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<p>Journal of Affective Disorders 141 (2012) 55–62</p><p>Contents lists available at SciVerse ScienceDirect</p><p>Journal of Affective Disorders</p><p>j ourna l homepage: www.e lsev ie r .com/ locate / jad</p><p>Research report</p><p>Increased IgA and IgM responses against gut commensals in chronic</p><p>depression: Further evidence for increased bacterial translocation or</p><p>leaky gut</p><p>Michael Maes a,⁎, Marta Kubera b, Jean-Claude Leunis c, Michael Berk d,e,f,g</p><p>a Maes Clinics @ Tria, Bangkok, Thailand</p><p>b Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland</p><p>c Laboratory Ategis, Waver, Belgium</p><p>d Mental Health Research Institute, Parkville, Australia</p><p>e Deakin University, School of Medicine, Barwon Health, Geelong, Australia</p><p>f University of Melbourne, Department of Psychiatry, Parkville, Australia</p><p>g Orygen Youth Health Research Centre, Parkville, Australia</p><p>a r t i c l e i n f o</p><p>⁎ Corresponding author at: Maes Clinics @ TRIA, P</p><p>Rimklongsamsen Road, Bangkok 10310, Thailand.</p><p>+32 3 4809282.</p><p>E-mail address: dr.michaelmaes@hotmail.com (M.</p><p>0165-0327/$ – see front matter © 2012 Elsevier B.V. A</p><p>doi:10.1016/j.jad.2012.02.023</p><p>a b s t r a c t</p><p>Article history:</p><p>Received 17 October 2011</p><p>Received in revised form 26 January 2012</p><p>Accepted 16 February 2012</p><p>Available online 11 March 2012</p><p>Background: Recently, we discovered that depression is accompanied by increased IgM and</p><p>IgA responses directed against gram negative gut commensals. The aim of this study was to</p><p>replicate these findings in a larger study group of depressed patients and to examine the asso-</p><p>ciations between the IgA and IgM responses to gut commensals and staging of depression as</p><p>well as the fatigue and somatic (F&S) symptoms of depression.</p><p>Methods: We measured serum concentrations of IgM and IgA against the LPS of gram-negative</p><p>enterobacteria, i.e. Hafnia alvei, Pseudomonas aeruginosa, Morganella morganii, Pseudomonas</p><p>putida, Citrobacter koseri, and Klebsiella pneumoniae in 112 depressed patients and 28 normal</p><p>controls. The severity of F&S symptoms was measured using the Fibromyalgia and Chronic Fa-</p><p>tigue Syndrome Rating Scale.</p><p>Results: The prevalences and median values of serum IgM and IgA against LPS of these com-</p><p>mensals were significantly higher in depressed patients than in controls. The IgM levels direct-</p><p>ed against the LPS of these commensal bacteria were significantly higher in patients with</p><p>chronic depression than in those without. The immune responses directed against LPS were</p><p>not associated with melancholia or recurrent depression. There was a significant correlation</p><p>between the IgA response directed against LPS and gastro-intestinal symptoms.</p><p>Discussion: The results indicate that increased bacterial translocation with immune responses</p><p>to the LPS of commensal bacteria may play a role in the pathophysiology of depression, partic-</p><p>ularly chronic depression. Bacterial translocation may a) occur secondary to systemic inflam-</p><p>mation in depression and intensify and perpetuate the primary inflammatory response once</p><p>the commensals are translocated; or b) be a primary trigger factor associated with the onset</p><p>of depression in some vulnerable individuals. The findings suggest that “translocated” gut</p><p>commensal bacteria activate immune cells to elicit IgA and IgM responses and that this phe-</p><p>nomenon may play a role in the pathophysiology of (chronic) depression by causing progres-</p><p>sive amplifications of immune pathways.</p><p>© 2012 Elsevier B.V. All rights reserved.</p><p>Keywords:</p><p>Depression</p><p>Inflammation</p><p>Cytokines</p><p>Chronic fatigue</p><p>Oxidative stress</p><p>Leaky gut</p><p>iyavate Hospital, 998</p><p>Tel.: +66 26602728,</p><p>Maes).</p><p>ll rights reserved.</p><p>1. Introduction</p><p>There is evidence that depression is a systemic disorder that</p><p>is accompanied by cell mediated immune (CMI) activation and</p><p>inflammation (Maes, 1993; Maes et al., 2012). Recently, two</p><p>http://dx.doi.org/10.1016/j.jad.2012.02.023</p><p>mailto:dr.michaelmaes@hotmail.com</p><p>http://dx.doi.org/10.1016/j.jad.2012.02.023</p><p>http://www.sciencedirect.com/science/journal/01650327</p><p>56 M. Maes et al. / Journal of Affective Disorders 141 (2012) 55–62</p><p>meta analyses showed consistent signs of inflammation</p><p>(Dowlati et al., 2010) and T cell activation (Liu et al., 2011) in</p><p>depression. CMI activation in depression is characterized by in-</p><p>creased levels of soluble interleukin-2 receptor (sIL-2R) and</p><p>neopterin and increased production of interferon (IFN)γ</p><p>(Maes et al., 1990; 2012). Inflammation is indicated by in-</p><p>creased