Altered IL-3 and lipocalin-2 levels are associated with the pathophysiology of major depressive disorder: a case-control study

Background Major Depressive Disorder (MDD) is a common mental ailment and is the primary reason for disability. It manifests a severe impact on moods, thoughts, and physical health. At present, this disorder has become a concern in the field of public health. Alteration of neurochemicals is thought to be involved in the pathogenesis of many psychiatric disorders. Therefore, we aimed to evaluate serum IL-3 and lipocalin-2 in MDD patients and healthy controls (HCs). Method We included a total of 376 participants in this study. Among them, 196 were MDD patients, and 180 were age-sex-matched HCs. MDD patients were recruited from the Psychiatry Department of Bangabandhu Sheikh Mujib Medical University (BSMMU), but the controls were from different parts of Dhaka. All study participants were evaluated by a psychiatrist using the DSM-5 criteria. To assess the severity of the depression, we used the Hamilton depression (Ham-D) rating scale. Serum IL-3 and lipocalin-2 levels were measured using commercially available enzyme-linked immune-sorbent assay kits (ELISA kits). Results According to this study, we observed elevated serum levels of IL-3 (1,024.73 ± 29.84 pg/mL) and reduced levels of serum lipocalin-2 (29.019 ± 2.073 ng/mL) in MDD patients compared to HCs (911.11 ± 20.55 pg/mL and 48.065 ± 3.583 ng/mL, respectively). No associations between serum levels of IL-3 and lipocalin-2 and depression severity were observed in patients. Conclusions According to the present findings, alterations of serum IL-3 and lipocalin might be associated with the pathogenesis of MDD. These results support that altered serum neurochemicals can serve as early risk assessment markers for depression. Further interventional studies are recommended for a better understanding of the role of IL-3 and lipocalin-2 in the pathophysiology of depression.


Background
Major depressive disorder (MDD) is a well-known, complex mood disorder with high rates of morbidity, recurrence, disability, and suicide that causes changes in mood, thoughts, behavior, and physical health [1,2].It is a widespread and severe mental disorder that can affect a person to enjoy life and reduce the ability to perform daily duties.It is also associated with other physical and mental illnesses [3].A study reported that 970 million people worldwide suffer from different mental disorders.Among them, 350 million suffer from MDD [4,5].The number of people living with depression worldwide increased by 50% from 1990 to 2017 [6].MDD is expected to be the leading cause of the burden of all medical disorders across the world by 2030 [7].Depression results from a complex interaction of genetic, social, psychological, biological, and environmental factors [8].People who have gone through adverse life events (unemployment, financial insecurity, chronic or life-threatening health problems, exposure to violence, social separation, sadness, etc.) are more likely to develop depression [9].The lifetime prevalence of depression is higher in women (20-25%) than in men (7-12%) [10].Evidence suggests that patients with a family history of MDD in first-degree relatives have up to 3 times higher risk of developing the disease than those who do not have it [11].MDD remains either undertreated or untreated even though it is a prevalent disease [12].The primary healthcare provider is the first person responsible for the diagnosis of depression.However, evidence suggests that it remains unrecognized at this level due to poor understanding of the disease mechanism by primary healthcare professionals.Since the diagnosis of depression mostly depends on statements by patients, this leads to misdiagnosis and confusion [13].To successfully treat depression, the first step is to recognize the problem and make a correct diagnosis [14].
Major depression is triggered by genetic, metabolic, endocrine, neurobiological, and environmental factors [15].Several earlier studies reported that depressed patients might have increased levels of cytokines or other inflammatory parameters in blood and cerebrospinal fluid [16].Though the etiology and pathophysiology of MDD remain unclear, numerous reports have suggested the association between neuroinflammation and depression [17,18].Inflammation is a complex biological response to infection, injury, and other organismal stress.It involves the activation process of immune systems that alter cytokine levels [15].Cytokines and other inflammatory molecules can cross the blood-brain barrier either by crossing through weak pores of the blood-brain barrier or by binding to the transport molecules on it.The pro-inflammatory mediators in the brain can affect brain function by several mechanisms [19].They can inhibit the release of neurotransmitters from the presynaptic neuron.These neurotransmitters include dopamine (DA), serotonin (5-hydroxytryptamine, 5HT), and norepinephrine (NE) play a fundamental role in mood and anxiety disorders [20].People experience pleasure mediated by the neurotransmitter dopamine.Many studies reported the decreased levels of DA, 5HT, and NE metabolites in serum and cerebrospinal fluid of depressed patients [21][22][23].The increased level of inflammatory cytokine alters the metabolism of dopamine, serotonin, and noradrenaline which activate the hypothalamic-pituitary-adrenal (HPA) axis to cause depression [15].The HPA axis is a neuroendocrine stress response system for maintaining stability and health [24].In contrast to pro-inflammatory cytokines, the levels of anti-inflammatory cytokines (IL-4, IL-10, IL-13, etc.) are reduced in MDD patients [25].Several studies reported that the altered levels of some pro-inflammatory and anti-inflammatory cytokines are responsible for the pathogenesis of depression [15].However, the knowledge about biological markers for understanding the pathophysiology of MDD is limited [26].
Altered levels of cytokines, for example, IL-3, lipocalin-2, tumor necrosis factor-alpha (TNF-α), IL-1β, and IL-6, have been reported in patients with MDD [17].IL-3 is a cytokine with multiple biological functions in immune response, including in the suppression of apoptosis, stimulation of cell growth, and development and differentiation of all hematopoietic cell types [27].IL-3 may affect inflammatory responses in the developing or mature brain [28].Abnormal serum levels of IL-3 have been observed in patients with mood disorders [29,30].Furthermore, according to previous studies, IL-3 and IL-3R are expressed in the brain cells of patients with mood disorders [31,32].Lipocalin-2 is known as neutrophil gelatinase-associated lipocalin (NGAL) which has recently been identified as a newly discovered adipokine (cytokine) that is highly expressed in the white adipose tissue [33].Lipocalin-2 has association with neuroinflammation.Therefore, it might play a role in developing depression, mild cognitive impairment, and neurological disorders [34,35].Interestingly, the induction of a peripheral inflammatory response and psychological stressors cause cerebral expression of lipocalin-2, which may consequently reduce spinal synaptic density in the hippocampus.Together, these data suggest that increased levels of lipocalin-2 in the central nervous system may lead to decreased neuroplasticity [33,34,36].Altered cytokines may lead to synaptic degeneration and neuronal cell death.The association between cytokine dysregulation and neurological disorders is evident [37].Therefore, altered serum levels of IL-3 have been observed in patients with mood disorders [29,30] and lipocalin-2 in patients with late-life depression [33,34,36].Thus, serum IL-3 and lipocalin-2 levels can serve as tools for diagnosis and early risk assessment of MDD.However, there are limited studies on the effects of IL-3 and lipocalin-2 in MDD patients.The present study aimed to evaluate serum IL-3 and lipocalin-2 in MDD patients and healthy controls (HCs) to understand their role in the pathophysiology and development of depression.

