The Effect Of Herpes Simplex Virus Type 1 On Human Melanocytes And Its Mechanism

Background and Objective: Herpes simplex virus type 1(HSV-1)is an ubiquitous pathogen that principally diffuses between epithelial and neuronal cells.HSV-1 infects humans through skin or mucocutaneous regions and often induces latent infections of sensory neurons.In the case of a weakened immune system,it can cause clinical symptoms.The nectin-1 and human herpes virus entry mediator(HVEM)are two important cellular receptors that interact with envelope glycoprotein D(g D)of HSV-1.A cell surface inhibitor receptor called paired immunoglobulin-like type 2 receptor alpha(PILRA)works with the g B of the virus.HSV-1 is functionally linked to these receptors and binds to them to infect cells.Vitiligo,as a common clinical acquired depigmentation disease,is mainly characterized by the loss of functional melanocytes and melanin of the skin and/or mucous membranes.Vitiligo is a complicated and multifactorial disease,which often exerts considerable mental and psychological burdens upon its patients.Currently,the recognized pathogenic factors have been proposed for vitiligo including genetic,autoimmune,biochemical,oxidant-antioxidant,neural and possibly viral.However,the specific etiology is still unclear and needs to be further explored.Since the pathogenesis of vitiligo is still unclear and complex,many researchers have been trying to find other possibilities about vitiligo.On this basis,the relationship between various viral infections and vitiligo has been concerned.According to previous reports,Turkey herpesvirus(THV)was a strong environmental candidate for vitiligo in The Smyth line(SL)chicken,establishing a causative link between THV and SL vitiligo.The presence of cytomegalovirus(CMV)DNA in skin biopsies from patients with vitiligo suggested that vitiligo may be caused by CMV infection.In addition,the association between vitiligo and other viruses has also been reported,such as human immunodeficiency virus,hepatitis C virus,etc.HSV infection has also been studied in melanocytes of a mouse model.In an ex vivo infection study with murine skin samples,HSV-1 has been shown to invade the basal epidermal layer,infect murine melanocytes and aberrantly activate bulge stem cells.HSV-1 inhibited cell proliferation and induced apoptosis in a dose-dependent manner in a murine melanocytes Melan-A cell line.At the same time,HSV infection is a relatively important factor implicated in the occurrence and development of erythema multiforme.HSV-specific DNA was detected in keratinocytes of erythema multiforme lesions.Erythema multiforme is a self-limited inflammatory disease that primarily affects the hands,feet,and mucosal areas of the face,which is similar to acro-facial vitiligo.Whether HSV-1 plays a role in the onset of melanocyte-associated disease vitiligo,the mechanism remains to be established.In this study,the effects of HSV-1 on human epidermal melanocyte cell line with the underlying mechanisms were studied through a series of experiments,thus providing some novel insights into the pathogenesis and treatment of vitiligo.Methods: The subjects of this study were immortalized human normal epidermal melanocyte cell line PIG1 and immortalized human vitiligo melanocyte cell line PIG3 V.PIG1 was infected by a recombinant virus of HSV-1 gene and enhanced green fluorescent protein gene(r EGFP-HSV-1).Set different virus infection concentrations and time points.PIG1 cells and PIG3 V cells were used as controls.The groups were specifically named as: test group(PIG1 cells infected with HSV-1),negative control group(PIG1 cells,free of HSV-1 infection)and PIG3 V control(PIG3V cells,free of HSV-1 infection).To observe whether r EGFP-HSV-1 could infect PIG1 cells and morphological changes,the fields of vision were randomly selected to take photos by inverted fluorescence microscope.At the same time,the numbers and length of PIG1 cell dendrits were analyzed by Image-Pro Plus 6.0 software to detect the morphological changes.Melanin synthesis and tyrosinase activity were determined by NAOH melanin content measurement and tyrosinase activity assay.The expression of nectin-1 and melanosome structural protein gp100 on cells were detected by cellular Immunofluorescence assay.Real-time quantitative PCR and western blot were used to detect the proteins associated with melanin synthesis,such as microphthalmia-associated transcription factor(MITF),tyrosinase(TYR),tyrosinase-associated protein 1(TRP-1)and gp100.Isobaric tags for relative and absolute quantitation(i TRAQ)-based quantitative proteomics methods and western blot were used to detect differential proteins after HSV-1 infection.Gene knockdown was performed using RNA interference technique,and gene suppression was performed using inhibitor SCH772984.Data was analyzed using Graph Pad Prism