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Soluble CD163 was linked to galectin-3, diabetic retinopathy and antidepressants in type 1 diabetes

Objective Depression has been associated with diabetic retinopathy and increased plasma levels of galectin-3, a lectin expressed in activated macrophages. Increased levels of sCD163, the soluble form of a macrophage expressed scavenger receptor involved in several inflammatory processes, have been demonstrated in the vitreous of the eye in type 1 diabetes (T1D) patients with severe diabetic retinopathy. The aim was to explore whether circulating sCD163 was associated with diabetic retinopathy, depression and/or galectin-3 in T1D patients, controlling for gender, metabolic factors, other diabetes complications, life style and medication. Design Cross sectional. Methods Two hundred eighty-seven T1D patients, men 56%, age 18–59 years, diabetes duration ≥1 year, were consecutively recruited from one specialist diabetes clinic. Depression was assessed by Hospital Anxiety and Depression Scale-Depression subscale. Blood samples, anthropometrics and blood pressure values were collected, supplemented with data from electronic medical records and the Swedish National Diabetes Registry. High plasma sCD163 was defined as ≥0.575 mg/L (corresponding to the 80th percentile) and high plasma galectin-3 as ≥4.659 µg/L (corresponding to the 95th percentile). Results The prevalence of depression was 10%, antidepressant medication 8%, diabetic retinopathy 72%, high sCD163 20% and high galectin 3 5%. High galectin-3 (AOR 9.7), antidepressants (AOR 3.8), diabetic retinopathy (AOR 2.4) and systolic blood pressure (per mmHg) (AOR 1.03) were associated with high sCD163. Conclusions This is the first study to show that circulating sCD163 was independently associated with galectin-3, the use of antidepressants and diabetic retinopathy, in patients with T1D. Depression was not associated with sCD163.


Introduction
Diabetic retinopathy (DR) is the most common complication of diabetes (1,2) and is a major cause of blindness (3,4). DR progresses over time, but fortunately only a limited number of patients acquire one of the two major vision-threatening complications, proliferative DR or macular edema (3). The necessity of optimal metabolic control in the prevention of DR is well documented, but some patients develop vision loss despite good metabolic control (2,3,5). The pathogenesis of DR occurs by alterations in multiple biochemical pathways, which involve several metabolic, inflammatory and endocrine responses (1,2,4,6).
Depression is the most common psychiatric disorder worldwide with great impact not only on functioning and quality of life, but also on somatic health due to metabolic, inflammatory, autonomic and hypothalamicpituitary-adrenal (HPA) axis dysregulations (7,8,9,10,11). Longitudinal studies have shown that depression was associated with the incidence and progression of DR, particularly progression to proliferative DR (12,13).
CD163 is a macrophage and monocyte expressed scavenger receptor (14,15). It was initially identified as a pattern recognition receptor in bacterial infections, and it was shown that interaction between bacterial structures and CD163 led to an increase of proinflammatory cytokines (16). The soluble form, sCD163, is shed during inflammation from CD163 by ADAM-17, the same metalloproteinase that sheds TNF-α (17), which is another inflammatory molecule involved in the development of DR (6). Increased concentrations of sCD163 have been demonstrated in the vitreous of the eye in patients with proliferative DR, and the sCD163 levels were particularly high in patients that had developed preretinal fibrovascular membranes (18). Increased levels of plasma sCD163 have been demonstrated in T1D (19), and in metabolic conditions such as obesity, dyslipidemia, hypertension and insulin resistance (15,20,21,22). There are currently no reports on sCD163 in diabetes patients with depressive disorders.
In these patients with T1D, we have previously shown that depression was associated with increased galectin-3 (23), a soluble β-galactoside-binding lectin, which is expressed in several cells such as activated macrophages and microglia, and is implicated in a variety of biological processes including neuroinflammation and neurodegeneration (24,25,26).
We hypothesize (1) there are immunological links between depression and DR, (2) high levels of plasma sCD163 could reflect the presence of DR and (3) macrophage activation could lead to simultaneous increase in galectin-3 and sCD163 levels. The primary aim was to explore whether high plasma sCD163 levels were associated with depression and with DR. The secondary aim was to explore whether high levels of sCD163 were associated with high galectin-3 levels. While exploring these aims, we controlled for gender, metabolic factors, creatinine, other diabetes complications, life style and medication.

