key: cord-0301088-h812isaj
authors: Rachayon, M.; Jirakran, K.; Sodsai, P.; Klinchanhom, S.; Sughondhabirom, A.; Plaimas, K.; Suratanee, A.; Maes, M.
title: Effects of cannabidiol on activated immune-inflammatory pathways in major depressive patients and healthy controls.
date: 2022-02-06
journal: nan
DOI: 10.1101/2022.02.04.22270489
sha: 93f371611ec8f320df9fb9fa802d40eef1e17d1e
doc_id: 301088
cord_uid: h812isaj

Background Major depressive disorder and a major depressive episode (MDD/MDE) are characterized by activation of the immune-inflammatory response system (IRS) and the compensatory immune-regulatory system (CIRS). Cannabidiol (CBD) is a phytocannabinoid isolated from the cannabis plant which was reported to have antidepressant-like and anti-inflammatory effects. The aim of the present study is to examine the effects of CBD on IRS, CIRS, M1, T helper (Th)-1, Th-2, Th-17, T regulatory (Treg) profiles, and growth factors in depression and healthy controls. Methods Culture supernatant of stimulated (5 microg/mL of PHA and 25 micro956;g/mL of LPS) whole blood of 30 depressed patients and 20 controls was assayed for cytokines using the LUMINEX assay. The effects of three CBD concentrations (0.1 microg/ml, 1 microg/mL, and 10 microg/mL) were examined. Results Depression was characterized by significantly increased Th-1, Th-2, Th-17, Treg, IRS, CIRS and neurotoxicity profiles. CBD 0.1 microg/mL did not have any immune effects. CBD 1.0 microg/mL decreased CIRS activities but increased growth factor production, while CBD 10.0 microg/mL suppressed Th-1, Th-17, IRS, CIRS, and a neurotoxicity profile and enhanced T cell growth and growth factor production. CBD 1.0 to 10.0 microg/mL dose-dependently decreased sIL-1RA, IL-8, IL-9, IL-10, IL-13, CCL11, G-CSF, IFN-gamma;, CCL2, CCL4, and CCL5, and increased IL-1beta;, IL-4, IL-15, IL-17, GM-CSF, TNF-alpha;, FGF, and VEGF. Conclusion CBD has very complex immunomodulatory effects which depend on the CBD dose. CBD does not normalize the activated immune profiles observed in depression while higher concentrations can worsen inflammatory processes.

Activated immune-inflammatory pathways are among the most important pathways involved in the pathophysiology of major depressive disorder (MDD) and a major depressive episode (MDE) in bipolar disorder [1] [2] [3] [4] [5] . Activation of the immune-inflammatory response system (IRS) in MDD/MDE is shown by a pro-inflammatory cytokine profile including activated M1 macrophage cells as indicated by elevated levels of interleukin (IL)-1β, IL-6, , and tumor necrosis factor-α (TNF-α); T helper-1 (Th-1) cells with increased interferon-γ (IFN-γ), IL-2, and IL-12 levels; and activated Th-17 cells with increased IL-17 and IL-6 [1, 2] . Depression (MDD/MDE) is not only accompanied by activation of the IRS but also by activation of the compensatory immune-regulatory system (CIRS), which tends to downregulate the primary immune response and hyperinflammation [1] . Activation of CIRS profiles in MDD/MDE is indicated by activated Th-2 cells with increased IL-4 and IL-5 levels, and activated T regulatory (Treg) cells, characterized by increased IL-10, a major negative immunoregulatory cytokine [1] .