Study design and participants
This prospective case-control study enrolled 196 MDD patients and 180 HCs matched by age and sex.We recruited MDD patients between 18 and 60 years suffering from depressive symptoms for at least two weeks or longer in this study from the Department of Psychiatry, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh.BSMMU is a tertiarylevel health care center in the capital city that provides general and specialized treatments to mass people.Patients of every age, sex, educational status, and financial condition from all over Bangladesh come here to seek medical aid.HCs were from different parts of Dhaka city.We included the study subjects through randomization.The study participants were evaluated by a qualified psychiatrist according to the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5).A predesigned structured questionnaire was used to record the socio-demographic and biographical profiles of subjects.The severity of depression was measured by the Hamilton depression (Ham-D) rating scale.A Ham-D score of 7 or greater was considered as case.Subjects with comorbid psychiatric illness or other neuropsychiatric disorders, abnormal body mass index (BMI), immune disorders, infectious diseases, and alcohol or drug dependency were excluded from this study.We also excluded subjects currently on psychiatric medications.Exclusion criteria were equally applied to both cases and controls.

Blood sample collection
Following an overnight fast, 5 ml of blood sample was drawn from the cephalic vein of each subject by venipuncture between 8:00 AM and 9.00 AM and collected into falcon tubes.After collection, the blood samples were kept in a fixed place at room temperature for one hour without shaking or agitation and allowed to clot.Then clotted blood samples were centrifuged at 1000×g for 15 min, and serum samples were collected into an Eppendorf tube and stored at − 80 0 C until further analysis.

Serum analysis
Serum IL-3 and lipocalin-2 levels were measured using commercially available human enzyme-linked immunosorbent assay (ELISA) kits (BosterBio, USA) following the instructions provided by the manufacturer.The sensitivity or minimum detectable range of IL-3 and lipocalin-2 were < 1 pg/ml and < 10 pg/ml, respectively.There was no cross-reactivity with the other cytokines present in the serum.Analysis of all samples was performed by a person who was blind to the clinical status of the participants.

Statistical analysis
All data were processed by Microsoft Excel and SPSS software package version 25.0 (IBM Corporation, Armonk, USA) was used to conduct all statistical analyses.An Independent sample t-test was applied for numerical variables to see the group differences.Whereas the chi-square test was applied for categorical variables.Also, Spearman's correlation test was used to determine the relationship among the variables.Bonferroni-corrected p-values were calculated for pairwise comparisons in correlation analysis.We constructed scatterplot and boxplot graphs to see the distribution pattern of the analyzed parameters.All analyses were considered significant if the p-value was less than or equal to 0.05.

