Psychiatry Research 220 (2014) 283–286
Contents lists available at ScienceDirect
Psychiatry Research journal homepage: www.elsevier.com/locate/psychres
Association between toll-like receptors expression and major depressive disorder Yi-Yung Hung a,b,c, Hong-Yo Kang b,c, Kai-Wei Huang d, Tiao-Lai Huang a,e,n a
Department of Psychiatry, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, ROC Graduate Institute of Clinical Medical Sciences, Chang Gung University, College of Medicine, Kaohsiung, Taiwan, ROC c Center for Menopause and Reproductive Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan, ROC d Department of Nursing, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, ROC e Genomic & Proteomic Core Laboratory, Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan, ROC b
art ic l e i nf o
a b s t r a c t
Article history: Received 11 March 2014 Received in revised form 14 July 2014 Accepted 25 July 2014 Available online 13 August 2014
Accumulating evidences suggest that Toll-like receptors (TLRs) were involved in the pathophysiology of major depressive disorder. TLR4 was thought to be associated with major depressive disorder in animal model, but the others were still unknown. In order to examine TLR1-9 mRNA expression levels in peripheral blood and their relationships with the psychopathology of major depressive disorder, 30 patients with major depressive disorder were compared with 29 healthy controls. The 17-item Hamilton Depression Rating Scale (HAMD-17) was used to assess the severity of major depression. The mRNA expression levels of TLRs were examined in parallel with a housekeeping gene using real-time polymerase chain reaction (RT-PCR). Analysis of covariance with age and body mass index adjustment revealed a significantly higher expression of TLR3, 4, 5 and 7 mRNA but lower expression of TLR1 and 6 in patients with major depressive disorder as compared with healthy controls. Multiple linear regression analysis revealed that TLR4 was an independent risk factor relating to severity of major depression. These findings suggest that TLRs, especially TLR4, may be involved in the psychopathology of major depression. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Depression Innate immunity TLR4 LPS Hamilton depression rating scale
1. Introduction Toll-like receptors (TLRs) are pattern recognition receptors proposed as main agents of the innate immune response, constituting the first line of defense against invading microorganisms (Akira and Takeda, 2004). TLRs recognize endogenous ligands, termed as damage-associated molecular patterns, and mediate an inflammatory response in the host to injury and stress (Piccinini and Midwood, 2010). There are various types of TLRs, and each have different cellsurface receptors; these include: TLR4, which recognizes lipopolysaccharide (LPS) present in Gram-negative bacteria; TLR2 and its co-receptors TLR1/TLR6, which sense bacterial lipoproteins and lipoteichoic acid; and TLR5 that recognizes bacterial flagellin. Another subtype of receptors are localized in the endosomal cell compartment and include TLR3, which recognizes doublestranded (ds) RNA; TLR9, which senses unmethylated CpG motifs
n Corresponding author at: Department of Psychiatry, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, ROC. Tel.: þ 886 7 7317123x6317; fax: þ886 7 7326817. E-mail address: [email protected] (T.-L. Huang).
http://dx.doi.org/10.1016/j.psychres.2014.07.074 0165-1781/& 2014 Elsevier Ireland Ltd. All rights reserved.
present in viral and bacterial genome and parasites; and TLR7, which binds single-stranded (ss) DNA (Kawai and Akira, 2007). TLRs are expressed in various cell types in the central nervous system, including microglia (TLR1-9), astrocytes (TLR1-5; TLR9), and neurons (TLR3). Neurodevelopment and neurodegeneration are associated with TLR activation. (Yuan et al., 2010; Okun et al., 2011). TLRs are expressed in the developing as well as the adult brain and play a significant role in plasticity ( Larsen et al., 2007; Kaul et al., 2012). TLR8 activation induces neuronal apoptosis through inhibition of neurite outgrowth (Ma et al., 2006), while induction of TLR3-mediated immunity during gestation inhibits cortical neurogenesis and synaptic transmission (Yuan et al., 2010). TLRs are also potential therapeutic targets against neuroinflammation. A number of these compounds are currently undergoing different phases of clinical trials (Kanzler et al., 2007). mRNA expression of TLRs in peripheral blood mononuclear cell (PBMC) was used to detect changes in innate immunity in many physical diseases including asthma (Sykes et al., 2013), chronic hepatitis B (Huang et al., 2013) or sepsis (Cejkova et al., 2012). The relationship between TLRs and psychiatric diseases were investigated before. A systematic analysis of TLRs revealed an enhanced response in TLR2 and TLR4 activation in patients with schizophrenia or bipolar disorder (McKernan et al., 2011a). In autism, an
284
Y.-Y. Hung et al. / Psychiatry Research 220 (2014) 283–286
enhanced TLR2 and TLR4 responsivity to its ligand can be measured, while TLR9 showed a decreased response in the blood (Enstrom et al., 2010). Alcohol abuse was also found to be associated with the TLR2, 3 and 4 complex (Kanuri et al., 2009; Crews et al., 2013). In clinical depression, translocation of the Gram-negative enterobacteria, which contain LPS in their bacterial wall, was thought to be associated with the onset of a number of inflammatory and oxidative/nitrosative stress pathways and play an important role in major depressive disorder (Maes et al., 2008). It has been demonstrated that after stress exposure or during certain episodes of depression, an innate immune response is strongly activated (Raison et al., 2006). Elevated TRIF and MYD88, intracellular adapter proteins for TLR signaling, were found in peripheral blood mononuclear cells isolated from depressed patients when compared to healthy subjects (Hajebrahimi et al., 2014). However, the systematic analysis of TLR expression in humans is still absent. Therefore, the present study aims to investigate whether TLRs expression levels are altered in patients with major depressive disorder, and how these changes correlate with the severity of depressive symptoms. 2. Method 2.1. Subjects Thirty outpatients or inpatients with major depressive disorder and 29 healthy controls were recruited from August 2013 to December 2013 at Kaohsiung Chang Gung Memorial Hospital Medical Center in Taiwan. Institutional Review Board approval was obtained from the hospital ethics committee (101-5012A3). Thirty patients with major depression were evaluated by one psychiatrist using the Structured Clinical Interview for DSM-IV Axis I Disorders. The severity of depression was assessed using the 17-item Hamilton Depression Rating Scale (HAMD-17) (Hamilton, 1960) by the same psychiatrist. Patients who had alcohol dependence or any immune inflammatory disease history were excluded from the study. The 29 healthy controls, who had neither a personal history nor a first degree relative with psychiatric disorder, were recruited from community. The Chinese health questionnaire-12 was done to assess the healthy control group by the same psychiatrist to rule out psychiatric disease according to Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria. All patients received blood pressure checks, chest X-rays, electrocardiogram examinations, and routine blood tests in order to exclude any chronic physical illness including heart, lung, liver, kidney, or metabolic diseases. They were also checked for acute infections or allergic reactions; moreover, they reported not taking any antidepressants for at least 1 week before they entered into this study. 2.2. Real-Time Polymerase Chain Reaction (RT-PCR) analysis
difference of age and BMI. Analysis of covariance (ANCOVA) with age, and BMI adjustment was used to compare the difference of TLRs expression levels. Multiple linear regression (stepwise method) was used to judge factors that correlates with HAMD-17 scores. All statistical analyses were performed using Statistical Product and Service Solutions (SPSS), version 22. For each test, po 0.05 was considered significant.
