Herpes simplex virus-induced upregulation of inflammatory cytokines in human gingival fibroblasts

Abstract

Herpes simplex virus type 1 (HSV-1) is the leading pathogen in the maxillo-facial region, affecting millions of individuals worldwide. Its periodic reactivation aligns with the most common course pattern of periodontal disease. The present study used RNA sequencing to investigate the transcriptomes of human gingival fibroblasts (HGFs) following HSV-1 infection from the early to late stages (12–72 h). At the early stage of infection (12 h post-infection), the most upregulated genes were interferon (IFN) regulatory factor family members, toll-like receptor (TLR) family members, IFN-β1, interleukin (IL)-1, C-C motif ligands, chemokine (C-X-C motif) ligands (CXCLs), and tumor necrosis factor (TNF). The strongest differential expression was observed in TNF, nucleotide-binding oligomerization domain-like receptor (NLR), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways. At the late stage of infection, the most upregulated genes were CXCLs and ILs. The differentially expressed genes were divided into nine clusters, according to the time series expression trend. Next, the prominent activation of TLRs, retinoic acid-inducible gene I-like receptor signaling, NLRs, and downstream IFNAR-JAK-STAT signaling pathways were observed via a modified HSV-1 infection map. The HSV-1-induced upregulation of inflammatory cytokines in HGFs may drive inflammatory processes in periodontitis. The dynamic variations in mRNAs in HGFs from the early to late stages after HSV-1 infection can provide an analytical framework for determining the host anti-viral defense response to antagonize HSV-1 infection in periodontal tissues.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12985-024-02595-5.

Introduction

Periodontitis is an inflammatory condition which leads to the degeneration of the periodontal ligament and alveolar bone. Approximately 50% of the global population is affected by periodontitis[1]. Importantly, periodontitis is the leading cause of tooth mobility and tooth loss worldwide[2, 3]. In addition to local factors (such as plaque), various systemic diseases and conditions, such as pregnancy, diabetes mellitus, cardiovascular disease, dementia, rheumatic fever, stroke, and cancer, are associated with periodontitis[4–8]. Furthermore, the pathogenesis of periodontitis remains complex and multifactorial, in which various systemic, environmental, and genetic factors contribute to its development[9]. In terms of the oral environment, specific microbial species present in the oral flora (such as Porphyromonas gingivalis and active herpesviruses) and proinflammatory cytokines are the main determinants of severe periodontitis[8]. For instance, active herpesviruses induce a powerful stimulus for dysbiosis, and facilitate the overgrowth of key pathogens associated with periodontitis.

Human herpes simplex virus (HSV) belongs to the alpha (α) subfamily of Herpesviridae, and can be classified as HSV-1 and HSV-2[10]. HSV-1 and HSV-2 comprise of a linear double-stranded DNA genome, which is shielded by an icosahedral capsid enclosed by tegument (a proteinaceous layer), and enveloped in viral glycoproteins[11]. There is a high global prevalence of HSV-1 (67%) and HSV-2 (13%) in humans[12]. Furthermore, most people acquire HSV-1 early in their life through the orolabial mucosa[11]. The complex interaction between HSV and the host immune system determines the consequences of viral infections[8, 13]. Since HSV can infect any body organ or system, a potential linkage among HSV, periodontitis, and various systemic diseases can be established.

On the other hand, periodontal herpesviruses may infect other organs through systemic circulation. For example, HSV infection may enhance the risk of cardiovascular diseases associated with periodontal pathogens[14]. Furthermore, HSV may lead to latent infections, where periodontitis may be aggravated upon periodic reactivation[10].

Barroso et al. compared the prevalence of the Herpesviridae family virus between periodontally healthy subjects (6%) and subjects who had stage II, III, and IV periodontitis (60%). The prevalence of HSV-1 DNA was reported in all periodontitis patients, regardless of the stage and grade[15]. Furthermore, a recent meta-analysis revealed the association of HSV-1 with periodontitis[16]. Moreover, HSV-1 viral DNA can be detected in periodontal and necrotic pulpal tissues[17]. These findings indicate the vital pathological role of HSV in the pathogenesis of periodontitis.

