Nina BaileyBSc (Hons) MSc PhD ANutr

Modulating inflammation by targeting NFkB

Mozaffarian D & Wu JH. Omega-3 Fatty Acids and Cardiovascular Disease: Effects on Risk Factors, Molecular Pathways, and Clinical Events J Am Coll Cardiol. 2011;58(20):2047-2067.

© CNELM

An omega-3 dose 1 g/d, (as Omacor) in the study by Rupp et al, increased omega-3 index from a mean of 3.6 % to 5.4 % but approximately 16 % of the subjects achieved an EPA + DHA blood level less than 4.8 %.

The AA to EPA ratio was reduced from 20 to 7.2

This is clinically relevant since it has been noted that an omega-3 index > 5 % is the range in which dramatic reduction in sudden coronary death reduction can be observed

Rupp H, Wagner D, Rupp T, Schulte LM, Maisch B. Risk stratification by the “EPA + DHA level” and the “EPA/AA ratio” Herz. 2004;29:673–685.

Mozaffarian D & Wu JH. Omega-3 Fatty Acids and Cardiovascular Disease: Effects on Risk Factors, Molecular Pathways, and Clinical Events J Am Coll Cardiol. 2011;58(20):2047-2067.

© CNELM

The role of transcription factors in health and disease

Inflammation is a complex and multisystem event affecting a wide range of cells, tissues and organs

Numerous mediators, like leukocyte adhesion molecules (ICAM-1 & VCAM-1), various proinflammatory cytokines (TNF & IL-1b), chemokines (such as IL-8 and monocyte chemoattractant protein-1 - MCP-1)), and reactive oxygen species (ROS, such as superoxide and peroxynitrite) are involved in both the generation and propagation of the inflammatory response

The transcription factors activator protein-1 (AP1) and nuclear factor-kappaB (NFkB) play key roles in the expression of immuno-modulatory genes and whose activity and expression are elevated in inflammatory conditions

These transcription factors regulate gene expression in response to a variety of stimuli, including cytokines, growth factors, stress and bacterial and viral infections

IL-1, TNFα, LPS

Two signalling pathways lead to the activation of NF-κB, known as the classical (canonical) pathway and the alternative (non-canonical) pathway

The common regulatory step in both of these cascades is activation of an IκB kinase (IKK)

The role of transcription factors in health and disease

NF-κB is one of the principal inducible transcription factors whose modulation triggers a cascade of signalling events involving an integrated sequence of protein-regulated steps, some of which are potential key targets for intervention in treating inflammatory conditions

The ‘canonical’ pathway is triggered by microbial products (via Toll-like microbial pattern recognition receptors [TLRs]) and proinflammatory cytokines such as TNFα and IL-1

• IL-1 and TNFα represent the archetypal proinflammatory cytokines that are rapidly released on tissue injury or infection

• Endogenous ligands may trigger TLRs during tissue injury and certain disease states, which may act to promote inflammation in the absence of infection

NF-κB activation is widely implicated in inflammatory diseases and much attention has focused on the development of anti-inflammatory drugs targeting NF-κB

NF-kB

Inflammation TNF, IL-1,

chemokines

AngiogenesisVEGF, TNF, IL-

1, IL-8

Metastasis ICAM-1, VCAM,

ELAM-1

Tumour promotion COX-2 iNOS, MMP-9

Anti-apoptosis/survival

Bcl-xl, cIAP, XIAP, cFLIP

Proliferation TNF, IL-1, IL-6 cyclin D1, cMyc

NF-kB in carcinogenesis

Toll-like receptors (TLRs) are membrane-bound sensors that detect and respond to microbial infection

The survival of multicellular organisms is dependent on their ability to recognise invading microbial pathogens and to induce a variety of defence reactions

The family of Toll-like receptors play a crucial role in the detection of microbial infection and the induction of immune and inflammatory responses

TLRs recognise highly conserved structural motifs known as pathogen-associated microbial patterns (PAMPs), which are exclusively expressed by microbial pathogens, or danger-associated molecular patterns (DAMPs) that are endogenous molecules released from necrotic or dying cells

Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol. 2001 Nov;1(2):135-45. Review.