3. Results 3.1. Baseline characteristics Table 1 shows baseline characteristics regarding participant sex, age, and BMI (depressive patients: 11 male/19 female, mean age 45.8 710.02 years, BMI 22.96 73.7 kg/m2; health controls: 11 male/18 female, mean age 42.72 712.49 years, mean BMI 22.58 7 2.93 kg/m2). There were no significant differences in the mean age, sex, or BMI between the two groups. Duration of illness and 17-item Hamilton Depression Rating Scale in patients with major depressive disorder are also shown Table 1. 3.2. Differential TLR mRNA expression in peripheral blood from patients with major depressive disorder and healthy controls We analyzed TLR mRNA expression and GADPH was used as a housekeeping gene to assess the relative abundance of mRNA in peripheral blood. Similar TLR expression profiles, but differences in transcript levels, were observed in depressive patients as compared to healthy controls (Table 2). Quantitative analysis revealed significantly higher TLR3, 4, 5 and 7 mRNA expression levels in patients with major depression (TLR3: patients 1.57 70.82, controls 1.08 7 0.86, F(1,50)¼ 4.2, p ¼0.046; TLR4: patients 1.75 71.18, controls 1.26 70.85, F(1,53) ¼4.31, p ¼0.043;TLR5: patients 2.317 1.96, controls 1.14 70.77, F(1,51) ¼7.83, p ¼0.007; TLR7: patients 1.54 70.66, controls 1.01 70.48, F(1, 50) ¼10.25, p ¼ 0.002), whereas no significant differences for TLR2, 8, or 9 were observed. In contrast, significant lower expressions of TLR1 and TLR6 in patients with major depressive disorder were found, compared to healthy controls (TLR1: patients 1.737 1.18, controls 2.977 2.37, F(1, 50) ¼9.55, p ¼ 0.003; TLR6: patients 1.60 71.25, controls 2.86 72.35, F(1, 51) ¼8.55, p ¼ 0.005). Table 1 Demographic findings and clinical data of health controls and patients with major depressive disorder.
Venous blood (5 mL) samples were drawn into PAXgene Blood RNA Tube between 8:00 am and 10:00 am, after the patients had fasted for 9 h. The tubes were stored in a 80 1C refrigerator immediately after collection and remained there until they were assayed. RNA was isolated using TRIzol reagent, and RT-PCR was performed using following sets of primers (López et al., 2012): TLR1 (sense 50 -GGGTCAGCTGGACTTCAGAG-30 , anti-sense 50 -AAAATCCAAATGCAGGAACG- 30 ); TLR2 (sense 50 -TCAGCCTCTCCAAGGAAGAA-30 , anti-sense 50 -AATGTTCAAGACTGCCCAGG-30 ); TLR3 (sense 50 -AGCCTTCAACGACTGATGCT-30 , anti-sense 50 -TTTCCAGAGCCGTGCTAAGT-30 ); TLR4 (sense 50 -TGAGCAGTCGTGCTGGTATC-30 , anti-sense 50 -CAGGGCTTTTCTGAGTCGTC-3); TLR5 (sense 50 -GGAACCAGCTCCTAGCTCCT-30 , anti-sense 50 -AAGAGGGAAACCCCAGAGAA-30 ); TLR6 (sense 50 -CCCTTTAGGATAGCCACTGC-30 , anti-sense 50 -CTCACAATAGGATGGCAGGA-30 ); TLR7 (sense 50 -CCTTGAGGCCAACAACATCT-30 , anti-sense 50 -GTAGGGACGGCTGTGACATT-30 ); TLR8 (sense 50 -TCCTTCAGTCGTCAATGCTG-30 , anti-sense 50 -CGTTTGGGGAACTTCCTGTA-30 ); and TLR9 (sense 50 -GGACACTCCCAGCTCTGAAG-30 , anti-sense 50 -TTG GCTGTGGATGTTGTTGT-30 ). The QuantiTect Reverse Transcription kit (Qiagen) was used for the reverse transcription of RNA. The PTC-200 apparatus (BioRad) and the QuantiTect SYBR Green PCR kit (Qiagen) were applied for the complete analysis. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as a housekeeping gene to assess the relative abundance of mRNA.
Age (years) male female Gender (M/F) BMI male female HAMD-17 male female Duration of illness (years) male female Smoking Education duration (year) Antidepressant use History None SSRI SNRI NDRI
2.3. Statistical analysis Outliers of TLRs were excluded initially based on a boxplot outlier detection rule. All results are presented as means 7standard deviation. Chi-square was used to compare the difference between sexes. Student's t-test was used to compare the
Major depression
Healthy controls
p Value
45.8 7 10.02 43.187 8.92 47.32 710.54 11/19 22.96 7 3.70 21.56 7 2.64 23.777 4.03 27.83 7 4.97 28.36 7 6.41 27.52 7 4.08 4.25 7 3.59 3.91 73.93 4.37 73.54 12 11.677 3.14
42.727 12.49 46.55 7 14.73 40.39 7 10.68 11/18 22.58 7 2.93 24.137 3.34 21.63 7 2.26 – – – – – – 3 15.077 3.18
0.30 0.53 0.055 0.92 0.67 0.06 0.055 – – – – – – – –
3 11 10 6
– – – –
– – – –
Data are presented as mean 7S.D. p Value o 0.05; HAMD-17 ¼ 17-item Hamilton Depression Rating Scale; BMI, body mass index.
n
Y.-Y. Hung et al. / Psychiatry Research 220 (2014) 283–286
Table 2 Toll-like receptors 1-9 mRNA expression level of health controls and patients with major depressive disorder. Major depression
Healthy controls
F and p Value
TLR1
1.737 1.18
2.977 2.37
TLR2
1.62 7 0.82
1.55 7 0.79
TLR3
1.577 0.82
1.08 7 0.86
TLR4
1.75 7 1.18
1.26 7 0.85
TLR5
2.317 1.96
1.147 0.77
TLR6
1.60 7 1.25
2.86 7 2.35
TLR7
1.54 7 0.66
1.017 0.48
TLR8
1.05 7 0.48
0.89 7 0.43
TLR9
1.80 7 1.45
1.277 0.76
F(1,50) ¼9.55 p ¼ 0.003n F(1,53) ¼1.88 p ¼ 0.666 F(1,50) ¼4.20 p ¼ 0.046n F(1,53) ¼4.31 p ¼ 0.043n F(1,51)¼ 7.83 p ¼ 0.007n F(1,51)¼ 8.55 p ¼ 0.005n F(1,50) ¼10.25 p ¼ 0.002n F(1,55) ¼2.12 p ¼ 0.15 F(1,53) ¼2.93 p ¼ 0.09
Analysis of covariance with age, and BMI adjustment was also done to compare the difference of TLRs; Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as a housekeeping gene to assess the relative abundance of mRNA; TLR, Toll-like receptor. n
p Value o 0.05
Table 3 Stepwise multiple linear regression model between HAMD-17 score and TLR. Independent factors
TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9
HAMD-17 score Standardized coefficients
t
p Value
0.043 0.169 0.027 0.438 0.102 0.044 0.217 0.442 0.090
0.19 0.66 0.12 2.29 0.31 0.22 1.12 1.70 0.24
0.855 0.516 0.903 0.032n 0.763 0.828 0.275 0.104 0.814
n p Value o 0.05; HAMD-17 ¼ 17-item Hamilton Depression Rating Scale; TLR, Toll-like receptor; BMI, body mass index.
3.3. Severity of depression and TLRs We also analyzed the correlation of age, BMI, gender, duration of illness, and TLR1-9 with severity of major depression. Linear regression revealed that TLR4 was an independent risk factor which explained 19.2% the total variance in depression. (r2 ¼0.192; p ¼ 0.032) (Table 3).
