Immune indicators and depression in adolescents: Associations with monocytes, lymphocytes, and direct bilirubin

Abstract

BACKGROUND

Depression is a significant psychiatric disorder with particularly high prevalence among adolescents. This mental health condition can have severe consequences, including academic failure, social withdrawal, and suicidal behavior. Given the increasing rate of depression in this age group, understanding the underlying biological mechanisms is essential for early detection and intervention. Recent studies have suggested that immune markers play a role in the pathophysiology of depression, prompting further investigation of their potential association with depressive symptoms in adolescents.

AIM

To investigate the relationship between immune markers (monocytes, lymphocytes, and direct bilirubin) and the incidence and severity of depression among adolescents.

METHODS

This cross-sectional study recruited 145 adolescent patients with depression [male (M)/female (F) = 38/107] from Jiangbin Hospital in Guangxi, Zhuang and 163 healthy controls (M/F = 77/86) from routine health check-ups. Blood samples were collected after an overnight fast. Depression severity was measured using the Zung Self-Rating Depression Scale. The inclusion criteria were age 12-24 years, diagnosis of depressive disorder (ICD-10), and no recent antidepressant use. The exclusion criteria included psychiatric comorbidities and serious somatic diseases. Key statistical methods included group comparisons and correlation analyses.

RESULTS

There was a higher prevalence of females in the depression group (P < 0.001). Significant age differences were observed between the groups (Z = 9.43, P < 0.001). The depression group had higher monocyte (Z = 3.43, P < 0.001) and lymphocyte (t = 2.29, P < 0.05) counts, and higher serum direct bilirubin levels (Z = 4.72, P < 0.001). Monocyte count varied significantly according to depression severity, with lower counts in the mild group (Z = -2.90, P < 0.05). A negative correlation between age and lymphocyte counts was observed (ρ = -0.22, P < 0.01). Logistic regression analysis showed that serum direct bilirubin levels significantly predicted depression.

CONCLUSION

The potential role of elevated levels of immune markers in the early detection of depression in adolescents has been highlighted. Therefore, it is necessary to explore further the relationships between these immune markers and depression.

Key Words: Depression; Adolescents; Immune markers; Monocyte; Lymphocyte; Direct bilirubin

Core Tip: This study investigated the relationship between immune markers, specifically monocyte and lymphocyte counts and direct bilirubin levels, and depression in adolescents. By comparing 145 adolescents with depression with 163 healthy controls, we found significantly elevated levels of immune markers in patients with depression, particularly in severe cases. These findings suggest that elevated direct bilirubin levels may serve as potential immune markers for detecting depression, highlighting the importance of immune responses in understanding the mental health of adolescents.

INTRODUCTION

Depression is a widespread and debilitating psychiatric disorder among adolescents, often leading to severe consequences, such as academic failure, social withdrawal, and suicidal behavior[1-5]. Its prevalence is increasing more rapidly among adolescents than among adults, attracting considerable scholarly attention[6]. Adolescence, a critical period of emotional and neurobiological development, makes this population particularly susceptible to the adverse effects of depression[7]. Despite advances in understanding its neurobiological foundations, the pathophysiology of depression remains only partially understood, and further research is required to improve identification methods[7,8]. Throughout the progression of major depressive disorder (MDD), the overactivity and underactivity of certain immune markers contribute to the onset of systemic complications, ultimately leading to a cascade of comorbid conditions[9]. Bidirectional communication between the neuroendocrine and immune systems is the key to understanding depression and post-illness damage[10]. Emerging evidence associates the immune system, particularly inflammation, with the onset and progression of depression, highlighting the potential of immune markers as diagnostic tools[11,12].