PAMPs include various bacterial cell wall components such as lipopolysaccharide (LPS), peptidoglycan (PGN) and lipopeptides, as well as flagellin, bacterial DNA and viral double-stranded RNA

DAMPs include intracellular proteins such as heat shock proteins as well as protein fragments from the extracellular matrix

The TLR4 signalling pathway culminates in activation of the transcription factor nuclear factor-kappaB (NF-kB)

NF-kB controls the expression of an array of inflammatory cytokine genes

TLR4 agonists:• Lipopolysaccharide (LPS)

gram negative bacteria

• Saturated fat/high fat diets• Palmitic acid• Stearic acid

Rocha DM, Caldas AP, Oliveira LL, Bressan J, Hermsdorff HH. Saturated fatty acids trigger TLR4-mediated inflammatory response. Atherosclerosis. 2016 Jan;244:211-5.

Elevated LPS is directly related to increased intestinal permeability

This phenomenon occurs due to reduced expression and activity of tight junction proteins, such as zonula occludens-1 (ZO-1) and occludin, that, together with gut epithelial cells, create a barrier that separates the intestinal lumen and its bacterial population and products from peritoneal tissues

SFA SFA Omega-3 Omega-3 Omega-3Palmitic Stearic EPA DPA DHA

16:0 18:0 20:5 22:5 22:6g/100g lipid

Cross bred steers Grass 18.42 17.54 2.23 2.56 0.2Grain 20.79 14.96 0.47 0.91 0.11

g/100g lipidMixed cattle Grass 26.9 17.0 0.31 0.24 n/a

Grain 26.3 13.2 0.19 0.06 n/amg/100g muscle tissue

Angus steers Grass 508 273 24.5 36.5 4.2Grain 899 463 13.1 31.6 3.7

% total fatCrossbred steers Grass 22.0 19.1 tr 0.6 tr

Grain 25.0 18.2 tr 0.4 tr% fatty acid within intramuscular fat

Hereford steers Grass 21.61 17.74 0.96 1.04 0.09Grain 24.26 15.77 0.3 0.56 0.09

Daley CA, Abbott A, Doyle PS, Nader GA, Larson S. A review of fatty acid profiles and antioxidant content in grass-fed and grain-fed beef Nutr J. 2010; 9: 10.

DISRUPTED METHYLATION/SULFATION

GENETICS

ANTIBIOTICSVACCINATIONS

INFLAMMATION

GUT PERMEABILITY

GUT DYSBIOSIS

TOXICITY

Gluten/Casein (opiates)

Food sensitivities(endorphins)

Gut inflammation

Toxins (bacterial/yeast)

Compromised detoxification

Compromised digestion

Dysregulated immune function

Heavy metal toxicity Increased virus exposure

DIET (i.e. high fat)

LPS

The pro-inflammatory effect of a high-fat diet has mainly been attributed to the inflammatory properties of dietary fatty acids (e.g. palmitic acid)

Recently, it has been proposed that such fatty acids trigger an inflammatory response by acting via LPS receptor (Toll-like receptor-4 [TLR-4]) signalling in adipocytes and macrophages, which might contribute to the inflammation of adipose tissue in obesity

High fat diets affect the composition of the gut flora with an increase in Firmicutes and a reduction in both Bacteroidetes and Bifidobacterium

Therefore excess dietary fat not only increases systemic exposure to potentially pro-inflammatory free fatty acids (e.g. palmitic acid) but also facilitates the development of metabolic endotoxaemia ( increased plasma LPS) triggering systemic low grade inflammatory responses in a range of tissues

Conlon MA, Bird AR. The impact of diet and lifestyle on gut microbiota and human health. Nutrients. 2014 Dec 24;7(1):17-44.

