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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.


RNA-Seq results reveal α7-impact on cell-specific changes in transcription following LPS challenge.A) CD45- interstitial cells were isolated and RNA-Seq performed. Transcript data were converted to CDS values and these were then compared as labeled between α7-genotypes following challenge with i.n. saline or i.n. LPS as indicated. The lines indicate a 2-fold threshold difference in expression between genotypes and the number (N) of gene transcripts exceeding the 200 average read depth minimal cut-off (see S1 Table). Genes achieving a 4-fold or greater are colored, and some of these genes that exhibit among the greatest change in relative expression between control (Saline) and i.n. LPS (LPS) treatments are identified by their gene name. B) GeneMANIA derived plots [23,24] based upon gene transcript read averages between α7-genotypes in response to i.n. LPS. Gene clusters to the left include transcripts exceeding a genotype-based average read depth of 2-fold or 4-fold expression for the gene clusters to the right. Diagrams were generated using the default settings (Max resultant genes and attributes were set to zero). Subsets of key functional gene groupings as defined by GeneMANIA are indicated. In the α7G control the i.n. LPS response is dominated by two highly significant functional groups inclusive of ‘innate immune response’ and ‘regulators of cytokine production’ gene sets. The ‘innate immune response’ groups are retained when the 4-fold stringency cut off analysis was applied. In contrast, the same analysis of the i.n. LPS response enhanced specifically in the α7E260A:G lung epithelium reveals three different gene groups. These include genes of ‘extracellular matrix’, ‘inorganic substance response’ and ‘lung epithelium secretions’ of which the ‘extracellular matrix’ genes, and ‘lung epithelial secretions’ are retained after increasing stringency to greater than 4-fold. C) Quantitative average CDS read depth measures for each α7 genotype and treatment group are compared for some of the major genes defined both in the GeneMANIA analysis as epithelial secretions and from the plot in (A). The inverse relationship between gene expression in response to i.n. LPS that is related to α7G-genotypes is apparent and highly significant (** = p>0.01; *** = p>0.0001). D) Genes were subgrouped into defined cell-specific transcripts for Club (blue), ciliated (red), ATI (violet) and ATII (green) cells (Table 3 and S1 Table). The relative shift in α7E260A:G expression after LPS is shown and some genes are identified that exhibit particularly robust shifts in expression by Club and ATII cells (relative increase by α7E260A:G to the control) versus genes transcripts that were increased in ciliated cells for the same comparisons but relatively unchanged for ATI cells as shown by their grouping around 1.0 when compared to the expression differences in saline exposed (control tissues). E) Polar plots of the same cell-specific genes plotted in order of the difference in expression between saline and LPS exhibits the most dysregulation in Club cell gene expression followed by ciliated cells and ATII cells. ATI cells, which exhibit no α7-expression or number differences between genotypes, were again grouped around the expected 1.0 coordinates indicating no change in expression.
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pone.0175367.g005: RNA-Seq results reveal α7-impact on cell-specific changes in transcription following LPS challenge.A) CD45- interstitial cells were isolated and RNA-Seq performed. Transcript data were converted to CDS values and these were then compared as labeled between α7-genotypes following challenge with i.n. saline or i.n. LPS as indicated. The lines indicate a 2-fold threshold difference in expression between genotypes and the number (N) of gene transcripts exceeding the 200 average read depth minimal cut-off (see S1 Table). Genes achieving a 4-fold or greater are colored, and some of these genes that exhibit among the greatest change in relative expression between control (Saline) and i.n. LPS (LPS) treatments are identified by their gene name. B) GeneMANIA derived plots [23,24] based upon gene transcript read averages between α7-genotypes in response to i.n. LPS. Gene clusters to the left include transcripts exceeding a genotype-based average read depth of 2-fold or 4-fold expression for the gene clusters to the right. Diagrams were generated using the default settings (Max resultant genes and attributes were set to zero). Subsets of key functional gene groupings as defined by GeneMANIA are indicated. In the α7G control the i.n. LPS response is dominated by two highly significant functional groups inclusive of ‘innate immune response’ and ‘regulators of cytokine production’ gene sets. The ‘innate immune response’ groups are retained when the 4-fold stringency cut off analysis was applied. In contrast, the same analysis of the i.n. LPS response enhanced specifically in the α7E260A:G lung epithelium reveals three different gene groups. These include genes of ‘extracellular matrix’, ‘inorganic substance response’ and ‘lung epithelium secretions’ of which the ‘extracellular matrix’ genes, and ‘lung epithelial secretions’ are retained after increasing stringency to greater than 4-fold. C) Quantitative average CDS read depth measures for each α7 genotype and treatment group are compared for some of the major genes defined both in the GeneMANIA analysis as epithelial secretions and from the plot in (A). The inverse relationship between gene expression in response to i.n. LPS that is related to α7G-genotypes is apparent and highly significant (** = p>0.01; *** = p>0.0001). D) Genes were subgrouped into defined cell-specific transcripts for Club (blue), ciliated (red), ATI (violet) and ATII (green) cells (Table 3 and S1 Table). The relative shift in α7E260A:G expression after LPS is shown and some genes are identified that exhibit particularly robust shifts in expression by Club and ATII cells (relative increase by α7E260A:G to the control) versus genes transcripts that were increased in ciliated cells for the same comparisons but relatively unchanged for ATI cells as shown by their grouping around 1.0 when compared to the expression differences in saline exposed (control tissues). E) Polar plots of the same cell-specific genes plotted in order of the difference in expression between saline and LPS exhibits the most dysregulation in Club cell gene expression followed by ciliated cells and ATII cells. ATI cells, which exhibit no α7-expression or number differences between genotypes, were again grouped around the expected 1.0 coordinates indicating no change in expression.