The imbalance between pro-inflammatory and anti-inflammatory cytokines plays an important role in the onset of depression [1] . Importantly, some cytokines or chemokines which are increased in MDD/MDE have neurotoxic effects including IRS cytokines (e.g., IL-1, IL-2, IL-6, IL8, TNF-α, IFN-γ), some chemokines (e.g., CCL1, CCL2, CCL11, CCL5, CXCL8, tissue damage [15, 26] . On the other hand, activation of peripheral CB1 receptors may aggravate inflammation via increasing ROS and activation of nuclear factor (NF)-κB [15] . As such, endocannabinoids have complex effects on immune system functions and may promote as well as inhibit inflammatory processes through the CB2 receptors. In vivo treatment with a low dose of CBD may decrease TNF-α production in LPS-treated mice [27] and CBD at higher doses highly significantly attenuate the expression of pro-inflammatory cytokines by CD3+ T cells including TNF-α, IL-2, IL-17, IFN-γ and GM-CSF (granulocyte colony stimulating factor) [28] .

Thus, it is plausible that CBD may exert antidepressive effects by reducing M1, Th-1 or Th-17 pathways or increasing CIRS functions (e.g. IL-10 production) thereby reducing neuroaffective toxicity. Nevertheless, so far there is no evidence whether CBD may modulate the IRS and CIRS and M1, Th-1, Th-2 and Th-17 activities, and the production of chemokines and growth factors in patients with MDD/MDE. Hence, the aim of the study is to examine the effects of CBD on M1, Th-1, Th-2, Th-17, Treg, IRS and CIRS profiles and cytokines, immune-related neurotoxicity, chemokines and growth factors. The specific hypotheses are a) CBD has anti-inflammatory effects by decreasing M1, Th-1, Th-17 and IRS profiles and enhancing Treg and CIRS functions; and b) CBD attenuates the increased M1, Th-1, Th-17 and IRS responses in depression.

We included 30 depressed outpatients recruited at the outpatient clinic of the Department of Psychiatry, King Chulalongkorn Memorial Hospital, Bangkok, Thailand. We included participants of both sexes, aged 18 to 65 years old. They were diagnosed as suffering from MDD . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Whole blood cultures were used for the assay of IL-1β, sIL-1RA, IL-2, IL-4, IL-5, IL-6,   IL-7, CXCL8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, CCL10, FGF, GM-CSF, IFN-γ, CXCL10, CCL2, CCL3, PDGF, CCL4, CCL5, TNF-α, VEGF, and G-CSF. Electronic Supplementary File (ESF) Table 1 shows a list with the names, abbreviations, and official gene symbols of all cytokines/chemokines/growth factors assayed in the present study. ESF, Table 2 shows a list of the different immune profiles examined in the current study.

The effects of CBD on these cytokines/growth factors were studied by stimulating whole blood with PHA and LPS and analyzing the levels of cytokine production in culture supernatant. [8, 9, 32] . RPMI-1640 medium (Gibco Life Technologies, USA) with L-glutamine and phenol red and containing 1% of penicillin (Gibco Life Technologies, USA) were employed with (stimulated) or without (unstimulated) 5 μg/mL PHA (Merck, Germany) + 25 μg/mL lipopolysaccharide (LPS; Merck, Germany). 1.8 mL of either one of these two media was placed into 24-well sterile plates with 0.2 mL of whole blood, 1/10 diluted. Whole blood was seeded in the 24-well culture plates with CBD which dissolved with dimethyl sulfoxide (DMSO) to make 1 mg/ml of stock and kept at -80C. For each subject, the specimens could be divided into 4 conditions, namely (1) stimulated with LPS+PHA and incubated 72 hours, and (2) (3) (4) stimulated with LPS+PHA and incubated 72 hours with three different concentration of CBD, namely CBD 0.1 µg/mL, 1 µg/mL and 10.0g/mL. Samples were incubated for 72 h in a humidified atmosphere at 37°C, 5% CO2. After incubation, the plates were centrifuged at 1500 rpm for 8 min.

Supernatants were taken off carefully under sterile conditions, divided into Eppendorf tubes, and frozen immediately at −70°C until thawed for assay of the cytokines/growth factors in culture supernatant. These CBD concentrations were chosen based on prior research in humans showing therapeutic CBD serum/plasma levels in the 0.1-1.0 µg/dL range and additionally a 10-fold . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 higher dose (10.0 µg/dL) which is obtained in the intestinal lymphatic system after more targeted treatment [28] .