Subjects and methods
This study has a cross-sectional design and is one of seven baseline analyses (7,11,23,27,28,29) for a randomized controlled trial (ClinicalTrials.gov: NCT01714986) where 'Affect School with Script Analysis' was tried against 'Basic Body Awareness Therapy' for persons with diabetes, inadequate glycemic control and psychological symptoms (30). Patients who attend the clinic every 6 months for regular follow-up visits were consecutively recruited by specialist diabetes physicians or diabetes nurses during a nine-month period, 03/25/2009 to 12/28/2009, from one hospital diabetes outpatient clinic. The catchment population was 125,000 in Region Kronoberg, Sweden. Inclusion and exclusion criteria, number of participants and missing values are shown in Figure 1. Inclusion criteria were T1D with ≥1-year duration, age 18-59 years. Exclusion criteria were cancer, hepatic failure, endstage renal disease, psychotic disorder, bipolar disorder, severe personality disorder, severe substance abuse, cognitive deficiency (due to stroke, dementia or mental retardation), or inadequate knowledge of Swedish. There were sCD163 measurements available for 287 patients out of 433 eligible patients (Fig. 1). Thus, 287 patients were included in this study. A questionnaire was used to assess self-reported depression. Blood samples, anthropometrics and blood pressure (BP) were collected. Data were collected from electronic medical records, and from the Swedish National Diabetes Register (S-NDR) (5). All participants provided written informed consent. The study was conducted in accordance with the principles of the Declaration of Helsinki as revised in 2008 and was approved by the Regional Ethical Review Board of Linköping University, Linköping (Registration no. M120-07, T89-08).

Self-reported depression
Self-reported depression was assessed by the Hospital Anxiety and Depression Scale-Depression subscale (HADS-D) which consists of seven statements, with four response alternatives from 0 to 3. The cut-off level, ≥8 points, was used (31).

Biochemical analyses
Blood samples were collected into EDTA plasma tubes (BD, Franklin Lakes, NJ, USA). Plasma sCD163 was analyzed using commercially available human enzymelinked immunosorbent assay (ELISA) DuoSet kits and supplementary ancillary kits (R&D Systems). Plasma samples from the participants were diluted 1:200 in PBS with 1% BSA and run in duplicates according to the manufacturer's instructions. Absorbance was measured at 450-580 nm in a FLOUstar optima plate reader (BMG Labtech GmbH, Ortenberg, Germany). A 7-point standard curve was used to create a 4-parameter logistic regression curve from which concentrations of unknown samples were calculated. The intra-assay coefficient of variation for the sCD163 ELISA analysis was 2.0%.
The plasma sCD163 (mg/L) were dichotomized at five levels, the 75th; the 80th; the 85th; the 90th; and the 95th percentiles, which were all tried against DR and depression.
The plasma galectin-3 was analyzed using commercial ELISA DuoSet kits and supplementary ancillary kit (R&D Systems). Patient samples were diluted 1:2 in PBS supplemented with 1% BSA and run in duplicates. The ELISA analysis was performed according to the manufacturer's instructions. Absorbance was measured at 450-580 nm in a FLOUstar optima plate reader (BMG Labtech GmbH, Ortenberg, Germany). A 7-point standard curve was used to create a 4-parameter logistic regression curve from which concentrations of unknown samples were calculated. The intra-assay coefficient of variation for the analysis was 4.3%. Galectin-3 (µg/L) was available for 282 patients and was dichotomized at the 75th; the 80th; the 85th; the 90th and the 95th percentiles, which were tried against the 80th percentile of sCD163.

Anthropometrics and BP
Weight, length and BP were measured according to standard procedures by a nurse and BMI (kg/m 2 ) was calculated.

Episodes of hypoglycemia
A severe episode of hypoglycemia was defined as needing help from another person. Episodes during the last 6 months prior to recruitment were registered.

Smoking and physical inactivity
Smokers were defined as having smoked any amount of tobacco during the last year.
Physical inactivity was defined as moderate activities, such as 30 min of walking, less than once a week.

Diabetic retinopathy
DR was defined as non-proliferative or proliferative retinopathy with microangiopathy changes as viewed by fundus photography through a dilated pupil. Data regarding DR were collected from the S-NDR, where DR disease severity levels were not described in 2009.

Cardiovascular complications
Cardiovascular complications were defined as ischemic heart disease, cardiac failure, stroke or transient ischemic attack.