4. Discussion The present study demonstrates that TLR expression levels in peripheral blood of patients with major depressive disorder are different from these of healthy controls. Moreover, the severity of major depressive disorder is correlated with the expression level of certain TLRs. Our study is the first broad analysis examining how major depressive disorder influences systemic innate immunity by evaluating levels of TLR expression in peripheral blood. Here, we found that TLR3, 4, 5 and 7 mRNA levels were significantly higher in subjects with acute major depressive disorder than in healthy controls; however, TLR1 and 6 mRNA levels were lower. The findings of TLR3 and 4 mRNA overexpression in peripheral blood
285
correspond to previous animal experimental models of depression, postmortem study and human study (Garate et al., 2011; Pandey et al., 2014; Kéri et al., 2014); however, the findings that TLR 5 and 7 have higher mRNA expression levels and TLRs 1 and 6 have lower mRNA expression levels are novel in patients with major depressive disorder and the mechanism that causes these expression difference are unknown. There are some indirect evidence that connect the expression level of TLR4 and major depressive disorder. Some regiment such as N-acetylcysteine and melatonin which can attenuate levels of TLR4 mRNA or protein expression can ameliorate chronic mild stress induced behavioral dysfunctions in mice or depressive symptoms in human (Kang et al., 2011; Lee et al., 2012; Carvalho et al., 2013; Haridas et al., 2013). Besides, some models of stress show increased intestinal permeability and resultant bacterial translocation to the systemic circulation (Collins, 2001; Ponferrada et al., 2007). These circulating Gram-negative enterobacteria, which are a major source of LPS, may activate TLR4 expression (Garate et al., 2014). In contrast to TLR4, the role of TLR 3, 5 and 7 have received little attention with regards to major depression. TLR5 has been associated with irritable bowel syndrome (Brint et al., 2011), and TLR5-deficient mice exhibit hyperphagia and develop hallmark features of metabolic syndrome, including hyperlipidemia, hypertension, insulin resistance, and increased adiposity (Okun et al., 2011). TLR3 and 7-induced IL-8 release was found in irritable bowel syndrome (McKernan et al., 2011b). Combination of Poly-inosinic/cytidylic acid (Poly I:C), a TLR3 agonist, and restrain stress could cause GABAergic abnormalities in the prefrontal cortex and striatum (Deslauriers et al., 2013). Besides, Poly I:C-induced activation of the immune response is reported to be accompanied by depression and anxiety-like behaviors (Gibney et al., 2013). These diseases or phenomenon were associated with major depressive disorder. However, the real mechanism behind this association is still unknown. There are several limitations of this study. First, the possible effect of antidepressants on TLRs expression levels cannot be excluded. However, only fluoxetine and citalopram were shown to inhibit the signaling of TLR3, 7, 8, and 9 in one study (Sacre et al., 2010) rather than to increase expression level. Second, the effects of distribution of different types of leukocytes on TLRs mRNA expression levels are unclear in this work. In previous studies, the decreased percentages of both lymphocytes and the absolute number of lymphocytes among patients with major depressive disorder were found (Kronfol et al., 1985). Lymphocyte, especially in B lymphocyte which expressed relative high levels of TLR1 and 6, may confound the finding of TLR1 and 6 in this work (Hornung et al., 2002). The increased percentage of neutrophil, which expressed TLR 1 - 9, may contribute to some difference in TLRs expression (Hayashi et al., 2003). However, no difference in expression of TLR 2, 8 and 9 found in this work showed opposite finding. Third, an increase in the content of TLR-specific mRNA may not cause an increase in TLR-specific surface expression in consequence. The dissociation between the receptors' protein and mRNA was noted in patients with sepsis or in some cell line (Cohen, 2002; Erdinest et al., 2014). Fourth, total RNA was isolated from whole blood with the PAXgene proprietary blood collection system rather than isolated leukocytes. Increased noise and reduced responsiveness in the gene expression profiles were previously reported (Feezor et al., 2004). Finally, the sample size of current study is small, and further analyses using larger sample sizes are needed to confirm these results. Taken together, the findings from the present study give evidence to the importance of TLRs expression level changes in patients with major depressive disorder. Expression level increases may increase the immune system's response to ligands, including
286
Y.-Y. Hung et al. / Psychiatry Research 220 (2014) 283–286
LPS and flagellin. Determination of TLR receptor expression ratios may help in designing novel therapeutic strategies based on TLRs signaling in major depression. Our findings should warrant further investigation on the relationship between TLRs and major depressive disorder. References Akira, S., Takeda, K., 2004. Toll-like receptor signalling. Nature Reviews Immunology 4, 499–511. Brint, E.K., MacSharry, J., Fanning, A., Shanahan, F., Quigley, E.M., 2011. Differential expression of toll-like receptors in patients with irritable bowel syndrome. The American Journal of Gastroenterology 106, 329–336. Carvalho, A.F., Macedo, D.S., Goulia, P., Hyphantis, T.N., 2013. N-acetylcysteine augmentation to tranylcypromine in treatment-resistant major depression. Journal of Clinical Psychopharmacology 33, 719–720. Cejkova, P., Chroma, V., Cerna, M., Markova, M., Marek, J., Lacinova, Z., Haluzik, M., 2012. Monitoring of the course of sepsis in hematooncological patients by extrapituitary prolactin expression in peripheral blood monocytes. Physiological Research/Academia Scientiarum Bohemoslovaca 61, 481–488. Cohen, J., 2002. The immunopathogenesis of sepsis. Nature 420, 885–891. Collins, S.M., 2001. Stress and the Gastrointestinal Tract IV. Modulation of intestinal inflammation by stress: basic mechanisms and clinical relevance. American Journal of Physiology: Gastrointestinal and Liver Physiology 280, G315–G318. Crews, F.T., Qin, L., Sheedy, D., Vetreno, R.P., Zou, J., 2013. High mobility group box 1/ Toll-like receptor danger signaling increases brain neuroimmune activation in alcohol dependence. Biological Psychiatry 73, 602–612. Deslauriers, J., Larouche, A., Sarret, P., Grignon, S., 2013. Combination of prenatal immune challenge and restraint stress affects prepulse inhibition and dopaminergic/GABAergic markers. Progress in Neuro-psychopharmacology & Biological Psychiatry 45, 156–164. Enstrom, A.M., Onore, C.E., Van de Water, J.A., Ashwood, P., 2010. Differential monocyte responses to TLR ligands in children with autism spectrum disorders. Brain, Behavior, and Immunity 24, 64–71. Erdinest, N., Aviel, G., Moallem, E., Anteby, I., Yahalom, C., Mechoulam, H., Ovadia, H., Solomon, A., 2014. Expression and activation of toll-like receptor 3 and toll-like receptor 4 on human corneal epithelial and conjunctival fibroblasts. Journal of Inflammation 11, 3. Feezor, R.J., Baker, H.V., Mindrinos, M., Hayden, D., Tannahill, C.L., Brownstein, B.H., Fay, A., MacMillan, S., Laramie, J., Xiao, W., Moldawer, L.L., Cobb, J.P., Laudanski, K., Miller-Graziano, C.L., Maier, R.V., Schoenfeld, D., Davis, R.W., Tompkins, R.G., 2004. Whole blood and leukocyte RNA isolation for gene expression analyses. Physiological Genomics 19, 247–254. Garate, I., Garcia-Bueno, B., Madrigal, J.L., Bravo, L., Berrocoso, E., Caso, J.R., Mico, J.A., Leza, J.C., 2011. Origin and consequences of brain Toll-like receptor 4 pathway stimulation in an experimental model of depression. Journal of Neuroinflammation 8, 151. Garate, I., Garcia-Bueno, B., Madrigal, J.L., Caso, J.R., Alou, L., Gomez-Lus, M.L., Leza, J. C., 2014. Toll-like 4 receptor inhibitor TAK-242 decreases neuroinflammation in rat brain frontal cortex after stress. Journal of Neuroinflammation 11, 8. Gibney, S.M., McGuinness, B., Prendergast, C., Harkin, A., Connor, T.J., 2013. Poly I:Cinduced activation of the immune response is accompanied by depression and anxiety-like behaviors, kynurenine pathway activation and reduced BDNF expression. Brain, Behavior, and Immunity 28, 170–181. Hajebrahimi, B., Bagheri, M., Hassanshahi, G., Nazari, M., Bidaki, R., Khodadadi, H., Arababadi, M.K., Kennedy, D., 2014. The adapter proteins of TLRs, TRIF and MYD88, are upregulated in depressed individuals. International Journal of Psychiatry in Clinical Practice 18, 41–44. Hamilton, M., 1960. A rating scale for depression. Journal Neurology Neurosurgery & Psychiatry 23, 56–62. Haridas, S., Kumar, M., Manda, K., 2013. Melatonin ameliorates chronic mild stress induced behavioral dysfunctions in mice. Physiology & Behavior 119, 201–207. Hayashi, F., Means, T.K., Luster, A.D., 2003. Toll-like receptors stimulate human neutrophil function. Blood 102, 2660–2669. Hornung, V., Rothenfusser, S., Britsch, S., Krug, A., Jahrsdorfer, B., Giese, T., Endres, S., Hartmann, G., 2002. Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. Journal of Immunology 168, 4531–4537.