During the primary HSV infection, the virus infects skin or mucosal epithelial cells, followed by latency in neurons, primarily those of the peripheral nervous system[11]. The major hallmark of HSV-1 is its ability to modify the metabolism and content of cellular RNA at multiple levels. The activation of various host pathways and progression of viral infection would lead to cellular heterogeneity. However, this hypothesis remains not fully understood, and requires further investigation. Human gingival fibroblasts (HGFs) are among the most abundant cells in gingival tissues. HGFs could not only respond to oral bacteria that penetrate the epithelial barrier or cytokines that are present in inflamed gingival tissues, but may also adopt imprinted proinflammatory phenotypes, contributing to the chronicity of inflammation[18]. HGFs have been confirmed the ability to drive inflammatory processes in periodontitis[19, 20]. Qin et al. reported that Bacterial lipoteichoic acid and lipopolysaccharide can upregulate the expression of heparin sulfate, which may promote the entry of herpesvirus-8 into the periodontal ligament and HGFs[21]. Although the scientific literature suggests that HSV-1 has a significant influence on the pathogenesis of periodontal diseases, little is known on how periodontal tissues react to virus invasion.

The present study aimed to determine how HGFs respond to HSV-1 invasion during the progression of infection. Furthermore, the deep transcriptomes of HGFs harvested before and from the early to late stages after HSV-1 infection were profiled. The present study provides insight through an analytical framework and investigates HSV-infected periodontal tissues through RNA sequencing (RNA-Seq). In addition, the relationships between the infection course, and specific host cell genes and pathways were predicted via unspliced messenger RNA (mRNA).

Materials and methods

Experimental ethics

The present research was conducted after ethics approval was obtained from the Institutional Ethical Review Board of the Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, China (Ref no: SH9H-2019-T76-2). The present study conformed to the STROBE guidelines. The study protocol was explained to all recruited participants, and an informed written consent was obtained from each study participant. The ethics guidelines of the Declaration of Helsinki were followed.

Cell culture

Five healthy subjects (two female and three male subjects) donated HGFs. The detailed inclusion and exclusion criteria are supplied in Table S1. These subjects underwent gingivectomy during the surgical extraction of their third molar. To extract the HGFs, the gingival tissues were gently rinsed with ethanol (80% v/v) for 30 s and Dulbecco’s phosphate-buffered saline. For digestion, the gingival tissues were incubated overnight in Dulbecco’s modified Eagle’s medium (DMEM; SH30243.01, HyClone; + 1% pen/strep), which contained 2.5 g/L of dispase II (Solarbio, Beijing, China) and 10% fetal bovine serum (FBS; 10,099,141, Gibco). To grow fibroblasts, the connective tissue was isolated and cut into small Sects. (1 mm³) before being transferred to a T25 cell culture flask that contained 1 mL of DMEM (2 mM L-glutamine, + 10% FBS, + 1% pen/strep, and + 1% non-essential amino acid). Then, the fibroblasts were split in a T75 cell culture flask after approximately two weeks, and cultured until further experimentation for HSV-1 infection. The cultures were tested to confirm that these were negative for infections, including HSV, mycoplasma, bacteria, yeast, and fungi. Next, the HGFs were cultured at 37 °C in a humidified incubator with 5% CO₂. When the cell density reached approximately 80–90%, the cells were passaged on a sterile workbench. The cells at the third to sixth logarithmic growth phase were used for further experiments.

Infection of HGFs with HSV-1

The HSV-1 strain F was supplied by Dr. Ling Zhao (Huazhong Agricultural University, Wuhan, China). In order to prepare the virus stocks, 100% confluent monolayers of Vero cells were infected and incubated in DMEM, which contained 2% FBS (Gibco). These were infected with HSV-1 at a multiplicity of infection (MOI) of 0.1. After 60 min, the cells were washed thrice with phosphate-buffered saline (PBS), and incubated in DMEM supplemented with 2% FBS for variable time periods (12, 24, 36, 48, and 72 h, respectively) before collection. Successively, the cultured cells were transferred into sterile 2 mL Eppendorf tubes, and centrifuged for 10 min at 12,000 × g. Then, the supernatant was collected. The virus titration information for variable time periods (12, 24, 36, 48, and 72 h, respectively) was provided in Table S2.

