Deficiencies of the T and natural killer cell system in major depressive disorder

Brain, Behavior, and Immunity xxx (2015) xxx–xxx

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Deficiencies of the T and natural killer cell system in major depressive disorder T regulatory cell defects are associated with inflammatory monocyte activation Laura Grosse a,b,⇑, Thomas Hoogenboezem c, Oliver Ambrée a, Silja Bellingrath d, Silke Jörgens a, Harm J. de Wit c, Annemarie M. Wijkhuijs c, Volker Arolt a, Hemmo A. Drexhage c a

Department of Psychiatry, University of Muenster, Germany Radiology Morphological Solutions, Rotterdam, The Netherlands c Department of Immunology, Erasmus MC, Rotterdam, The Netherlands d Department of Psychology, University of Duisburg-Essen, Germany b

a r t i c l e

i n f o

Article history: Received 24 August 2015 Received in revised form 25 November 2015 Accepted 3 December 2015 Available online xxxx Keywords: T regulatory cells T helper cells Natural killer cells Lymphocytes Monocyte gene expression Inflammation Major depressive disorder

a b s t r a c t Background: In a previous study, we found an up-regulated inflammatory monocyte gene expression profile in major depressive disorder (MDD) patients aged P 28 years and a down-regulated inflammatory gene expression profile in MDD patients aged < 28 years. In the same sample of patients, we aimed to investigate immune dysregulation in the lymphocyte arm of the immune system, particularly in the context of the described monocyte (de-)activation states. Methods: From deep frozen leukocytes, circulating percentages of monocytes, lymphocytes, B, T, and natural killer (NK) cells, and various functional subsets of T and T helper (Th) cells (Th1, Th2, Th17, and natural T regulatory cells) were measured in N = 50 MDD patients and N = 58 age- and gender-matched healthy controls (HC). In addition, serum levels of interleukin (IL)-6, sCD25, IL-7, IL-3, SCF, IGF-BP2, and EGF were evaluated. Results: MDD patients were in general characterized by an impaired maturation of Th2 cells, Th17 cells, and NK cells and by decreased serum levels of IL-7 and sCD25. MDD patients aged P 28 years additionally exhibited decreased percentages of CD4+CD25highFoxP3+ T regulatory cells, next to signs of the above described partial T cell defects. Natural T regulatory cells were inversely associated with the pro-inflammatory state of the monocytes (r = .311; p = .034) that characterized this patient subgroup. Conclusions: Deficiencies of the NK and T (regulatory) cell system and inflammatory monocyte immune activation co-occur as partly interrelated phenomena within the same MDD patients. Ó 2015 Published by Elsevier Inc.

1. Introduction There is accumulating evidence that cell-mediated immunity plays an important role in the pathogenesis of major mood disorders (Beumer et al., 2012; Drexhage et al., 2010; Miller, 2010; Toben and Baune, 2015). In support of this view, we recently described an up-regulation of a coherent set of immune activation and inflammation-related genes in circulating monocytes of subgroups of patients with major depressive disorder (Carvalho et al., 2014; Grosse et al., 2015b). More precisely, monocyte inflammatory gene expression enabled us to stratify major depressive ⇑ Corresponding author at: Universitätsklinikum Münster, Klinik für Psychiatrie und Psychotherapie, Albert-Schweitzer-Campus 1, Geb. A9, 48149 Münster, Germany. E-mail address: [email protected] (L. Grosse).

disorder (MDD) patients in two subgroups with different age and depression characteristics: An up-regulated monocyte inflammatory gene expression profile characterized MDD patients aged P 28 years with a recurrent disease, while a down-regulated monocyte gene expression profile was typical of younger MDD patients in a first episode depression (Grosse et al., 2015b). Apart from cells of the monocyte/macrophage lineage, T cells and natural killer (NK) cells are important separate contributors and regulators to the cell-mediated immune response. Abnormal numbers and functions of T cells and NK cells have been reported in MDD patients (Blume et al., 2011; Eyre et al., 2014; Miller, 2010). Similar to the gene expression in monocytes described above, two divergent sets of observations have been established in MDD. One concerns immune suppression, such as reduced proliferative responsiveness of T cells and reduced levels and functions of

http://dx.doi.org/10.1016/j.bbi.2015.12.003 0889-1591/Ó 2015 Published by Elsevier Inc.

