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. 2022 Dec 22;239:112632. doi: 10.1016/j.jphotobiol.2022.112632

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

Phil-Sun Oh 1, Yeon-Hee Han 1, SeokTae Lim 1, Hwan-Jeong Jeong 1,⁎
PMCID: PMC9771843  PMID: 36608399

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, Viral replication, Inflammatory factors

1. Introduction

Herpes simplex virus type-I (HSV-1), a double-stranded DNA virus, is a pathogen that primarily infects the oral mucosal surfaces of humans [1,2]. Following infection and replication, HSV-1 invades sensory neurons [1]. It is transported to the ganglion cells of trigeminal nerves, subsequently leading to a lifelong latent infection. It can be easily reactivated by transportation back to epithelial cells around the mouth due to events such as stress, hormonal changes, and dental treatment, among others [3,4]. >70% of the infected people worldwide have confirmed viral reactivation since no vaccine is available. Despite therapeutic options with antiviral drugs including acyclovir, famciclovir, and valacyclovir, which are known to inhibit viral genome replication by targeting virus DNA polymerase [5,6], many studies have reported that people with low immunity can develop resistance to acyclovir and its derivatives and can easily experience recurrent infections [[7], [8], [9]].

HSV-1 viral infection is initiated by the interaction between viral envelop proteins such as glycoprotein (g) B, gH/gL, and gD, and molecules such as syndecan, nectin, and integrin in the plasma membrane of host cells, which allow viral entry, subsequently leading to the release of tegument proteins and viral capsids into the cytosol [10,11]. After viral entry, a tegument protein called virion host shutoff (VHS) protein begins to degrade cellular mRNA molecules to inhibit translational processes in the host cells. Another tegument protein, VP-16, protects viral mRNAs and the transcriptional activation of viral genes. Meanwhile, the viral capsid is transported to the nuclear membrane by a scaffold of microtubules, and the herpes chromosome enters the nucleus. VP-16 protein then activates the transcription of immediate-early (IE) mRNA by binding with imported-viral DNA. IE mRNAs are exported from the nucleus to the cytoplasm where they are translated by ribosomes for the synthesis of proteins such as ICP0 and ICP4. For lytic infection, translated proteins of the IE genes return to the nucleus for the transcription of the early (E) gene. E gene mRNAs are translocated to the cytoplasm for translation by ribosomes that are highly regulated. Since E proteins include viral DNA polymerase, the E proteins imported into the nucleus replicate many copies of viral DNA, leading to the transcription of late (L) genes. Some L genes encode capsid proteins or envelope proteins and are translated in the cytoplasm (capsid protein) and endoplasmic reticulum (envelope proteins). The L proteins produced re-enter the nucleus to assemble viral capsids, followed by the insertion of the viral genome into the capsid. The assembled capsids emerge from the nuclear membrane by enveloping/de-enveloping steps. Viral proteins are translated and modified in the rough-endoplasmic reticulum (rough ER) by glycosylation. They are further processed in the trans-Golgi compartment system, and eventually transported and fused with the plasma membrane of cells. Completed virions are released into the extracellular space by exocytosis [4,5,12,13].

Inflammation is a generally well-known immune defense response of host cells against infectious agents including viruses, microbes, and various physical stimuli [14]. Upon viral infection, macrophages and dendritic cells are stimulated to produce pro-inflammatory cytokines such as interleukin (IL)-18 and IL-1β, which are tightly controlled by multi-protein complex called inflammasome. The secretion of cytokines activates lymphocytes and facilitates their infiltration into the primarily infected site by activating the expression of interferon and other pro-inflammatory cytokines, which generates an inflammatory response [15,16]. The cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS)/stimulator of the IFN gene (STING) pathway controls type I IFN production with downstream NF-κB activation signaling, which, in turn, facilitates the secretion of other pro-inflammatory mediators [[17], [18], [19]].