Huang, Y.W., Lin, S.C., Wei, S.C., Hu, J.T., Chang, H.Y., Huang, S.H., Chen, D.S., Chen, P.J., Hsu, P.N., Yang, S.S., Kao, J.H., 2013. Reduced Toll-like receptor 3 expression in chronic hepatitis B patients and its restoration by interferon therapy. Antiviral Therapy 18, 877–884. Kang, J.W., Koh, E.J., Lee, S.M., 2011. Melatonin protects liver against ischemia and reperfusion injury through inhibition of toll-like receptor signaling pathway. Journal of Pineal Research 50, 403–411. Kanuri, G., Weber, S., Volynets, V., Spruss, A., Bischoff, S.C., Bergheim, I., 2009. Cinnamon extract protects against acute alcohol-induced liver steatosis in mice. The Journal of Nutrition 139, 482–487. Kanzler, H., Barrat, F.J., Hessel, E.M., Coffman, R.L., 2007. Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nature Medicine 13, 552–559. Kaul, D., Habbel, P., Derkow, K., Kruger, C., Franzoni, E., Wulczyn, F.G., Bereswill, S., Nitsch, R., Schott, E., Veh, R., Naumann, T., Lehnardt, S., 2012. Expression of Tolllike receptors in the developing brain. PloS One 7, e37767. Kawai, T., Akira, S., 2007. TLR signaling. Seminars in Immunology 19, 24–32. Kéri S., Szabó C., Kelemen O., 2014. Expression of Toll-Like Receptors in peripheral blood mononuclear cells and response to cognitive-behavioral therapy in major depressive disorder. Brain, Behavior, and Immunity. Kronfol, Z., Nasrallah, H.A., Chapman, S., House, J.D., 1985. Depression, cortisol metabolism and lymphocytopenia. Journal of Affective Disorders 9, 169–173. Larsen, P.H., Holm, T.H., Owens, T., 2007. Toll-like receptors in brain development and homeostasis. Science's STKE: Signal Transduction Knowledge Environment 2007 (pe47). Lee, S.I., Park, K.H., Kim, S.J., Kang, Y.G., Lee, Y.M., Kim, E.C., 2012. Mechanical stressactivated immune response genes via Sirtuin 1 expression in human periodontal ligament cells. Clinical and Experimental Immunology 168, 113–124. López, J., Fernández-Pisonero, I., Dueñas, A.I., Maeso, P., Román, J.A., Crespo, M.S., García-Rodríguez, C., 2012. Viral and bacterial patterns induce TLR-mediated sustained inflammation and calcification in aortic valve interstitial cell. International Journal of Cardiology 158, 18–25. Ma, Y., Li, J., Chiu, I., Wang, Y., Sloane, J.A., Lu, J., Kosaras, B., Sidman, R.L., Volpe, J.J., Vartanian, T., 2006. Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis. The Journal of Cell Biology 175, 209–215. Maes, M., Kubera, M., Leunis, J.C., 2008. The gut-brain barrier in major depression: intestinal mucosal dysfunction with an increased translocation of LPS from gram negative enterobacteria (leaky gut) plays a role in the inflammatory pathophysiology of depression. Neuro Endocrinology Letters 29, 117–124. McKernan, D.P., Dennison, U., Gaszner, G., Cryan, J.F., Dinan, T.G., 2011a. Enhanced peripheral toll-like receptor responses in psychosis: further evidence of a proinflammatory phenotype. Translational Psychiatry 1, e36. McKernan, D.P., Gaszner, G., Quigley, E.M., Cryan, J.F., Dinan, T.G., 2011b. Altered peripheral toll-like receptor responses in the irritable bowel syndrome. Alimentary Pharmacology & Therapeutics 33, 1045–1052. Okun, E., Griffioen, K.J., Mattson, M.P., 2011. Toll-like receptor signaling in neural plasticity and disease. Trends in Neurosciences 34, 269–281. Pandey, G.N., Rizavi, H.S., Ren, X., Bhaumik, R., Dwivedi, Y., 2014. Toll-like receptors in the depressed and suicide brain. Journal of Psychiatric Research 53, 62–68. Piccinini, A.M., Midwood, K.S., 2010. DAMPening inflammation by modulating TLR signalling. Mediators of Inflammation 2010. Ponferrada, A., Caso, J.R., Alou, L., Colon, A., Sevillano, D., Moro, M.A., Lizasoain, I., Menchen, P., Gomez-Lus, M.L., Lorenzo, P., Cos, E., Leza, J.C., Menchen, L., 2007. The role of PPARgamma on restoration of colonic homeostasis after experimental stress-induced inflammation and dysfunction. Gastroenterology 132, 1791–1803. Raison, C.L., Capuron, L., Miller, A.H., 2006. Cytokines sing the blues: inflammation and the pathogenesis of depression. Trends in Immunology 27, 24–31. Sacre, S., Medghalchi, M., Gregory, B., Brennan, F., Williams, R., 2010. Fluoxetine and citalopram exhibit potent antiinflammatory activity in human and murine models of rheumatoid arthritis and inhibit toll-like receptors. Arthritis and Rheumatism 62, 683–693. Sykes, A., Edwards, M.R., Macintyre, J., Del Rosario, A., Gielen, V., Haas, J., Kon, O.M., McHale, M., Johnston, S.L., 2013. TLR3, TLR4 and TLRs7-9 induced interferons are not impaired in airway and blood cells in well controlled asthma. PloS One 8, e65921. Yuan, T.M., Sun, Y., Zhan, C.Y., Yu, H.M., 2010. Intrauterine infection/inflammation and perinatal brain damage: role of glial cells and Toll-like receptor signaling. Journal of Neuroimmunology 229, 16–25.
