From Immune Activation to Cell Death: Investigating the Pro-inflammatory Phenotype of PBMCs in Major Depressive Disorder

From Immune Activation to Cell Death: Investigating the Pro-inflammatory Phenotype of PBMCs in Major Depressive Disorder | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article From Immune Activation to Cell Death: Investigating the Pro-inflammatory Phenotype of PBMCs in Major Depressive Disorder Krzysztof Pietruczuk, Katarzyna Rückemann-Dziurdzińska, Szymon Bierzanowski, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9336198/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Depression's complex pathogenesis involves neurotransmission disorders, chronic inflammation, and immune dysregulation. Supporting the inflammatory theory, this study analyzed lymphocyte subpopulations and surface molecule expression in patients to investigate immunological mechanisms and provide evidence for this clinical perspective. The study involved twenty-seven patients with newly diagnosed depression and ten healthy volunteers. Level of T-lymphocyte subpopulations, B-lymphocytes and NK-cells, as well as the expression of activation markers (CD25, CD69), inflammatory markers (TLR-4), apoptosis markers (CD95, TRAIL-R1) and maturity markers (CD10, CD40, CD5). A reduced proportion of NK-cells was observed in patients. An increase in number of immature B-cells CD19 + CD10+ and CD19 + CD5+ cells was observed, accompanied by a decrease in CD40 expression. TLR-4 expression on CD4 + and CD19 + lymphocytes in patients was more than twice as high compared with the group of healthy volunteers. There was also an increase in the expression of the activation markers CD25 and CD69 on T-lymphocytes, and CD95 and TRAIL-R1 on T and B-lymphocytes. Some parameters correlated with severity of depressive symptoms. The results confirm that chronic inflammation influences depression through changes in lymphocytes. This suggests that immunomodulatory and anti-inflammatory treatments should be implemented as an important adjunct to standard clinical management of this condition. Health sciences/Diseases Biological sciences/Immunology depression disorders inflammation lymphocyte TLR-4 apoptosis activation markers Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Depression is one of the most common mental health conditions. In 2019, around 280 million people worldwide were suffering from depression. Depending on the country, this accounted for 2–6% of the population 1 . The Covid-19 pandemic has also taken its toll, and depression is now one of the most pressing health and economic issues worldwide 2 , 3 . According to current scientific understanding, depression is a systemic disorder. Its symptoms go beyond psychological aspects and include disturbances in the regulation of the nervous, endocrine and immune systems 4 , 5 . Unfortunately, the exact cause of depression remains unknown, although several theories have emerged, each of which sheds some light on the complex interactions between the psychological, social and biological factors that contribute to the development of depressive disorders. The best known of these include the monoamine theory, the inflammatory theory and the theory of HPA axis dysregulation. In recent years, the theory of gut-brain axis dysregulation has been added to these. The best-known and most widely accepted of these is the monoamine theory of depression, which posits that there is a reduced concentration of neurotransmitters such as dopamine, serotonin and norepinephrine in the synaptic cleft 6 , 7 . It has also been demonstrated that depression is accompanied by inflammation, with changes in certain parameters of the immune system observed in patients. Elevated white blood cell counts, accelerated erythrocyte sedimentation rate 8 , elevated CRP levels 8 , 9 and increased levels of inflammatory markers such as IL-1beta, IL-6, TNF-alpha, IL-8, IL-16, as well as numerous chemokines in the blood and cerebrospinal fluid 8 , 10 have been observed. All of these constitute elements of the inflammatory theory of depression. In turn, the involvement of the neurohormonal system – such as the HPA axis – in the stress response has led to the formulation of theories regarding its role in the development of depressive symptoms 11 . This involvement manifests itself through the effect of elevated cortisol levels on mood regulation, as demonstrated in studies involving patients and in observations regarding the increased frequency of depressive episodes in individuals undergoing cortisol therapy 12 . Numerous studies point to the potential involvement of the gut-brain axis in the pathophysiology of depression, as it mediates complex interactions between the gut microbiota, the immune system and the brain 13 . Components of the gut microbiota may influence brain function and behaviour by producing various metabolites, neurotransmitters and signalling molecules, which also act directly on the vagus nerve 14 . Studies on the functioning of the gut-brain axis in patients with depression have indicated the presence of dysbiosis 15 . These studies have contributed to the formulation of a further theory regarding the causes of depression, namely the theory of gut-brain axis dysregulation. Despite numerous studies conducted over the years, the pathophysiology of depression is still not fully understood. Research findings and emerging theories point to a complex interplay of many factors; however, the core principles of the monoamine theory remain a key reference point in the search for new targets for pharmacotherapy. Based on the monoamine theory, new antidepressants are being developed that act on neurotransmitter systems such as serotonin, noradrenaline and dopamine, with the aim of increasing the concentration of these substances in the synaptic cleft 16 , 17 . However, patients often exhibit high resistance to these drugs 18 , 19 and the therapeutic effect is delayed, with clinical response frequently taking several weeks to achieve 20 . It is suggested that, in the case of a condition with such a multifactorial a etiology as depression, this therapeutic approach is not sufficiently effective. There is ample evidence supporting the validity of the theories mentioned above, which are not mutually exclusive; however, we still do not know which of them is of key importance and is capable of bringing all the others together into a coherent whole. In our study, we focused on the inflammatory theory of depression. However, going beyond classic markers of inflammation, such as cytokines or acute-phase proteins, we sought to determine, by broadening our understanding of the general functioning of the immune system in the course of depression, the potential cause of this condition, as well as the interrelationships and influence of the other theories. Thanks to this holistic view of the pathophysiology of mood disorders, which still harbours many unknowns, we have succeeded in demonstrating the potential for future developments in new, more effective therapies for depressive disorders. Results Analysis of the main lymphocyte subpopulations revealed comparable levels of CD3 + T cells in patients with depression and healthy individuals. In patients, a positive correlation was observed between CD3 + cell levels and the severity of depressive symptoms. This applies to the assessment of their mental state using both the Beck Depression Scale and the Hamilton Depression Rating Scale (Fig. 1A). Patients also exhibited a statistically significant reduction in the number of CD16+/CD56 + NK cells compared to healthy individuals, which was inversely correlated with measures of mental state (Fig. 1B). Analysis of the CD19 + B-lymphocyte population indicates a reduction in the number of these cells in patients with depression (Fig. 2A). No statistically significant association was found between this parameter and disease severity. Analysis of T-lymphocyte subpopulations revealed no statistically significant differences between the numbers of CD4 + and CD3+/CD16+/CD56 + cells in the compared groups. In the case of CD8 + T lymphocytes, a statistically significant difference was observed between the patient group and the healthy control group (Fig. 1C). An analysis was then carried out of the surface markers of CD4+, CD8 + and CD19 + lymphocyte populations. The aim of the study was to identify the characteristic antigens of these cells, the presence of activation markers, the TLR-4 molecule indicating ongoing inflammation in the body, and two markers indicating the cells’ readiness to undergo apoptosis (CD95, TRAIL-R1). Analysis of B-cell surface markers revealed a higher number of CD19 + CD5+ and CD19 + CD10+ cells and a decrease in the level of CD19 + CD40+ cells in patients with depression compared with those in the control group (Fig. 2B). The number of CD19 + lymphocytes expressing CD20, CD22 or Baff-R antigens was comparable in both study groups (Fig. 2B). In patients, a positive correlation was observed between the severity of depression and the presence of CD19 + CD20+ cells (Fig. 2C). An analysis of activation markers was carried out for both CD4 + and CD8 + T cells. In both types of T cells, the expression of TCR (alpha/beta) and CD28 molecules was comparable between