Low levels of soluble TWEAK, indicating on-going inflammation, were associated with depression in type 1 diabetes: a cross-sectional study

Background Low levels of the soluble tumour necrosis factor-like weak inducer of apoptosis (sTWEAK) and depression are linked to cardiovascular disease. Galectin-3, inadequate glycemic control and low high-density lipoprotein (HDL)-cholesterol levels were previously linked to depression in these patients with type 1 diabetes mellitus (T1DM). The main aim was to explore whether sTWEAK was associated with depression. A secondary aim was to explore diabetes related variables associated with low sTWEAK. Methods Cross-sectional design. T1DM patients (n = 283, men 56%, age18–59 years) were consecutively recruited from one specialist diabetes clinic. Depression was defined as Hospital Anxiety and Depression Scale-Depression sub scale ≥8 points. Blood samples, anthropometrics and blood pressure were collected, supplemented with data from electronic health records. Enzyme linked immunosorbent assays were used to measure sTWEAK and galectin-3. Low sTWEAK was defined as < 7.2 ng/ml and high galectin-3 as ≥2.6 ng/ml. Multiple logistic regression analyses were performed, calibrated and validated for goodness of fit. We adjusted for age, sex, diabetes duration, galectin-3, metabolic variables, serum-creatinine, smoking, physical inactivity, medication, and cardiovascular complications. Results For 29 depressed versus 254 non-depressed patients the prevalence rates were for low sTWEAK: 93 and 68% (p = 0.003) and for high galectin-3: 34 and 13% (p = 0.005) respectively. HDL-cholesterol levels were lower for the depressed (p = 0.015). Patients with low sTWEAK versus high sTWEAK had lower usage of continuous subcutaneous insulin infusion (CSII) (6% versus 17%, p = 0.005). Low sTWEAK (adjusted odds ratio (AOR) 9.0, p = 0.006), high galectin-3 (AOR 6.3, p = 0.001), HDL-cholesterol (per mmol/l) (AOR 0.1, p = 0.006), use of antidepressants (AOR 8.4, p < 0.001), and age (per year) (AOR 1.05, p = 0.027) were associated with depression. CSII (AOR 0.3, p = 0.003) and depression (AOR 7.1, p = 0.009) were associated with low sTWEAK. Conclusions Lower levels of sTWEAK and HDL-cholesterol and higher levels of galectin-3 were independently associated with depression in T1DM. These factors might all contribute to the increased risk for cardiovascular disease and mortality previously demonstrated in patients with depression. CSII (inversely) and depression were independently associated with low sTWEAK levels.