As a promising phototherapeutic platform, phototherapy with visible light wavelengths has been clinically applied for treating various medical conditions including inflammation, pain, and tissue injury by inducing photophysical and photochemical changes [[20], [21], [22]]. It is well known that red light wavelengths (>600 nm) with the highest penetration depth can decrease erythema, edema, and pain in patients with burn wounds [23,24]. Its combination with near-infrared light has been shown to be more effective for wound repair by stimulating proliferation and collagen synthesis in fibroblasts [25]. Recently, we published experimental data using a phototherapeutic approach with blue light without photosensitizers in various cancer cells both in vitro and in vivo [[26], [27], [28], [29], [30], [31]]. Especially, low-level BL exerted anti-proliferative, anti-inflammatory, and anti-metastatic effects, whereas high-level BL induced the apoptosis of cancer cells depending on the number of irradiating photons. More recently, photo-biogoverning properties and the relevant mechanism of the action of BL on the replication of the SARS-CoV-2 virus have been confirmed [32]. However, the interaction between photo-energy and immune cells engaged in viral infection has not been reported.

In this study, we explored the anti-viral and anti-inflammatory effects of BL on viral replication and the activation of the HSV-1 virus. For this, we set out to determine whether BL irradiation could inhibit plaque formation, viral copy numbers, and HSV-1 gene expression, and reduce canonical inflammasome activation in human M1-type macrophage-differentiated THP-1 cells for subsequent clinical application.

2. Materials and Methods

2.1. Cells and Chemicals

Vero cells, an African monkey kidney cell line and human THP-1 monocytes cells were grown in DMEM and RPMI1640 containing 10% fetal bovine serum (FBS, Serana, Australia) and antibiotics (Gibco), respectively. ICP0 (sc-53,070), ICP4 (sc-69,809), gD (sc-21,719), and cGAS (sc-515,777) antibodies were acquired from Santa Cruz Biotech (CA, USA). STING (#13647), phospho-NF-κB (#3033) and phospho-IRF3 (#37829) antibodies were purchased from Cell Signaling Tech (MA, USA). DMEM (#12-604F) was obtained from Lonza (Basel, Swiss). Alexa Fluor 488-conjugated IgG antibody (A21202), human IL-1β ELISA kit (BMS224–2), and human IL-18 ELISA kit (BMS267–2) were acquired from ThermoFisher Scientific (MA, USA).

2.2. Virus Infection

HSV-1 virus (KBPV-VR-52) was obtained from the Korea Bank for Pathogenic Viruses (KBPV, Seoul, Korea). Vero cells (1.3 × 105 cells/well) were grown in 12 well plates and infected with HSV-1 (multiplicity of infection (MOI) = 1) at 37 °C for 1 h according the protocol of KBPV. Following viral absorption, the viral infection medium was aspirated, and the cells were washed twice with PBS. Subsequently, fresh medium containing 2% heat-inactivated FBS and 1% antibiotics was added. BL (10 J/cm2) irradiation at a power of 8.25 mW/ cm2 was performed once or twice daily after replacing with fresh DMEM media. THP-1 cells (1.5 × 105 cells/well) were grown in 12-well plates and incubated with phorbol 12-myristate 13-acetate (PMA, 100 nM) overnight. The next day, M0 phase resting cells were differentiated into M1 macrophages after treatment with lipopolysaccharide (LPS, 0.1 μg/ml) and interferon (IFN)-γ (20 ng/ml) for 24 h. After HSV-1 absorption (MOI = 1), the cells were irradiated with BL (10 J/cm2) and further incubated for 24 h to collect cell culture supernatant for ELISAs.

2.3. Plaque Reduction Assay

Vero cells (1.3 × 105 cells/well) were grown in 12-well plates and infected with HSV-1 (MOI = 1) at 37 °C for 1 h. 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 BL irradiation once or twice per day, the infected cells were incubated for 72 h and fixed with 3.7% formaldehyde for 20 min. Finally, the cells were visualized after staining with 1% crystal violet solution for 10 min as relatively clear zone. Data presented as plaque forming units (PFU)/mL after calculating plaque numbers.