levels of pro-inflammatory cytokines (PICs), including</p><p>IL-1, IL-6 and tumor necrosis factor (TNF)α (Maes et al.,</p><p>2012). These systemic CMI and inflammatory responses in</p><p>depression may be associated with consequent neuroinflam-</p><p>mation and neuroprogression, that is neurodegeneration, de-</p><p>creased neurogenesis and neuronal apoptosis (Berk et al.,</p><p>2011; Catena-Dell'Osso et al., 2011; Maes et al., 2009; 2011b)</p><p>and with the onset of melancholic symptoms, e.g. anhedonia,</p><p>anorexia, weight loss, psychomotor retardation; anxiety; and</p><p>fatigue and somatic symptoms (e.g. hyperalgesia, gastro-</p><p>intestinal symptoms, fatigue) (Leonard and Maes, 2012). The</p><p>above pathways are activated in depression even in the ab-</p><p>sence of a clear inflammatory lesion and the source of this in-</p><p>flammation has to date been opaque.</p><p>One mechanism that could contribute to systemic inflam-</p><p>mation and CMI activation in the absence of an inflammatory</p><p>injury is bacterial translocation due to increased gut perme-</p><p>ability or leaky gut. In the gut, there are 300–600 different</p><p>types of commensal bacteria. Some of these are potentially</p><p>toxic, i.e. the gram negative enterobacteria, because they</p><p>contain endotoxin or LPS, a toxic substance that is part of</p><p>the bacterial wall. LPS is recognized by the CD14-Toll-like</p><p>receptor-4 (TLR4) complex that is expressed not only by pe-</p><p>ripheral blood mononuclear cells (PBMCs), but also by neu-</p><p>rons, microglia, and astrocytes. LPS recognition induces an</p><p>immune response that proceeds through activation of nucle-</p><p>ar factor κB (NFκB) and mitogen activated protein kinases</p><p>(MAPK), which eventually activate the production of pro-</p><p>inflammatory cytokines (PICs). In a pilot study we found</p><p>that depression is characterized by higher IgA and IgM-</p><p>mediated immune responses directed against the LPS of</p><p>some commensal gut bacteria, such as Hafnia alvei, Pseudo-</p><p>monas aeruginosa, Morganella morganii, Pseudomonas putida,</p><p>Citrobacter koseri, and Klebsiella pneumoniae, suggesting that</p><p>depression is accompanied by increased bacterial transloca-</p><p>tion (Maes et al., 2008).</p><p>Under normal conditions, the immune system is not</p><p>primed against these gram negative commensals because</p><p>the systemic immune system is geographically and function-</p><p>ally separated from mucosal defenses. The mucosal and tight</p><p>junction barrier and mesenteric lymph nodes (MLNs) segre-</p><p>gate enterobacteria from the interstitium (Macpherson and</p><p>Uhr, 2004). However, when the tight junction barrier is loos-</p><p>ened, poorly invasive enterobacteria may exploit lipid raft-</p><p>mediated transcytotic pathways or enlarged spaces between</p><p>the epithelial cells to cross the gut wall from the epithelial</p><p>mucosa into the lamina propria and the MLNs (Berg and</p><p>Garlington, 1979; Chavez et al., 1999; Clark et al., 2005;</p><p>Wiest and Garcia-Tsao, 2005; Yang et al., 2003). In the</p><p>MLNs, a site of antigen presentation, the “translocated” com-</p><p>mensal enterobacteria can both prime and activate immune</p><p>cells. This phenomenon may explain why the gut may be-</p><p>come an organ that produces inflammatory mediators even</p><p>in the absence of an inflammatory lesion. Moreover, if there</p><p>are defects in the local or systemic immune defenses the</p><p>translocated gram-negative bacteria may spread systemically</p><p>(Wiest and Garcia-Tsao, 2005). There is now evidence that</p><p>this mechanism, called leaky gut, may drive gut-derived in-</p><p>flammation (Wischmeyer, 2006). For example, in patients</p><p>undergoing abdominal surgery and in thermally injured pa-</p><p>tients, treatment of leaky gut decreases serum LPS concentra-</p><p>tions and signs of CMI activation (Quan et al., 2004</p><p>Zhou et al.,</p><p>2003), the latter being a potent driver of oxidative stress and</p><p>mitochondrial dysfunction; both being well described in de-</p><p>pression (Galley, 2011; Scaglia, 2010). On the other hand,</p><p>the inflammatory and oxidative processes in depression may</p><p>cause injuries to the tight junction barrier and thus induce in-</p><p>creased gut permeability with bacterial translocation (Chavez</p><p>et al., 1999; Clark et al., 2005; Yang et al., 2003). The latter</p><p>mechanism, even when secondary to depression, may inten-</p><p>sify and perpetuate the primary inflammatory response and</p><p>thus contribute to the pathophysiology of depression.