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Inflammatory Diseases and Vitamin E—What Do We Know
and Where Do We Go?
Maria Wallert, Lisa Börmel, and Stefan Lorkowski*
Inflammation-driven diseases and related comorbidities, such as the metabolic syndrome, obesity, fatty liver disease, and cardiovascular diseases cause significant global burden. There is a growing body of evidence that nutrients alter inflammatory responses and can therefore make a decisive contribution to the treatment of these diseases. Recently, the inflammasome, a cytosolic multiprotein complex, has been identified as a key player in inflammation and the development of various inflammation-mediated disorders, with nucleotide-binding domain and leucine-rich repeat pyrin domain (NLRP) 3 being the inflammasome of interest. Here an overview about the cellular signaling pathways underlying nuclear factor
“kappa-light-chain-enhancer” of activated B-cells (NF-𝜿B)- and NLRP3-mediated inflammatory processes, and the pathogenesis of the inflammatory diseases atherosclerosis and non-alcoholic fatty liver disease (NAFLD) is provided; next, the current state of knowledge for drug-based and dietary-based interventions for treating cardiovascular diseases and NAFLD is discussed. To date, one of the most important antioxidants in the human diet is vitamin E. Various in vitro and in vivo studies suggest that the different forms of vitamin E and also their derivatives have anti-inflammatory activity. Recent publications suggest that vitamin E—and possibly metabolites of vitamin E—are a promising therapeutic approach for treating inflammatory diseases such as NAFLD.
1. Regulation of (Chronic) Inflammatory Processes
Inflammation is a double-edged sword. The interaction of the innate and adaptive immune system in response to invadedDr. M. Wallert, L. Börmel, Prof. S. Lorkowski
Department of Nutritional Biochemistry and Physiology Institute of Nutritional Science
Friedrich Schiller University Jena Jena 07743, Germany
E-mail: [email protected] Dr. M. Wallert, L. Börmel, Prof. S. Lorkowski
Competence Cluster for Nutrition and Cardiovascular Health (nutriCARD)
Halle-Jena-Leipzig, Germany
The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/mnfr.202000097 © 2020 The Authors.Molecular Nutrition & Food Research published by
Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
DOI: 10.1002/mnfr.202000097
pathogens is an essential part of the heal-ing process. However, excessive acute as well as chronic low-grade inflam-mation are driving forces for many chronic diseases. Typical diseases hall-marked by excessive inflammation are rheumatoid arthritis and inflammatory bowel diseases, such as Crohn’s dis-ease and ulcerative colitis. The total bur-den caused by inflammatory diseases includes cancer, Alzheimer’s disease as well as metabolic syndrome-related dis-eases like atherosclerosis, non-alcoholic fatty liver disease (NAFLD), and diabetes mellitus type 2 (DMT2) (Figure 1). In ad-dition, these diseases are related to an un-healthy lifestyle or rather an unbalanced diet, with obesity being an indepen-dent risk factor, especially for NAFLD.[1]
Obese people suffer from increased ox-idative and inflammatory stress,[2]which
in turn reinforces progression of inflam-matory diseases.
1.1. NF-𝜿B—The Canonical Signaling Pathway Involved in Pro-Inflammatory Re-sponses
One of the most prominent transcription factors, abundantly expressed by almost all cell types, is the nuclear factor “kappa-light-chain-enhancer” of activated B-cells (NF-𝜅B).[3] NF-𝜅B
is a central cellular mediator of the inflammatory response that controls the expression of a number of target genes in-volved in immune and stress responses, regulation of cell proliferation as well as apoptosis.[4,5] Due to its central role,
it is not surprising that the target genes of NF-𝜅B include regulators of NF-𝜅B itself. This allows an auto-regulatory
feed-back loop that is vital for the regulation of cellular immune homeostasis.[4,6] One of these is the A20 deubiquitinase, which
is a major inhibitor of NF-𝜅B; consequently, A20-deficient mice die spontaneously as a result of uncontrolled multi-organ inflammation.[6]
Structurally, NF-𝜅B forms a dimer with two of the five mem-bers of the Rel protein family, namely NF-𝜅B1 (p50), NF-𝜅B2
(p52), RelA (p65), c-Rel or RelB.[7] Each of these proteins has
an N-terminal Rel homology domain, which allows for dimeriza-tion, DNA-binding, nuclear translocadimeriza-tion, and interaction with inhibitory Inhibitor𝜅B (I𝜅B) proteins.[7,8]In the basal state,
Figure 1. Impact of inflammation on major chronic diseases. A variety of diseases are characterized and triggered by chronic low-grade inflammation or acute excessive inflammatory responses, including Alzheimer’s disease, cancer, metabolic syndrome-related diseases as well as rheumatoid arthritis and inflammatory bowel diseases, respectively. DMT2, diabetes mellitus type 2; NAFLD, non-alcoholic fatty liver disease.