The cytokines were measured using a LUMINEX assay (BioRad, California, United States of America) which is bead-based multiplex method. In brief, supernatants were fourfold diluted with media and incubated with coupled magnetic beads for 30 min. Detection antibodies were then added followed by streptavidin-PE for 30 min and 10 min, respectively, before measuring the fluorescence intensities (FI) using the Luminex 200 machine. In the present study we used the (blank analyte subtracted) FI values in the statistical analysis because FI are generally a more appropriate choice than absolute concentration especially when multiple plates are used [33] . Table 1 shows the range of the FI values that fall within the concentration curve.

All samples of all cytokines were well measurable, except IL-7, which showed too many values below the sensitivity of the assay and was, therefore, excluded. IL-13 showed the acceptable limit of 30% values lower than the sensitivity and could be included in the statistical analysis.

The inter-assay CV values for all analyses are less than 11%.

Chi-square tests were performed to determine relationships between categorical variables, while analysis of variance (ANOVA) was employed to compare scale variables across categories. We employed generalized estimating equations (GEE) analysis, repeated measures, to determine the impact of CBD treatment on the cytokine profiles and all cytokines/growth factors separately. The pre-specified GEE analyses, repeated measures (AR working correlation matrix, a linear scale response, and maximum likelihood estimate as scale parameter method), included fixed categorical effects of time (treatment with vehicle and 3 CBD concentrations), groups . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 (depression versus controls) and time-by-treatment interaction, sex, smoking, and continuous fixed covariates, namely age and BMI. Moreover, the unstimulated values of the respective immune profiles and separate cytokines/growth factors were used as additional covariates but this did not change the results (because there were no significant differences in the unstimulated values between time and groups; results not shown). The immune profiles were the primary outcome variables and if these yielded significant outcomes we also examined the separate cytokines/growth factors. The multiple effects of time or groups on the immune profiles were subjected to false discovery rate (FDR) p correction [34] . Moreover, we also entered the drug state, age, sex, BMI and smoking in the GEE analysis as additional predictors to rule out any effects of these possible confounders. The GEE technique enables us to account for important interactions and confounders when analyzing treatment effects at the subject level, while avoiding biased imputations caused by incomplete evaluations. Nevertheless, there were no missing values in any of the demographic, clinical or cytokines/growth factor data (except IL-7, which was deleted from the analyses) assessed in this study. We computed the estimated marginal mean values of the treatment and diagnostic groups and the treatment X group interactions and used (protected) pairwise contrasts (least significant difference at p=0.05) to examine differences between the treatment conditions (CBD compared with vehicle) and the group x treatment interactions. The tests were two-tailed, and statistical significance was defined as p < 0.05. IBM SPSS windows version 28 was used to perform the statistical analyses. The statistical studies followed the International Conference on Harmonisation's E9 statistical guidelines (November 2005) . Using a two-tailed test with a significance level of 0.05 and assuming a power of 0.80, an effect size of 0.2, two groups, 4 measurements with a . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 intercorrelations of around 0.4 showed that the estimated sample size for a repeated measurement design ANOVA would be around 44.

GO (biological processes, geneontology.org) enrichment analyses were performed separately on the upregulated and downregulated differentially expressed proteins (DEPs) using the R package clusterProfiler [35] and GO.db [36] . The dotplots and the results of the enrichment analysis are always shown using FDR corrected p-values and q-values. The PPI was constructed based on our DEP list and the gene members of the obtained GO terms. All interactions among the DEPs were retrieved from STRING database version 11.5 [37] using a high confidence score > 0.900. In addition, we constructed an expanded PPI network around the upregulated DEPs using OmicsNet 2.0 (OmicsNet) and the IntAct Molecular Interaction Database (https://www.ebi.ac.uk/intact/ (accessed on 2-2-2022) and examined the PPI network for its enrichment terms using the REACTOME (European Bio-Informatics Institute Pathway Database) pathways (https://reactome.org, accessed on 19 September, 2021). Table 1 shows the socio-demographic data and clinical data of the patients and controls in this study. There were no significant differences in sex distribution, education, and smoking between the study groups. Patients were somewhat younger and showed a higher BMI.

