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Updated: Jun 3, 2025
Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages
Published on: November 3, 2014
Differential TLR-ERK1/2 Activity Promotes Viral ssRNA and dsRNA Mimic-Induced Dysregulated Immunity in Macrophages
Rakshya Shrestha1, Paige Marie Johnson1, Roshan Ghimire1
1Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078, USA.
Abstract:
RNA virus-induced excessive inflammation and impaired antiviral interferon (IFN-I) responses are associated with severe disease. This innate immune response, also referred to as "dysregulated immunity" is caused by viral single-stranded RNA (ssRNA)- and double-stranded-RNA (dsRNA)-mediated exuberant inflammation and viral protein-induced IFN antagonism. However, key host factors and the underlying mechanism driving viral RNA-mediated dysregulated immunity are poorly defined. Here, using viral ssRNA and dsRNA mimics, which activate toll-like receptor 7 (TLR7) and TLR3, respectively, we evaluated the role of viral RNAs in causing dysregulated immunity. We observed that murine bone marrow-derived macrophages (BMDMs), when stimulated with TLR3 and TLR7 agonists, induced differential inflammatory and antiviral cytokine response. TLR7 activation triggered a robust inflammatory cytokine/chemokine induction compared to TLR3 activation, whereas TLR3 stimulation induced significantly increased IFN/IFN stimulated gene (ISG) response relative to TLR7 activation. To define the mechanistic basis for dysregulated immunity, we examined cell-surface and endosomal TLR levels and downstream mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) activation. We identified significantly higher cell-surface and endosomal TLR7 levels compared to TLR3, which were associated with early and robust MAPK (p-ERK1/2, p-P38, and p-JNK) and NF-kB activation in TLR7-stimulated macrophages. Furthermore, blocking EKR1/2 and NF-kB activity reduced TLR3/7-induced inflammatory cytokine/chemokine levels, whereas only ERK1/2 inhibition enhanced viral RNA mimic-induced IFN/ISG responses. Collectively, our results illustrate that high cell-surface and endosomal TLR7 expression and robust ERK1/2 activation drive viral ssRNA mimic-induced excessive inflammatory and reduced IFN/ISG response and blocking ERK1/2 activity would likely mitigate viral-RNA/TLR-induced dysregulated immunity.
Insights
Viral RNA triggers excessive inflammation and impairs interferon responses by activating Toll-like receptors (TLRs). High TLR7 expression drives inflammation, while blocking ERK1/2 may mitigate this dysregulated immunity.
Area of Science:
- Immunology
- Virology
- Molecular Biology
Background:
- Excessive inflammation and impaired interferon responses during RNA virus infections contribute to severe disease.
- This
- dysregulated immunity
- stems from viral RNA activating Toll-like receptors (TLRs) and viral proteins interfering with interferon production.
- Key host factors and mechanisms underlying viral RNA-mediated dysregulated immunity remain unclear.
Purpose of the Study:
- To investigate the role of viral single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) in causing dysregulated immunity.
- To elucidate the mechanistic basis of viral RNA-mediated immune responses, focusing on TLR3 and TLR7 signaling pathways.
Main Methods:
- Utilized viral ssRNA and dsRNA mimics to stimulate murine bone marrow-derived macrophages (BMDMs).
- Assessed inflammatory and antiviral cytokine responses, cell-surface and endosomal TLR levels, and downstream signaling pathways (MAPK, NF-kB).
- Investigated the effects of blocking ERK1/2 and NF-kB pathways on cytokine production and interferon responses.
Main Results:
- TLR7 activation induced significantly higher inflammatory cytokine/chemokine responses compared to TLR3 activation.
- TLR3 activation resulted in a greater interferon (IFN) and IFN-stimulated gene (ISG) response than TLR7 activation.
- Higher cell-surface and endosomal TLR7 levels correlated with robust MAPK and NF-kB activation.
- Blocking ERK1/2 and NF-kB reduced inflammation; ERK1/2 inhibition enhanced IFN/ISG responses.
Conclusions:
- High cell-surface and endosomal TLR7 expression and robust ERK1/2 activation drive excessive inflammation and reduced IFN/ISG response to viral ssRNA mimics.
- Blocking ERK1/2 activity presents a potential therapeutic strategy to mitigate viral RNA/TLR-induced dysregulated immunity.