The monocyte-to-lymphocyte ratio (MLR), a key indicator of the immune system, has garnered attention for its potential role in the pathophysiology of depression[13,14]. Monocytes are integral to the innate immune system, serve as the first line of defense against pathogens, and differentiate into macrophages or dendritic cells during infection[15,16]. In contrast, lymphocytes are central to the adaptive immune system, and coordinate and execute immune responses. The MLR, which reflects the balance between innate and adaptive immunity, often indicates a pro-inflammatory state when elevated, suggesting increased innate activity at the expense of adaptive functions. Emerging evidence links an elevated MLR with systemic inflammation, a key contributor to neuroinflammation in depressive disorders, thereby supporting the neuroinflammatory hypothesis of depression[17]. However, research on the role of the MLR across depressive phases remains inconclusive. For instance, Koureta et al[18] demonstrated that the MLR was significantly higher during the manic phase of bipolar disorder, with no significant differences observed between the unipolar and bipolar depressive phases, while Dionisie et al[19] reported a slight elevation in the MLR in bipolar depression compared to unipolar depression. A meta-analysis showed that MLR levels were higher in patients with depression than in healthy controls[20], but a 2022 cross-sectional study found a non-linear relationship between MLR and depressive symptoms[13]. These inconsistencies underscore the need for further research to clarify the role of the MLR in depressive states and assess its reliability as a biomarker.

The link between direct bilirubin level and depression has gained attention in recent psychiatric studies. Direct bilirubin, a water-soluble form conjugated with glucuronic acid in the liver, is an excreted waste product from the breakdown of heme, a hemoglobin component. As an antioxidant, direct bilirubin neutralizes reactive oxygen species (ROS) and mitigates oxidative stress[21,22]. Recent studies have highlighted its relevance to psychiatric disorders, particularly depression. Emerging evidence suggests that oxidative stress, marked by an imbalance between ROS production and antioxidant defense, contributes to depressive symptoms[23]. A large cross-sectional study reported lower bilirubin levels in individuals with depression, indicating that oxidative stress markers such as bilirubin may serve as potential immune markers of depression[24]. Although these findings are promising, further research is required to explore the relationship between direct bilirubin levels and depression in adolescents. Replication and additional studies are essential to confirm these results and evaluate the potential of direct bilirubin as a biomarker of adolescent depression.

Given the urgent need for reliable immune markers for the early diagnosis of adolescent depression, this study employed a retrospective case-control design to examine the relationship between monocyte and lymphocyte counts and direct bilirubin levels and depressive symptoms in adolescents. By elucidating these relationships, this study seeks to deepen the understanding of the immune mechanisms underlying adolescent depression, which may provide valuable insights for the early screening of at-risk adolescents.

MATERIALS AND METHODS

Study population

The study population consisted of adolescent patients with depression recruited from Guangxi Zhuang Autonomous Region Jiangbin Hospital between July and August, 2024. Based on previous studies[17,19], a total of 145 patients met the inclusion criteria. The control group consisted of 163 healthy adolescents who underwent routine physical examinations at the Health Examination Center of Guangxi Zhuang Autonomous Region Jiangbin Hospital.

Inclusion and exclusion criteria

The inclusion criteria were as follows: (1) Patients who met the diagnostic criteria for depressive disorder as defined by the ICD-10; (2) Patients aged 12-24 years; (3) No use of antidepressants, antipsychotics, or corticosteroids within one month prior to enrollment, with normal menstrual cycles for female patients; and (4) Both patients and their families provided informed consent and signed confidentiality agreements. The exclusion criteria were as follows: (1) Comorbid psychiatric disorders; (2) A history of electroconvulsive therapy or substance abuse; (3) Presence of cardiovascular or serious somatic diseases; and (4) Missing data.

Variables and laboratory tests

Blood collection: Blood samples for the study group were collected from the antecubital vein after overnight fasting (8-10 mL) at 7 a.m. on the second day of hospital admission and sent to the laboratory for analysis. Similarly, fasting venous blood samples (8-10 mL) were obtained from the control group at 7 a.m. during routine health examinations and sent to the laboratory for testing.

Assessment of the severity of depression: The severity of depression was evaluated using the Zung Self-Rating Depression Scale (SDS)[25]. The SDS consists of 20 items, each rated on a 4-point Likert scale. The total raw score for the scale ranges from 20 to 80. The raw score was then multiplied by 1.25 to obtain the SDS score. Based on the SDS score, participants were classified as normal (< 53), mildly depressed (53-62), moderately to severely depressed (63-72), or severely to extremely severely depressed (> 72).