Intestinal microbes altered by high-fat diet suppress fasting induced adipose factor (Fiaf) expression, promoting lipoprotein lipase (Lpl), a regulatory enzyme that enhances the absorption of fatty acid and the build-up of adipocyte triglyceride thereby increasing fat storage

https://microbewiki.kenyon.edu/index.php/Gut_Microbiota_and_Obesity

An increase in glucagon-like peptide-2 (GLP-2) increases gut permeability

Firmicutes significantly contribute to increased LPS activate TLR4 and upregulate the expression of pro-inflammatory cytokines

As obesity is considered a chronic inflammatory disease, the increased production of pro-inflammatory cytokines will trigger an inflammatory response that ultimately leads to weight gain

Lactobacillus rhamnosus - helps repair leaky gut, reduces candida cell numbers

Lactobacillus reuteri reduces candida cell numbers

Lactobacillus plantarum reduces gut wall permeability. This bacterium adheres to reinforce the barrier function of the intestinal mucosa, thus preventing the attachment of the pathogenic bacteria to the intestinal wall

Lactobacillus fermentum - antimicrobials that inhibits the growth of some harmful pathogens

Lactobacillus bulgaricus and lactobacillus breve ferment sugars into lactic acid, thereby increasing the acidity of the intestine, inhibiting the reproduction of harmful microbes (e.g. candida, that prefers an alkaline environment) and strains with known microbial activity

http://www.probiotic.org

Probiotic treatment reduces epithelial barrier dysfunction

Commensal and probiotic strains modulate the amount of tight junction proteins at the cell boundaries and can prevent or reverse adverse effects of pathogens

Several probiotic strains such as Lactobacillus Bacteroides thetaiotaomicron, Bifidobacterium longum and Lactobacillus rhamnosus, Bifidobacterium infantis, Lactobacillus plantarum shown to have beneficial impacts on tight junction- and intestinal barrier function

Increasing zonula occludens-1 (ZO-1) increased transcription of occludin and cingulin genes decreased faecal zonulin levels (a marker indicating enhanced gut

permeability) Decreased proinflammatory cytokines

Ulluwishewa D, Anderson RC, McNabb WC, Moughan PJ, Wells JM, Roy NC Regulation of tight junction permeability by intestinal bacteria and dietarycomponents. Nutr. 2011 May;141(5):769-76.

Lamprecht M, Bogner S, Schippinger G, Steinbauer K, Fankhauser F, Hallstroem S, Schuetz B, Greilberger JF Probiotic supplementation affects markers of intestinal barrier, oxidation, and inflammationin trained men; a randomized, double-blinded, placebo-controlled trial. J Int Soc Sports Nutr. 2012 Sep 20;9(1):45.

Beneficial bacteria favour an acidic environment of pH5.9 to 6.9 (the ascending colon) whilst less beneficial bacteria create an alkaline environment and are most active at pH 7.1 to 7.9 (descending colon)

Some genera of bacteria, such as Bacteroides and Clostridium, have been associated with an increase in colonic tumour growth rate, while other genera like Lactobacillus and Bifidobacterium are known to prevent tumour formation

Bifidobacterium are associated with reduced cell proliferation and a reduction in carcinogenic-promoting substances such as ammonia and faecal pH

Lactobacillus can decrease cresol and bile levels, decrease urinary and faecal mutagenicity as well as carcinogen-promoting enzymes

Lactobacillus are also associated with the modification of the immune system

Guarner F, Malagelada JR Gut flora in health and disease. Lancet. 2003 8:512-9. Review.

Beneficial bacteria favour an acidic environment of pH5.9 to 6.9 (the ascending colon) whilst less beneficial bacteria create an alkaline environment and are most active in at pH 7.1 to 7.9 (descending colon)

Guarner F, Malagelada JR Gut flora in health and disease. Lancet. 2003 8:512-9. Review.

Valdés L, Cuervo A, Salazar N, Ruas-Madiedo P, Gueimonde M, González S. The relationship between phenolic compounds from diet and microbiota: impact on human health.Food Funct. 2015 Aug;6(8):2424-39.

