Lung eosinophilia induced by house dust mites or ovalbumin is modulated by nicotinic receptor α7 and inhibited by cigarette smoke

Abstract

Eosinophilia (EOS) is an important component of airway inflammation and hyperresponsiveness in allergic reactions including those leading to asthma. Although cigarette smoking (CS) is a significant contributor to long-term adverse outcomes in these lung disorders, there are also the curious reports of its ability to produce acute suppression of inflammatory responses including EOS through poorly understood mechanisms. One possibility is that proinflammatory processes are suppressed by nicotine in CS acting through nicotinic receptor α7 (α7). Here we addressed the role of α7 in modulating EOS with two mouse models of an allergic response: house dust mites (HDM; Dermatophagoides sp.) and ovalbumin (OVA). The influence of α7 on EOS was experimentally resolved in wild-type mice or in mice in which a point mutation of the α7 receptor (α7^E260A:G) selectively restricts normal signaling of cellular responses. RNA analysis of alveolar macrophages and the distal lung epithelium indicates that normal α7 function robustly impacts gene expression in the epithelium to HDM and OVA but to different degrees. Notable was allergen-specific α7 modulation of Ccl11 and Ccl24 (eotaxins) expression, which was enhanced in HDM but suppressed in OVA EOS. CS suppressed EOS induced by both OVA and HDM, as well as the inflammatory genes involved, regardless of α7 genotype. These results suggest that EOS in response to HDM or OVA is through signaling pathways that are modulated in a cell-specific manner by α7 and are distinct from CS suppression.

Keywords: chemokine; cigarette smoke; eosinophils; eotaxin; house dust mites; nicotine; nicotinic receptor α7; ovalbumin.

Fig. 1.

Experimental design for allergen [house dust mite (HDM) or ovalbumin (OVA)] sensitizations and challenges used in these experiments. i.n., intranasal; i.p., intraperitoneal.

Fig. 2.

Modulation of house dust mite (HDM)-induced eosinophilia (EOS) by nicotinic receptor α7 (α7). A: average bronchial alveolar lavage fluid (BALF) total cell number of α7^G or α7^E260A:G lungs subsequent to sensitization and challenge with HDM before or after exposure to sidestream cigarette smoke (CS) exposure. B: flow cytometry (FC) scatterplot (left, boxed area) and marker analysis (right) of α7^G control BALF (non-HDM treated) reveals that >95% are alveolar macrophages (AMs) (CD11c⁺/SiglecF⁺; asterisks). Smaller granular cells (CD11c⁻/SiglecF⁺, arrows) were infrequent. SSC, side scatter; FSC, forward scatter. C: FC analysis of α7^G BALF 96 h after HDM challenge and increased EOS (>75%). Left: high granularity (SSC) and smaller size (FSC) (arrows). Right: CD11c⁻/SiglecF⁺ (arrows). Note reduced proportion of AMs (CD11c⁺/SiglecF⁺; asterisks). D–G: histology of α7^G lung after HDM confirms the presence of small granular cells (D, arrowhead) in terminal bronchioli and alveolar spaces that are absent from controls (not shown; see Refs. 9, 10). E: at increased magnification the classic size and lobed nuclear appearance of EOS are seen (arrowheads). An AM is also present (asterisk). F: Giemsa-stained eosinophils (red nuclei; arrowheads) and an AM (asterisk). G: increased magnification (Giemsa stained) shows the common EOS (arrowhead) association with an AM (asterisk). Bars, 50 μm (D), 10 μm (E–G). H: quantitation of serum IgE from α7^G mice not receiving HDM (Con) and after HDM. I: FC analysis of nontreated α7^E260A:G lung BALF. J: α7^E260A:G BALF after HDM. EOS (arrows) is identified by size and granularity (left) and marker confirmation (CD11c⁻/SiglecF⁺; right). AMs identified by asterisks. K: histology of the α7^E260A:G lung after HDM challenge confirms cell infiltrates (EOS, arrowhead). L: Giemsa staining shows accumulation of EOS (red nuclei) mostly along alveolar cell linings (asterisk) and in association with AMs (arrowheads). Bars, 50 μm (K), 10 μm (L). M: FC results of BALF from α7^G or α7^E260A:G after exposure to CS for 4 mo (see text and Ref. 9). Size and granularity (boxed areas, left) and AM content of >95% (CD11c⁺/SiglecF⁺, right) are shown (compare with B and I). N: FC of BALF from CS-treated mice that were sensitized and challenged with HDM. EOS (arrows) and AM (asterisks) are identified. O: average EOS response of each α7 genotype after HDM challenge in either the presence or the absence of CS. CS suppression of HDM EOS is highly significant for both genotypes (P < 0.0001). N.S., not significant (P > 0.05). Error bars in A, H, and O = SEM.