Immunofluorescence assay

The HGF monolayers were infected with HSV-1 at an MOI of 0.1 for one hour at 37 °C, and washed thrice with PBS. Then, fresh medium was added to the cells, which were incubated for specific time points at 37 °C. Next, the cells were rinsed with PBS before being fixed in cold methanol at − 20 °C for 10 min. Then, these cells were treated with Tween-20 (0.1%) in PBS for five minutes to permeabilize the cells at room temperature. Afterwards, the cells were blocked with 5% bovine serum albumin in PBS (60 min, 37 °C), and incubated at 37 °C for one hour with the mouse anti-HSV1 gD monoclonal antibody (Abcam) at a 1:50 dilution of PBS and 1% bovine serum albumin. Subsequently, these cells were rinsed with PBS thrice prior to incubation in the dark for one hour using the fluorescently labeled secondary antibody[22]. Finally, the nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; C1002, Beyotime Biotechnology), and analyzed under a fluorescence microscope with an objective lens (Carl Zeiss, Oberkochen, Germany).

RNA-Seq

For the RNA-Seq, TRIzol reagent (Invitrogen Life Technologies) was used to isolate total RNA. The NanoDrop spectrophotometer (Thermo Scientific) was used to determine the quality and integrity of the samples. The NovaSeq 6000 platform (Illumina, Shanghai Personal Biotechnology Cp. Ltd.) was used to sequence the generated sequencing libraries. The sequence information of the present project was submitted to the National Center for Biotechnology Information BioSample database (Accession number: PRJNA1062757).

Bioinformatics and statistical analysis

After quality control, the HISAT2 (v2.1.0) computer software was used to map the filtered reads to the reference genome. The read count values for each gene (as an original expression) were compared via HTSeq (v0.9.1) statistics. Then, the expression was calibrated via the fragments per kilobase of exon per million mapped reads method. The differentially expressed genes (DEGs) were determined via DESeq (v1.38.3) under the following screening conditions: expression difference multiple |log2FoldChange|> 1 and significant p-value < 0.05. Simultaneously, the bi-directional clustering analysis of various genes in the samples was performed via the R language Pheatmap (v1.0.12) software. All genes were mapped to terms in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and Gene Ontology (GO) database, and the number of diversely enriched genes for each term was computed. The GO enrichment analysis for differential genes (all DEGs/upregulated DEGs/downregulated DEGs) was conducted via top GO (v2.50.0), which identified the GO term with remarkably enriched differential gene key biological functions that corresponded to the differential genes. The enrichment analysis of the KEGG pathways of the DEGs was conducted via the ClusterProfiler (v4.6.0) software, which focused on the significantly enriched pathways (p-value < 0.05).

Results

Human gingival fibroblast mRNA profiles changed with time during HSV-1 infection

In order to evaluate the effectiveness of the HSV-1 infection on periodontal tissues, HGFs were infected at an MOI of 0.1, and total RNA-Seq was performed at various post-infection intervals (12, 24, 36, 48, and 72 h) with three biological replicates (Fig. 1A). Then, the mRNA was quantified, and the expression of a total of 13,378 genes was detected (Fig. 1B). The principal component analysis revealed sound clustering in the samples at each time point. The different time points were clearly isolated, suggesting the good reproducibility of the samples within each group (Fig. 1C).

Fig. 1.

Characterization of HSV-1-infected human gingival fibroblast mRNAs. A HSV-1-infected human gingival fibroblasts (MOI = 0.1) at different post-infection intervals of 12, 24, 36, 48, and 72 h. Cellular labeling was accomplished via the use of specific antibodies. DAPI (4,6-diamidino-2-phenylindole) labeling was used to label the cellular nuclei, and the fluorescent signals were monitored via a confocal immunofluorescence microscope (scale bars = 50 μm). B Overview of the mRNA expression via the UpSet plot. C Principal component analysis of the common set of sequenced genes at different time points, at post-infection (12, 24, 36, 48, and 72 h)

