Immune targets for novel depression therapeutics DRAFT Revised submission to Nature Reviews Drug Discovery, 18 August 2021 Immune targets for therapeutic development in depression: towards precision medicine   Wayne C. Drevets1*, Gayle M. Wittenberg2*, Edward T. Bullmore3,4, Husseini K. Manji5 1 Neuroscience, Janssen Research & Development, LLC, San Diego, CA, USA 2 Data Science, Janssen Research & Development, LLC, Titusville, NJ, USA 3 Department of Psychiatry, University of Cambridge, Cambridge, United Kingdom 4 Cambridgeshire & Peterborough NHS Foundation Trust, Cambridge, United Kingdom 5 Science for Minds, Johnson & Johnson, New Brunswick, NJ, USA *Contributed equally as co-lead authors. Corresponding Author: Husseini K Manji, MD, FRCPC Global Head J&J Science for Minds hmanji@its.jnj.com Keywords: Title: 85/90 characters w spaces Abstract: 206/200 words (unstructured) Main Text: 10,189 words Display Items (Figures=3 + Tables=1 + boxes=1): 5/7 References: 178/(150-200). Level 1 Headings: 38 characters include spaces Level 2 Headings: 39 characters include spaces Level 3 Headings: ~80 ABSTRACT Over the past two decades compelling evidence has emerged indicating that immune mechanisms can contribute to the pathogenesis of major depressive disorder (MDD), and that drugs with primary immune targets can improve depressive symptoms. These data may serve to illuminate paths to novel therapeutics. Among patients with MDD, heterogeneity exists with respect to symptoms, treatment responses, and biological correlates. A narrowing of the phenotypic description toward a more biologically defined patient group that achieves higher treatment response rates would constitute a major advance in clinical psychiatry. Moreover, the extant data indicate that depressed patients who manifest elevated pro-inflammatory biomarkers are less likely to respond to conventional antidepressant drugs, so in such individuals it is conceivable that novel immune-based therapeutics hold potential to address severe unmet clinical needs. This article outlines a framework for developing drugs targeted to a novel patient subtype within MDD, while reviewing the current state of neuroimmune drug development for mood disorders, including evidence for a causal role of immune mechanisms in the pathogenesis of depression, together with targets under investigation in randomized controlled trials, biomarker evidence elucidating the link to neural mechanisms, biological and phenotypic patient selection strategies, and the unmet clinical need among MDD patients manifesting biomarker evidence of immune-associated depression. INTRODUCTION Currently available therapeutics for major depressive disorder (MDD) lead to remission in approximately one third of patients and partial remission in another third1. This heterogeneity in treatment outcomes is thought to reflect biological heterogeneity within the MDD diagnostic category. Among characteristics of patients with poorer treatment outcomes are various forms of immune biomarker abnormalities2-4, which has raised interest in targeting the immune system in the quest for developing precision medicine approaches in psychiatry. The relationship between immune dysregulation and depression cuts across a fault-line in conventional diagnostic categorization of depression5. On one side of the line is a large group of patients with MDD, defined by a clinical checklist comprising at least one of two core symptoms (low mood and anhedonia), and at least 3 of a list of 7 other symptoms, for at least 2 weeks, to a distressing or disabling degree of severity. Formally, the diagnosis of MDD is excluded if the patient has a medical or physiological condition that could explain the depression. On the other side is a second large group of patients who manifest major depressive symptoms in the context of primary immune disorders, with examples including rheumatoid arthritis, Crohn’s Disease, or Systemic Lupus Erythematosus. These depressed patients are ineligible for a diagnosis of MDD. Instead, they are typically diagnosed as cases of “co-morbid depression”, and coded more formally using the DSM-5 category, “depressive disorder due to another medical condition”, based partly on the criterion of “evidence from the history, physical examination, or laboratory findings that the disturbance is the direct pathophysiological consequence of another medical condition.” In this context, the immunology of depression is diagnostically disruptive, because it points to immune dysfunction as a dimension of pathogenesis that could contribute to both co-morbid depression (associated with clinical inflammation) and a proportion of cases who receive the diagnosis of MDD but also manifest laboratory evidence of associated low-grade inflammation. Moreover, additional heterogeneity likely exists within the larger subset of MDD patients manifesting immune-associated depression. For example, one group accrued evidence for an "immunometabolic” subtype of MDD based on the clustering of immunological and metabolic (e.g., leptin, insulin sensitivity) dysregulations along with specific behavioral symptoms reflecting altered energy intake/expenditure balance (hyperphagia, weight gain, hypersomnia, fatigue). This subgroup of MDD patients showed persistence of this cluster of clinical features and biomarker abnormalities across a several year follow-up period6. Nevertheless, other MDD subgroups have also been described which show distinct or partly overlapping clusters of immune biomarker abnormalities, along with different phenotypic patterns of symptoms and non-responsiveness to conventional antidepressants, especially those with predominantly serotonergic mechanisms4. Therefore, immune targets could provide both new approaches to antidepressant efficacy for a proportion of cases of MDD defined by biomarker evidence of immune dysfunction related to the targeted pathway, as well as for co-morbid depressive symptoms, which are commonly ranked among the greatest unmet needs by patients with rheumatoid arthritis and other immunological diseases7. To pursue these ideas, we address the criteria required to develop a therapeutic for a novel subtype for patients within the broader MDD diagnostic class (Box 1). First, we consider evidence for a causal association between immune dysregulation and depression, and the potential targets implicated thus far. Second, we walk through the set of randomized, placebo-controlled trials (RCT) run to date. We next examine emerging evidence of biomarkers associated with MDD for their potential as tools for patient stratification in clinical trials as well as in clinical treatment algorithms, and as endpoints in proof-of-concept studies of novel agents. We finally discuss the unmet need today among patients whose MDD appears mediated through immune mechanism. EVIDENCE FOR CAUSALITY There is overwhelming evidence that immune dysregulation and depression are associated or correlated with each other8-10. MDD has been associated with an elevation of pro-inflammatory cytokines and acute phase proteins in blood and cerebrospinal fluid (CSF), a decreased adaptive immune response, differences in the relative abundance of specific immune cell types, a bias towards autoimmunity, increased activation of microglia (a type of neuroglia which constitute resident macrophage cells within the central nervous system (CNS) and more (Figure 1). But regardless of the evidence for an association between immune dysregulation and depression, the most compelling rationale for considering the development of immune-targeted drugs for depression is if immune dysregulation can cause depression in susceptible individuals. For full validation, we must also understand the causal chains that mechanistically mediate the proximal effects of a peripheral or central immune target on the functional organization of the CNS, thus distally causing depressive changes in emotion, cognition and behavior. Here we review almost exclusively human studies, since these data are most directly relevant to the critical phase of early clinical development of novel immunotherapeutics for patients with depression. Nevertheless, strong preclinical evidence also indicates that peripheral and central immune mechanisms can have adverse effects on neuronal function and cause anhedonia, social isolation, and other depressive-like behaviors in animals11-15. Finally, we note that while immune pathways are complex and interconnected (Figure 1), most studies in MDD have focused on the role of pro-inflammatory actors of the innate immune system, and their therapeutic modulation. In contrast, the potential for harnessing mechanisms within the adaptive immune system to discover further therapeutic innovation in neuropsychiatry has remained relatively unexplored. Longitudinal and Challenge Studies Longitudinal studies have tested the hypothesis that inflammation can precede depression, which is a necessary condition for inflammation to be causal. There is supportive evidence for inflammation anticipating the later emergence of depression or depressive symptoms over a wide range of time scales. Blood concentration of IL-6 at age 9 years predicted an elevated risk of depression at 18 years in a UK birth cohort16. Middle-aged women who were not depressed but had C-reactive protein (CRP) greater than the upper limit of the normal range (3 mg/L) when tested in 2004 and 2008 were significantly more likely to be depressed for the first time in 2012 than women who had normal CRP levels over the previous years17. Finally, among women depressed at baseline, in the Netherlands Study of Depression and Anxiety (NESDA), higher IL-6 levels predicted a chronic course of MDD based on two, and six year follow-up evaluations18. Challenge studies have demonstrated that an intervention causing immune activation can lead to increased rates of depressive symptoms on short to medium timescales. For example, about a third of hepatitis patients clinically treated with the pro-inflammatory cytokine interferon-alpha (IFN) had developed major depressive symptoms when re-assessed 4-48 weeks later19,20. In many patients who manifested the new onset of a major depressive episode following IFN therapy, the administration of selective serotonin reuptake inhibitor (SSRI) antidepressants ameliorated these depressive symptoms21. As another example, the experimental administration of typhoid vaccine caused increased blood IL-6 and transient