2.4. Viral DNA Analysis by Quantitative PCR

At 48 h post-infection, viral DNA from cell culture media was isolated using an AccuPrep® Viral DNA/RNA Extraction Kit (K-3033, Bioneer, Korea) and analyzed using AccuPrep® 2× GreenStar™ qPCR Master Mix. The viral DNA was amplified at 95 °C for 3 min, 95 °C for 15 s, 60 °C for 20 s for 40 cycles using a Step One Plus PCR system (ABI, USA). Viral DNA copy numbers were determined by PCR analysis with viral-specific primers for the major capsid protein (F, 5’-TTTCGTCGATCCGACAGC-3′; R, 5’-CTGGGTGAGCGTGAAGTTTA-3′).

2.5. Transmission Electron Microscopy (Bio-TEM)

Sample preparation for transmission electron microscopy (TEM) was performed as described previously [27]. In brief, HSV-1-infected Vero cells were harvested at 24 h post-infection and fixed in 0.25% glutaraldehyde in 50 mM phosphate buffer (pH 6.8) followed by post-fixation in 1% OsO4 overnight. The fixed cells were dehydrated at increasing ethanol concentrations from 30% to 99.9% ethanol and propylene oxide. The dehydrated cells were finally embedded in Embed 812 resin mixture. Ultrathin sections were stained with uranyl acetate and lead citrate. Viruses in HSV-1-infected cells were observed on a Zeiss EM10 transmission electron microscope installed in the center for University-wide Research Facilities (CURF) at Jeonbuk National University.

2.6. RNA Isolation and RT-PCR

Vero cells were infected with HSV-1 for 1 h and irradiated with BL (10 J/cm2) twice per day after aspirating the inoculum. At 24 h post-infection, total RNA was isolated from infected-cells using an AccuPrep® Universal RNA Extraction Kit (K-3140, Bioneer, Korea) according to the manufacturer's protocol. Two hundred nanograms of total RNA were reacted at 60 °C for 15 min for reverse transcription, and amplified at 95 °C for 5 min, 95 °C for 15 s, 56 °C for 20 s for 40 cycles using AccuPower® GreenStarTM RT-qPCR Premix (K-6403, Bioneer, Korea) with specific primers. The primer sequences used in the current study were as follows: HSV-1 UL19 F (5’-CTTCAAGATCAGTCCCGTGG-3′), UL19 R (5’-TTCTCAGTCACAAAGCGGTC-3′); UL27 F (5’-ACACAAGGCCAAGAAGAAGG-3′), UL27 R (5’-GGGAACTTGGGTGTAGTTGG-3′); UL52 F (5’-GGAGTTTGCCTACAGGTTCC-3′), UL52 R (5’-GACAGAATCTCCGCCGTTAG-3′); US6 F (5’-AACAACATGGGCCTGATCG-3′), US6 R (5’-CATCCAGTACACAATTCCGC-3′); GAPDH F (5’-ACTTTGGTATCGTGGAAGGAC-3′), and GAPDH R (5’-GCAGGGATGATGTTCTGGAG-3′).

2.7. Protein Extraction and Western Blot Analysis

Protein isolation and Western blot analysis were performed as described previously [26]. The cells were harvested in RIPA buffer and subjected to 10–12% SDS-PAGE gel. The subjected proteins were transferred to PVDF membranes, and the transferred blots were blocked in 5% a non-fat dry milk solution for 1 h. After incubation with specific primary antibodies overnight and horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h, protein band was visualized using enhanced chemoiluminescence (ECL) reagents (DG-WP250, DoGenBio, Korea) in a chemiluminescence detection system (Alliance Q9 Micro, Uvitec, UK). The relative intensity of the protein bands was quantified using Image-J software (NIH, MD, USA).

2.8. Immunofluorescence

The infected-cells were fixed in 4% paraformaldehyde (PFA), permeabilized in 0.2% Triton X-100 solution for 5 min and incubated in 10% conventional serum for blocking. After washing with ice-cold PBS three times, the cells were treated to ICP0 antibody at 4 °C overnight and Alexa Fluor 488-conjugated secondary antibody for 90 min. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 4 min. ICP0 expression was determined using a confocal microscope installed in the CURF at Jeonbuk National University.

