Ketones and Autism Part 4 � Inflammation, Activated Microglia, CtBP, the NLRP3 Inflammasome and IL-1�




This series of posts on ketones and the ketogenic diet (KD) is nearly finished and I am glad that I made favourable comments about the KD earlier on in this blog, before I knew all the nitty-gritty of the science. (no re-editing required)




Inflammasome Inhibition: Putting Out the Fire                                                                                                                       


There is more than one anti-inflammatory mechanism involved in the ketogenic diet (KD); in Part 3 we covered Niacin Receptor HCA2, today in Part 4 we look at NLRP3 and CtBP.
The reason I am going into all this detail is because if you knew why someone responds to ketones in a favourable way, there might actually be an even more potent therapy using an entirely different substance.
CtBP represses the transcription of certain tumour supressing genes and some other genes involved in the development of cancer, i.e. they promote tumorigenesis.  CtBP is often overexpressed in certain cancers and indicates a worse prognosis. In these cancers you would want to inhibit CtBP.
Just to complicate matters, CtBP also supresses the activity of certain inflammatory genes. So, in certain diseases like diabetes you might benefit from keeping CtBP permanently in its active state. In particular, this would apply to when the microglia are activated, which is the case in much autism.
The coconut oil doctors have the idea that the key problem in autism is activated microglia in the brain.  Microglia mediate immune responses in the central nervous system, clearing cellular debris and dead neurons via a process called phagocytosis. These doctors propose coconut oil to calm the microglia.
Microglia can be in a resting or activated state, the research suggests that in much autism the microglia are permanently activated.
Some research suggests that microglia act like an �immunostat� reflecting not just what is going on in the brain, but elsewhere in the body.  I favour this view.
A small trial using a drug to calm the microglia did not impact autism.
Personally, I believe that microglia being activated is not a good thing, but that it is part of a much more complex picture than the coconut doctors suggest. 
As we learn later in this post, to get the CtBP benefit to microglia, it appears that you need the kind of ketosis you achieve only in the full ketogenic diet, not the transient mild ketosis that you achieve from two heaped tablespoons of coconut oil, or any of the keto supplements. 

NLRP3 inflammasome
The complicated-sounding NLRP3 inflammasome relates to diseases where the proinflammatory cytokine IL-1� is elevated; this includes Alzheimer�s, MS, Inflammatory Bowel Disease (IBD) and often autism.
For the details of how NLRP3 works see below; the important thing to note is that the result is elevated levels of IL-1�, which, at least in blood, is easy to measure.  It is an open question whether this represents the level inside the brain.  If your child has elevated IL-1� then it is worth studying NLRP3.




Schematic illustration of the NLRP3 inflammasome activation. Upon exposure to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), Toll-like receptors (TLRs) are phosphorylated and subsequently activate NF-?B. In the nucleus, NF-?B promotes the transcription of NLRP3, proIL-1�, and proIL-18, which, after translation, remain in the cytoplasm in inactive forms. Thus, this signal (depicted in red as �Signal 1�) is a priming event. A subsequent stimulus (shown as �Signal 2� in black) activates the NLRP3 inflammasome by facilitating the oligomerization of inactive NLRP3, apoptosis-associated speck-like protein (ASC), and procaspase-1. This complex, in turn, catalyzes the conversion of procaspase-1 to caspase-1, which contributes to the production and secretion of the mature IL-1� and IL-18. Three models have been proposed to describe the second step of inflammasome activation: (1) Extracellular ATP can induce K+/potassium e?ux through a purogenic P2X7-dependent pore, which, leads to the assembly and activation of the NLRP3 inflammasome. Calcium flux is also involved in this process. (2) PAMPs and DAMPs trigger the generation of ROS that promote the assembly and activation of the NLRP3 inflammasome. (3) Phagocytosed environmental irritants form intracellular crystalline or particulate structures leading to lysosomal rupture (magenta box) and release of lysosomal contents like cathepsin B. These induce NLRP3 inflammasome assembly and activation. In addition, other factors and mechanisms have been implicated in the assembly and activation of the NLRP3 inflammasome, including mitochondrial damage, autophagic dysfunction, and thioredoxin-interacting protein (TXNIP).