Mentions: Comparisons between the α7G and α7E260A:G i.n. LPS transcriptional response as measured using RNA-Seq are summarized graphically in Fig 5A. Genes exhibiting a 2-fold or greater difference between α7G and the α7E260A:G i.n. saline (control) samples include 144 gene total transcripts of which 40 genes were consistently expressed more in the α7G and 104 genes were expressed more in the α7E260A:G (Table 1 (see S1 Table) and Fig 5A). These values were increased after i.n. LPS to 362 total genes exceeding a 2-fold difference between α7 genotypes. This included 152 genes expressed preferentially in the α7G samples and 210 in the α7E260A:G (Table 2 (see S1 Table) and Fig 5A). Some examples of the genes that were reduced in constitutive expression in the α7E260A:G include Postn (periostin), whose gene product imparts cell adhesion and extracellular matrix remodeling [50], Retnla (resistin-like alpha), which impacts on IL-6 secretion and allergic inflammatory responses [51,52], Cyp2a5 (cytochrome P450 family 2 subfamily a5) that modulates the LPS response and has been suggested to degrade nicotine [53], and Ltf (lactotransferrin) which participates in antimicrobial activity [54,55]. Also, a recurring gene that is suppressed in the α7E260A:G is Lcn2 (lipocalin2), which can also contribute to modulating the host defenses to multiple bacterial species [54,55]. One striking aspect of the difference in gene expression between these genotypes is revealed after i.n. LPS. As highlighted for a subset of these genes in Fig 5A, their expression was substantially greater in the α7G versus α7E260A:G lung, but completely reversed in terms of their response and expression levels following i.n. LPS. Some of these are identified by gene name in Fig 5A and they are recognizable as genes important to lung secretory and epithelial function. This issue is returned to in greater detail below.


Nicotinic alpha 7 receptor expression and modulation of the lung epithelial response to lipopolysaccharide
RNA-Seq results reveal α7-impact on cell-specific changes in transcription following LPS challenge.A) CD45- interstitial cells were isolated and RNA-Seq performed. Transcript data were converted to CDS values and these were then compared as labeled between α7-genotypes following challenge with i.n. saline or i.n. LPS as indicated. The lines indicate a 2-fold threshold difference in expression between genotypes and the number (N) of gene transcripts exceeding the 200 average read depth minimal cut-off (see S1 Table). Genes achieving a 4-fold or greater are colored, and some of these genes that exhibit among the greatest change in relative expression between control (Saline) and i.n. LPS (LPS) treatments are identified by their gene name. B) GeneMANIA derived plots [23,24] based upon gene transcript read averages between α7-genotypes in response to i.n. LPS. Gene clusters to the left include transcripts exceeding a genotype-based average read depth of 2-fold or 4-fold expression for the gene clusters to the right. Diagrams were generated using the default settings (Max resultant genes and attributes were set to zero). Subsets of key functional gene groupings as defined by GeneMANIA are indicated. In the α7G control the i.n. LPS response is dominated by two highly significant functional groups inclusive of ‘innate immune response’ and ‘regulators of cytokine production’ gene sets. The ‘innate immune response’ groups are retained when the 4-fold stringency cut off analysis was applied. In contrast, the same analysis of the i.n. LPS response enhanced specifically in the α7E260A:G lung epithelium reveals three different gene groups. These include genes of ‘extracellular matrix’, ‘inorganic substance response’ and ‘lung epithelium secretions’ of which the ‘extracellular matrix’ genes, and ‘lung epithelial secretions’ are retained after increasing stringency to greater than 4-fold. C) Quantitative average CDS read depth measures for each α7 genotype and treatment group are compared for some of the major genes defined both in the GeneMANIA analysis as epithelial secretions and from the plot in (A). The inverse relationship between gene expression in response to i.n. LPS that is related to α7G-genotypes is apparent and highly significant (** = p>0.01; *** = p>0.0001). D) Genes were subgrouped into defined cell-specific transcripts for Club (blue), ciliated (red), ATI (violet) and ATII (green) cells (Table 3 and S1 Table). The relative shift in α7E260A:G expression after LPS is shown and some genes are identified that exhibit particularly robust shifts in expression by Club and ATII cells (relative increase by α7E260A:G to the control) versus genes transcripts that were increased in ciliated cells for the same comparisons but relatively unchanged for ATI cells as shown by their grouping around 1.0 when compared to the expression differences in saline exposed (control tissues). E) Polar plots of the same cell-specific genes plotted in order of the difference in expression between saline and LPS exhibits the most dysregulation in Club cell gene expression followed by ciliated cells and ATII cells. ATI cells, which exhibit no α7-expression or number differences between genotypes, were again grouped around the expected 1.0 coordinates indicating no change in expression.