2.9. ELISA

THP-1 cells (1.5 × 105 cells/well) were grown in 12-well plates and incubated with PMA (100 nM) overnight for M0 resting cells. For differentiation into M1 macrophages, the cells were treated with LPS (0.1 μg/ml) and IFN-γ (20 ng/ml) for 24 h. Following HSV-1 absorption, the cells were washed twice and incubated in fresh serum-free RPMI1640 medium. At 24 h post-infection, human IL-1β and IL-18 in the cell culture media were measured with the human IL-1β ELISA kit and human IL-18 ELISA kit according to the manufacturer's instructions.

2.10. Statistics

All data represent at least triplicates independent experiments and shown as the means ± standard error (S.E.). Unpaired two-tailed Student's t-test was used to evaluate statistical significance between groups. *P < 0.05, **P < 0.01.

3. Results

3.1. Anti-Viral Activity of Accumulated-BL Irradiation on HSV-1 Infection

Previously, we examined the inhibitory effect of BL on SARS-CoV-2 viral replication, which showed no cytotoxic effects. To assess the anti-viral activity of accumulated -BL irradiation on HSV-1 infection, we applied BL to HSV-1-infected cells twice daily, at 0 h and 24 h post-infection. As shown in Fig. 1 , BL irradiation reduced the number of plaque formed by HSV-1. BL irradiation twice significantly inhibited by 53% and 76.2% the viral titers (Fig. 1B), as well as viral genomic replication (Fig. 1C), respectively, compared to the control group. To investigate the specificity of BL, we further tested the effect of green light (GL) at a wavelength of 550 nm on HSV-1 viral replication. As shown in Fig. 1D, the viral copy number after GL irradiation was not decreased compared to the control group, indicating the specificity of BL's anti-viral activity.

Fig. 1.

Fig. 1

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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.

To confirm the ant-viral effect of BL on HSV-1 infection, TEM images were obtained. Results revealed that BL irradiation reduced the number of HSV-1 viral particles and conserved the nucleolus in HSV-1-infected cells. As shown in Fig. 2 , syncytium formation with neighboring cells, nuclear expansion, and nucleolus fragmentation were observed in HSV-1-infected cells. Additionally, some viral nucleocapsids adjacent to the nuclear envelope were present in the nucleus, and one nuclear capsid showed budding into the perinuclear space for delivery into the cytoplasm (red arrows). However, following accumulated- BL irradiation, the nucleolus was clearly conserved and viral nucleocapsids were not observed in the nucleus. Specifically, the number of autophagosomes in HSV-1-infected cells was increased after BL irradiation (blue arrows).

Fig. 2.

Fig. 2

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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.

3.2. BL Irradiation Inhibits Viral Genes and Replication-Related Proteins Expression

At 24 h post-infection, the RT-qPCR results showed that the mRNA level of viral early gene UL52 (helicase primase subunit), late gene UL27 (glycoprotein B), and leaky-late genes UL19 (major capsid protein VP5) and US6 (glycoprotein D) expression was considerably higher in HSV-1-infected cells than in normal uninfected cells. However, BL irradiation twice considerably decreased the levels of UL19, UL27, UL52, and US6 gene expression by 36.3, 29.1, 629.8, and 1981.0-fold, respectively, compared to HSV-1-infected cells (Fig. 3 ). In addition to viral gene expression, we further examined the anti-viral effect of BL on the expression of the viral replication-related proteins ICP0 and ICP4 in HSV-1-infected cells (Fig. 4 ). After HSV-1 infection, the ICP0 green signal was localized in the cytoplasm, but it was almost completely diminished after BL irradiation. The Western blotting data showed that the levels of ICP0 and ICP4 protein expression were considerably higher at 30 h − 48 h post-infection than in uninfected cells. Consistent with the immunofluorescence results, accumulated- BL irradiation decreased ICP4 and ICP0 protein expression by 20.4 and 20.8-fold, respectively, at 48 h post-infection, compared to HSV-1-infected cells.

Fig. 3.

Fig. 3

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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.

Fig. 4

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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.

