Adopting a low-carb ketogenic diet will induce the state of ketosis, in which most of the body’s energy supply comes from ketone bodies in the blood, rather than from blood glucose or sugar. However, the role of ketones bodies in the regulation of the immune system is largely unknown. This is critically important because excess inflammation has been shown to be involved in the development of major diseases, such as cancer, heart disease, Alzheimer’s disease, diabetes, obesity, Parkinson’s disease and inflammatory bowel diseases. The purpose of this article is to explain a recent groundbreaking study that examined whether ketone bodies, specifically β-hydroxybutyrate, can reduce the severity of inflammation in the immune system.

Background on Inflammation and Inflammasomes

Inflammation is a protective immune response mounted by the evolutionarily conserved innate immune system in response to harmful stimuli. This process is very important, as it allows the body to get rid of infectious agents, dead or damaged cells, as well as toxins. Deregulation of the immune response can be harmful, as insufficient inflammation can lead to persistent infection by pathogens, while excessive inflammation can cause a wide variety of autoinflamma¬tory and autoimmune diseases.

Two main recognition patterns are involved in the innate immunity activation, namely the pathogen-associated molecular patterns (PAMPs), derived from invading pathogens, and the damage-associated molecular patterns (DAMPs), linked to molecules released in the body. Upon recognition of PAMPs or DAMPs, multimeric protein complexes called inflammasomes are assembled in the cell [1, 2]. After its activation, the assembled inflammasome is then responsible for an inflammatory cascade response. It notably binds and activates the inflammatory caspase-1, which is then responsible for a variety of cellular processes related to inflammation, such as the maturation of proinflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-18 (IL-18) [2]. IL-1β is an important mediator of the inflammatory response involved in many related cellular processes, while IL-18 is able to induce severe inflammatory reactions potentially involved in some inflammatory disorders.

There are many types of inflammasomes that are commonly named according to the main protein forming their scaffold and responsible to recruit other proteins in order to assemble the complex. The ligands of five different types of inflammasomes have been clearly identified until now, notably NLRP1, NLRP2, NLRP3, AIM2 and IPAF/NLRC4. NLRP1 [2, 3] and IPAF/NLRC4 [4, 5]. Inflammasomes are involved in response to different types of bacteria, while other types of bacteria or viruses can trigger AIM2 inflammasomes [6]. Recently, NLRP2 inflammasomes has been linked to the response involved in central nervous system trauma [7]. However, NLRP3 inflammasome remains the most studied one, as it can be involved in the recognition of certain types of bacteria, viruses and fungi [8-11], as well as in the recognition of many types of DAMPs involved in several chronic autoimmune diseases [12-16].

Balance seems to be the key in the innate immune response triggered by the inflammasome formation. On one hand, inflammasomes can be beneficial to fight against numerous types of pathogens. However, on the other hand, inflammasome activation can be linked to an excess of inflammation involved in the development of several diseases, mainly autoinflammatory and autoimmune diseases. As such, activation of NLRP3 inflammasome in our body in response to diverse DAMPs has been linked to several diseases such as some types of cancer (reviewed in [17]), atherosclerosis (corresponding to a hardening of the arteries leading to heart attack and stroke) [18], multiple sclerosis [19], Alzheimer’s disease [20], Parkinson’s disease [21], type 2 diabetes [22, 23], age-related functional decline [24], gout [13], obesity [22], and inflammatory bowel diseases, such as ulcerative colitis and Crohn’s disease [25-27]. Notably, NLRP3 inflammasome has also been linked to neuroinflammation involved in chronic mild stress-induced depression [28].

