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Identification and characterization of poly(I:C)-induced molecular responses attenuated by nicotine in mouse
Wen-Yan Cui1, Shufang Zhao, Renata Polanowska-Grabowska
1Department of Psychiatry and Neurobehavioral Sciences University of Virginia 1670 Discovery Drive, Suite 110, Charlottesville, VA 22911, USA.
Abstract:
To further our understanding of the effects of nicotine on the molecular responses of macrophages during virus or virus-like infections, poly(I:C)-stimulated macrophage-like RAW264.2 cells or mouse primary peritoneal macrophages were challenged with nicotine; and their molecular responses were evaluated using a qRT-PCR array, antibody array, ELISA, Western blotting, and Ca(2+) imaging. Of 51 genes expressed in the Toll-like receptor (TLR) and RIG-I-like receptor (RLR) pathways, mRNA expression of 15 genes in RAW264.7 cells was attenuated by nicotine, of which mRNA expression of IL-6, TNF-α, and IL-1β was confirmed to be attenuated in peritoneal macrophages. Concurrently, nicotine treatment attenuated the release of IL-6 and TNF-α from poly(I:C)-stimulated macrophages. However, when poly(I:C)-stimulated macrophages were challenged with nicotine plus α-bungarotoxin (α-BTX), secretion of IL-6 and TNF-α was found to be in a level seen with poly(I:C) stimulation only, indicating that α7-nAChR, a highly Ca(2+) permeable ion channel sensitive to blockade by α-BTX, is involved in this process. Furthermore, results from an antibody array indicated that nicotine treatment attenuated the phosphorylation of 82 sites, including Thr286 on CaMKIIα, from poly(I:C)-stimulated RAW264.7 cells, of which 28 are expressed in the downstream cascade of Ca(2+) signaling. Coincidentally, poly(I:C)-stimulated macrophages showed attenuated expression of phosphorylated CaMKIIα when pretreated with nicotine. In addition, nicotine attenuated intracellular Ca(2+) signal from poly(I:C)-stimulated RAW264.7 cells. Collectively, these results indicate that poly(I:C)-induced molecular responses of macrophages could be significantly attenuated by nicotine.
Insights
Nicotine dampens the immune response of macrophages during viral infections by reducing key inflammatory signals like IL-6 and TNF-α. This effect is mediated through the α7-nicotinic acetylcholine receptor (α7-nAChR) pathway.
Area of Science:
- Immunology
- Pharmacology
- Cell Biology
Background:
- Macrophages play a crucial role in innate immunity against viral infections.
- Nicotine's impact on macrophage immune responses, particularly during viral challenges, requires further elucidation.
- Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) are key pathways in antiviral immunity.
Purpose of the Study:
- To investigate the effects of nicotine on the molecular responses of macrophages stimulated with poly(I:C), a viral mimic.
- To identify specific molecular pathways and signaling molecules modulated by nicotine in macrophages.
- To determine the role of the α7-nicotinic acetylcholine receptor (α7-nAChR) in nicotine's immunomodulatory effects.
Main Methods:
- Utilized poly(I:C)-stimulated RAW264.7 cells and primary mouse peritoneal macrophages.
- Employed qRT-PCR arrays, antibody arrays, ELISA, Western blotting, and Ca(2+) imaging to assess molecular responses.
- Investigated the involvement of α7-nAChR using α-bungarotoxin (α-BTX) as a blocking agent.
Main Results:
- Nicotine attenuated mRNA expression of 15 genes in TLR and RLR pathways in RAW264.7 cells, including IL-6, TNF-α, and IL-1β in primary macrophages.
- Nicotine reduced the release of IL-6 and TNF-α from poly(I:C)-stimulated macrophages.
- α-BTX blocked nicotine's inhibitory effect, implicating α7-nAChR in the modulation of cytokine secretion.
- Nicotine attenuated the phosphorylation of CaMKIIα and intracellular Ca(2+) signaling in response to poly(I:C).
Conclusions:
- Nicotine significantly attenuates poly(I:C)-induced molecular responses in macrophages.
- The α7-nAChR pathway is critically involved in mediating nicotine's immunomodulatory effects on macrophages.
- These findings provide insights into the complex interplay between nicotine and the innate immune system during viral infections.