Proinflammatory cytokine IL-1� 
My public enemy number 1 cytokine is actually IL-6, today we primarily look at IL-1�, which for many people with a neurological disorder is a big part of their problem. IL-6 and IL-1� are actually interrelated, as we see later.
For a summary of the role of this cytokine in autism, I will leave it to Paul Ashwood:-  


Interleukin (IL)-1B

IL-1? is an inflammatory cytokine expressed very early in immune responses. In tissue, IL-1? propagates inflammation by activating local immune cells and the vascular endothelium. Systemically, IL-1? stimulates IL-6 production and eventually an acute phase response in the liver. Systemic IL-1? can cross the blood brain barrier and stimulate its own expression in the hypothalamus, which leads to neuroendocrine changes associated with fever and sickness behavior . IL-1? receptors are structurally related to toll-like receptors (TLRs), and signaling is achieved through NF-?B and MAP kinase (MAPK) signaling cascades. IL-1? belongs to an evolutionarily conserved family of proteins that function beyond immunity. It shares structural homology with fibroblast growth factors, which are critical in embryonic neurodevelopment, and are implicated in autism and schizophrenia.
Genes for IL-1?, its receptor, and its receptor-associated proteins are associated with intellectual disability, schizophrenia, and autism. Children and adults with autism have increased plasma IL-1? and skewed cellular IL-1? responses following stimulation. Compared to controls, monocytes from children with ASD produce excessive IL-1? following LPS exposure, and lower levels following exposure to TLR 9 agonists. The IL-1 antagonist, IL-1ra, is also increased among ASD subjects. IL-1ra reduces inflammation by competing for the IL-1? receptor, and increased levels may represent an attempt to counteract inflammation in ASD. Postmortem brains from ASD subjects had normal IL-1? levels, but given that peripheral IL-1? can enter the brain, increased systemic levels could directly impact neurological processes
IL-1? disruption can have a variety of neurological consequences relevant to autism. The cytokine and its receptors are found throughout the nervous system during critical developmental periods. IL-1? induces neural progenitor cell proliferation in some CNS regions, while inhibiting it in others. This could contribute to the region-specific overgrowth and undergrowth observed in the ASD brain. Excitatory synapse formation is partially mediated by the IL-1 receptor and receptor-associated proteins.
Altering these proteins can tip the balance between excitatory and inhibitory signaling, which might underlie neurological features of autism. Increased IL-1ra in autism suggests an attempt to counterbalance IL-1? and may or may not be beneficial. Following brain injury, IL-1ra upregulation serves a neuroprotective role by dampening excessive inflammation. However, if administered during critical windows of neurodevelopment, IL-1ra can negatively impact neurogenesis, brain morphology, memory consolidation, and behavior. This shows that some level of IL-1B signaling is essential during development. In adulthood, IL-1? is implicated in CNS disorders like Alzheimer�s disease and the advancement of amyloid-containing plaques. While excessive IL-1B contributes to pathology in some cases, it may have a protective role in others. For example, IL-1? limits neuronal damage following excitotoxic exposures, and mice lacking IL-1? fail to undergo remyelination following experimental autoimmune encephalitis (EAE) induction. IL-1? is involved in higher order brain processes and is induced in the hippocampus during learning processes, and is critical for maintenance of long-term potentiation (LTP) Both over expression and under expression of IL-1 beta are associated with impairments in memory and learning.



At the table in the kitchen, there were three bowls of porridge.  Goldilocks was hungry.  She tasted the porridge from the first bowl.
"This porridge is too hot!" she exclaimed.
So, she tasted the porridge from the second bowl.
"This porridge is too cold," she said
So, she tasted the last bowl of porridge.
"Ahhh, this porridge is just right," she said happily and she ate it all up. 

In summary, IL-1? participates in neurological processes, and appears to have a role in both CNS pathology and healing. Normal, homeostatic levels of IL-1? and its antagonist IL-1ra are necessary for proper brain development and function. This �Goldilocks� state is typical of many cytokines, where too much or too little is not desirable. Alterations in IL-1? systems due to genetic mechanisms or environmental exposures may contribute to autism. 


