Fig 1.
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.
Fig 2.
Immunohistochemical examination of the α7 reporter by lung neuronal and neuroepithelial cells.
Panels A through F show images of the α7 reporter tGFP co-expression in lung sections co-stained with various neuronal and neuroepithelial markers. A) Expression of neuropeptide Y (NPY) is observed occasionally in infrequent cell clusters associated with brachial epithelium as reported by others [37]. When present, cells reactive for GFP were adjacent to (arrow head), but did not overlap with, cells expressing NPY (arrow). B) Tyrosine hydroxylase (TH) identifies cells capable of catecholamine synthesis usually in association with neuroendocrine functions. These cell clusters were in part identified by TH immunostaining (arrow heads) and localization to sites of bronchial bifurcation consistent with their identity as Pulmonary neuroepithelial bodies [38] and they contained a mix of GFP stained cells (arrow) and cells co-expressing TH (arrow heads). C) Protein-G product 9.5 (Pgp9.5, aka; ubiquitin C-terminal hydrolase, Uchl1) immunofluorescence in lung sections labeled nerve fibers (arrow head) and occasional solitary cells (inset, arrow). No co-expression of this marker with tGFP was observed. D) Calcitonin-gene related peptide (CGRP) labeled lung sensory fibers did not exhibit detectable tGFP co-expression. E,F) Sensory innervation of the lung by peripherin labeled fibers was prevalent and as shown were particularly prominent in processes of the alveolar compartments that did not label with GFP (arrow). Some of these processes may interact with tGFP-labeled ATII cells (asterisk; see Fig 3). The interaction between AMs that are in contact with alveolar cell linings and peripherin labeled processes is notable (arrowhead). This includes the appearance of specialized neuronal process end points that form a complex of interactions with these stationary AMs. F) Increased magnification shows a strongly tGFP labeled AM (arrows) that is in association with the alveoli and are richly associated with peripherin positive fibers (arrowheads). These process endings often wrap the AMs and multiple process swellings that are interacting with the AM (arrowheads) and in some cases form indentations on the AM (right panel, asterisk). Bars = A-E, 15 microns; F, 5 microns.
Fig 3.
tGFP (GFP) expression by major lung cell subtypes.
A) The definitive Club cell marker, CC10, is shown to co-localize with GFP (arrows). Other GFP staining in the bronchial lining includes neuronal axons (arrow head; see Fig 2). Bar = 15 microns. B) Examination of Club cell anti-CC10 staining revealed by a peroxidase stained section of both α7G and α7E260A:G distal bronchial sections is shown at two magnifications. The panels to the right show greater magnification. In the lower magnification images (panels to the left) the typical appearance of the relatively smooth surface of the α7G bronchia and including evenly distributed Club cells (arrows) is observed. In contrast, the α7E260A:G bronchial lining is more uneven and Club cells appear to protruded from the bronchial surface (arrows). In the α7E260A:G lung this includes aggregate-like clusters (arrowheads) of these cells that are rare or absent from the α7G lung. This is particularly evident in the images of increased magnification to the right. The CC10-labeled Club cells (arrows) are evident and again in the α7E260A:G commonly protrude into the airway space. Relatively large aggregates (arrow heads) are also evident. Bars = 25 microns (left panels) and (right panels) = 15 microns. C) Merged images of immune-localization of GFP (green) and acylated-α-tubulin (acTub, red) shows ciliated cells (arrows) typical of α7G (left panel) or in the α7E260A (right panel) lung. No co-expression of tGFP and acTub was observed. Also the ciliated cells of the α7E260A mouse appeared to be fewer and of altered morphology relative to the control. Bar = 15 microns. D) Sections similar to those from (C) but stained using peroxide to reveal acTub in ciliated cells. Consistent with fluorescent immunostaining, ciliated cells are relatively evenly dispersed in the α7G lung bronchial linings and prominent well-defined cilium are present. In the α7E260A:G lung similar staining shows ciliated cells that tend to be dispersed further apart that often lack well defined cilia (arrows). E) A longitudinal section reveals the bronchial surface facing the airway and the distribution of acTub stained ciliated cells (arrows). Their distribution and the cilium complexity differ between these α7-genotypes and the regular networks formed by ciliated cells (asterisk) in the α7G lung are absent in the α7E260A:G. Bar = 15 microns. F) Quantitation of ciliated cell number comparing the α7G to the α7E260A:G mouse. The average percent of ciliated cells per unit measure was made from 5 sections from each of 3 mice of the identified α7 genotype. A highly significant difference shows that α7E260A:G lung harbors approximately one-third fewer ciliated cells relative to the α7G lung. G) Co-expression of GFP and the ATII cell marker, surfactant protein c (Sftpc; arrows). The arrow head identifies an AM. H) The GFP staining is compared to aquaporin 5 (Aqp5), a marker of ATI cells (asterisk). Essentially no overlap in expression between these markers was observed. An ATII cell is identified by an arrow and an AM by an arrowhead. Staining for these cells was essentially identical between α7G (shown) and α7E260A:G mice with differences to be discussed. For G and H the Bars = 15 microns.