</p><p>The aim of the present study is to replicate the results of</p><p>our earlier pilot study showing increased immune responses</p><p>against the LPS of gram negative bacteria in a larger study</p><p>sample of depressed individuals. The specific aims are to</p><p>examine a) whether depression is characterized by increased</p><p>IgM and IgA levels against the LPS of 6 enterobacteria</p><p>(H. alvei, P. aeruginosa, M. morganii, P. putida, C. koseri, and</p><p>K. pneumoniae); b) whether this phenomenon is related to</p><p>the staging or subtype of depression, i.e. chronicity of depres-</p><p>sion, recurrent or melancholic depression; and c) the associ-</p><p>ations between increased IgM/IgA responses and the fatigue</p><p>and somatic symptoms of depression.</p><p>2. Subjects and methods</p><p>2.1. Subjects</p><p>One hundred forty subjects participated in the study (112</p><p>depressed patients and 28 normal volunteers). The depressed</p><p>patients were admitted to the Maes Outpatient Clinics,</p><p>Belgium. They were classified according to the DSM-IV-R di-</p><p>agnostic criteria (American Psychiatric Association, 2000)</p><p>and complied with the diagnostic criteria of “major depres-</p><p>sion” or “major depression with melancholia”. The diagnostic</p><p>criteria were assessed by senior psychiatrists employing a</p><p>semistructured interview based on the SCID-I, research ver-</p><p>sion. Subsequently, the depressed patients were divided in</p><p>subgroups, i.e. major depression with melancholic features;</p><p>chronic depression, i.e. duration>2 year; and recurrent de-</p><p>pression, i.e. number of previous depressive episodes, which</p><p>was dichotomized into those with >2, >3, >4 and >5 de-</p><p>pressive episodes. Normal controls were laboratory personnel</p><p>or their family members. Both controls and depressed pa-</p><p>tients belonged to the higher middle class in the Benelux</p><p>countries, where differences between social classes are mini-</p><p>mal. Subjects with other medical illness, e.g. inflammatory or</p><p>autoimmune disorders, COPD, diabetes, etc., and with actual</p><p>and life-time diagnosis of other psychiatric DSM-IV-TR disor-</p><p>ders, e.g. schizophrenia, substance use disorders and organic</p><p>mental disorders, were excluded. We also excluded subjects</p><p>who had been taking psychotropic drugs during the last year</p><p>prior to the studies and those who were treated with mood</p><p>stabilizers; immunomodulatory drugs, including glucocorti-</p><p>coids; statins; β-blockers; and antioxidant supplements, and</p><p>57M. Maes et al. / Journal of Affective Disorders 141 (2012) 55–62</p><p>subjects with alcohol abuse and smoking. Subjects with acute</p><p>inflammatory and allergic reactions for at least 1 month prior</p><p>to the study were not allowed to participate. Healthy controls</p><p>were included if they did not suffer from an axis-I DSM-IV-R</p><p>diagnosis. All subjects gave written informed consent after</p><p>the study protocol was explained. The study was approved</p><p>by the local ethical committee.</p><p>2.2. Methods</p><p>Severity of depression was measured using the Hamilton</p><p>Depression Rating Scale (HDRS) (Hamilton, 1960). The sever-</p><p>ity of fatigue and somatic symptoms was measured with the</p><p>Fibromyalgia and Chronic Fatigue (FF) Syndrome Rating</p><p>Scale (Zachrisson et al., 2002). This scale measures 12 items</p><p>reminiscent for chronic fatigue and fibromyalgia: pain,</p><p>muscular tension, fatigue, concentration difficulties, failing</p><p>memory, irritability, sadness, sleep disturbances, autonomic</p><p>disturbances, gastro-intestinal symptoms, headache, and sub-</p><p>jective experience of infection. The total sum of the FF scale is</p><p>used as an index for severity of the fatigue and somatic symp-</p><p>toms in depression (Maes, 2009a).</p><p>Fasting blood was collected between 8.30 a.m. and</p><p>11.30 a.m. for the assay of serum IgA and IgM-mediated re-</p><p>sponses directed against the LPS of commensal bacteria</p><p>(Hafnia alvei, P. aeruginosa, M. morganii, P. putida, C. koseri,</p><p>and K. pneumoniae). An indirect ELISA method was employed</p><p>to assay the IgM and IgA responses against the LPS of the</p><p>commensal bacteria (GEMAC-IDRPHT, The Ultimate Biophar-</p><p>maceuticals, Talence, France). Each plasma sample was mea-</p><p>sured in duplicate and tested simultaneously with three</p><p>standard dilutions of a known antibody containing serum.</p><p>The optical densities (OD) of the three standards are</p><p>expressed as Z values and from this the reference linear</p><p>curve is calculated. The Z ratio equals: the optical density</p><p>(OD) of the patients serum minus the OD of control serum</p><p>assayed at the same time and in the same microplate divided</p><p>by the standard deviation of the ODs of the control samples.</p><p>The analytical interassay CV values were b6%. We computed</p><p>the peak values of the 6 IgA (peak IgA) and 6 IgM (peak</p><p>IgM) responses as indices of the overall severity of IgA and</p><p>IgM-mediated immune responses, respectively. We comput-</p><p>ed the peak values of the 12 IgM and IgA responses (peak</p><p>IgA/IgM) as an overall index of bacterial translocation. We di-</p><p>chotomized the peak IgA and peak IgM responses and the</p><p>peak IgA/IgM values using a threshold of >3.0 SDs in order</p><p>to delineate patientswith increased IgA versus IgM responses,</p><p>and increased bacterial translocation.