CtBP (C-terminal-binding protein) 
In 2017 research led by Dr Raymond Swanson, a professor of neurology at the University of California, San Francisco, suggested CtBP as an additional possible mechanism by which the ketogenic diet can reduce brain inflammation.   CtBP activation turns off key inflammatory genes.
In the case of CtBP, I doubt that the very partial ketosis achieved with BHB and C8 supplements will be enough, I think you would need the full ketogenic diet. 
Restricting the glucose metabolism with the ketogenic diet lowers the NADH/NAD+ ratio which activates CtBP. There is no direct role played by ketones in this process, it is just the presence of large amounts of ketones reduces the role of glucose.



Factors that reduce glucose flux through glycolysis, such as reduced glucose availability or glycolytic inhibitors, reduce NADH levels and thereby reduce NADH:NAD+ ratio, whereas factors that inhibit oxidative metabolism, such as hypoxia and mitochondrial inhibitors, have the opposite effect. Glutamine provides ketone bodies (a-ketoglutarate) to fuel mitochondrial ATP production in the absence of glycolysis. Lactate dehydrogenase (LDH) maintains the lactate:pyruvate ratio in equilibrium with the cytosolic NADH:NAD+ ratio.


BHB is not directly a CtBP activator.
A drug that acts as an CtBP activator would be great for diabetes and anyone with brain inflammation.
Using BHB and C8 you would need to create enough ketones in your blood to reduce the glucose metabolism substantially, not by a trivial amount.
The easy to read version:- 

New research uncovers and replicates the mechanism by which a ketogenic diet curbs brain inflammation. The findings pave the way for a new drug target that could achieve the same benefits of a keto diet without having to actually follow one.

A keto state lowers brain inflammation
A keto diet changes the metabolism, or the way in which the body processes energy. In a keto diet, the body is deprived of glucose derived from carbs, so it starts using fat as an alternative source of energy.

In the new study, Dr Swanson and his colleagues recreated this effect by using a molecule called 2-deoxyglucose (2DG).
The 2DG molecule stopped glucose from metabolizing and created a ketogenic state in rodents with brain inflammation as well as in cell cultures. Levels of inflammation were drastically reduced - almost to healthy levels - as a result.
"We were surprised by the magnitude of our findings," said Dr Swanson. "Inflammation is controlled by many different factors, so we were surprised to see such a large effect by manipulating this one factor. It reinforces the powerful effect of diet on inflammation."
The restricted glucose metabolism lowered the so-called NADH/NAD+ ratio
"Cells convert NAD+ to NADH, as an intermediary step in generating energy from glucose, and thus increase the NADH/NAD+ ratio," he added.
When this ratio is lowered, the CtBP protein gets activated and attempts to turn off inflammatory genes. As Dr. Swanson told us, "CtBP is a protein that senses the NADH/NAD ratio and regulates gene expression depending on this ratio."
So, the scientists designed a molecule that stops CtBP from being inactive. This keeps the protein in a constant "watchful" state, blocking inflammatory genes in an imitation of the ketogenic state. Dr. Swanson said, "Our findings show that it is [...] possible to get the anti-inflammatory effect of a ketogenic diet without actually being ketogenic
The findings could apply to other conditions that are characterized by inflammation. In diabetes, for example, the excessive glucose produces an inflammatory response, and the new results could be used to control this dynamic.
"[The] ultimate therapeutic goal would be to generate a [drug] that can act on CtBP to mimic the anti-inflammatory effect of [the] ketogenic diet," Dr. Swanson concluded. 

Full Paper:- 


The innate inflammatory response contributes to secondary injury in brain trauma and other disorders. Metabolic factors such as caloric restriction, ketogenic diet, and hyperglycemia influence the inflammatory response, but how this occurs is unclear. Here, we show that glucose metabolism regulates pro-inflammatory NF-?B transcriptional activity through effects on the cytosolic NADH:NAD+ ratio and the NAD(H) sensitive transcriptional co-repressor CtBP. Reduced glucose availability reduces the NADH:NAD+ ratio, NF-?B transcriptional activity, and pro-inflammatory gene expression in macrophages and microglia. These effects are inhibited by forced elevation of NADH, reduced expression of CtBP, or transfection with an NAD(H) insensitive CtBP, and are replicated by a synthetic peptide that inhibits CtBP dimerization. Changes in the NADH:NAD+ ratio regulate CtBP binding to the acetyltransferase p300, and regulate binding of p300 and the transcription factor NF-?B to pro-inflammatory gene promoters. These findings identify a mechanism by which alterations in cellular glucose metabolism can influence cellular inflammatory responses.

