Immune Dysregulation in Acute Herpes Zoster: Predictive Factors for Postherpetic Neuralgia

Introduction

Varicella zoster virus (VZV) is transmitted through droplets or direct contact. The initial infection mainly causes chickenpox, and then VZV can ascend along sensory nerve axons or fuse with neurons through infected T cells, and transfer to the posterior root ganglia or cranial ganglia of the spinal cord. After the recovery from primary infection, VZV will lie dormant in the human body. When the host’s immune function is impaired, the VZV lurking in the ganglia can be activated, replicate, and transfer to the skin along sensory nerve fibers, resulting in typical unilateral aggregated blisters and pustules, known as herpes zoster (HZ) [2,3]. HZ can cause a series of complications, including postherpetic neuralgia (PHN), keratitis, decreased vision, blindness, hearing impairment, facial paralysis, and central nervous system (CNS) infections. Among these, PHN is the most common complication [4]. The incidence and prevalence of HZ and PHN are significantly increasing [5–8]. Epidemiological data show that the prevalence of HZ in China is 7.7%, with 29.8% of HZ patients developing PHN [9]. Once PHN occurs, if not treated properly, the pain can persist for several years or even decades, causing huge suffering for patients. In severe cases, they may become unable to work or perform activities of daily living, causing heavy healthcare and economic burdens to families and society [10,11]. At present, there are many clinical methods for treating PHN, but the treatment effects are mostly unsatisfactory. However, early diagnosis and standardized treatment of HZ patients can significantly reduce the incidence of PHN [12–14]. Therefore, early identification and intervention of PHN are extremely important.

HZ is an immune-related disease, and PHN is closely related to the host’s immune response. Immunocompromised HZ patients have a significantly increased probability of developing PHN [15–17]. However, there are still many individuals with immunocompetent who undergo HZ and PHN in clinical practice. As an important component of the cellular immune system, lymphocyte subpopulations participate in various important immune responses in the body, exert immune functions, and participate in the occurrence of PHN [18,19]. Humoral immunity, which is an important component of the host’s immune system, plays a unique role and cooperates with cellular immunity to jointly exert immune responses. While the role of cellular immunity is established, it is unclear whether humoral immunity affects the incidence of PHN. The study of immune response in PHN is important for exploring its specific pathogenesis, early identification of PHN patients, and searching for potential therapeutic targets. Therefore, to provide reference for clinical physicians to identify, treat, and reduce the occurrence of PHN in the early stage, this study explored the roles of cellular and humoral immune functions in the occurrence and development of PHN in immunocompetent acute HZ patients.

Material and Methods

SUBJECTS:

We enrolled 188 patients with acute HZ who were admitted in our hospital from April 2021 to June 2023 as the HZ group, and 79 individuals who underwent physical examinations at our hospital during the same period were selected as the control group.

Inclusion criteria: Presence of clinical symptoms, signs, and auxiliary examination results that meet the diagnostic criteria for HZ [20], and have not received relevant treatment. The diagnosis of HZ was made by dermatologists.

Exclusion criteria: 1) Age <18 years; 2) Zoster sine herpete; 3) Recurrent herpes zoster; 4) Women during lactation or pregnancy; 5) Immunocompromised patients (eg, solid-organ malignant tumors, hematological malignancies, solid-organ transplantation, hematopoietic stem cell transplantation, HIV infected individuals, congenital immunodeficiency, and autoimmune diseases); 6) Severe infections in other parts of the body; 7) Patients with mental illness or severe dementia who cannot accurately express pain; 8) Individuals who received VZV vaccination.

This study was approved by the Ethics Committee of the Third people’s Hospital of Hangzhou (NO.2021KA013). All patients signed the informed consent forms.

DATA COLLECTION:

We collected data on patient sex, age, course of disease, location of herpes, and comorbidities. Based on their ages, HZ patients were divided into 3 groups: <50 years, 50–79 years, and ≥80 years.

PERIPHERAL BLOOD HUMORAL AND CELLULAR IMMUNE INDEX DETECTION:

Levels of Serum Immunoglobulin IgA, IgG, IgM, and Complement C3 and C4

We drew 5 mL of fasting venous blood from all enrolled acute HZ patients and the control group on the morning of the second day of admission, centrifuged at 3000 at rpm for 10 minutes, and serum samples were taken. Turbidimetric inhibition immunoassay was used to detect the levels of serum immunoglobulin IgA, IgG, IgM, and complement C3 and C4 using a fully automated biochemical analyzer (Hitachi 7600–220 biochemical analyzer, Japan), strictly following the instructions of the reagent kit (DiaSys Co. LTD, Germany).