Contents lists available at ScienceDirect
Psychiatry Research journal homepage: www.elsevier.com/locate/psychres
Association between toll-like receptors expression and major depressive disorder Yi-Yung Hung a,b,c, Hong-Yo Kang b,c, Kai-Wei Huang d, Tiao-Lai Huang a,e,n a
Department of Psychiatry, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, ROC Graduate Institute of Clinical Medical Sciences, Chang Gung University, College of Medicine, Kaohsiung, Taiwan, ROC c Center for Menopause and Reproductive Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan, ROC d Department of Nursing, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, ROC e Genomic & Proteomic Core Laboratory, Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan, ROC b
art ic l e i nf o
a b s t r a c t
Article history: Received 11 March 2014 Received in revised form 14 July 2014 Accepted 25 July 2014 Available online 13 August 2014
Accumulating evidences suggest that Toll-like receptors (TLRs) were involved in the pathophysiology of major depressive disorder. TLR4 was thought to be associated with major depressive disorder in animal model, but the others were still unknown. In order to examine TLR1-9 mRNA expression levels in peripheral blood and their relationships with the psychopathology of major depressive disorder, 30 patients with major depressive disorder were compared with 29 healthy controls. The 17-item Hamilton Depression Rating Scale (HAMD-17) was used to assess the severity of major depression. The mRNA expression levels of TLRs were examined in parallel with a housekeeping gene using real-time polymerase chain reaction (RT-PCR). Analysis of covariance with age and body mass index adjustment revealed a significantly higher expression of TLR3, 4, 5 and 7 mRNA but lower expression of TLR1 and 6 in patients with major depressive disorder as compared with healthy controls. Multiple linear regression analysis revealed that TLR4 was an independent risk factor relating to severity of major depression. These findings suggest that TLRs, especially TLR4, may be involved in the psychopathology of major depression. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Depression Innate immunity TLR4 LPS Hamilton depression rating scale
1. Introduction Toll-like receptors (TLRs) are pattern recognition receptors proposed as main agents of the innate immune response, constituting the first line of defense against invading microorganisms (Akira and Takeda, 2004). TLRs recognize endogenous ligands, termed as damage-associated molecular patterns, and mediate an inflammatory response in the host to injury and stress (Piccinini and Midwood, 2010). There are various types of TLRs, and each have different cellsurface receptors; these include: TLR4, which recognizes lipopolysaccharide (LPS) present in Gram-negative bacteria; TLR2 and its co-receptors TLR1/TLR6, which sense bacterial lipoproteins and lipoteichoic acid; and TLR5 that recognizes bacterial flagellin. Another subtype of receptors are localized in the endosomal cell compartment and include TLR3, which recognizes doublestranded (ds) RNA; TLR9, which senses unmethylated CpG motifs
n Corresponding author at: Department of Psychiatry, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, ROC. Tel.: þ 886 7 7317123x6317; fax: þ886 7 7326817. E-mail address: [email protected] (T.-L. Huang).
http://dx.doi.org/10.1016/j.psychres.2014.07.074 0165-1781/& 2014 Elsevier Ireland Ltd. All rights reserved.
present in viral and bacterial genome and parasites; and TLR7, which binds single-stranded (ss) DNA (Kawai and Akira, 2007). TLRs are expressed in various cell types in the central nervous system, including microglia (TLR1-9), astrocytes (TLR1-5; TLR9), and neurons (TLR3). Neurodevelopment and neurodegeneration are associated with TLR activation. (Yuan et al., 2010; Okun et al., 2011). TLRs are expressed in the developing as well as the adult brain and play a significant role in plasticity ( Larsen et al., 2007; Kaul et al., 2012). TLR8 activation induces neuronal apoptosis through inhibition of neurite outgrowth (Ma et al., 2006), while induction of TLR3-mediated immunity during gestation inhibits cortical neurogenesis and synaptic transmission (Yuan et al., 2010). TLRs are also potential therapeutic targets against neuroinflammation. A number of these compounds are currently undergoing different phases of clinical trials (Kanzler et al., 2007). mRNA expression of TLRs in peripheral blood mononuclear cell (PBMC) was used to detect changes in innate immunity in many physical diseases including asthma (Sykes et al., 2013), chronic hepatitis B (Huang et al., 2013) or sepsis (Cejkova et al., 2012). The relationship between TLRs and psychiatric diseases were investigated before. A systematic analysis of TLRs revealed an enhanced response in TLR2 and TLR4 activation in patients with schizophrenia or bipolar disorder (McKernan et al., 2011a). In autism, an
284
Y.-Y. Hung et al. / Psychiatry Research 220 (2014) 283–286
enhanced TLR2 and TLR4 responsivity to its ligand can be measured, while TLR9 showed a decreased response in the blood (Enstrom et al., 2010). Alcohol abuse was also found to be associated with the TLR2, 3 and 4 complex (Kanuri et al., 2009; Crews et al., 2013). In clinical depression, translocation of the Gram-negative enterobacteria, which contain LPS in their bacterial wall, was thought to be associated with the onset of a number of inflammatory and oxidative/nitrosative stress pathways and play an important role in major depressive disorder (Maes et al., 2008). It has been demonstrated that after stress exposure or during certain episodes of depression, an innate immune response is strongly activated (Raison et al., 2006). Elevated TRIF and MYD88, intracellular adapter proteins for TLR signaling, were found in peripheral blood mononuclear cells isolated from depressed patients when compared to healthy subjects (Hajebrahimi et al., 2014). However, the systematic analysis of TLR expression in humans is still absent. Therefore, the present study aims to investigate whether TLRs expression levels are altered in patients with major depressive disorder, and how these changes correlate with the severity of depressive symptoms. 2. Method 2.1. Subjects Thirty outpatients or inpatients with major depressive disorder and 29 healthy controls were recruited from August 2013 to December 2013 at Kaohsiung Chang Gung Memorial Hospital Medical Center in Taiwan. Institutional Review Board approval was obtained from the hospital ethics committee (101-5012A3). Thirty patients with major depression were evaluated by one psychiatrist using the Structured Clinical Interview for DSM-IV Axis I Disorders. The severity of depression was assessed using the 17-item Hamilton Depression Rating Scale (HAMD-17) (Hamilton, 1960) by the same psychiatrist. Patients who had alcohol dependence or any immune inflammatory disease history were excluded from the study. The 29 healthy controls, who had neither a personal history nor a first degree relative with psychiatric disorder, were recruited from community. The Chinese health questionnaire-12 was done to assess the healthy control group by the same psychiatrist to rule out psychiatric disease according to Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria. All patients received blood pressure checks, chest X-rays, electrocardiogram examinations, and routine blood tests in order to exclude any chronic physical illness including heart, lung, liver, kidney, or metabolic diseases. They were also checked for acute infections or allergic reactions; moreover, they reported not taking any antidepressants for at least 1 week before they entered into this study. 2.2. Real-Time Polymerase Chain Reaction (RT-PCR) analysis
difference of age and BMI. Analysis of covariance (ANCOVA) with age, and BMI adjustment was used to compare the difference of TLRs expression levels. Multiple linear regression (stepwise method) was used to judge factors that correlates with HAMD-17 scores. All statistical analyses were performed using Statistical Product and Service Solutions (SPSS), version 22. For each test, po 0.05 was considered significant.
