MOODSTRATIFICATION has now come to its end and ran from 2018-2023. The hypothesis of MOODSTRATIFICATION was that a large proportion of the severe mood disorders (i.e. major depressive disorder (MDD) and bipolar disorder (BD)), are in part caused by deviant immune reactions. These deviant immune reactions are based on(inborn and acquired) defects in the function of immune cells, in particular in the functions of monocytes/macrophages and T cells. Since these immune cells are essential for a proper development and function of the brain, in particular the emotional brain, this results in

 

1. Mal development and malfunctioning of the brain, in particular of the part of the old brain that regulates emotions (the limbic system), and
2. Flares of low grade chronic inflammation of the brain , further impacting functioning of the brain.

 

Objectives of the project

• The firstobjective of MOODSTRATIFICATION was to test and refine this novel concept by further exploring the earlier collected large datasets of the previous EU funded projects of the coordinator MOODINFLAME (2008–2012) and PSYCH-AID (2013-2017).
• The secondobjective of MOODSTRATIFICATION was to develop simple blood tests to measure the above mentioned immune defects in clinical practice.
• The thirdobjective was to carry out proof-of-principle clinical studies with T cell enforcing therapies (low dose IL-2 and thymus hormone) to correct the immune defects and see whether this improves depression in the patients (WP2),
• The fourthobjective was to select (in later phases of the project) depressed patients – with a specific immune profile and to treat patients with this profile with an intervention targeting the abnormality of the profile to see whether such preselection would improve therapeutical outcome. For this we would in particular use the simple blood tests developed in the second objective. We also would calculate the cost-benefit of such approach.

 

Results

With regard to objective 1 we have found that there definitely are immune defects in patients with MDD and BD. We also found that these immune defects slightly differ between MDD and BD:

• MDD is characterized by a premature aging (senescence) of the T cell and monocyte/macrophage system leading to mild low grade inflammation, which under circumstances of  childhood trauma and obesity will lead to more signs of low grade inflammation (monocyte inflammatory activation, higher serum levels of pro-inflammatory cytokines). Genetic factors and chronic viral infections play a role in the premature aging of the immune system in MDD.
• BD is characterized by a reduced maturation of a certain subpopulation of T cells important for building immune memory. There also is a reduced level of IL-7 in serum. IL-7 is a T cell maturation factor. Other subpopulations of T cells mature better, including a population of T cells that stimulates low grade inflammation (Th17 cells). During depressive or manic episodes a stronger low grade inflammation occurs. This is induced by monocyte/macrophage activation and a rise in serum inflammatory cytokines.

Collectively this means that both mood disorders are characterized by T cell and monocyte/macrophage abnormalities which were shown in other investigations of our group and those of others to be capable of influencing the function of our emotional brain.

 

With regard to objective 2 we have developed simplified reliable blood tests to measure these T cell and monocyte/macrophage abnormalities via our commercial partners, APD (Belgium) and PRONTO (Israel). The latter firm has also developed a relatively easy to perform finger prick test.

Collectively this means that easy to perform assays are in principle available

 

With regard to objective 3 we completed:

• Two double blind placebo-controlled trials using injections with low dose IL-2, one in Paris (AP-HP, the DEPIL trial) and one in Milano (OSR, the IL2REG trial). The trials ran from 2019-2023 and unfortunately recruitment was hampered by the corona pandemic.

Nevertheless we have been able to recruit sufficient numbers in both trials to reach meaningful and statistically reliable numbers of treated patients.

Outcomes of both low dose IL-2 trials are a breakthrough.

In both trials the depression of the patients improved due to the low dose IL-2 treatment. The improvement went in parallel with the stimulation of a special T cell population that dampens low grade inflammation reactions and also has effects on our limbic system. Also the low dose IL-2 treatment was very safe with few, if any, adverse side-effects.

Data also show that in particular patients with high signs of low grade inflammation and resistant to regular anti-depressant treatments benefit from this immune therapy. These outcomes urge for larger placebo-controlled trials to confirm the data.

• The open label TIDAM study (running in 2022 and 2023) in Rotterdam (EMC) to treat depressed patients with thymus hormone (Thymalfasin). The depressed patients belonged to a group of patients with an already existing immune deficiency. We established that in these patients with this immune deficiency depression is more common.