Please cite this article in press as: Grosse, L., et al. Deficiencies of the T and natural killer cell system in major depressive disorder. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.003

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L. Grosse et al. / Brain, Behavior, and Immunity xxx (2015) xxx–xxx

NK cells (Miller, 2010). The other concerns immune hyperactivation, defined as enhanced proliferative responses of T cells and an increased production of pro-inflammatory cytokines (Dowlati et al., 2010). Both sets of observations are well supported and raise questions whether these opposing processes are part of a single pathophysiological pathway within the same individuals (e.g. immune activation in one arm of the immune system and immune suppression in the other) or whether they represent two (or more) different processes occurring in either different individuals, e.g. with different forms of depression, or even occur within the same individuals but in different stages of the disease (Blume et al., 2011; Eyre et al., 2014). Clinical studies investigating a direct potential interrelationship between these opposing types of immune dysregulation within the same individuals are scarce (Blume et al., 2011; Pike and Irwin, 2006), although some studies do suggest the concurrence and a role of T cells defects, and in particular of T regulatory cells, in the pro-inflammatory state of MDD patients (Himmerich et al., 2010; Ho et al., 2015; Miller, 2010; Sanna et al., 2014). For the present study, we used the same sample of randomly collected MDD patients in whom we previously had studied monocyte gene activation and had found up-regulated monocyte gene expression in patients aged 28 years and older and downregulated expression in patients younger than 28 years (Grosse et al., 2015b). In the present study, we aimed at aimed at investigating a different aspect of immunity, e.g. lymphocyte populations, important in the regulation of the cell-mediated immune response. We therefore quantified selected lymphocyte subsets and related cytokines and growth factors in the circulation. Using the same subjects as in the previous study offers the crucial opportunity to analyze the interrelationships of the two different biological processes in MDD. We therefore also analyzed post-hoc outcomes of the present lymphocyte subset study in relation to the previous findings on inflammatory monocyte gene expression. From deep frozen leukocytes, circulating percentages of lymphocytes (B, T, and NK cells), various functional subsets of T and T helper (Th) cells (Th1, Th2, Th17, and natural T regulatory cells), and monocytes were measured in N = 50 MDD patients and in N = 58 age- and gender-matched healthy controls (HC). Next to these cellular analyses, we evaluated serum levels of the shed T cell factor soluble interleukin-2 receptor (sCD25), which is highly expressed on activated T cells and on CD4+CD25highFoxP3+ regulatory T cells, of the NK and T cell growth factor IL-7, of the myeloid growth factor IL-3, of stem cell factor (SCF), of the insulin-like growth factor-binding protein 2 (IGF-BP2), of the epidermal growth factor (EGF), and of the cytokine IL-6. The listed growth factors/cytokines were chosen since they play a role in macrophage and/or lymphocyte development and since abnormal levels have been found in multi-analyte screening studies in the serum of psychiatric patients (Schwarz et al., 2012).

or tetracyclic antidepressants (TCA/TeCA), 23 (32%) received seroto nin–norepinephrine reuptake inhibitors (SNRI), 17 (24%) received selective serotonin reuptake inhibitors (SSRI), 11 (16%) received benzodiazepines, 9 (13%) received non-benzodiazepines, 9 (13%) received a melatonergic agonist, 3 (4%) received lithium, and 1 (1%) received a norepinephrine reuptake inhibitor (NRI). 7 (10%) received electroconvulsive therapy (ECT). In total, 93% of the patients were medicated. From those, 68% received a combination pharmacotherapy consisting of at least 2 different types of medication and 30% consisting of at least 3 different types of medication. Not selected were patients with another primary psychiatric disorder, pregnancy, recent (4 weeks) or acute infections or allergic reactions, recent (4 weeks) vaccinations, suffering from any immune system disorder, untreated metabolic disorders or currently under immune-modulatory medication. Seventy-one ageand gender-matched healthy controls (HC) without any psychiatric history were recruited from the community. Somatic exclusion criteria for HC were the same as for the patients. While clinical information were available for all recruited N = 71 patients and N = 71 control subjects, immune laboratory data were determined in subsamples of this larger sample (Suppl. Fig. S1), e.g. for leukocyte subsets: N = 50 patients and N = 58 controls; for monocyte gene expression: N = 55 patients and N = 57 controls (of these 75%, N = 41, and 84%, N = 48, respectively, overlap with leukocyte subsets); and serum cytokines and growth factors: N = 71 patients and N = 69 controls (of these 70%, N = 50, and 81%, N = 56, respectively, overlap with leukocyte subsets). 2.2. Clinical instruments MDD diagnoses according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) (American Psychiatric Association, 2000) were confirmed using the MiniInternational Neuropsychiatric Interview (M.I.N.I.) (Sheehan et al., 1998), a structured standard instrument in epidemiological studies. Severity of depression was assessed using the Inventory of Depressive Symptomatology (IDS) (Rush et al., 1996) which covers melancholic and atypical depression features. Patients were assessed with the clinician-rated version (IDS-C; Crohnbach’s a = .76 .82), HC with the self-rate version (IDS-SR; a = .92 .94). 2.3. Laboratory methods 2.3.1. Blood collection and preparation Blood was collected in sodium-heparin tubes for immune cell preparation. From the heparinized blood, peripheral blood mononuclear cell (PBMC) suspensions were prepared by lowdensity gradient centrifugation via Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden), as described previously in detail (Knijff et al., 2006), within 8 h to avoid erythrophagy-related activation of the monocytes. PBMCs were frozen in 10%-dimetylsulfoxide and stored in liquid nitrogen. This enabled us to test immune cells of patients and controls concomitantly at a later stage.