The innate inflammatory response contributes to secondary injury in brain trauma and other disorders. Metabolic factors such as caloric restriction, ketogenic diet, and hyperglycemia influence the inflammatory response, but how this occurs is unclear. Here, we show that glucose metabolism regulates pro-inflammatory NF-?B transcriptional activity through effects on the cytosolic NADH:NAD+ ratio and the NAD(H) sensitive transcriptional co-repressor CtBP. Reduced glucose availability reduces the NADH:NAD+ ratio, NF-?B transcriptional activity, and pro-inflammatory gene expression in macrophages and microglia. These effects are inhibited by forced elevation of NADH, reduced expression of CtBP, or transfection with an NAD(H) insensitive CtBP, and are replicated by a synthetic peptide that inhibits CtBP dimerization. Changes in the NADH:NAD+ ratio regulate CtBP binding to the acetyltransferase p300, and regulate binding of p300 and the transcription factor NF-?B to pro-inflammatory gene promoters. These findings identify a mechanism by which alterations in cellular glucose metabolism can influence cellular inflammatory responses.

One way that CtBP regulates gene transcription is through interactions with the histone acetyltransferase HDAC1. 

Taken together, our findings indicate that metabolic influences that alter the cytosolic NADH:NAD+ ratio regulate NF-?B transcriptional activity through an NADH-dependent effect on CtBP dimerization. Conditions that reduce glycolytic flux, such as ketogenic diet and caloric restriction, can thereby suppress NF-?B activity, while conditions that increase glycolytic flux may increase it. These interactions provide a mechanism for the suppressive effects of ketogenic diet and caloric restriction on brain inflammation after brain injury. By extension, these interactions may also contribute to the pro-inflammatory states associated with diabetes mellitus and metabolic syndrome. 



Inhibiting NLRP3 and/or activating CtBP

You do not need to be a genius to see that inhibiting NLRP3 and/or activating CtBP, using the ketogenic diet, is likely to benefit some people with autism.
On the flipside, someone with colon cancer, where CtBP is over-expressed to the point where the cancer depends on it for growth, certainly would not want the ketogenic diet.
This cancer flipside we have seen before, antioxidants like NAC and Sulforaphane (via activating the redox switch Nrf2) are chemoprotective for healthy people, but bad for you if you have developed cancer.  Oxidative stress is very damaging to cancer cells and so it becomes a good thing. Some people who develop cancer then choose to improve their diet to include new healthy foods, sadly for some people this may actually be counterproductive.
Estrogen is another case in point, it has many positive effects and has been suggested to be one reason why women like longer than men. If you develop estrogen positive breast cancer, more estrogen is the last thing you would want.  

Other NLRP3 inhibitors 

                          

Coll et al. (2015) discovered that MCC950, a diarylsulfonylurea-containing compound known to inhibit caspase-1-dependent processing of IL-1�, also inhibits both canonical and non-canonical activation of the NLRP3 inflammasome. MCC950 inhibits secretion of IL-1� and NLRP3-induced ASC oligomerization in mouse and human macrophages. It reduces secretion of IL-1� and IL-18, alleviating the severity of EAE and CAPS in mouse models. Coll et al. (2015) further showed that MCC950 acts specifically on the NLRP3 inflammasome

Note that MCC950 is the new name for a drug Pfizer originally called CP-456773 or CRID3, which was not successful as a treatment for arthritis, but now has a second chance