3.3. BL Irradiation Inhibits Viral Absorption during HSV-1 Infection

To investigate whether BL could disturb viral entry into host cells, we irradiated cells with BL during viral absorption. As shown in Fig. 5A, 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. As a result, both Co-BL15 and Co-BL30 significantly inhibited by 88.1% and 65.9% the number of plaques formed and by 95% and 73.4% the viral copy numbers, respectively, compared to control group, showing the most effective treatment in the early phase of the infection (Fig. 5B and C). Additionally, the level of ICP0, ICP4, and gD protein expression was significantly inhibited by 0.99, 0.97, and 0.99-fold at Co-BL15 compared to HSV-1-infected cells (Fig. 5D). In addition to viral protein expression, the level of UL27, UL52, and US6 was almost completely lower in Co-BL15 group than in HSV-1-infected group (Fig. 5E). These results indicate that BL could play a role as a virucide by interfering with viral entry, subsequently inhibiting the viral replication and plaque formation of HSV-1. In deeds, the virucidal effect of BL was determined by pre-irradiating the HSV-1 virus with BL alone and then treating this virus to the cells. As a result, BL (10 J/cm2) irradiation was sufficient for HSV-1 virus inactivation as achieved by plaque assay and PCR (Supplementary Fig. 2).

Fig. 5.

Fig. 5

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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.

3.4. BL Irradiation Has an Anti-Inflammatory Effect on HSV-1-Infected M1 Macrophages

Since a number of viruses are known to activate inflammatory responses, we investigated whether BL irradiation could inhibit inflammatory mediators in macrophages after HSV-1 infection. Following treatment with LPS and IFN-γ, THP-1 cells were polarized toward M1-macrophages. As shown in Fig. 6A, the concentration of IL-18 and IL-1β was significantly increased in both HSV-1 infected- and uninfected-M1-macrophage, compared to M0 macrophages. However, increases in IL-18 and IL-1β were prevented by BL irradiation, indicating that BL irradiation could be used to treat inflammation caused by viruses. To determine how to inhibit the inflammatory response, we focused on DNA-sensing or binding molecules including cGAS/STING and interferon-inducible protein (IFI16) in HSV-1-infected M1-macrophages. As shown in Fig. 6B, the protein expression levels of cGAS, STING, and IFI16 in HSV-1-infected macrophages were significantly inhibited by 0.48, 0.95, and 5.02-fold after BL irradiation, compared to HSV-1-infected-M1 macrophages.

Fig. 6.

Fig. 6

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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.

4. Discussion

Advances in anti-viral treatments and agents have focused on targeting various dynamic factors in host cells by inhibiting viral entry or viral replication, and/or targeting the virus itself in infectious diseases. Regulating the immune response caused by a viral infection could be also a beneficial strategy for finding promising new anti-viral therapeutics. In the present study, we used two different approaches for inhibiting HSV-1 activation; to determine whether BL irradiation could inhibit viral replication and relevant biochemical components in replication cycles and/or regulate viral inflammation in HSV-1-infected cells. Our results showed that BL irradiation twice daily exerted significant anti-viral and anti-inflammatory effects in HSV-1 infected cells, shown by the inhibition of plaque formation and viral replication without cell toxicity. Additionally, TEM images showed the induction of autophagosomal formation in the cytoplasm after the BL irradiation of HSV-1-infected cells. Since the BL wavelength (450 nm) used in the present study could absorb GL, we further tested the anti-viral effect of a 550 nm GL wavelength in HSV-1-infected cells, which showed no inhibitory effects on plaque forming numbers and viral copy numbers. In addition to the specific inhibition caused by the BL wavelength, BL irradiation during viral absorption might have either inhibited of viral entry or induced a killing effect.