2. Methods and materials 2.1. Participants This study was approved by the ethics committee of the medical association Westphalia-Lippe, Germany (reference 2009019-f-S). After study procedures had been fully explained, subjects provided written informed consent. A total of N = 71 patients with MDD were recruited from the Department of Psychiatry of the University Hospital Münster, Germany. All patients were naturalistically treated and received psychiatric medication according to their doctor’s choice: 5 (7%) were medication-free, 37 (52%) received atypical antipsychotics, 25 (35%) received tricyclic and/

2.3.2. Flow cytometric analyses Fluorescence-activated cell sorting (FACS) analysis was used to determine percentages of T lymphocytes (CD3+), T helper lymphocytes (CD3+CD4+), T cytotoxic lymphocytes (CD3+CD8+), natural killer cells (CD3 CD56+), B cells (CD19+), and monocytes (CD14+). Membrane staining was performed on 50.000 thawed noncultured peripheral blood mononuclear cells (PBMC), using anti CD45Pacific Orange (Invitrogen, Carlsbad, California), CD3-PercP-Cy5.5, CD4-Pacific Blue (BD Biosciences, California, USA), CD8-PC7 (Beckman Coulter, Brea, California), CD19-APC, CD14-APC-H7 (BD Biosciences, California, USA), CD56-PE (Cytognos, Salamanca, Spain).

Please cite this article in press as: Grosse, L., et al. Deficiencies of the T and natural killer cell system in major depressive disorder. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.003

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For the enumeration of Th1, Th2, Th17, and CD4+CD25highFoxP3+ regulatory T cells, 1⁄106 PBMC were suspended in complete culture medium. Cell suspensions were then stimulated with PMA (Sigma Aldrich, Missouri, USA), ionomycin (Sigma Aldrich, Missouri, USA) in the presence of Golgistop (BD Biosciences, California, USA) for 4 h in 37 °C under a 5% CO2 environment. Cells were harvested; membrane staining was done with CD3-APC-H7 and CD25-APC (BD Biosciences, California, USA) via standard protocol. Following membrane staining, the cells were fixed and permeabilized according to the manufacturer’s instructions (eBioscience, California, USA) and then stained for CD4-PercP-Cy5.5 and intracellular IFN-c-Horizon V500 (BD Biosciences, California, USA), IL-4PE-Cy7 (eBioscience, California, USA), IL-17A-BV421 (BioLegend, San Diego, USA), and FoxP3-PE. Stained cells were analyzed by eight color flowcytometry (FACS Canto, BD Biosciences) and analyzed using FlowJo (Tree star Inc, Ashland, Oregon, USA). The gating strategies are shown in Suppl. Fig. S2. Specificity was controlled using five isotype controls provided by the manufacturer (BD) and background positivity was negligible (between 0.2% and 1% of the specific staining depending on the isotype control, both in patient samples and controls). 2.3.3. mRNA expression in monocytes The definition and determination of the mRNA gene expression in monocytes and the method of quantifying monocyte immune gene expression has been previously described in detail (Drexhage et al., 2010; Grosse et al., 2015b). 2.3.4. Serum cytokine and growth factor determinations We studied a panel of seven analytes: IL-6, IL-7, IL-3, sCD25, SCF, IFGBP-2, and EGF. IL-6 was measured by high sensitive ELISA with signal amplification (eBioscience, BenderMedSystems GmbH, 1030 Vienna, Austria) according to the manufacturer’s protocol. For sCD25 a commercially available Enzyme Linked Immunosorbent Assay (ELISA) was used according to the manufacturer’s protocol (Gen-Probe Diaclone SAS, Besancon Cedex, France). Serum concentrations of IL-3, IL-7, SCF, IGFBP-2, and EGF were measured using the bead-based Luminex system. These multiplexed sandwich immunoassays were developed from commercially available capture and detection antibodies and standard proteins, validated and approved at Myriad-EDI-GmbH (Reutlingen, Germany) according to methods described previously (Schmohl et al., 2012). Control samples with low, medium and high concentrations for each analyte were run in duplicate on each plate. Patient and healthy control samples were run singular. Assays were measured on either the Luminex FlexMap-3D or Luminex 200 system. The coefficients of variation (CV) of the positive and negative control samples were at acceptable levels: 2.9% for IL-3, 8.2% for IL-7, 6.2% for SCF, 3.2% for IFGBP-2, 5.7% for EGF, 12.5% for sCD25, and 12.8% for IL-6 (manufacturer’s values 5–10%). 2.4. Statistical analyses Analyses were performed using IBM SPSS version 22 and Microsoft Excel for Mac 2011. Continuous sample characteristics, cell percentages, and serum protein levels are reported as mean ± SD. For sample characteristics, continuous data were analyzed using Mann–Whitney U test, categorical data were analyzed using Pearson’s chi-square (v2) test. Group differences of cell percentages and serum protein levels were tested using ANCOVA controlling for age, gender, BMI, and smoking, or in case of age-stratified analyses, controlling for gender, BMI, and smoking. IL-6, EGF, and sCD25 were measureable in all samples, whereas for IL-3, 51%, for IL-7, 24%, for SCF, 9%, and for IGFBP-2, 3%, of the values were below the detection limit. Since the majority of IL-3 assays were below the detection limit, we decided to discard these