</p><p>2.3. Statistics</p><p>We used analyses of variance (ANOVA) or covariance</p><p>(ANCOVA) to ascertain group mean differences. The Dunn–</p><p>Scheffe test was used to check multiple comparisons among</p><p>treatment means (with Bonferroni p-correction for multiple</p><p>testing). Associations between two classification systems,</p><p>e.g. diagnosis and gender, were assessed by means of ana-</p><p>lyses of Contingence Tables (χ2-test). Relationships between</p><p>the biomarkers and clinical data, e.g. the HDRS and FF rating</p><p>scale, were checked with Pearson's product moment correla-</p><p>tion coefficients, Spearman's rank order correlations, and</p><p>regression analyses. The diagnostic performance of the bio-</p><p>markers was ascertained with receiver operating characteris-</p><p>tics (ROC) analysis with computation of the area under the</p><p>ROC curve, sensitivity, specificity and predictive value of a</p><p>positive test result (PV+) with κ statistics. The significance</p><p>was set at α=0.05 (two tailed).</p><p>3. Results</p><p>There were no significant differences in the male/female</p><p>ratio between depressed patients (53/59) and normal controls</p><p>(10/18; χ2=1.2, df=1, p=0.3). There were no significant</p><p>gender differences in any of the IgMor IgA-mediated responses</p><p>directed against LPS of the 6 commensals. There were no sig-</p><p>nificant differences in age between depressed patients (mean</p><p>age=44.2±11.4 years) and normal controls (mean age=</p><p>39.6±11.4 years; F=3.7, df=1/138, p=0.053). There were</p><p>no significant relationships between age and serum IgM or</p><p>IgA levels directed against the LPS of the different commensals.</p><p>Nevertheless, we have adjusted the intergroup differences in</p><p>the IgMand IgA responses for possible effects of age and gender</p><p>by entering age as a covariate and gender as second group in</p><p>ANCOVAs.</p><p>Table 1 shows that the IgM responses to H. alvei, P. aerugi-</p><p>nosa, M. morganii and P. putida were significantly higher in</p><p>depressed patients than in controls. There were no significant</p><p>differences in the IgM responses against C. koseri and</p><p>K. pneumoniae between depressed patients and controls.</p><p>ANCOVAs showed that the IgA responses directed against</p><p>H. alvei, P. aeruginosa,M. morganii and K. pneumoniaewere sig-</p><p>nificantly higher in depressed patients than in controls, while</p><p>there were no significant differences in the IgA responses to</p><p>P. putida and C. koseri between both groups. ANCOVAs showed</p><p>that the peak IgM and IgA responses were significantly higher</p><p>in depressed patients than</p><p>in controls. ROC analysis performed</p><p>on the peak IgM and IgA values showed that the areas under</p><p>the ROC curve (AUC) were 69.1% and 74.2%, respectively.</p><p>The diagnostic performance of the peak IgM values was mar-</p><p>ginally significant; using a cut off value>3.0 Z the diagnostic</p><p>performance was: sensitivity=69.1%, specificity=74.2%, and</p><p>PV+=89.2% (κ=0.16, t=2.08, p=0.04). The diagnostic per-</p><p>formance of the peak IgA values was significant: using a cut off</p><p>value of peak IgA>3.0 Z we found sensitivity=56.3%, specific-</p><p>ity=75.0%, and PV+=90.0% (κ=0.20, t=2.42, p=0.02).</p><p>The AUC of the peak IgA/IgM values was 81.1% and the</p><p>diagnostic performance when using a threshold value>3.0</p><p>SD was: sensitivity=62.5%, specificity=85.7% and PV+=</p><p>85.7% (κ=0.32, t=3.89, p=0.0004). At a threshold</p><p>value>3.5 Z we found: sensitivity=56.2%, specificity=96.4%,</p><p>and PV+=98.4% (κ=0.32, t=4.16, p=0.0002).</p><p>Table 2 shows the peak IgM and IgA values in subgroups</p><p>of depressed patients. Shown are the results of ANCOVAs</p><p>with age as covariate and gender as second factor. Dunn</p><p>Sheffe test (used at p=0.0166) showed that the peak IgM,</p><p>but not IgA, responses were significantly higher in depressed</p><p>patients with chronic depression versus those without. There</p><p>were significant differences in the IgM responses between</p><p>patients with chronic depression and normal controls</p><p>(t=4.23, p=0.0001), but not between depressed patients</p><p>with a duration of illness b2 year and controls (t=1.92,</p><p>p=0.05). There were no significant differences in the peak</p><p>Table 1</p><p>Measurements of serum IgM and IgA levels against the LPS of Hafnia alvei, Pseudomonas aeruginosa, Morganella morganii, Pseudomonas putida, Citrobacter koseri</p><p>and Klebsiella pneumoniae in 28 normal controls and 112 depressed patients.