Statistical analysis

All statistical analyses were performed using R version 4.4.1. The normality of data distribution was evaluated using the Shapiro-Wilk test, and variance homogeneity was checked using Levene’s or Bartlett’s test. For data with non-normal distributions or unequal variances, log or square root (sqrt) transformations were applied, and normality was reassessed after transformation. If the transformations did not normalize the data, non-parametric tests were used. Variables with homogeneous distributions were compared using the t-test for two groups or one-way analysis of variance (ANOVA) for multiple groups. For non-homogeneous distributions, the Mann-Whitney U test was used for two groups, and the Kruskal-Wallis test was used for multiple groups. Following the Kruskal-Wallis test, Dunn’s test with Bonferroni correction was used for post-hoc analyses to control for multiple comparisons. Correlations between the parameters and age were analyzed using Spearman’s correlation tests, as appropriate, when normality or linearity assumptions were not met. Multinomial logistic regression analysis was employed to identify the factors contributing to depression, with age and sex as controlled variables to mitigate confounding effects. Receiver operating characteristic (ROC) curve analysis was used to evaluate the sensitivity and specificity of the immune markers in distinguishing patients with depression from healthy controls. In cases of group imbalance, adjustments and weighted ROC were considered. Statistical significance was set at P < 0.05.

RESULTS

Comparison of variables between patients with depression and healthy controls

This study included 145 patients with depressive disorder and 163 healthy controls. The difference in sex between the depression and the healthy control groups was statistically significant (χ² = 13.62, P < 0.001), with females more likely to experience depressive disorder (OR = 2.51, 95%CI: 1.56-4.09). The Mann-Whitney U test showed that the difference in age was also statistically significant, which ages of patients with depression are significantly lower than ages of healthy controls (Z = 9.43, P < 0.001), and further revealed that both monocyte count and direct bilirubin levels in the depressive disorder group were significantly higher than those in the healthy control group (Z = 3.43, P < 0.001; Z = 4.72, P < 0.001). For lymphocyte count, t-test analysis of the log-transformed data confirmed a significant difference (t = 2.29, P < 0.05). These data are presented in Table 1.

Table 1 Comparison of sociodemographic and laboratory variables between healthy controls and patients with depression.

Variables	Group		Test
	DEP (n = 145)	HC (n = 163)
Sex, n (%)			χ2 = 13.62, P < 0.001, OR = 2.51 (95%CI: 1.56- 4.09)
Male	38 (26.21)	77 (47.24)
Female	107 (73.79)	86 (52.76)
Age (year)			Z = 9.43; P < 0.001
Min/Max	12.0/24.0	13.0/26.0
Median (P25, P75)	17 (16, 20)	22 (21, 23)
Monocyte count (× 109/L)			Z = 3.43; P < 0.001
Min/Max	0.2/2.0	0.2/1.7
Median (P25, P75)	0.52 (0.43, 0.61)	0.45 (0.37, 0.55)
Lymphocyte count (× 109/L)			t = 2.29; P < 0.05
Min/Max	1.1/6.8	1.0/4.8
Mean (SD)	2.60 ± 0.89	2.38 ± 0.66
Direct bilirubin concentration (μmol/L)			Z = 4.72; P < 0.001
Min/Max	0.4/11.4	0.9/10.0
Median (P25, P75)	3.30 (2.30, 4.65)	2.41 (1.90, 3.26)

DEP: Depression; HC: Healthy control; Min: Minimum; Max: Maximum.

Comparison of variables among different degrees of depression

ANOVA showed no significant differences in log-transformed lymphocyte count and direct bilirubin levels among patients with varying degrees of depression [F (2, 142) = 2.27, P = 0.107; F (2 ,142) = 0.64, P = 0.530, respectively]. The Kruskal-Wallis test revealed that the differences in monocyte count were significant for different degrees of depression [H (2) = 8.52, P = 0.014]. These data are presented in Table 2.

Table 2 One-way analysis of variance analysis of immune markers in different degrees of depression.