Nevertheless, all those variables were controlled for by entering these data in the GEE analysis.

The mean HAM-D and STAI scores were significantly higher in patients as compared with . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 controls, indicating that most patients suffered from medium to severe clinical depression and anxiety. Table 2 shows the results of GEE analyses with the effects of treatment. CBD 0.1 µg/mL did not have significant effects on the immune profiles. CBD 1.0 µg/mL significantly increased the growth factor and reduced the CIRS profile. At 10.0 µg/mL, CBD significantly decreased Th-1, Th-17, CIRS and the neurotoxicity profiles but increased the growth factor and T cell growth profiles. The effects of time on these 7 immune profiles remained significant after FDR p correction (at the p=0.0012 level).

There were no significant effects of any of drugs on any of the immune profiles (even without FDR p correction). For example, there were no significant effects of sertraline (p=0.528), other antidepressants (0.982), benzodiazepines (p=0.944), and atypical antipsychotics (p=0.172) on the Th-1 profile while the effects of treatment remained significant at p<0.001. In addition, we have also examined the interaction between the drug state and the 4 conditions (vehicle and 3 CBD concentrations) but none of these was significant. For example, when examining the Th-1 profile, there were no significant interactions between time x sertraline (p=0.955), other antidepressants (p=0.905), benzodiazepines (p=0.545), and antipsychotics (p=0.608). Moreover, gender, age, BMI, and smoking did not affect any of the immune profiles even without FDR p correction. Table 3 shows the impact of CBD on the different cytokines/growth factors. CBD 0.1 µg/mL had no significant effects on any of the cytokines/growth factors. CBD 1.0 µg/mL significantly decreased the production of sIL-1RA, CXCL8, IL-9, IL-10, IL-13, CCL11, IFN-γ, . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101/2022.02.04.22270489 doi: medRxiv preprint CCL2, CCL4, and CCL5, and increased the production of IL-1β, IL-2, IL-4, IL-15, FGF, GM-CSF, CXCL10, TNF-α, and VEGF. CBD 10.0 µg/mL had a major impact on most immune markers (except IL-2). CBD 10.0 µ/mL dose-dependently (from CBD 1.0 to 10.0 µg/mL) increased IL-1β, IL-4, IL-15, IL-17, GM-CSF, TNF-α, FGF, VEGF, and decreased sIL-1RA, IL-6, CXCL8, IL-9, IL-10, IL-13, CCL11, G-CSF, IFN-γ, CCL2, CCL3, CCL4, and CCL5. Such a dose-response was not found in the case of IL-2, CXCL10, and PDGF.

Differences depression versus controls. Table 4 shows the results of the same GEE analyses as described in table 2 but with the focus on the group differences and time X group interactions (the latter are shown only when they are significant). Table 4 shows that the Th-2, Th-17, Th-2, IRS, CIRS, Tcell activation, and neurotoxicity profiles were all higher in depression than in controls, while all interaction terms were non-significant. These intergroup differences remained significant after applying FDR p correction (at p=0.0484). Table 5 shows the differences in the separate cytokines/growth factors between both groups: sIL-1RA, IL-5, IL-9, IL-12, IL-15, IL-17, FGF, G-CSF, IFN-γ, CXCL10, TNF-α, and VEGF were significantly higher in depression than in controls. CCL3 was significantly lower in depression than in controls. There were significant interaction patterns for CXCL8, IL-10, GM-CSF, and CCL5. ESF, Figure 1 shows that depression patients have at the 3 CBD conditions lower GM-CSF than controls, whereas in control conditions there are no differences. ESF, Figure 2 shows that CCL5 levels were higher in patients than in controls in all 4 conditions. ESF Figure 3 shows the interaction pattern for CXCL8 indicating that this chemokine was higher in depressed patients than in the control, CBD 0.1 and 1.0 µg/mL conditions, whereas those intergroup differences almost disappeared in the CBD 10.0 µg/mL . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10. 1101 /2022 condition. This indicates that the latter concentration tends to normalize the increased CXCL8 values. IL-10 levels were not significantly different between both groups at any condition, but while there was a trend towards higher levels in the control, 0.1 and 1.0 µg/mL conditions, there was a trend towards lowered levels in the 10.0 µg/mL condition in depression as compared with controls.