Table 1 Sample characteristics. Clinical characteristics

MDD N = 71

HC N = 71

MDD vs. HC

v2

p

Female n (%) Smoking n (%)

44 (62) 37 (53)

47 (66) 17 (24)

0.275 12.470

.600 <.001

M ± SD

M ± SD

U

p

Age Body mass index IDS score Duration of current episode (weeks) Number of prev. episodes Age of first depression onset

33 ± 12 25 ± 5 35 ± 12 38 ± 43 3±3 24 ± 12

31 ± 11 23 ± 3 6±4 – – –

2358 2000 42 – – –

.506 .045 <.001 – – –

Abbreviations: MDD: major depressive disorder; HC: healthy controls; IDS: Inventory of Depressive Symptomatology.

data. All other values lower than the detection limit were set to half of the lowest value observed (e.g. IL-7: 0.07 pg/ml; SCF: 0.13 pg/ml; IGFBP-2: 7384 pg/ml). To approximate normality, levels of IGFBP-2 were transformed with natural logarithm and levels of IL-7 were transformed with square root. Of all serum parameters, only sCD25 was measured in two different runs. To control for potential inter-assay variation, sCD25 levels were transformed into fold change (FC) values relative to the mean of healthy controls per run. Thus, in the case of sCD25, healthy controls mean was set to 1 and patient values >1 indicate higher and patient values <1 indicate lower levels of sCD25 versus healthy controls. 3. Results 3.1. Sample characteristics MDD patients were on average 33 (±12) years old, 62% were female, 53% were current smokers, and the mean body mass index (BMI) was 25 (±5), indicating normal weight at the threshold to overweight. Patients had a significantly higher BMI and a significantly higher prevalence of smoking compared to HC, while age and gender distribution were similar (Table 1). The patients’ mean IDS score (35 ± 12) indicated a moderate depression severity. Patients reported being in the current MDD episode since 38 (±43) weeks on average, having had 3 (±3; range: 0–14) previous episodes of MDD in their lifetime with an average reported first depression onset of 24 years of age (±12; range: 6–54). All patients were naturalistically treated. Further analyses were controlled for age, gender, BMI, and smoking. 3.2. Lymphocyte subsets and serum growth factor/cytokine concentrations Table 2 shows that percentages of NK cells, Th2 cells, and Th17 cells were significantly decreased in MDD patients (n = 50) compared to HC (n = 58). Moreover, serum concentrations of sCD25 and IL-7 were significantly decreased in MDD patients compared to HC. Because inflammatory monocyte gene expression was shown to be strongly age-associated in this sample (Grosse et al., 2015b), further lymphocyte and growth factor analyses were performed age-stratified. 3.2.1. MDD patients aged < 28 years As previously described (Grosse et al., 2015b), inflammatory monocyte gene expression was decreased in MDD patients < 28 years as compared to HC, particularly for subcluster 1 inflammatory genes (e.g. IL1B, IL6, and TNF). With regard to the here studied lymphocyte populations and related serum parameters, in this younger MDD subgroup, the

Please cite this article in press as: Grosse, L., et al. Deficiencies of the T and natural killer cell system in major depressive disorder. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.003

4

Total

Cell populations +

CD14 monocytes Lymphocytes CD19+ (B cells) CD3 CD56+ (NK cells) CD3+ (T cells) CD3+CD4 CD8+ (Tc) CD3+CD4+CD8 (Th) CD3+CD4+IFN-y+ (Th1) CD3+CD4+IL-4+ (Th2) CD3+CD4+IL-17A+ (Th17) CD3+CD4+CD25highFoxP3+ (Treg)

HC n = 58

M

(SD)

M

15.17 80.28 6.87 8.03 63.08 19.62 40.28 9.13 0.40 0.62 3.91

(6.12) (6.87) (1.62) (3.42) (10.11) (6.87) (8.93) (4.20) (0.27) (0.36) (1.32)

14.81 80.05 6.42 11.39 60.43 18.76 37.14 10.92 0.56 0.79 4.00

n = 71 Serum proteins IL-6 pg/ml sCD25 (FC) IL-7 pg/ml SCF pg/ml IGFBP-2 pg/ml EGF pg/ml

M 0.73 0.95 2.03 14.49 81,593 157.1

Subgroup P 28 years

Subgroup < 28 years

MDD n = 50

MDD n = 21 (SD) (5.57) (6.27) (2.45) (5.61) (8.50) (5.34) (7.68) (6.57) (0.32) (0.42) (1.33)

(8.2) (0.37) (2.42) (9.96) (49,626) (73.1)