After a lengthy discussion with the ethical review board of EMC (in which we had first to proof that depression is more common in this group of immune deficient patients, and that the immune defects are similar to the premature senescence we found in MDD patients), we were allowed to treat up to 20 patients to monitor a putative improvement of the depression and the T cell senescence.

We have only been able to treat 5 depressed patients in this non-placebo controlled trial. Nevertheless outcomes are promising and urge for further studies.

Outcomes are that Thymalfasin treatment induced in all 5 patients an improvement of depression and also a change in the parameters of senescence of T cells. The latter correlated to the improvements found in the depression scores. Since the trial is not placebo controlled we are not sure whether these improvements are due to the drug or linked to the natural course of the disease. Very mild and transient adverse side effects were only seen in one patient

Collectively this means that immune drugs (which directly target the T cell system and indirectly low grade inflammation) are novel drugs capable of treating depression with high safety and minimal side effects. Further studies are urgently needed.

 

With regard to objective 4 we have started (after a discussion with all partners regarding outcomes of WP1 and considering the literature) the MOODSTRATA trial, in which severely depressed patients do or do not undergo a physical training scheme (spinning). Both groups are treated as usual (TAU) with regular medication.

1. The primary aim is to find an immune senescence parameter which predicts favorable outcome (it is known that physical activity, like spinning, combats immune senescence).
2. Secondary aims involve clinical improvement and predictive capability/change of other immune parameters.

The trial started in 2021 and ran in Muenster (WWU), Leuven (KU-Leuven), Ulm (UULM) and Groningen (UMCG). The separate studies are designed so that they can be pooled for some of the immune parameters. We have reached the required 90 patients in July 2023 to enable us to have meaningful statistically significant data..

Outcomes are that we identified a group of patients with certain T cell senescence characteristics (high CD4+ central memory T helper cells) who benefitted in particular of the spinning therapy.

Spinning therapy did change apportioning in the CD4+ memory T cell subgroups.

In the last half of 2023 we will also evaluate the predictive capability of the other immune parameters which have not been fully characterized.

 

In a special work package we also estimate the economic costs and benefits of the physical endurance therapy described above, particularly in patients with recurrent and drug-resistant depression. These patients were shown by AU (partner in Denmark) in this project to have the strongest adverse social consequences of their disease.

AU has developed an R-based algorithm to calculate the cost-benefit of preselecting MDD patients on the basis of the senescence characteristics (lab tests) and the better outcomes of treatment  with less disease burden for patient and society.

AU has calculated that the approach of preselecting patients is cost-effective.

 

Conclusion

Despite considerable delays, due to the lock downs of the corona pandemic and (administrative) struggles with our ethical review boards, we have successfully ended the MOODSTRATIFICATION project in M66 and we have reached virtually all of our objectives and have delivered important new break-through data regarding:

• The abnormal immune (T cell and monocyte/macrophage) profiles of MDD and BD and differences in these abnormal immune profiles between these disorders, facilitating
  1. An early differential diagnosis between the disorders with the clinical applicable (easy) assays developed in this project.
  2. The preselection possibility to target specific subgroups with a specific immune therapy (see underneath).
• A drug intervention (low dose IL-2) increasing the percentages of T regulatory cells in depressed patients with correlating improvements in depression severity in two placebo-controlled trials. Particularly patients with clear signs of low grade inflammation benefit from this intervention.
• A drug intervention (Thymalfasin) mildly improving signs of premature immune T cell senescence in depressed patients with correlating improvements in depression severity in a limited non-placebo controlled trial.
• A specific T cell senescence profile in severely depressed patients (likely testable with relatively simple methods), which is in particular sensitive to react in a clinical and socio-economic beneficial way to spinning therapy.
• Data show that antidepressant treatments (be it immune or non-immune) “do not fit all”, but that depressed patients need to be preselected to enable personalized immune and non-immune treatment schemes for better results.

