. Note the overall agreement between these measures that was typical for these results. Further, neither the IL-1β or Cxcl10 gene transcript in the α7E260A:G lung CD45- fraction was responsive relative to the substantial increase in transcription in the control following LPS exposure. F) Plots comparing the result of RNA samples analyzed by RNA-Seq versus the Affymetrix based array platform. Overall good agreement between these methods was achieved although the expected compression of the dynamic range in Affymetrix samples is evident.

Mentions: Previously we found that α7E260A:G mice respond to i.n. LPS (as measured by elevated numbers of inflammatory cells in the blood) but fail to recruit hematopoietic cells into the lung, a result that was associated with altered transcriptional signaling profiles by non-hematopoietic resident lung cells [13,14]. To increase the resolution of how α7-expression impacts the CD45- cell-specific response to LPS, we compared the α7 and α7E260A:G distal lung epithelial transcriptional response to i.n. saline or i.n. LPS using a polyadenylated transcriptome strategy combined with RNA-Seq quantification. This approach is greatly facilitated by two key experimental factors. First, the α7E260A:G mouse was constructed in the α7G background to provide a genetically defined experimental system in which the α7-coupled response by specific cell subtypes to inflammatory challenge can be directly measured. Second, the detection of a cell-specific impact by α7 is greatly enhanced by the ability to enrich cells of a desired phenotype using specific markers (Methods). This is particularly important in the lung where the contribution by CD45+ cells of bone marrow origin can dominate the detection of often more subtle changes in the transcriptional response to i.n. LPS by other cell types such as those in the epithelium. As before [14], mice were treated with 250 μg LPS/mouse in 30 μl saline and control mice received only 30 μl i.n. saline. The systemic response to LPS was confirmed by removing a small blood sample to measure the influx of inflammatory cells into the blood (Methods). Upon confirmation of an LPS response, the mice were sacrificed 24 hours following i.n. LPS, the lungs cleared of BALF content by lavage, the distal lung tissue enriched by dissection and removal of the trachea and the remaining interstitial cells dissociated to facilitate the selective enrichment of epithelial cells. To do this the CD45- cells were collected from the flow-through of a CD45+ magnetic bead column that routinely produced >90% enrichment after a single pass (Fig 4A). Of the enriched CD45- cells the majority (>95%) were epCAM+ (Fig 4B), a marker defining epithelial cells including Club cells, ciliated cells, ATI and ATII cells. Further analysis revealed the samples used contained few endothelial cells (<1% CD31+; not shown, but see Fig 4A). The epithelial enriched population was then extracted, poly-adenylated RNA collected and these samples were processed further for analysis by RNA-Seq (Methods). Tests of reproducibility and the possible contributions from gender effects were examined by comparing different experimental samples and samples taken from male and female lung epithelium of both α7 mouse genotypes (Fig 4C). This analysis revealed highly significant correspondence in the results between both independently replicated preparations as well as between genders (Y-chromosome specific transcripts were removed). For example, the expression correlation between α7G male and female was highly significant (R2 = 0.97). The α7E260A:G mouse gender comparisons were also highly significant albeit slightly reduced correlation (R2 = 0.88). The results demonstrated that in addition to attaining reproducibility between independent samples, significant differences among transcripts in α7 and α7E260A:G mice associated with gender were not evident.