depressive or dysphoric symptoms within 24-48 hours22. Genetics and Epigenetics Genetic research is fundamental to target validation since the discovery that DNA variation in a particular gene is associated with a disease provides evidence implicating that gene in the pathogenic pathway. Genetically validated targets have improved probability of success for drug development in many therapeutic areas23. Genetic evidence that immune related genes are causally implicated in depression is just beginning to emerge. Only in the last few years have genome-wide association studies (GWAS) and meta-analyses become sufficiently powerful, with sample sizes in the order of 100,000s, to report statistically robust associations between allelic variation and MDD. In one of the larger studies, pathway analysis of 44 single nucleotide polymorphisms (SNPs) from an MDD GWAS gene list indicated enrichment for 19 pathways, including cytokine and immune response pathways24. Many genetic variants significantly associated with MDD by GWAS are in non-coding regions of the genome and were enriched at epigenetically active regulatory sites in fetal and adult brain tissue, and in peripheral lymphoid cells, indicating that genetic risks for MDD are likely to be phenotypically expressed through the adaptive immune system as well as the central nervous system25. In a candidate gene Mendelian randomization analysis, a functional SNP in the IL-6 receptor promoter region predicted levels of IL6, CRP and depressive symptom severity in a population cohort26. In another candidate gene study of a distinct functional polymorphism in the IL-6 receptor promoter region (rs1800795), the 'low IL-6' synthesizing genotype (CC) was associated with fewer depressive symptoms during IFN- treatment27. Further, coheritability between CRP levels and individual depressive symptoms has been demonstrated28. Finally, genetic variation in P2RX7, which encodes the purinergic P2X7 receptor (P2X7R; an ATP gated cation channel abundantly expressed in myeloid cells, including microglia, where P2X7R stimulation mediates activation of the NLRP3 inflammasome complex29) was implicated as being causally related to depression in an integrated depression GWAS with human brain proteome-wide association study (PWAS) in depression30. This finding was replicated in an independent depression GWAS and PWAS dataset and extended using cis-regulated mRNA levels associated with depression in an integrated depression GWAS plus human brain transcriptomic analysis. In candidate gene studies, haplotypes containing gain-of-function SNPs of P2RX7 (which increase P2X7R pore activity)31 were associated with increased time spent depressed32 and increased depression severity among patients with bipolar disorder (BD)33,34, MDD, and type-1 diabetes mellitus35,36,37. Epigenetics will also be important for validation of immune targets for depression and other stress-related disorders. In epigenetic studies in humans, as well as in animal models of MDD, DNA methylation of the glucocorticoid receptor gene (GR), and the GR-chaperone protein-coding gene FKBP5, caused under-expression of cortisol-responsive signals, releasing some innate immune mechanisms from the negative feedback normally mediated by the HPA axis and thereby increasing proinflammatory biomarkers in response to social and behavioral stress38,39. Such epigenetic “memories” of early life stress may reflect a persistent allostatic change related to the state of glucocorticoid resistance previously associated with MDD40 and with the clinically well-recognized risk of MDD in adult life following an episode of adversity in childhood41. These epigenetic data thus converge with the results of gene expression studies in depressed patients in providing evidence that immune system dysregulation and reduced GR signaling (which restrains the effects of some pro-inflammatory mediators including P2X7R signaling) contribute to the pathophysiology of MDD42. Genome-wide DNA methylation profiling in brain tissue obtained post mortem from individuals with mood disorders who died of suicide and in blood sampled in vivo from depressed patients with suicide ideation implicated additive epigenetic and genetic association with suicide risk at a locus in SKA2, which regulates GR transactivation43. Another study of tissue obtained post mortem from the prefrontal cortex of depressed suicide completers found differentially methylated regions versus non-psychiatric controls in the psoriasis susceptibility gene, PSORS1C3, and in TAPBP, a gene located in the major histocompatibility complex that encodes a glycoprotein which mediates interaction between major histocompatibility complex class I molecules and the transporter associated with antigen processing42. Finally, genome-wide DNA methylation and gene expression analyses performed in patients prospectively-defined as either responders or non-responders to escitalopram treatment reported two genes that exhibited increased DNA methylation and mRNA expression in the non-responders: JAK2, which activates both innate and adaptive immunity, and CHN2, which may affect hippocampal neurogenesis44. Interestingly, whole methylome screening in a small case-control study of MDD demonstrated that methylation of peripheral white blood cells was highly correlated with the methylome of frontal cortical and other brain regions45, suggesting that stress disorder-related epigenetic changes may be coupled across peripherally measurable (distal) immune cells and clinically unmeasurable (proximal) neuronal and glial cells. In summary, while emerging genetic data support the hypothesis that immune mechanisms contribute to the pathogenesis of mood disorders, highly penetrant mutations have not yet been identified which are clearly causal. These data instead corroborate the broader conclusion from genetic studies of psychiatric disorders that their etiology involves daunting polygenicity and phenotypic complexity, posing significant challenges for the elucidation of potential therapeutic targets46. Nevertheless, the genetic data converge with other evidence to implicate specific immune related gene networks in the pathophysiology of MDD, which has guided early efforts to evaluate drugs targeting these immune pathways as potential therapeutics for depression. IMMUNE TARGETS EVALUATED IN DEPRESSION Several immune system targeted compounds have been evaluated for potential antidepressant effects. The studies assessing these effects represent three approaches: first, meta-analyses of changes in depressive symptoms within aggregate data from clinical trials of primary immunological disorders; second, analyses of mood effects in immunology clinical trials where item-level patient data were available; and third, RCTs in MDD patients (Table 1). The former two approaches were opportunistic insofar as each study’s primary outcome measure assessed the efficacy of the compound tested on improving cardinal symptoms of a primary immunological disease, and the post hoc analyses of effects on depression ratings were exploratory. In contrast, the third approach provides the most direct exploration of the effect of a compound, or evaluation of a mechanism of action, as a potentially viable antidepressant pharmacotherapy. A range of targets and mechanisms have been evaluated, modulating different points within the complex immune networks. These include the production of prostaglandins targeted by non-steroidal anti-inflammatory drugs (NSAIDs); a variety of pro-inflammatory cytokines (TNF-, IL-6, IL-23/23, IL-23) targeted using monoclonal antibodies; p-38 MAP kinase, which orchestrates not only innate immunity, but is a key regulator of the adaptive immune system including T- and B-cell mediated functions; and B-cell related targets (CD20, BLS). Additionally, several classes of compounds have been evaluated which show anti-inflammatory effects among their pleiotropic actions (glucocorticoids, statins, minocycline, pioglitazone). For example, statins, in addition to their lipid lowering effects, have immunomodulatory and anti-inflammatory effects that include inhibition of monocyte expression of pro-inflammatory cytokines and reduction in CRP levels47,48. However, a limitation of most of these studies of anti-inflammatory treatments in depression is that target engagement was insufficiently assessed to address causality (i.e., which would require that the decrease in inflammation should be apparent and precede the improvement in symptoms). Another limitation of this research is its bias towards modulating inflammation and the innate immune response, leaving considerable room for innovation in targeting aspects of adaptive immunity, a wider range of immune cell types, and autoantibody-driven pathologies. Immunology Clinical Trials The ability of anti-inflammatory treatments to produce antidepressant effects was initially demonstrated in secondary analyses of studies in patients with immunological disorders which included depressive symptom ratings. These studies focused predominantly on non-steroidal anti-inflammatory drugs (NSAIDs) and cytokine inhibitors, and are included in several meta-analyses49-52 (Figure 2). Kohler et al. reported results for 6,262 participants across 14 trials, of which 10 evaluated the efficacy of NSAIDs and 4 evaluated cytokine inhibitors targeting TNF- or IL12/2349. They found significant pooled effects for NSAIDs (SMD=0.27, 95% CI=0.08-0.45) and cytokine inhibitors (SMD=0.38, 95% CI=0.22-0.88). Kappelmann et al performed a random effect meta-analysis of seven RCTs of 2,370 participants and found a significant effect of cytokine inhibitors on depressive symptoms compared with placebo (SMD=0.4, 95% CI: 0.22-0.59), and specifically for TNF- inhibitors (SMD=0.33, 95% CI: 0.06-0.6)50. Kohler-Forsberg et al. evaluated a wider range of drug classes and combined studies evaluating treatment effects in comorbid depression as well as in MDD. Overall, they reported significant effects across pooled NSAIDs (SMD=0.4, 95% CI: 0.18-1.62) and cytokine inhibitors (SMD=0.56, 95% CI=0.29-0.93), as well as glucocorticoids (SMD=0.9, 95% CI=0.36-1.44), statins (SMD=0.26, 95% CI=0.04-0.48), and minocycline (SMD=0.87, 95% CI=0.29-1.45). A sixth drug class composed of pioglitazone did not show a significant antidepressant effect (SMD=0.43, 95% CI=-0.7-1.56)51 (Figure 2). There are several challenges to the interpretation of improvements in comorbid depressive symptoms in the