Socio-demographic characteristics of study population
The socio-demographic profiles of the study population are summarized in Table 1.There was no significant difference between MDD patients and HCs regarding age (MDD patients: 30.86 ± 0.69 years; HCs: 30.83 ± 0.70 years).Both study groups had a higher number of participants from the 18-25 and 23-35 age groups, where MDD patient counts were 36.22% and 37.76%, respectively, and healthy control counts were 35.00% and 36.67%,respectively, in the stated age ranges.The proportion of male (MDD patients: 33.16%; HCs: 29.44%) and female participants (MDD patients: 66.84%; HCs: 70.56%) was similar in both groups.We found that two-thirds of the MDD patients were married (66.33%), whereas the ratio is slightly less than half in HCs (47.22%).BMI was normal in the majority of the study subjects (MDD patients: 53.57%; HCs: 56.11%).In terms of education, the maximum number of participants in the MDD groups had a secondary education level (38.27%), whereas HCs had a graduation or higher degree (43.89%).Higher numbers of study subjects in MDD groups were unemployed (27.55%), but HCs subjects were commonly students (31.11%) among other occupational groups.Most study subjects, both MDD patients and HCs, had a medium economic impression (MDD patients: 72.96%; HCs: 53.33%).A superior number of the study subjects were from urban areas (MDD patients: 57.14%; HCs: 76.11%) and primarily nonsmokers (MDD patients: 90.82%; HCs: 91.11%).Despite having a lower family history of MDD (27.04%), most MDD patients were previously diagnosed with MDD (55.61%).None of the study subjects from HCs had a previous history of MDD, and very few had a family history of MDD.

Laboratory findings of the study
The clinical outcomes and laboratory findings of this study are shown in Table 2.We found higher serum IL-3 levels in MDD patients than HCs (1,024.73± 29.84 pg/mL vs. 911.11± 20.553; p = 0.002).Additionally, we observed serum IL-3 levels of females were significantly higher in MDD patients than in HCs.However, the MDD patients had lower mean serum lipocalin-2 levels than HCs (38.03 ± 2.07 ng/mL vs. 47.10 ± 3.58 ng/ mL; p = 0.026).Also, serum lipocalin-2 levels of females were significantly lower in MDD patients than in HCs. Figure 1 shows the alteration of serum IL-3 and lipocalin-2 levels.Patients with MDD display greater mean IL-3 concentrations than HCs in both sexes.Alterations in serum IL-3 and lipocalin-2 were not linked to severity scores (Ham-D score) in MDD patients.According to Spearman's correlation, we observed only serum IL-3 levels were positively associated with BMI among all pairwise comparisons after Bonferroni correction of p values (r = 0.216; p = 0.018) (Table 3).Female MDD patients had higher serum IL-3 and lower serum lipocalin-2 levels at higher Ham-D scores, but regarding lipocalin-2, male MDD patients had the opposite pattern, according to sex-specific scatter plot graphs (Fig. 2).