Levels of Peripheral Blood Lymphocyte Subsets (CD3+, CD4+, CD8+, CD4+/CD8+, CD19+, CD56+)

On the morning of the second day of admission, 2 mL of venous blood was drawn and anticoagulated with EDTA. Each flow cytometry sample was divided into 2 tubes for testing. Two branch tubes were used, one tube is filled with 20 μL of fluorescein-labeled monoclonal antibodies (CD45+/CD3+/CD4+/CD8+) (BD Company, USA), and the other tube was filled with 20 μL of fluorescein-labeled monoclonal antibodies (CD45+/CD3+/CD19+/CD56+) (BD Company, USA), then we added 50 μL of anticoagulant whole-blood cell mixture to each tube. After mixing well, samples were incubated at room temperature in the dark for 20 minutes, then we added 1 mL of red blood cell lysis solution, centrifuged samples at 2000 rpm for 4 minutes, discarded the supernatant, washed with PBS solution once, mixed well, centrifuged again, and added 500 uL of PBS solution for sample resuspension. The resuspended specimens were placed on a flow cytometer (FACSCAlibur, BD company, USA) for detection, with CD45/SSC as the gate. CELLQuest software was used for cell acquisition and data analysis, and the percentage of lymphocyte subsets was measured. We used a fully automated blood cell analyzer (Mindray BC-7500, China) to determine the total number of lymphocytes in each blood sample. The cell count of each lymphocyte subgroup in each specimen was determined by multiplying the total number of lymphocytes in each specimen with the corresponding percentage of lymphocyte subgroups.

PATIENT FOLLOW-UP AND PHN ASSESSMENT:

After discharge, patients underwent outpatient or telephone follow-up, and their pain status was evaluated using the numerical rating scale (NRS). HZ patients were divided into an HZ-PHN group and an HZ-control group based on whether they had neuralgia more than 1 month after herpes healing in the herpes distribution area.

STATISTICAL ANALYSIS:

Data were processed and analyzed using SPSS 17.0 statistical software. Count data are expressed in frequency and percentage, and inter-group comparisons were conducted using the chi-square (χ²) test. Quantitative data that conformed to a normal distribution are expressed as mean ± standard deviation (χ±SD). Analysis of variance (ANOVA) was used for comparison between multiple groups, and the LSD t test was used for comparisons between groups. Factors with statistically significant differences in single-factor analysis were selected for multivariate logistic regression analysis, and P<0.05 indicates statistically significant differences.

Results

BASELINE CHARACTERISTICS OF 2 GROUPS:

According to the inclusion and exclusion criteria, a total of 188 acute HZ patients were included: 102 males and 86 females, aged 19–96 years (60.0±21.6 years). There were 79 patients in the control group, including 44 males and 35 females, aged 24–89 years (64.3±15.5 years). There were no significant differences between the 2 groups in terms of age, sex, or comorbidities (eg, hypertension, diabetes, coronary heart disease, stroke) (P>0.05). See Table 1.

COMPARISON OF PERIPHERAL BLOOD HUMORAL AND CELLULAR IMMUNE INDEX LEVELS BETWEEN THE 2 GROUPS:

There was no statistically significant difference in the levels of serum C3, C4, IgG, IgA, and IgM between the 2 groups (P>0.05). Compared with the control group, patients in the HZ group had lower levels of CD3+ and CD4+ and CD4+/CD8+ ratios (P<0.05), but there was no significant difference in the levels of CD8+, CD19+, and CD56+ (P>0.05). See Table 2.

COMPARISON OF HUMORAL AND CELLULAR IMMUNE FUNCTIONS IN HZ PATIENTS WITH DIFFERENT AGE GROUPS:

According to the age difference of HZ patients, there are 73 cases in the <50 years group, 55 cases in the 50–79 years group, and 60 cases in the ≥80 years group. There was no statistically significant difference in serum levels of C3, C4, IgG, IgA, and IgM among the 3 groups (P>0.05). As age increased, the levels of CD3+, CD19+, CD4+, and CD8+ gradually decreased, with significant differences (P<0.01). Inter-group comparisons showed that the levels of CD3+, CD4+, and CD8+ in patients ≥80 years were significantly lower than those in patients aged <50 years and 50–79 years (P<0.05, P<0.01), while there was no significant difference in the levels of CD56+ and CD4+/CD8+ ratio (P>0.05). See Table 3.

COMPARISON OF BASELINE CHARACTERISTICS AND HUMORAL/CELLULAR IMMUNE FUNCTIONS BETWEEN HZ-PHN GROUP AND HZ-CONTROL GROUP:

All 188 patients completed follow-up without shedding detectable levels of virus. According to the follow-up results, among 188 HZ patients, 91 (48.4%) (HZ-PHN group) developed PHN (aged 64.6±21.4 years, 49 males and 42 females). Among them, 52 had herpes lesions in the head and neck, 17 in the chest and back, 11 in the waist, 9 in the limbs, and 2 were disseminated. There were 97 HZ patients (51.6%) who did not develop to PHN (HZ-control group) (aged 55.7±21.1 years, 52 males and 45 females). Herpes lesions were located in the head and neck in 65, chest and back in 11, waist in 13, limbs in 6, and disseminated in 2. The age of patients in the HZ-PHN group was significantly older than in the HZ-control group (P<0.01), and there was no significant difference in sex, disease duration, herpes location, and comorbidities between the 2 groups (P>0.05), as shown in Table 4.