context of a medical immune or inflammatory disorder that is responsive to the same treatment. First, most patients with primary immunological disorders enter studies with depression ratings in the non-depressed range, so sensitivity for detecting an antidepressant effect is limited to the minority of patients who manifest clinically significant depression at baseline. Second, improvement in primary disease symptoms, including pain and fatigue, poses a potentially confounding variable when rating improvement in mood50. To address these concerns, Wittenberg et al. reported a mega-analysis of patient-level data combined from 18 RCTs conducted by Janssen or GlaxoSmithKline for one of nine disorders, and for nine drugs against seven different molecular targets (N=10,743 participants)52. Access to patient-level data enabled stratification of patients at baseline into a high-depressive symptom cohort, and a low-depressive symptom cohort, the former being the appropriately enriched population for detecting an antidepressant effect. Access to patient-level data also enabled the statistical adjustment for potential confounding factors including changes in symptom severity for the primary indication (e.g., changes in joint pain in trials of rheumatoid arthritis). The greatest effects were seen among anti-IL6 antibodies (SMD=0.8, 95% CI [0.20-1.41]) and an anti-IL12/23 antibody (SMD=0.48, 95% CI [0.26-0.70])(Figure 2). Antibodies targeting BLS and TNF- demonstrated nonsignificant trend-level efficacy for depressive symptoms, and small molecules targeting p38 MAPK or cyclooxygenase-2 (COX-2) demonstrated no significant antidepressant effect (Figure 2). While most attention on cytokine antagonists as antidepressants has focused on antibodies against TNF- and IL-6, some studies noted the positive effect of the IL-12/23 antagonist ustekinumab on depressive symptoms52,53. Newer compounds such as guselkumab, more selectively target IL-23. In patients with moderate-severe plaque psoriasis, guselkumab treatment was associated with significant improvement in anxiety and depression symptoms relative to placebo54. In summary, in seeking targets for which some hint of a potential causal link to MDD exists, a number of proteins have been suggested through longitudinal studies, inflammatory challenges, genetics and epigenetics, and studies in patients with comorbid immunological disorders. These include TNF-, IL-6, IL-23, COX enzymes, and P2X7R. To date, only a subset of these has been tested in patients with primary mood disorders. Immune Drug Development in MDD Of the relatively few studies that tested whether immunomodulatory drugs produce antidepressant efficacy in patients with MDD or BD, most used drugs that reportedly improved depressive symptoms arising comorbidly in a primary immunological disorder or influenced immunological processes that affect synaptic function in preclinical models. For example, several cytokines are considered “neuroactive” (e.g., TNF-, IL-6, IL-1β) because they modulate synaptic plasticity mechanisms including neurogenesis or dendritic remodeling, and/or mediate neuronal-glial interactions. The drug targets assessed to date include monoclonal antibodies (mAb) that neutralize cytokines or cytokine receptors, molecular factors downstream of cytokines (e.g., cyclo-oxygenases, prostaglandins, kynurenine metabolites), mechanisms affecting release and effects of cytokines, signaling molecules involved in the production and effects of cytokines (e.g., MAP kinases), and molecules involved in the maturation or processing of cytokines (e.g., P2X7R). Anti-cytokine or cytokine receptor antibodies The first anti-cytokine antibody tested in MDD patients was infliximab, an anti-TNF- antibody, studied in two RCTs. Raison et al focused on patients (N=60) who were moderately treatment resistant, and stratified patients based on high-sensitivity CRP levels ≥2 mg/L)55. Infliximab was studied as an augmentation therapy (N=37) or in medication free subjects (N=23). The primary outcome measure (change in Hamilton Depression Rating Scale 17 (HDRS-17) at 12 weeks) did not differ between the treatment and placebo arms. However, a post hoc analysis suggested that for patients with baseline CRP≥ 5 mg/L, infliximab’s treatment effect exceeded that of placebo. Infliximab was also tested by McIntyre et al. in 60 participants studied during the depressed phase of BD who had been non-responsive to conventional antidepressants56. . Infliximab or placebo was administered adjunctively. The primary outcome measure (change in Montgomery-Åsberg Depression Rating Scale (MADRS) at 12 weeks) did not differ between treatment and placebo arms; however, a post hoc analysis suggested a treatment effect among patients with a childhood history of physical abuse. Notably, peripherally administered antibodies, are expected to exert effects predominantly in peripheral blood, due to the limited permeability of the blood brain barrier (BBB) to large molecules, and the ability of infliximab to impact CNS targets remains unclear. The anti-IL-6 antibody, sirukumab, was tested as an adjunctive treatment in MDD patients who were inadequately responsive to conventional antidepressants, and had plasma CRP levels ≥ 3 mg/L at screening and baseline57. The change in HDRS-17 at week 12 did not differ significantly between the sirukumab versus placebo arms. However, secondary analyses showed that the mean Snaith–Hamilton Pleasure Scale (SHAPS) anhedonia rating improved in the sirukumab group versus the control group (nominal p=0.014). Moreover, the mean HDRS-17 score decreased to a greater extent under sirukumab versus placebo for participants with baseline CRP values ≥ 8 mg/L (although the subsamples with such CRP levels were relatively small). The observation that sirukumab, a monoclonal antibody that is poorly brain penetrant, may improve anhedonia has interesting implications. Potentially consistent with this observation, studies in experimental animals showed that anti-IL6 antibodies, which directly neutralize IL-6 in the periphery, can reduce the amount of IL-6 in the brain, and have profound effects on anhedonia-like behaviors in rodent depression models14. However, an open label study in rheumatoid arthritis patients using tocilizumab, an mAb that targets the IL-6 receptor, found no significant change in depression symptom ratings on the Hospital Anxiety and Depression Scale (HADS), which is sensitive to anhedonia58. Taken together, these data suggest the hypothesis that by blocking IL-6 receptors in the periphery but not the CNS (due to poor brain penetrance), tocilizumab would not have reduced IL-6 receptor signaling in the CNS. In contrast, by neutralizing IL-6 cytokines in the periphery, sirukumab may have reduced IL-6 receptor signaling in the CNS as well as in the periphery59. Molecular factors downstream of cytokines Several clinical trials in mood disorders assessed potential antidepressant effects of small molecule drugs targeting molecular factors downstream of cytokines. The most commonly used agents have been NSAIDs known to be brain penetrant, including selective COX-2 inhibitors (i.e., celecoxib), dual COX-1 and COX-2 inhibitors (naproxen, ibuprofen, high dose aspirin), or predominantly COX-1 inhibitors (low dose aspirin). Basic research showed that COX-1 and COX-2 inhibition can differentially affect neuroplasticity and neuroprotection mechanisms in some preclinical models60,61. These effects are mediated partly by modulatory effects on the arachidonic acid pathway, which is implicated in the pathophysiology of BD62, and by preventing neurotoxicity associated with inflammation-driven activation of the kynurenine metabolic pathway (see below), which has been associated with reduced volumes of the hippocampus and other subcortical structures in MDD63,64. As detailed elsewhere65,66 the sample sizes of these studies have been small, so meta-analyses have been conducted to increase statistical power (Figure 2). Among four RCTs evaluating celecoxib as an augmentation therapy in MDD, three found the celecoxib arm superior, although in one the beneficial effect at 4 weeks was no longer significant at the 8-week endpoint (Table 1). None of these studies stratified patients based on immune biomarkers, but in one the baseline serum IL-6 predicted changes in depression ratings at 6 weeks67. The results of studies assessing the antidepressant effects of adjunctive anti-inflammatory agents in bipolar depression also are mixed. A meta-analysis of eight RCTs assessing adjunctive NSAIDs, omega-3 fatty acids, N-acetylcysteine, and pioglitazone showed a moderate antidepressant effect compared with standard therapy, with significant effects observed for the omega-3 and N-acetyl cysteine arms, but not for NSAIDs (SMD=-0.02, 95% CI=-0.56-0.52)68. These data showed a significant overall benefit of small-to-moderate effect size for combined results across NSAIDs, but no significant benefit for studies considering selective COX-2 inhibitors alone (Figure 2). Results of studies exploring whether more selective inhibition of COX-1 may prove beneficial in mood disorders are similarly mixed. A pharmacoepidemiological study found that low dose aspirin showed benefits in stabilizing illness course in BD (fewer medication changes), while high dose aspirin, non-selective NSAIDs, selective COX-2 inhibitors, and glucocorticoids were associated with an increase in medication changes69. Similarly, a pharmacoepidemiological study of risk for developing MDD70 found that continued use of low-dose aspirin, as well as of statins, decreased the rate of incident depression, whereas continued use of non-aspirin NSAIDs or high-dose aspirin increased the rate of incident depression. Nevertheless, in a trial comparing health outcomes on low dose aspirin vs placebo in older adults (n=19,114, with 1879 depressed at baseline), the depression symptoms increased to a greater extent in the aspirin arm versus the placebo arm across a median of 4.7 years66; among individuals depressed at baseline, aspirin was associated with a worsening in depression ratings. Finally, low dose aspirin failed to reduce the incidence of new cases of depression71. Mechanisms affecting cytokine release Another brain-penetrant, small molecule drug tested for potential antidepressant effects is the tetracycline antibiotic, minocycline, which exhibits pleiotropic anti-inflammatory effects in the periphery and the brain72. Minocycline