or𝛾) of the NF-𝜅B inhibitory protein I𝜅B in the cytoplasm. The canonical mechanism to activate the transcription factor NF-𝜅B
is by degradation of I𝜅B through its phosphorylation via I𝜅B ki-nase (IKK) complex, which is comprised of two catalytic subunits, IKK𝛼, and IKK𝛽, and a regulatory subunit (IKK𝛾).[5,9]The IKK
complex is activated by various stimuli, such as tumor necro-sis factor alpha (TNF𝛼), interleukin (IL)-1𝛽 as well as ligands of
the toll-like receptors (TLRs) 2 and 4,[10]such as
lipopolysaccha-rides (LPS).[11]The phosphorylated and released I𝜅B is
polyubiq-uitinated, which causes proteasomal degradation of I𝜅B.[7]The
resulting free NF-𝜅B dimer can then translocate from the cyto-plasm into the nucleus where it activates the expression of spe-cific target gene (Figure 2).[12]
1.2. NLRP3 Inflammasome—Another Look at Inflammatory Processes
1.2.1. Structure and Function
In 2002, a research group around Jürg Tschopp identified and characterized a multiprotein complex, which serves as a central regulator in inflammatory processes.[13–15] The so-called
“in-flammasome” activates a proteolytic cascade driven by caspase-1, which cleaves proteins of the interleukin family, namely IL-1𝛽 as well as IL-18, and triggers a form of cell death known as pyroptosis.[16–18] Structurally, the inflammasome is comprised
of a sensor complex, an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) complex and pro-caspase-1. Today, different types of inflammasomes are known. Which inflammasome is activated is mainly mediated by the sensor complex. On the one hand, nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are
involved and on the other hand there are the non-NLRs like pro-teins which are interferon-𝛾-inducible protein (IFI) 16, absent
in melanoma 2 (AIM2), and pyrin.[19,20] The best studied
in-flammasome is the nucleotide-binding domain and leucine-rich repeat pyrin domain (NLRP) 3, which seems to involve the most complex signaling of the known inflammasomes.[19]
When the inflammasome complex is fully assembled, pro-caspase-1 is cleaved into its active fragments p20 and p10. These fragments further cleave the pro-forms of the cytokines pro-IL-1𝛽, pro-IL-18, and pro-IL-33 to their biologically active forms.[18,21,22]The role of IL-33 in the NLRP3 inflammasome
sig-naling pathway has not been fully elucidated and studies revealed controversial results.[23]On the one hand, Schmitz et al. postulate
that the cleavage of pro-IL-33 into its mature form by caspase-1 is essential for its optimal biologic activity.[22] On the other
hand, Ohno et al. found that caspase-1-deficient macrophages are able to release IL-33 after stimulation with LPS.[23,24] IL-1𝛽
and IL-18 are classical pro-inflammatory cytokines. IL-33 is a lig-and of the orphan IL-1 family receptor T1/suppression of tu-morigenicity (ST2). Consequently, IL-33 triggers the production of pro-inflammatory mediators, in response to the activation of mast cells and their effect on T-helper 2 cells.[25]Besides the
acti-vation of the pro-inflammatory cytokines, caspase-1 activates the pro-pyroptotic factor gasdermin D (GSDMD) by cleavage,[21,26]
and the N-terminal fragment of GSDMD oligomerizes in the cell membrane and forms pores, which lead to pyroptosis.[27]
Figure 2. Canonical and non-canonical activation of the NLRP3 inflammasome. Activation of the NLRP3 inflammasome occurs via a two-step process. A) First, priming of the canonical pathway is initiated by PAMPs via TLR4 resulting in the activation of the NF-𝜅B pathway. Consequently, the expression of NLRP3, ASC, pro-caspase-1 and the pro-forms of IL-1𝛽 and IL-18 is induced. In the context of NLRP3 inflammasome activation, the role of IL-33 is not yet fully understood (see Section 1.2.1). Alternatively, priming occurs via the non-canonical pathway: LPS activates the TRIF/type I IFN cascade via TLR4, resulting in