Foot complications
These were defined as neuropathy, angiopathy, earlier or present diabetes foot ulcer, foot infection, foot deformity, arthropathy or amputation of the lower limb.
Diabetes-specific treatment was divided into two groups: Insulin only (multiple daily insulin injections or continuous subcutaneous insulin infusion) or insulin combined with oral antidiabetic drugs (OAD) (ATC code A10BA02). The indications for OAD prescription in addition to insulin were obesity and insulin resistance.

Statistical analysis
Analysis of data distribution using histograms and calculation of skewness was performed and revealed that plasma sCD163 and galectin-3 were not normally distributed. Data were presented as median values (quartile (q) 1 , q 3 ), and analyses were performed with Mann-Whitney U test. Fisher's Exact Test (two-tailed) was used to analyze categorical data. Log-transformation was performed for both sCD163 and galectin-3 when entered in linear regression analyses. Plasma sCD163 and galectin-3 were dichotomized at five cut-off levels corresponding to the 75th, 80th, 85th, 90th and 95th percentiles of each molecule. To establish cut-off levels for defining high levels of sCD163 and galectin-3, two backward elimination multiple logistic regression analyses were performed. The results with the lowest P values were defined as high levels. First, the five cut-off levels for sCD163 were tested against DR. The lowest P value was found for the 80th percentile of sCD163 (≥0.575 mg/L) which was defined as high sCD163. Second, the five cut-off levels for galectin-3 were tested against sCD163 ≥0.575 mg/L. The lowest P value was found for the 95th percentile of galectin-3 (≥4.659 µg/L), which was defined as high galectin-3.
Crude odds ratios (CORs) were calculated and variables with P < 0.10, and gender, age and self-reported depression independent of P values, were entered in multiple logistic regression analyses (Backward: Wald) with high sCD163 and use of antidepressants as dependent variables. The Hosmer and Lemeshow test for goodness of fit and Nagelkerke R 2 were used to evaluate the multiple logistic regression analyses models. Linear regression was performed with log-transformed galectin-3 and sCD163 data. CIs of 95% were used. Imputation for one missing value in the HADS-D scale was performed using multinomial regression on the other variables in the scale in one case. P < 0.05 was considered statistically significant. SPSS version 18 (IBM) was used to perform all statistical analyses.

Results
Baseline characteristics are presented for all the 287 T1D patients and gender specified in Table 1. The prevalence of foot complications was higher in the 162 men than in the 125 women (P = 0.023). Otherwise there were no gender differences.
The test results from HADS-D, the laboratory measurements, the anthropometrics and BP measurements are presented in Table 2 for all and gender specified. S-creatinine, SBP and DBP were higher (all P < 0.001), and HDL was lower (P = 0.003) in the men.
In Table 3 the associations between DR and five cut-off values for sCD163 are presented. High sCD163 (≥0.575 mg/L) (corresponding to the 80th percentile) was associated with DR (AOR 2.4, P = 0.028). The associations between high sCD163 and five cut-off levels for galectin-3 are also presented in Table 3. High galectin-3 (≥4.659 µg/L) (corresponding to the 95th percentile) was associated with high sCD163 (≥0.575 mg/L) (AOR 6.1, P = 0.001).
The five cut-off values for sCD163, corresponding to the 75th, 80th, 85th, 90th and 95th percentiles, were tried against depression, but none of these cut-off levels was associated with depression. The lowest P value was found for the 75th percentile (AOR 1.9 (0.8-4.1), P = 0.13).
Median  smoking; cardiovascular complications/no cardiovascular complications; foot complications/no foot complications; LLD/no LLD; physical inactivity/no physical inactivity.
Of the 22 patients that used antidepressants, 10 (46%) had self-reported depression and 12 (54%) patients did not and of the 265 patients that did not use antidepressants, 20 (8%) had self-reported depression and 245 (92%) did not.

Discussion
The main findings in this study of depression, diabetes retinopathy, sCD163 and galectin-3 in patients with T1D, were that DR, high levels of galectin-3 (≥4.659 µg/L), use of antidepressants and increasing levels of SBP,