modulates immune function by inhibiting the activation, migration, and/or proliferation of T-cells, neutrophils and microglia, inhibiting the release of pro-inflammatory cytokines, and increasing the release of anti-inflammatory and anti-apoptotic molecules73,74. In addition, minocycline indirectly reduces the pro-inflammatory activation of the kynurenine pathway by attenuating the expression of cytokines under pro-inflammatory challenge, thereby reducing release of neuroactive kynurenine (KYN) metabolites65 75. In rodent models the depression-like behaviors induced via inflammatory challenge with lipopolysaccharide (LPS) are blocked by genetic or pharmacological (using minocycline or the IDO antagonist 1-methyltryptophan) knockdown of the enzyme, indoleamine-2,3-dioxygenase (IDO), which converts tryptophan to KYN75,76. Cytokines such as interferon-gamma and TNF-α activate the KYN pathway by increasing IDO expression, thereby shunting tryptophan away from serotonin synthesis and toward the KYN pathway, leading to increased production of neuroactive KYN metabolites. Depression has been associated with an imbalance in KYN metabolites, such that increased metabolism down the 3-hydroxykynurenine (3HK) branch leads to increased levels of the putatively neurotoxic metabolite, quinolinic acid (QA)77. A post mortem study of patients with MDD or BD who died by suicide showed elevated QA concentrations in microglia in rostral and subgenual portions of the anterior cingulate cortex (ACC), a key region in the neurocircuitry of depression78. In MRI studies the relative imbalance between the putatively neuroprotective KYN metabolite, kynurenic acid, versus the putatively neurotoxic KYN metabolites, 3HK and QA in blood mediated the relationship between reductions in grey matter thickness in this cortical region and having MDD, as well as that between reductions in cortex thickness and elevated CRP levels79. The relative reduction in levels of kynurenic acid versus QA also has been associated with reductions in hippocampal and amygdala volumes64, as well as with altered hippocampal function during emotion-dependent autobiographical memory recall in patients with MDD80. Based on minocycline’s effects of reducing cytokine release and indirectly reducing IDO function, several RCTs assessed its antidepressant efficacy in MDD and BD. Husain et al examined minocycline as an adjunctive therapy for patients who were treatment resistant to conventional antidepressants81. The minocycline arm (n=21) showed a decrease in HDRS-17 scores compared with the placebo arm (n=20) at week 12 (standardized effect size 1.21, p<0.001). In contrast, Dean et al studied the efficacy of adjunctive minocycline treatment in depressed patients selected from the general MDD population82, and found no significant change in MADRS scores, although improvements were observed in secondary outcomes related to global functioning and quality of life. Nevertheless, the negative primary outcome in participants from the general MDD population may highlight the importance of selecting patients based on biomarkers or treatment resistance to conventional antidepressants. Finally, an open-label study adding minocycline to SSRI antidepressants in patients with MDD with psychotic features merits comment, since the antidepressant effect appeared profound and this study population is generally nonresponsive to SSRI alone83. Two studies assessed the efficacy of adjunctive minocycline for improving depressive symptoms in BD. Husain et al84 compared the efficacy of minocycline plus active celecoxib, minocycline alone, and celecoxib alone versus placebo. The mean change in HDRS-17 scores at 12 weeks did not differ across groups. In contrast, Savitz et al tested the efficacy of low dose aspirin and minocycline as augmentation therapy for bipolar depression85, in patients randomized to receive minocycline plus aspirin, minocycline alone, aspirin alone, or placebo. The primary outcome measure was treatment response at week 6, defined conventionally as >50% decrease in MADRS score. The minocycline plus aspirin group showed a significantly greater response rate than the placebo alone group. When all four arms were included in the analysis, the main effect of aspirin (but not minocycline) on treatment response was significant. Nevertheless, a significant interaction with serum IL-6 level was observed, indicating greater response to minocycline versus placebo among participants with higher IL-6 levels. Thus, while the nonsignificant effect of minocycline in the entire sample was consistent with the results of Husain et al, the preliminary observation that bipolar depressives who manifest elevated basal IL-6 levels may benefit from minocycline warrants further testing14,86,87. Signaling molecules involved in cytokine production. The p38 MAP kinase plays a key role in pro-inflammatory cytokine synthesis and was implicated as a hub in a network of white blood cell genes for which expression differed between MDD vs healthy subjects in a meta-analysis88. A small molecule inhibitor of P38 MAP kinase, losmapimod, was studied in two RCTs in participants with MDD89. Patients were selected for loss of energy/interest and psychomotor retardation but were excluded for previous antidepressant treatment resistance. Neither study showed a significant advantage for losmapimod versus placebo. The extent of target engagement within the CNS was not established in these studies. Molecules involved in cytokine maturation Recently a focus in CNS drug development has turned towards targets that modulate microglial activation, especially via the P2X7R-NLRP3 axis29. As an ATP-gated ion channel, P2X7Rs respond to extracellular ATP release by stressed or dying cells, or by psychosocial stress.90 During innate immune responses, damage-associated molecular patterns (DAMPs) or pathogen-associated molecular pattern (PAMPs) activate pattern recognition receptors (i.e., Toll-like receptors—TLRs) that induce ATP release, activating P2X7Rs. The TLR-mediated NF-κB pathway activation acts as a “first signal” promoting transcription of genes encoding inflammatory mediators, such as pro-IL-1β, and inflammasome components, such as NLRP3; P2X7R stimulation represents the “second signal” to inflammasome activation and subsequent caspase-1 activation, which processes pro-IL-1β into its mature form, enabling IL-1β release91. P2X7R activation also promotes IL-6 release, and stimulates free radical production, phospholipase activation, cell cycle regulation and apoptosis. During adaptive immune responses P2X7R stimulation participates in T cell activation, and ATP-P2X7R signaling decreases the suppressive activity and viability of Treg cells, favoring polarization of T cells into Th17 cells91. A pathological role of microglial P2X7R activation is reported in neuroinflammatory diseases (e.g., multiple sclerosis) and neuropsychiatric disorders, including MDD12,92-95. In MDD the P2RX7 mRNA levels were abnormally increased in patients who were treatment resistant, irrespective of medication status, but reduced in medicated-responsive patients in remission.96 Compatible with these data, abnormal increases in the expression of TLR proteins and mRNA transcripts were reported in depressed patients who died by suicide97. Notably, in mice, stress-induced depression-like behaviors were reversed by brain penetrant (but not by non-brain penetrant) P2X7R antagonists.29 An RCT is underway to test the antidepressant efficacy of a brain penetrant P2X7R antagonist, JNJ-54175446, in MDD. Enhancement of anti-inflammatory cytokine function. Another area of emerging interest in therapeutics discovery involves mechanisms that enhance the release or function of anti-inflammatory cytokines, such as IL-10, which participate in the immune system’s inherent compensatory mechanisms for restraining pro-inflammatory cytokine signaling. In preclinical models, the resolution of inflammation-induced depression-like behaviors required T lymphocytes acting via an IL-10-dependent pathway to decrease the expression of the enzyme, IDO, suggesting that novel therapeutics targeting the T lymphocyte/IL-10 pathway could promote recovery from MDD15. The importance of inherent anti-inflammatory systems to recovery from depression also was suggested by a longitudinal study of MDD that assessed immune biomarkers pre- and post- antidepressant pharmacotherapy; while treatment responders and non-responders both manifested elevations in pro-inflammatory cytokines pre-treatment and increases in anti-inflammatory cytokines post-treatment (IL-4, IL-5, and IL-10), the pro-inflammatory cytokine levels continued to increase in the non-responders98. These data suggested the hypothesis that MDD patients who respond poorly to conventional antidepressants manifest a defective anti-inflammatory response. Nevertheless, despite these encouraging leads there has been no study yet on MDD with the objective of increasing anti-inflammatory activity. ASSESSMENT OF MECHANISM While most studies associating immune dysregulation and clinical depression have focused on the peripheral immune system, understanding CNS mechanisms whereby altered function mediates depressive symptomatology is important for prioritizing potential targets and characterizing patient subgroups most likely benefit from new treatments. Neuroimaging biomarkers assessed using PET, MRI and magnetic resonance spectroscopy (MRS) provide approaches to elucidate CNS effects of altered immune function. Each represents a different trade-off between immune specificity of the imaging signal, spatial resolution, cost, and accessibility. PET Imaging PET is the most target-specific biomarker of human brain inflammation, but its use in research is limited by higher cost and lower accessibility; PET also involves exposure to relatively low levels of radioactivity, limiting its repeatability. To date, studies of neuroinflammation in MDD have relied on radioligands, (e.g., [11C]-PK11195, [11C]-PBR28) that bind to the translocator protein (TSPO) expressed on mitochondrial membranes. The TSPO binding potential increases in multiple conditions involving neuroinflammation. However, the expression of TSPO protein on microglia as well as on other glial cells and neurons limits the specificity of TSPO binding. Also a SNP in the TSPO gene (rs6971) causes an amino acid substitution in TSPO and alters the affinity of binding to ligands other than TSPO-binding