patients and the control group. Patients with depression showed statistically significantly higher levels of CD25 + and CD69 + T cells among helper cells, and a positive correlation with the severity of depressive symptoms in both the Beck and Hamilton tests (Fig. 3A and 3B). The number of CD8 + CD25+ cells in patients did not show any statistical differences compared with the control group; however, a positive correlation with the patients’ mental state was observed (Fig. 3C). In the case of the TLR-4 antigen, a significant increase in its expression was observed on CD4 + and CD19 + cells across various lymphocyte populations, with the differences in the patient group being more than twice as high as in the healthy control group (Fig. 4A). The increased activation of lymphocytes and the rise in the proportion of lymphocytes expressing the inflammatory marker (TLR-4) on their surface were also accompanied by an increased susceptibility of the cells to enter the apoptotic pathway. In patients, significantly higher levels of cells expressing the CD95 antigen were observed in the CD8 + and CD19 + populations (Fig. 4B and 4C). TRAIL-R1 expression on T lymphocytes was comparable in both study groups. Only the patients’ B lymphocytes showed increased TRAIL-R1 expression (Fig. 4D). Discussion Investigating the link between depression and the immune system is like putting together a jigsaw puzzle where the number of pieces is unknown. We will show how our findings fit into the overall picture that has already emerged, and in which areas our research provides entirely new insights. The results of our study unequivocally confirm the validity of the inflammatory theory of depression, which, however, many researchers consider solely in the context of elevated levels of pro-inflammatory cytokines such as IL-1, IL-6, TNF and the acute-phase protein CRP 21 – 23 . Advances in and wider availability of cytometric methods in recent years have enabled a more detailed investigation of this phenomenon through the analysis of the main immune cell populations involved in inflammatory processes. An increase in leukocytosis and monocytosis has been reported in patients with depression, and an elevated ratio of both neutrophils to lymphocytes and CD4 + to CD8 + lymphocytes has been observed 24 – 26 In our study, we went a step further: we examined not only the basic PBMC cell populations but also characteristic markers indicating cell activation, their role in promoting inflammation, as well as their readiness for apoptosis. In the main PBMC populations, we found elevated levels of CD8 + lymphocytes; however, with a comparable number of CD3 cells, we did not observe an elevated CD4/ CD8 ratio indicated by Soresen in his meta-analysis, in which he states that in 16 of the 26 studies included in his publication, patients with depressive disorders are characterised by an elevated CD4/CD8 ratio 27 . The group of patients we studied was also characterised by a lower proportion of NK cells with the CD3-/CD16+/CD56 + phenotype compared with healthy individuals. The observed lower proportion of classical NK cells lacking the CD3 molecule on their surface indicates impaired cytotoxic capacity of the patients’ immune system, which may contribute to the chronic inflammation observed in these patients in other studies 28 . Numerous studies have examined the levels and activity of NK cells in patients suffering from depression 29 – 31 . So far, the results are inconclusive. Some studies indicate a significantly lower proportion of cells in this subpopulation among patients with major depressive disorder compared with the control group, and link this finding to the presence of chronic stress, which is thought to lead to a reduction in the cytotoxic activity of NK cells 29 , 30 . Other studies indicate changes in the activity of NK cells, but not in their numbers 31 . However, discrepancies in study results may stem from a number of factors, such as differences in diagnostic criteria, symptom severity or the patients’ treatment status; the distinction between NK cell count and functional activity is of particular importance here. Even if the number of NK cells remains unchanged, patients with depression may have an impaired ability to destroy target cells or produce cytokines 29 . The impaired ability of NK cells to regulate the body’s inflammatory response may be responsible for the disease becoming chronic. The co-occurrence of impaired NK cell function and elevated levels of pro-inflammatory cytokines in people suffering from depression confirms the link between the state of NK cells and the inflammatory theory of depression. On the other hand, when analysing the theory of depression based on HPA axis dysregulation, we also observe certain patterns. The elevated cortisol levels observed in patients with depression may have an inhibitory effect on both the number and function of NK cells 29 . Immune disorders caused by inflammation and hormonal imbalances, such as HPA axis dysregulation 32 may be the cause of the increase in the proportion of CD19 + CD10+ cells that we have observed. The CD10 antigen is found mainly on immature B lymphocytes and disappears as they mature 33 , 34 . Inflammation, which is characteristic of depression and chronic stress, can disrupt the maturation and differentiation of B cells in the bone marrow, leading to the release of a greater number of immature B cells into the peripheral circulation 35 . The increase in CD19 + CD10+ cells is not the only difference in B-cell subpopulations that we observed. We also observed an increase in the CD19 + CD5+ B-cell subpopulation. CD5 + B-lymphocytes constitute a population with regulatory potential and are involved in both normal immune responses and autoimmunity 36 . Changes in the number of these cells may therefore have a significant impact on immune homeostasis in the course of depression. The disruption of immune homeostasis in patients with depression is also indicated by the reduction in the proportion of B-cell subpopulations expressing the CD40 molecule on their surface, as demonstrated in our study. The presence of this molecule is of crucial importance for B cells, particularly in the context of their T-cell-mediated activation 37 . The interaction between the CD40 receptor and its ligand CD154, which is present on activated T cells, is crucial for the humoral immune response. Abnormalities in CD40 receptor expression can disrupt these interactions and affect antibody production and other effector functions of B cells 38 . This is also of fundamental importance for B-cell proliferation and differentiation, a finding that appears to be indirectly supported by our study, as the reduced proportion of the CD19 + CD40+ population is accompanied by an increased proportion of immature B-cells, i.e. CD19 + CD10+ 38 . Our findings confirm the presence of an inflammatory process in the course of depression. This is indicated both by an increase in the number of cells expressing the activation markers CD25 and CD69 on lymphocytes, and by the presence of the TLR-4 molecule, which is recognised as a marker of inflammation. T-cell activation, which is a key step in triggering an immune response 39 . The regulation of T-cell populations is essential for maintaining immune homeostasis, and apoptosis, or programmed cell death, is a key process in this regulation 40 . It is believed that an imbalance between T-cell activation and regulation, including alterations in normal apoptotic processes, contributes to the chronic inflammation observed in depression. In our study, we observed an increase in the number of CD4 + CD25+ cells. However, no statistically significant differences were found in CD8 + cytotoxic lymphocytes in patients compared with the control group of healthy individuals. The contradictions found in the literature regarding the CD25 activation marker can, however, be explained by the behaviour of the CD25 cell subpopulation, i.e. CD4 + CD25+FoxP3 + Treg lymphocytes, to which the authors of other studies most frequently refer 41 , 42 . It also appears that the presence of inflammation may depend on the subtype of depression. For example, in treatment-resistant depression (TRD), a lower proportion of CD4 + CD25+ cells has been observed, suggesting reduced activation of CD4 lymphocytes in this group of patients 43 . The TLR-4 molecule, which we observed in increased quantities on CD4 + T cells and CD19 + B cells, is responsible for detecting pathogen-associated molecular patterns (PAMPs), primarily lipopolysaccharide (LPS) – a component of the outer membrane of Gram-negative bacteria. Furthermore, this receptor recognises damage-associated molecular patterns (DAMPs), which are released from damaged tissues, thereby also playing a role in inflammatory processes unrelated to infection 44 . This suggests its involvement in aseptic inflammation, which is of significant importance in the context of depression, where the role of aseptic inflammation triggered by stress or cellular damage is increasingly emphasised. Aseptic inflammation, i.e. an immune response triggered by tissue damage in the absence of pathogens, relies heavily on the recognition of DAMPs by TLRs, particularly TLR4. This recognition initiates the