Compared with the HZ-control group, there was no significant difference in serum levels of C3, C4, IgA, IgG, and IgM in the HZ-PHN group. However, the levels of CD3+, CD4+, and CD8+ and CD4+/CD8+ ratios were significantly lower (P<0.05, P<0.01). See Table 4.

Compared with the control group, the CD4+ level and CD4+/CD8+ ratio in the HZ-control group were lower (P<0.05, P<0.01), and there was no difference in the ratios of CD3+, CD19+, CD8+, and CD56+. However, the CD3+ and CD4+ levels and CD4+/CD8+ ratios in the HZ-PHN group were significantly lower than those in the control group (P<0.05, P<0.01), and the ratios of CD19+, CD8+, CD56+ were not different (P>0.05).

INDEPENDENT RISK FACTOR ANALYSIS OF HZ CONCURRENT PHN:

Using the presence of PHN as the dependent variable and statistically significant factors (CD3+, CD4+, CD8+, CD4+/CD8+) as independent variables in univariate analysis, a multivariate binary logistic regression analysis was conducted. After adjusting the results of lymphocyte subtype counts according to age, we found that age and CD4+/CD8+ ratios were independent risk factors of concomitant PHN in acute HZ patients (P<0.05), while the levels of CD3+, CD4+, and CD8+ were not independent risk factors (P>0.05). See Table 5.

ROC CURVE ANALYSIS FOR PREDICTING THE CCCURRENCE OF PHN IN HZ PATIENTS:

The ROC curve for predicting PHN was established based on age and CD4+/CD8+ ratios. The results showed that age predicted PHN occurrence (AUC=0.623, sensitivity 54.0%, and specificity 75.3%). The predictive value of CD4+/CD8+ ratios for occurrence of PHN was AUC=0.578, with sensitivity 47.9% and specificity 80.2%. The combinations of age and CD4+/CD8+ ratios had a better predictive value for occurrence of PHN, with AUC=0.663, sensitivity 61.2%, and specificity 77.9% (Figure 1).

Discussion

The host’s immune response includes humoral and cellular immunity, both of which jointly determine the host’s immune function. Lymphocytes are composed of T (CD3+), B (CD19+), and NK (CD56+) cells, each with different characteristics and functions, and they jointly undertake the host’s cellular and humoral immune functions in the immune response. Among them, B lymphocytes participate in humoral immunity, while T lymphocytes participate in cellular immunity, and they are the main cells involved in cellular immunity. They are divided into different subgroups according to their functions and surface markers, such as CD19+ representing B cells, CD56+ representing natural killer cells, CD4+ indicating helper T cells, and CD8+ indicating cytotoxic T cells. Flow cytometry can detect various lymphocyte subsets based on different surface markers of lymphocytes. Studies have shown that immunocompromised patients are at greater risk of HZ-related health problems [7,21,22], which may be related to the weakened function or decreased number of host T lymphocytes [23–25]. Our study used immunoglobulin and complement protein as humoral immune markers, and selected T lymphocytes, B lymphocytes (CD19+), and natural killer (NK) cells as cellular immune markers. The results showed that the levels of T lymphocyte subsets CD3+ and CD4+ in HZ patients were significantly lower in the control group, while there was no difference in the levels of C3, C4, IgG, IgA, and IgM, which reflect humoral immune function. These results indicate that HZ patients generally exhibit cellular immune dysregulation and low cellular immune status, while their humoral immune status remains unchanged.

PHN is the most common complication of HZ, with pain lasting for weeks, months, or even years. There are currently different opinions internationally on the definition of PHN [26–28]. Many scholars believe that PHN is a pain that lasts for at least 3 months after the healing of herpes lesions [20,29]. According to some scholars and the Chinese expert consensus on the diagnosis and treatment of PHN, PHN is defined as pain that lasts for more than 1 month after herpes lesions have healed [30,31]. The incidence and prevalence of PHN vary with the definition of pain duration and intensity. In this study, the incidence of PHN was 48.4%, higher than previously reported [4,9,32], which may be related to the small number of patients we included, and the diagnostic standard of PHN we used was the Chinese standard.