Fig. 3.

Modulation of ovalbumin (OVA)-associated eosinophilia (EOS) by nicotinic receptor α7 (α7). A: bronchial alveolar lavage fluid (BALF) average total cell number from control α7^G or α7^E260A:G (Con) or subsequent to sensitization and challenge with OVA before or after exposure to sidestream cigarette smoke (CS). B: typical EOS of BALF from both α7 genotypes as measured by flow cytometry (FC). Scatterplots (left) and marker analysis (right) show EOS (arrows) and alveolar macrophage (AM; asterisks) content. SSC, side scatter; FSC, forward scatter. C–J: histology of α7 lung after OVA inhalation. The accumulation of small granular cells (EOS) was particularly strong in perivascular regions (C and G; arrows) and in focal clusters in the lung parenchyma (D and H; arrowheads) that are more extensive in the α7^E260A:G. Increased magnification shows infiltrating cells distributed differently within alveoli of α7^G (adjacent to alveolar walls; E) or α7^E260A:G (alveolar spaces; I). F and J: Giemsa stain of EOS (red nuclei, arrowheads). Note different distributions between α7 genotypes and association with AMs (asterisk). Leukocytes not identified by Giemsa staining are also present (v). Bars, 50 μm (C, D, G, and H), 10 μm (E, F, I, and J). K: BALF from CS- and OVA-treated mice. FC of BALF from CS- and OVA-treated lungs shows AMs in scatterplots (left; asterisks) or marker analysis (right). L: average EOS response for each α7 genotype after OVA challenge in either the presence or the absence of CS and statistical significance. N.S., not significant (P > 0.05). Error bars in A and L = SEM.

Fig. 4.

Impact of α7 on gene expression associated with house dust mite (HDM) or ovalbumin (OVA) challenges. A and B: RNA sequencing (RNA-Seq) results (Glimma plots; materials and methods) showing the results for cigarette smoke (CS)-exposed mice of both genotypes and their response to HDM (A) or OVA (B). Genes exhibiting a significant difference in expression [false discovery rate (FDR) P < 0.05] are highlighted in red (upregulated) or blue (downregulated). Number of genes retained after greater stringency FDR of P < 0.01 is indicated and underlined (see Supplemental Tables S1 and S2). Ccl11 and Ccl24 are identified. CPM, count per million. C: diagram generated in GeneMANIA (biological process based, no attributes) shows the relationship between genes that were upregulated in instances of increased eosinophilia (EOS) associated with α7 (α7^G-HDM and α7^E260A:G-OVA). D–F: α7^G (G) or α7^E260A:G (EA) Ccl11 (eotaxin1) and Ccl24 (eotaxin2) transcriptional response to either HDM or OVA. The epithelium was measured with RNA-Seq (fold change; D) or quantitative PCR (E). F: RT²-PCR array quantitated alveolar macrophage (AM) response. G: HDM or OVA AM transcription response by IL-13, IL-4, or IL-12α in α7^G or α7^E260A:G. These transcripts were not detected in the epithelium fraction. Error bars = ±10% of mean. H: diagrams generated with GeneMANIA show interactions among the most responsive gene transcripts increased in response to α7^G-HDM and α7^E260A:G-OVA. The core group (from C) is outlined or identified with an arrowhead. Genes in red  = EOS associated; yellow = cell chemotaxis; blue = extracellular matrix.

Fig. 5.

Cigarette smoke (CS) suppresses transcriptional responses by distal lung epithelium to ovalbumin (OVA). A: RNA sequencing (RNA-Seq) results showing results for CS-exposed mice of both genotypes and their response to OVA (Glimma plots; materials and methods). Genes exhibiting a significant difference in expression [false discovery rate (FDR) P < 0.05] are highlighted in red (upregulated) or blue (downregulated), and the number with an increased stringency of FDR of P < 0.01 is indicated and underlined. Ccl11 and Ccl24 are identified. CPM, count per million. B: average protein coding sequence (CDS) values from RNA-Seq for the individual responses. G, α7^G; EA, α7^E260A:G. Error bars = ±10% of mean.