ligands other than [11C]-PK1119599, a limitation which must be addressed in the study design. Several case-control PET studies of MDD reported small-moderate sized, but statistically significant increases in TSPO binding in the ACC and other regions where post mortem studies had more specifically shown greater activation of microglia in individuals with MDD or BD who died by suicide100,78(Figure 3). Future development of ligands with greater specificity for microglia or other central immune targets is needed to optimize the potential of PET biomarkers of neuroinflammation in depression101. fMRI Imaging Functional MRI (fMRI) data collected during performance of simple cognitive tasks, or in the “resting state”, can be used to measure functional activity in various brain regions, as well as the functional connectivity matrix (connectome) of dynamic coherence between the resting state fMRI signals simultaneously measured at each possible pair of regions. This is a relatively safe, affordable and accessible imaging biomarker, which is suitable for repeated assessments. However, the hemodynamic signals measured via fMRI are nonspecific and limited in the extent they can inform the neurobiological mechanisms underlying any difference observed between experimental and control samples. A review of human fMRI studies of the effects of inflammation on cerebral function defined two broad strategies across this literature: observational and experimental102. Observational studies measured the correlation between fMRI metrics and blood concentration of immune markers. Experimental studies measured fMRI metrics before and after a controlled pro-inflammatory stimulus (e.g., typhoid vaccination). Meta-analysis of these data identified regions where functional activity was significantly changed in observational and experimental studies, including the amygdala, hippocampus, hypothalamus, striatum, insula, midbrain, and brainstem, as well as prefrontal and temporal cortices. This observation is consistent with emerging evidence that systemic inflammation can be correlated with, or could cause, changes in functional connectivity between the areas of limbic, prefrontal and temporal cortices, and anatomically related subcortical nuclei103,104 that form the “medial prefrontal network” which mediates and regulates emotional behavior, and comprises the neurocircuitry of mood disorders105. These data thus provide evidence that interactions of immune activation of neural and humoral interoceptive mechanisms can mediate discrete changes in brain and behavior within the context of infection and inflammation106. Microstructural MRI Imaging Alternative MR modalities may achieve greater neurobiological specificity than fMRI. Micro-structural MRI enables measures of physical parameters, such as T1 or T2 relaxation times and magnetization transfer (MT), that can be interpreted in terms of extracellular fluid, neurite density, myelination, or other microscopic properties of brain tissue within an image voxel107. Micro-structural MRI metrics sensitive to the proportion of free water in a voxel seem intuitively more likely to be indicative of central inflammation than a macro-structural MRI metric like cortical volume. Recent studies reported changes in micro-structural MRI data related to blood-based biomarkers associated with systemic inflammation in patients with MDD108,109. Magnetic Resonance Spectroscopy (MRS) Imaging MRS assesses the brain tissue concentration of molecules such as GABA and glutamate. MRS studies have shown that peripheral administration of IFN was associated with increased glutamate levels in the basal ganglia and cingulate cortex that correlated with fatigue and anhedonia ratings. Basal ganglia glutamate concentration was also increased in MDD patients with high levels of CRP (>3 mg/L) compared to patients with low CRP (< 1 mg/L)104. While the glutamate signal is predominantly intracellular, these data appear compatible with evidence from preclinical models that pro-inflammatory cytokines and microglial activation can alter glutamate release and transport, resulting in increased extracellular glutamate concentrations110. Neuroinflammation thus has been hypothesized to play a role in neurotoxicity via its downstream effects on glutamate metabolism111. However, many other factors besides inflammation can alter glutamate levels, and MRS in vivo can only resolve the minor peak of glutamate resonance in relatively large (~1 cm3) volumes of tissue. In summary, noninvasive biomarker measures of central immune status that can provide anatomically fine-grained information on specific molecular or cellular targets in the human brain are not yet available. The increasing use and technical refinement of functional and micro-structural MRI, and MRS, conceivably may serve as “bridging” biomarkers that can be used to investigate relationships between immune mechanisms and depression. Further developments in PET radiochemistry, single cell analysis of circulating immune cells, and greater assay sensitivity for studies of CSF and blood also can be expected to inform more precise insights into immune mechanisms for small numbers of patients and prioritized immune targets. Nevertheless, peripheral blood biomarkers remain the most accessible and comprehensive platform for assessing immune status in large numbers of patients. PATIENT STRATIFICATION The discovery and development of novel antidepressants targeting immune dysregulation will require multidisciplinary partnerships between researchers with expertise in psychiatry, experimental medicine, biomarker and diagnostic development, and neuroimmunology, and success will likely depend on patient selection or stratification criteria. It is clear in reviewing the diverse approaches taken to both inclusion criteria and within-trial stratification, among the studies reviewed above, that this remains an area of uncertainty in the field. A combination of varying levels of treatment resistance and thresholds set on CRP have been used, along with symptoms related to anhedonia, in patients with either primary mood disorders or depression arising in the context of primary immunological diseases. Many other small studies focused on “all-comers” with MDD or BD, which might have resulted in Type II error, as an experimental treatment that did not separate statistically from placebo conceivably may prove effective in a biologically distinct patient subgroup (Table 1). One approach to identifying potential options for patient stratification is to outline the different immune axes along which depressed patients separate statistically from non-depressed controls. As potential diagnostics, blood biomarkers are more accessible than imaging-based markers and provide finely detailed mechanistic information about the circulating compartment of the peripheral immune system, although the extent to which central immune function can be inferred from peripheral immune markers remains unclear. Nevertheless, the output of an immune-dysregulated brain circuits may manifest as observable symptoms and behaviors. Characterizing phenotypic aspects of “immune-mediated depression” conceivably may offer another dimension for quantification of a treatment cohort. Too often, especially for molecular analyses, focus is placed on differentiation of effects based on MDD diagnosis or outcomes based on more general clinical ratings rather than on specific dimensional constructs, e.g., anhedonia. Ultimately, refinement towards precision medicine will require collection of data from patient cohorts that are both phenotypically rich and biomarker dense. Understanding case-control differences may prove helpful in generating hypotheses for candidate diagnostics, and stratifying patients in early trial phases. But refinement through clinical trials, where treatment outcome is characterized, will be needed to identify the diagnostic strategy that enables a compound to be accessed by the widest range of patients likely to benefit, while also enriching the target population for individuals with a higher likelihood to benefit than is offered by the current standard of care. Biomarkers with Diagnostic Potential Immune markers demonstrating some ability to distinguish depressed patients from healthy controls, and quantify the level of immune dysregulation within depressed patients include CRP112, cytokines113,114, genome-wide gene expression88,115,116 and qPCR of specific mRNAs96,117, ex vivo LPS-stimulated cytokine or gene expression levels118-121, and quantitative immune cell counts122-126(Figure 1). Studies that have derived CRP thresholds predictive of treatment response in the context of clinical trials have, to date, only identified putative ideal cutoff values post hoc55,57. However, these cutoffs identified subgroups that represented a small proportion of depressed subjects. Baseline IL-6 levels predicted response to both minocycline85 and celecoxib67. Cytokines have been increasingly measured using multiplex assays for dozens of proteins, and both aggregated summary scores119 and algorithmically derived scores117,127 have been considered as an approach to increasing the robustness and precision of the measurements. As diagnostic biomarkers some cytokines are difficult to measure because what is considered elevated in the context of MDD is often near the limit of quantification of available assays. Further, the intrinsic lability of cytokines poses a concern for their use as diagnostic biomarkers, as they fluctuate over a wide range over the course of a day and in response to activity. Just as HbA1c provides a weeks-long average of blood sugar control compared to glucose, more stable measure of immune dysregulation may prove more clinically viable. Whole genome mRNA expression has been measured in whole blood and in peripheral blood mononuclear cells (PBMCs) in multiple MDD case-control studies and allows a more comprehensive assessment of peripheral immune pathways associated with depression88,115,116. Meta-analyses have identified an over-expression of innate immune genes coupled to under-expression of genes involved in adaptive immunity88. Depression-related impairments of adaptive immunity are consistent with previous findings128,129. Notably, the inverse relationship between innate and adaptive immune gene expression was evident at the level of individual subjects, i.e., MDD cases with increased expression of innate immune genes had decreased