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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

Immunostaining for α7G tGFP reporter expression in lung immune cells co-expressing Iba-1.A) In both the α7G and α7E260A:G mouse tGFP is produced as a bicistronic reporter of α7 transcript expression. Immunohistochemistry of the lungs from these animals reveals strong GFP expression by alveolar macrophages (AM; arrow) that are co-labeled by Iba-1 (red) in the control (saline) or i.n. LPS treated lungs of both α7-genotypes. Also present in interstitial regions adjacent to alveoli are Iba-1 labeled cells that are not co-stained for tGFP expression (arrow head). These cells exhibit a more flat in morphology and reduced Iba-1 signaling compared to AMs. In lungs 24 hours after i.n. LPS exposure, cells strongly labeled by Iba-1 of amoeboid-like morphology are evident (asterisks). These prominent cells suggestive of activated macrophages do not co-label with tGFP. AMs are also present in both α7-genotypes as identified by co-expression of Iba-1 and tGFP, their location in alveolar spaces and the retention of their spheroid morphology. Other cells in the alveolar lining also labeled with tGFP (^ and see Fig 3). B) Increased magnification of an α7G control alveolar region identifies two Iba-1 staining macrophages of differing morphology that are either within or associated in part with the interstitial region (arrow) or only in the alveolar space (arrow head). Co-expression of tGFP reveals α7-expression only by the AM cell. Results were similar in the α7E260A:G lung (not shown). Bars = 30 microns.
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pone.0175367.g001: Immunostaining for α7G tGFP reporter expression in lung immune cells co-expressing Iba-1.A) In both the α7G and α7E260A:G mouse tGFP is produced as a bicistronic reporter of α7 transcript expression. Immunohistochemistry of the lungs from these animals reveals strong GFP expression by alveolar macrophages (AM; arrow) that are co-labeled by Iba-1 (red) in the control (saline) or i.n. LPS treated lungs of both α7-genotypes. Also present in interstitial regions adjacent to alveoli are Iba-1 labeled cells that are not co-stained for tGFP expression (arrow head). These cells exhibit a more flat in morphology and reduced Iba-1 signaling compared to AMs. In lungs 24 hours after i.n. LPS exposure, cells strongly labeled by Iba-1 of amoeboid-like morphology are evident (asterisks). These prominent cells suggestive of activated macrophages do not co-label with tGFP. AMs are also present in both α7-genotypes as identified by co-expression of Iba-1 and tGFP, their location in alveolar spaces and the retention of their spheroid morphology. Other cells in the alveolar lining also labeled with tGFP (^ and see Fig 3). B) Increased magnification of an α7G control alveolar region identifies two Iba-1 staining macrophages of differing morphology that are either within or associated in part with the interstitial region (arrow) or only in the alveolar space (arrow head). Co-expression of tGFP reveals α7-expression only by the AM cell. Results were similar in the α7E260A:G lung (not shown). Bars = 30 microns.

Mentions: Macrophages of the lungs reside in both the parenchymal tissue and the bronchial and alveolar spaces. To determine if these cells express α7, we co-labeled lung tissue sections to identify the α7-expression reporter (anti-GFP antibody) with an antibody to Iba1. The Iba1 marker was selected because it is present on both AM and interstitial macrophages. Other markers such as CD11c, which is present on AM, and CD11b [13,14] which is expressed by interstitial macrophages have also been used with results consistent with Iba1 staining and location of cells (not shown). Iba1 intensely co-stained with tGFP in mice of both α7 genotypes (Fig 1) which is notable in that staining is effectively restricted to alveolar macrophages (AM). In parallel sections the identity of these cells as AMs was further supported by the absence of co-labeling with CD11b (not shown). Interstitial (parenchymal) macrophages (IM; Iba1+) in normal lung sections were confirmed by CD11b co-staining, but these cells do not exhibit co-staining with tGFP over background (not shown, but see Fig 1A and 1B).