Stage-specific mRNA expression alterations in HSV-1-infected HGFs

In order to determine the HGF gene expression changes after HSV-1 infection, and the differences between the late and early stages, DEG analysis was conducted using |log2FoldChange|> 1 and p-value < 0.05 at 12, 24, 36, 48, and 72 h, post-infection vs. mock infection, and at 24, 36, 48, and 72 h, post-infection vs. 12 h, post-infection (Fig. 2A and Table S3). A total of 9,841 DEGs were identified in all the comparisons. The Venn diagram of the DEG matrix was used to analyze the co-DEGs. That is, the DEGs among groups were compared to non-infected cells (Fig. 2B). The UpSet plot depicted the remarkable differences in mRNAs that occurred between the various infection time points and control groups (Fig. 2C).

Fig. 2.

Stage-specific alterations in the mRNA expression of HSV-1-infected human gingival fibroblasts (HGFs). A Overview of differentially expressed genes (DEGs) identified in the present study. In order to determine the inflammatory cytokine time-variant changes in HGFs, the mRNA changes in HSV-1 infection at various time points were compared to non-infection controls, as well as to cells at the early stages. B and C) The co-DEGs and specific DEGs at various infection time points vs. non-infected cells are depicted via the Venn diagram B and UpSet plot C. (D–H) The DEGs at different time points, post-infection (12, 24, 36, 48, and 72 h) vs. non-infected cells are depicted via the volcano plot (LDA score [log10] > 1 and p-value < 0.05 were considered significant). I Distribution of differentially expressed transcription factors at 12 h, post-infection

Compared to the mock infection cells, 2,685 DEGs, which included the interferon (IFN) regulatory factor (IRF) family members (IRF3 and IRF7), toll-like receptor (TLR) family members (TLR2, TLR3, and TLR7), myeloid differentiation factor 88 (MyD88), IFN-β1, tumor necrosis factor (TNF), B-cell scaffold protein with ankyrin repeats (BANK1), chemokine (C-X-C motif) ligand 11 (CXCL11), interleukin-1α (IL-1α), IL-1β, gamma-IFN-inducible protein 16 (IFI16), and C-C motif ligand 5 (CCL5), were upregulated at 12 h post-infection. Furthermore, 1,501 DEGs, such as mitochondrial antiviral signal proteins (MAVS), were downregulated (Fig. 2D and Table S4). Moreover, the expression of TNF superfamily members, IRF, and interferon-inducible protein genes was significantly upregulated at the early stage of infection (12 h, post-infection). Other mRNA expression alterations were identified via volcano plots (Figs. 2E-2H). At the late stage (72 h, post-infection), there was a marked upregulation of CXCLs, IFNβ1, and IL11, while the tripartite motif-containing (TRIM) genes were downregulated. Next, the distribution of differentially expressed transcription factors (TFs) was analyzed (Figs. 2I and S1). A number of DEGs were identified to be regulated by TFs in the TRRUST database. Compared with those in control cells, both downregulated and upregulated TFs were enriched in the zf-C2H2, bHLH and homeobox families at 12 h post-infection. In addition, the upregulated TFs were enriched in the IRF and TF_bZIP families.

In order to more intuitively observe the DEGs after HSV-1 infection, the development trend of HGF activity over time was analyzed through expression clustering. The analysis results of the two-way clustering heatmap were used (Fig. S2), and these genes were further divided into different clusters (nine clusters in the present study) on the basis of the similarity of gene expression patterns (Fig. 3). Notably, most of the DEGs belonged to cluster 1, such as those that encoded TGF-β2, TANK binding kinase 1 (TBK1), BANK1, MAVS, and metalloproteinase 8 (MMP8). These genes were remarkably downregulated at the early stage of HSV-1 infection. Then, these were upregulated over time, after which these remained stable in the late stage. The DEGs of cluster 2, cluster 5, and cluster 8 increased during the initial period of viral infection, but gradually decreased from 24 to 72 h, post-infection. Furthermore, IL-23α, IRF1, IRF2, IRF3, JAK2, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and TLR3 belonged to cluster 2, while signal transducer and activator of transcription 1 (STAT1), IL-7R, fibroblast growth factor 7 (FGF7), IFI16, and IL-1R1 belonged to cluster 8. The DEGs in cluster 7, such as CXCL8, were rapidly upregulated during the first 12 h, but the expression subsequently decreased. The expression remained upregulated, when compared to that in noninfected cells. Furthermore, the mRNA expression levels of cluster 9, which included IL-6, IL-11, CXCL2, CXCL3, CXCL5, and FGF2, continuously increased over time within the first 36 h post-infection, and this trend tended towards stability.