M 0.96 1.00 3.84 14.83 82,550 136.5

(1.74) (0.42) (3.82) (11.59) (46,868) (79.7)

HC n = 23

M

(SD)

M

(SD)

p

M

(SD)

M

.944 .683 .975 .003 .101 .483 .069 .215 .003 .002 .222

15.77 79.98 6.94 7.55 64.21 20.69 39.59 7.81 0.30 0.59 4.13

(6.97) (7.61) (1.67) (3.23) (9.09) (4.86) (9.66) (3.93) (0.14) (0.38) (1.33)

14.65 80.47 6.58 10.45 61.69 19.19 37.51 9.26 0.55 0.77 3.90

(5.91) (6.51) (2.80) (5.38) (8.34) (5.08) (6.78) (4.90) (0.32) (0.42) (1.27)

.817 .921 .962 .140 .224 .162 .543 .368 .002 .051 .674

14.70 80.51 6.80 8.41 62.22 18.81 40.82 10.09 0.48 0.63 3.76

(5.48) (6.38) (1.61) (3.57) (10.92) (8.09) (8.49) (4.20) (0.32) (0.36) (1.32)

15.10 79.29 6.12 13.08 58.18 17.99 36.48 13.44 0.58 0.82 4.15

(SD)

p

n = 28 (SD)

MDD n = 29

p

n = 69 (SD)

HC n = 35

p .833 .028 .014 .895 .690 .146

M 0.47 0.91 1.91 12.64 59,645 157.8

n = 38 (SD) (0.40) (0.34) (2.16) (8.96) (37,824) (77.7)

M 0.88 1.01 4.68 14.44 65,665 148.4

n = 43

(1.88) (0.42) (4.96) (11.7) (44,715) (95.0)

.860 .052 .016 .923 .872 .718

M 0.92 0.98 2.11 15.69 95,885 156.6

(SD) (5.06) (5.91) (1.68) (5.74) (8.53) (5.84) (9.23) (7.98) (0.32) (0.42) (1.43)

p .853 .582 .875 .008 .259 .963 .087 .311 .208 .016 .032

n = 31 (SD) (0.97) (0.39) (2.60) (10.49) (51,523) (70.9)

M 1.05 0.99 3.47 15.33 104,502 121.4

(SD) (1.58) (0.42) (3.93) (11.63) (40,605) (52.2)

p .631 .195 .202 .652 .529 .044

Abbreviations: MDD: Major depressive disorder; HC: healthy controls. Lymphocyte subsets are presented as percentages of live CD45+ peripheral blood mononuclear cells (PBMC). T helper (Th) subsets are presented as percentages of total CD4+ T helper cells. All analyses between major depressive disorder (MDD) patients and healthy controls (HC) were controlled for age, gender, body mass index, and smoking. Compared to HC, MDD patients showed decreased percentages of natural killer (NK) cells, Th2 cells, and Th17 cells. T regulatory (Treg) cell percentages were particularly decreased in the MDD subgroup aged P 28 years, which was previously characterized by increased monocyte gene activation. With regard to growth factors, MDD patients exhibited decreased concentrations of sCD25 and IL-7 compared to HC.

L. Grosse et al. / Brain, Behavior, and Immunity xxx (2015) xxx–xxx

Please cite this article in press as: Grosse, L., et al. Deficiencies of the T and natural killer cell system in major depressive disorder. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.003

Table 2 Lymphocyte subsets and serum protein concentrations in major depressive disorder patients and controls.

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overall pattern of decreased percentages of Th2 cells (p = .002) and (tendentially) decreased percentages Th17 (p = .051) cells and decreased levels of IL-7 (p = .016) and (tendentially) decreased levels of sCD25 (p = .052) was similar to the T cell deficiency pattern of the total group of MDD patients (Table 2).

3.2.5. Monocyte activation and lymphocyte suppression As is visualized and summarized in Suppl. Fig. S3, both increased monocyte inflammatory gene expression and deficiencies of several lymphocyte subset populations (e.g. NK cells, T helper cells) co-occurred within the same MDD patients.

3.2.2. MDD patients P 28 years As previously described (Grosse et al., 2015b), in this older (P28 years) MDD patients, monocyte gene expression was increased in MDD patients compared to HC, again this particularly applied to the inflammatory subcluster 1 gene expression profile. With regard to the here studied lymphocyte and serum parameters, again, similar to the total group of MDD patients, percentages of NK cells and Th17 cells were significantly decreased in MDD patients vs. HC aged P 28 years (Table 2). However, additionally a significant decrease in the percentages of T regulatory cells was observed specifically in the MDD patients of this subgroup (Fig. 1A), while this pattern could not be observed in the younger MDD patients. Moreover, levels of EGF were significantly increased in this patient subgroup as compared to HC.