In this analysis we entered the DEPs which were upregulated and downregulated by 1.0 µg/mL. When performing annotation and enrichment analyses, the same GO terms with large gene members were often found in both the upregulated and downregulated networks. To avoid GO terms with a large member of genes, only enriched GOs with a size of their gene members < 90 genes were of interest. After that, we filtered to select the enriched GOs which contain our genes > 7% with a gene ratio > 25%. Table 6 and Figures 1 and 2 show the results and the dotplots of the GO enrichment analyses performed on the upregulated and downregulated genes.

To delineate the relationship between these two sets of DEPs, we examined the protein interactions. The gene members in the GO:0042531 and GO:0048245 terms were aggregated and included in the up and downregulated DEP sets. All interactions among these genes were retrieved using STRING database version 11.5 [37] with a high confidence score > 0.9. Some genes that had no interactions with this criterion were then discarded from the plot; except VEGFA and IL9 which are in our DEP list. Figure 3 displays the plot of all protein-protein interactions among these genes. VEGFA and IL9 were not found to interact with the other DEPs.

IL-4 and CCL5 were present in both GO terms. The results show that CBD increases the regulation of tyrosine phosphorylation of STAT protein while it decreases eosinophil . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101/2022.02.04.22270489 doi: medRxiv preprint chemotaxis. Table 7 shows the REACTOME pathways that are over-represented in the enlarged PPI networks of the upregulated and downregulated DEPs.

The first major finding of this study is that CBD 0.1 µg/mL has no significant effects on any of the immune profiles, CBD 1.0 µg/mL decrease CIRS activity and increases growth factor production, and that CBD 10.0 µg/mL significantly suppresses Th-1, Th-17, IRS, CIRS, and neurotoxicity profiles, while increasing the growth factor and T cell growth profiles. Millar et al.

[38] in a systematic review showed that the administration of CBD (oral route) used in human studies ranged between <1 to 50 mg/kg/day [38] . The same authors [39] also published another systematic review on the pharmacokinetics of CBD administration and found that administration Nevertheless, in the rodent, the concentrations in mesenteric lymph nodes were more than 50fold higher than in the spleen and 250-fold higher than in plasma [28] . The latter authors ascertain that oral CBD coupled with dietary lipids may considerably increase delivery of CBD to the plasma and mesenteric lymphatic system. As such, lymphatic PBMCs may be exposed to high CBD concentrations in the 5-20 µg/mL range when using this targeted approach, which will eventually be applied in patients with inflammatory disorders [28] . Accordingly, in our study we employed not only the 0.1 and 1.0 µg/mL concentrations but also the 10.0 µg/mL concentration.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10. 1101 /2022 It is important to note that, in animal studies, there is a significant association between plasma and brain CBD concentrations whereby a higher intake results in higher plasma and brain concentrations [40] . Therefore, we may conclude that CBD treatments, which normally use the low oral dosages, will not result in any significant effects on M1, Th-1, Th-2, Th-17, IRS, CIRS or the neurotoxic profiles and on the cytokines/growth factors measured here. Nevertheless, when using the higher intravenous dosage, CIRS functions may be suppressed as indicated by a significant suppression of IL-10, IL-13 and sIL-1RA production, while the dose-response effects of CBD 1.0 to 10.0 µg/mL indicate a reduced production of all CIRS products, and an increased production of IRS products including IL-6, CXCL-8, IL-9, CCL11, G-CSF, IFN-γ, CCL2, CCL3, CCL4, and CCL5.