Variables	Mild depression (n = 20), median (P25, P75)/mean (SD)	Moderate depression (n = 37), median (P25, P75)/mean (SD)	Severe depression (n = 88), median (P25, P75)/mean (SD)	F/χ2	P value
Monocyte count (× 109/L)	0.445 (0.35, 0.52)	0.53 (0.43, 0.56)	0.54 (0.438, 0.68)	8.52	0.014a
Lymphocyte count (× 109/L)	2.24 ± 0.59	2.58 ± 0.81	2.69 ± 0.97	2.27	0.107
Direct bilirubin concentration (μmol/L)	3.72 ± 1.69	3.90 ± 2.00	3.59 ± 2.09	0.64	0.530

^aP < 0.05.

Dunn's test (with Bonferroni correction) showed that the difference between the mild and severe groups was significant (Z = -2.90, P < 0.05), the difference between the mild and moderate groups was not significant (Z = -1.78, P = 0.23), and the difference between the moderate and severe groups was also significant (Z = -1.15, P = 0.75). The data are shown in Figure 1.

Figure 1 Comparison of monocyte counts among patients with mild, moderate, and severe depression.

Impact of sex and age on immune markers among patients with depression

In the depressive disorder group, sex did not have an effect on lymphocyte and monocyte count, or on serum direct bilirubin levels. There were no significant differences in log-transformed lymphocyte count or sqrt-transformed direct bilirubin levels between male and female patients with depressive disorders (t = 0.42, P = 0.68; t = 1.99, P > 0.05). Similar monocyte counts were also found in male and female patients (Mann-Whitney U test, W = 1897.5, P = 0.54).

There was no significant correlation, in patients with depressive disorder, between age and either monocyte count (Spearman correlation, ρ = 0.03, P = 0.73) or direct bilirubin level (ρ = 0.12, P = 0.15). However, lymphocyte count showed a significant negative correlation with age (ρ = -0.22, P = 0.007). The scatter plot in Figure 2A illustrates the relationship between age and lymphocyte count in patients with depression, with the regression line indicating a general trend.

Figure 2 Correlation of age and lymphocyte count and correlation of age and direct bilirubin level in patients. A and B: Correlation of age and lymphocyte count in patients of depression (A) and severe depression (B); C: Correlation of age and direct bilirubin level in patients with severe depression.

In patients with the most severely depressive disorder, there was a significant negative correlation between age and lymphocyte count (Spearman correlation, ρ = -0.28, P = 0.007). In addition, direct bilirubin level showed a significant positive correlation with age (ρ = 0.23, P = 0.034). Figure 2B shows the relationship between age and lymphocyte counts in patients with severe depression, and Figure 2C shows the relationship between age and direct bilirubin levels. In both cases, the regression line indicated a general trend.

The immune marker predictors of the diagnosis of depressive disorder

We developed a multivariable logistic regression model to assess the association between depression and three immune markers–monocyte count, lymphocyte count, and direct bilirubin level–with sex and age as control variables (Table 3).

Table 3 Multivariable logistic regression.

Variable	Coefficient	OR (95%CI)	SE	Z	P value
Intercept	6.52	680.18 (60.10-8666.00)	1.27	5.14	0.000a
Monocyte count	1.17	3.22 (0.49-24.90)	1.00	1.17	0.243
Lymphocyte count	-0.03	0.97 (0.60-1.57)	0.24	-0.11	0.911
Direct bilirubin concentration	0.40	1.49 (1.23-1.83)	0.10	3.91	0.000a
Sex (male)	-1.09	0.34 (0.18-0.61)	0.31	-3.50	0.000a
Age	-0.41	0.67 (0.60-0.73)	0.05	-7.93	0.000a

^aP < 0.001.

In the multivariable model, direct bilirubin (P < 0.001) with a regression coefficient of 0.40 (OR = 1.49, 95%CI: 1.23-1.83), was significantly associated with depression, while monocyte count (P = 0.243) and lymphocyte count (P = 0.911) were not. Sex and age were included as control variables and were also significantly associated with depression (P < 0.001) with a regression coefficient of -1.09 (OR = 0.34, 95%CI: 0.18-0.61) for sex, and a regression coefficient of -0.41 (OR = 0.67, 95%CI: 0.60-0.73) for age.