 

 

Pronto MOODGDx KIT

The Fingerprick (FP in short) is a quick, patient-friendly method for collection of minor volumes of blood for genetic analysis. It is performed on the patient’s finger using a dedicated pressure-activated sterile lancet. The collected capillarial blood can be directly collected from the finger top for downstream applications.

This method has multiple major advantages over traditional blood collection methods. To mention a few:

• Enabling Point-of-Care collection of blood
• Safety and minimal invasiveness
• Ease and readiness of performance

Pronto provides the centers participating in the Mooodstratification project with ready-to-use FP kits so that these centers can easily collect finger blood from patients taking part in the study.

Each provided kit contains the following components:

1. Lancet device for finger-pricking
2. MICROSAFE capillary tube for blood drop collection
3. Tempus blood RNA 5 mL microcentrifuge tube
4. Sterile alcohol swab
5. Instructions manual

The lancet designed to reach a 2.4 mm depth, ensuring obtaining enough capillarial blood for the

downstream assay, with minimal suffering from the patient’s side.

Microsafe capillary tube is a single use plastic capillary used for accurate, volumetric collection and dispensing of small amounts of blood. After pricking the patient finger, the capillary is immediately applied to the blood drops. Filling of the capillary occurs automatically. The capillary provided with the kit designed to collect 75-80 ul (-0%-+10%) of blood. Once the blood reaches this volume, suction ceases automatically. For dispensing the collected blood, the bulb of the capillary should be squeezed firmly.

This collection step should be performed quickly in order to ensure that the blood does not clot inside the capillary.

The Tempus reagent is a powerful stabilizer used for total RNA isolation from whole blood for downstream gene expression analysis. Upon dispensing blood drop in the tube containing the Tempus reagent, whole blood cells are immediately lysed, cellular RNases are inactivated and the comprising RNA is selectively precipitated and stabilized in a single step, without the need for red blood cells lysis or proteinase K treatments.

After dispensing the blood into the tube, the mixture must be immediately vigorously mixed for at least 15 seconds, optimally using a Vortex mixer, to ensure that the stabilizing reagent is thoroughly mixed with the sample. Failure to do so leads to the formation of micro-clots and disrupts the gene expression-profile assay.

Briefly centrifuge the 1.5 mL microcentrifuge tube to remove drops from the inside of the lid.

Freeze the 1.5 mL microcentrifuge tube at -80°C until shipping it to Pronto Diagnostics for gene expression assays.

Safety guidelines

• Please read the Tempus Blood RNA Tube safety data sheet before using the
• Avoid any skin and eye contact, inhalation and ingestion of the Tempus reagent filled in the 5 mL microcentrifuge tubes.
• Blood drawing from finger should be handled by professional medical staff with appropriate personal protective equipment (gloves, gown, etc.).
• Ensure hand washing and fingertip cleaning using a sterile alcohol swab prior to finger
• Discard the lancet device and MICROSAFE capillary tube in an appropriates biohazard container following the FP

Additional safety concerns and procedure-success tips to consider:

• The FP should always be performed on the middle or ring fingers, minimizing the likelihood of bone puncturing.
• FP is ought to be avoided on the same side of the body of patients underwent mastectomy on that
• FP lancet is latex-free to minimize allergenicities.
• Its advised that the whole collection process take no more than 30 seconds to avoid blood
• Try covering the puncture site with a warm, moist towel for 4-5 min prior to puncturing to increase arterial blood flow to the
• Its recommended to refrain from collecting the first drop of blood that oozes out of the punctured finger, as this drop can contain contaminants from the finger surface such as remnants of alcohol from the sterilization swab, skin surface bacteria and other

Using the Tempus-fixed material it is possible to determine (as has been reported before in D1.5):

 

1. Cluster 1 activity in monocytes via TGFBR3 and NFATC2 expression
2. Cluster 2 activity in monocytes via CCL2 expression
3. Apoptosis activity in monocytes via BAX expression
4. M(hb) state/cholesterol metabolism in monocytes via CD206, CD163, LXR-a, ABCG1 and HMOX1. ABCA1 can be measured via its correlate CAMP
5. MVK activity in monocytes might be measured via its correlate SERPINE1 (PAI-1)
6. T reg state via FOXP3
7. Th1 state via TBX1
8. Th17 state via ROR-γ