These findings extend those of previous papers showing that CBD has a dose-related effect on immune cells [41] . Different studies established that CBD 1.0 -10.0 µg/ml may suppress IFN-γ production by PBMCs [42] [43] [44] . Kozela et al. [44] found that CBD 1µM had an dose-related inhibitory effect on IL-6 production and Anil et al. [45] reported that CBD 4 µg/ml can reduce CXCL8 levels more than dexamethasone in a model of lung epithelial cells, although treatment with CBD in macrophages may result in increased CXCL8 levels [45] . Zgair et al. [28] observed that the expression of IFN-γ and GM-CSF by CD3+ T cells of multiple sclerosis patients, but not normal controls, was dose-dependently reduced by CBD 2.5-20 µg/mL.

On the other hand, our study established that CBD 1.0 to 10.0 µg/mL dose-dependently increased the production of growth factors including FGF and VEGF, and IL-1β, IL-4, IL-15, IL-17, GM-CSF, and TNF-α. As such, the higher therapeutic dosages and the targeted treatment with lipid-associated CBD may induce pro-inflammatory effects which linked to the anti-CIRS effects may have clinical relevance.

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Our results extend those of previous papers showing that CBD 1 µM had no effect on TNF-α production while CBD 10 µM induced its production. [46] . On the other hand, an animal model revealed that higher concentrations of CBD (50 µg/mL, 100 µg/mL) can inhibit TNF-α production from microglia cells [47] . CBD 1.0 µg/mL and 10 µg/mL may enhance plasma IL-4 levels, whereas rodent studies showed that CBD 20 mg/kg/day (IV) may reduce plasma IL-4 levels [48] . Other studies established that CBD 1 µM, but not 0.5 µM, can inhibit IL1-β secretion from monocytes [46] and that the dose-response curve of CBD 2.5 -20 µg/dL indicated suppressant effects on IL-17, GM-CSF and TNF-α. In our study, CBD 1.0 µg/mL, but not 10.0 µg/mL, significantly increased IL-2 production. In this respect, Chen et al. reported that CBD may enhance or suppress IL-2 depending on the stimulus condition and that in strongly stimulated cells, CBD may suppress IL-2, but following lower stimuli CBD may enhance cytokine production [49] . Kaplan et al. investigated the effects of CBD in stimulated splenocytes and splenic T cells and found that CBD 1-10 µM dose-dependently suppresses IL-2 production

[43].

The above differences between studies may be explained by the different stimuli and models used to assess the immune network. We used a combination of LPS + PHA to stimulate diluted whole blood and analyzed cytokine and growth factor production in culture supernatant [8, 9, 50, 51] . This diluted whole blood method more adequately reflects the in vivo cytokine production than assays on isolated PBMCs [8, 9, 50, 51] The method used in the present study retains all natural cell-to-cell connections, whereas techniques that isolate PBMC subtypes change the lymphocyte/monocyte ratio thereby affecting cytokine production [8, 9, 50, 51] .

Moreover, because some cytokines (including the master immunoregulatory cytokine IL-10) are . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101/2022.02.04.22270489 doi: medRxiv preprint produced by macrophages, Th-0, Th-1, Th-2, and Treg cells [52] , it is critical to assess IL-10 in whole blood cultures with conserved cell-to-cell interactions.

The second major finding of this study is that depression is accompanied by increased production of the Th-1, Th-2, Th-17, and Treg cytokines and a partial activation of the M1 phenotype (increased production of TNF-α, sIL-1RA, and CXCL8) as compared with heathy controls. These findings support the results of a recent review showing that depression is accompanied by increased activities of M1, Th-1, Th-2, Th-17 and Treg cytokines [1] .