Improving gut health

Probiotics from the following three families have been found to be beneficial for gut function: Bifidobacteria, Lactobacilli, and Saccharomyces

Short-chain non-digestible carbohydrates (inulin-type fructans, fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS)) are the quintessential prebiotics (occurring naturally in cereals, fruits and vegetables) and the target bacterial groups are typically Bifidobacterium and Lactobacillus

Fermented foods like sauerkraut, kimchi, yogurt, kefir

Free radical damage and

oxidative stressSkin

LungsInflammation

Cardiovascular

BrainImmunity

Organs

Oxidative stress/ROS/free radicals is an important inducer of NF-kB

Low antioxidant status leads to increased free radical generation

Numerous antioxidants have been shown to reduce inflammation via NF-kB

Lipoic acid, vitamin c, vitamin E CoQ10 (ubiquinol) Polyphenols (resveratrol, curcumin, EGCG, quercetin) Carotene (beta carotene, lycopene) Xanthophylls (astaxanthin, lutein, zeaxanthin)

Martin KR. Targeting apoptosis with dietary bioactive agents.Exp Biol Med (Maywood). 2006 Feb;231(2):117-29. Review.

Martin KR. Targeting apoptosis with dietary bioactive agents.Exp Biol Med (Maywood). 2006 Feb;231(2):117-29. Review.

Kundu JK, Surh YJ. Cancer chemopreventive and therapeutic potential of resveratrol: mechanistic perspectives.Cancer Lett. 2008 Oct 8;269(2):243-61.

Resveratrol!

Kundu JK, Surh YJ. Cancer chemopreventive and therapeutic potential of resveratrol: mechanistic perspectives. Cancer Lett. 2008 Oct 8;269(2):243-61.

A free radical has an electron missing from its outer shell

X

Ubiquinol donates anelectron to a free radical

X

Ubiquinol donates electrons to other

antioxidants

‘Recharged’ antioxidants (i.e. vitamins C & E, lipoic acid) can

donate electrons to free radical

Ubiquinol – the antioxidant/antioxidant recycler

Bioavailability of CoQ10 supplements depends on the formulation taken

Ubiquinol is more bioavailable than ubiquinone

The addition of two hydrogen atoms increases the polarity, making ubiquinol more water-soluble and therefore more bioavailable than ubiquinone

Bhagavan HN, Chopra RK. Coenzyme Q10: absorption, tissue uptake, metabolism and pharmacokinetics. Free Radic Res. 2006 40:445-53.

• Absorption occurs in the small intestine directly into the lymphatic system followed by absorption into the bloodstream

• Absorption is generally poor (large lipophilic molecule) and fat soluble molecules must cross the ‘unstirred’ water layer for effective uptake

• ~60% of oral supplements may be excreted via the faeces

• High variability with absorption depending on the dosage form

• Higher bioavailability when taken with lipid-containing food but need to increase bioavailability!

Liu ZX, Artmann C.Relative bioavailability comparison of different coenzyme Q10 formulations with a novel delivery system. Altern Ther Health Med. 2009 15:42-6.

VESIsorb® is a novel delivery technology that mimics this natural absorption process to improve bioavailability of large lipophilic molecules

Ubiquinol studies coming out on top!

Supplementation with 150 mg/ day ubiquinol for 14 days reduces inflammatory processes via gene expression

Oral intake of ubiquinol increased its proportion significantly (P < 0.001), with the highest increase in those persons having a low basal serum ubiquinol content (<92.3%)

Ubiquinol status significantly correlated to the concentration of the inflammation marker monocyte chemotactic protein 1 (involved in the accumulation of inflammatory cells).CoQ10 redox state predicts the concentration of C-reactive protein (CRP)

People with lower ubiquinol status, higher BMI, and low grade inflammation may benefit from ubiquinol supplementation

Fischer A1, Onur S1, Niklowitz P2, Menke T2, Laudes M3, Döring F1. Coenzyme Q10 redox state predicts the concentration of c-reactive protein in a large caucasian cohort. Biofactors. 2016 Feb 23. doi: 10.1002/biof.1269. [Epub ahead of print]

Fischer A, Onur S, Schmelzer C, Döring F. Ubiquinol decreases monocytic expression and DNA methylation of the pro-inflammatory chemokine ligand 2 gene in humans. BMC Res Notes. 2012 Oct 1;5:540. doi: 10.1186/1756-0500-5-540.

Ubiquinol not ubiquinone for Parkinson’s

Parkinson’s disease is a progressive disease, and established drug treatments can slow but not halt disease process.