increased expression of GBP, IRGB10, caspase-11 (nomenclature refers to the murine proteins; see text for details on the human nomenclature) and GSDMD. B) During activation via the non-canonical pathway, GBP and IRGB10 lysate gram-negative bacteria and the released LPS activates caspase-11, which in turn cleaves GSDMD. C) A second stimulus with DAMPs, such as ATP, causes oligomerization of the NLRP3 inflamma-some, the cleavage of the active p20 and p10 fragments of caspase-1, and the respective formation of active interleukins. Like capsase-11, caspase-1 can cleave GSDMD. In turn, the N-terminal fragments of GSDMD form pores to release the matured interleukins and to provoke pyroptosis. ASC, apoptosis-associated speck-like protein containing a caspase-recruitment domain; DAMPs, damage-associated molecular patterns; GSDMD, gasder-min D; GBP, guanylate-binding protein; IFN, interferon; IFNAR1/2, IFN-𝛼/𝛽 receptor 1/2; IKK, I𝜅B kinase; I𝜅B, inhibitor 𝜅B; IRAK1/4, interleukin-1
receptor-associated kinase 1 or 4; IRF, interferon regulatory factor; IRGB10, immunity-related GTPase family member b10; MyD88, myeloid differentia-tion primary response 88; NF-𝜅B, nuclear factor “kappa-light-chain-enhancer” of activated B-cells; NLRP3, nucleotide-binding domain and leucine-rich repeat pyrin domain 3; PAMPs, pathogen-associated molecular patterns; P2XR4/7, P2X purinoreceptor 4/7; STAT1/2, signal transducer and activator of transcription 1/2; TAK1, transforming growth factor beta-activated kinase 1; TLR4, toll-like receptor 4; TRAF6, TNF receptor associated factor 6; TRIF, TIR-domain-containing adapter-inducing interferon-𝛽.
1.2.2. NLRP3 Inflammasome Activation—Canonical and Non-Canonical Signaling Pathways
Two major pathways have been described that activate the NLRP3 inflammasome, the canonical and the non-canonical pathways.
Even though the pathways differ in their course of activation, the outcome, that is, the release of the mature forms of the above-mentioned interleukins, are the same (Figure 2).
Canonical NLRP3 Inflammasome Activation: Canonical
process. The first hit, known as priming, mediates the induction of the expression of key components of the inflammasome, such as NLRP3, ASC, pro-caspase-1, and the pro-forms of the inter-leukin protein family members IL-1𝛽 and IL-18.[29] To initiate
this step, extracellular stimulation must occur via TLR ligands by pathogen-associated molecular patterns (PAMPs) such as LPS.[30]This triggers the association of the myeloid
differentia-tion primary response (MyD) 88 adaptor protein, which results in the recruitment of the IL-1 receptor-associated kinases (IRAK) 1 or 4.[31]Following phosphorylation of IRAK1 or 4, TNF receptor
associated factor (TRAF) 6 is activated by ubiquitination.[6,31]
In turn, transforming growth factor 𝛽-activated kinase (TAK) 1 performs its function as an IKK activator, thus activating the NF-𝜅B pathway (see Section 1.1).[32]As NLRP3 and the pro-ILs
are direct target genes of NF-𝜅B, their expression is induced to prepare the cells’ response to the inflammasome activation.[5]
The second hit is provoked by damage-associated molecu-lar patterns (DAMPs), such as adenosine triphosphate (ATP), nigericin, viral RNAs, pore-forming toxins, crystalline sub-stances, as well as reactive oxygen species (ROS), and re-sults in the oligomerization and final activation of the NLRP3 inflammasome.[21,33] None of the second hit mediators are
di-rectly bound to the NLRP3 complex, but these rather initiate path-ways that activate the NLRP3 inflammasome.[19] Platnich and
Muruve described the importance of potassium efflux for inflam-masome activation. For example, ATP activates the P2X puri-noreceptors (P2XR) 4 and 7 which causes potassium efflux; in turn, the resulting lower intracellular potassium level activates the NLRP3 inflammasome.[34]Swanson and co-workers reported