4. Discussion This study shows various signs of NK and T cell defects in patients with major depressive disorder. Specifically, decreased percentages of NK cells, an impaired capability of T helper cells to mature into Th2 cells and Th17 cells, and decreased serum levels of the NK/T cell growth factor IL-7 and of the shed T cell receptor for IL-2 (sCD25) were found in MDD patients, all features of an underperformance of the NK and T cell system. Taken the findings of this and the previous study (Grosse et al., 2015b) together, MDD patients aged < 28 years were characterized by both defects in the T cell arm and the monocyte arm of the immune system, i.e. an impaired maturation of Th2 and Th17 cells, decreased serum levels of IL-7 and sCD25, and an under expression of monocyte inflammatory genes (Grosse et al., 2015b). On the other hand, MDD patients aged P 28 years particularly exhibited decreased percentages of CD4+CD25highFoxP3+ T regulatory cells (next to signs of the above described partial NK and T cell defects) and monocyte pro-inflammatory activation. Interestingly, the levels of the natural T regulatory cells were inversely associated with the activation state of the monocytes. As a further sign of the increased activity of the monocytes in this older (P28 years) subgroup, serum EGF levels were increased in these patients as well and correlated with the inflammatory monocyte activation, as did serum IL-6 levels in this subgroup. Raised levels of EGF have been considered as a sign of monocyte activity before (Schipper et al., 2012). In essence, our observations show that immune suppression (NK cell deficiencies, T helper cell maturation deficiencies) and immune activation co-occur in the same MDD patients. Recently, signs of both immune suppression and immune activation were also found in peripheral blood cells of a larger sample of MDD patients (Jansen et al., 2015). Most noteworthy, depressed patients were characterized by an upregulated expression of genes associated with IL-6 signaling and a downregulated expression of genes associated with NK cell pathways. Our data also point in the direction that these abnormalities might be (st)age dependent: Over-expression of monocyte immune genes and natural T regulatory cell defects were not noticeable in

3.2.3. Natural T regulatory cells Since regulatory T cells are known to regulate inflammation, we tested the hypothesis that there is an association of this regulatory population with the inflammatory monocyte state. We found a significant inverse correlation of T regulatory cell percentages with the inflammatory monocyte activation profile in MDD patients, as represented by the previously determined total gene expression score (r = .331, p = .034; Fig. 1B) and also by the pro-inflammatory subcluster 1 gene expression score (r = .346, p = .027). In exploratory sub-analyses we further found that percentages of T regulatory cells were associated with smoking, however only significantly in HC (r = .399, p = .002), while interestingly, this correlation was not observed in MDD patients. In contrast to HC, in MDD patients aged P 28 years, smoking did hardly increase the generally decreased percentages of T regulatory cells (Fig. 1C). 3.2.4. IL-6, EGF, and monocyte gene expression score It is also worthy to note that serum levels of the proinflammatory cytokine IL-6 were significantly associated with the monocyte gene expression score (r = .300, p = .029) in MDD patients. Moreover, also serum levels of EGF correlated significantly and positively with the inflammatory gene score (r = .289, p = .032) and negatively with T regulatory cell percentages (r = .338, p = .016) in MDD patients.

(A)

HC MDD

5

(B)

2

4

4 3 2

2

1

**

5

% Treg

3

HC MDD

*

7

6

% Treg

% Treg

(C) 6

4

0

r = −.331* N = 41

8

*

1

N<=28 35 years N = 21

< 28 years

N≥=28 23 years N = 29

>= 28 years

0

0 -20

0

20

40

Monocyte gene expression MDD < 28 years

MDD ≥ 28years

N = 17 N = 11

HC

N=6

Non-smokers

N = 18

Smokers

≥ 28 years

Fig. 1. T regulatory cells in association with age, monocyte inflammatory activation, and smoking. Abbreviations: MDD: Major depressive disorder, HC: healthy controls, Treg: T regulatory cells. A. Only in the subgroup of P28 years, percentages of Treg cells were decreased in MDD patients vs. HC (F = 4.828; p = .032). B. Percentages of T regulatory cells were associated with inflammatory monocyte gene expression in MDD patients (r = .311; p = .034). C. In the age group of P28 years, HC smokers had significantly increased percentages of Treg cells compared to HC non-smokers (F = 8.455; p = .009), while this difference between smokers and non-smokers was not observed in MDD patients.

Please cite this article in press as: Grosse, L., et al. Deficiencies of the T and natural killer cell system in major depressive disorder. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.003