</p><p>Variables Response Normal controls Depression F p</p><p>Hafnia alvei IgM</p><p>IgA</p><p>−0.52 (0.9)</p><p>−0.89 (0.67)</p><p>0.45 (1.71)</p><p>0.03 (1.82)</p><p>9.6</p><p>7.8</p><p>0.003</p><p>0.006</p><p>Pseudomonas aeruginosa IgM</p><p>IgA</p><p>−0.36 (0.98)</p><p>−0.55 (1.09)</p><p>0.61 (1.63)</p><p>0.41 (1.74)</p><p>9.4</p><p>11.2</p><p>0.003</p><p>0.001</p><p>Morganella morganii IgM</p><p>IgA</p><p>−0.15 (0.88)</p><p>−0.86 (0.78)</p><p>0.66 (2.13)</p><p>0.12 (2.05)</p><p>4.5</p><p>8.0</p><p>0.03</p><p>0.005</p><p>Pseudomonas putida IgM</p><p>IgA</p><p>−0.21 (0.85)</p><p>−0.62 (0.95)</p><p>0.69 (1.76)</p><p>0.27 (2.27)</p><p>8.1</p><p>2.7</p><p>0.005</p><p>0.09</p><p>Citrobacter koseri IgM</p><p>IgA</p><p>−0.26 (0.98)</p><p>−0.63 (0.67)</p><p>0.42 (1.79)</p><p>0.38 (2.80)</p><p>3.6</p><p>1.4</p><p>0.06</p><p>0.2</p><p>Klebsiella pneumoniae IgM</p><p>IgA</p><p>−0.49 (0.74)</p><p>−0.91 (1.29)</p><p>0.06 (1.72)</p><p>0.42 (2.62)</p><p>2.4</p><p>8.1</p><p>0.11</p><p>0.005</p><p>Peak responses IgM</p><p>IgA</p><p>0.37 (0.86)</p><p>0.07 (1.10)</p><p>1.74 (2.39)</p><p>2.22 (3.17)</p><p>10.2</p><p>15.3</p><p>0.002</p><p>0.0003</p><p>Peak IgM or IgA response – 0.67 (0.91) 3.35 (3.13) 27.1 0.00002</p><p>All results are expressed in standard deviations and are shown as mean (±SD).</p><p>All results of ANCOVAs (df=1/136) with age as covariate and gender as second factor.</p><p>58 M. Maes et al. / Journal of Affective Disorders 141 (2012) 55–62</p><p>IgA or peak IgM responses between patients with melanchol-</p><p>ic versus those with simple major depression. There were no</p><p>significant differences between patients who suffered from</p><p>more than >2, >3, >4 and >5 depressive episodes than</p><p>those who did not (Table 2 shows the differences between</p><p>patients who suffered from >3 versus b3 episodes). There</p><p>was a non-significant trend towards lower peak IgA, but not</p><p>IgM, responses in patients who had been treated with antide-</p><p>pressants versus those who were drug free (p=0.08).</p><p>In all subjects combined, there was a significant albeit weak</p><p>correlation between the peak IgA and peak IgM responses</p><p>(r=0.25, p=0.004, n=140). In controls (r=0.23, p=0.2,</p><p>n=28) and in depressed patients (r=0.09, p=0.7, n=112)</p><p>no significant correlations could be found between the peak</p><p>IgA and peak IgM responses. There were no significant correla-</p><p>tions between the HDRS and the peak IgM (r=−0.09, p=0.6)</p><p>and IgA (r=−0.04, p=0.7) responses. There were no signifi-</p><p>cant correlations between the FF score and the peak IgM</p><p>(r=0.09, p=0.7) and IgA (r=0.04, p=0.6) responses. There</p><p>was a significant correlation between item 10 on the FF scale</p><p>Table 2</p><p>Measurements of serum peak IgM and IgA responses directed against the LPS of 6 d</p><p>morganii, Pseudomonas putida, Citrobacter koseri and Klebsiella Pneumoniae, in 112 de</p><p>i.e. >2 year duration; depressed patients who experienced more than three episode</p><p>antidepressants (ADs).</p><p>Peak response Depression</p><p>IgM</p><p>IgA</p><p>Durationb2 years (n=77)</p><p>1.30 (1.95)</p><p>2.13 (2.99)</p><p>IgM</p><p>IgA</p><p>Number episodesb3 (n=50)</p><p>1.49 (1.88)</p><p>2.87 (3.72)</p><p>IgM</p><p>IgA</p><p>Simple major depression (n=84)</p><p>1.87 (2.46)</p><p>2.18 (3.12)</p><p>IgM</p><p>IgA</p><p>Without ADs (n=72)</p><p>1.69 (1.59)</p><p>2.60 (3.35)</p><p>All results are expressed in standard deviations and are shown as mean (±SD).</p><p>All results of ANCOVAs (df=2/133) with normal controls and the 2 depressive sub</p><p>Shown are the results of t-test (Dunn–Sheffe test, performed at p=0.0166) testing</p><p>(gastro-intestinal symptoms) and the peak IgA responses</p><p>(r=0.35, p=0.0007). No other correlations between peak</p><p>IgA and any other FF scale itemswere found. Therewere no sig-</p><p>nificant associations between any of the FF item scores and the</p><p>peak IgM values.</p><p>4. Discussion</p><p>The first major finding of this study is that depression is</p><p>accompanied by increased IgA and IgMmediated immune re-</p><p>sponses directed against the LPS of gram negative, commen-</p><p>sal bacteria. These findings confirm those of our previous</p><p>pilot study (Maes et al., 2008). As explained in the introduc-</p><p>tion, these findings point towards a loss of integrity of the in-</p><p>testinal barrier function whereby gram negative commensal</p><p>enterobacteria are no longer contained by the tight junction</p><p>barrier and thus may be translocated from the epithelial mu-</p><p>cosa into the lamina propria and the MLNs. In the MLNs the</p><p>gram negative commensals may activate the PBMCs to be-</p><p>come inflammatory cells. If only the IgA responses directed</p><p>ifferent enterobacteria, i.e. Hafnia Alvei, Pseudomonas aeruginosa, Morganella</p><p>pressed patients subdivided into different subgroups, i.e. chronic depression</p><p>s; patients with melancholia; and depressed patients who were treated with</p><p>Subtype t p</p><p>Duration>2 years (n=35)</p><p>2.70 (2.94)</p><p>2.43 (3.56)</p><p>3.19</p><p>0.52</p><p>0.002</p><p>0.6</p><p>Number episodes>3 (n=62)</p><p>1.94 (2.72)</p><p>1.69 (2.55)</p><p>0.65</p><p>1.80</p><p>0.5</p><p>0.07</p><p>Melancholic depression (=28)</p><p>1.33 (2.14)</p><p>236 (3.37)</p><p>1.35</p><p>0.30</p><p>0.2</p><p>0.8</p><p>ADs (n=40)</p><p>1.83 (2.00)</p><p>1.56 (2.73)</p><p>0.79</p><p>1.76</p><p>0.6</p><p>0.08</p><p>groups as first treatment, gender as second treatment, and age as covariate</p><p>the differences between the two depressive subgroups.