that efflux of chloride and influx of calcium ions are both equally involved.[35]The oligomerization of NLRP3 allows for
inflamma-some complex formation with ASC and pro-caspase-1, which in turn results in cleavage to the active fragments of pro-caspase-1 and the mature forms of IL-1𝛽 and IL-18.[36]
Non-Canonical NLRP3 Inflammasome Activation: The
non-canonical pathway is an alternative route for activating the NLRP3 inflammasome. During an infection gram-negative bacteria (PAMPs) activate the TLR4 receptor via the toll/ interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-𝛽 (TRIF) protein.[21] In this context,
in-terferon regulatory factor (IRF) 3 and 7 are recruited and induce the expression of type I interferons (IFN) IFN-𝛼 and IFN-𝛽.[37]
The IFN-𝛼/𝛽 receptor (IFNAR) 1 and 2 binds to type I IFNs and
activates downstream the phosphorylation of the transcription factors signal transducer and activator of transcription (STAT) 1 and 2 and their nuclear translocation.[37,38]Hence, the expression
of guanylate-binding protein (GBP), immunity-related GTPase family member b10 (IRGB10), caspase-11 and GSDMD is induced.[21,39]The task of GBP and IRGB10 is to orchestrate the
lysis of gram-negative bacteria, whereby LPS is released from the bacterial membrane into the cytoplasm.[21,40]Murine caspase-11
and its human orthologs caspase-4 and -5 are activated by LPS.[41]Consequently, activated caspase-11 cleaves GSDMD; the
N-terminal GSDMD fragment oligomerizes and this initiates the activation of the NLRP3 inflammasome by mediating potassium efflux.[35]As caspase-11 is unable to directly activate IL-1𝛽 and
IL-18, it has to cleave GSDMD and the derived fragments in turn activate the NLRP3-caspase-1-depended pathway for the activation of the respective IL family members.[41]
1.2.3. Limitations of Cell Models for Studying the NLRP3 Inflammasome
A well-established cell model for investigating inflammatory pro-cesses is the murine RAW264.7 macrophage cell line. However, recently it has been shown that this cell line is inappropriate for studies regarding the NLRP3 inflammasome as the vital compo-nent of the NLRP3 inflammasome complex, the ASC is insuffi-ciently expressed.[42,43]Thus, the important link between NLRP3
and pro-caspase-1 for complete complex formation is lacking.[44]
By interrupting the NLRP3-caspase-1 induced formation of ac-tive IL-1𝛽 and IL-18, RAW264.7 macrophages can only form the pro-forms of IL-1𝛽 and IL-18.[42]As an alternative to RAW264.7
cells, the murine macrophage-like cell line J774 and primary macrophages can be used. These cells express the ASC and form a full NLRP3 inflammasome complex.[43] However, there are
studies which have demonstrated the expression of the ASC at both the mRNA and protein level in RAW264.7 cells.[45]Despite
that, it is possible that cells use ASC-independent pathways of inflammasome activation, whereby IL-1𝛽 and IL-18 as well as
pyroptosis can be induced by the inflammasomes NLRC4 and NLRP1b.[46]It remains therefore unknown whether the ASC
de-tected in these studies is fully functional and RAW264.7 cells are a suitable cell line for NLRP3 research.
Pelegrin and co-workers pointed out an interesting question for the selection of the “right” cell model: “Thus, the question does not seem to be, what is the correct model of release of IL-1′ but rather, which model corresponds to which release state?”[42]
In this context, RAW264.7 cells might be used to study the prim-ing of the NLRP3 inflammasome (see Section Canonical NLRP3 Inflammasome Activation), since the TLR4 cascade is fully func-tional and priming can be therefore studied independently from the activation of the NLRP3 inflammasome. For investigating the activation cascades RAW264.7 cells might be not the best choice, since the functionality of the ASC is not clear; J774 or primary cells might be the better cell model.