Youm et al. (2015) discovered that the ketone metabolite �-hydroxybutyrate (BHB), but not acetoacetate or the short-chain fatty acids butyrate and acetate, reduced IL-1�, and IL-18 production by the NLRP3 inflammasome in human monocytes. Like MCC950, BHB appears to block inflammasome activation by inhibiting NLRP3-induced ASC oligomerization. Their in vivo experiments showed that BHB or a ketogenic diet alleviate caspase-1 activation and caspase-1-mediated IL-1� production and secretion, without affecting the activation of NLRC4 or AIM2 inflammasomes. BHB inhibits NLRP3 inflammasome activation independently of AMP-activated protein kinase, ROS, autophagy, or glycolytic inhibition. These studies raise interesting questions about interactions among ketone bodies, metabolic products, and innate immunity. BHB levels increase in response to starvation, caloric restriction, high-intensity exercise, or a low-carbohydrate ketogenic diet. Vital organs such as the heart and brain can exploit BHB as an alternative energy source during exercise or caloric deficiency. Future studies should examine how innate immunity, particularly the inflammasome, is influenced by ketones and other alternative metabolic fuels during periods of energy deficiency 
Although both MCC950 and BHB inhibit NLRP3 inflammasome activation, their mechanisms differ in key respects. BHB inhibits K+ e?ux from macrophages, while MCC950 does not. MCC950 inhibits both canonical and non-canonical inflammasome activation, while BHB affects only canonical activation. Nevertheless both inhibitors represent a significant advance toward developing therapies that target IL-1� and IL-18 production by the NLRP3 inflammasome in various diseases. 

Type I Interferon (IFN) and IFN-�

In contrast to these newly described, NLRP3-specific inflammasome inhibitors, type I interferons (IFNs), including IFN-a and IFN-�, have been used for some time to inhibit the NLRP3 and other inflammasomes in various auto-immune and auto-inflammatory diseases. These diseases include multiple sclerosis, systemic-onset juvenile idiopathic arthritis caused by gain-of-function NLRP3 mutations, rheumatic diseases and familial-type Mediterranean fever.

These studies highlight the efficacy of type I IFN therapy and the need for future studies to elucidate the mechanisms of NLRP3 inflammasome inhibition. This work may improve clinical approaches to treating multiple sclerosis and other auto-immune and auto-inflammatory diseases.

Other Kinds of NLRP3 Inflammasome Inhibitors
Several additional ways for inhibiting the NLRP3 inflammasome have opened up in recent years. Autophagy, a self-protective catabolic pathway involving lysosomes, has been shown to inhibit the NLRP3 inflammasome, leading researchers to explore the usefulness of autophagy-inducing treatments  

Cannabinoid receptor 2 (CB2R) is an already demonstrated therapeutic target in inflammation-related diseases (Smoum et al., 2015). Work from our own laboratory (Shao et al., 2014) has shown that autophagy induction may help explain why activation of the anti-inflammatory CB2R leads to inhibition of NLRP3 inflammasome priming
Thus CB2R agonists similar to the HU-308 used in our work may become an effective therapy for treating NLRP3 inflammasome-related diseases by inducing autophagy.
Several other microRNAs have been reported to be involved in the activation of the NLRP3 inflammasome, including microRNA-155, microRNA-377, and microRNA-133a-1. Reducing the levels of these factors may be useful for treating inflammasome-related disease 


Conclusion regarding NLRP3 inhibitors

At this point in time BHB is clearly the best choice; at some point it would be expected that Pfizer will commercialize MCC950. 

 Further relevant papers: 

Inflammasomes are newly recognized, vital players in innate immunity. The best characterized is the NLRP3 inflammasome, so-called because the NLRP3 protein in the complex belongs to the family of nucleotide-binding and oligomerization domain-like receptors (NLRs) and is also known as �pyrin domain-containing protein 3�. The NLRP3 inflammasome is associated with onset and progression of various diseases, including metabolic disorders, multiple sclerosis, inflammatory bowel disease, cryopyrin-associated periodic fever syndrome, as well as other auto-immune and auto-inflammatory diseases. Several NLRP3 inflammasome inhibitors have been described, some of which show promise in the clinic. The present review will describe the structure and mechanisms of activation of the NLRP3 inflammasome, its association with various auto-immune and auto-inflammatory diseases, and the state of research into NLRP3 inflammasome inhibitors. 

