Blue light irradiation exerts anti-viral and anti-inflammatory properties against herpes simplex virus type 1 infection

Abstract

The aim of this study was to investigate the antiviral and anti-inflammatory functions of blue light (BL) in cutaneous viral infections. Previously, we examined the photo-biogoverning role of 450 nm BL in SARS-CoV-2-infected cells, which showed that photo-energy could inhibit viral activation depending on the number of photons. However, the communication network between photo-energy irradiation and immune cells involved in viral infections has not been clarified. We verified viral activation, inflammatory responses, and relevant downstream cascades caused by human simplex virus type I (HSV-1) after BL irradiation. To examine the antiviral effect of BL, we further tested whether BL could disturb viral absorption or entry into host cells. The results showed that BL irradiation, but not green light (GL) exposure, specifically decreased plaque-forming activity and viral copy numbers in HSV-1-infected cells. Accumulated BL irradiation inhibited the localization of viral proteins and the RNA expression of characteristic viral genes such as UL19, UL27, and US6, thus exerting to an anti-viral effect. The results also showed that BL exposure during viral absorption interfered with viral entry or destroyed the virus, as assessed by plaque formation and quantitative PCR assays. The levels of the pro-inflammatory mediators interleukin (IL)-18 and IL-1β in M1-polarized macrophages were increased by HSV-1 infection. However, these increases were attenuated by BL irradiation. Importantly, BL irradiation decreased cGAS and STING expression, as well as downstream NF-κB p65, in M1-polarized HSV-1-infected macrophages, demonstrating anti-viral and anti-inflammatory properties. These findings suggest that BL could serve as an anti-viral and anti-inflammatory therapeutic candidate to treat HSV-1 infections.

Keywords: Blue light; Herpes simplex virus-1; Inflammatory factors; Viral replication.

Fig. 1

Inhibitory effect of BL irradiation on HSV-1 replication. Vero cells (1.3 × 10⁵ cells/well) were infected with HSV-1 (MOI = 1) for 1 h (A). Following viral absorption, Vero cells were washed twice with PBS and overlaid with DMEM media containing 0.3% agarose, 2% heat-inactivated FBS and 1% antibiotics after washing with PBS. After BL (10 J/cm²) irradiation once or twice per day, infected cells were maintained for 72 h and then fixed with 3.7% formaldehyde. Fixed cells were visualized and stained with crystal violet to count plaque numbers. Viral titers were determined by plaque forming assay (B). For viral DNA extraction, Vero cells were replaced with new DMEM media without agarose and irradiated with BL or GL, then further incubated for 48 h. At 48 h post-infection, viral DNA from cell culture media was harvested and amplified using Step One Plus PCR system (C, D). *P < 0.05, **P < 0.01 vs. HSV-1 infected group.

Fig. 2

Inhibitory effect of BL irradiation on HSV-1 virions. Vero cells were infected with HSV-1 (MOI = 1) for 1 h. After BL (10 J/cm²) irradiation twice per day, the infected cells were incubated for 24 h, and fixed in glutaraldehyde-phosphate solution as described in Materials and Methods section. TEM images showed the syncytium with neighboring cells, nucleus expansion, and nucleolus fragmentation after HSV-1 infection (A). Red arrows indicate virus particles in nucleus and blue arrows indicate the autophagosomal formation. C, cytoplasm; N, Nucleus.

Fig. 3

Inhibitory effect of BL irradiation on HSV-1 specific genes expression. Vero cells were infected with HSV-1 (MOI = 1) for 1 h. Total RNA was extracted from cells and subjected to RT-qPCR to determine mRNA levels of UL19, UL27, UL52, and US6. Expression levels of these genes were normalized by GAPDH expression. **P < 0.01 vs. HSV-1 infected group.

Fig. 4

Inhibitory effect of BL irradiation on HSV-1 specific proteins expression. (A) HSV-1-infected Vero cells were incubated with ICP0 antibody at 4 °C overnight and Alexa Fluor 488-conjugated antibody for 90 min, as described in Materials and Methods. Cellular nuclei were counterstained with DAPI for 4 min. (B) Expression levels of intracellular ICP0 and ICP4 were determined by western blot analysis.

Fig. 5

Inhibitory effect of BL irradiation on HSV-1 virus absorption. To investigate whether BL could disturb viral entry into host cells, the experimental schedule was modified by irradiating BL during virus absorption. BL (10 J/cm²) irradiation was carried out from 15 min (Co-BL15) or from 30 min (Co-BL30) after the onset of viral absorption, followed by washing the cells with PBS and changing the media (A). Plaque formation determined by staining with crystal violet as described in Materials and Methods section (B). Viral DNA from cell culture media was harvested and amplified using Step One Plus PCR system (C). Expression levels of intracellular ICP4, ICP0, gD, and GAPDH were determined by western blot analysis (D). Total RNA was extracted from cells to determine mRNA levels of UL19, UL27, UL52, and US6. Expression levels of these genes were normalized by GAPDH expression (E). *P < 0.05, *P < 0.01 vs. HSV-1 infected group.

Fig. 6

Inhibitory effect of BL irradiation on production of pro-inflammatory cytokines upon HSV-1 infection. THP-1 cells (1.5 × 10⁵ cells/well) were grown into 12-well plates and incubated with PMA (100 nM) overnight. Next day, M0-resting cells were differentiated into M1 macrophage after treatment with LPS (0.1 μg/ml) and IFN-γ (20 ng/ml) for 24 h. Following HSV-1 absorption, BL (10 J/cm²) was irradiated and further incubated for 24 h to collect cell culture supernatant for ELISA. **P < 0.01 vs. HSV-1 infected group.

Fig. S1

Inhibitory effect of BL irradiation on enterovirus-71. Vero E6 cells (1.5 × 10⁵ cells/well) were infected with enterovirus-71 (MOI = 0.01) for 1 h. Following viral absorption, Vero E6 cells were washed twice with PBS and overlaid with DMEM media containing 0.5% agarose, 5% heat-inactivated FBS and 1% antibiotics. After BL (10 J/cm²) irradiation once or twice per day, the infected cells were maintained for 72 h and then fixed with 3.7% formaldehyde. Fixed cells were visualized and stained with crystal violet to count plaque numbers. Viral titers were determined by plaque forming assay. *P < 0.05, **P < 0.01 vs. HSV-1 infected group.

Fig. S2

Virucidal effect of BL irradiation. HSV-1 was irradiated with BL (10 J/cm²) once and then infected to the Vero cells for 1 h (A). For viral DNA extraction, Vero cells were replaced with new DMEM media without agarose, then further incubated for 48 h. At 48 h post-infection, viral DNA from cell culture media was harvested and amplified using Step One Plus PCR system (B). For plaque assay, Vero cells were washed twice with PBS after viral absorption and overlaid with DMEM media containing 0.3% agarose, 2% heat-inactivated FBS and 1% antibiotics. At 72 hpi, viral titers were determined by plaque forming assay (C). *P < 0.05, **P < 0.01 vs. HSV-1 infected group.