In 2002, a small study (n=80) published findings suggesting that taking 1,200 mg/day of CoQ10 (as oxidised ubiquinone) may benefit those with early Parkinson’s disease

Less disability developed in subjects assigned to CoQ10 than in those assigned to placebo

This study gathered much excitement, resulting in a follow on phase III trial that recruited more than 600 participants who were randomly assigned to receive placebo, or CoQ10 at doses 1200 mg/d or 2400 mg/d.

However the trial was stopped early because although CoQ10 levels increased, there was some worsening of symptom severity

The findings of this latter study were finally published in 2014 with two additional, relatively small studies published in the interim also failing to report any significant benefit from ubiquinone intervention in early Parkinson’s disease

In contrast, more recent evidence suggests that the reduced, ubiquinol form of CoQ10 may offer benefits for Parkinson’s patients

Yoritaka A, Kawajiri S, Yamamoto Y, Nakahara T, Ando M, Hashimoto K, Nagase M, Saito Y, Hattori N. Randomized, double-blind, placebo-controlled pilot trial of reduced coenzyme Q10 for Parkinson's disease. Parkinsonism Relat Disord. 2015 Aug;21(8):911-6. May 29.

• Igennus is the only independent manufacturer of specialist Fatty Acid in the UK. Based in Cambridge the medical innovation hub for the UK:

- Seven Seas Merck Pharma Germany- Minami Atrium Pharma Canada- Biocare Elder Pharma India- Eskimo 3 Bringwell Pharma Sweden- Equizen Vifor Pharma Swiss

VESIsorb® Ubiquinol-QHVESIsorb® Ubiquinol-QH is the most bioavailable source of the activated form of coenzyme Q10 (CoQ10). The patented VESIsorb® delivery system provides colloidal, highly solubilised ubiquinol that passes easily through the water layer barrier in the gut and into the bloodstream for unprecedented absorption, optimal blood CoQ10 levels and sustained action.

'Bioactive’ form of CoQ10 with dual benefits as a coenzyme and antioxidant

Solubilised ubiquinol improves absorption and uptake

Patented VESIsorb® delivery system mimics the natural transport of the intestine, pre-digesting and emulsifying the ubiquinol into microscopic water-soluble particles that are easily absorbed into the bloodstream for optimal tissue distribution

Pre-digested ubiquinol is absorbed faster, reaches higher blood plasma concentrations & offers sustained action over oil-based forms

Unprecedented bioavailability 1-a-day dosing offers therapeutic benefits Offers benefits for cardiovascular health,

energy production within the heart, brain & muscles and protection from free radicals

Shanmugam MK, Rane G, Kanchi MM, Arfuso F, Chinnathambi A, Zayed ME, Alharbi SA, Tan BK, Kumar AP, Sethi G. The multifaceted role of curcumin in cancer prevention and treatment. Molecules. 2015 Feb 5;20(2):2728-69.

Curcumin

Curcumin is the most active constituent of turmeric (50-60%) and it has been shown to exhibit antioxidant and anti-inflammatory properties

However, studies regarding absorption, transportation, assimilation and elimination of curcumin have revealed low absorption and its rapid metabolism and short half life which leads to relatively low bioavailability

After absorption, curcumin undergoes conjugations like sulfation and glucuronidation

When metabolised in the liver, the major metabolic products of curcumin are glucuronides of Tetrahydrocurcumin (THC) and Hexahydrocurcumin (HHC)

Studies have shown that these metabolites are actually less active compared to curcumin itself and so when metabolised the activity of curcumin seems to be lost

Ghalandarlaki N, Alizadeh AM, Ashkani-Esfahani S: Nanotechnology-applied curcumin for different diseases therapy. BioMed research international 2014, 2014:394264.

Ghalandarlaki N, Alizadeh AM, Ashkani-Esfahani S: Nanotechnology-applied curcumin for different diseases therapy. BioMed research international 2014, 2014:394264.

Ghalandarlaki N, Alizadeh AM, Ashkani-Esfahani S: Nanotechnology-applied curcumin for different diseases therapy. BioMed research international 2014, 2014:394264.