HSV-1 infection involves complicated steps consisting of viral attachment and penetration for viral entry, genomic replication and transcription, protein synthesis, assembly, and virion release [4]. The blockage of viral gene transcription is correlated with the inhibition of viral protein synthesis. To assess the anti-viral activity of BL in HSV-1 infection, we examined the transcription levels of several viral genes including UL19, UL27, UL52, and US6 in HSV-1-infected cells. The results showed that increases in UL19, UL27, UL52, and US6 gene expression were inhibited by accumulated- BL irradiation. Viral IE proteins ICP0 and ICP4 are commonly found on both the virion envelope and the surface of infected cells to help evade the host immune network. As a nuclear phosphoprotein, ICP4 plays a critical role in initiating downstream gene expression to drive viral replication. Especially, the DNA-binding domain (DBD), one of the major domains of ICP4, can induce DNA oligomerization to enhance DNA affinity or specificity for E or L genes, promoting transcriptional switching from the host genome to the viral genome by regulating host RNA polymerase II [[33], [34], [35]]. Extensive biochemical studies have shown that the mutation of ICP4 could disrupt DNA interaction by causing binding failure or lower affinity for DNA, subsequently negatively affecting the function of ICP4, along with viral replication [35,36]. To establish lifelong latency in infected hosts, ICP0 regulates the latent states of HSV-1 by the proteasomal degradation of cellular target proteins because ICP0 is encoded within the HSV-1 latency-associated locus [[37], [38], [39]]. Replication of the ICP0 null virus was rapidly suppressed by the innate immune response of host cells, indicating that ICP0 is essential for viral replication [39]. Both the expression levels of ICP0 and ICP4 were highly increased by HSV-1 infection, however, accumulated- BL irradiation decreased the expression levels compared to the controls.

To further investigate whether BL irradiation could affect viral entry during viral absorption, we set up an experimental irradiation schedule for BL. The results showed decreased numbers of plaques and viral copies by Co-BL15 and Co-BL30 irradiation, which were groups exposed to BL from 15 min (Co-BL15) or from 30 min (Co-BL30) after the onset of viral absorption, indicating either the inhibition of viral entry or a killing effect of BL irradiation. In addition, Co-BL15 showed the most effective inhibition of viral envelope gD protein and IE protein (ICP0 and ICP4) expression in HSV-1 infection, and inhibited the viral gene expression of UL27 and US6 compared to HSV-1 infected cells, suggesting the preventive potential of the BL irradiation at the early stage of infection. However, cells pre-irradiated with BL before viral absorption did not show significant changes in plaque numbers or viral replication compared to the HSV-1-infected group (Data not shown). From obtained above results, we presume that BL irradiation might have promising anti-viral therapeutic potential by specifically inhibiting viral replication, plaque formation, and viral protein expression at the early stage of infection.

One possible mechanism by which BL irradiation exerts its anti-viral effect in HSV-1 infection may be due to activation of autophagy. Autophagy is a catabolic process to maintain cellular function in host cells upon stimulation by viruses and other pathogens, although the role of autophagy is controversial as a double-edged sword during infection [40]. Functional autophagic responses can be beneficial for regulating viral replication, but they also act as host factors for inhibiting viral distribution by degrading viral proteins [41,42]. It is also evident that ICP34.5 polypeptide encoded by HSV-1 restricts host beclin-1, which is an autophagic marker that inhibits autophagy and supports viral replication. ICP34.5 mutant-HSV-1 was shown to have reduced pathogenicity in mice [43]. Previously, our results revealed continued induction of the autophagic process and prolonged activation of ER stress after the BL irradiation in SARS-CoV-2-infected cells [32]. As the difference between previous and present data relies on targeting intracellular components and viral species (e.g. DNA or RNA virus), BL irradiation could participate as an activator or repressor of autophagosomal conditions during viral infection. For instance, during SARS-CoV-2 infection, BL irradiation inhibited cell apoptosis and viral replication by stimulating survival pathways via the AKT-MAPK axis, leading to translational inhibition by increasing phosphorylated eukaryotic initiation factor-2 alpha (eIF-2α) caused by sustained ER stress, and consequently, inhibiting viral replication and the activation of SARS-CoV-2, although an experiment for viral entry was not performed. In this study, TEM images showed autophagosome formation in BL-irradiated cells after HSV-1 infection, indicating that BL could act as an activator of autophagy in the early phase of infection.