In order to treat or alleviate the symptoms related to many diseases associated with uncontrolled inflammation, the scientific community and drug companies have been researching pharmacological inhibitors targeting constituents of the NLRP3 inflammasomes and its related activated cytokines, such as IL-1β and IL-18 (reviewed in [29]). For instance, the inhibition of IL-1β using antibodies or receptor antagonists is currently used to treat multiple diseases (reviewed in [30]). However, this type of drug is associated with multiple adverse side effects, mainly related to the increase susceptibility to infections, as the drug targets an essential component of the immune system. An antibody to suppress IL-18 has also been developed by GlaxoSmithKline (Brentford, UK), but it is still at the clinical trial stage and not ready to be commercialized at present. Another promising therapeutic avenue would be to block the inflammasome prior to the release of the deleterious proinflammatory cytokines IL-1β and IL-18, in order to limit the damages caused by uncontrolled inflammation. As such, some molecules potentially inhibiting the formation of the inflammasome, by targeting for instance NLRP3 or the caspase-1, are currently under clinical trial. Furthermore, as a reduction in the potassium level inside the cell is required for the induction of the activation of caspase-1 by NLRP3 [1, 31], inhibitors of the ATP receptor P2X7, responsible for lowering potassium level, is also a promising therapeutic avenue. However, inhibitors of P2X7 tested to date in clinical trials have been proved to be ineffective (reviewed in [29]).

This leads us to a review of the following study examining an alternative inhibitor of the NLRP3 inflammasome that can be triggered by a ketogenic diet.

The Study

Prolonged fasting, high-intensity exercise and a ketogenic diet (KD) are known to increase the concentration of β-hydroxybutyrate (BHB), the first ketone produced from acetoacetate in the liver in order to serve as an alternative source of energy instead of glucose when the body is in a fasting state [32]. This phenomenon has been linked to the reduction of oxidative stress [33], to an increase in the AMPK activity [34], an enzyme involved in cellular energy homeostasis, and to autophagy [35], a process involved in the degradation and recycling of altered cellular components. It is interesting to note that these three mechanisms are involved in the regulation of NLRP3 inflammasome [1], meaning that BHB plays at least an indirect role in this process. Furthermore, it has been suggested that BHB may act as a signaling molecule through the binding of the G protein coupled receptor GPR109a [36] or by inhibiting the histone deacetylase (HDAC) molecule [33].

Recently, Youm et al. (2015) [37] reported that BHB suppresses the activation of NLRP3 inflammasome in response to several different NLRP3 activators, with no impact on the activation of other types of inflammasomes. This finding suggests that an elevation in the circulating BHB level, such as the one obtained with a KD, might be beneficial against NLRP3-mediated proinflammatory diseases. The following sections will summarize the main results obtained in this article by Youm et al. (2015) [37].

Methods and Results

First, the authors tested whether BHB impacts the activation of the inflammasome by treating activated mouse bone marrow derived macrophages (BMDMs), a key cell type in the immune system responsible for the release of many cytokines, with the NLRP3 activator ATP, a known potassium efflux agent, in the presence of BHB. Then, they measured the activation of caspase-1.

They found that the activation of caspase-1 induced by ATP was inhibited by BHB at doses similar to the ones induced by strenuous exercise or 2-day fasting. However, other molecules structurally similar to BHB did not affect the activation of NLRP3 induced by ATP, meaning that the observed effect is specific for BHB only.

Furthermore, using human monocytes, the authors also showed that BHB is able to inhibit the release of IL-1β and IL-18. The same experiment with the BMDMs was conducted using seven other NLRP3 activators, namely monosodium urate crystal, particulate matter, nigericin, silica particles, lipotoxic fatty acids palmitate, ceramides and sphingosine. For all of them, BHB specifically blocked the activation of the inflammasome.

Next, the authors investigated whether the inhibition effect of BHB on NLRP3 inflammasome activation was specific for this type of inflammasome only. To do so, they conducted an experiment similar to the previous one in presence of different bacteria known to induce either AIM2 or NLRC4 inflammasomes, as well as with LPS, a constituent of the outer membrane of some bacteria known to induce the activation of the inflammasome independently of the classical scaffolding proteins by the activation of caspase-11. BHB failed to inhibit inflammasome activation in all those cases, meaning that BHB acts specifically on the NLRP3 inflammasome and not on the other types of inflammasomes in response to either PAMPs or DAMPs.

The authors also wanted to test the relevance of the indirect effects of BHB on the regulation of NLRP3 inflammasome observed in other studies. To do so, they reversed independently the cellular effects of an elevation of BHB observed by others. They showed that, taken separately, an increase in oxidative stress, a decrease in the AMPK activity and an inhibition of autophagy did not alter the inhibition of inflammasome activation mediated by BHB. These findings suggest that BHB alone, and not its associated effects on those cellular processes, is responsible for the inhibition of the inflammasome activation. They also found that previously reported interactions of BHB with G protein coupled receptor GPR109a or with the histone deacetylase (HDAC) did not affect the inhibition of inflammasome activation mediated by BHB.