</p><p>,</p><p>.</p><p>59M. Maes et al. / Journal of Affective Disorders 141 (2012) 55–62</p><p>to LPS would be increased it could be concluded that the IgA-</p><p>mediated immune response is confined to the mucosal im-</p><p>mune system (Fanous et al., 2007). However, the increased</p><p>IgA levels together with increased IgM responses indicate</p><p>that a systemic immune response is mounted against the</p><p>LPS from various gram-negative bacteria. This may indicate</p><p>that the gram negative commensals have spread beyond the</p><p>MLNs into the systemic circulation.</p><p>Increased bacterial translocation could play a role in the</p><p>inflammatory pathophysiology of depression. It is well</p><p>known that bacterial translocation causes acute inflammato-</p><p>ry responses and contributes to subchronic inflammation</p><p>(Manukyan et al., 2008). For example, in abdominal postop-</p><p>erative patients increased gut permeability is a cause of sys-</p><p>temic inflammation, while an attenuation of leaky gut is</p><p>accompanied by reduced systemic inflammation (Quan</p><p>et al., 2004). A new theory has been put forward explaining</p><p>the role of the intestinal barrier in the onset of extra-</p><p>intestinal inflammatory-autoimmune disorders (Fasano and</p><p>Shea-Donohue, 2005). Elevated IgA or IgM antibodies against</p><p>commensal microbiota occur in inflammatory disorders, such</p><p>as multiple sclerosis, ankylosing spondylitis, alcohol-induced</p><p>liver disease and chronic fatigue syndrome (Geffard et al.,</p><p>2002; Maes et al., 2007; Nishimura et al., 2001; Tani et al.,</p><p>1997).</p><p>Since LPS is known to elicit depressive-like behaviors it is</p><p>tempting to speculate that the translocated LPS of commen-</p><p>sal bacteria is involved. LPS administration elicits anhedonia</p><p>and suppresses</p><p>food consumption, body weight, social inter-</p><p>action and activity in the open-field test (Bluthé et al., 1994;</p><p>De La Garza et al., 2005; Plata-Salamán and Borkoski, 1993;</p><p>Yirmiya, 1996). LPS is also able to induce anxiety-like behav-</p><p>iors, including reduced exploratory behavior and social inter-</p><p>actions (Engeland et al., 2003). Of course, it is difficult to</p><p>differentiate LPS-induced depressive- and anxiety-like be-</p><p>haviors from LPS-induced sickness behavior (Swiergiel and</p><p>Dunn, 2007). Nevertheless, Frenois et al. (2007) were able</p><p>to show that there is a dissociation between LPS-induced</p><p>sickness behavior and LPS-induced depressive-like behaviors.</p><p>Different pathways may translate the systemic inflammatory</p><p>responses induced by bacterial translocation to the brain:</p><p>1) signals leading to microglial activation, e.g. proceeding</p><p>through the vagus nerve; and 2) the “neuraxes” that translate</p><p>information about the body's internal state to brain areas that</p><p>coordinate anxiety, dysphoria, arousal, vigilance, appetite,</p><p>coping with stressors, etc. (Maes et al., 2011a). Another</p><p>mechanisms are direct stimulation of the blood–brain-barrier</p><p>endothelium by circulating bacteria and/or their toxins re-</p><p>leasing pro-inflammatory and oxidative signals that affect</p><p>the underlying neuropil and neuronal functions (Grab et al.,</p><p>2011). For example, systemic LPS results in rapid increases</p><p>in brain TNFα levels, which may remain elevated for</p><p>10 months (Qin et al., 2007). LPS instillations in the nasal</p><p>cavities elicit transcriptional regulation of TNFα in different</p><p>brain regions (Tonelli et al., 2008). Moreover, in depression</p><p>there could be an interaction between increased LPS translo-</p><p>cation and the effects of psychological stressors on TLR ex-</p><p>pression. Thus, social stress elicits higher TLR expression on</p><p>macrophages (Bailey et al., 2007), while chronic unpredict-</p><p>able stress aggravates LPS-induced NFκB activation in the</p><p>hippocampus and frontal cortex (Munhoz et al., 2004). A</p><p>better behavioral outcome and lower inflammatory re-</p><p>sponses in the brain in response to immobilization stress</p><p>are observed in TLR4-deficient mice (Caso et al., 2008).