Our results also revealed that BL irradiation inhibited pro-inflammatory cytokines IL-1β and IL-18 known to be byproducts of inflammasome activation during HSV-1 infection. To investigate whether BL irradiation could inhibit the inflammatory response in clinical applications, we used the human THP-1 monocyte cell line and differentiated the cells into M1-polarized macrophages by treatment with LPS and IFN-γ before HSV-1 infection. The first step in response to pathogens is monocyte infiltration and macrophage activation to drive innate immunity in inflamed tissue [14]. Macrophages activated during viral infection can stimulate high levels of pro-inflammatory cytokines such as IL-1β, IL-18, IL-6, and TNF-α, and induce the activation of other immune cells, while the virus acquires the ability to evade and escape host immune responses [16]. IL-1β and IL-18 production was increased in M1-polarized macrophages irrespective of HSV-1 -infection but significantly inhibited after BL irradiation, indicating the anti-inflammatory effect of BL in viral infection. Specifically, BL irradiation also inhibited cGAS and STING expression in HSV-1 infection. The cGAS-STING signaling pathway has been implicated in infectious diseases by activating and producing type I IFN as host defense responses [18,19]. After HSV-1 infection, viral DNA can be recognized by various DNA-sensing factors including IFI16 and cGAS. Interaction with cytoplasmic cGAS and dsDNA allows a conformational change for synthesizing 2′3’ cyclic GMP-AMP (cGMP-AMP), a second messenger, subsequently activating STING and downstream cascade. Recent studies identified several critical functions of the cGAS-STING pathway, especially its role in anti-viral mechanism [19,44]. U37 of HSV-1 is a generally well-known protein for viral survival and the inhibition of type I IFN production. The deamidation of cGAS by U37 resulted in decreases in cGAMP synthesis and downstream innate immune activation, facilitating viral replication in cGAS-knock-out mice [44]. The ER membrane protein STING can be detected by cGAMP, and then oligomerized and phosphorylated with extensive conformational changes for translocation from the ER via ERGIC to the Golgi compartment in a process called trafficking [18]. The activated-STING mediates the recruitment of tank-binding kinase 1 (TBK1) and IFN-regulatory factor 3 (IRF3). Phosphorylated IRF3 via the STING-TBK1 complex leads to translocation into the nucleus for the gene expression of IFN-I and other pro-inflammatory cytokines by activating NF-κB signaling [17]. The activation of IFN results in the secretion of numerous pro-inflammatory cytokines. Paradoxically, our results showed decreases in cGAS and STING expression levels, as well as downstream NF-κB p65, in M1-polarized macrophages after BL irradiation in HSV-1 infection. The reason why BL irradiation decreased cGAS-STING expression upon HSV-1 might be that BL predominantly exerts anti-inflammatory effect in virus-infected macrophages. Indeed, the levels of pro-inflammatory cytokines and cGAS-STING expression were not significantly affected by HSV-1 infection, meaning that BL irradiation itself could inhibit macrophage activation and the relevant byproducts.

The inhibition of viral replication at an early stage of HSV-1 infection is obviously important for host cells. At the same time, the control of the early immune reaction is essential for inhibiting infectious progression. Our data revealed the biogoverning property of BL irradiation in viral activation and inflammatory responses by decreasing cytopathic effect and viral replication, as well as inflammatory cytokine production, in HSV-1 infection. The dual effect of BL irradiation could benefit patients with stomatitis and other skin eruption-causing pathogens (e.g. acne, enterovirus-71, and measles) alone or in combination with other therapeutics. The plaque-forming ability induced by enterovirus-71 was inhibited by BL irradiation (Supplementary Fig. 1), indicating anti-viral ability independent of the viral species, although the precise mechanism involved in the effect of BL irradiation has not been clarified for DNA or RNA viruses. The inhibition of viral entry or virucidal effects in the early stages of infection, and anti-inflammatory responses after BL irradiation could consequently reduce the pathogenic process period after viral infection, suggesting potential in clinical applications.

The following are the supplementary data related to this article.

Fig. S1.

Fig. S1

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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.

Fig. S2

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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.

Author Statement

Phil-Sun Oh: Conceptualization, Investigation, Methodology, Formal analysis, Visualization, and Writing-Original Draft; Phil-Sun Oh, Yeon-Hee Han, and SeokTae Lim: Validation, Formal analysis, Writing-Review & Editing; Hwan-Jeong Jeong: Conceptualization, Supervision, Funding acquisition. All authors accepted the final manuscript.

Declaration of Competing Interest

All authors declare that they have no conflicts of interest.

Acknowledgements

This Work was supported by National University Development Project in 2020.

Data availability

Data will be made available on request.

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