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pone.0175367.g005: RNA-Seq results reveal α7-impact on cell-specific changes in transcription following LPS challenge.A) CD45- interstitial cells were isolated and RNA-Seq performed. Transcript data were converted to CDS values and these were then compared as labeled between α7-genotypes following challenge with i.n. saline or i.n. LPS as indicated. The lines indicate a 2-fold threshold difference in expression between genotypes and the number (N) of gene transcripts exceeding the 200 average read depth minimal cut-off (see S1 Table). Genes achieving a 4-fold or greater are colored, and some of these genes that exhibit among the greatest change in relative expression between control (Saline) and i.n. LPS (LPS) treatments are identified by their gene name. B) GeneMANIA derived plots [23,24] based upon gene transcript read averages between α7-genotypes in response to i.n. LPS. Gene clusters to the left include transcripts exceeding a genotype-based average read depth of 2-fold or 4-fold expression for the gene clusters to the right. Diagrams were generated using the default settings (Max resultant genes and attributes were set to zero). Subsets of key functional gene groupings as defined by GeneMANIA are indicated. In the α7G control the i.n. LPS response is dominated by two highly significant functional groups inclusive of ‘innate immune response’ and ‘regulators of cytokine production’ gene sets. The ‘innate immune response’ groups are retained when the 4-fold stringency cut off analysis was applied. In contrast, the same analysis of the i.n. LPS response enhanced specifically in the α7E260A:G lung epithelium reveals three different gene groups. These include genes of ‘extracellular matrix’, ‘inorganic substance response’ and ‘lung epithelium secretions’ of which the ‘extracellular matrix’ genes, and ‘lung epithelial secretions’ are retained after increasing stringency to greater than 4-fold. C) Quantitative average CDS read depth measures for each α7 genotype and treatment group are compared for some of the major genes defined both in the GeneMANIA analysis as epithelial secretions and from the plot in (A). The inverse relationship between gene expression in response to i.n. LPS that is related to α7G-genotypes is apparent and highly significant (** = p>0.01; *** = p>0.0001). D) Genes were subgrouped into defined cell-specific transcripts for Club (blue), ciliated (red), ATI (violet) and ATII (green) cells (Table 3 and S1 Table). The relative shift in α7E260A:G expression after LPS is shown and some genes are identified that exhibit particularly robust shifts in expression by Club and ATII cells (relative increase by α7E260A:G to the control) versus genes transcripts that were increased in ciliated cells for the same comparisons but relatively unchanged for ATI cells as shown by their grouping around 1.0 when compared to the expression differences in saline exposed (control tissues). E) Polar plots of the same cell-specific genes plotted in order of the difference in expression between saline and LPS exhibits the most dysregulation in Club cell gene expression followed by ciliated cells and ATII cells. ATI cells, which exhibit no α7-expression or number differences between genotypes, were again grouped around the expected 1.0 coordinates indicating no change in expression.
Mentions: Comparisons between the α7G and α7E260A:G i.n. LPS transcriptional response as measured using RNA-Seq are summarized graphically in Fig 5A. Genes exhibiting a 2-fold or greater difference between α7G and the α7E260A:G i.n. saline (control) samples include 144 gene total transcripts of which 40 genes were consistently expressed more in the α7G and 104 genes were expressed more in the α7E260A:G (Table 1 (see S1 Table) and Fig 5A). These values were increased after i.n. LPS to 362 total genes exceeding a 2-fold difference between α7 genotypes. This included 152 genes expressed preferentially in the α7G samples and 210 in the α7E260A:G (Table 2 (see S1 Table) and Fig 5A). Some examples of the genes that were reduced in constitutive expression in the α7E260A:G include Postn (periostin), whose gene product imparts cell adhesion and extracellular matrix remodeling [50], Retnla (resistin-like alpha), which impacts on IL-6 secretion and allergic inflammatory responses [51,52], Cyp2a5 (cytochrome P450 family 2 subfamily a5) that modulates the LPS response and has been suggested to degrade nicotine [53], and Ltf (lactotransferrin) which participates in antimicrobial activity [54,55]. Also, a recurring gene that is suppressed in the α7E260A:G is Lcn2 (lipocalin2), which can also contribute to modulating the host defenses to multiple bacterial species [54,55]. One striking aspect of the difference in gene expression between these genotypes is revealed after i.n. LPS. As highlighted for a subset of these genes in Fig 5A, their expression was substantially greater in the α7G versus α7E260A:G lung, but completely reversed in terms of their response and expression levels following i.n. LPS. Some of these are identified by gene name in Fig 5A and they are recognizable as genes important to lung secretory and epithelial function. This issue is returned to in greater detail below.

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.