Regarding the M1 macrophage profile, increased TNF-α, sIL-1RA and CXCL8 levels were also established in recent meta-analyses [53, 54] . However, in the current study, the stimulated production of IL-6 was not elevated in depression, whereas increased serum/plasma IL-6 was established in MDD/MDE in reviews and a meta-analysis [4, 53] while an early study found increased IL-6 in culture supernatant of PBMCs [55] . The current study and a recent metaanalysis [53] did not detect any changes in the stimulated production or serum/plasma levels of IL-1β, whereas an early study reported a higher stimulated production of IL-1β in MDD with melancholic features as compared with controls [55]. Köhler et al. also examined CXCL8 but they could not find any changes in MDD [53] . While, in the current study, CCL3 was significantly lower in depression, Köhler et al. [53] did not detect any differences in this chemokine. Nevertheless, as explained above, differences among studies may be explained by differences in the media used, e.g., culture supernatant of diluted whole blood versus serum/plasma, and also by differences in depression phenotypes, e.g. melancholia versus simple major depression.

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The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10. 1101 /2022 Regarding the Th-1 profile, we found highly significant elevations in culture supernatant IFN-γ and IL-12, but not IL-2, production in MDD/MDE. Some previous studies also reported increased IFN-γ levels in MDD patients [56, 57] We found significant increases in Th-2 cytokine production with increased levels of IL-5 and IL-9 in MDD patients, whereas there were no differences in IL-4 and IL-13. A previous meta-analysis [53] found that serum/plasma IL-13 levels were significantly elevated in MDD but that IL-4 and IL-5 did not significantly differ between groups. Other authors found increased IL-5 levels in medication-free MDD patients and in MDD patients with or without obesity as a . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. Here we report that activation of the Th-17 T cell line as indicated by increased supernatant IL-17 production. Both the review by Maes and Carvalho [1] and the meta-analysis by Kohler et al. [53] showed that IL-17 levels are increased in depression. In the present study we identified that the IRS as well as CIRS immune profiles were higher in depression than in controls, which supports the review of Maes and Carvalho [1] . However, it should be stressed that the master regulatory cytokine IL-10 was not significantly higher in depression versus controls indicating that those CIRS functions are insufficient to damp IRS activation.

Importantly, in the current study we found that the neurotoxicity profile was significantly higher in patients than controls which was attributable to increased CXCL8, IL-12, IL-15, IL-17, TNF-α, IFN-γ, CXCL10, and CCL5 levels. Such findings further support the neurotoxicity theory of both MDD/MDE which conceptualizes that increased levels of neurotoxic cytokines/chemokines exert neuro-affective toxicity on brain functions leading to depression [1] .

These effects are additionally aggravated by increased nitro-oxidative stress, hypernitrosylation, and bacterial load, and lowered levels of neuroprotective antioxidants [67, 68] .

Interestingly, depression was also accompanied by increased levels of growth factors, including FGF, PDGF and VEGF. Previously, Wu et al., in a meta-analysis, found higher peripheral FGF in MDD as compared with healthy controls [69] . They hypothesized that increases in peripheral FGF2 may be a compensatory effect in order to counter the increased oxidative stress and dysfunctional blood-brain-barrier [69] . Moreover, MDD patients who received treatments had lower levels of peripheral FGF [70] . We also found elevations of in PDGF and VEGF levels in MDD/MDE patients which extends the results of previous studies . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101/2022.02.04.22270489 doi: medRxiv preprint [71] [72] [73] . Increased PDGF may constitute a compensatory response since exposure to prolonged stress and higher levels of pro-inflammatory cytokines may damage the blood-brain-barrier resulting in increased PDGF production [74, 75] . However, Raymond et al. found no differences or even lower VEGF levels in MDD patients as compared with healthy controls [71].

