The monocyte-to-lymphocyte ratio and depression in diabetes patients

Background: The purpose of this study was to determine the association between The monocyte-to-lymphocyte ratio (MLR) and depression with diabetes mellitus. Method: We examined data from the US National Health and Nutrition Examination Survey from 2009 to 2016. Cox proportional hazard models were used to calculate the associations between MLR and depression. For precise investigation of the relationship, we also plotted the smooth curve t and generated a two-piecewise linear regression model using the penalized spline method. Result: We enrolled 2820 diabetes patients in the database. Diabetes patients who had high MLR tended to be young, female, obese, unmarried, and had low levels of education. For tertile analysis, the ORs and 95% CIs of clinically relevant depression in tertile analysis were 1.03 (0.91, 1.17) for the second group and 1.62 (1.44, 1.82) for the third group in the unadjusted model compared to the control group. A similar trend was observed for the adjusted model and the quartiles analysis. We found the inection point of MLR was 2.7. There is a positive association between MLR and depression above the threshold, and no relationship was found when MLR was below the threshold in diabetes patients. Conclusion: There is a nonlinear relationship between MLR and depression in diabetes patients. High level of MLR more than the inection point may add prognostic information for depression in diabetes patients.


Background
Depression is associated with diabetes mellitus (DM) [1]. Depression in diabetes patients is a condition that negatively impacts patient engagement and adherence to medication, leading to reduced quality of life, inadequate glucose control, increased functional disability, elevated risk of mortality, and increased health expenditures [2][3][4]. Greater risk of developing depressive symptoms is found in DM patients, and depression patients are also susceptible to DM [5]. A study found that almost 30% of diabetes patients suffered from depression[6], however, the morbidity of depression in diabetes is usually underestimated.
There are various tests for diagnosing and monitoring depression disorders. The Back Depression Inventory (BDI-II) and the 9-item Patient Health Questionnaire (PHQ-9) are usually used to diagnose major depression disorder (MDD) [7,8]. However, these tools are not easy to use by non-psychiatrists, and they may be inaccurate in patients with both with depression and DM because there are few symptom overlaps between the conditions [9]. Therefore, it is important to identify early biomarkers to diagnose depression in diabetes patients.
Several lines of evidence support the notion that the immune dysfunction and in ammation activation play signi cant roles in the pathogenesis of MDD and DM [10,11]. Increased serum levels of proin ammation cytokines and chemokines are found in MDD patients [12]. Depression can be attenuated in a diabetes mouse model with decreased levels of in ammatory biomarkers Interleukin-1 (IL-1) and Interleukin-6 (IL-6) [13]. These data suggest that changes in levels of in ammation may be used to predict depression in patients with DM. The monocyte-to-lymphocyte ratio (MLR) may be a biomarker of systemic in ammation to predict the severity and prognosis in malignant tumors and cardiovascular diseases. MLR is a low-cost biomarker that can be calculated simply from complete blood counts [14,15].
Higher neutrophil-to-lymphocyte ratio (NLR) and MLR are strongly association with increased multiple sclerosis -related neurological disability and brain atrophy [16]. In patients with DM, higher MLR is an independent risk factor for diabetic retinopathy [17]. However, there are no data about the association between MLR and depression in DM patients. Therefore, investigating the usage of LMR in DM patients with depression is worthwhile.

Data source
The National Health and Nutrition Examination Survey (NHANES) is directed by the Centers for Disease Control and Prevention (CDC) [18]. It was initiated from 1999 and is updated in 2-year cycles. NHANES is a strict, long-term, and large-scale survey representative of the civilians of the United States. Through interviews, examinations, questionnaires and anthropometry, NHANES monitors the health and nutrition status of the general American population. Other detail information regarding sampling, design, and components can be found at http://www.cdc.gov/nchs/nhanes. For our analyses, we combined data from four cycles of the NHANES survey (2009-2010, 2011-2012, 2013-2014, and 2015-2016).

MLR and depression in diabetes patients
Monocytes and lymphocytes counts were analyzed on an automated hematology analyzing device and were expressed as 1000 cells/µL. The MLR was calculated as monocyte count/lymphocytes count. We also evaluated the morbidity rate of depression in diabetes patients based on each value of MLR. To identify associations between MLR and depression in diabetes patients, we treated them as continuous variables and tertiles in order to apply the available data more e ciently and exibly.