Nicotinic alpha 7 receptor expression and modulation of the lung epithelial response to lipopolysaccharide
Isolation of CD45- lung epithelium and characterization of RNA transcript expression.A) BALF was collected from groups of 3–5 mice, pooled, and blocked with anti-CD16/CD32 antibodies (FcRγ) to prevent non-specific binding. These cells were then stained with monoclonal antibodies directed against mouse CD45 (marker of bone marrow derived cells) and CD31 (endothelial cell marker) and analyzed using flow cytometry. In the BALF 97% or more of the cells were CD45+. B) The lung tissue was removed, trachea and proximal structures removed by dissection, and the remaining tissue (interstitium) was digested. These single cell suspensions Fc-blocked and stained as above and described in the Methods. Stained samples were analyzed using flow cytometry. More than 94% of the cells were CD45- and greater than 90% were epCam positive defining epithelial cells. C) Cells from CD45- fractions were then collected for each α7-genotype, RNA prepared and RNA-Seq performed (Methods and text). The results show a log(2) plot comparison of transcript expression between the α7g male-female or α7E260A:G male female analyses. Linear regression (R2) of the results demonstrates high correspondence between the respective genders of each genotype. D) Comparisons of RNA-Seq results (Log(2) plots) between CD45- distal lung cell fractions from α7G or α7E260A:G genotypes both following exposure to intranasal saline (control) or i.n. lipopolysaccharide (i.n.LPS). E) Examples of RNA-Seq results for two genes (interleukin1-β (IL-1β) and chemokine Cxcl10) overlaid with the UCSC RefSeq. The read coverage graphs taken from the UCSC browser reflect the results of raw counts (total) per sample. Because the library sizes for each sample were similar, differences in magnitude do not account for shifts in the reported relative transcript values (not shown). The quantitation (shown adjacent to the plot for each sample) after different treatments is also compared with the results of qPCR analysis for these gene transcripts. Note the overall agreement between these measures that was typical for these results. Further, neither the IL-1β or Cxcl10 gene transcript in the α7E260A:G lung CD45- fraction was responsive relative to the substantial increase in transcription in the control following LPS exposure. F) Plots comparing the result of RNA samples analyzed by RNA-Seq versus the Affymetrix based array platform. Overall good agreement between these methods was achieved although the expected compression of the dynamic range in Affymetrix samples is evident.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383308&req=5

pone.0175367.g004: Isolation of CD45- lung epithelium and characterization of RNA transcript expression.A) BALF was collected from groups of 3–5 mice, pooled, and blocked with anti-CD16/CD32 antibodies (FcRγ) to prevent non-specific binding. These cells were then stained with monoclonal antibodies directed against mouse CD45 (marker of bone marrow derived cells) and CD31 (endothelial cell marker) and analyzed using flow cytometry. In the BALF 97% or more of the cells were CD45+. B) The lung tissue was removed, trachea and proximal structures removed by dissection, and the remaining tissue (interstitium) was digested. These single cell suspensions Fc-blocked and stained as above and described in the Methods. Stained samples were analyzed using flow cytometry. More than 94% of the cells were CD45- and greater than 90% were epCam positive defining epithelial cells. C) Cells from CD45- fractions were then collected for each α7-genotype, RNA prepared and RNA-Seq performed (Methods and text). The results show a log(2) plot comparison of transcript expression between the α7g male-female or α7E260A:G male female analyses. Linear regression (R2) of the results demonstrates high correspondence between the respective genders of each genotype. D) Comparisons of RNA-Seq results (Log(2) plots) between CD45- distal lung cell fractions from α7G or α7E260A:G genotypes both following exposure to intranasal saline (control) or i.n. lipopolysaccharide (i.n.LPS). E) Examples of RNA-Seq results for two genes (interleukin1-β (IL-1β) and chemokine Cxcl10) overlaid with the UCSC RefSeq. The read coverage graphs taken from the UCSC browser reflect the results of raw counts (total) per sample. Because the library sizes for each sample were similar, differences in magnitude do not account for shifts in the reported relative transcript values (not shown). The quantitation (shown adjacent to the plot for each sample) after different treatments is also compared with the results of qPCR analysis for these gene transcripts. Note the overall agreement between these measures that was typical for these results. Further, neither the IL-1β or Cxcl10 gene transcript in the α7E260A:G lung CD45- fraction was responsive relative to the substantial increase in transcription in the control following LPS exposure. F) Plots comparing the result of RNA samples analyzed by RNA-Seq versus the Affymetrix based array platform. Overall good agreement between these methods was achieved although the expected compression of the dynamic range in Affymetrix samples is evident.
Mentions: Previously we found that α7E260A:G mice respond to i.n. LPS (as measured by elevated numbers of inflammatory cells in the blood) but fail to recruit hematopoietic cells into the lung, a result that was associated with altered transcriptional signaling profiles by non-hematopoietic resident lung cells [13,14]. To increase the resolution of how α7-expression impacts the CD45- cell-specific response to LPS, we compared the α7 and α7E260A:G distal lung epithelial transcriptional response to i.n. saline or i.n. LPS using a polyadenylated transcriptome strategy combined with RNA-Seq quantification. This approach is greatly facilitated by two key experimental factors. First, the α7E260A:G mouse was constructed in the α7G background to provide a genetically defined experimental system in which the α7-coupled response by specific cell subtypes to inflammatory challenge can be directly measured. Second, the detection of a cell-specific impact by α7 is greatly enhanced by the ability to enrich cells of a desired phenotype using specific markers (Methods). This is particularly important in the lung where the contribution by CD45+ cells of bone marrow origin can dominate the detection of often more subtle changes in the transcriptional response to i.n. LPS by other cell types such as those in the epithelium. As before [14], mice were treated with 250 μg LPS/mouse in 30 μl saline and control mice received only 30 μl i.n. saline. The systemic response to LPS was confirmed by removing a small blood sample to measure the influx of inflammatory cells into the blood (Methods). Upon confirmation of an LPS response, the mice were sacrificed 24 hours following i.n. LPS, the lungs cleared of BALF content by lavage, the distal lung tissue enriched by dissection and removal of the trachea and the remaining interstitial cells dissociated to facilitate the selective enrichment of epithelial cells. To do this the CD45- cells were collected from the flow-through of a CD45+ magnetic bead column that routinely produced >90% enrichment after a single pass (Fig 4A). Of the enriched CD45- cells the majority (>95%) were epCAM+ (Fig 4B), a marker defining epithelial cells including Club cells, ciliated cells, ATI and ATII cells. Further analysis revealed the samples used contained few endothelial cells (<1% CD31+; not shown, but see Fig 4A). The epithelial enriched population was then extracted, poly-adenylated RNA collected and these samples were processed further for analysis by RNA-Seq (Methods). Tests of reproducibility and the possible contributions from gender effects were examined by comparing different experimental samples and samples taken from male and female lung epithelium of both α7 mouse genotypes (Fig 4C). This analysis revealed highly significant correspondence in the results between both independently replicated preparations as well as between genders (Y-chromosome specific transcripts were removed). For example, the expression correlation between α7G male and female was highly significant (R2 = 0.97). The α7E260A:G mouse gender comparisons were also highly significant albeit slightly reduced correlation (R2 = 0.88). The results demonstrated that in addition to attaining reproducibility between independent samples, significant differences among transcripts in α7 and α7E260A:G mice associated with gender were not evident.