Fig. 3.

The time series of mRNA expression trends in HSV-1-infected HGFs. The differentially expressed genes (DEGs) were divided into nine clusters in the present study A–I on the basis of the similarity of gene expression patterns. Genes with greater expression correlations in the samples were ordered into one category. The values represent the FPKM values averaged over the samples of DEGs with Z score normalization

HSV-1-associated stage-specific changes in HGF function profiles

In order to determine the functional alterations in HSV-1-infected HGFs, DEG functional annotation was conducted via the KEGG database. Figure 4 presents the significant enrichment of the KEGG pathways of DEGs at 12 and 72 h, post-infection. This may determine the main biological functions that DEGs perform at the early and late infection stages. At 12 h, post-infection, the pathways, such as the NF-κB signaling pathway and cytokine-cytokine receptor interaction signaling pathway, were significantly activated (Fig. 4A). In particular, the upregulated DEGs were markedly enriched in the apoptosis, necroptosis, and TNF signaling pathways, the cytokine-cytokine receptor interaction pathway, the JAK-STAT signaling pathway, the viral protein interaction pathway with cytokines, the NF-κB signaling pathway, the cytokine receptor and cytokine-cytokine receptor interaction pathway, the TLR signaling pathway, the nucleotide-binding oligomerization domain-like receptor (NLR) signaling pathway, and the antigen processing and presentation pathway (Fig. 4B). For example, there were 47 upregulated genes and three downregulated genes in the NF-κB signaling pathway, while 34 upregulated genes and two downregulated genes were identified in the TLR signaling pathway (Table S5). It is noteworthy that the downregulated DEGs were markedly enriched in the HSV-1 infection pathway and cell cycle (Fig. 4C). However, at 72 h, post-infection, the upregulated DEGs were significantly enriched in the HSV-1 infection and antigen processing signaling pathways (Figs. 4D-4F). The enriched KEGG pathways of DEGs at other infectious time points are presented in Supplemental figures S3, S4, and S5. The DEGs were markedly enriched in the MAPK signaling pathway and calcium signaling pathway at 24 h, post-infection, in the IL-17 signaling pathway at 36 h, post-infection, and in the TGF-β signaling pathway at 48 h, post-infection.

Fig. 4.

HSV-1-associated stage-specific changes in HGF function profiles. A–C Functional enrichment analysis to establish key biological functions via differentially expressed genes (DEGs) at 12 h, post-HSV-1 infection vs. non-infected cells, as determined via the KEGG database. A For all DEGs. B The upregulated DEGs. C The downregulated DEGs. D–F KEGG enrichment analysis of DEGs at 72 h vs. non-infected cells. D For all DEGs. E The upregulated DEGs. F The downregulated DEGs

The HSV-1-associated host antiviral signaling pathway was altered in HGFs

HSV-1 has progressed through different approaches to evade the host antiviral innate immunity and cellular survival-associated pathways. In order to downregulate the signal transduction, HSV-1 proteins indirectly or directly target adaptors in antiviral innate immunity signaling pathways, which include the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling and TLR signaling pathways[23]. The HSV-1-associated changes in DEGs associated with the enriched KEGG pathways at various infection stages were further analyzed. The variations in gene abundance are presented in a pathway representation (Fig. 5). Modified KEGG pathway maps were used to manually construct the pathway modules, according to map05168 (HSV-1 infection) in the KEGG database (Fig. 5A).

Fig. 5.

