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

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.

High-fat diet-induced obesity affects alpha 7 nicotine acetylcholine receptor expressions in mouse lung myeloid cells

“…These cells were able to migrate from the blood following LPS challenge 52 . Contrasting to previous studies 49 , we found that only a small fraction of interstitial macrophages but not alveolar macrophages expressed α7nAChR in normal conditions. Meanwhile, a study by Mikulski W et.al showed that no α7nAChR mRNA expression was observed in bronchoalveolar lavage cells 53 , and we did not observe either.…”

“…Previous studies have shown the infiltration of M1 type macrophage was enhanced in adipose tissue in α7nAChR knockout mice 48 . Literature shows that approximately 20% of CD45 + cells expressed α7nAChR in the lung 49 . In contrast to CD45 + , CD11b + , CD8 + and B220 + cells, F4/80 + , Gr1 + and CD11c + cells exhibited different expression levels of α7nAChR 50 , 51 .…”

Lung epithelial response to cigarette smoke and modulation by the nicotinic alpha 7 receptor

“…This also means that the nicotine-α7 interaction imparts several parallel and potentially very different effects depending upon the target tissues and cells as well as the challenging agent (inflammagen). In the lung the cell specific expression of α7 includes neuronal cells (e.g., autonomic nervous system) as well as non-neuronal cells (including alveolar macrophages (AM), club cells and Type II alveolar cells [ 7 ]). The anti-inflammatory impact role of α7 has been best characterized in terms of the response to inflammagens such as lipopolysaccharide (LPS).…”

“…In this case α7 tends to suppress the inflammatory responses acting through the vagal nerve as well as directly inhibiting macrophage pro-inflammatory NF-κB signaling and activation of cytokine cascades (e.g., TNFα; [ 2 , 3 , 8 ]). Additionally, effects by α7 on lung epithelial cells include modifying their response to LPS [ 7 ] through alterations to subsequent signaling processes leading to recruitment and infiltration of bone marrow cells into the lung as well as changes to local production of certain mucins (e.g., Muc5b), surfactant proteins and genes of fibrosis. An important issue that has been raised by these studies is what role does α7 have in modulating inflammatory processes in response to CS and whether or not these are distinct from those activated by LPS.…”

“…To elucidate the mechanistic role of the α7 receptor in inflammatory processes, we developed a mouse model [ 7 , 9 – 12 ] in which a point mutation was introduced to limit α7 calcium coupling to cell signaling pathways (termed α7 E260A:G ) that when compared to the control mouse (α7 G ) provides a method to discriminate how α7 signaling in different cells types modifies the host response to an inflammatory challenge (for details see [ 7 , 11 , 12 ]). For example, the α7 E260A:G mouse lung exhibits a reduced response to LPS due in part both to the reduced inflammatory response by alveolar macrophages (AM; relative to control α7 G mice) and a decrease in the transcriptional response by epithelial cells that includes reduced signaling for recruitment of cells from the bone-marrow into the lung.…”