To improve the bioavailability of curcumin, numerous approaches have been undertaken including:

• use of adjuvant that interferes with glucuronidationPiperine (black pepper extract)

• Inclusion of turmeric volatile oils BCM-95®

• nanoformulations (liposomes, micelles, etc) Novasol® Longvida®

• the use of curcumin phospholipid complex Meriva-SR®

Micelles and phospholipid complexes increase the absorption resulting in higher blood plasma concentration and lower elimination thus increasing the bioavailability

Shanmugam MK, Rane G, Kanchi MM, Arfuso F, Chinnathambi A, Zayed ME, Alharbi SA, Tan BK, Kumar AP, Sethi G. The multifaceted role of curcumin in cancer prevention and treatment. Molecules. 2015 Feb 5;20(2):2728-69.

Shanmugam MK, Rane G, Kanchi MM, Arfuso F, Chinnathambi A, Zayed ME, Alharbi SA, Tan BK, Kumar AP, Sethi G. The multifaceted role of curcumin in cancer prevention and treatment. Molecules. 2015 Feb 5;20(2):2728-69.

Shanmugam MK, Rane G, Kanchi MM, Arfuso F, Chinnathambi A, Zayed ME, Alharbi SA, Tan BK, Kumar AP, Sethi G. The multifaceted role of curcumin in cancer prevention and treatment. Molecules. 2015 Feb 5;20(2):2728-69.

Peroxisome proliferator-activated receptors (PPARs) are activated by fatty acids and eicosanoids and are the target for drugs such as fibrates (PPARa; lipid lowering) thiazolidinediones (PPARg; diabetes) and NSAIDs

After activation by ligand binding, PPARs heterodimerize with retinoid X receptor (RXR) and bind to specific PPAR response elements (PPRE) on the promoter of target genes to regulate glucose and lipid metabolism (right)

This aspect of PPAR-α and PPAR-γ action has cardiovascular protective effects through effects on atherosclerosis and diabetes

PPAR-α and PPAR-γ also interact with different transcription factors to repress proinflammatory genes (left)

Inflammatory regulation via PPARs

Schiffrin EL. Peroxisome proliferator-activated receptors and cardiovascular remodeling.Am J Physiol Heart Circ Physiol. 2005 Mar;288(3):H1037-43. Epub 2004 Sep 16. Review.

Down regulate the expression of membrane receptors (5)

Increase IΚBα expression (4)

Inhibition translocation of NF-kB to the nucleus (3)

Decrease transcription factor expression levels (2)

Interfere with the activation of the transcription initiation complex (1)

Omega-3

Zambon, A., P. Gervois, et al. (2006). "Modulation of hepatic inflammatory risk markers of cardiovascular diseases by PPAR-alpha activators: clinical and experimental evidence." Arteriosclerosis, thrombosis, and vascular biology 26(5): 977-986.

Omega-3 and NF-κB!Omega-3 inhibits the activity of NF-κB by attenuating phosphorylation and degradation of the inhibitory factor, IκB

Inflammtory regulating pathways, cell proliferation, differentiation and apoptosis are modified by components of diet

Reactive oxygen species (ROS), such as superoxide

and peroxynitrite

Activation of AP-1 (TPA-response

element) and NF-κB (IκB kinase, PKC, ROS)

The MAPK kinase signalling cascades

(ERKs, JNK, SAPKs, p38 kinases)

COX & LOX enzymes

c-Jun, c-Fos, c-Myc, Bax, and Cdks

(oncogene activation)

p53, Rb, Bcl-2, p21, and p27

(tumour suppressor inactivation)

PKC, calcium release, and calcium channel

activation

Toll-like receptors

PPARs

NF-kB & AP1

Adhesion molecules (ICAM-1 & VCAM-1)

Inflammatory regulation

Cytokines (such as TNF & IL-1b), chemokines (such as IL-8 and MCP-1)

Therapeutic

Wellbeing

Additional nutrients

Our products range

Education Technical

Sophie TullyNutrition Education Manager

sophiet@igennus.com

Dr Nina Bailey Head of Nutrition

ninab@igennus.comTwitter @DrNinaBailey

An inflammatory reaction can be regulated in a number of different ways…

The detection and interpretation of inflammatory signals

The response of the activated immune cells (such as cytokine release)

The reaction of the surrounding tissue to inflammatory agents and to immune mediators as well as the resolution/repair phase

These are all targets of intervention