Nicotinic alpha 7 receptor expression and modulation of the lung epithelial response to lipopolysaccharide
Immunostaining for α7G tGFP reporter expression in lung immune cells co-expressing Iba-1.A) In both the α7G and α7E260A:G mouse tGFP is produced as a bicistronic reporter of α7 transcript expression. Immunohistochemistry of the lungs from these animals reveals strong GFP expression by alveolar macrophages (AM; arrow) that are co-labeled by Iba-1 (red) in the control (saline) or i.n. LPS treated lungs of both α7-genotypes. Also present in interstitial regions adjacent to alveoli are Iba-1 labeled cells that are not co-stained for tGFP expression (arrow head). These cells exhibit a more flat in morphology and reduced Iba-1 signaling compared to AMs. In lungs 24 hours after i.n. LPS exposure, cells strongly labeled by Iba-1 of amoeboid-like morphology are evident (asterisks). These prominent cells suggestive of activated macrophages do not co-label with tGFP. AMs are also present in both α7-genotypes as identified by co-expression of Iba-1 and tGFP, their location in alveolar spaces and the retention of their spheroid morphology. Other cells in the alveolar lining also labeled with tGFP (^ and see Fig 3). B) Increased magnification of an α7G control alveolar region identifies two Iba-1 staining macrophages of differing morphology that are either within or associated in part with the interstitial region (arrow) or only in the alveolar space (arrow head). Co-expression of tGFP reveals α7-expression only by the AM cell. Results were similar in the α7E260A:G lung (not shown). Bars = 30 microns.
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getmorefigures.php?uid=PMC5383308&req=5

pone.0175367.g001: Immunostaining for α7G tGFP reporter expression in lung immune cells co-expressing Iba-1.A) In both the α7G and α7E260A:G mouse tGFP is produced as a bicistronic reporter of α7 transcript expression. Immunohistochemistry of the lungs from these animals reveals strong GFP expression by alveolar macrophages (AM; arrow) that are co-labeled by Iba-1 (red) in the control (saline) or i.n. LPS treated lungs of both α7-genotypes. Also present in interstitial regions adjacent to alveoli are Iba-1 labeled cells that are not co-stained for tGFP expression (arrow head). These cells exhibit a more flat in morphology and reduced Iba-1 signaling compared to AMs. In lungs 24 hours after i.n. LPS exposure, cells strongly labeled by Iba-1 of amoeboid-like morphology are evident (asterisks). These prominent cells suggestive of activated macrophages do not co-label with tGFP. AMs are also present in both α7-genotypes as identified by co-expression of Iba-1 and tGFP, their location in alveolar spaces and the retention of their spheroid morphology. Other cells in the alveolar lining also labeled with tGFP (^ and see Fig 3). B) Increased magnification of an α7G control alveolar region identifies two Iba-1 staining macrophages of differing morphology that are either within or associated in part with the interstitial region (arrow) or only in the alveolar space (arrow head). Co-expression of tGFP reveals α7-expression only by the AM cell. Results were similar in the α7E260A:G lung (not shown). Bars = 30 microns.
Mentions: Macrophages of the lungs reside in both the parenchymal tissue and the bronchial and alveolar spaces. To determine if these cells express α7, we co-labeled lung tissue sections to identify the α7-expression reporter (anti-GFP antibody) with an antibody to Iba1. The Iba1 marker was selected because it is present on both AM and interstitial macrophages. Other markers such as CD11c, which is present on AM, and CD11b [13,14] which is expressed by interstitial macrophages have also been used with results consistent with Iba1 staining and location of cells (not shown). Iba1 intensely co-stained with tGFP in mice of both α7 genotypes (Fig 1) which is notable in that staining is effectively restricted to alveolar macrophages (AM). In parallel sections the identity of these cells as AMs was further supported by the absence of co-labeling with CD11b (not shown). Interstitial (parenchymal) macrophages (IM; Iba1+) in normal lung sections were confirmed by CD11b co-staining, but these cells do not exhibit co-staining with tGFP over background (not shown, but see Fig 1A and 1B).

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