3. Results 3.1. Baseline characteristics Table 1 shows baseline characteristics regarding participant sex, age, and BMI (depressive patients: 11 male/19 female, mean age 45.8 710.02 years, BMI 22.96 73.7 kg/m2; health controls: 11 male/18 female, mean age 42.72 712.49 years, mean BMI 22.58 7 2.93 kg/m2). There were no significant differences in the mean age, sex, or BMI between the two groups. Duration of illness and 17-item Hamilton Depression Rating Scale in patients with major depressive disorder are also shown Table 1. 3.2. Differential TLR mRNA expression in peripheral blood from patients with major depressive disorder and healthy controls We analyzed TLR mRNA expression and GADPH was used as a housekeeping gene to assess the relative abundance of mRNA in peripheral blood. Similar TLR expression profiles, but differences in transcript levels, were observed in depressive patients as compared to healthy controls (Table 2). Quantitative analysis revealed significantly higher TLR3, 4, 5 and 7 mRNA expression levels in patients with major depression (TLR3: patients 1.57 70.82, controls 1.08 7 0.86, F(1,50)¼ 4.2, p ¼0.046; TLR4: patients 1.75 71.18, controls 1.26 70.85, F(1,53) ¼4.31, p ¼0.043;TLR5: patients 2.317 1.96, controls 1.14 70.77, F(1,51) ¼7.83, p ¼0.007; TLR7: patients 1.54 70.66, controls 1.01 70.48, F(1, 50) ¼10.25, p ¼ 0.002), whereas no significant differences for TLR2, 8, or 9 were observed. In contrast, significant lower expressions of TLR1 and TLR6 in patients with major depressive disorder were found, compared to healthy controls (TLR1: patients 1.737 1.18, controls 2.977 2.37, F(1, 50) ¼9.55, p ¼ 0.003; TLR6: patients 1.60 71.25, controls 2.86 72.35, F(1, 51) ¼8.55, p ¼ 0.005). Table 1 Demographic findings and clinical data of health controls and patients with major depressive disorder.
Venous blood (5 mL) samples were drawn into PAXgene Blood RNA Tube between 8:00 am and 10:00 am, after the patients had fasted for 9 h. The tubes were stored in a 80 1C refrigerator immediately after collection and remained there until they were assayed. RNA was isolated using TRIzol reagent, and RT-PCR was performed using following sets of primers (López et al., 2012): TLR1 (sense 50 -GGGTCAGCTGGACTTCAGAG-30 , anti-sense 50 -AAAATCCAAATGCAGGAACG- 30 ); TLR2 (sense 50 -TCAGCCTCTCCAAGGAAGAA-30 , anti-sense 50 -AATGTTCAAGACTGCCCAGG-30 ); TLR3 (sense 50 -AGCCTTCAACGACTGATGCT-30 , anti-sense 50 -TTTCCAGAGCCGTGCTAAGT-30 ); TLR4 (sense 50 -TGAGCAGTCGTGCTGGTATC-30 , anti-sense 50 -CAGGGCTTTTCTGAGTCGTC-3); TLR5 (sense 50 -GGAACCAGCTCCTAGCTCCT-30 , anti-sense 50 -AAGAGGGAAACCCCAGAGAA-30 ); TLR6 (sense 50 -CCCTTTAGGATAGCCACTGC-30 , anti-sense 50 -CTCACAATAGGATGGCAGGA-30 ); TLR7 (sense 50 -CCTTGAGGCCAACAACATCT-30 , anti-sense 50 -GTAGGGACGGCTGTGACATT-30 ); TLR8 (sense 50 -TCCTTCAGTCGTCAATGCTG-30 , anti-sense 50 -CGTTTGGGGAACTTCCTGTA-30 ); and TLR9 (sense 50 -GGACACTCCCAGCTCTGAAG-30 , anti-sense 50 -TTG GCTGTGGATGTTGTTGT-30 ). The QuantiTect Reverse Transcription kit (Qiagen) was used for the reverse transcription of RNA. The PTC-200 apparatus (BioRad) and the QuantiTect SYBR Green PCR kit (Qiagen) were applied for the complete analysis. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as a housekeeping gene to assess the relative abundance of mRNA.
Age (years) male female Gender (M/F) BMI male female HAMD-17 male female Duration of illness (years) male female Smoking Education duration (year) Antidepressant use History None SSRI SNRI NDRI
2.3. Statistical analysis Outliers of TLRs were excluded initially based on a boxplot outlier detection rule. All results are presented as means 7standard deviation. Chi-square was used to compare the difference between sexes. Student's t-test was used to compare the
Major depression
Healthy controls
p Value
45.8 7 10.02 43.187 8.92 47.32 710.54 11/19 22.96 7 3.70 21.56 7 2.64 23.777 4.03 27.83 7 4.97 28.36 7 6.41 27.52 7 4.08 4.25 7 3.59 3.91 73.93 4.37 73.54 12 11.677 3.14
42.727 12.49 46.55 7 14.73 40.39 7 10.68 11/18 22.58 7 2.93 24.137 3.34 21.63 7 2.26 – – – – – – 3 15.077 3.18
0.30 0.53 0.055 0.92 0.67 0.06 0.055 – – – – – – – –
3 11 10 6
– – – –
– – – –
Data are presented as mean 7S.D. p Value o 0.05; HAMD-17 ¼ 17-item Hamilton Depression Rating Scale; BMI, body mass index.
n
Y.-Y. Hung et al. / Psychiatry Research 220 (2014) 283–286
Table 2 Toll-like receptors 1-9 mRNA expression level of health controls and patients with major depressive disorder. Major depression
Healthy controls
F and p Value
TLR1
1.737 1.18
2.977 2.37
TLR2
1.62 7 0.82
1.55 7 0.79
TLR3
1.577 0.82
1.08 7 0.86
TLR4
1.75 7 1.18
1.26 7 0.85
TLR5
2.317 1.96
1.147 0.77
TLR6
1.60 7 1.25
2.86 7 2.35
TLR7
1.54 7 0.66
1.017 0.48
TLR8
1.05 7 0.48
0.89 7 0.43
TLR9
1.80 7 1.45
1.277 0.76
F(1,50) ¼9.55 p ¼ 0.003n F(1,53) ¼1.88 p ¼ 0.666 F(1,50) ¼4.20 p ¼ 0.046n F(1,53) ¼4.31 p ¼ 0.043n F(1,51)¼ 7.83 p ¼ 0.007n F(1,51)¼ 8.55 p ¼ 0.005n F(1,50) ¼10.25 p ¼ 0.002n F(1,55) ¼2.12 p ¼ 0.15 F(1,53) ¼2.93 p ¼ 0.09
Analysis of covariance with age, and BMI adjustment was also done to compare the difference of TLRs; Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as a housekeeping gene to assess the relative abundance of mRNA; TLR, Toll-like receptor. n
p Value o 0.05
Table 3 Stepwise multiple linear regression model between HAMD-17 score and TLR. Independent factors
TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9
HAMD-17 score Standardized coefficients
t
p Value
0.043 0.169 0.027 0.438 0.102 0.044 0.217 0.442 0.090
0.19 0.66 0.12 2.29 0.31 0.22 1.12 1.70 0.24
0.855 0.516 0.903 0.032n 0.763 0.828 0.275 0.104 0.814
n p Value o 0.05; HAMD-17 ¼ 17-item Hamilton Depression Rating Scale; TLR, Toll-like receptor; BMI, body mass index.
3.3. Severity of depression and TLRs We also analyzed the correlation of age, BMI, gender, duration of illness, and TLR1-9 with severity of major depression. Linear regression revealed that TLR4 was an independent risk factor which explained 19.2% the total variance in depression. (r2 ¼0.192; p ¼ 0.032) (Table 3).
