Ketone bodies and in ﬂ ammation modulation: A mini-review on ketogenic diet ’ s potential mechanisms in mood disorders

: Mental disorders such as depression and anxiety in ﬂ ict signi ﬁ cant burdens on individuals and society. Commonly prescribed treatments often involve cognitive therapy and medications. However, for patients resistant to these conventional methods, alternative therapies like the Ketogenic Diet (KD) offer a promising avenue. KD and its key metabolite, β -hydroxybutyrate (BHB), have been hypothesized to alleviate mental disorders through anti-in ﬂ ammatory actions, a crucial pathway in the pathophysiology of depression. This mini-review examines 15 clinical trials exploring the in ﬂ uence of KD and BHB on in ﬂ ammation and their potential roles in managing mental disorders. Both human and animal studies were scrutinized to elucidate possible cellular and molecular mechanisms. Out of the 15 trials, 10 reported reduced levels of at least one in ﬂ ammatory mediator or mRNA post KD or BHB treatment, while two observed an elevation in anti-in ﬂ ammatory agents. These ﬁ ndings suggest that KD and BHB could modulate cellular in ﬂ ammatory pathways, highlighting their potential for therapeutic application in mental disorders.


List of Abbreviations 5-HT
Phosphoinositide 3-kinases PRISMA Preferred reporting items for systematic reviews and meta-analyses SCOT Succinyl-CoA:3-ketoacid-CoA transferase TNF Tumor necrosis factor TRP Tryptophan

Introduction
Common mental disorders (CMD) refer to two main diagnostic categories: depressive and anxiety disorders. According to the World Health Organization, in 2015, the proportion of the global population with depression was estimated to be 4.4%. The prevalence was more common among females (5.1%) than males (3.6%) (World Health Organization, 2017). The proportion of anxiety disorders was estimated to be 3.6%, which also appears to be more common among females (4.6%) than males (2.6%) (World Health Organization, 2017). Both these mental disorders are a leading cause of disability, imposing a substantial burden on individuals and society (Friedrich, 2017;Stein and Craske, 2017). While cognitive therapy and medications are the firstline treatment for CMD, 10%-30% of the patients do not respond to antidepressant medicines. Additionally, they exhibit symptoms such as declined physical health and social/occupational function, or even suicidal intentions (Pilkington, 2018). Therefore, alternative and complementary therapies for the prevention and treatment of CMD are urgently needed.
Though the pathology of CMD is not fully understood, the mutual and functional role of the immune system in the development of CMD symptomology has been highlighted by recent findings. Inflammation, mainly mediated by cytokines and chemokines that are secreted by immune cells, is generally considered a defense mechanism upon infection. However, chronic and excessive inflammation may interfere with synaptic remodeling, transcription, and epigenetics. It may also harm the integrity of neuronal function, influence neurocircuitry and/or neurotransmitter systems, and produce behavioral alternations (van Velzen et al., 2017). Mounting evidence indicates that inflammatory cytokines are responsible for the development of CMD. For instance, epidemiological studies using data collected from 147,478 individuals from the UK Biobank and 2,905 from the Netherlands Study of Depression and Anxiety indicate that the inflammation level was associated with core depressive and anxiety symptoms of low mood, anhedonia, and other symptoms (van Eeden, 2022;Milaneschi et al., 2021). In animal model studies, evidence shows that activated immune responses are observed in many CMD animal models, and treatment with several kinds of cytokines could produce depressive and anxiety-like behaviors (Camara et al., 2013;Murray et al., 2013). Furthermore, CMD occurs more frequently in those who already have medical disorders associated with immune dysfunction (Gałecki and Talarowska, 2018). Therefore, anti-inflammatory agents and treatments are considered effective ways to fight CMD.
The ketogenic diet (KD) is a dietary regimen that contains high fat and low carbohydrate content. It is an established treatment for refractory epilepsy, including some inflammation-induced epileptic encephalopathies (Ma and Suzuki, 2019).
Nowadays, ketone supplementations are available for use, and people can use the supplementations to achieve nutritional ketosis easily (Kovács et al., 2019). There are two primary forms of ketone supplements, ketone salts, and ketone esters. Both will be metabolized to β-hydroxybutyrate (BHB) after absorption (O'Malley et al., 2017;Hashim and VanItallie, 2014). The anti-inflammatory properties of BHB are attracting increasing attention. However, the efficacy and underlying mechanisms of KD and BHB are not fully understood. Therefore, this review aims to discuss the therapeutic utility of KD and ketone supplementations, as substitutions for KD, in CMD treatment, focusing on their anti-inflammatory properties.