A Model Disease for an Incorrect Regulated NLRP3 Inflam-masome: CAPS: The relevance of a chronic activation of
the NLRP3 inflammasome is highlighted by a group of rare auto-inflammatory diseases that is caused by defects of the inflammasome and is known as cryopyrin-associated periodic syndrome (CAPS); CAPS includes the entities known as familial cold auto-inflammatory syndrome (FCAS), Muckle–Wells syn-drome (MWS), and neonatal onset multisystem inflammatory disorder (NOMID).[14,16] About 60% of the patients affected by
this type of diseases carry a mutation in the NALP3/CIAS1 gene, which encodes the cryopyrin/NLRP3 protein.[15,47]A
char-acteristic of this autosomal dominant hereditary unusual fever syndrome leads to a constitutively active or hyperactive NLRP3 inflammasome and in turn to an increased release of IL-1𝛽 and
a systemic inflammatory reaction of skin, joints, eyes, bone, muscles and the central nervous system.[14,16,48] Gattorno and
co-workers found that in peripheral blood monocytes obtained from carriers of CIAS1 mutations treatment with LPS leads to an increased release of mature IL-1𝛽, whereby cells from non-carriers usually require an additional stimulation with ATP to induce the release of mature IL-1𝛽.[49]Further studies have
Figure 3. Clinical spectrum of CAPS. This overview shows the facets and different clinical manifestations of CAPS, which is an auto-inflammatory disease that is caused by mutations of the NLRP3 gene. Based on the severity of the clinical symptoms, CAPS is distinguished as FCAS, MWS, and NOMID. CAPS, cryopyrin-associated periodic syndrome; FCAS, familial cold auto-inflammatory syndrome; MWS, Muckle–Wells syndrome; NOMID, neonatal onset multisystem inflammatory disorder.
MWS, and NOMID exhibit a number of similarities, but differ in their manifestation and the severity of symptoms (Figure 3).
2. Vitamin E and its Metabolites Attenuate the
Inflammatory Burst
2.1. Vitamin E Characteristics and Metabolism
In 1922, vitamin E was found as a cofactor responsible for fertility in rats. In the following years, vitamin E was described as a chain-breaking antioxidant preventing lipoxygenation. Next to its anti-oxidative capacity, Azzi and colleagues reported additional gene-regulatory properties of vitamin E, more pre-cisely𝛼-tocopherol (𝛼-TOH). In the following sections, the term vitamin E will be used for simplicity. However, we are aware that 𝛼-TOH has been announced as the only vitamin E form mediating the actual vitamin function.[51]
The term vitamin E describes a group of chromanols, which can be distinguished as tocopherols (TOHs), tocotrienols (T3s), and vitamin E-like structures such as tocomonoenols as well as marine-derived TOHs (MDTs). Tocopherols are characterized by a saturated phytyl side-chain, whereas tocomonoenols, MDTs, and T3s are unsaturated at either the terminal isoprene unit or have three double bonds within the side-chain.[52]Each of these
vitamin E groups can occur in four different forms, namely
𝛼-, 𝛽-, 𝛾-, and 𝛿-forms, which are classified depending on the
methylation pattern of the hydroxychromanol ring system. While methylation at position C-8 is a hallmark of all forms of vitamin E, the different forms differ in their methylation pattern at positions C-5 and C-7. In addition to position C-8, the
𝛼-form is methylated at both, positions C-5 and C-7, whereas 𝛽- and 𝛾-forms are methylated at positions C-5 (𝛽) and C-7 (𝛾),
respectively. The 𝛿-form is methylated only at position C-8.
The different forms of vitamin E occur in oily plants, such as
nuts and seeds, with 𝛼-TOH being the most abundant form
in human nutrition. Almonds and hazelnuts as well as germ oil and sunflower oil contain high amounts of𝛼-TOH. Within
the group of non-𝛼-TOHs, 𝛾-TOH is the most abundant form predominantly occurring in walnuts, palm oil, and soybean.[53]
Other non-𝛼-TOH are found in some cereals, in palm oil and rice bran oil, coconut oil, cocoa butter, soybeans, barley, and wheat germ.[54] Vegetables such as avocados,[55] olives,[56] and
legumes[57]contain relevant amounts of𝛼-TOH.
The properties of vitamin E depend on its molecular struc-ture which can be divided in three functionally distinct units, as shown in Figure 4: the functional unit (I), the signaling unit (II), and the hydrophobic domain (III).𝛼-TOH, is the most po-tent form of vitamin E in terms of its properties as an antioxi-dant and ensuring fertility. Units I and II are responsible for the biological activity of𝛼-TOH, and unit III, the side-chain, is con-sidered as a passive domain that is responsible for the hydropho-bicity of the molecule and the integration into lipoproteins and membranes.[58]
As a lipophilic nutrient, vitamin E follows more or less the metabolic routes of other lipophilic molecules in the body, whereby the liver is the central organ for uptake, distribution, and metabolism, and storage. In contrast to other fat-soluble vi-tamins, vitamin E does not accumulate in the liver to toxic levels, since excessive vitamin E intake results in increased vitamin E metabolism which in turn results in the excretion of the catabolic products of the vitamin E molecule via urine and feces. Within the group of vitamin E, 𝛼-TOH is most abundant in animals and humans, as a result of the postulated discrimination by the
𝛼-TOH transfer protein (𝛼-TTP) or rather enhanced metabolic
degradation of the non-𝛼-TOH congeners by cytochrome P450 enzymes and their subsequent elimination via urine, as reviewed in ref. [59]. Hepatic𝛼-TTP preferentially promotes the
Figure 4. Structure of𝛼-TOH. Functional units of vitamin E are based on its molecular structure. The functional unit (I) and the signaling unit (II) are responsible for the biological activity. The hydrophobic unit (III) is considered as a more or less passive domain that is mediating the hydrophobicity of the molecule.