Study variables
We used structure query language (SQL) to extract data from the database. The variables included age, sex, race, education, and marital status. Vital signs variables included systolic blood pressure (SBP), diastolic blood pressure (DBP), body mass index (BMI), and waist circumference. Laboratory variables included total cholesterol, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), triglycerides, red cell distribution width (RDW), cholesterol, glucose, and HbA1c. Diabetes-related variables included diabetes mellitus, family history of diabetes and the use of insulin. Comorbidities included coronary artery disease (CAD), chronic heart failure (CHF), diabetic retinopathy (DR) and stroke.
The total PHQ-9 score was also presented.

Statistical analyses
We enrolled 2820 diabetes patients in NHANES database from 2009 to 2016. According to value of MLR, we divided all patients into three subgroups. Patients with MLR value < 2.7 were regarded as the low group, 2.7-3.45 were the medium group, and > 3.45 were the high group. Continuous variables were expressed as mean ± standard deviation or interquartile range (IQR), and frequencies for categorical data.
Differences betwen groups were compared using the Kruskal-Wallis test for continuous variables and the χ2 test or Fisher's exact test (expected frequency < 10) for categorical variables. A value of p < 0.05 was considered statistically signi cant.
We used the multivariate Cox proportional hazards model to analyze the association between MLR and depression in DM patients. To analyze the data in detail, we divided the MLR into tertials or quartiles.
or(ORs) with 95% con dence intervals (CIs) was used to express the results of statistical analyses. Model 1 was adjusted for the confounders age, sex, and race. Model 2 was adjusted for age, sex, race, marital status, education, CHF, CHD, and stroke.
To identify the nonlinear relationship between MLR and depression in diabetes patients, we established a weighted generalized additive model and plotted a smooth curve t (using the penalized spline method).
We calculated the point of in ection by applying a recursive algorithm. Later, we established a weighted two-piecewise linear regression model. R software (http://www.R-project.org) was used to perform the statistical analyses. A value of p < 0.05 was considered statistically signi cant.

Characteristics of enrolled participants
We enrolled 2820 participants in the analysis and divided them into three groups according to the value of MLR: < 2.7 were regarded as the low group, 2.7-3.45 were the medium group, and > 3.45 were the high group. The general characteristics of each group are summarized in Table 1, including demographics, vital signs, laboratory parameters, diabetes-related variables, and comorbidities. In summary, diabetes patients who had high MLR tended to be young, female, obese, unmarried, and had low levels of education. They were more likely to have high levels of triglycerides and RDW, low levels of HDL, and higher risk of co-morbid DR and depression.

Association between MLR and clinically relevant depression in diabetics
We established two models to measure the independent effects of MLR and CRD in diabetes patients.
ORs and 95% CIs are displayed in Table 2

Nonlinear correlation between MLR and clinically relevant depression in diabetics
The relationship between MLR and clinically relevant depression in diabetes patients appeared to be nonlinear. For precise investigation of the relationship, we plotted the smooth curve t (Fig. 1). We generated a two-piecewise linear regression model using the penalized spline method ( Table 3). The in ection point of MLR was 2.7. When value of MLR was higher than the in ection point, the HR (95% CI) was 1.4 (1.3, 1.5) (P = 0.006). To the left of the in ection point, the relationship was not signi cant [HR 1.0 (0.8, 1.2)].