production of inflammatory mediators such as TNF-α, IL-1β, IL-6 and IL-8, as well as chemokines 45 , 46 . The presence of TLR-4 on CD4 + lymphocytes may influence their ability to trigger inflammatory responses. Experiments in mice have shown that TLR4 expression increased under conditions of chronic stress, whilst its deficiency protected against stress-induced lymphocyte depletion and disruption of the Th1/Th2 cytokine balance. This indicates the involvement of the TLR-4 molecule in modulating lymphocyte responses under stress, suggesting the key role of this molecule in the pathophysiology of depression 47 . Conversely, the presence of TLR-4 on the surface of B cells is associated with impaired humoral responses and autoimmunity, which may be signalled by the binding of the TCR to LPS, leading in turn to the increased production of pro-inflammatory cytokines observed in depression 48 . The impaired B-cell function mentioned earlier, as demonstrated by our research, and the role of the TLR-4 receptor in inflammatory and autoimmune processes may provide evidence of ongoing chronic inflammation in depression 49 . As chronic inflammation associated with depression may be responsible for increased apoptosis of immune cells, we also investigated this aspect of depression. We assessed two molecules whose presence indicates a greater susceptibility to apoptosis. One of these was the CD95 molecule, which plays a key role in maintaining the body’s immune homeostasis. Studies on the state of the immune system in depression do not yield clear-cut results, as there are also reports of a lower proportion of CD3 + CD8+CD95 + cells in treatment-resistant patients 50 . This discrepancy highlights the complexity of CD95 regulation in depression and may suggest that CD95 expression depends on specific characteristics of the patient population studied, i.e. on the various subtypes of the condition and on the use of specific antidepressants. Apoptosis is also a key mechanism regulating the CD19 + B-cell population, influencing both its size and composition. Apoptosis eliminates B cells that are autoreactive and could potentially lead to autoimmune diseases, or those that have been activated but have already fulfilled their function. Proper regulation of B-cell apoptosis is therefore essential for maintaining immune tolerance and preventing excessive or uncontrolled immune responses 51 , 52 . As we also demonstrated in our study, a decrease in the total number of CD19 + B lymphocytes, accompanied by an increase in the proportion of immature and regulatory subpopulations, suggests that mature B lymphocytes may undergo preferential apoptosis due to increased expression of CD95 and TRAIL-R1, as we observed. In the case of the CD95 molecule, it is suggested that there is a bidirectional relationship between inflammation and apoptosis 53 . The second molecule we studied, TRAIL-R1, is also recognised as a death receptor. It has been shown that the expression of TRAIL-R1 on B cells is not constant, but is modulated during their development and activation. B cells from proliferation centres, which are strongly activated and undergo selection, exhibit higher levels of TRAIL-R1. Furthermore, TRAIL-R1 undergoes rapid overexpression following activation, particularly in memory B cells 54 . The results of our study unequivocally confirm that depression is accompanied not only by an impairment of immune system function, but also by a chronic inflammatory process. Consequently, traditional treatment of depression, based primarily on the monoamine theory and the correction of neurotransmission disorders, may prove insufficient or partially ineffective. Our observations point to the need to incorporate the inflammatory theory into the diagnostic and therapeutic process. Treatment of depression should therefore involve not only the regulation of neurotransmitters, but also the reduction of chronic inflammation and the normalisation of immune dysfunction. Considering anti-inflammatory or immunomodulatory therapies, in parallel with conventional antidepressants, may be crucial for more effective treatment of depression, particularly in cases resistant to standard methods. In the context of our findings, it therefore becomes justified to seek new therapeutic targets that would simultaneously suppress inflammation and restore normal neurotransmission. Despite the promising results, this study has certain limitations which point to avenues for future research. The relatively small size of the study group limits the statistical power of the analyses and makes it difficult to generalise the findings to a wider patient population. Further research involving larger, representative cohorts is essential to validate the observed associations. The high clinical heterogeneity of depression also remains a significant challenge. The data obtained suggest only the existence of a so-called inflammatory biotype in which immune processes play a leading role. This means that the changes described may not occur in all patients, which necessitates rigorous stratification of participants in future studies to precisely identify subgroups with different pathophysiological mechanisms. Focusing solely on the phenotype of immune cells represents only a fragment of the disease’s complex aetiology. To fully confirm the central role of inflammation, an integrative approach is required, combining immunophenotyping with other key theories of depression. A multisystemic approach will allow for a full understanding of the interactions between the immune system and the central nervous system in the context of affective disorders. Materials and methods Patients and healthy volunteers. 27 patients diagnosed with depression (15 female and 12 male; mean age 40,81 ± 6,53) and 10 healthy volunteers (5 female and 5 male; mean age 43,40 ± 7,34) participated in the study. All participants underwent basic physical examination and psychiatric interview in the Adult Psychiatry Clinic of the Medical University of Gdansk. Patients were diagnosed with the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders Fifth Edition (DSM-5) 55 . Mental status of patients with depression and healthy individuals was assessed with two tests: Beck Depression Inventory 56 (mean score for the patients: 30,56 ± 1,45 and for the control group: 2,50 ± 0,97) and Hamilton Depression Rating Scale 57 (mean score for the patients: 39,41 ± 3,41 and for the control group: 3,80 ± 1,03). For all of the patients included into the study it was the first diagnosis of depression and blood was collected before they started any antidepressive treatment. Exclusion criteria for the control group included any diagnose of a mental disorder and/or incidence of mental illness in the family. Exclusion criteria for both patients and control group included: diagnosis of an autoimmune disorder, chronic inflammatory disorder, diabetes or any allergy. None of the study participants declared use of recreational drugs. In both groups an occasional alcohol consumption was reported. The study has been approved by the Bioethical Committee for Scientific Research at the Medical University of Gdansk. All participants were informed about the purpose of the study and gave their informed consent in a written form. Relevant guidelines and regulations were observed throughout the study. Peripheral blood collection. 3 ml of peripheral venous blood was collected from patients and healthy controls after overnight fasting into EDTA tubes for the cytometric analysis of lymphocytes’ subpopulations. Determination of T and B-cell subpopulations. 50 µl blood samples were transferred into cytometric tubes for red blood cell (RBC) lysis with 0,8% NH4Cl and 0,1% KHCO3 buffer and subsequent staining with monoclonal antibodies. After RBC lysis step the cells were washed with PBS (phosphate buffered saline) buffer and stained with the following monoclonal antibodies (all from Becton Dickinson USA). [Table 1 . Fluorochromes used and their corresponding antigens in the cytometric analysis of lymphocyte subpopulations.] Table 1 Fluorochrom Target antigen APC TLR-4 PerCP TLR2, CD4 FITC CD95, CD25, CD20, CD22, CD3, CD5, CD10 PE TRAILR1, CD69, TCRαβ, CD28, BAFFR, CD40, CD16CD56 V450 CD8, CD19 Cells were incubated with antibodies for 30 min at 4°C in the dark, washed with PBS and suspended in 200 µl of PBS for flow cytometry analysis. Cytometric analysis. Quantitative fluorescence analysis was performed with FACSVerse (Becton Dickinson, USA). Ten thousand lymphocytes (based on their forward and side scatter gating) were acquired for each sample. Cytometric data were analysed with FlowJo X 10.0.7 (Tree Star; USA). Statistical analysis. Statistical analysis was performed using libraries for data exploration, analysis and visualization available in Python, such as: Pandas, Numpy, Matplotlib, Seaborn and Scikit-learn using Datalore 2023.4 JetBrains. Normally distributed data were analysed using the Student's t-test, while the non-parametric Mann-Whitney test for independent samples was used to analyse non-normally distributed data. Statistical significance was assumed at p < 0.05. Declarations Funding This work was supported by the Polish National Science Centre project “Miniature 2” granted to Krzysztof Pietruczuk and by the statutory funds of the Medical University of Gdansk (02-0058/07/262) granted to Jacek M. Witkowski. Declaration of Compliance with Regulations I hereby declare that all studies were conducted in accordance with the regulations, and that patients provided informed consent to participate in the studies. The study has been approved by the Bioethical Committee for Scientific Research at the Medical University of Gdansk. All participants were informed about the purpose of the study and gave their informed consent in a written form. Relevant guidelines and regulations were observed throughout the study. Statement on Data Availability The dataset used and analysed during the current study are available from the corresponding author on reasonable request. Acknowledgements Cytometric analyses were performed using the instrument acquired within the Network for Imaging of Structural and Functional Pathology of Cells of the University and Medical University of Gdansk. 