are the three methyl groups of the chromanol ring system, es-pecially at position C-5, the free hydroxyl group and the phythyl side-chain.[60] In addition, Grebenstein et al. showed that the
metabolism of vitamin E but not𝛼-TTP may discriminate mostly
non-𝛼-TOH forms, as 𝛼-TTP protects the side-chain of the vi-tamin E molecules from𝜔-hydroxylase-initiated degradation.[61]
The long-chain metabolites (LCMs) of vitamin E have been con-sidered as ligands for𝛼-TTP and/or other hepatic TOH-binding
proteins. Therefore, the importance of these proteins for the transfer of the LCMs to mitochondria needs to be studied.[61]
In the liver, the metabolism of vitamin E, mainly𝛼-TOH, likely occurs at or in three cell compartments: endoplasmic reticulum, peroxisomes, and mitochondria. So far, it is not known how the metabolites enter the peroxisome or leave it. At the endoplasmic reticulum, the truncation of the phytyl side-chain is initiated by CYP4F2/3A4-dependent𝜔-hydroxylation which is the initial step resulting in the formation of the alcohol derivative 𝛼-13′-OH (13′-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2,6,10-trimethyltridecanol).[62]Subsequent𝛼-oxidation in peroxisomes
forms the acid derivative𝛼-13′-COOH
(13′-(6-hydroxy-2,5,7,8,-tetramethylchroman-2-yl)-2,6,10-trimethyltridecanoic acid) by alcohol and aldehyde dehydrogenases. In the peroxisomes, two rounds of 𝛽-oxidation result in the elimination of either propionyl-CoA or acetyl-CoA and formation of the intermediate-chain metabolites; this is followed by three rounds of mitochon-drial𝛽-oxidation resulting in the sequential shortening of the side chain. This results first in short-chain metabolites (SCM) and finally in 𝛼-carboxyethyl-hydroxychroman (𝛼-CEHC), the catabolic end-product of the degradation of both 𝛼-TOHs and 𝛼-T3s.[63]Metabolism of non-𝛼-TOH forms of vitamin E follows
the path of 𝛼-TOH, forming respective 𝛽-, 𝛾-, 𝛿-metabolites.
In contrast to TOHs, the double bonds of T3s remain in the side-chain while entering the𝛽-oxidation pathway which implies the involvement of further enzymes, possibly two auxiliary enzymes, namely 2,4-dienoyl-CoA reductase and 3,2-enoyl-CoA isomerase, which are known to be involved in the metabolic degradation of unsaturated fatty acids.[63]
Initially, 𝛼-CEHC was considered as a marker for
(su-per)optimal 𝛼-TOH supply in humans, since an increase of
𝛼-CEHC excretion via urine was found only after exceeding an
individual𝛼-TOH threshold of 30–50 µM in plasma.[64]
Devel-opment of more precise analytical tools allowed the detection of increased urinary𝛼-CEHC concentrations after intake of >9 mg
𝛼-TOH d−1.[65] A daily intake of 12–15 mg 𝛼-TOH is
recom-mended. Hence, a daily excretion of >1.39 mmol 𝛼-CEHC/g creatinine is a marker for a higher than adequate𝛼-TOH status
in healthy humans.[65,66]
During hepatic vitamin E catabolism, the LCMs 13′-hydroxychromanol and 13′-carboxychromanol (13′-OH and 13′-COOH) are formed, of which most forms have been found in serum of humans, mice, and rats.[67–69]
2.2. Anti-Inflammatory Effects of Vitamin E
Beyond its vitamin function, vitamin E is one of the most promi-nent lipid soluble radical chain-breaking antioxidants. Within the group of the different vitamin E forms, RRR-𝛼-TOH is assigned
as the most potent member, mainly due to its protection against peroxidation of PUFA in phospholipids in cell membranes and plasma lipoproteins at least in vitro.[70,71]Hence, the activities of
the other vitamin E forms are expressed as effect equivalents (to-copherol equivalents, TE [%]) compared to RRR-𝛼-TOH and are as follows:𝛽-TOH 50%, TOH 10%, 𝛿-TOH 3%, 𝛼-T3 30%, 𝛾-T3 8%, and𝛽-T3 5%.[72]For𝛿-T3 no calculation coefficient is
de-fined.