Other experiments were done in order to better understand how BHB affects the inflammasome activation in the macrophage. These experiments confirm that neither the metabolism of BHB through the TCA cycle nor BHB oxidized metabolites are involved in the inhibition of the inflammasome activation mediated by BHB. As mentioned earlier, the reduction in the potassium level of the cell is known to activate the NLRP3 inflammasome. As such, the authors tested whether BHB is able to prevent the decrease in potassium level. They found that BHB indeed helps to maintain the potassium level within the cell in presence of three different NLRP3 activators, namely ATP, monosodium urate and ceramides. They also found that BHB inhibits the ATP-induced ASC oligomerization and speck formation, two common mechanisms known to be involved in the activation of NLRP3 inflammasome [38, 39].

Next, the authors did in vivo experiments in mice in order to assess the potential of BHB as an inhibitor of the NLRP3 inflammasome activation. To do so, they used BHB nanolipogels (nLGs) formula in order to improve its bioavailability in vivo [39]. They activated the NLRP3 inflammasome in mice using monosodium urate crystal injection and they administered them either BHB nLGs or nLGs alone as a control. BHB nLGs seems to have direct effects in vivo on the NLRP3-driven neutrophil influx to the site of injection, as it reduces the number of neutrophils recruited to this site without directly affecting the neutrophil migration. Neutrophils are part of the innate immune system and are the first cells to migrate to the inflammation site at the very beginning of the inflammation process. BHB nLGs also reduces the concentration of IL-1β in the serum of the mice following the monosodium urate crystal injection. Furthermore, another experiment showed that BHB nLGs inhibits the inflammasome activation in BMDMs derived from genetically modified mice where NLRP3 inflammasomes are always activated.

Finally, an experiment was conducted in order to assess the potential of a KD in the treatment of NLRP3 inflammasome-mediated diseases in mice. To do so, genetically modified mice were fed with either 1,3-butanediol ketone diesters to mimic a KD, thereby producing an increase in their BHB level or normal mice food as a control. After one week of this diet, a mutation was induced in the mice to provoke the Familial Cold Autoinflammatory syndrome (FCAS), a disease characterized by a mutation in NLRP3 eliminating the need for a NLRP3 activator, meaning that the NLRP3 inflammasome is always activated. They showed that KD protects the mice from common symptoms for this disease, such as neutrophilia and hyperglycemia, without impacting the overall frequency of the main immune cells. This result suggests that KD elevating BHB levels might be beneficial for patients by inhibiting the NLRP3 inflammasome activation.

Conclusion

The authors confirmed that BHB, but not other structurally similar molecules, is able to specifically block the activation of the NLRP3 inflammasome mediated by many known NLRP3 activators. They also found that this effect was specific to NLRP3 inflammasome, as BHB did not affect the activation of the other types of inflammasome. These findings are promising, as chronic inflammatory diseases could be theoretically treated with BHB without affecting others inflammasomes necessary to fight against infections. Furthermore, they showed that BHB is able to inhibit the secretion of IL-1β and IL-18 by human immune cells. They also demonstrated that this BHB-related process is mediated by preventing the decrease of potassium level in the cell, as well as by inhibiting ASC oligomerization, speck formation and, consequently, the formation of the active inflammasome.

To summarize, the authors state that “these findings provide insight into immunological functions of metabolic signals such as BHB and suggest that dietary or pharmacological approaches to elevate BHB, without inducing the generalized starvation response, holds promise in reducing the severity of multiple NLRP3 mediated chronic inflammatory diseases.”

Study Editor

Marie-Christine Brotherton holds a Ph.D. in Cellular and Molecular Biology with specific expertise in Parasitology, Proteomics, Drug Resistance and Genomics. She also holds a MBA with a major in Corporate Social and Environmental Responsibility. She has strong experience with the scientific publication process, including author guidelines requirements, as well as with the medical and social/environmental fields. She can be reached by email at marie-christine.brotherton.1@ulaval.ca

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