version 8.0 software.The changes of cell dendrites,fluorescence intensity and western blot bands were analyzed using Image-Pro Plus 6.0.Data were analyzed by Unpaired two-tailed Student's t-test.Each experiment was repeated for at least three times.The data were presented as mean ± SD.P<0.05 was considered to be statistically significant.The pictures in this study were stitched using Photoshop 2019.Results: PIG1 cells were infected with r EGFP-HSV-1,morphological changes were observed under inverted microscope.After HSV-1 infection,PIG1 cells shrank and acquired a rounded shape,accompanied by decreases in the number and length of dendrites(compared to PIG1 cells free of HSV-1 infection).We found that PIG1 cells showed scattered and weak degree of fluorescence at 24 hours in response to all concentrations tested except for that of the 11:1 concentration,which did not begin to show fluorescence until 36 hours.The number of cells showing fluorescence and intensities of fluorescence at all concentrations were further increased from 48 hours to72 hours.The optimal HSV-1 infection condition was found with use of a concentration of 7:1 at 72 hours.Some cells died and floated to the surface of the medium with the extension of infection time exposures.HSV-1 induced inhibition of melanin content and tyrosinase activity at 72 hours in PIG1 cells.Specifically,cellular immunofluorescence assay indicated that the fluorescence intensity of gp100 in PIG1 cells infected with HSV-1 decreased compared to negative control group.RT-q PCR and western blot showed that the expression of MITF,TYR,TRP-1 and gp100 was decreased at both RNA and protein levels.To understand the mechanism by which HSV-1 infected PIG1 cells,we detected three important viral receptors(nectin-1,HVEM and PILRA)on both PIG1 cells and PIG3 V cells that were closely related to HSV-1 invasion.Cellular immunofluorescence assay confirmed the location of nectin-1 in two cell lines.Western blot showed that the expression of three receptors in PIG1 cells infected with HSV-1 decreased compared with PIG1 cells free of HSV-1 infection at 72 hours.Results from the i TRAQ assay revealed that 33 proteins showed a statistically significant differential expression,with 18 being downregulated and 15 upregulated in HSV-1 infected PIG1 cells.Among all the 33 proteins,VN1R5 changed most,being significantly increased in the test group at 72 hours,and our western blot also confirmed this phenomenon.Meanwhile,our western blot results showed that the expression of p-ERK and Nrf2/HO-1 was significantly decreased while the expression of p-p38 was increased at 72 hours in HSV-1 infected PIG1 cells.The p-JNK expression did not change significantly.VN1R5-si RNA1 was used to knock down VN1R5.Nrf2-si RNA1 was used to knock down Nrf2.The expression of ERK was inhibited with inhibitor SCH772984 at the concentration of 30 n M.The results suggested that knockdown of VN1R5 could increase the expression of p-ERK and MITF in test group.Inhibition of ERK reduced the expression of VN1R5,but had no effect on the expression of Nrf2 in test group.Knockdown of Nrf2 increased the expression of p-ERK in test group.Knockdown of VN1R5 had no effect on Nrf2,nor did the knockdown of Nrf2 affect VN1R5 in test group.Conclusion: 1)HSV-1 could infect human normal melanocytes,causing pyknosis and roundness of cell morphology with their dendrites showing significantly decreased lengths and numbers.The optimal infection condition was culture medium: virus suspension = 7:1,and the time point was 72 hours.Receptors of HSV-1 named nectin-1,HVEM and PILRA were confirmed to exist on both PIG1 cells and PIG3 V cells.Compared with the negative control group,the protein expression of the three receptors was significantly decreased in the test group,suggesting that they might play some roles in the process of virus infection.HSV-1 infection on PIG1 cells induced inhibition of melanogenesis.Compared with the negative control group,the RNA and protein levels of MITF,TYR,TRP-1 and gp100 in the test group and PIG3 V control were significantly decreased.2)Compared with the negative control group,the protein expression of VN1R5 in the test group was up-regulated,the protein expression of Nrf2/HO-1 in the test group was down-regulated at 72 hours.The protein expression of p-ERK in the test group decreased gradually with the duration of HSV-1 infection,while the expression of p-p38 protein increased gradually.3)HSV-1 reduced melanin production through the VN1R5/ERK pathway.In this process,ERK had a protective feedback on VN1R5 to resist HSV-1 infection.ERK/Nrf2 pathway didn't participate in the biological process of HSV-1 infection on PIG1 cells.VN1R5 was not associated with Nrf2 in the test group.This study showed that HSV-1 can infect human epidermal melanocyte,and this process may be related to the VN1R5/ERK pathway.