The third major finding of our study is that CBD did not normalize the aberrations in the Receptor signaling, which all play a role in depression [77] . As such, higher CBD concentrations may aggravate the immune pathophysiology of depression (and other inflammatory disease). In addition, the 1.0 µg/mL concentration also decreases eosinophil chemotaxis as well as the phosphatidylinositol signaling system (converting extracellular to intracellular signals and mediating cell metabolism, survival and proliferation), the forehead box (FoxO) signaling pathway (mediating cell-cycle control and oxidative stress) and the sphingolipid signaling . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101/2022.02.04.22270489 doi: medRxiv preprint pathways (producing sphingolipids which are involved in signal transduction) (https://reactome.org). Moreover, since CBD significantly decreases IL-10 production, we may conclude that treatments with CBD concentrations ranging from 1.0-1.0 µg/mL should be avoided in depression and probably in most immune and autoimmune disorders. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101/2022.02.04.22270489 doi: medRxiv preprint secured financial support. All authors contributed to interpretation of the data and writing of the manuscript and agreed to publish the final version of the manuscript

AMERI-ASIA MED CO, Ltd, supported this work

The study was conducted according to International and Thai ethics and privacy laws.

Approval for the study was obtained from the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (#528/63)

All controls and patients provided written informed consent prior to participation in this study

The dataset generated during and/or analyzed during the current study will be available from the corresponding author (M.M.) upon reasonable request and once the dataset has been fully exploited by the authors

The authors have no conflict of interest with any commercial or other association in connection with the submitted article . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101/2022.02.04.22270489 doi: medRxiv preprint

Ogłodek . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted February 6, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. (1) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101/2022.02.04.22270489 doi: medRxiv preprint differential T cell response to HIVgp120. J. Leukoc. Biol. 2012, 92 (5) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 6, 2022. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted February 6, 2022. ; https://doi.org/10.1101/2022.02.04.22270489 doi: medRxiv preprint Results of GEE analyses with immune profiles as dependent variables and time, group (depression versus controls) and time by group interactions as explanatory variables, and age, sex, and body mass index as covariates. Shown are the time effects (Wald) with a,b,c,d ,indicating pairwise comparisons among the treatment conditions. All data are shown as estimated marginal means (mean ±SE). See ESF Table 2 for explanation of the profiles and cytokines measured in this study. IRS: immune-inflammatory response system, CIRS: compensatory immunoregulatory response system, Tcell: T cell growth, GF: growth factors, NT: neurotoxicity Table 2 for explanation of the profiles and cytokines measured in this study Results of GEE analyses with immune profiles as dependent variables and time, group (depression versus controls) and time by group interactions as explanatory variables. Only the effects of groups are shown as the interactions between group and time were always non-significant (the time effects are shown in Table 2 ). All data are shown as estimated marginal means (mean ±SE) See ESF Table 2 for explanation of the profiles and cytokines measured in this study 

The Compensatory Immune-Regulatory Reflex System

Mechanistic explanations how cell-mediated immune activation, inflammation and oxidative and nitrosative stress pathways and their sequels and concomitants play a role in the pathophysiology of unipolar depression

Differences in the immune-inflammatory profiles of unipolar and bipolar depression

The effects of groups and/or the interactions between group and time are shown when significant (the time effects are shown in Table 3)

Effects of cannabidiol on the activated immune-inflammatory pathways in major depressive patients and healthy controls

Advanced Virtual and Intelligent Computing

ESF Figure 1. Effects of the Interaction between diagnosis and cannabidiol (CBD) administration on the production of granulocytemacrophage colony-stimulating factor (GM-CSF) in culture supernatant

 Table 6 Results of GO enrichment analysis of biological processes performed on the upregulated and downregulated protein-protein interaction networks of the differently expressed proteins (DEPs) induced by cannabidiol (CBD, 1 µg/mL)