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Increased CD95 (Fas) and PD-1 expression in peripheral blood T lymphocytes in COVID-19 patients. Br. J. Haematol. 191 , 207–211 (2020). Kim, S. J. et al. CD4 + CD25+ regulatory T cell depletion modulates anxiety and depression-like behaviors in mice. PloS One . 7 , e42054 (2012). Yu, X. et al. The alterations in CD4 + Treg cells across various phases of major depression. J. Affect. Disord . 362 , 485–492 (2024). Mancuso, E. et al. Biological correlates of treatment resistant depression: a review of peripheral biomarkers. Front. Psychiatry . 14 , 1291176 (2023). Kim, H. J., Kim, H., Lee, J. H. & Hwangbo, C. Toll-like receptor 4 (TLR4): new insight immune and aging. Immun. Ageing A . 20 , 67 (2023). Lucas, K. & Maes, M. Role of the Toll Like receptor (TLR) radical cycle in chronic inflammation: possible treatments targeting the TLR4 pathway. Mol. Neurobiol. 48 , 190–204 (2013). Kahan, R. et al. Sterile inflammation in liver transplantation. Front. Med. 10 , 1223224 (2023). Zhang, Y. et al. Toll-like receptor 4 mediates chronic restraint stress-induced immune suppression. J. Neuroimmunol. 194 , 115–122 (2008). Rip, J. et al. Toll-Like Receptor Signaling Drives Btk-Mediated Autoimmune Disease. Front. Immunol. 10 , 95 (2019). Blume, J., Douglas, S. D. & Evans, D. L. Immune suppression and immune activation in depression. Brain Behav. Immun. 25 , 221–229 (2011). Szałach, Ł. P., Cubała, W. J. & Lisowska, K. A. Changes in T-Cell Subpopulations and Cytokine Levels in Patients with Treatment-Resistant Depression-A Preliminary Study. Int. J. Mol. Sci. 24 , 479 (2022). Mayer, C. T. et al. An apoptosis-dependent checkpoint for autoimmunity in memory B and plasma cells. Proc. Natl. Acad. Sci. U. S. A. 117, 24957–24963 (2020). Simpson, M. J. et al. Peripheral apoptosis and limited clonal deletion during physiologic murine B lymphocyte development. Nat. Commun. 15 , 4691 (2024). Peshes-Yaloz, N., Rosen, D., Sondel, P. M., Krammer, P. H. & Berke, G. Up-regulation of Fas (CD95) expression in tumour cells in vivo. Immunology 120 , 502–511 (2007). Staniek, J. et al. TRAIL-R1 and TRAIL-R2 Mediate TRAIL-Dependent Apoptosis in Activated Primary Human B Lymphocytes. Front. Immunol. 10 , 951 (2019). Kaltenboeck, A., Winkler, D. & Kasper, S. Bipolar and related disorders in DSM-5 and ICD-10. CNS Spectr. 21 , 318–323 (2016). Shafer, A. B. Meta-analysis of the factor structures of four depression questionnaires: Beck, CES-D, Hamilton, and Zung. J. Clin. Psychol. 62 , 123–146 (2006). Bech, P., Allerup, P., Larsen, E. R., Csillag, C. & Licht, R. W. The Hamilton Depression Scale (HAM-D) and the Montgomery-Åsberg Depression Scale (MADRS). A psychometric re-analysis of the European genome-based therapeutic drugs for depression study using Rasch analysis. Psychiatry Res. 217 , 226–232 (2014). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9336198","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":634226160,"identity":"9962db2e-629e-43e6-abcf-5dcbf5737a66","order_by":0,"name":"Krzysztof Pietruczuk","email":"data:image/png;base64,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","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":true,"prefix":"","firstName":"Krzysztof","middleName":"","lastName":"Pietruczuk","suffix":""},{"id":634226161,"identity":"45b921c3-ef82-4ad1-82ab-8a70c9dc169b","order_by":1,"name":"Katarzyna Rückemann-Dziurdzińska","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Katarzyna","middleName":"","lastName":"Rückemann-Dziurdzińska","suffix":""},{"id":634226163,"identity":"1bafe32c-3293-4c59-b7c0-50b668bc3f04","order_by":2,"name":"Szymon Bierzanowski","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Szymon","middleName":"","lastName":"Bierzanowski","suffix":""},{"id":634226170,"identity":"176ea6b3-93f8-40b6-96db-d7cb90f80e30","order_by":3,"name":"Karol Grabowski","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Karol","middleName":"","lastName":"Grabowski","suffix":""},{"id":634226171,"identity":"1111c0c7-5f3a-45d4-9fdc-4f2dffaa2092","order_by":4,"name":"Ewa Bryl","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Ewa","middleName":"","lastName":"Bryl","suffix":""},{"id":634226172,"identity":"2044aecf-896e-4e96-8eca-f24c51126743","order_by":5,"name":"Jacek M Witkowski","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Jacek","middleName":"M","lastName":"Witkowski","suffix":""}],"badges":[],"createdAt":"2026-04-06 17:24:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9336198/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9336198/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108977161,"identity":"5e43680a-9460-4a67-9652-e4c29c68fb53","added_by":"auto","created_at":"2026-05-11 11:30:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":412344,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of lymphocyte subpopulations in patients with depression compared with healthy individuals, and correlation with the severity of depressive symptoms. (A) Comparison of CD3+ cell levels (T lymphocytes) in patients and healthy individuals, and the correlation of these cells with the severity of depressive symptoms assessed using the Beck Depression Inventory (BDI) and the Hamilton Depression Rating Scale (HDRS). (B) Comparison of NK cell (CD16+/CD56+) levels in patients and healthy individuals, and a negative correlation between the percentage of NK cells and the severity of depressive symptoms. (C) Comparison of the number of CD4+, CD8+ and NK-T cells in patients and control subjects. A statistically significant difference was observed for CD8+ lymphocytes.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9336198/v1/ff6a5d2fa458635ac4cf9012.png"},{"id":108976837,"identity":"c6daed67-cb76-4083-859b-20866f87bc41","added_by":"auto","created_at":"2026-05-11 11:29:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":308566,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the expression of B-cell surface markers (CD19+) in patients with depression compared with healthy individuals, and correlation with the severity of depressive symptoms. (A) Comparison of CD19+ B-cell levels (statistically significant difference) and CD19+CD22+ and CD19+Baff-R cells (B) Comparison of the number of B-cells expressing CD5, CD10 and CD40 markers in patients and healthy individuals. (C) Correlation between CD19+CD20+ lymphocyte levels and the severity of depression assessed using the BDI (Beck Depression Inventory) and HDRS (Hamilton Depression Rating Scale), and a positive correlation with mental state\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9336198/v1/81258941bb74da148dd7c385.png"},{"id":108839553,"identity":"6f242a30-bb30-4786-aedd-c5c6e2915a6a","added_by":"auto","created_at":"2026-05-09 00:48:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":529572,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of activation markers on CD4+ and CD8+ T cells in patients with depression compared with a control group, and the correlation of selected markers with the severity of depressive symptoms. (A–B) A statistically significant difference in CD69 and CD25 markers on CD4+ T cells and a positive correlation between these cells and mental state. (C) No significant difference in the CD8+CD25 population and a positive correlation between these cells and the severity of depressive symptoms\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9336198/v1/f28d5b0c7792f756708b7ffc.png"},{"id":108977162,"identity":"bbcfc2b3-7aa9-460b-ac96-ee077e73515b","added_by":"auto","created_at":"2026-05-11 11:30:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":601122,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Expression of the TLR-4 receptor on the surface of T cells and CD19+ B cells in patients with depression compared with healthy individuals, and its correlations with markers of apoptotic readiness. (B–D) Markers of increased susceptibility to apoptosis, CD95 and TRAIL-R1; the difference was statistically significant for CD8+CD95+ cells and for CD19+ B cells and CD19TRAIL-R1\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9336198/v1/8ab36da99954e78fef1212bf.png"},{"id":108979586,"identity":"6087e46b-2c70-4d0b-9ded-3f16488c3e53","added_by":"auto","created_at":"2026-05-11 11:59:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1972907,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9336198/v1/3a90f939-ff2c-45d7-9d88-7e089b0a2493.