expression of adaptive immune genes, enabling the development of a single bioscalar representing immune dysregulation that could be used for patient selection88. Studies focusing on transcription of a few, pre-selected candidate genes have reported differential expression of pro-inflammatory, glucocorticoid receptor, and neuroplasticity-related genes96,117. Genome wide studies may guide the deliberate selection of a small number of stable mRNA representative of key immune processes, pathways, and cell types, that can be assessed by quantitative polymerase chain reaction (qPCR) based assays; these may enable more refinement of the immune compartments prioritized for patient selection more specifically around an individual drug and its mechanism of action. For example, one study using this approach showed that MDD patients who either were currently depressed and treatment-resistant or currently depressed and drug-free could be differentiated by signatures of mRNA transcripts that putatively reflected inflammasome activation and glucocorticoid resistance from healthy controls and drug treated patients who had remitted during treatment. Moreover, a signature of six mRNAs (P2RX7, IL1B, IL6, TNF, CXCL12 and GR) distinguished antidepressant treatment-resistant versus responsive patients. These data appeared similar to those from a longitudinal study that showed drug-free depressed patients have increased mRNA expression of IL1B, IL6 and TNF, together with reduced expression of GR and increased expression of the FKBP5 mRNA, and the pattern of mRNA expression of these and/or other inflammation related genes predicted or correlated with responsiveness to antidepressant drug treatment117. Beyond proteins and genes, another easily accessible snapshot of the state of the immune system is provided through immune cell counts. Studies of depressed cases compared to controls have consistently reported increased total white blood cell count, increased neutrophils, decreased T or B lymphocytes130, increased neutrophil/lymphocyte ratio, and increased CD4+/CD8+ T cell ratio122-126. Effects of depression on CD4+ cell differentiation have been implicated by decreased T regs, and increased Th1 and Th17 counts in MDD131-133 (Figure 1). In a recent study, on the basis of absolute counts of 14 blood cell types in MDD cases, it was possible to identify sub-groups of inflamed depression, characterized by increased myeloid (neutrophil, monocyte) or lymphoid (CD4+ T) cell counts, which correlated with increased CRP and IL6, and increased depression severity134. Moreover, increased precision in understanding the complexities of the immune system at a cellular level is being facilitated by single cell sequencing, which may further elucidate key drivers of depressive symptoms, and identify potential therapeutic targets. One study applying this approach in BD patients found remarkably robust biomarker abnormalities among mRNA transcripts measured specifically in monocytes. An inflammatory gene expression signature composed of these transcripts discriminated both a large proportion of the BD patients and the offspring of BD parents from age-matched controls135. The interplay between innate and adaptive systems modulates not only the protective immune response to microbial infection and other non-self-antigens, but also the pathogenic generation of autoantibodies directed against self-antigens. For example, one study found that while patients with BD showed both pro-inflammatory activation in monocytes and elevated percentages of anti-inflammatory T cells (specifically, CD4(+)CD25(high)FoxP3(+) regulatory T cells), the two sets of abnormalities occurred independently from each other, and patients who manifested co-morbid autoimmune thyroid disease (which is common in BD) showed fewer anti-inflammatory T cells compared to patients without this condition136. Furthermore, the potential for autoantibodies to play a pathogenic role in psychiatric syndromes has been seen in patients with autoimmune encephalitis resulting from autoantibodies against NMDA receptors137 or potassium channel complex proteins138,139. While unlikely to drive symptoms in more than a small minority of patients, higher levels of overall autoantibody burden are seen among patients with depression and other psychiatric disease compared with healthy patients, suggesting this is an area that merits further exploration as novel treatment options to regulate IgG levels emerge140,141. Target specific, rather than disease specific, patient selection strategies may emerge early where a direct hit to the functioning of a target can be observed, which could come in the form of genetic mutations, epigenetic modifications, or even autoantibodies. The utility of the biomarker as a patient selection strategy ultimately will depend both on its predictive power and the prevalence within the population. Clinical Phenotypes with Diagnostic Potential While a definitive standard has not yet emerged to optimize selection of depressed patients for immune directed therapeutics, some patterns around common phenotypic traits of patients with elevated inflammatory markers are apparent within the more heterogenous set of symptoms comprising MDD. Most compelling has been evidence pointing towards inflammation having dimensional effects on anhedonia and the cognitive processing of rewarding stimuli rather than having an equally weighted impact across DSM5 symptoms. Like depressed mood and anxiety, anhedonia is observed trans-diagnostically, and among individuals with a variety of psychiatric, neurological or primary inflammatory disorders142. Ultimately, a patient selection approach based on clinical phenotype would be more widely accessible and economical than a biological measurement, if sufficiently accurate. Evidence suggesting a link between anhedonia, depression, and the function of the mesolimbic dopaminergic circuits that underlie reward processing and motivated behavior is converging across informatics, clinical and imaging studies. Inflammatory cytokines have been shown to exert a direct effect on mesolimbic dopamine (DA) transmission that is associated with reduced willingness to expend effort for reward as well as with the capacity for reward learning142. This effect at least partly involves a reduction in the availability of dopamine precursors, as neuroimaging and in vivo microdialysis studies in humans and/or nonhuman primates indicate that inflammatory cytokines such as IFNα reduce dopamine release in the ventral striatum, in association with depressive symptoms including anhedonia and psychomotor slowing111. In a non-human primate model this effect could be reversed by administering the dopamine precursor, levodopa143. By impacting motivated behavior and psychomotor activity, the effects of inflammation on central dopaminergic transmission may contribute to the reduction in physical activity manifest in depressed patients, which increases the risk for developing obesity and associated metabolic disturbances. Obesity further drives systemic inflammation, as adipose tissue actively secretes cytokines and obesity is associated with changes in the adipocyte secretome leading to increased production of proinflammatory cytokines144. Notably, positive genetic correlations with body mass and Mendelian randomization analysis were consistent with BMI being causal or correlated with causal risk factors for depression24. Moreover, a longitudinal study of 3,809 non-depressed adults (age ≥50 years) found participants with higher baseline levels of inflammatory markers more likely to report low levels of physical activity four years later, and low activity partially mediated the relationship between systemic low-grade inflammation and subsequent depressive symptoms145. Interrupting the effects of inflammation on central dopaminergic pathways and physical activity via pharmacological and nonpharmacological (e.g., exercise) approaches conceivably may reduce or prevent depressive symptoms and associated medical co-morbidities. A noteworthy study in this regard profiled drugs for their side-effect similarity across 4,201 different side effects, cytokine inhibitors were found closest in the high-dimensional side-effect space to drugs with a dopaminergic effect, but distant from other antidepressants146. This finding suggested the possibility that dopaminergic compounds influencing reward-guided behaviors may be more effective in patients with an inflammatory component to their depression. Clinically, this hypothesis was confirmed in the Combining Medications to Enhance Depression Outcomes (CO-MED) study147, which showed that with increasing CRP levels, SSRI monotherapy became less effective, and augmentation with the dopamine and norepinephrine reuptake inhibitor, bupropion, became more effective148. In clinical trials, IL-6 antagonists showed a potential antidepressant signal in patients with rheumatoid arthritis (sirukumab) and multicentric Castleman’s disease (MCD, siltuximab), but notably these post hoc analyses relied upon a depressive symptom score which weighted anhedonia by 50%149. This may explain the findings in the sirukumab study in MDD cited above in which significant improvement was not observed on the HDRS-17 (of which only one of 17 items assesses anhedonia), but benefit was observed on a scale that rated anhedonia severity (SHAPS).57 Recently, in the NESDA study, the association of both basal and ex vivo LPS stimulated inflammatory markers with individual depressive symptoms was explored in a large, longitudinal cohort. In both cases, inflammatory markers were most strongly associated with “sickness behavior” symptoms, including deficits in general interest and capacity for enjoying previously pleasurable activities or stimuli (i.e., anhedonic symptoms), at up to a 9-year follow up121. Convergently, multimodal neuroimaging studies of inflammation-related changes in brain structure, function and biochemistry have implicated components of the mesolimbic reward circuitry, including increased hemodynamic activity in the subgenual ACC22, increased basal ganglia glutamate104, decreased cortico-striatal functional connectivity104, decreased striatal volume63, increased markers of inflammation in insular cortex108 and microglial activation in the subgenual ACC and other regions100,78 (Figure 3). While in principle, a symptom-based patient selection approach should be simpler than a biological diagnostic, it