4. Discussion The present study demonstrates that TLR expression levels in peripheral blood of patients with major depressive disorder are different from these of healthy controls. Moreover, the severity of major depressive disorder is correlated with the expression level of certain TLRs. Our study is the first broad analysis examining how major depressive disorder influences systemic innate immunity by evaluating levels of TLR expression in peripheral blood. Here, we found that TLR3, 4, 5 and 7 mRNA levels were significantly higher in subjects with acute major depressive disorder than in healthy controls; however, TLR1 and 6 mRNA levels were lower. The findings of TLR3 and 4 mRNA overexpression in peripheral blood
285
correspond to previous animal experimental models of depression, postmortem study and human study (Garate et al., 2011; Pandey et al., 2014; Kéri et al., 2014); however, the findings that TLR 5 and 7 have higher mRNA expression levels and TLRs 1 and 6 have lower mRNA expression levels are novel in patients with major depressive disorder and the mechanism that causes these expression difference are unknown. There are some indirect evidence that connect the expression level of TLR4 and major depressive disorder. Some regiment such as N-acetylcysteine and melatonin which can attenuate levels of TLR4 mRNA or protein expression can ameliorate chronic mild stress induced behavioral dysfunctions in mice or depressive symptoms in human (Kang et al., 2011; Lee et al., 2012; Carvalho et al., 2013; Haridas et al., 2013). Besides, some models of stress show increased intestinal permeability and resultant bacterial translocation to the systemic circulation (Collins, 2001; Ponferrada et al., 2007). These circulating Gram-negative enterobacteria, which are a major source of LPS, may activate TLR4 expression (Garate et al., 2014). In contrast to TLR4, the role of TLR 3, 5 and 7 have received little attention with regards to major depression. TLR5 has been associated with irritable bowel syndrome (Brint et al., 2011), and TLR5-deficient mice exhibit hyperphagia and develop hallmark features of metabolic syndrome, including hyperlipidemia, hypertension, insulin resistance, and increased adiposity (Okun et al., 2011). TLR3 and 7-induced IL-8 release was found in irritable bowel syndrome (McKernan et al., 2011b). Combination of Poly-inosinic/cytidylic acid (Poly I:C), a TLR3 agonist, and restrain stress could cause GABAergic abnormalities in the prefrontal cortex and striatum (Deslauriers et al., 2013). Besides, Poly I:C-induced activation of the immune response is reported to be accompanied by depression and anxiety-like behaviors (Gibney et al., 2013). These diseases or phenomenon were associated with major depressive disorder. However, the real mechanism behind this association is still unknown. There are several limitations of this study. First, the possible effect of antidepressants on TLRs expression levels cannot be excluded. However, only fluoxetine and citalopram were shown to inhibit the signaling of TLR3, 7, 8, and 9 in one study (Sacre et al., 2010) rather than to increase expression level. Second, the effects of distribution of different types of leukocytes on TLRs mRNA expression levels are unclear in this work. In previous studies, the decreased percentages of both lymphocytes and the absolute number of lymphocytes among patients with major depressive disorder were found (Kronfol et al., 1985). Lymphocyte, especially in B lymphocyte which expressed relative high levels of TLR1 and 6, may confound the finding of TLR1 and 6 in this work (Hornung et al., 2002). The increased percentage of neutrophil, which expressed TLR 1 - 9, may contribute to some difference in TLRs expression (Hayashi et al., 2003). However, no difference in expression of TLR 2, 8 and 9 found in this work showed opposite finding. Third, an increase in the content of TLR-specific mRNA may not cause an increase in TLR-specific surface expression in consequence. The dissociation between the receptors' protein and mRNA was noted in patients with sepsis or in some cell line (Cohen, 2002; Erdinest et al., 2014). Fourth, total RNA was isolated from whole blood with the PAXgene proprietary blood collection system rather than isolated leukocytes. Increased noise and reduced responsiveness in the gene expression profiles were previously reported (Feezor et al., 2004). Finally, the sample size of current study is small, and further analyses using larger sample sizes are needed to confirm these results. Taken together, the findings from the present study give evidence to the importance of TLRs expression level changes in patients with major depressive disorder. Expression level increases may increase the immune system's response to ligands, including
286
Y.-Y. Hung et al. / Psychiatry Research 220 (2014) 283–286
LPS and flagellin. Determination of TLR receptor expression ratios may help in designing novel therapeutic strategies based on TLRs signaling in major depression. Our findings should warrant further investigation on the relationship between TLRs and major depressive disorder. References Akira, S., Takeda, K., 2004. Toll-like receptor signalling. Nature Reviews Immunology 4, 499–511. Brint, E.K., MacSharry, J., Fanning, A., Shanahan, F., Quigley, E.M., 2011. Differential expression of toll-like receptors in patients with irritable bowel syndrome. The American Journal of Gastroenterology 106, 329–336. Carvalho, A.F., Macedo, D.S., Goulia, P., Hyphantis, T.N., 2013. N-acetylcysteine augmentation to tranylcypromine in treatment-resistant major depression. Journal of Clinical Psychopharmacology 33, 719–720. Cejkova, P., Chroma, V., Cerna, M., Markova, M., Marek, J., Lacinova, Z., Haluzik, M., 2012. Monitoring of the course of sepsis in hematooncological patients by extrapituitary prolactin expression in peripheral blood monocytes. Physiological Research/Academia Scientiarum Bohemoslovaca 61, 481–488. Cohen, J., 2002. The immunopathogenesis of sepsis. Nature 420, 885–891. Collins, S.M., 2001. Stress and the Gastrointestinal Tract IV. Modulation of intestinal inflammation by stress: basic mechanisms and clinical relevance. American Journal of Physiology: Gastrointestinal and Liver Physiology 280, G315–G318. Crews, F.T., Qin, L., Sheedy, D., Vetreno, R.P., Zou, J., 2013. High mobility group box 1/ Toll-like receptor danger signaling increases brain neuroimmune activation in alcohol dependence. Biological Psychiatry 73, 602–612. Deslauriers, J., Larouche, A., Sarret, P., Grignon, S., 2013. Combination of prenatal immune challenge and restraint stress affects prepulse inhibition and dopaminergic/GABAergic markers. Progress in Neuro-psychopharmacology & Biological Psychiatry 45, 156–164. Enstrom, A.M., Onore, C.E., Van de Water, J.A., Ashwood, P., 2010. Differential monocyte responses to TLR ligands in children with autism spectrum disorders. Brain, Behavior, and Immunity 24, 64–71. Erdinest, N., Aviel, G., Moallem, E., Anteby, I., Yahalom, C., Mechoulam, H., Ovadia, H., Solomon, A., 2014. Expression and activation of toll-like receptor 3 and toll-like receptor 4 on human corneal epithelial and conjunctival fibroblasts. Journal of Inflammation 11, 3. Feezor, R.J., Baker, H.V., Mindrinos, M., Hayden, D., Tannahill, C.L., Brownstein, B.H., Fay, A., MacMillan, S., Laramie, J., Xiao, W., Moldawer, L.L., Cobb, J.P., Laudanski, K., Miller-Graziano, C.L., Maier, R.V., Schoenfeld, D., Davis, R.W., Tompkins, R.G., 2004. Whole blood and leukocyte RNA isolation for gene expression analyses. Physiological Genomics 19, 247–254. Garate, I., Garcia-Bueno, B., Madrigal, J.L., Bravo, L., Berrocoso, E., Caso, J.R., Mico, J.A., Leza, J.C., 2011. Origin and consequences of brain Toll-like receptor 4 pathway stimulation in an experimental model of depression. Journal of Neuroinflammation 8, 151. Garate, I., Garcia-Bueno, B., Madrigal, J.L., Caso, J.R., Alou, L., Gomez-Lus, M.L., Leza, J. C., 2014. Toll-like 4 receptor inhibitor TAK-242 decreases neuroinflammation in rat brain frontal cortex after stress. Journal of Neuroinflammation 11, 8. Gibney, S.M., McGuinness, B., Prendergast, C., Harkin, A., Connor, T.J., 2013. Poly I:Cinduced activation of the immune response is accompanied by depression and anxiety-like behaviors, kynurenine pathway activation and reduced BDNF expression. Brain, Behavior, and Immunity 28, 170–181. Hajebrahimi, B., Bagheri, M., Hassanshahi, G., Nazari, M., Bidaki, R., Khodadadi, H., Arababadi, M.K., Kennedy, D., 2014. The adapter proteins of TLRs, TRIF and MYD88, are upregulated in depressed individuals. International Journal of Psychiatry in Clinical Practice 18, 41–44. Hamilton, M., 1960. A rating scale for depression. Journal Neurology Neurosurgery & Psychiatry 23, 56–62. Haridas, S., Kumar, M., Manda, K., 2013. Melatonin ameliorates chronic mild stress induced behavioral dysfunctions in mice. Physiology & Behavior 119, 201–207. Hayashi, F., Means, T.K., Luster, A.D., 2003. Toll-like receptors stimulate human neutrophil function. Blood 102, 2660–2669. Hornung, V., Rothenfusser, S., Britsch, S., Krug, A., Jahrsdorfer, B., Giese, T., Endres, S., Hartmann, G., 2002. Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. Journal of Immunology 168, 4531–4537.