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"From Immune Activation to Cell Death: Investigating the Pro-inflammatory Phenotype of PBMCs in Major Depressive Disorder","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDepression is one of the most common mental health conditions. In 2019, around 280\u0026nbsp;million people worldwide were suffering from depression. Depending on the country, this accounted for 2\u0026ndash;6% of the population\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The Covid-19 pandemic has also taken its toll, and depression is now one of the most pressing health and economic issues worldwide\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAccording to current scientific understanding, depression is a systemic disorder. Its symptoms go beyond psychological aspects and include disturbances in the regulation of the nervous, endocrine and immune systems\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnfortunately, the exact cause of depression remains unknown, although several theories have emerged, each of which sheds some light on the complex interactions between the psychological, social and biological factors that contribute to the development of depressive disorders. The best known of these include the monoamine theory, the inflammatory theory and the theory of HPA axis dysregulation. In recent years, the theory of gut-brain axis dysregulation has been added to these.\u003c/p\u003e \u003cp\u003eThe best-known and most widely accepted of these is the monoamine theory of depression, which posits that there is a reduced concentration of neurotransmitters such as dopamine, serotonin and norepinephrine in the synaptic cleft\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. It has also been demonstrated that depression is accompanied by inflammation, with changes in certain parameters of the immune system observed in patients. Elevated white blood cell counts, accelerated erythrocyte sedimentation rate\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, elevated CRP levels\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and increased levels of inflammatory markers such as IL-1beta, IL-6, TNF-alpha, IL-8, IL-16, as well as numerous chemokines in the blood and cerebrospinal fluid\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e have been observed. All of these constitute elements of the inflammatory theory of depression.\u003c/p\u003e \u003cp\u003eIn turn, the involvement of the neurohormonal system \u0026ndash; such as the HPA axis \u0026ndash; in the stress response has led to the formulation of theories regarding its role in the development of depressive symptoms\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This involvement manifests itself through the effect of elevated cortisol levels on mood regulation, as demonstrated in studies involving patients and in observations regarding the increased frequency of depressive episodes in individuals undergoing cortisol therapy\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNumerous studies point to the potential involvement of the gut-brain axis in the pathophysiology of depression, as it mediates complex interactions between the gut microbiota, the immune system and the brain\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Components of the gut microbiota may influence brain function and behaviour by producing various metabolites, neurotransmitters and signalling molecules, which also act directly on the vagus nerve\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Studies on the functioning of the gut-brain axis in patients with depression have indicated the presence of dysbiosis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These studies have contributed to the formulation of a further theory regarding the causes of depression, namely the theory of gut-brain axis dysregulation.\u003c/p\u003e \u003cp\u003eDespite numerous studies conducted over the years, the pathophysiology of depression is still not fully understood. Research findings and emerging theories point to a complex interplay of many factors; however, the core principles of the monoamine theory remain a key reference point in the search for new targets for pharmacotherapy. Based on the monoamine theory, new antidepressants are being developed that act on neurotransmitter systems such as serotonin, noradrenaline and dopamine, with the aim of increasing the concentration of these substances in the synaptic cleft\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, patients often exhibit high resistance to these drugs\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and the therapeutic effect is delayed, with clinical response frequently taking several weeks to achieve\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. It is suggested that, in the case of a condition with such a multifactorial a etiology as depression, this therapeutic approach is not sufficiently effective.\u003c/p\u003e \u003cp\u003eThere is ample evidence supporting the validity of the theories mentioned above, which are not mutually exclusive; however, we still do not know which of them is of key importance and is capable of bringing all the others together into a coherent whole. In our study, we focused on the inflammatory theory of depression. However, going beyond classic markers of inflammation, such as cytokines or acute-phase proteins, we sought to determine, by broadening our understanding of the general functioning of the immune system in the course of depression, the potential cause of this condition, as well as the interrelationships and influence of the other theories. Thanks to this holistic view of the pathophysiology of mood disorders, which still harbours many unknowns, we have succeeded in demonstrating the potential for future developments in new, more effective therapies for depressive disorders.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAnalysis of the main lymphocyte subpopulations revealed comparable levels of CD3\u0026thinsp;+\u0026thinsp;T cells in patients with depression and healthy individuals. In patients, a positive correlation was observed between CD3\u0026thinsp;+\u0026thinsp;cell levels and the severity of depressive symptoms. This applies to the assessment of their mental state using both the Beck Depression Scale and the Hamilton Depression Rating Scale (Fig.\u0026nbsp;1A). Patients also exhibited a statistically significant reduction in the number of CD16+/CD56\u0026thinsp;+\u0026thinsp;NK cells compared to healthy individuals, which was inversely correlated with measures of mental state (Fig.\u0026nbsp;1B). Analysis of the CD19\u0026thinsp;+\u0026thinsp;B-lymphocyte population indicates a reduction in the number of these cells in patients with depression (Fig.\u0026nbsp;2A). No statistically significant association was found between this parameter and disease severity. Analysis of T-lymphocyte subpopulations revealed no statistically significant differences between the numbers of CD4\u0026thinsp;+\u0026thinsp;and CD3+/CD16+/CD56\u0026thinsp;+\u0026thinsp;cells in the compared groups. In the case of CD8\u0026thinsp;+\u0026thinsp;T lymphocytes, a statistically significant difference was observed between the patient group and the healthy control group (Fig.\u0026nbsp;1C).\u003c/p\u003e \u003cp\u003eAn analysis was then carried out of the surface markers of CD4+, CD8\u0026thinsp;+\u0026thinsp;and CD19\u0026thinsp;+\u0026thinsp;lymphocyte populations. The aim of the study was to identify the characteristic antigens of these cells, the presence of activation markers, the TLR-4 molecule indicating ongoing inflammation in the body, and two markers indicating the cells\u0026rsquo; readiness to undergo apoptosis (CD95, TRAIL-R1).\u003c/p\u003e \u003cp\u003eAnalysis of B-cell surface markers revealed a higher number of CD19\u0026thinsp;+\u0026thinsp;CD5+ and CD19\u0026thinsp;+\u0026thinsp;CD10+ cells and a decrease in the level of CD19\u0026thinsp;+\u0026thinsp;CD40+ cells in patients with depression compared with those in the control group (Fig.\u0026nbsp;2B). The number of CD19\u0026thinsp;+\u0026thinsp;lymphocytes expressing CD20, CD22 or Baff-R antigens was comparable in both study groups (Fig.\u0026nbsp;2B). In patients, a positive correlation was observed between the severity of depression and the presence of CD19\u0026thinsp;+\u0026thinsp;CD20+ cells (Fig.\u0026nbsp;2C).\u003c/p\u003e \u003cp\u003eAn analysis of activation markers was carried out for both CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells. In both types of T cells, the expression of TCR (alpha/beta) and CD28 molecules was comparable between patients and the control group. Patients with depression showed statistically significantly higher levels of CD25\u0026thinsp;+\u0026thinsp;and CD69\u0026thinsp;+\u0026thinsp;T cells among helper cells, and a positive correlation with the severity of depressive symptoms in both the Beck and Hamilton tests (Fig.\u0026nbsp;3A and 3B). The number of CD8\u0026thinsp;+\u0026thinsp;CD25+ cells in patients did not show any statistical differences compared with the control group; however, a positive correlation with the patients\u0026rsquo; mental state was observed (Fig.