The potential roles of herpesvirus and cytomegalovirus in the exacerbation of pemphigus vulgaris

Background Among exogenous etiologies, the critical role of microbial agents such as herpesviruses (HSV1/2) and cytomegalovirus (CMV) in triggering and flaring autoimmune conditions such as pemphigus vulgaris (PV) has been recently discovered. Objectives The present study aimed to investigate the plausible role of these viruses in the exacerbation of PV using serological and molecular methods. Patients/Methods Sixty patients with PV (30 with relapse type and 30 with remission type) were recruited for the purpose of this case-control study. Skin, mucosal, and throat specimens were obtained and examined for viruses by reverse transcriptase polymerase chain reaction. To determine the immunoglobulin G (IgG) titer, enzyme-linked immunosorbent assay was used. Results Desmoglein1-specific IgG was positive in 56.7% of patients with the relapse form and in 20.0% of those with the remission form indicating a significant difference across the 2 groups (P = 0.003), but the rate of positivity for desmoglein3-specific IgG in the relapse and remission types was 76.7% and 63.3%, respectively, with no significant difference (P = 0.260). There was no difference in the mean levels of HSV-IgG and CMV-IgG in the relapse and remission groups. HSV and CMV positivity in PV patients was independent of the site of the samples. Using the multivariable linear regression model, the level of CMV-IgG in PV patients was directly affected by female sex and advanced ages. Conclusions Our study could not demonstrate the role of HSV1/2 and CMV as triggering factors for PV exacerbation. Further studies are needed to evaluate the potential role of these viruses in PV exacerbation especially considering demographic variables.

Atypical HSV infections have been described in immunosuppressed patients [16]. Several studies have suggested the activation or exacerbation of pemphigus after HSV or CMV infections [17]. More recently, Japanese researchers have detected high levels of HSV in the saliva samples of PV patients [18]. In this regard, several studies have shown the benefits of adding antiviral therapies to an immunosuppressive therapy in recalcitrant disease [19,20]. Several studies also failed to detect herpesviruses in pemphigus patient specimens [21,22], suggesting that viruses may have only a transient role for exacerbation of pemphigus disease, although yet to be further defined.
The number of patients with PV in Iran appears to be increasing, and little information is available with regard to the role of HSV1/2 and CMV in triggering PV among the Iranian population. The present study aimed to investigate the plausible role of these viruses (HSV1/2 and CMV) in the exacerbation of PV using serological and molecular methods.

Disease Definitions in PV Patients
PV patients were categorized as follows: relapse (development of more than 3 new lesions/month that do not heal within a week without treatment, or development of established lesions in a patient whose disease was controlled) and complete remission (absence of new or established lesions in a patient whose all-systemic therapy is discontinued for ≥2 months, or while the patient is receiving minimal therapy) [23]. Scoring of disease was recorded based on the pemphigus disease area index.

Introduction
Pemphigus disease is an autoimmune disease that is characterized by secretion of autoantibodies that act against surface glycoproteins of epithelial cells [1]. The immunological basis for pemphigus disease includes autoantibodies targeted against keratinocyte surface antigens desmoglein1 and des-moglein3 (DSG1, DSG3) [2]. These proteins are primarily involved in intercellular cell-to-cell adhesion structures. Pemphigus vulgaris (PV) is a common type of pemphigus disease with painful blistering on the skin and mucous membranes [3]. Overall, the close link between autoimmune diseases and both endogenous (genetic) and exogenous (environmental) factors has been clearly understood. Exogenous factors include thiol drugs, physical trauma such as burn, ultraviolet exposure, X-ray, neoplasm, hormones and pregnancy, nutritional factors, and emotional stress [4][5][6][7].
Among exogenous etiologies, the critical role of microbial agents such as viruses in triggering and flaring autoimmune conditions has been recently discovered. In this regard, the causative role of herpesviruses (HSVs) as the most important human pathogens in the pathogenesis of PV has been recently suggested [8]. Thus, efforts have been made to clarify viral etiologies for PV and in this way the role of HSVs and cytomegalovirus (CMV) is taken into consideration [9,10]. The main common features of both pointed viruses include their ability to survive in the host body for a long time as well as activating periodically [11]. The human HSVs including herpesviruses 1 and 2 (HSV1/2) and CMV infect between 60% and 90% of the adult population worldwide [12,13]. After primary infection, the virus establishes lifelong latency, with periodic reactivations that are effectively controlled by a robust immune response in most infected individuals despite the virus-producing proteins that interfere with adaptive and innate immunity. The activation of T cells is the important aspect of adaptive immunity. Tissue-resident memory (Trm) T cells are a subtype of memory lymphocytes that enter nonlymphoid tissues such as skin and become permanently established without recirculating. Local immune control of viruses can be mediated by Trm T cells through direct killing of infected cells and recruiting circulating memory CD8+ to the skin. Also, Trm T cells maintain HSV1 latency by secreting granzyme B, which degrades the early protein ICP4 that is important in viral replication [14,15]. CMV positivity in PV patients was independent of the site of the samples. Using the multivariable linear regression model, the level of CMV-IgG in PV patients was directly affected by female sex and advanced ages.