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Conclusions: Lower levels of sTWEAK and HDL-cholesterol and higher levels of galectin-3 were independently associated with depression in T1DM. These factors might all contribute to the increased risk for cardiovascular disease and mortality previously demonstrated in patients with depression. CSII (inversely) and depression were independently associated with low sTWEAK levels.
Keywords: Cardiovascular complications, Continuous subcutaneous insulin infusion, Depression, Galectin-3, HbA1c, High-density lipoprotein-cholesterol, Inflammation, Soluble tumour necrosis factor-like weak inducer of apoptosis, Type 1 diabetes mellitus Background Type 1 diabetes (T1DM) is an autoimmune disease, characterized by insulin deficiency due to pancreatic beta cell loss leading to hyperglycaemia [1]. The introduction of intensive insulin therapy for patients with T1DM has resulted in an increased prevalence of the components of the metabolic syndrome, contributing to an amplified prevalence of cardiovascular complications [2]. Inflammatory disturbances also contribute to cardiovascular disease [3].
In patients with diabetes, depression is associated with increased prevalence of all diabetes-related complications [4], and with increased cardiovascular and allcause mortality [5]. Depression has been linked to metabolic, autonomic and hypothalamic-pituitary-adrenal (HPA)-axis dysregulations with subsequent disturbances of cortisol secretion [6,7], and there is growing evidence that immuno-inflammatory changes contribute to the development of depression [6][7][8][9][10]. Insulin deficiency, hyperglycemia as well as episodes of hypoglycemia all have impact on the brain, and might contribute to the development of depression in patients with T1DM [7].
The tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is a transmembrane protein, which is a member of the tumour necrosis factor (TNF)-receptor super family [11]. TWEAK is proteolytically processed by furin which leads to the release of soluble (s)TWEAK [11]. In the reverse process, sTWEAK binds to the functional receptor of TWEAK, Fn14 [12]. The receptor Fn14 is highly upregulated in systemic inflammatory states, which leads to increased sTWEAK binding and subsequently lower levels of sTWEAK [12]. The TWEAK/Fn14 axis plays a beneficial role in tissue repair after acute injury. However, it has been shown that chronic TWEAK/Fn14 axis activation is implicated in the development of cardiovascular disease [12]. High levels of sTWEAK are released by normal arteries, but are diminished in people with chronic vascular damage such as carotid stenosis, coronary artery disease and heart failure [12,13], resulting in an increased risk for cardiovascular mortality [14]. Proinflammatory effects of TWEAK on astrocytes in vitro implies that TWEAK could play a significant role in brain inflammation [15]. Low sTWEAK levels have been demonstrated in people with T1DM [16], type 2 diabetes mellitus (T2DM) [17], and gestational diabetes [18]. Low sTWEAK levels have also been demonstrated in depressed people without diabetes [19], and in people with bipolar disorder during ongoing manic episodes [20]. Galectin-3 is a beta-galactoside-binding lectin involved in several inflammatory processes [21]. Increased galectin-3 levels have been linked to coronary artery disease [22,23], heart failure [24,25], prolonged inflammatory responses in the brain [26], and to cardiovascular and allcause mortality [25,27]. Systemic inflammation causes decreased high-density lipoprotein (HDL)-cholesterol levels which contribute both to reduced capacity for reverse cholesterol transport, and to reduced capacity to protect lowdensity lipoprotein (LDL)-cholesterol from oxidation [28]. Low HDL-cholesterol levels were previously associated with high levels of galectin-3 binding protein in these patients [29]. Inadequate glycemic control contributes to increased cardiovascular and all-cause mortality [30].
We hypothesised that one biological link between depression and cardiovascular complications in depressed patients with T1DM is a chronic inflammatory state due to TWEAK activation with subsequent low levels of sTWEAK. The main aim was to explore whether low levels of sTWEAK were associated with depression in T1DM patients. A secondary aim was to explore diabetes related variables associated with low sTWEAK.

Participants and study design
The study has a cross sectional design and included 287 patients with T1DM. For inclusion and exclusion criteria, included and missing variables, see Fig. 1. Inclusion criteria were T1DM with ≥1-year duration, in patients 18-59 years of age. Exclusion criteria were pregnancy, severe somatic and psychiatric disorders such as cancer, hepatic failure, end-stage renal disease, Cushing's disease, severe autoimmune disorders such as systemic lupus erythematosus, psychotic disorders, bipolar disorder, severe personality disorders, severe substance abuse, cognitive deficiency (due to stroke, dementia or mental retardation), or inadequate knowledge of the Swedish language [29,[31][32][33][34][35][36]. As patients with these disorders were excluded, no specific medications for any of these disorders were used by the included patients.

Self-reported depression
Depressive symptoms were assessed by the Hospital Anxiety and Depression Scale -the depression subscale (HADS-D), which consists of 7 statements. Each statement has four response alternatives with scores from 0 to 3. The recommended cut-off level was used to define depression: ≥ 8 points as in our previous studies [29,[31][32][33][34][35][36]38]. HADS was developed to detect symptoms of depression and anxiety in patients repeatedly searching medical care for somatic complaints, where no somatic disorders were confirmed that could explain their symptoms [38]. No higher levels of emotional awareness are necessary to respond to the statements. A major characteristic of HADS-D is that potential symptoms of somatic disease are not included [38]. According to previous research HADS-D is a useful instrument for detecting symptoms of depression, both at an individual and a collective level, and has been demonstrated to have a good reliability and discriminant validity [39].

Biochemical analyses
Plasma levels of sTWEAK and galectin-3 were measured using commercially available DuoSet enzyme linked immunosorbent assays (ELISA) kits (R&D Systems, Minneapolis, Mn, USA) and optimised for human plasma. The analyses were run according to the manufacturer's instructions. The samples were diluted 1:5 and 1:2, and the intra-assay coefficients of variation were 1.8 and 4.3% respectively for sTWEAK and galectin-3. All samples were run as duplicates. High galectin-3 levels were defined as ≥2.6 ng/ml as in our previous research [33].