View Article: PubMed Central - PubMed

ABSTRACT

Nicotine modulates multiple inflammatory responses in the lung through the nicotinic acetylcholine receptor subtype alpha7 (&alpha;7). Previously we reported that &alpha;7 modulates both the hematopoietic and epithelium responses in the lung to the bacterial inflammogen, lipopolysaccharide (LPS). Here we apply immunohistochemistry, flow cytometry and RNA-Seq analysis of isolated distal lung epithelium to further define &alpha;7-expression and function in this tissue. Mouse lines were used that co-express a bicistronic tau-green fluorescent protein (tGFP) as a reporter of &alpha;7 (&alpha;7G) expression and that harbor an &alpha;7 with a specific point mutation (&alpha;7E260A:G) that selectively uncouples it from cell calcium-signaling mechanisms. The tGFP reporter reveals strong cell-specific &alpha;7-expression by alveolar macrophages (AM), Club cells and ATII cells. Ciliated cells do not express detectible tGFP, but their numbers decrease by one-third in the &alpha;7E260A:G lung compared to controls. Transcriptional comparisons (RNA-Seq) between &alpha;7G and &alpha;7E260A:G enriched lung epithelium 24 hours after challenge with either intra-nasal (i.n.) saline or LPS reveals a robust &alpha;7-genotype impact on both the stasis and inflammatory response of this tissue. Overall the &alpha;7E260A:G lung epithelium exhibits reduced inflammatory cytokine/chemokine expression to i.n. LPS. Transcripts specific to Club cells (e.g., CC10, secretoglobins and Muc5b) or to ATII cells (e.g., surfactant proteins) were constitutively decreased in in the &alpha;7E260A:G lung, but they were strongly induced in response to i.n. LPS. Protein analysis applying immunohistochemistry and ELISA also revealed &alpha;7-associated differences suggested by RNA-Seq including altered mucin protein 5b (Muc5b) accumulation in the &alpha;7E260A:G bronchia, that in some cases appeared to form airway plugs, and a substantial increase in extracellular matrix deposits around &alpha;7E260A:G airway bronchia linings that was not seen in controls. Our results show that &alpha;7 is an important modulator of normal gene expression stasis and the response to an inhaled inflammogen in the distal lung epithelium. Further, when normal &alpha;7 signaling is disrupted, changes in lung gene expression resemble those associated with long-term lung pathologies seen in humans who use inhaled nicotine products.

No MeSH data available.


Related in: MedlinePlus