TLR7 Induces CCL2 Expression in Neurons via NF-κB

Research Background

Toll-like receptors (TLRs) are one of the important mechanisms for recognizing specific molecular structures of microbes. In humans, ten TLRs (TLR1-10) are known, among which TLR3 and TLR7 are located on the endosomal membrane and play important roles in sensing virus-derived nucleic acids and inducing antiviral immune responses (Nishiya et al., 2005). TLR3 recognizes double-stranded RNA (dsRNA), while TLR7 can bind to single-stranded RNA (ssRNA) and imidazoquinoline derivatives (Alexopoulou et al., 2001; Diebold et al., 2004; Hemmi, 2002). Currently, the immune response of TLR7 in the central nervous system (CNS) is not fully understood, but recent studies have shown that TLR7 plays a key role in innate immunity against neurotropic viruses such as West Nile virus (WNV) and Langat virus (Town et al., 2009; Baker et al., 2013). Major cell types in the central nervous system, such as astrocytes, microglia, and neurons, all express TLR7 (Gern et al., 2021). The activation of TLR7 signaling pathways in astrocytes and microglia is crucial for the immune response against neurotropic viruses (Luo et al., 2019; Mukherjee et al., 2019). Furthermore, the TLR7/8 agonist Resiquimod (an imidazoquinoline compound) activates microglia to produce proinflammatory chemokines including C-C motif chemokine ligand 2 (CCL2) (Kwon et al., 2022). Similar to glial cells, neurons also express TLR7 (Gern, 2021; Lehmann et al., 2012), suggesting that they can activate the immune system in response to TLR7 ligands. However, the significance of TLR7 expression in neurons remains unclear. Therefore, this study aims to determine whether Resiquimod can induce the expression of inflammatory chemokines in SH-SY5Y human neuroblastoma cells.

Research Source

This article, titled “Resiquimod Induces CCL2 Expression via NF-κB in SH-SY5Y Human Neuroblastoma Cells,” was written by researchers Masatoshi Kaizuka, Shogo Kawaguchi, Tetsuya Tatsuta, Mayuki Tachizaki, Yuri Kobori, Yusuke Tanaka, Kazuhiko Seya, Tomoh Matsumiya, and Tadaatsu Imaizumi, affiliated with the Department of Gastroenterology and Hematology, Institute of Brain Science, Department of Vascular Biology, Department of Respiratory Medicine, and Department of Health Sciences at Hirosaki University Graduate School of Medicine. The paper was published in 2024 in the journal “Neuromolecular Medicine,” Volume 26, Article 16. The corresponding author is Shogo Kawaguchi, with the contact email kawaguchi.s@hirosaki-u.ac.jp.

Research Content and Process

This study gradually revealed the mechanism of Resiquimod-induced CCL2 expression in SH-SY5Y cells through detailed experimental design and data analysis.

Experimental Procedure

1. Cell Culture: SH-SY5Y human neuroblastoma cell line was cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) and 100mg/ml penicillin-streptomycin. To obtain differentiated SH-SY5Y cells, cells were cultured in medium supplemented with 10µM all-trans retinoic acid for 6 days, changing the medium every 2 days.

2. Immunofluorescence Microscopy (IF): TLR7 expression in SH-SY5Y cells was determined by IF microscopy. After fixation, cells were immunostained with specific antibodies to observe the localization and expression of TLR7, TLR3, and nuclear factor-κB (NF-κB) p65 under light microscopy.

3. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR): Total RNA was extracted from cells, single-stranded cDNA was synthesized, and qRT-PCR analysis was performed with specific primers to assess mRNA levels of CCL2, CXCL8, and CXCL10.

4. Western Blotting: The expression of IκB-α, p-p65, p65, and Actin in cell lysates was detected by Western blotting to evaluate the activation of the NF-κB signaling pathway.

5. Enzyme-Linked Immunosorbent Assay (ELISA): Cell culture supernatants were collected, and protein levels of CCL2, CXCL8, and CXCL10 were determined using ELISA kits.

6. RNA Interference (RNAi): p65 siRNA or negative control siRNA was transfected into SH-SY5Y cells using lipid transfection reagent in buffer to detect the effect of p65 knockdown on Resiquimod-induced CCL2 expression.

Research Results

1. Basal Expression: IF microscopy results showed that TLR7 and TLR3 were constitutively expressed in SH-SY5Y cells and localized near the cell nucleus. After stimulation with Resiquimod and Poly IC, mRNA levels of CCL2, CXCL8, and CXCL10 were rapidly upregulated.

2. Gene Expression Upregulation: Resiquimod induced the expression of CCL2, CXCL8, and CXCL10 in a concentration-dependent manner. ELISA results indicated that Resiquimod significantly increased the secretion of CCL2, while changes in CXCL8 and CXCL10 were minimal.

3. NF-κB Dependency: Western blot results showed that Resiquimod significantly increased the expression of p-p65 and led to nuclear translocation of p65. RNAi experiments further demonstrated that knockdown of p65 significantly reduced Resiquimod-induced CCL2 expression and secretion.

Research Conclusions

This study revealed for the first time that Resiquimod significantly induces CCL2 expression in SH-SY5Y neuroblastoma cells by activating the NF-κB pathway. CCL2 plays a key role in the immune response of the central nervous system through its receptor CCR2. The results suggest that Resiquimod has potential application prospects as a therapeutic agent in central nervous system viral infections through its immunostimulatory effects.

Research Highlights

The main highlights of this study include: 1. Verification of basal TLR7 expression and Resiquimod stimulatory effects in SH-SY5Y cells. 2. Detailed elucidation of the molecular mechanism by which Resiquimod induces CCL2 through activation of the NF-κB pathway. 3. Experimental data supporting the value of Resiquimod as a potential immunotherapeutic agent in central nervous system viral infections.

Research Value

This study provides a new perspective for understanding the function of TLR7 in neurons and lays the foundation for developing immunotherapies for neurotropic viral infections. Additionally, the research points to future directions for studying TLR7 stimulation interactions between neurons and glial cells, emphasizing the importance of fully exploring the responses of non-primary neuron-like cell lines.

The study provides important theoretical support and experimental basis for further research in the fields of neuroscience and immunology.