Conclusions:
Our study could not demonstrate the role of HSV1/2 and CMV as triggering factors for PV exacerbation. Further studies are needed to evaluate the potential role of these viruses in PV exacerbation especially considering demographic variables.

ABSTRACT Statistical Analysis
The results were presented as mean ± standard deviation for quantitative variables and were summarized by absolute frequencies and percentages for categorical variables. Normality of data was analyzed using the Kolmogorov-Smirnoff test. Categorical variables were compared using chi-squared test or Fisher's exact test when more than 20% of cells with an expected count of less than 5 were observed. Quantitative

Results
In total, 30 patients with the relapse type of PV and 30 patients with the remission type were assessed. The demographic and clinical features of patients are presented in Table 1. Comparison of the baseline characteristics of the 2 groups (Table 2) showed no difference in gender, mean age, subtypes of disease

Sample Preparation
For viral detection and determination of specific antibodies, we used blood, skin biopsy, and throat swab samples from patients. After patient consent forms were obtained, one skin specimen or mucosal specimens were obtained by punch biopsy (3 mm) and blood samples were taken from all patients. In addition, a sample from the throat was collected by swabbing. All samples were transferred in cold chain conditions to the virology laboratory. As soon as samples were received, preparation and storage were carried out. Sera (for serology) and buffy coat (for virus detection) were isolated from whole blood and stored at −20°C. DNA extraction from buffy coat, skin biopsy, and throat swab were performed using DNA Isolation Kit for Cells and Tissues according to the manufacturers' instructions (Roche, Berlin, Germany).
After DNA extraction, it was eluted in 50 µL of buffer and then adjusted to a definitive concentration of 500 ng/µL.

DSG-Specific IgG
The levels of autoantibody to DSG1 and DSG3 were measured by ELISA. The cutoff values were 20 U/mL for both.

Determination of Viral Load
The absolute quantification of HSV1/2 and CMV-specific

Discussion
Several studies have investigated the relationship between viral infections and the initiation or exacerbation of autoimmune As indicated in Table 4 and Figure 1 and using the multivariable linear regression model, the level of HSV-IgG was not associated with the baseline characteristics including demographics, underlying comorbidities, disease duration, and form of PV. However, as shown in Table 5

Conclusions
In conclusion, our study could not demonstrate the role of HSV1/2 and CMV as triggering factors for PV exacerbation.
Further studies are needed to evaluate the potential role of these viruses in PV exacerbation.
diseases such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and diabetes mellitus [24]. In this Also, overproduction of IL4 and IL10 causes a shift of TH1 to TH2 response which potentiates antibody response [26,27].
The role of viruses in the pathogenesis of pemphigus has been assessed in some previous studies. 'The importance of viral infections as an exacerbating factor of PV is neglected by some clinicians. Concerning this, in our current case-control study, we evaluated the probable role of HSV1/2 and CMV in the exacerbation of PV. In our study, a significantly higher number of PV patients (regardless of disease activity) were positive for HSV-and CMV-IgG antibodies, similar to the studies by Tufano et al and Sagi et al [28,29]. This may show a history of HSV and CMV exposure in most patients and may indicate more frequent recrudescence of latent viruses [30]. A significant increase in IgG antibody levels, discovered by comparing relapse and remission samples, indicates an active infection. According to a general consensus, relatively higher IgG levels are associated with a higher probability of having an active viral infection and relatively lower IgG levels represent a previous viral infection rather than an active viral infection. In this regard, Senger et al showed higher levels of HSV1 antibody in patients in the active phase of disease than in remission patients [31]. In contrast to our study, there was no significant difference in antibody levels between relapse and remission patients. In the studies of Marzano et al and Rah-