Alterations in the HSV-1-associated host antiviral signaling pathway in HGFs. A The pathway modules were modified from the KEGG pathway maps according to map05168 (HSV-1 infection), and these modules were created through BioRender.com. The symbols in red font present the viral proteins of HSV-1. B The relative abundance changes of genes in the toll-like receptor signaling pathway. C The relative abundance changes of genes in the retinoic acid-inducible gene I-like receptor signaling pathway. D The relative abundance changes of genes in the antigen processing and presentation signaling pathway. E The relative abundance changes of genes in the IFNAR-JAK-STAT signaling pathway. The relative gene abundance was evaluated for significant elevation or depletion at different time points, post-infection (12, 24, 36, 48, and 72 h) vs. non-infected cells via the LDA score [log10] > 1 and the significant p-value < 0.05. The bar plots present the FPKM values averaged over the samples of DEGs with Z score normalization. *p < 0.05, **p < 0.01, ***p < 0.001

In the TLR signaling pathway (Fig. 5B), the present results revealed that both the levels of surface proteins TLR2 and TLR3, and the level of TLR7 in endosomes significantly increased at 24 and 48 h, post-infection. These TLRs can sense HSV-1, and transduce signals through MyD88 and TRIF, which activate IRFs and NF-κB. Furthermore, the MyD88 and IRF5 mRNA expression markedly increased after HSV-1 infection, inducing the production of IL-6 and IL-8 for antiviral immunity in HGFs. In addition, the IL-1β expression gradually increased after HSV-1 infection until 72 h, post-infection.

In the RLR signaling pathway (Fig. 5C), the expression of melanoma differentiation-associated gene 5 (MDA5) and RIG-I exhibited an instant significant increase in HGFs after HSV-1 infection. However, the expression level slightly decreased with the increase in infection time. Interestingly, the mRNA expression of downstream adaptor protein MAVS decreased, and reached the lowest point at 48 h. This value subsequently increased at the late stage of viral infection. However, the expression level remained lower than that in uninfected cells. The HSV-1 infection induced an immediate increase in the mRNA expression of IRF3, IRF7, and NF-κB, and this remained at elevated levels for up to 72 h. The expression levels of IκB kinase (IKK)-related kinases IKKε and TBK1 exhibited a declining trend after an initial increase, and this increased after viral infection. The IFN-β and TNF-α expression was induced for antiviral immunity, which rapidly increased during the first 24 h, and subsequently and consistently decreased at 72 h. For the antigen processing signaling pathway (Fig. 5D), the analysis results revealed that the TAP1/2 mRNA expression remarkably increased after viral infection, but this slightly decreased with the infection time. The IFNAR-JAK-STAT signaling pathway (Fig. 5E), as the downstream of the NLR sensor signaling pathway, plays a vital role in restricting the systemic spread of HSV-1 through type I interferons (IFN-I). These findings reveal that the key host factors (IRF9, STAT1, and STAT2) were obviously upregulated in HGFs at different post-infection time points, while IFNAR2 was significantly upregulated at 36–72 h, post-infection.

Discussion

HSV-1 is associated with periodontitis, as reported by the recent National Health and Nutrition Examination Survey[24]. HGFs are abundant in periodontal tissues, and contribute to the pathogenesis of periodontitis by responding to associated pathogens or inflammatory cytokines. The present results revealed the dynamic variations in mRNAs in HGFs from the early to late stages of post-HSV-1 infection via RNA-Seq. The relationships between the infection course, and specific host cell genes and pathways were analyzed, providing information on the host anti-viral defense response to antagonize HSV-1 infection in the periodontal environment.

The virulence of pathogenic microbes, along with other factors, including demographic, immune, and environmental factors, determines the progression of periodontitis. Herpesviridae contributes to the development of periodontitis and tissue degeneration through various mechanisms, such as facilitating the colonization of pathogenic microorganisms, decreasing the effectiveness of the host’s immune response, altering inflammatory mediators, and producing antibodies to combat neutrophils that lead to secondary infections[15, 25]. Since HSV-1 DNA can be detected in periodontitis tissues, regardless of the stage and grade[15], HSV-1 and HGFs were selected for the present study to determine the response of periodontal tissues to viral invasion. There is evidence that HSV-1 alters the content and metabolism of RNA at the cellular level[26]. The synthesis of cellular proteins is inhibited through the activation of viral genome transcription, which interferes with splicing regulation. Similarly, viral exposure affects various cellular pathways, which may lead to the repression of the downstream or transcriptional activation of target genes[27, 28].