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MDD patients of less than 28 years. We assume that against the backdrop of maturation defects of the Th2 and Th17 cells which were already present in the younger patients, defects additionally develop in the generation of T regulatory cells when young MDD patients grow older and/or have had recurrent episodes of depression. We further assume that these T regulatory cell defects in MDD might be less responsive to environmental factors such as smoking, since our data suggest that smoking is not related to increases in T regulatory cells in MDD patients as it is in healthy individuals. We can only speculate that an aging and/or episode induced defect in T regulatory cells would then unleash the inflammatory capacity of the monocyte/macrophage system in MDD patients aged 28 years and older. Indeed, a recent review on phasespecific neuroimmune alterations in depression indicates that dysregulations of T cell populations may be important precursors to depressive disorders and other immune alterations in MDD (Eyre et al., 2014). It is worthy to note that we have found decreased percentages of natural T regulatory cells in a different sample of older (52 ± 9 years) MDD patients before (Grosse et al., 2015a), strengthening the view of the importance of defects of this regulatory system in (older) MDD. Interestingly, in this other sample, the previously decreased percentages of T regulatory cells normalized in MDD patients after antidepressant therapy (Grosse et al., 2015a). However, potential effects of aging, course of the disease, or antidepressant treatment on natural T regulatory cells need to be formally tested and replicated in longitudinal and functional studies before any firm conclusions can be drawn. Nonetheless, an age-related weakening of the natural T regulatory cell system due to T cell generating failures has been noticed before in individuals prone to auto-inflammatory conditions (d’Hennezel et al., 2010). In contrast, in healthy organisms, aging actually strengthens the natural T regulatory system, but may negatively influence inducible T regulatory cells (Jagger et al., 2014). With regard to the here found maturation defects of the T helper/regulatory system in MDD patients, our data are reminiscent of what is presently known on T cell defects in the 22q11 deletion syndrome (Gennery, 2012). In this syndrome, partial T cell defects are due to thymus mal-development and hypoplasia’s caused by heterozygous deletions of variable parts of chromosome 22q11. Patients with a 22q11 deletion are not only characterized by variable and partial T cell defects, but also by a variety of autoimmune diseases and psychiatric syndromes. The most commonly reported psychiatric disorders are attention deficit/hyperactivity disorder (ADHD), anxiety, autism spectrum disorders, mood disorders including major depression and bipolar disorder, and psychotic disorders (Squarcione et al., 2013). Although the association with psychiatric diseases has in part been explained by the contribution of genes on chromosome 22q11 important for normal brain function and neuronal development (such as COMT, PRODH and TBX1), the awareness is growing that T cell defects per se could explain psychiatric dysfunction and the high prevalence of autoimmunity in the syndrome. The focus in this novel perspective lies on the defects in the natural T regulatory system in this syndrome (McLeanTooke et al., 2008), leading to a putative high inflammatory state resulting in auto-inflammatory conditions such as psychiatric diseases and autoimmunity. Our finding of the defect in natural T regulatory cells in association with the pro-inflammatory state of monocytes in the ‘‘older” MDD subgroup is noteworthy in this concept of T regulatory cell defects being central to the often described mild inflammatory state of psychiatric patients. With regard to the here found decreased percentages of NK cells in MDD patients, there are early meta-analyses showing a lower activity and quantity of NK cells in MDD (Zorrilla et al., 2001). Yet, normal and higher activities of the NK cell system have also been found, as reviewed by Blume et al. (2011) and there are data indicating that the NK cell system and T cell system might be

differentially affected in melancholic and non-melancholic subtypes of MDD (Rothermundt et al., 2001). However, since the classical function of NK cells is the combat of tumours and viral infections, decreased activity of the NK cell system in MDD patients has traditionally been linked to a potential higher susceptibility of MDD patients for infections and malignancies. However, a novel function for NK cells in the brain has recently been reported (Blume et al., 2011). NK cells can act as immune regulatory cells and are able to migrate to the brain under specific inflammatory conditions. In the brain, they are capable of dampening down microglial inflammatory activity and brain inflammation (Shi et al., 2011). In fact, NK cells can thus act as a kind of brainspecific ‘‘suppressor” cells. Reduced circulating levels of NK cells – as described here – may therefore be detrimental, because they are no longer capable of dampening down the high inflammatory activity of microglia and peri-vascular macrophages in the brain of MDD patients. Indeed, longitudinal data from our group from another sample of MDD patients demonstrate that lower percentages of NK cells prior to treatment are associated with a poor antidepressant response (Grosse et al., 2015a), highlighting the significance of these lymphocyte system in MDD. 4.1. Limitations of the study Since frozen PBMC were used and since the total number of leukocytes had not been determined following the MOODINFLAME study protocol, only percentages of the various lymphocyte subsets (of total lymphocytes or of total T helper cells) were determined and no absolute values in relation to the number of leukocytes per ml blood could be given. Moreover, for this study, no longitudinal data of the participants were available. Therefore, we cannot conclude with certainty from our cross-sectional data whether ‘‘growing older” indeed affects levels of T regulatory cells. To prove if lymphocyte subsets change during aging or stages of the disease, follow-up studies are needed. Another important limitation of our study is that all MDD patients were hospitalized and received some form of treatment. Thus, there is a possibility that the observed immune dysregulations were not or not solely due to MDD but to antidepressant treatment. In case of the here studied Treg cells, both phenomena have been described before: decreased levels of these T cell subsets in MDD patients free of antidepressant medication (Grosse et al., 2015a), but also antidepressant-induced increases of these cell subsets (Grosse et al., 2015a; Himmerich et al., 2010). Furthermore, in this study, we were not able to control for additional factors that might also be associated with dysregulated cellular immunity, such as hormonal status, i.e. during menstrual cycle phases (Oertelt-Prigione, 2012), physical activity (Eyre and Baune, 2012) or other lifestyle- or environment-associated factors. When correcting our analyses for multiple testing as described by Hochberg and Benjamini (1990) with q = 0.10, and also taking the three monocyte scores of our previous report as tests into account, all reported group differences remained significant, except for differences in growth factors and for the group difference in T regulatory cells in the older (P28 years) subgroup. However, since this study investigates a separate aspect of immunity compared to our previous study, it can be discussed whether this statistical control taking all parameters of both studies into account is strictly necessary. Nevertheless, this underscores the notion that confirmation studies are needed with larger samples of MDD patients before firm conclusions can be drawn. 4.2. Conclusion In conclusion, our observations show that immune deficiencies and inflammatory immune activation co-occur as partly