\u0026nbsp;3C).\u003c/p\u003e \u003cp\u003eIn the case of the TLR-4 antigen, a significant increase in its expression was observed on CD4\u0026thinsp;+\u0026thinsp;and CD19\u0026thinsp;+\u0026thinsp;cells across various lymphocyte populations, with the differences in the patient group being more than twice as high as in the healthy control group (Fig.\u0026nbsp;4A).\u003c/p\u003e \u003cp\u003eThe increased activation of lymphocytes and the rise in the proportion of lymphocytes expressing the inflammatory marker (TLR-4) on their surface were also accompanied by an increased susceptibility of the cells to enter the apoptotic pathway. In patients, significantly higher levels of cells expressing the CD95 antigen were observed in the CD8\u0026thinsp;+\u0026thinsp;and CD19\u0026thinsp;+\u0026thinsp;populations (Fig.\u0026nbsp;4B and 4C). TRAIL-R1 expression on T lymphocytes was comparable in both study groups. Only the patients\u0026rsquo; B lymphocytes showed increased TRAIL-R1 expression (Fig.\u0026nbsp;4D).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInvestigating the link between depression and the immune system is like putting together a jigsaw puzzle where the number of pieces is unknown. We will show how our findings fit into the overall picture that has already emerged, and in which areas our research provides entirely new insights.\u003c/p\u003e \u003cp\u003eThe results of our study unequivocally confirm the validity of the inflammatory theory of depression, which, however, many researchers consider solely in the context of elevated levels of pro-inflammatory cytokines such as IL-1, IL-6, TNF and the acute-phase protein CRP\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Advances in and wider availability of cytometric methods in recent years have enabled a more detailed investigation of this phenomenon through the analysis of the main immune cell populations involved in inflammatory processes. An increase in leukocytosis and monocytosis has been reported in patients with depression, and an elevated ratio of both neutrophils to lymphocytes and CD4\u0026thinsp;+\u0026thinsp;to CD8\u0026thinsp;+\u0026thinsp;lymphocytes has been observed\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003eIn our study, we went a step further: we examined not only the basic PBMC cell populations but also characteristic markers indicating cell activation, their role in promoting inflammation, as well as their readiness for apoptosis. In the main PBMC populations, we found elevated levels of CD8\u0026thinsp;+\u0026thinsp;lymphocytes; however, with a comparable number of CD3 cells, we did not observe an elevated CD4/ CD8 ratio indicated by Soresen in his meta-analysis, in which he states that in 16 of the 26 studies included in his publication, patients with depressive disorders are characterised by an elevated CD4/CD8 ratio\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe group of patients we studied was also characterised by a lower proportion of NK cells with the CD3-/CD16+/CD56\u0026thinsp;+\u0026thinsp;phenotype compared with healthy individuals. The observed lower proportion of classical NK cells lacking the CD3 molecule on their surface indicates impaired cytotoxic capacity of the patients\u0026rsquo; immune system, which may contribute to the chronic inflammation observed in these patients in other studies\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNumerous studies have examined the levels and activity of NK cells in patients suffering from depression\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. So far, the results are inconclusive. Some studies indicate a significantly lower proportion of cells in this subpopulation among patients with major depressive disorder compared with the control group, and link this finding to the presence of chronic stress, which is thought to lead to a reduction in the cytotoxic activity of NK cells\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Other studies indicate changes in the activity of NK cells, but not in their numbers\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, discrepancies in study results may stem from a number of factors, such as differences in diagnostic criteria, symptom severity or the patients\u0026rsquo; treatment status; the distinction between NK cell count and functional activity is of particular importance here. Even if the number of NK cells remains unchanged, patients with depression may have an impaired ability to destroy target cells or produce cytokines\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The impaired ability of NK cells to regulate the body\u0026rsquo;s inflammatory response may be responsible for the disease becoming chronic. The co-occurrence of impaired NK cell function and elevated levels of pro-inflammatory cytokines in people suffering from depression confirms the link between the state of NK cells and the inflammatory theory of depression. On the other hand, when analysing the theory of depression based on HPA axis dysregulation, we also observe certain patterns. The elevated cortisol levels observed in patients with depression may have an inhibitory effect on both the number and function of NK cells\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImmune disorders caused by inflammation and hormonal imbalances, such as HPA axis dysregulation\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e may be the cause of the increase in the proportion of CD19\u0026thinsp;+\u0026thinsp;CD10+ cells that we have observed. The CD10 antigen is found mainly on immature B lymphocytes and disappears as they mature\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Inflammation, which is characteristic of depression and chronic stress, can disrupt the maturation and differentiation of B cells in the bone marrow, leading to the release of a greater number of immature B cells into the peripheral circulation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe increase in CD19\u0026thinsp;+\u0026thinsp;CD10+ cells is not the only difference in B-cell subpopulations that we observed. We also observed an increase in the CD19\u0026thinsp;+\u0026thinsp;CD5+ B-cell subpopulation. CD5\u0026thinsp;+\u0026thinsp;B-lymphocytes constitute a population with regulatory potential and are involved in both normal immune responses and autoimmunity\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Changes in the number of these cells may therefore have a significant impact on immune homeostasis in the course of depression.\u003c/p\u003e \u003cp\u003eThe disruption of immune homeostasis in patients with depression is also indicated by the reduction in the proportion of B-cell subpopulations expressing the CD40 molecule on their surface, as demonstrated in our study. The presence of this molecule is of crucial importance for B cells, particularly in the context of their T-cell-mediated activation\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The interaction between the CD40 receptor and its ligand CD154, which is present on activated T cells, is crucial for the humoral immune response. Abnormalities in CD40 receptor expression can disrupt these interactions and affect antibody production and other effector functions of B cells\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. This is also of fundamental importance for B-cell proliferation and differentiation, a finding that appears to be indirectly supported by our study, as the reduced proportion of the CD19\u0026thinsp;+\u0026thinsp;CD40+ population is accompanied by an increased proportion of immature B-cells, i.e. CD19\u0026thinsp;+\u0026thinsp;CD10+\u003csup\u003e38\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur findings confirm the presence of an inflammatory process in the course of depression. This is indicated both by an increase in the number of cells expressing the activation markers CD25 and CD69 on lymphocytes, and by the presence of the TLR-4 molecule, which is recognised as a marker of inflammation. T-cell activation, which is a key step in triggering an immune response\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The regulation of T-cell populations is essential for maintaining immune homeostasis, and apoptosis, or programmed cell death, is a key process in this regulation\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. It is believed that an imbalance between T-cell activation and regulation, including alterations in normal apoptotic processes, contributes to the chronic inflammation observed in depression.