Discussion
Clinically relevant depression in diabetes patients correlated with MLR in a nonlinear manner. Higher MLRs were found in diabetes patients with depression than those without in a cohort of the US population when the level of MLR was more than 2.7. Increased MLR might predict high risk of depression in diabetes patients.
In ammation plays a critical role in the initiation and progression of depression in diabetes patients. High levels of cytokines and chemokines induced insulin resistance, and interfered with the function of pancreatic cells [19]. Proin ammatory cytokines play important roles in the pathophysiology of depression, including downregulated neurotransmitter levels, impaired synaptic plasticity, and disturbed neuroendocrine function [20]. We found that increased MLR in DM patients was association with high risk of depression, possibly representing a proin ammatory response. Elevated MLR is a biomarker for endothelial dysfunction and system in ammation in malignancies [21], while MLR was used to predict poor prognosis in psychiatric diseases. Ikbal et al. found that MLR was higher in patients in the manic state of bipolar disorder than the control group [22]. Mario et al. found higher MLR in both major depression disorder and the depressive phase bipolar disorder than in the bipolar disorder manic phase [23]. These results were similar to those of our study, in that the high value of MLR was associated with elevated risk of depression. We might explain the role of MLR in depression in DM patients from two aspects: monocytes and lymphocytes.
Monocytes are derived from the bone marrow (BM). BM-derived monocytes were shown to be tra cked and recruited into the central nervous system (CNS) under conditions of psychological stress [24,25]. Accumulation of BM-derived monocytes in the brain ampli ed pro-in ammation signaling [26]. Finally, increased levels of in ammation cytokines and chemokines (IL-1β, TNF-α, IL-6, CXCL) are implicated in depressive behavior [27]. In support of this idea, Torres et al. found that depressed patients who committed suicide had higher levels of monocyte marker Iba-1 than did the control group without depression [28]. Researchers also found increased numbers of CD11b + CD45 hi cells, marker of monocytes, in the brains of a depression mouse model caused by repeated social defeat [29]. These data suggest that elevated levels of monocytes might mediate stress-induced in ammation responses in DM patients. On the other hand, BM-derived monocytes mediated depression-related functions activated by microglia [30].
Microglia activation is implicated in in ammatory responses and depression-like behavior caused by stress [31]. For instance, Elaine al et. performed a PET imaging study and found that duration of untreated major depressive disorder was signi cantly associated with levels of a biomarker of microglial activation, translocator protein (TSPO). They found that TSPO total distribution volume in brain predicted the duration of untreated major depressive disorder; it was greater in depressed patients with long duration disease than in those with short duration [32]. Taken together, the data suggest that microglia activation and neuroin ammatory might be the reason why diabetes patients with depression have high levels of monocytes.
Lymphocytes are a subgroup of leukocytes and mediate immune regulatory roles in in ammatory diseases. Activation and disturbance of stress systems in DM patients mediated the activation of the Hypothalamic-pituitary-adrenal axis (HPA-axis) and sympathetic nervous system (SNS) [33]. Chronic stress increased the number of leukocytes, while it was a selective increase for myeloid cells, not for lymphocytes in the BM. With proliferating and expanding of myeloid progenitor cells in the BM, chronic stress induced increased monocyte release from BM and reductions in lymphocytes and erythrocytes [34]. For example, Heidt and colleagues found that chronic variable stress elevated proliferation of hematopoietic stem cell and selective increased output of in ammatory monocytes in the periphery [35]. Furthermore, sustained activation of the HPA-axis caused by chronic stress was associated with promoted apoptosis of lymphocytes [36]. The reduction caused by abnormal monocyte proliferation and increased apoptosis of lymphocytes might explain the greater degree of lymphocytopenia in DM patients with depression than in those without. In general, high MLR in patients with combined depression and DM might represent monocyte activation and neuroin ammation induced by chronic stress.
There are some limitations in our study. First, as a cross-sectional survey, NHANES cannot provide longitudinal follow-up, and temporal alterations in MLR cannot be evaluated in diabetes patients. Second, it is less rigorous to diagnose depression only using PHQ-9, because this depends on clinical and methodological settings. Therefore, designs of experimental research have been more suitable to solve this problem. Third, it is di cult to distinguish whether patients are depressed after diabetes, or whether they have depression before diabetes. In other words, the train of causation cannot be determined.

Conclusion
We found a nonlinear relationship between MLR and depression in patients with diabetes patients. When the level of MLR was above the in ection point (2.7), high MLR is associated with increased risk of clinically relevant depression in diabetes patients. Further research with longitudinal follow-up of MLR in patients of diabetes combined with depression is needed.

Declarations
Ethics approval and consent to participate Not applicable

Authors' contributions
Depu Zhou: Date analyze and writing. Jie Wang: Data collection. Xiaokun Li: Writing-Reviewing and Editing.

Availability of data and materials
All the data used to support this study are available from the corresponding author upon request.

Con icting Interest
The authors declare that there is no con ict of interest.

Funding Statement
There are no funding supporting this article.

Figure 1
Association of MLR levels with prevalence of depression in diabetes. Dashed lines are 95% con dence intervals.