Based on ROC curve analysis for depressive disorder diagnosis, the area under the curve (AUC) for individual predictors was 0.864. The ROC curve is shown in Figure 3.

Figure 3 Receiver operating characteristic curve of the multivariable model. AUC: Area under the curve.

DISCUSSION

The present study found that patients with depression exhibited higher peripheral blood monocyte and lymphocyte counts, and higher serum direct bilirubin levels than the healthy controls. Significant differences in age and sex were also observed between patients with depression and healthy controls. Further analysis revealed that monocyte count varied between patients with mild and severe depression, with patients in the severe depression group exhibiting significantly higher monocyte counts than those in the mild depression group.

Among patients with depression, a correlation was observed between lymphocyte count and age, with older patients showing lower lymphocyte counts. Specifically, lymphocyte count decreased with age in patients with severe depression, whereas direct bilirubin levels increased with age.

Direct bilirubin levels demonstrated a stronger predictive value for depressive status than lymphocyte and monocyte counts. Furthermore, when monocyte and lymphocyte counts, and direct bilirubin levels were combined into a single predictive model with age and sex as control variables, the model showed good collective predictive power (AUC = 0.864) to distinguish between patients with depression and healthy controls.

Monocytes are leukocytes involved in immune responses and inflammatory processes, whereas lymphocytes are important components of adaptive immunity[26]. Changes in the immune cell profiles are indicative of systemic inflammation and immune dysregulation[20]. The elevation of monocyte and lymphocyte counts in patients with depression reflects the complex interplay between chronic psychological stress and immune dysregulation. Elevated monocyte and lymphocyte counts can trigger depression. On the one hand, depression leads to chronic inflammation characterized by elevated counts of monocytes and lymphocytes[27]. Chronic stress, a typical symptom of depression, can activate the hypothalamic-pituitary-adrenal axis, leading to sustained release of cortisol, which disrupts immune function[28]. However, the inflammatory state may negatively affect mood regulation, cognitive function, motivation, appetite, and sleep in patients with depression. Thus, elevated counts of monocytes and lymphocytes can further drive depression by exacerbating symptoms.

Immune dysregulation in depression is a dynamic process. Effective depression interventions can normalize immune markers, suggesting a link between the treatment response and immune dysregulation[29]. A longitudinal study[30] indicated that patients with depression who showed significant clinical improvement during hospitalization had decreased monocyte counts. In contrast, patients with slower recovery maintained higher monocyte counts than healthy controls, suggesting that monocyte counts can be reduced by interventional therapy. Another study found that the levels of prostaglandin E2 produced by monocytes in response to endotoxin challenge were partially normalized in patients after eight months of antidepressant treatment, such as escitalopram[31].

In a study on the relationship between age and immune markers, lymphocyte counts were negatively correlated with age in patients with depression. Lymphocyte activity is an important indicator of the immune system which may decrease with patient age, although the average lymphocyte count in the depression group was higher than that in the healthy controls. The same result was observed in patients with severe depression who also exhibited a positive correlation between age and direct bilirubin levels.

Following improvement from depression intervention, lymphocyte levels may appear to increase or decrease, both of which may reflect improvements in depressive symptoms. A longitudinal study indicated that patients with MDD who showed significant clinical improvement during treatment experienced a greater decrease in lymphocyte counts compared to those with slower recovery[32]. This suggests that a reduction in lymphocyte levels may be associated with better treatment response and recovery from depression. Conversely, some studies have reported an increase in the absolute counts of certain lymphocyte subsets, particularly CD4+ helper T cells, in patients with depression after effective treatment[29]. This suggests that treatment can restore lymphocyte function and improve their activation status, which is often impaired in depression.

Bilirubin is an important biomarker that is closely associated with oxidative stress and the antioxidant defense system. In the present study, serum direct bilirubin concentration was found to be a robust predictor of depression. It is produced during the breakdown of hemoglobin from red blood cells. Although traditionally regarded as a metabolic waste product, recent studies have demonstrated that bilirubin possesses potent antioxidant properties[33-35]. Its antioxidant effects are well documented, and bilirubin is regarded as a crucial endogenous molecule that maintains cellular redox balance and prevents oxidative damage[36]. Elevated bilirubin levels have been widely reported in various oxidative stress-related conditions such as metabolic syndrome and cardiovascular diseases[37].