remains unclear how patients with anhedonia/reward processing deficits can optimally be selected for clinical trials. Measurement of anhedonia is imprecise, and the available rating scales variably address different aspects of this complex phenomenon which includes deficits in motivation, consummatory pleasure, and reward learning, taking into account both direct and indirect rewards150. New clinical scales which address these challenges, are under development and validation, such as the Dimensional Anhedonia Rating Scale (DARS)150. As an alternative to symptom scales, functioning of the dopaminergic reward circuitry can be probed through cognitive testing that uses reward-related tasks, which may provide a more specific metric for patient selection142. These are currently being tested and validated in human studies, with different tasks measuring distinct aspects of reward learning151,152. Further, with emerging trends in the digital health sector, both cognitive tests and symptom assessments could be deployed as digital diagnostics to provide on-line platforms for screening large numbers of potential participants for trials of new agents focusing on anhedonia or other specific dimensions of depression. UNMET CLINICAL NEED For a putative immune subtype of MDD to be viable for drug development, this patient subpopulation should be demonstrably under-served by current therapeutic options, and the introduction of a new therapeutic should result in fewer patients suffering from depression. The unmet need has been demonstrated for patients with high inflammatory burden in several ways. For example, in the longitudinal NESDA study, MDD patients recently starting an antidepressant who had more than four indicators of chronic inflammation or metabolic dysregulation at baseline had a 6.85 fold (95% CI=1.95–24.06) increased odds of chronicity of MDD at year 2153. In addition, during antidepressant treatment, higher pre-treatment levels of inflammation measured by CRP, IL-6 and TNF- were associated with poorer response to first-line antidepressants 2-4, and after treatment the patients who proved non-responsive showed deficits in the ability of anti-inflammatory cytokine release to restrain further increases in pro-inflammatory cytokine release98. Further, in longitudinal studies, depressed patients with suicidality manifest persistently elevated CSF levels of KYN pathway metabolites (e.g., QA), and IL-6 was elevated in the CSF of suicide attempters and correlated with the severity of depression and suicide ideation154,155,156. Finally, in vitro evidence shows that inflammatory cytokines change expression of the tryptophan metabolic pathway enzymes and serotonin transporters, which conceivably may contribute to resistance to SSRI antidepressants157. Beyond the impact of psychiatric symptoms on the morbidity and mortality associated with MDD, depressed patients also manifest a 2 to 4 fold higher risk for cardiovascular disorders (CVD) 158, and poorer outcomes during comorbid heart failure, stroke, and peripheral artery disease159-161. Moreover, MDD is associated with exaggerated biological aging as reflected by premature decreases in leukocyte telomere length (a marker of cellular age that predicts several aging-related diseases and early mortality) and increases in senescence related gene transcription and molecular secretion patterns in physiological stress and inflammatory systems162-164. Dysregulation of the interactions between physiological stress systems that include inflammation, HPA-axis hyperactivity and metabolic dysregulation partly underlie these associations165-167. In addition, early life stress, a risk factor for MDD, is associated with high levels of inflammation168, and increased risk of cardiovascular-related and all-cause mortality169. Breaking these links could yield positive outcomes for patients beyond mental health. For example, metformin (a drug used in the treatment of type 2 diabetes) has been proposed as a mechanism for producing anti-senescence effects by modulating intracellular pathways (e.g., NFκB and p53) which can reduce senescence associated secretory proteins, including pro-inflammatory cytokines, and mitigate negative health outcomes in overweight depressed patients163. Moreover, elevated release of pro-inflammatory cytokines together with reactive oxygen species putatively leads to development of the mitochondrial dysfunction and oxidative metabolism alterations evident in many patients with mood disorders, which contributes to biological aging170,171. Chronic oxidative stress, characterized by elevated reactive oxygen and nitrogen species and/or reduced glutathione levels, is a particularly consistently finding in BD and other neuropsychiatric or neuroimmune disorders that are associated with elevated pro-inflammatory cytokine levels170. Activation of microglia by pro-inflammatory cytokines in turn leads to further release of reactive oxygen intermediates, proinflammatory cytokines, complement proteins and proteinases, driving a chronic inflammatory state that can trigger or maintain neurodegenerative processes.172,173 Such processes may underlie progression in the cognitive impairment and illness morbidity manifest in some BD patients174. Elucidating the interactions between inflammation, oxidative stress, mitochondrial dysfunction, and impaired neuroplasticity may prove critical for identifying therapeutic targets that prevent illness progression and improve outcomes. Non-pharmacological treatments also may prove beneficial in countering the effects of inflammation and oxidative stress in mood disorders; for example, exercise (running therapy) has been shown to reduce oxidative stress, inflammation, cortisol release and depressive symptoms, and to have a beneficial impact on biological aging (telomere length)175. Among the unmet psychiatric needs associated with biomarkers of inflammation111, anhedonia itself predicts antidepressant nonresponse, as observed in the Genome-Based Therapeutic Drugs for Depression (GENDEP)2 and the Sequenced Treatment Alternatives to Relieve Depression studies176. Beyond anhedonia, other clinical phenotypes have been dimensionally associated with different aspects of inflammation. For example, in patients with MDD and anxious distress, basal levels of inflammatory cytokines were not associated with anxiety symptoms. Upon LPS-stimulation, however, an increased production capacity for pro-inflammatory cytokines was revealed, with higher levels in patients with anxious symptoms120. Fatigue is associated with MDD as well as being a commonly comorbid and non-specific symptom in primary immunological disorders. In a meta-analysis of immune trials for effect on depressive symptoms52, treatment effects on fatigue did not remain significant after correction for concomitant improvement in primary illness. Changes in appetite in MDD have been mapped to the association between blood CRP and connectivity between the striatum and areas of the brain implicated in food hedonics177. Sleep changes have also been associated with inflammation in MDD178. As troubling symptoms for patients, assessment of these dimensions may prove viable both as stratification approaches and as outcome measures for trials of immune based therapies. A somewhat different example of unmet need is provided by the recent emergence of depression and related symptoms (fatigue, anxiety, mild cognitive impairment) as part of the “long COVID” syndrome of physical and mental health disorders experienced by many patients following infection with SARS-CoV2179. The role of immune mechanisms in causing post-COVID depressive syndromes remains to be elucidated but is suggested by recent reports highlighting the association between MRI changes in brain structure and peripheral immune cell counts in patients following discharge from hospital after treatment for severe COVID180. Ultimately, the development and adoption of immune-targeted therapeutics for depression may disrupt the conventional, syndromic categorization of MDD and focus instead on trans-diagnostic symptom dimensions, such as anhedonia, that may be particularly salient in some clinically defined subgroups of patients and more specifically related to tractable immune mechanisms111. ADDITIONAL CONSIDERATIONS Safety In addition to addressing unmet medical need, new compounds targeting immune function to treat MDD will require a risk-benefit ratio that supports their differentiation from alternative treatments, increasing the need to identify patient subpopulations most likely to benefit. For example, an increased risk of infections has been cited in association with TNFantagonism, including reactivation of latent tuberculosis and opportunistic infections181. Depending on the safety profile for an immune targeted therapeutic, patient pre-screening for specific infections may be warranted182. Nevertheless, such risks must be balanced against the potential benefit of helping patients with treatment resistant depression, which is associated with higher morbidity and mortality rates than the general MDD population183. In addition, most antidepressants include a black box warning indicating they may increase the risk of suicidal thinking in children and adolescents. Recently the anti-IL-17 receptor monoclonal antibody, brodalumab, was approved as a treatment for psoriasis with a warning that it has been linked to suicidal ideation184. Effects on suicidal ideation will need to be quantified for any new immune targeted therapeutic in an at-risk population. Treatment Paradigms Most commonly, novel therapeutics for MDD are clinically tested as adjunctive therapies to standard-of-care antidepressants. The expectation is that they will augment the response to available antidepressant therapies, which often are partially beneficial even in those patients who have experienced inadequate response. Nevertheless, for any immune-targeted therapeutic in MDD, it will need to be determined whether the interaction with SSRIs is synergistic as is often assumed, or whether there might be a detrimental effect of augmentation compared to monotherapy. For example, while evidence shows that patients with elevated inflammatory markers respond more poorly to SSRIs/SNRIs104, in the CO-MED trial148 patients with low CRP responded better to SSRIs alone than to a combination of bupropion and SSRI, while in patients with higher CRP, the combination proved superior to SSRIs alone. As another