Insulin resistance
A ratio between triglycerides and HDL-cholesterol was calculated for the estimation of insulin resistance [41].

Episodes of hypoglycemia
A severe episode of hypoglycemia was defined as hypoglycemia to such a degree that the patient needed help from another person. Episodes during the last 6 months prior to recruitment were registered [29,[31][32][33][34][35][36].

Smoking and physical inactivity
Smokers were defined as having smoked any amount of tobacco during the last year. Physical activity was dichotomized into physical inactivity which was defined as less than 30 min of moderate activities once a week, and physical activity which represents all other levels of physical activity [29,[31][32][33][34][35][36].
Indications for antihypertensive drugs were systolic blood pressure > 130 mmHg and/or diastolic blood pressure > 80 mmHg according to the Swedish national guidelines in 2009 [42].

Statistical analysis
Analysis of data distribution using histograms revealed that age, diabetes duration, sTWEAK, galectin-3, triglycerides, systolic and diastolic blood pressure, were not normally distributed. Data were presented as median (quartile (q) 1 , q 3 ), and analyses were performed with Mann-Whitney U test. Fisher's Exact Test (two-tailed) was used to analyse categorical data, and data were presented as N (%). The 60th, 65th, 70th and 75th percentiles of sTWEAK and log-transformed sTWEAK (Lg10) were tried against depression in a backward elimination multiple logistic regression analysis, and the percentile with the highest association was defined as low sTWEAK and was therefore used in the further analyses. Crude odds ratios (CORs) for the associations with depression and with low sTWEAK (< 7.2 ng/ml) were calculated for all included variables. Variables with p < 0.10 for the CORs were entered into multiple logistic regression analyses (Backward: Wald) with depression and low sTWEAK as dependent variables [43]. The Hosmer and Lemeshow test for goodness-of-fit and Nagelkerke R 2 were used to evaluate each multiple logistic regression analysis model. Confidence intervals (CIs) of 95% were used. P < 0.05 was considered statistically significant. SPSS® version 25 (IBM, Chicago, Il, USA) was used.
Comparisons between patients with low sTWEAK levels (< 7.2 ng/ml), and high sTWEAK levels (≥ 7.2 ng/ ml) are presented in Table 4. Fourteen percent of the patients with low sTWEAK were depressed and 2% of the patients with high sTWEAK were depressed (p = 0.003). Patients with low sTWEAK used CSII (6%) to a lower extent than patients with high sTWEAK (17%) (p = 0.005).