Materials and Methods
Literature search strategy Abstracts of publications identified in PubMed (http://www. ncbi.nlm.nih.gov/pubmed) and Ovid (including Medline, PsychINFO, NURSING, and EMBASE), Scopus, and EBSCO (including CINAHL) were searched and reviewed for relevant papers. The search took place on 22 August 2022 and was not restricted by publication date. Two searches included (1) evaluations on the status of inflammation in mood disorders using animal models, and (2) the antiinflammation properties of KD or BHB in patients. The exact search terms were depression (Title/Abstract) OR depressive symptom (Title/Abstract) OR anxiety (Title/ Abstract) OR anxiety symptom (Title/Abstract) AND (ketogenic diet (Title/Abstract) OR ketone body (Title/ Abstract) OR ketone ester (Title/Abstract) OR ketone diester (Title/Abstract) OR ketone salt (Title/Abstract) OR ketone supplements (Title/Abstract) OR ketosis (Title/ Abstract) for (1), and, inflammation (Title/Abstract) OR inflammatory (Title/Abstract) OR cytokine (Title/Abstract) OR anti-inflammation (Title/Abstract)) AND (ketogenic diet (Title/Abstract) OR ketone body (Title/Abstract) OR ketone ester (Title/Abstract) OR ketone diester (Title/Abstract) OR ketone salt (Title/Abstract) OR ketone supplements (Title/ Abstract) OR ketosis (Title/Abstract)) for (2) and each part was carried out in a single search. Due to limited evidence, the first search was concluded using narrative descriptions. The second search was carried out based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) search strategy (Page et al., 2021), and subsequent reference narrowing is described in Fig. 1. The Prism checklist can be found as Suppl. Table S1 in the supplementary file. Two authors carried out the search and screen separately using the Covidence system (https://www. covidence.org/). The flow chart of the selection of studies is shown in Fig. 1.

Inclusion and exclusion criteria
For search (2), randomized crossover or parallel controlled trials assessing the impact of KD or BHB on human inflammation status measures were included in the current analysis. There were no exclusion criteria for study duration, diet, ketone supplement type or dose, or participants' sex and age, sample size, or health status. Studies assessing the impact in animal models were separated from human trials and analyzed separately. Studies of case reports were excluded.
Data extraction Data were extracted from 15 studies that were selected to meet the inclusion and exclusion criteria. Intervention type and duration, sample characterization, subject status, study design, assay, and inflammatory mediators were extracted from each study to provide descriptive characteristics of participants. For animal studies, the findings have been discussed in a non-systematic way as an elaboration on the proposed mechanisms.

Results
For the results of search (2), based on the PRISMA principles shown in Fig. 1, we retrieved 15 articles that have been summarized in Table 1.
Among the studies, 10 of 15 (66.7%), reported at least one inflammatory mediator (C-reactive protein (CRP), leptin, interleukin (IL)-1β, IL-2, IL-6, IL-12p40, interferon (IFN)-γ and/or tumor necrosis factor (TNF)-α) (Bosco et al., 2018;Castaldo et al., 2021;Cipryan et al., 2020bCipryan et al., , 2020aKong et al., 2020;Shaw et al., 2020;Khodabakhshi et al., 2021, Monda et al., 2020 or the mRNA of the indicated inflammatory mediators (IFN-γ mRNA, Shaw et al., 2020) was decreased by KD or BHB intervention. However, 3 studies reported that no changes were observed (Bock et al., 2018;Rhyu and Cho, 2014;). Further, 2 reported that an anti-inflammation mediator (adiponectin and/or IL-10) was increased (Rosenbaum et al., 2019;Monda et al., 2020;Bertoli et al., 2015). The selected studies conducted on humans showed that KD has great potential in treating inflammatory diseases, showing the potential to be utilized as a treatment for CMD based on the inflammation hypothesis of the pathology of CMD. However, the evidence of BHB is far from convincing, and further studies are warranted. The details of the above studies are listed as Table 1.