\u003c/p\u003e \u003cp\u003eIn our study, we observed an increase in the number of CD4\u0026thinsp;+\u0026thinsp;CD25+ cells. However, no statistically significant differences were found in CD8\u0026thinsp;+\u0026thinsp;cytotoxic lymphocytes in patients compared with the control group of healthy individuals. The contradictions found in the literature regarding the CD25 activation marker can, however, be explained by the behaviour of the CD25 cell subpopulation, i.e. CD4\u0026thinsp;+\u0026thinsp;CD25+FoxP3\u0026thinsp;+\u0026thinsp;Treg lymphocytes, to which the authors of other studies most frequently refer\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. It also appears that the presence of inflammation may depend on the subtype of depression. For example, in treatment-resistant depression (TRD), a lower proportion of CD4\u0026thinsp;+\u0026thinsp;CD25+ cells has been observed, suggesting reduced activation of CD4 lymphocytes in this group of patients\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe TLR-4 molecule, which we observed in increased quantities on CD4\u0026thinsp;+\u0026thinsp;T cells and CD19\u0026thinsp;+\u0026thinsp;B cells, is responsible for detecting pathogen-associated molecular patterns (PAMPs), primarily lipopolysaccharide (LPS) \u0026ndash; a component of the outer membrane of Gram-negative bacteria. Furthermore, this receptor recognises damage-associated molecular patterns (DAMPs), which are released from damaged tissues, thereby also playing a role in inflammatory processes unrelated to infection\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. This suggests its involvement in aseptic inflammation, which is of significant importance in the context of depression, where the role of aseptic inflammation triggered by stress or cellular damage is increasingly emphasised. Aseptic inflammation, i.e. an immune response triggered by tissue damage in the absence of pathogens, relies heavily on the recognition of DAMPs by TLRs, particularly TLR4. This recognition initiates the production of inflammatory mediators such as TNF-α, IL-1β, IL-6 and IL-8, as well as chemokines\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe presence of TLR-4 on CD4\u0026thinsp;+\u0026thinsp;lymphocytes may influence their ability to trigger inflammatory responses. Experiments in mice have shown that TLR4 expression increased under conditions of chronic stress, whilst its deficiency protected against stress-induced lymphocyte depletion and disruption of the Th1/Th2 cytokine balance. This indicates the involvement of the TLR-4 molecule in modulating lymphocyte responses under stress, suggesting the key role of this molecule in the pathophysiology of depression\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Conversely, the presence of TLR-4 on the surface of B cells is associated with impaired humoral responses and autoimmunity, which may be signalled by the binding of the TCR to LPS, leading in turn to the increased production of pro-inflammatory cytokines observed in depression\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe impaired B-cell function mentioned earlier, as demonstrated by our research, and the role of the TLR-4 receptor in inflammatory and autoimmune processes may provide evidence of ongoing chronic inflammation in depression\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs chronic inflammation associated with depression may be responsible for increased apoptosis of immune cells, we also investigated this aspect of depression. We assessed two molecules whose presence indicates a greater susceptibility to apoptosis. One of these was the CD95 molecule, which plays a key role in maintaining the body\u0026rsquo;s immune homeostasis. Studies on the state of the immune system in depression do not yield clear-cut results, as there are also reports of a lower proportion of CD3\u0026thinsp;+\u0026thinsp;CD8+CD95\u0026thinsp;+\u0026thinsp;cells in treatment-resistant patients\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. This discrepancy highlights the complexity of CD95 regulation in depression and may suggest that CD95 expression depends on specific characteristics of the patient population studied, i.e. on the various subtypes of the condition and on the use of specific antidepressants.\u003c/p\u003e \u003cp\u003eApoptosis is also a key mechanism regulating the CD19\u0026thinsp;+\u0026thinsp;B-cell population, influencing both its size and composition. Apoptosis eliminates B cells that are autoreactive and could potentially lead to autoimmune diseases, or those that have been activated but have already fulfilled their function. Proper regulation of B-cell apoptosis is therefore essential for maintaining immune tolerance and preventing excessive or uncontrolled immune responses\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. As we also demonstrated in our study, a decrease in the total number of CD19\u0026thinsp;+\u0026thinsp;B lymphocytes, accompanied by an increase in the proportion of immature and regulatory subpopulations, suggests that mature B lymphocytes may undergo preferential apoptosis due to increased expression of CD95 and TRAIL-R1, as we observed. In the case of the CD95 molecule, it is suggested that there is a bidirectional relationship between inflammation and apoptosis\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe second molecule we studied, TRAIL-R1, is also recognised as a death receptor. It has been shown that the expression of TRAIL-R1 on B cells is not constant, but is modulated during their development and activation. B cells from proliferation centres, which are strongly activated and undergo selection, exhibit higher levels of TRAIL-R1. Furthermore, TRAIL-R1 undergoes rapid overexpression following activation, particularly in memory B cells\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe results of our study unequivocally confirm that depression is accompanied not only by an impairment of immune system function, but also by a chronic inflammatory process. Consequently, traditional treatment of depression, based primarily on the monoamine theory and the correction of neurotransmission disorders, may prove insufficient or partially ineffective. Our observations point to the need to incorporate the inflammatory theory into the diagnostic and therapeutic process. Treatment of depression should therefore involve not only the regulation of neurotransmitters, but also the reduction of chronic inflammation and the normalisation of immune dysfunction. Considering anti-inflammatory or immunomodulatory therapies, in parallel with conventional antidepressants, may be crucial for more effective treatment of depression, particularly in cases resistant to standard methods. In the context of our findings, it therefore becomes justified to seek new therapeutic targets that would simultaneously suppress inflammation and restore normal neurotransmission.\u003c/p\u003e \u003cp\u003eDespite the promising results, this study has certain limitations which point to avenues for future research. The relatively small size of the study group limits the statistical power of the analyses and makes it difficult to generalise the findings to a wider patient population. Further research involving larger, representative cohorts is essential to validate the observed associations.\u003c/p\u003e \u003cp\u003eThe high clinical heterogeneity of depression also remains a significant challenge. The data obtained suggest only the existence of a so-called inflammatory biotype in which immune processes play a leading role. This means that the changes described may not occur in all patients, which necessitates rigorous stratification of participants in future studies to precisely identify subgroups with different pathophysiological mechanisms. Focusing solely on the phenotype of immune cells represents only a fragment of the disease\u0026rsquo;s complex aetiology. To fully confirm the central role of inflammation, an integrative approach is required, combining immunophenotyping with other key theories of depression. A multisystemic approach will allow for a full understanding of the interactions between the immune system and the central nervous system in the context of affective disorders.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003ePatients and healthy volunteers.\u003c/p\u003e \u003cp\u003e27 patients diagnosed with depression (15 female and 12 male; mean age 40,81\u0026thinsp;\u0026plusmn;\u0026thinsp;6,53) and 10 healthy volunteers (5 female and 5 male; mean age 43,40\u0026thinsp;\u0026plusmn;\u0026thinsp;7,34) participated in the study. All participants underwent basic physical examination and psychiatric interview in the Adult Psychiatry Clinic of the Medical University of Gdansk. Patients were diagnosed with the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders Fifth Edition (DSM-5)\u003csup\u003e55\u003c/sup\u003e. Mental status of patients with depression and healthy individuals was assessed with two tests: Beck Depression Inventory\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e (mean score for the patients: 30,56\u0026thinsp;\u0026plusmn;\u0026thinsp;1,45 and for the control group: 2,50\u0026thinsp;\u0026plusmn;\u0026thinsp;0,97) and Hamilton Depression Rating Scale\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e (mean score for the patients: 39,41\u0026thinsp;\u0026plusmn;\u0026thinsp;3,41 and for the control group: 3,80\u0026thinsp;\u0026plusmn;\u0026thinsp;1,03). For all of the patients included into the study it was the first diagnosis of depression and blood was collected before they started any antidepressive treatment.\u003c/p\u003e \u003cp\u003eExclusion criteria for the control group included any diagnose of a mental disorder and/or incidence of mental illness in the family. Exclusion criteria for both patients and control group included: diagnosis of an autoimmune disorder, chronic inflammatory disorder, diabetes or any allergy. None of the study participants declared use of recreational drugs. In both groups an occasional alcohol consumption was reported.