Huang, Y.W., Lin, S.C., Wei, S.C., Hu, J.T., Chang, H.Y., Huang, S.H., Chen, D.S., Chen, P.J., Hsu, P.N., Yang, S.S., Kao, J.H., 2013. Reduced Toll-like receptor 3 expression in chronic hepatitis B patients and its restoration by interferon therapy. Antiviral Therapy 18, 877–884. Kang, J.W., Koh, E.J., Lee, S.M., 2011. Melatonin protects liver against ischemia and reperfusion injury through inhibition of toll-like receptor signaling pathway. Journal of Pineal Research 50, 403–411. Kanuri, G., Weber, S., Volynets, V., Spruss, A., Bischoff, S.C., Bergheim, I., 2009. Cinnamon extract protects against acute alcohol-induced liver steatosis in mice. The Journal of Nutrition 139, 482–487. Kanzler, H., Barrat, F.J., Hessel, E.M., Coffman, R.L., 2007. Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nature Medicine 13, 552–559. Kaul, D., Habbel, P., Derkow, K., Kruger, C., Franzoni, E., Wulczyn, F.G., Bereswill, S., Nitsch, R., Schott, E., Veh, R., Naumann, T., Lehnardt, S., 2012. Expression of Tolllike receptors in the developing brain. PloS One 7, e37767. Kawai, T., Akira, S., 2007. TLR signaling. Seminars in Immunology 19, 24–32. Kéri S., Szabó C., Kelemen O., 2014. Expression of Toll-Like Receptors in peripheral blood mononuclear cells and response to cognitive-behavioral therapy in major depressive disorder. Brain, Behavior, and Immunity. Kronfol, Z., Nasrallah, H.A., Chapman, S., House, J.D., 1985. Depression, cortisol metabolism and lymphocytopenia. Journal of Affective Disorders 9, 169–173. Larsen, P.H., Holm, T.H., Owens, T., 2007. Toll-like receptors in brain development and homeostasis. Science's STKE: Signal Transduction Knowledge Environment 2007 (pe47). Lee, S.I., Park, K.H., Kim, S.J., Kang, Y.G., Lee, Y.M., Kim, E.C., 2012. Mechanical stressactivated immune response genes via Sirtuin 1 expression in human periodontal ligament cells. Clinical and Experimental Immunology 168, 113–124. López, J., Fernández-Pisonero, I., Dueñas, A.I., Maeso, P., Román, J.A., Crespo, M.S., García-Rodríguez, C., 2012. Viral and bacterial patterns induce TLR-mediated sustained inflammation and calcification in aortic valve interstitial cell. International Journal of Cardiology 158, 18–25. Ma, Y., Li, J., Chiu, I., Wang, Y., Sloane, J.A., Lu, J., Kosaras, B., Sidman, R.L., Volpe, J.J., Vartanian, T., 2006. Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis. The Journal of Cell Biology 175, 209–215. Maes, M., Kubera, M., Leunis, J.C., 2008. The gut-brain barrier in major depression: intestinal mucosal dysfunction with an increased translocation of LPS from gram negative enterobacteria (leaky gut) plays a role in the inflammatory pathophysiology of depression. Neuro Endocrinology Letters 29, 117–124. McKernan, D.P., Dennison, U., Gaszner, G., Cryan, J.F., Dinan, T.G., 2011a. Enhanced peripheral toll-like receptor responses in psychosis: further evidence of a proinflammatory phenotype. Translational Psychiatry 1, e36. McKernan, D.P., Gaszner, G., Quigley, E.M., Cryan, J.F., Dinan, T.G., 2011b. Altered peripheral toll-like receptor responses in the irritable bowel syndrome. Alimentary Pharmacology & Therapeutics 33, 1045–1052. Okun, E., Griffioen, K.J., Mattson, M.P., 2011. Toll-like receptor signaling in neural plasticity and disease. Trends in Neurosciences 34, 269–281. Pandey, G.N., Rizavi, H.S., Ren, X., Bhaumik, R., Dwivedi, Y., 2014. Toll-like receptors in the depressed and suicide brain. Journal of Psychiatric Research 53, 62–68. Piccinini, A.M., Midwood, K.S., 2010. DAMPening inflammation by modulating TLR signalling. Mediators of Inflammation 2010. Ponferrada, A., Caso, J.R., Alou, L., Colon, A., Sevillano, D., Moro, M.A., Lizasoain, I., Menchen, P., Gomez-Lus, M.L., Lorenzo, P., Cos, E., Leza, J.C., Menchen, L., 2007. The role of PPARgamma on restoration of colonic homeostasis after experimental stress-induced inflammation and dysfunction. Gastroenterology 132, 1791–1803. Raison, C.L., Capuron, L., Miller, A.H., 2006. Cytokines sing the blues: inflammation and the pathogenesis of depression. Trends in Immunology 27, 24–31. Sacre, S., Medghalchi, M., Gregory, B., Brennan, F., Williams, R., 2010. Fluoxetine and citalopram exhibit potent antiinflammatory activity in human and murine models of rheumatoid arthritis and inhibit toll-like receptors. Arthritis and Rheumatism 62, 683–693. Sykes, A., Edwards, M.R., Macintyre, J., Del Rosario, A., Gielen, V., Haas, J., Kon, O.M., McHale, M., Johnston, S.L., 2013. TLR3, TLR4 and TLRs7-9 induced interferons are not impaired in airway and blood cells in well controlled asthma. PloS One 8, e65921. Yuan, T.M., Sun, Y., Zhan, C.Y., Yu, H.M., 2010. Intrauterine infection/inflammation and perinatal brain damage: role of glial cells and Toll-like receptor signaling. Journal of Neuroimmunology 229, 16–25.