The results of this study indicated that direct bilirubin levels in patients with depression were significantly higher than those in healthy controls. These findings suggest that elevated bilirubin levels reflect increased oxidative stress in patients with depression. Although bilirubin generally acts as an antioxidant defence mechanism, its significant elevation may indicate that the antioxidant system responds to excessive oxidative stress. Such a physiological imbalance might not only be a response to depression-related oxidative stress, but also play a crucial role in the pathophysiology of depression.

The elevated bilirubin levels observed in patients with depression can be attributed to several factors. Firstly, depression is closely associated with increased oxidative stress and inflammation in the body, both of which can compromise normal antioxidant defense mechanisms, leading to higher bilirubin levels[38-40]. This bilirubin upregulation may be a physiological response aimed at counteracting oxidative damage to the central nervous system and other tissues. However, persistent oxidative stress can overwhelm the protective capacity of bilirubin, further exacerbating physiological imbalances and depressive symptoms[41].

Moreover, chronic inflammation commonly observed in patients with depression may interfere with the ability of the liver to metabolize and clear direct bilirubin, leading to its abnormal accumulation in the blood. This elevation in bilirubin levels could be both a consequence of and a contributing factor to depression. As some antidepressants (for example, paroxetine, fluoxetine, and fluvoxamine) can affect the renal function of patients with depression[42], the increase in direct bilirubin with age may also be attributed to the use of these medications. Rapid social changes have increased stressors in adolescents, highlighting the urgent need to update detection methodologies for depression[43]. The correlation between inflammatory immune markers and the severity of depression highlights their potential utility in assessing the functional status of patients with depression. These immune markers may serve as supplementary indicators of disease severity when used alongside more formal clinician- and patient-rated assessment tools[44].

Strengths and limitations

This study focused specifically on adolescents, a group that is particularly vulnerable to depression. This study offers a detailed analysis of multiple immune markers (peripheral blood monocyte and lymphocytes counts, and serum direct bilirubin concentration), presents an enlightening view of the immune markers associated with the severity and prevalence of depression, and suggests the value of a convenient blood test to examine immune markers associated with depression.

However, this study has some limitations. First, potential confounding factors such as medication status, diet, duration, and other health conditions that might have influenced the levels of the immune markers studied were not considered, potentially affecting the results. Second, although this study included a significant number of participants, the sample may not be fully representative of all adolescents with depression, which limits the generalizability of the findings to a broader population. The analysis in this study was limited by insufficient inclusion of background variables. While factors such as ethnicity, personal residence history, and family medical history were considered, relevant variables identified in previous studies were omitted. This resulted in inaccuracies in the comparison between the healthy and depressed groups. Future studies should incorporate additional factors, including detailed medication use, timing of the first onset and relapse, family mental health history, and environmental factors. By considering the factors that may affect patients' conditions, future research can yield more detailed and accurate results. Finally, because this was a cross-sectional study, we could not establish a causal relationship between these immune markers and depression. Future research could focus on longitudinal or intervention studies grounded in rigorous theoretical frameworks to enhance the applicability of the conclusion that immune factors serve as risk factors for depression.

CONCLUSION

Adolescents with depression exhibited higher peripheral blood monocyte and lymphocyte counts, and higher serum direct bilirubin concentrations than healthy controls. Among these immune markers, direct bilirubin levels demonstrated a stronger predictive value for depression. When these three immune markers with control variables (sex and age) were combined into a single predictive model, the ability to distinguish between individuals with and without depression was significant. These findings suggest that immune dysregulation is an important factor in the pathophysiology of depression in adolescents and that these immune markers could potentially be used for early screening and diagnosis. However, further research, particularly longitudinal studies, is necessary to confirm these findings and better understand the causal relationships.

ACKNOWLEDGEMENTS

We would like to thank all the medical staff of Guangxi Zhuang Autonomous Region Jiangbin Hospital for their assistance with data collection.