Figure 2
Associations between log-transformed levels of galectin-3 and sCD163.
were independently associated with high sCD163 (≥0.575 mg/L). Depression was not associated with high sCD163. Gender sub-analyses showed that DR, high galectin-3 and increasing levels of SBP were independently associated with high sCD163 in the men. In the women, the use of antidepressants was independently associated with high sCD163.
Strengths of the study were that we can demonstrate new significant findings. The association between plasma sCD163 and plasma galectin-3 was strong and has not to our knowledge been demonstrated previously. This is of interest as both molecules are released from macrophages, and both indicate increased inflammatory responses (15,21,24,25,26,32). Another new finding is the   association between circulating sCD163 and DR, which has not been explored previously. This is important as high levels of sCD163 have in previous research been demonstrated in the vitreous of the eye, particularly in patients with severe proliferative types of DR (18). A third new finding is the association between the use of antidepressants and sCD163. Another strength is that precise ELISA techniques were used. The commercial ELISA assay showed a low intra-assay coefficient of variation for both sCD163 and galectin-3.
Further, the population of patients with T1D was well defined, since persons with severe somatic and psychiatric comorbidities and severe substance abuse were excluded. We have systematically controlled for potential confounders, and we have explored gender differences.
One difficulty we had to address was that there is no established consensus regarding harmful plasma sCD163 levels. Therefore, we explored the associations between DR and five cut-off levels (from the 75th to the 95th percentile) of sCD163 to find the optimal cut-off level for sCD163, which could have a clinical implication. We also systematically explored the associations between high sCD163 and five cut-off levels for galectin-3. Here we found that high galectin-3, using a cut-off level corresponding to the 95th percentile, was associated with high sCD163. Previously we have found that a lower cut-off level, corresponding to the 85th percentile of galectin-3, was associated with depression (23). Finally, we explored the associations between depression and the five cut-off levels for sCD163, without finding any association at any level.
The main limitation of our study was that the DR was not categorized according to severity. The reason is that we collected the information regarding DR from the S-NDR, where disease severity levels were not reported in 2009. We still consider that the fact that high levels of plasma sCD163 did reflect the presence of DR is worth presenting, even without classification of DR severity. Another weakness was the limited number of patients within certain sub groups. There were only 22 (8%) patients using antidepressants which prevents analyses of associations between different types of antidepressants and sCD163. The association between the use of antidepressants and high sCD163 did not reach significance for the 11 men, but the P value was below 0.10, which indicates that this is a possible type 2 error. Depression was not confirmed by a structured interview, but it was recently shown that 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 (31).
We found that high levels of sCD163 was associated with increasing SBP, but not with increasing DBP, which differs from previous research (22). The patients with high levels of sCD163 had a higher prevalence of AHD which might have lowered the DBP more effectively than the SBP. We found no association between BMI and high sCD163, contrarily to in previous research where associations between sCD163 and obesity were described (21). Neither did we find any association between HbA1c and high sCD163, which could have been possible due to the previously demonstrated impact of sCD163 on insulin resistance (20).
There are several subjects for further research. As this is a cross-sectional study, we have no information regarding sCD163 levels at the initiation of antidepressant medication. To be able to draw any conclusions about whether one or several types of antidepressants are causing high sCD163 or not, further research will be necessary. The associations between different types of antidepressants, depression and sCD163 must be further explored in prospective studies. It was unexpected to find a positive association between the use of antidepressants and sCD163, as in previous research it has been demonstrated that antidepressant medication attenuated inflammatory activity (10,33).
In further research we wish to explore whether increasing sCD163 levels are associated with increased severity of DR. We plan a 10-year follow-up of this study, and we will then use information from the medical records to categorize DR according to severity levels both at baseline and at follow-up. It would also be of interest to measure the levels of sCD163 in the vitreous of the eye and in the plasma simultaneously. It is important to find biomarkers that can contribute to the understanding of the pathology of DR, that can be used for early detection, to check the pharmacological response during therapeutic intervention, or that can be used as treatment targets (2,6,18). In previous research it has been shown that glucagon-like peptide 1 (GLP-1) analog therapy directly modulates innate immune-mediated inflammation in individuals with type 2 diabetes mellitus (34). If sCD163 is causative in the development of DR, one potential treatment for reducing sCD163 could be a GLP-1 analog, which would be another subject for further research.
Our main hypothesis was that there are immunological links between depression and DR. We found no evidence that sCD163 was one of these immunological links. Instead we found that the use of antidepressants was associated with high levels of sCD163. Our second hypothesis that high levels of plasma sCD163 could reflect the presence of DR was confirmed. This is of great importance as plasma sCD163, but not vitreous sCD163, might be used to check the pharmacological response during therapeutic interventions. Our third hypothesis was that macrophage activation could lead to simultaneous increase in galectin-3 and sCD163 levels, and we did find a strong association between high galectin-3 and high sCD163. We did not find any association between the use of antidepressants and DR. To our knowledge, this is the first study to explore and to show that circulating sCD163 was associated with circulating galectin-3, the use of antidepressants, DR and SBP in patients with T1D.