Discussion
The main finding of this study of adult patients with T1DM was that the depressed patients had lower levels of sTWEAK than the non-depressed patients. Lower levels of sTWEAK and HDL-cholesterol, higher levels of galectin-3, the use of antidepressants, and age were independently associated with depression. The depressed patients had also higher levels of HbA1c, but the association between high HbA1c and depression was not independent in this context. The use of CSII was inversely associated with low sTWEAK.
Depression is a serious disease with somatic implications, including cardiovascular disease and all-cause mortality [4][5][6][7]. Three independent risk factors or risk markers for cardiovascular disease were demonstrated in these depressed patients with T1DM. Low sTWEAK levels [12][13][14] and high galectin-3 levels [22][23][24][25]27] have previously been linked to the development of cardiovascular disease and increased mortality. HDL-cholesterol levels decrease in inflammatory states [28,29]. HDL-cholesterol is an established risk marker for cardiovascular disease, but the role of HDL-cholesterol as a causal factor in cardiovascular disease is disputed [44]. In previous research, we have not demonstrated any higher prevalence of metabolic disturbances in the depressed than in the nondepressed, except for increased HbA1c and lower HDLcholesterol in the depressed patients [31,34]. In this study we added a proxy for insulin resistance [41], which was not associated with depression. Increased HbA1c levels without associated obesity or signs of insulin resistance in the depressed patients could be due to inadequate insulin supply. Insulin deficiency has been suggested as one reason for the development of depression in patients with T1DM [7,45]. Users of MDII compared to users of CSII had more often low levels of sTWEAK. We haven't found any previous research exploring the associations between sTWEAK and CSII.
One difficulty we had to address was that there is no established consensus regarding normal sTWEAK levels. Therefore, we explored and compared the associations between log transformed sTWEAK, four different definitions of low sTWEAK levels and depression. Low sTWEAK levels, defined as levels below the 70th percentile (< 7.2 ng/ml), showed the highest association with depression, and this cut-off level was therefore chosen in the further analyses. To our knowledge, a potential association between sTWEAK and depression hasn't previously been explored in patients with T1DM. Neither has it been explored whether low sTWEAK, high galectin-3, high HbA1c and low HDL-cholesterol were independently associated with depression. We only found one previous study exploring the association between sTWEAK and depression, and that study was performed in a population without diabetes [19]. Their findings of an association between low sTWEAK levels and depression is in accordance with our findings. Another study showed that manic episodes were linked to low levels of Table 2 Elimination analyses between log transformed sTWEAK, 4 levels of dichotomized sTWEAK and depression   sTWEAK levels [20]. Both depressive states and manic episodes may be symptoms of brain inflammation. Our findings of low sTWEAK levels in the depressed patients imply activation of TWEAK, which has proinflammatory effects on the astrocytes in the brain, which have been demonstrated in vitro [15]. According to previous research, galectin-3 may contribute to microglia activation with sustained inflammatory responses in the brain [26]. These findings are potentially very important as it has been demonstrated in previous research that both astrocytes and microglia are involved in the development of depression [46,47]. There are several subjects for further research. As this is a cross-sectional study, we can't clarify whether the depressed state leads to immunological disturbances, or if these immunological disturbances lead to a depressed state. To answer this question, it will be necessary to perform longitudinal studies. We will perform a followup exploring the impact of sTWEAK and galectin-3 on cardiovascular complications, comparing with conventional diabetes related risk factors. According to previous research, some antidepressants attenuate immunoinflammatory changes [9]. Whether antidepressants may have impact on the levels of sTWEAK and galectin-3 is a subject for further exploration. As immunoinflammatory changes and the activation of the hypothalamic pituitary axis are part of the stress response, which is involved in depression, it would also be interesting to explore associations between sTWEAK, galectin-3 and cortisol secretion [9]. To compare TWEAK levels between users of MDII and users of CSII in a future larger study would be very interesting. If our findings would be confirmed in a larger study, it would be a very important finding supporting the choice of CSII over MDII. Finally, development of novel therapeutics against the TWEAK/Fn14 axis may be of value both for the treatment or prevention of depression and cardiovascular disease. Strengths of the study are that inclusion and exclusion criteria were well defined. No patients using systemic corticosteroids, or specific drugs for psychotic or bipolar disorders were included. The findings of this study are new as exploration of the association between sTWEAK and depression has not previously been performed in a clinically well-defined setting of T1DM. Neither has the mode of insulin distribution been explored against sTWEAK. The results were controlled for relevant variables which in previous research have been linked to either depression or cardiovascular complications, or to both. The logistic regression models were elaborated for the associations, and calibrated and validated for goodness of fit with the data variables. Precise ELISA techniques were used, and the assays showed low intra-assay coefficients of variation for both sTWEAK and galectin-3. One weakness was that there was no control group with persons without diabetes. Another weakness was that there were very few patients with cardiovascular complications, so no further explorations of associations with cardiovascular complications could be performed. A third weakness was that depression was not assessed by a clinical interview. However, the association between the use of antidepressants and selfreported depression in this study was high, indicating that depression assessed by HADS-D had clinical significance.

Conclusions
The hypothesis that the depressed patients with T1DM had lower levels of sTWEAK than the non-depressed was confirmed. Low levels of sTWEAK and HDL-cholesterol and high levels of galectin-3 were independently associated with depression in T1DM. These disturbances have previously been associated with cardiovascular disease and mortality, and might contribute to the increased risk for Sex (women) 1.5 (0.9-2.5) 0.14 -- cardiovascular disease and mortality previously demonstrated in T1DM patients with depression. We also found that the users of CSII had lower prevalence of low sTWEAK levels than the users of MDII.