NLRP3-inflammasome activates caspase-1 and processes pro-IL-1� and pro-IL-18 into the active cytokines. Two recent studies describe specific inhibitors of NLRP3 inflammasome that inhibit IL-1� release and inflammation. The specificity and potency of these compounds gives hope that a targeted approach to inhibit NLRP3-driven inflammation may be just around the corner



Activation of the inflammasome is implicated in the pathogenesis of an increasing number of inflammatory diseases, including Alzheimer�s disease (AD). Research reporting inflammatory changes in post mortem brain tissue of individuals with AD and GWAS data have convincingly demonstrated that neuroinflammation is likely to be a key driver of the disease. This, together with the evidence that genetic variants in the NLRP3 gene impact on the risk of developing late-onset AD, indicates that targeting inflammation offers a therapeutic opportunity. Here, we examined the effect of the small molecule inhibitor of the NLRP3 inflammasome, MCC950, on microglia in vitro and in vivo. The findings indicate that MCC950 inhibited LPS + A�-induced caspase 1 activation in microglia and this was accompanied by IL-1� release, without inducing pyroptosis. We demonstrate that MCC950 also inhibited inflammasome activation and microglial activation in the APP/PS1 mouse model of AD. Furthermore, MCC950 stimulated A� phagocytosis in vitro, and it reduced A� accumulation in APP/PS1 mice, which was associated with improved cognitive function. These data suggest that activation of the inflammasome contributes to amyloid accumulation and to the deterioration of neuronal function in APP/PS1 mice and demonstrate that blocking assembly of the inflammasome may prove to be a valuable strategy for attenuating changes that negatively impact on neuronal function. 

Scientists say new treatments for inflammatory diseases could be on the way

New treatments for inflammatory diseases could be on the way thanks to a significant discovery made by an international group of scientists, including some at Trinity College Dublin. 
The treatments could be used for a whole range of inflammatory disease including arthritis, Alzheimer's, multiple sclerosis, Parkinson's, gout, asthma and Muckle-Wells syndrome.

The researchers have found that a molecule, previously developed and then abandoned by a multinational pharmaceutical company, can block one of the key drivers of a plethora of inflammatory conditions.
The molecule, MCC950, was produced by Pfizer two decades ago as a possible treatment for arthritis.
However, the company discontinued its efforts to bring the drug to market, and the intellectual property rights on it subsequently lapsed.
Around eight years ago, scientists at Trinity's Biomedical Sciences Institute led by Professor of Biochemistry Luke O'Neill came across the compound and began to explore its potential uses.
They subsequently discovered that it could effectively block the NLRP3 inflammasome.
Inflammasomes are a complex of molecules that trigger inflammation when exposed to infection or stress.
They have been identified as promising therapeutic targets for researchers in recent years.
The NLRP3 inflammasome has been found to be a common activator of a key process in certain inflammatory diseases.
The discovery by the research team, details of which are published in the journal Nature Medicine, confirms that all inflammatory diseases share a common process, although the part of the body which experiences the inflammation might differ.
The scientists subsequently carried out trials on mice and found that the molecule stopped the progression of multiple sclerosis and sepsis.
They also carried out testing on samples taken from humans with Muckle-Wells syndrome, a rare auto-inflammatory disorder, and discovered it was equally effective.
The scientists also say that it is likely the drug could produce fewer common side-effects, such as susceptibility to infection, than other anti-inflammatory drugs, and could prove cheaper and capable of being administered orally.
The next stage will involve testing the compound on humans and a wider group of diseases.
The researchers say for certain conditions, like Muckle-Wells syndrome and asthma, such trials could take place as early as two to three years from now, as the drug had already undergone some human testing by Pfizer.
However, even if the trials prove the drug is safe and effective, they stress that it could be ten-15 years before it could be fully approved for use in humans for the treatment of more complex diseases like multiple sclerosis or Alzheimer's.
They also stress that while the molecule could become an effective treatment, it will not be a cure, though it is possible it could be effective in undoing some of the damage done by well progressed cases of certain diseases.
Prof O'Neill and his team now plan to form a company to further develop and test the compound.
MCC950 is also currently being tested on mice in the US for anti-ageing properties, as there is a growing school of thought that inflammation is responsible for much of the ageing process - a theory which has come to be known as "inflammaging".
The study, part funded by Science Foundation Ireland and the European Research Council, was carried out by a collaboration of six institutions, including the Universities of Queensland, Michigan, Massachusetts and Bonn. 

Conclusion

I am amazed at all the potentially good things that ketones and KD can do for many people�s health and it is all based on science from very serious institutions. 






Ketones and Autism Part 4 � Inflammation, Activated Microglia, CtBP, the NLRP3 Inflammasome and IL-1�

This series of posts on ketones and the ketogenic diet (KD) is nearly finished and I am glad that I made favourable comments about the KD ea...