Discussion
Inflammation is our body's defense system protecting us against infection, cellular damage, and other harmful agents, both exogenously and endogenously (Suzuki, 2018a;Suzuki et al., 2020). During inflammation, cytokines play a significant role in mediating cellular signaling and immunological responses. Cytokines are a category of small proteins (5~20 kDa) secreted by a broad range of cells, including immune cells such as macrophages, lymphocytes, mast cells, endothelial cells, fibroblasts, myocytes, adipocytes, and other kinds of cells Suzuki, 2018b). The subcategories of cytokines include chemokines,   (Aw et al., 2018). However, inflammation that acts against wrong stimuli may induce unwanted cytokine production, thus bringing undesired consequences. A large body of studies has reported that proinflammatory or inflammatory cytokines increase in CMD patients ( The evidence on the cytokine secretion profile collected from papers published in the recent five years is depicted in Fig. 2. According to a case-controlled study conducted in generalized anxiety disorder (GAD) patients, significantly higher ratios of TNF-α/IL-10, TNF-α/IL-4, IFN-γ/IL-10, and IFN-γ/IL-4 were found in the GAD group compared to the control group (patient group: n = 54; control group: n = 64) (Hou et al., 2017). In pregnant women with severe anxiety and depression, the levels of Th1-(IL-6, TNF-α, IL-2, IFNγ), Th17-(IL-17A), and Th2-(IL-9, IL-10, and IL-13) were higher than those in the control group (n = 139, patient group; n = 40, control group) (Gelman, 2019). In one report, patients with major depressive disorders had significantly higher levels of IL-1β, IL-10, and TNF-α but significantly lower levels of IL-8 (n = 117, patient group; n = 102, control group) (Zou et al., 2018). In another report, IL-1, IL-6, TNF-α, IFN-γ, and IL-10 were found to be associated with depression scores in people with alcohol and drug use disorder (n = 80, patient) (Martinez et al., 2018). Further, a significantly positive correlation between serum cortisol levels and Hamilton Depression Rating Scale scores was observed in 89 male depression patients (Jia et al., 2019). In women with postpartum depression, significant fluctuation of TNF-α and IL-18 were found (Petralia et al., 2019). Therefore, targeting pro-inflammatory and inflammatory cytokines and their signaling pathways might be novel strategies to treat CMD.
Although the mechanism of inflammation in CMD has not been fully explained, the loss of regulation of inflammatory agents and neurotransmitters seems to be the key. The imbalance of neurotransmitters, including serotonin (5-HT), norepinephrine, and dopamine has been reported and treated to be the reason for CMD (Yan, 2018;Naoi et al., 2018). Meanwhile, inflammatory cytokines trigger neuroinflammation, microglial activation, and disturbance of neurotransmitters, resulting in CMD pathophysiological process (Morris et al., 2016). Let us consider the loss of the regulation process of 5-HT as an example. According to the cytokine theory, the initiation of stress increases the production of cytokines, including TNFs, and ILs. IFNs may contribute to the pathophysiological process of CMD. Increased levels of the abovementioned inflammatory cytokines may activate the production of the indoleamine 2,3 dioxygenase (IDO), with subsequent production of tryptophan (TRP) catabolites along the IDO pathway, decreasing the availability of TRP and serotonin and contributing to the progress of CMD (Morris et al., 2016). Conversely, phytochemical supplementation that suppresses pro-inflammatory cytokines, thus inhibiting type A monoamine oxidase (MAOA), has been reported to be efficient in relieving depression (Yan, 2018). Therefore, the use of anti-inflammatory compounds and diets that exhibit anti-inflammatory properties may be a strategy for CMD treatment.
Introduced as a treatment for refractory epilepsy in the first place, the KD has been reported for its antiinflammatory properties in recent years. This provides a potential contributing role in treating CMDs, based on the inflammation hypothesis of the pathology of CMD. For example, in a lipopolysaccharide (LPS)-induced inflammation model in rats, a 2-week administration of KD attenuated LPS-induced fever, and reduced blood and hippocampal IL-1β concentration to alleviate inflammation (Barua et al., 2018). In another rodent inflammation model built by inducing a hind paw inflammation, a KD significantly reduced decreased paw swelling, plasma extravasation, and the peripheral inflammatory response (Ruskin et al., 2021). In a paper published in 2016, after a one-month feeding period of KD mice then received LPS by intraperitoneal injection it was reported that KD mice had significantly low expression of nuclear factor-kappa B (NF-κB), IL-6, and TNF-α (Nandivada et al., 2016). These  studies showed that a KD has excellent potential in serving as an anti-inflammation therapy for inflammation. In the neuroscience field, KD also attracts great attention. In a memory impairment and central nervous systeminflammation murine model, pre-feeding of a one-week KD (12.2 g polyunsaturated fatty acids in 100 g per KD) decreased circulating inflammatory cytokines including IL-1β, IL-6, TNF-α, IL-12, IL-17A, IFN-γ, MCP-1, MIP-1α and MIP-1β (Kim et al., 2012). In a spinal cord injury rat model, KD suppressed the NF-κB pathway and the expression of TNF-α, IL-1β, and IFN-γ (Lu et al., 2018). In another mouse model, motor dysfunction was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment, and pretreatment of KD decreased IL-1β, IL-6, TNF-α (Yang and Cheng, 2010). The anti-inflammatory properties of a KD may be attributed to the ability to activate peroxisome proliferatoractivated receptor-gamma (Jeong et al., 2011;Zhang et al., 2018), subsequently decreasing systematic inflammation . Furthermore, increasing evidence shows that the enhanced levels of BHB, the metabolite of fatty acids during a KD, may contribute to playing an anti-inflammatory role (Prattichizzo et al., 2018;Zitvogel et al., 2017).