example, a study testing the efficacy of infliximab as an adjunctive therapy in treatment resistant MDD patients observed that patients whose CRP levels were in the lower range pre-treatment showed greater improvement on adjunctive placebo than on adjunctive infliximab administration55. The development of a monotherapy is often considered a more challenging path for MDD from a regulatory and payer perspective. However, ultimately the path forward for a compound will be paved by data from RCTs, if it can be demonstrated that a well-defined subpopulation has a high likelihood of non-response to currently available monotherapies, and a high likelihood of response to a novel therapeutic. Sex Differences As is true for MDD, most autoimmune disorders are more prevalent in women than in men. Inherent biological differences between sexes also may impact the relationship between immunity and MDD. In the NESDA study, differences were observed in the association of basal cytokine levels with depression where currently depressed men, but not women had significantly higher CRP and IL-6 than healthy controls185. In a later study in the same cohort, IL-6 levels in depressed patients predicted a chronic course of depression in women, but not men18. In the EMBARC study, higher baseline CRP was predictive of antidepressant outcomes in women, but not men186. In the GENDEP study, higher baseline CRP levels correlated with greater depression severity, but the effect was only significant in women187. With the development of immune-targeted therapies for MDD, sex differences should be considered as playing a role not only in the potential response rate but also in relation to the patient selection strategy. OUTLOOK Collectively, these data imply that immune mechanisms play a role in the pathophysiology of depressive symptoms for at least a subtype of patients with MDD and provide clues toward the development of novel therapeutics and personalized medicine approaches in neuropsychiatry. Ultimately, the goal is to improve treatment outcomes by enabling patients experiencing depressive episodes to achieve and maintain remission. Immune mechanisms appear promising in this regard in that the impact of chronic inflammation is not unique to MDD, but extends to patients suffering from a variety of other medical conditions. Moreover, a pathophysiological role for the immune system has been demonstrated in some other psychiatric conditions (e.g., schizophrenia), as well as in neurodegenerative and other neurological disorders. As biology cuts across diagnostic boundaries, the development of precision medicine approaches based on immune biomarkers may connect a medication to biological deficits and/or psychological dimensions that span multiple DSM5 categories. Such approaches also hold the potential to redefine diagnostic boundaries in psychiatry, leading to a nosology based on pathophysiology rather than on symptom-based syndromes. Box 1. Criteria for an immune-targeted precision medicine in major depressive disorder (MDD) For successful drug development of an immune directed compound in MDD, several criteria must be met: Causality. The link between immune dysfunction and depression must be causal, or a contributing factor to the pathogenesis of depressive symptoms. Targetability. The causal biology or its up- or down-stream molecular pathways must be targetable, with consideration as to the necessity of peripheral versus central drug delivery. Diagnosability. A biologically sound approach to selecting patients for specific treatment must be identified, and ultimately the diagnostic approach must be scalable, affordable, and accessible. Notably, as a patient selection strategy emerges, a more precise understanding of the clinical profile associated with inflammation should be established; such a profile may be digitally measurable for stratification at scale and outcome assessment repeatedly over time. Unmet Need. In comparative analyses of patients who do or do not fulfill the criteria for treatment, a demonstrable unmet need under treatment as usual must exist for selected patients. Table 1: Summary of randomized, controlled trials assessing whether immune-targeted therapeutics produce antidepressant effects in primary mood disorders (major depressive disorder [MDD], bipolar disorder - currently depressed) Study (by 1st author surname)/Target Patient Selection / Inclusion Criteria Patient Stratification Primary Outcome Measure Top line results Post-hoc findings Raison55 TNF- infliximab MDD Treatment resistance: Score of 2 or higher on Massachusetts General Hospital Staging Consistent antidepressant regimen or medication free hs-CRP >2mg/L HDRS-17, 12 week No significant difference among treatment groups hs-CRP > 5mg/L favors infliximab McIntyre56 TNF- infliximab Bipolar Depression 2 prior treatments in episode Meets one of: -CRP>5mg/L -BMI>=30 AND either increased triglyceride levels, decreased HDL cholesterol OR elevated blood pressure; -type 1 or 2 diabetes; -inflammatory bowel disorder; -rheumatologic disorder; -daily cigarette smoking -migraine headaches Stable medication regimen none additional to inclusion MADRS, 12 week No significant difference among treatment groups Childhood history of physical abuse was associated with infliximab efficacy Salvadore57 IL-6 sirukumab MDD One partial antidepressant failure in current depressive episode Adjunctive to monaminergic antidepressant hs-CRP >3 mg/L HDRS-17, 12 week No significant difference among treatment groups Post-hoc threshold >=8mg/L Significant differentiation on SHAPS (anhedonia) scale Inamdar89 p38 MAPK losmapimod MDD One prior episode that was responsive to treatment. Current episode is a recurrence. Patients selected for loss of energy/interest and psychomotor retardation. Loss of energy/interest psychomotor retardation Bech 6-item depression subscale of the HDRS-17 First study terminated early. Effect was observed at termination. Second study effect not significant n/a Husain81 minocycline MDD Patients with non-response to two rounds of antidepressant treatment. Adjunctive treatment as usual: antidepressants, mood stabilizers, antipsychotics none HDRS-17 Minocycline superior to placebo. Effect size 1.21, p<0.001 n/a Dean82 minocycline MDD Adjunctive to treatment as usual none MADRS No significant difference among treatment groups Significant effects observed on Quality of life enjoyment and satisfaction questionnaire Savitz85 Minocycline, low dose aspirin Bipolar depression Allowed concurrent medications included antidepressants, anticonvulsants, antipsychotics and anxiolytics. none MADRS (response) Minocycline + aspirin significant over placebo/placebo Significant interaction between aspirin, minocycline, and IL-6, as Minocycline appeared to have a greater effect among patients with elevated IL-6, which then decreased in responders. Muller188 COX-2 Celecoxib augmentation of reboxetine MDD Other medications subject to 3-day washout none HDRS-17 Celecoxib plus reboxetine arm superior to reboxetine plus placebo (6 weeks) n/a Akhondzadeh189 COX-2 Celecoxib augmentation of fluoxetine MDD Free of psychotropic medications none HDRS-17 Celecoxib plus fluoxetine arm superior to fluoxetine plus placebo (6 weeks) n/a Abbasi67 COX-2 Celecoxib augmentation of sertraline MDD Free of psychotropic medications none HDRS-17 Celecoxib plus sertraline arm superior to sertraline plus placebo (6 weeks) Baseline serum IL-6 levels correlated with change in HDRS-17 at 6 weeks. Majd190 COX-2 Celecoxib augmentation of sertraline First episode MDD in women none HDRS-17 Celecoxib plus sertraline arm superior to sertraline plus placebo at 4 weeks. Not significant after 8 weeks. n/a Abbreviations: BMI – body mass index; COX-2 - cyclooxygenase-2; hs-CRP – high sensitivity C-reactive protein; HDRS-17 – Hamilton Depression Rating Scale, 17 item version; IL-6 – interleukin 6; MADRS – Montgomery-Asberg Depression Rating Scale; SHAPS – Snaith-Hamilton Pleasure Scale; TNF - tumor necrosis factor  FIGURE LEGENDS Figure 1. Peripheral and central immune dysregulation associated with Major Depressive Disorder (MDD). A. In the periphery, a variety of immune markers have been associated with MDD. These include elevation of pro-inflammatory cytokines, increases in innate immune cell types, changes in differentiation of T-cells and other immune cell types. Direction of change of expression or cell count in MDD is indicated with an arrow. B. In the central nervous system (CNS), elevation of pro-inflammatory cytokines is observed in the cerebrospinal fluid (CSF) which may come from the periphery, entering the CNS through the blood brain barrier or dural lymphatics, or produced from cells within the CNS, including glia. An increase in activated microglia, the resident macrophages in the CNS, in MDD has been implicated in imaging and postmortem studies and is a current biological target of interest. Abbreviations: HSC=Hematopoietic stem cell, NK=Natural Killer Cell, ILC=Innate Lymphoid Cell, DC=Dendritic Cell, BBB=Blood Brain Barrier. Figure 2. Summary of meta-analyses of antidepressant effects measured in immunology clinical trials. The Standardized Mean Difference (SMD) and 95% confidence intervals are shown aggregated by mechanism of action across the four primary meta-analyses complied to study the effects of immune-targeted compounds on depressive symptoms. Abbreviations: NSAIDs=Nonsteroidal anti-inflammatory drugs. Figure 3. Neuroimaging biomarkers of inflammation. Examples of findings linking inflammation (assessed by TSPO binding in brain or by basal C-reactive protein [CRP], typhoid vaccine induced increases in IL-6, or kynurenine-to-tryptophan [Kyn/TRP] levels in blood) to significant effects on neural circuit function, neurochemistry, or neural structure in healthy volunteers or participants with major depressive disorder (MDD). Abbreviations: TSPO=Translocator Protein, ACC=Anterior Cingulate Cortex, CRP=C-Reactive Protein, Kyn=Kynurenine, TRP=Tryptophan. ACKNOWLEDGEMENTS The authors thank Noel Derecki for assistance with early versions of this manuscript and artwork in Figure 1. COMPETING INTERESTS GMW, WCD are employees of Janssen Research & Development, LLC, of Johnson & Johnson, and own J&J stock. HKM is an employee and shareholder of Johnson & Johnson. ETB serves on the scientific advisory board of Sosei Heptares and as a consultant for GlaxoSmithKline. REFERENCES 1. 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