\u003c/p\u003e \u003cp\u003e The study has been approved by the Bioethical Committee for Scientific Research at the Medical University of Gdansk. All participants were informed about the purpose of the study and gave their informed consent in a written form. Relevant guidelines and regulations were observed throughout the study.\u003c/p\u003e \u003cp\u003ePeripheral blood collection.\u003c/p\u003e \u003cp\u003e3 ml of peripheral venous blood was collected from patients and healthy controls after overnight fasting into EDTA tubes for the cytometric analysis of lymphocytes\u0026rsquo; subpopulations.\u003c/p\u003e \u003cp\u003eDetermination of T and B-cell subpopulations.\u003c/p\u003e \u003cp\u003e50 \u0026micro;l blood samples were transferred into cytometric tubes for red blood cell (RBC) lysis with 0,8% NH4Cl and 0,1% KHCO3 buffer and subsequent staining with monoclonal antibodies. After RBC lysis step the cells were washed with PBS (phosphate buffered saline) buffer and stained with the following monoclonal antibodies (all from Becton Dickinson USA).\u003c/p\u003e \u003cp\u003e[Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Fluorochromes used and their corresponding antigens in the cytometric analysis of lymphocyte subpopulations.]\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFluorochrom\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTarget antigen\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTLR-4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePerCP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTLR2, CD4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFITC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCD95, CD25, CD20, CD22, CD3, CD5, CD10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTRAILR1, CD69, TCRαβ, CD28, BAFFR, CD40, CD16CD56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCD8, CD19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCells were incubated with antibodies for 30 min at 4\u0026deg;C in the dark, washed with PBS and suspended in 200 \u0026micro;l of PBS for flow cytometry analysis.\u003c/p\u003e \u003cp\u003eCytometric analysis.\u003c/p\u003e \u003cp\u003eQuantitative fluorescence analysis was performed with FACSVerse (Becton Dickinson, USA). Ten thousand lymphocytes (based on their forward and side scatter gating) were acquired for each sample. Cytometric data were analysed with FlowJo X 10.0.7 (Tree Star; USA).\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis.\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using libraries for data exploration, analysis and visualization available in Python, such as: Pandas, Numpy, Matplotlib, Seaborn and Scikit-learn using Datalore 2023.4 JetBrains. Normally distributed data were analysed using the Student's t-test, while the non-parametric Mann-Whitney test for independent samples was used to analyse non-normally distributed data. Statistical significance was assumed at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Polish National Science Centre project \u0026ldquo;Miniature 2\u0026rdquo; granted to Krzysztof Pietruczuk and by the statutory funds of the Medical University of Gdansk (02-0058/07/262) granted to Jacek M. Witkowski.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDeclaration of Compliance with Regulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI hereby declare that all studies were conducted in accordance with the regulations, and that patients provided informed consent to participate in the studies. The study has been approved by the Bioethical Committee for Scientific Research at the Medical University of Gdansk. All participants were informed about the purpose of the study and gave their informed consent in a written form. Relevant guidelines and regulations were observed throughout the study.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eStatement on Data Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCytometric analyses were performed using the instrument acquired within the Network for Imaging of Structural and Functional Pathology of Cells of the University and Medical University of Gdansk. This work was supported by the Polish National Science Centre project \u0026ldquo;Miniature 2\u0026rdquo; granted to Krzysztof Pietruczuk and by the statutory funds of the Medical University of Gdansk (02-0058/07/262) granted to Jacek M. Witkowski.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no conflict of interest.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eData, O. W. in. OWID Homepage. \u003cem\u003eOur World Data\u003c/em\u003e https://ourworldindata.org (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchou, T. M., Joca, S., Wegener, G. \u0026amp; Bay-Richter, C. Psychiatric and neuropsychiatric sequelae of COVID-19 - A systematic review. \u003cem\u003eBrain Behav. Immun.\u003c/em\u003e \u003cb\u003e97\u003c/b\u003e, 328\u0026ndash;348 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJarrott, B., Head, R., Pringle, K. G., Lumbers, E. R. \u0026amp; Martin, J. H. LONG COVID\u0026rsquo;-A hypothesis for understanding the biological basis and pharmacological treatment strategy. \u003cem\u003ePharmacol. Res. 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A psychometric re-analysis of the European genome-based therapeutic drugs for depression study using Rasch analysis. \u003cem\u003ePsychiatry Res.\u003c/em\u003e \u003cb\u003e217\u003c/b\u003e, 226\u0026ndash;232 (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"depression disorders, inflammation, lymphocyte, TLR-4, apoptosis, activation markers","lastPublishedDoi":"10.21203/rs.3.rs-9336198/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9336198/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDepression's complex pathogenesis involves neurotransmission disorders, chronic inflammation, and immune dysregulation. Supporting the inflammatory theory, this study analyzed lymphocyte subpopulations and surface molecule expression in patients to investigate immunological mechanisms and provide evidence for this clinical perspective.\u003c/p\u003e \u003cp\u003eThe study involved twenty-seven patients with newly diagnosed depression and ten healthy volunteers. Level of T-lymphocyte subpopulations, B-lymphocytes and NK-cells, as well as the expression of activation markers (CD25, CD69), inflammatory markers (TLR-4), apoptosis markers (CD95, TRAIL-R1) and maturity markers (CD10, CD40, CD5).\u003c/p\u003e \u003cp\u003eA reduced proportion of NK-cells was observed in patients. An increase in number of immature B-cells CD19\u0026thinsp;+\u0026thinsp;CD10+ and CD19\u0026thinsp;+\u0026thinsp;CD5+ cells was observed, accompanied by a decrease in CD40 expression. TLR-4 expression on CD4\u0026thinsp;+\u0026thinsp;and CD19\u0026thinsp;+\u0026thinsp;lymphocytes in patients was more than twice as high compared with the group of healthy volunteers. There was also an increase in the expression of the activation markers CD25 and CD69 on T-lymphocytes, and CD95 and TRAIL-R1 on T and B-lymphocytes. Some parameters correlated with severity of depressive symptoms.\u003c/p\u003e \u003cp\u003eThe results confirm that chronic inflammation influences depression through changes in lymphocytes. This suggests that immunomodulatory and anti-inflammatory treatments should be implemented as an important adjunct to standard clinical management of this condition.\u003c/p\u003e","manuscriptTitle":"From Immune Activation to Cell Death: Investigating the Pro-inflammatory Phenotype of PBMCs in Major Depressive Disorder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-09 00:47:52","doi":"10.21203/rs.3.rs-9336198/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"157646462857340579625042281564349162997","date":"2026-05-04T14:24:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"107563612930961822766126935940029310060","date":"2026-04-27T17:45:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257890945876059134698800422539106224376","date":"2026-04-27T13:54:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"268344790064368683533442776690389665499","date":"2026-04-27T05:43:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-27T02:19:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-19T01:04:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-13T12:59:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-10T18:47:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-10T17:09:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"97265a52-35e4-4ba4-9696-7080a2163202","owner":[],"postedDate":"May 9th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"157646462857340579625042281564349162997","date":"2026-05-04T14:24:16+00:00","index":65,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67495383,"name":"Health sciences/Diseases"},{"id":67495384,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2026-05-09T00:47:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-09 00:47:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9336198","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9336198","identity":"rs-9336198","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}