Although clinical trials on patients with common mental disorders aiming to elicit the anti-inflammatory properties of BHB have not yet been carried out, animal studies have provided some clues. For example, exogenous ketone body administration has been shown to decrease anxiety-related behaviors evaluated by an open-arm system in rodents (83days sub-chronic beta-hydroxybutyrate-mineral salt supplementation) (Kovács et al., 2018), possibly by interacting with the Adenosine A1 receptor (Kovács et al., 2018). In another study, BHB administration has also been shown to reduce depressive-like behaviors in rodents as evaluated by chronic unpredictable stress and sucrose preference experimental methods (Kovács et al., 2018). In situ BHB administration has been reported to suppress macrophage/microglia activation (Huang et al., 2018). Although the underlying mechanisms have not been elucidated yet, one of the possible mechanisms to ameliorate depressive and anxiety symptoms is by suppressing inflammation (Kong et al., 2017).
The anti-inflammatory properties of BHB have been reported extensively. A study conducted in a rodent model revealed the protective effect of BHB on neuroinflammation. In this study, repeated BHB administration reduced depressive and anxiety behaviors in animals undergoing chronic unpredictable stress (Kong et al., 2017). Additionally, pre-treatment of BHB reduced hippocampal IL-1β and TNF-α (Yamanashi et al., 2017). BHB could alleviate inflammation by attenuating NLR family pyrin domain containing (NLRP)-3 inflammasome formation in IL-1β/IL-18 over-expression mice (Yamanashi et al., 2017). Suppression of the activation of the NLRP3 inflammasome by preventing K+ efflux and reducing apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) oligomerization and speck formation in human monocytes has also been documented (Kajitani et al., 2020). In BV2 cells, BHB supplementation inhibited LPS-induced inflammatory responses and NLRP3 inflammasome protein level, shifting the activation of macrophages/microglia from the proinflammatory M1 to the anti-inflammatory M2a type (Deng et al., 2021). In both mice and human neutrophils separated from the blood, BHB suppressed IL-1β production by inhibiting both priming and assembling procedures in the activation of the NLRP3 inflammasome (Youm et al., 2015). On the other hand, when the ketolytic, rate-limiting enzyme SCOT (succinyl-CoA:3-ketoacid-CoA transferase 1; encoded by Oxct1) was deleted, markers of sterile inflammation and macrophage infiltration were shown to be attenuated in mice that underwent transverse aortic constriction surgery. Further, the NLRP3 expression was also reduced, indicating that elevated circulating BHB might be linked to reduced inflammation through an NLRP3mediating manner (Goldberg et al., 2017).
Calorie restriction is well known for its antiinflammation activities (Ottaviano and Zaman, 2023). Other studies also reported that BHB might exert its antiinflammatory effects as a calorie restriction mimic (Kim et al., 2019). In one report, BHB administration upregulated Forkhead Box1 (FoxO1) and its target genes catalase/ manganese superoxide dismutase (MnSOD). Both play critical roles in quenching inflammation-induced reactive oxygen species, thus ameliorating renal inflammation in aging rats (Kim et al., 2019) by down-regulating TNFSF6, TNF-α, PI3K, NF-κB and toll-like receptor 1 on LPSstimulated macrophages (Qiao et al., 2020). Another role of BHB during anti-inflammation might be as the inhibitor of histone deacetylase (HDAC), as HDAC inhibition is wellknown to have anti-inflammatory effects. For example, in HEK293 cells, HDAC activity decreased with the elevation of BHB concentration, indicating that BHB function as an HDAC inhibitor is dose-dependent (Shimazu et al., 2013). In the same study, it was also shown that dietary BHB exhibited protective effects on cells against oxidative stress via up-regulation of FoxO3a, which shares a similar function as FoxO1, and its target gene, catalase and mitochondrial MnSOD2 (Shimazu et al., 2013). To summarize, numerous studies also show the potential role of BHB in the treatment of CMD. However, since most of the studies are conducted in animals, and the mechanisms are speculative, further studies are urgently encouraged to validate the safety, effectiveness, and mechanisms of BHB application for human CMD treatment.

Conclusions
The anti-inflammatory properties of KD or BHB, the biomarker metabolite during KD administration, are attracting the attention of researchers. In this review, we summarized 15 clinical trials that employed KD or BHB to study their effects on the prevention and treatment of inflammation. Most of them documented favorable results. A KD or BHB-based therapy may thus contribute to relieving neuroinflammation and depressive and anxiety behaviors and have the potential to become a part of the CMD-treatment strategy. Availability of Data and Materials: All data generated or analyzed during this study are included in this published article and its supplementary information files.
Ethics Approval: Not applicable.
Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the present study.