</p><p>There is a new hypothesis relating a variety of microor-</p><p>ganisms to chronic inflammatory disorders that are increas-</p><p>ing rapidly in incidence in rich developed countries (Raison</p><p>et al., 2010; Rook and Lowry, 2008). Urbanization-induced</p><p>loss of exposure to the immunoregulatory effects of these</p><p>“old friend” microorganisms decreases tolerance to a wide</p><p>array of potentially proinflammatory stimuli. This mecha-</p><p>nism increases the vulnerability of mounting inappropriate</p><p>inflammatory reactions to gut commensals and therefore</p><p>promotes depression in vulnerable individuals by enhancing</p><p>the production of depressogenic cytokines (Raison et al.,</p><p>2010; Rook and Lowry, 2008). As such, the urbanization-</p><p>induced changes in microbiota and the consequent immuno-</p><p>regulatory deficits may lead to increased rates of depression</p><p>and other immune disorders in industrial countries.</p><p>Nevertheless, the major question is whether the increased</p><p>IgM and IgA antibody levels, which indicate bacterial transloca-</p><p>tion, are cause or consequence of depression. There is some ev-</p><p>idence that depression and also psychological stress, which can</p><p>be secondary to depression, may cause increased gut perme-</p><p>ability and thus bacterial translocation via inflammation and</p><p>oxidative stress, activation of the hypothalamic-pituitary-</p><p>adrenal (HPA) axis, etc. Psychological stressors may cause</p><p>increased gut permeability with an increased uptake of poten-</p><p>tially noxious stimuli, including antigens (Ferrier, 2008;</p><p>Söderholm and Perdue, 2001). Firstly, systemic inflammation</p><p>through an increased production of PICs, such as IL-1, IL-6,</p><p>TNFα, and IFNγ, aggravates the loss of epithelial barrier func-</p><p>tions (Chavez et al., 1999; Clark et al., 2005; Yang et al.,</p><p>2003). The PIC-induced loosening of the tight junction barrier</p><p>is mediated by cell signaling networks, e.g. NFκβ activation</p><p>(Al-Sadi and Ma, 2007; Ma et al., 2005; Ye et al., 2006). More-</p><p>over, increased reactive oxygen species and lipid peroxidation</p><p>may also weaken the mucosal barrier (Alptekin et al., 1996).</p><p>Secondly, depression, is accompanied by HPA-axis activation,</p><p>including increased levels of corticotropin-releasing hormone</p><p>(CRH) and glucocorticoids (Plotsky et al., 1998). CRH is an im-</p><p>portant mediator of stress responses in the brain-gut axis</p><p>(Fukudo, 2007). CRH as well as CRH1-receptor (CRH-R1) and</p><p>CRH-R2 mRNAs have been detected in human colonic mucosa</p><p>(Fukudo, 2007). CRH is known to regulate macromolecular</p><p>permeability in human colonic mucosa via CRH-R1 and CRH-</p><p>R2 receptor subtypes (Wallon et al., 2008). Increased glucocor-</p><p>ticoid levels may also weaken the mucosal barrier (Meddings</p><p>and Swain, 2000). Thirdly, barrier dysfunctions may be caused</p><p>by other depression or stress-related mechanisms, e.g. cholin-</p><p>ergic mechanisms (Castagliuolo et al., 1996) and activated</p><p>mast cells (Santos et al., 2000). Fourthly, there are also data</p><p>showing that stress can alter gut microbiota and that distur-</p><p>bances of the gut microbiota can alter brain function and</p><p>behavior. For example, stressors reduce the number of Lactoba-</p><p>cilli and increase the mucosal uptake of gram-negative patho-</p><p>gens, e.g. Pseudomonas (Lutgendorff et al., 2008). Alterations</p><p>in the composition of intestinal microbiotamay change explor-</p><p>atory behavior in association with changes in hippocampal</p><p>levels of brain-derived neurotropic factor (BDNF) (Bercik</p><p>et al., 2011). Germ-free mice show alterations in stress-</p><p>responsivity and behaviors indicating anxiolytic activities in</p><p>60 M. Maes et al. / Journal of Affective Disorders 141 (2012) 55–62</p><p>association with changes in 5-HT1A receptors and BDNF in</p><p>the brain (Cryan and O'Mahony, 2011; Neufeld et al., 2011).</p><p>Lactic acid bacteria, such as Lactobacillus rhamnosus, attenuate</p><p>stress-induced anxiety- and depression-related behaviors</p><p>(Bravo et al., 2011). However, even if the bacterial translocation</p><p>might be secondary to depression, the increased gut perme-</p><p>ability may amplify the inflammatory and CMI responses thus</p><p>aggravating the immune pathophysiology of depression.</p><p>The above pathways may also explain the comorbidity of</p><p>depression with medical disorders that are known to be asso-</p><p>ciated with the onset of depression (Maes et al., 2011a) and</p><p>are characterized by increased bacterial translocation. Exam-</p><p>ples are Parkinson's (PD) disease; multiple sclerosis; cardio-</p><p>vascular disorder; chronic fatigue syndrome; rheumatoid</p><p>arthritis; inflammatory bowel disease (IBD); psoriasis; and</p><p>HIV infection (Arai et al., 2004; Charalambous et al. 2007;</p><p>Gabrielli et al., 2011; Geffard et al., 2002; Gyurcsovics and</p><p>Bertók, 2003; Kamer et al., 2008; Krack et al. 2005; Lim</p><p>et al., 1993; Maes et al., 2007; Ochoa-Repáraz et al., 2011;</p><p>Sandek et al., 2007; 2008; Shanahan, 1994; Sundqvist et al.,</p><p>1982). The increased bacterial translocation that accom-</p><p>panies these medical disorders may amplify the inflammato-</p><p>ry pathways that may underpin comorbid depression.