Discussion
As far as we know, this is the first study on serum IL-3 and lipocalin-2 levels in MDD patients from Bangladesh.Identification and application of appropriate biomarkers are crucial for the successful diagnosis and treatment of MDD patients.Organized clinical interviewing is still  the mainstay of diagnosing depression, as a reliable risk evaluation marker has yet to be identified [38,39].In this study, we investigated the potential value of IL-3 and lipocalin-2 levels as a marker for early risk assessment of MDD.Our findings indicate that serum IL-3 was significantly higher and lipocalin-2 levels were lower in MDD patients compared to HCs.Several biomarkers depict various components of the inflammatory system, which is incredibly complex.Numerous mechanisms, including stimulation of the hypothalamic-pituitary-adrenal axis (HPA axis) [40], cytokine receptor stimulation in neurons [41], activation of the kynurenine pathway [42], elevated serotonergic expression [43,44], decreased neuronal growth factors, and changes in synthesis, release, and reuptake of neurotransmitter [45], could link cytokine-mediated immune activation to the pathophysiology of depression [46,47].Several studies on biomarkers, including pro-and anti-inflammatory cytokines, were  conducted in previous decades to determine their association with depressive disorders [48].These findings are supported by the connection between MDD and the neurological underpinnings of stress-induced alterations to the immune and nervous systems [49].
The Association between IL-3 and depression has not been investigated in Bangladeshi population yet but we found significantly higher IL-3 levels in the serum of MDD patients than HCs (p = 0.002) in our study.Some studies correlated elevated IL-3 levels in depressed patients of different populations [50].Also, our findings support a previous study that found a positive correlation between high IL-3 levels and depressive symptoms in schizophrenia [51].Antigen-activated T cells mostly produce IL-3, a cytokine that is the connection between immunity and hemopoietic system.It is related to various immune pathologies [52].
Additionally, other pro-inflammatory cytokines showed similar outcomes in depression like IL-1β, TNFα, and IL-18 [53][54][55].MDD patients had considerably higher levels of IL-1β and TNF-α than HCs, according to the findings of earlier studies [15,[56][57][58][59].In addition, serum levels of IL-6, IL-10, and IL-18 were significantly higher in MDD patients [13,60,61].Therefore, Increased IL-3 levels can contribute to the symptoms of neuropsychiatric illnesses like depression [51].Moreover, our study observed that female MDD patients had serum IL-3 levels with significant elevation than the female controls (p = 0.012).Similar findings were observed in depressed females regarding other inflammatory cytokines like IL-17 A, IL-6, TNF-α [62].The prevalence and severity of depression in women could be a contributing factor to our study findings.
Lipocalin-2 serves a variety of inflammation-related functions that are connected to autoimmune responses and persistent inflammation [63].In this study, we found decreased lipocalin-2 levels in MDD patients.Also, the decrease in serum lipocalin-2 was significant in female MDD patients compared to female controls (p = 0.005).Although the mean lipocalin-2 level was found elevated in male patients than in male controls, that was not statistically significant.Men are reported to have higher plasma lipocalin-2 levels than women [64].So, this gender-specific variability could be a reason for differences found in lipocalin-2 levels between males and females.Some studies showed that lipocalin-2 has anti-inflammatory activity [65] and play an antagonistic role in the effect of pro-inflammatory cytokines like IL-6 [66].Also, a study revealed that depressive behaviors increase in case of a complete absence of lipocalin-2 in the body [67].Moreover, the absence of lipocalin-2 makes the brain prone to inflammation [68].So, here decreased expression of lipocalin-2 in the body may be associated with increased inflammatory activities leading to depression.Some earlier studies found the opposite result to our findings and suggested a correlation between elevated lipocalin-2 levels and late-life depression [33][34][35][36].According to a different study, serum NGAL levels and depression indices are associated with persons with heart failure [33].
It has been demonstrated that high levels of cytokines cause symptoms similar to systemic depression, such as exhaustion, anorexia, weight loss, sleep difficulties, feelings of hopelessness, and decreased psychomotor activity [69,70], and numerous studies have identified a relationship between various inflammatory and immunological biomarkers and depressive symptoms, or MDD [71].Therefore, it is crucial to identify and define biological risk factors to understand the etiology and treatment of depression [72].The finding of altered peripheral levels of IL-3 and lipocalin-2 in MDD patients may indicate a strong relationship between the pathophysiology of depression and these biomarkers.Additionally, analysis of serum IL-3 and lipocalin-2 levels provided significant diagnostic value for MDD patients and can be an effective indicator of probable depression development.The fact that age, sex, BMI, and other socio-demographic profiles have been firmly controlled between the groups is a positive aspect of the current study.

Limitations
The current study has several drawbacks.We did not assess dietary habits, physical activity, and treatment outcomes on the analyzed parameters.However, the relatively small size of our study may generate results with statistical power.Here we measured serum IL-3 and lipocalin-2 levels at a one-time point in patients with MDD.Thus, we are uncertain whether the altered levels of IL-3 and lipocalin-2 are a state or trait marker.Moreover, evaluating serum IL-3 and lipocalin-2 levels does not reflect the entire neuroinflammatory process of MDD.Assessing other parameters within the same population and research facility setup would be suitable.Despite these limitations, our study has sufficient strengths.For instance, this is the first study conducted in Bangladesh to evaluate IL-3 and lipocalin-2.Only a few studies have been done about this pro-inflammatory cytokine globally, and some were with contradictory findings.This study ensured diversity and homogeneity among the study population.

Conclusions
Altered serum IL-3 and lipocalin-2 levels might be associated with the pathophysiology of MDD.Therefore, these findings can pave a novel way to evaluate the risk of depression and potential treatment modalities.An observational study assessing peripheral markers may have a few limitations.Considering the results of the present study as preliminary, we recommend further research to examine the impact of the aforementioned markers on depression using larger, and more homogeneous samples.

Fig. 2 Fig. 1
Fig. 2 Sex-specific scatter plot graphs showing the distribution of serum IL-3 and lipocalin-2 levels with Ham-D scores of MDD patients

Table 1
Socio-demographic profile of the study population

Table 2
Clinical profile and laboratory findings of the study population

Table 3
Spearman's correlation study among various research parameters among MDD patients