The dynamic process of viral infection and pathogenesis is driven by the complex interaction of antiviral cellular pathways and viral virulence[26, 29, 30]. HSV-1 can exist for a long time in humans, and the battle between the host immune system and virus never stops. Innate immunity represents the first line of host defense by limiting the viral spread, and regulating the adaptive response activation[23]. However, few scientific studies have reported the pathogenesis of periodontal tissues and its immune reactions to HSV-1. The present study revealed the prominent activation of the apoptosis, necroptosis, TNF, NF-κB, JAK-STAT, TLR, viral protein interaction with cytokines, and NLR signaling pathways at 12 h, post-infection. Furthermore, studies have reported that incoming HSV-1 virions activate cellular pathways, such as TLRs, RLRs, and NLRs, while downstream of the IFNAR-JAK-STAT signaling pathway, which may ultimately induce the production of inflammatory interferons and cytokines[28, 31]. Moreover, host pathogen recognition receptors (PRRs) may identify viral pathogen-associated molecular patterns (PAMPs), which trigger the activation of IRFs and NF-κB through associated signaling pathways[32]. These TFs moderate the IFN-I expression (13 subtypes of IFN-α and IFN-β) in humans, which in turn induces the expression of IFN-stimulated genes (ISGs)[29, 32–36]. Hypothetically, host immunity responds to primary viral infection, in order to limit viral infection and replication. However, HSV-1 has a strong immune evasion capability, and establishes a latent phase by evading and subverting host immune surveillance[23].

In HSV-1-infected HGFs, both the levels of TLR2 and TLR3 on the cell surface, and the level of TLR7 in endosomes significantly increased at 24 and 48 h, post-infection. The ability of TLR families to recognize HSV-1 PAMPs has been well-demonstrated. Various pathways, including the TLR2, TLR3, TLR4, TLR7, and TLR9 pathways, contribute to the recognition of HSV-1, and the induction of IFN-I throughout the viral entry and replication process[37, 38]. The signal transduction of TLRs is dependent on various MyD88 proteins (such as TLR1, 2, 7, and 9) or TRIF proteins (such as TLR3 and 4)[32]. Furthermore, the mRNA expression of MyD88 remarkably increased after HSV-1 infection, and remained high until the late stage of viral infection. In another key signaling pathway, RLR, RIG-I, and MDA5 recruit the MAVS protein present at the external mitochondrial membrane, which, upon activation, mediates signal transduction[23]. These results reveal that the expression of RIG-I and MDA5 is significantly elevated in HGFs after HSV-1 infection. However, this expression level slightly decreased with the increase in infection time. Furthermore, in HSV-1-infected HGFs, the degree of TBK1 and IKKε expression tended to decrease after the initial increase, and this ultimately increased after viral infection. The HSV-1 infection led to the significant increase in mRNA expression of IRF3, IRF7, and NF-κB, which remained at high levels for up to 72 h. PRRs recognize viral ligands to initiate a cascade of reactions, which eventually converge upon TBK-1/IKKε activation[26]. This process further continues with the phosphorylation and activation of IRF3 or IRF7, along with other transcription factors, thereby stimulating the expression of proinflammatory cytokines and IFN-I through HGFs. The HSV-1-induced upregulation of inflammatory cytokines in HGFs may drive inflammatory processes in periodontitis.

In order to determine the unidentified biological associations between genes via expression clustering, genes with a high expression correlation between specimens were ordered into one category. In general, these genes are involved in specific biological procedures, and certain metabolic or signaling pathways. It has been reported that the tegument protein US3 virally encodes a kinase that blocks the nuclear accumulation of NF-κB at the initial stage of infection, which leads to the inhibition of the TLR2 signaling pathway, and the reduction in levels of chemokine CCL2 and inflammatory cytokines, such as IL-6 and IL-8[39]. In the present study, the expression levels of IL-6 and IL-8 were remarkably upregulated at all stages. Interestingly, for HGFs, the mRNA expression of CCL2 in cluster 5 exhibited an immediate increase at 24 h, post-infection, and this steadily decreased up to 72 h, post-infection. Furthermore, the MAVS mRNA expression decreased, and reached the lowest point at 48 h, but this increased at the later stage of infection, indicating the potential of HSV-1 proteins to capture the RLR and TLR signaling pathways, and multiple downstream steps.