</p><p>Moreover, many causes of increased gut permeability may –</p><p>in theory – induce “secondary depression”, e.g. alcoholism,</p><p>chemotherapeutic agents, radiation, surgery, trauma, use of</p><p>non-steroid anti-inflammatory drugs, prolonged use of anti-</p><p>biotics, etc. (Maes, 2009b).</p><p>The second major finding of this study is that chronic de-</p><p>pression is associated with increased IgM, but not IgA, re-</p><p>sponses to gram negative bacteria. Therefore it may be</p><p>hypothesized that bacterial translocation may play a role in</p><p>the pathophysiology of chronic depression. For example, bac-</p><p>terial translocation may cause progressive amplifications of</p><p>the inflammatory pathways that are involved in depression.</p><p>A second pathway is that exposure to chronic depression in-</p><p>creases the likelihood to develop a secondary weakening of</p><p>the tight junction barrier and thus consequent gut-derived</p><p>inflammation which may further amplify the pathophysiolo-</p><p>gy of depression.</p><p>The third major finding of this study</p><p>is that the IgA re-</p><p>sponses to LPS of commensal bacteria were significantly cor-</p><p>related to one FF symptom, i.e. gastro-intestinal symptoms.</p><p>Overall, the strength of the correlation coefficients was</p><p>much lower than in our previous pilot study (Maes et al.,</p><p>2008). However, in the previous study more severely de-</p><p>pressed patients were included which could have accentuat-</p><p>ed the correlation coefficients. In any case, the significant</p><p>correlations between gastro-intestinal symptoms and the</p><p>IgA-mediated immune responses in both studies suggest</p><p>that the presence of gastro-intestinal symptoms reflect aber-</p><p>rations in gut permeability or the factors that cause leaky gut.</p><p>Examples in depression are small intestine bacterial over-</p><p>growth (SIBO) and gut inflammation (Maes et al., personal</p><p>data).</p><p>Future research on the role of bacterial translocation in de-</p><p>pression should examine the presence of LPS in the blood, and</p><p>inflammation or other ultrastructural changes in the gut of</p><p>depressed patients. For example, excessive extrusion of cell</p><p>cytoplasm and enterocytes, damage to the brush border and</p><p>partial villus atrophy are seen with electron microscopy in</p><p>those with functional bowel symptoms, in children with</p><p>non-specific intestinal symptoms (Poley, 1983). In addition,</p><p>the roles of SIBO and gut inflammation as well as the TLR</p><p>pathways should be examined. In addition, our findings that</p><p>bacterial translocation is correlated with gastro-intestinal</p><p>symptoms suggests that the data have relevance for the path-</p><p>ophysiology of irritable bowel syndrome (IBS). For example,</p><p>the IgA and IgM responses to LPS of commensal bacteria are</p><p>significantly higher in individuals with Myalgic Encephalo-</p><p>myelitis/Chronic Fatigue Syndrome (ME/CFS) with IBS than</p><p>in those without IBS (Maes et al., submitted). In addition,</p><p>the increased IgA and IgM responses to LPS in those individ-</p><p>uals are positively correlated to specific intestinal symptoms</p><p>used by the Rome II criteria for IBS (Thompson et al., 2000),</p><p>e.g. abdominal discomfort or pain relieved with defecation,</p><p>abnormal stool form, abnormal urgency, feeling of incomplete</p><p>bowel movement, and passing mucus during bowel move-</p><p>ment. A study is underway to examine the interrelationships</p><p>between depression, IBS and bacterial translocation.</p><p>In conclusion, this study shows increased IgM and IgA re-</p><p>sponses directed against LPS of commensal enterobacteria in</p><p>depression and, in particular, in chronic depression. These</p><p>findings suggest that in the MLNs, “translocated” commen-</p><p>sals have activated immune cells and/or that they have</p><p>spread into the systemic circulation to elicit IgA and IgM re-</p><p>sponses. 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JPEN Journal of Parenteral and Enteral Nutrition 27 (4),</p><p>241–245.</p><p>Increased IgA and IgM responses against gut commensals in chronic depression: Further evidence for increased bacterial translocation or leaky gut</p><p>1. Introduction</p><p>2. Subjects and methods</p><p>2.1. Subjects</p><p>2.2. Methods</p><p>2.3. Statistics</p><p>3. Results</p><p>4. Discussion</p><p>Role of the funding source</p><p>Conflict of interest</p><p>Acknowledgments</p><p>References</p>