Please cite this article in press as: Grosse, L., et al. Deficiencies of the T and natural killer cell system in major depressive disorder. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.003

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interrelated phenomena within the same MDD patients. MDD patients of less than 28 years were characterized by both deficiencies in the T cell arm and the monocyte arm of the immune system, whereas MDD patients aged P 28 years exhibited increased monocyte activation in the presence of NK cell and T (regulatory) cell deficiencies. Taken together, this study highlights interrelated dysregulated cellular immune responsiveness in the lymphocyte and monocyte arm in an unselected sample of MDD patients in routine clinical practice. 5. Financial disclosures This study was supported by European Union EU-FP7HEALTH-F2-2008-222963 ‘‘MOODINFLAME”. Laura Große, Thomas Hoogenboezem, and Annemarie Wijkhuijs were funded by EU-FP7PEOPLE-2009-IAPP ‘‘PSYCH-AID’’. Oliver Ambrée, Silja Bellingrath, Silke Jörgens, and Harm de Wit declare no potential conflicts of interest. Volker Arolt received grants from the German Ministry of Science and Education, from the Münster Interdisciplinary Center of Clinical Research, and from the European Union; he is a member of the advisory board of, or has given presentations on behalf of, the following companies: Astra-Zeneca, JanssenOrganon, Lilly, Lundbeck, Servier, Pfizer, Otsuka, and Trommsdorff. Hemmo A. Drexhage has received grants from the Netherlands Organisation for Health Research and Development, the European Union, the Stanley Medical Research Institute, the Dutch Diabetic Foundation and the JDRF; he has received speaker’s fees from Astra Zenica and he serves/has served in advisory boards of the Netherlands Organisation for Health Research and Development, the European Union and the JDRF. These supporters had no further role in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the paper for publication. Acknowledgments We are grateful to Rhea Balske, Kathrin Entrich, Franka Hanysek, Christina Uhlmann, and Rolf Voegler for recruitment of patients and controls. Further we thank Katrin Blaschei, Christiane Schettler, and Kordula Vorspohl for excellent technical assistance. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbi.2015.12.003. References American Psychiatric Association, 2000. Diagnostic and Statistical Manual of Mental Disorders, Washington, DC. Beumer, W., Gibney, S.M., Drexhage, R.C., Pont-Lezica, L., Doorduin, J., Klein, H.C., Steiner, J., Connor, T.J., Harkin, A., Versnel, M.A., Drexhage, H.A., 2012. The immune theory of psychiatric diseases: a key role for activated microglia and circulating monocytes. J. Leukoc. Biol. 92, 959–975. Blume, J., Douglas, S.D., Evans, D.L., 2011. Immune suppression and immune activation in depression. Brain Behav. Immun. 25, 221–229. Carvalho, L.A., Bergink, V., Sumaski, L., Wijkhuijs, J., Hoogendijk, W.J., Birkenhager, T. K., Drexhage, H.A., 2014. Inflammatory activation is associated with a reduced glucocorticoid receptor alpha/beta expression ratio in monocytes of inpatients with melancholic major depressive disorder. Transl. Psychiatry 4, e344. d’Hennezel, E., Kornete, M., Piccirillo, C.A., 2010. IL-2 as a therapeutic target for the restoration of Foxp3+ regulatory T cell function in organ-specific autoimmunity: implications in pathophysiology and translation to human disease. J. Transl. Med. 8, 113. Dowlati, Y., Herrmann, N., Swardfager, W., Liu, H., Sham, L., Reim, E.K., Lanctot, K.L., 2010. A meta-analysis of cytokines in major depression. Biol. Psychiatry 67, 446–457. Drexhage, R.C., van der Heul-Nieuwenhuijsen, L., Padmos, R.C., van Beveren, N., Cohen, D., Versnel, M.A., Nolen, W.A., Drexhage, H.A., 2010. Inflammatory gene expression in monocytes of patients with schizophrenia: overlap and difference

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Please cite this article in press as: Grosse, L., et al. Deficiencies of the T and natural killer cell system in major depressive disorder. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.003