In addition, the IRF9, STAT1, and STAT2 expression levels were obviously upregulated in HGFs at post-infection. However, the IFNAR2 expression started to increase at 48 h after infection. IFN-I binds to cognate receptors (IFNAR1 and IFNAR2), which trigger the JAK-STAT pathway, and inhibit antiviral activities. IFNAR1/IFNAR2 induces the direct phosphorylation and heterodimerization of STAT1/STAT2 by recruiting and phosphorylating tyrosine kinase 2 and JAK1[40]. Furthermore, this produces the IFN-stimulated gene factor 3 complex by attaching to IRF9, which in turn engages the ISRE to promote the transcription of various ISGs.

Our understanding of the pathophysiology of viral periodontitis can be further enhanced through ample evaluation of viral evasion strategies and the host’s antiviral innate immunity, which may facilitate the exploration of novel drug targets and immune-therapeutic modalities for the management of HSV-1-associated periodontitis. It remains unclear whether the findings related to the gene expression profiles of HGFs can be generalized to other types of fibroblasts or periodontal tissues. Hence, further investigations are needed.

Conclusion

In conclusion, the present study revealed that HSV-1 induced the upregulation of inflammatory cytokines in HGFs, which may drive inflammatory processes in periodontitis. The functional analysis suggested that HSV-1 may have periodontitis pathogenic potential through the modification of HGF inflammatory mediators, tissue degeneration, and damage to the efficacy of the host immune response. Classic host signaling pathways and countermeasures are vital for antiviral immunity, since these restrain the HSV infection in the periodontium. The dynamic variations in mRNAs in HGFs from the early to late stages after HSV-1 infection can provide an analytical framework for investigating the host anti-viral defense response to antagonize HSV-1 infection in periodontal tissues.

Abbreviations

HSV

Human herpes simplex virus

HGF

Human gingival fibroblasts

MOI

Multiplicity of infection

PBS

Phosphate-buffered saline

DEG

Differentially expressed gene

KEGG

Kyoto encyclopedia of genes and genomes

GO

Gene ontology

IFN

Interferon

IRF

Interferon regulatory factor

IFI

Interferon-inducible protein

IFN-I

Type I interferon

TLR

Toll-like receptor

MyD88

Myeloid differentiation factor 88

TNF

Tumor necrosis factor

IL

Interleukin

MAVS

Mitochondrial antiviral signal protein

TFs

Transcription factors

TBK1

TANK binding kinase 1

NF-κB

Nuclear factor kappa-light chain enhancer of activated B cells

NLR

Nucleotide-binding oligomerization domain-like receptor

RIG-I

Retinoic acid-inducible gene I

RLR

Retinoic acid-inducible gene I-like receptor

MDA5

Melanoma differentiation-associated gene 5

CCL

C-C motif ligand

CXCL

C-X-C motif ligand

Author contributions

Conceptualization: Y.Z., X.F., J.N., X.C.; Formal analysis: Y.Z., X.F., J.N.; Funding acquisition: Y.Z.; Investigation: Y.Z., K.L., C.W., G.Z; Methodology: Y.Z., K.L., C.W.; Software: Y.Z.; Supervision: X.C.; Resources: X.F., J.N.; Writing-original draft: Y.Z., K.L.; Writing-review & editing: Y.Z., K.L., C.W., X.F., J.N., X.C.; Visualization: Y.Z., K.L.; All authors reviewed the manuscript.

Funding

This work was supported by the Shanghai Municipal Health Commission (Grant number: 20234Y0103), and the Shanghai Ninth People’s Hospital Physicians in Clinical Research Training Program (Grant number: 2022hbyjxys-zy).

Availability of data and materials

All relevant data are presented in the article and additional file 1. The sequence information of the present project is available in the National Center for Biotechnology Information BioSample database (Accession number: PRJNA1062757, https://www.ncbi.nlm.nih.gov/).

Declarations

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethical Review Board of the Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, China (Protocol code: SH9H-2019-T76-2, Approval date: 2020/10/08). A written informed consent was obtained from all subjects involved in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflicts of interest.