Fig 4.
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.
Fig 5.
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.
Table 1.
Saline α7E260A:G versus α7G Response Difference (Gene Ratio >2-fold).
Table 2.
LPS α7E260A:G versus α7G Response Difference (Gene Ratio >2-fold).
Table 3.
Lung epithelium cell-specific transcript expression differences.
(α7E260A:G / α7G).
Fig 6.
Dysregulation and accumulation of mucins in airways of the α7E260A:G lung.
A) RNA-Seq results showing CDS quantitation of read-depth for the identified mucins in sample of α7G or α7E260A:G (E260A) genotypes with (+) and without (-) i.n. LPS challenge. Asterisk (*) designates a significant (P<0.05) difference from the paired genotype control. B) ELISA measuring Muc5b in mouse BALF from 3 independent samples (in replicate) of each genotype. As predicted by the RNA-Seq results, Muc5b was elevated significantly in the α7E260A:G compared to control samples. C) Lungs of each α7 genotype were fixed and prepared for immunohistochemical analysis as described in the Methods. The sections shown are longitudinal slices stained for immunoreactivity to Muc5b and visualized with a red fluorochrome. Immunostaining of bronchial pathways is apparent in the α7G mouse. However, in the α7E260A:G there are multiple deposits of Muc5b that coincide with many of these airway passages. D) Increased magnification of a bronchial lining of the α7G lung reveals immunostaining towards Muc5b lining the bronchia (arrow heads) but also associated with Club cells (arrow), which in the mouse are a major source of this mucin. AW = airway. E) Comparisons of the expression of Muc5b in the α7G (upper panels) and α7E260A:G (lower panels). Phase microscopy images reveal alveolar spaces (as) and the same section stained for Muc5b is shown to the right (merged image is also shown for the α7E260A:G section). The α7G staining is low or not detected as would be expected in these sites that lack Club cells and Muc5b production. In the α7E260A:G lung accumulations of Muc5b (asterisk) in similar spaces is seen and immunostaining in the matrix of the alveolar spaces appears to be enhanced over controls. F) Increased magnification of some sites of Muc5b accumulation in the α7E260A:G mouse shows the fibrous appearance often associated with these bodies in the phase microscopy image (arrow). G) An example of changes in the expression of transcripts that encode proteins involved in post-translation modifications of mucins and other secreted proteins. Shown is the RNA-Seq CDS count (numbers to the right) for the gene St6galnac2 whose protein is alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 2, an important O-linked glycosylation enzyme. In control α7G lung this gene is expressed at relatively low levels that are increased almost 8-fold after LPS stimulation. In the α7E260A:G lung the basal expression is already almost 9-fold over the α7G lung and its expression increased an additional 4-fold in in response to LPS. This dramatic increase in overall St6galnac expression is similar to that observed for Muc5b and other proteins targeted by the gene product activity. Bars (microns) = C, 700; D, 30; E, 45; F, 15.
Fig 7.
Disproportionate expression of fibrotic genes in the α7E260A:G and accumulation of connective-like tissue.
A) The results of RNA-Seq reveal enhanced expression in the α7E260A:G lung and disproportionate increases in their response to LPS relative to controls. Examples of this are shown for Col1a1 and the decorin gene (Dcn) whose gene product binds to secreted collagen and contributes to crosslinking of the extracellular matrix. These genes are particularly overexpressed in the α7E260A:G after challenge by LPS as reflected by the nearly 7-fold increase in expression relative to the α7G lung epithelium. B) When genes associated with the extracellular matrix exhibiting dysregulated expression (also see Fig 5B) were analyzed using Genemania, a strong interactive association is observed. C) Analysis of possible transcription factors, in-common among the dysregulated genes, using the PASTAA analysis program suggests a strong affiliation with NF1 (neurofibromin1) and the CCAAT-enhancer binding protein (C/ebp, C/ebp-alpha) family. D) Masson’s trichrome staining of whole lung mounts from an α7G or α7E260A:G age and gender matched mice. The α7E260A:G mouse exhibits notable accumulation of connective tissue along bronchial airways (arrowheads) that is not present or greatly reduced in the α7G lung. E) Increased magnification of similar sections as in D show the appearance of enhanced Masson’s trichrome staining comparing fibrotic-like depositions (arrow heads) at multiple sites in the α7G to the α7E260A:G lung. Deposits are normal but greatly reduced in the α7G. Bars (microns) = D, 700; E, 100