The interaction between the nervous system and the immune system plays an important role in the occurrence and development of neuropathic pain [33,34]. The mechanism of PHN is currently unclear, but many studies have shown that the host’s immune function is involved in the occurrence and development of PHN. Immunocompromised individuals typically have a history of solid-organ malignancies, hematological malignancies, solid-organ transplantation, hematopoietic stem cell transplantation, human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS), end-stage kidney disease, congenital immunodeficiency, or autoimmune diseases [35]. In patients with acute HZ, the probability of developing PHN is significantly higher in immunocompromised patients than in immunocompetent patients. A study carried out in China found that the incidence of HZ in immunocompromised people ≥50 years old was 15.94/1000 person years (95% CI: 14.77/1000~17.17/1000 person years), while that in immunocompetent people was only 5.97/1000 person years (95% CI: 5.78/1000~6.17/1000 person years), while the proportion of HZ patients with concurrent PHN was 8.38% and 7.04%, respectively [22]. A study in the UK showed that compared to immunocompetent individuals, those aged ≥18 years with HIV infection, leukemia, lymphoma, myeloma, or hematopoietic stem cell transplantation have higher risk of developing PHN, at 2.17, 2.07, 2.45, 2.17, and 5.91 times, respectively [15]. Although studies have shown an increased likelihood of developing PHN in immunocompromised individuals, the relationship between cellular immunodeficiency and PHN cannot be qualitatively expressed at present. People with HZ complicated with PHN generally have low cell-mediated immune function, mainly manifested by a decrease in the absolute number of T lymphocyte subsets. Sutherland et al [36] conducted histological studies on the ganglia of 2 HZ patients at different stages of onset, and compared them with autopsy results of 2 deceased patients who had not been infected with VZV, and found that there were different proportions and quantities of immune cells infiltrating the ganglia tissue of HZ patients. They found that immune cells infiltrated the ganglia during the development of HZ into PHN, and CD4+ and CD8+ T cells were key subgroups of cells involved in this process. Wei et al [37] also found that the number of T lymphocytes in PHN patients was significantly lower than that in the HZ patients, and concluded that T lymphocytes play an important role in the occurrence of PHN in HZ patients. Although humoral immune function is closely related to and coordinated with cellular immunity, studies have not found that humoral immunity is involved in the development of PHN. Our study also found that the levels of CD3+, CD4+, and CD8+ and CD4+/CD8+ ratios were significantly reduced in HZ-PHN patients, and the age and CD4+/CD8+ratios were both independent risk factors for concomitant PHN in HZ patients. After adjusting for confounding factors of age on lymphocyte subsets, CD4+/CD8+ratios remained an independent risk factor for concurrent PHN. This further demonstrates that immune function is involved in the occurrence and development of PHN in HZ patients, mainly through cellular immunity, while humoral immunity may not be involved in the development of PHN.

In this study, there were differences in cellular immune function among HZ patients of different age groups. As age increased, the levels of lymphocyte subsets CD3+, CD4+, CD8+, and CD19+ all significantly decreased, and age was an independent risk factor affecting the occurrence of PHN. This indicates that with age increased, host cell-mediated immunity decreases, and VZV latent in the dorsal root ganglia is more likely to be activated and develop disease [38]. At the same time, due to the reduced neural repair ability of elderly people, the slow process of neural tissue repair makes them more vulnerable to PHN [39,40]. This is also in line with the fact that HZ is a cellular immune-related disease, and any suppression of cellular immune function caused by any factor will increase the risk of HZ. Autopsy, skin biopsy, and other studies have shown irreversible damage to the nervous systems of PHN patients [41]. Previous studies have suggested that the viral load in the blood/cerebrospinal fluid of HZ patients may be related to the occurrence of central nervous system complications [42,43]. Some scholars also believe that pain may be related to abnormal neural impulses caused by the sustained replication of the virus in the nervous system, but this lacks strong evidence [44–46].

There are also some shortcomings in this study. Firstly, this was a single-center study with a small sample size, and most of the selected patients had severe symptoms, which may have led to patient selection bias. Secondly, there was no dynamic monitoring of the patient’s immune function indicators. Thirdly, our analysis did not include other prognostic factors that can affect the occurrence of PHN, such as herpes area and pain scores, and this may also lead to lower sensitivity of single-factor prediction of PHN, such as age and CD4+/CD8+ ratios. Multicenter studies with larger samples are needed to clarify the results and guide clinical practice.

Conclusions

HZ patients generally exhibit cellular immune dysfunction in the acute phase, manifested by a decrease in the levels of CD3+ and CD4+ and CD4+/CD8+ ratios, especially for older patients and those prone to PHN. Immune function is involved in the occurrence and development of PHN in HZ patients, mainly via T cells immune function, while humoral immune function is not involved. Age and CD4+/CD8+ ratios are independent risk factors for concomitant PHN in HZ patients, and the combination of the age and CD4+/CD8+ ratios have clinical value in predicting the occurrence and development of PHN.