Properties of vitamin E forms independent of the antioxida-tive function were first suggested in the early 1950s but were not pursued for the following decades.[73]Research of Angelo Azzi
and colleagues then brought forth several working hypotheses in non-antioxidative properties of vitamin E, or rather of𝛼-TOH. Thereby, the focus of vitamin E research changed toward the modulation of gene regulation and enzyme activities, signaling cascades within uptake, transport, degradation, metabolism, and excretion of TOH, lipoprotein uptake and inflammation, to name only a few.[70]
such as transcriptions factors (NF-𝜅B, peroxisome proliferator-activated receptor (PPAR) y, STAT3) and related signaling cas-cades (cyclooxygenases, COXs) and the production of signaling molecules such as prostaglandins (PGs).
In principle, the activation of the inflammasome can be mod-ulated by interaction during priming via the transcription factor NF-𝜅B and the actual activation of the inflammasome complex (see Section 1.2.2). In the following, the known interactions of vitamin E forms within both processes is described.
2.2.1. Interactions of Vitamin E and NF-𝜅B
The different forms of vitamin E regulate NF-𝜅B activation differ-ently depending on the saturation of the side chain (TOHs vs T3s) and the methylation pattern of the chromanol ring system (𝛼-, 𝛽-,
𝛾-, and 𝛿-forms). Whereas Azzi et al. showed that 50 µm 𝛼-TOH
and𝛽-TOH failed to preserve NF-𝜅B activation by phorbol-12-myristat-13-acetat (PMA),[74]treatment with very high doses of
𝛼-TOH (500 µm) in combination with vitamin C (10 mm)
de-creased the activity of NF-𝜅B in dendritic cells, without affecting the phosphorylation of I𝜅B𝛼.[75] Compared to TOHs, T3s,
ap-plied as both a mixture of the𝛼-, 𝛽-, 𝛾-, 𝛿-forms[76,77]or as single
compounds, have more pronounced effects on the activation of NF-𝜅B. A mixture of T3s at doses of 25, 50, and 100 mg/kg and a T3-rich fraction (100 and 300 mg/kg body weight) prevented damage of kidneys[76]and skeletal muscle[77] in
streptozotocin-induced diabetic rats, by reducing the expression of the NF-𝜅B subunit p65 in the nuclear fraction of respective tissue cells. In addition, effects on NF-𝜅B activity depend on the methylation pattern of the chromanol ring, with𝛿-T3 and 𝛾-T3 being the most
potent T3 forms, followed by𝛽-T3, whereas 𝛼-T3 failed to exert effects on NF-𝜅B activity.[78]𝛿-T3 (50 µm) inhibited NF-𝜅B/p65
activity (cytosol and nucleus) and consequently suppressed the expression of NF-𝜅B/p65 and phosphorylated I𝜅B𝛼 in pancreatic
cancer cells, MiaPaCa-2 and AsPc-1 cells and in tissues of mice xenografted with AsPc-1 cells.[78]Furthermore, 400 mg/kg𝛾-T3
daily per os sensitized pancreatic tumors in mice to gemcitabine treatment, a drug applied in clinical treatment of pancreatic cancer, by suppressing NF-𝜅B-mediated inflammatory pathways
linked to tumorigenesis.[79] However, concerns on the data in
this publication have been stated just recently.[80]𝛾-T3 has been
described to directly inhibit constitutive and TNF𝛼-induced NF-𝜅B activation dose- and time-dependently as well as the following phosphorylation of the p65 subunit and degradation of I𝜅B, without interfering with the binding of NF-𝜅B to DNA in various cancer cell lines at concentrations of 5–50 µm.[81]
2.2.2. Interactions of Vitamin E and the NLRP3 Inflammasome
The activation of the inflammasome NLRP3 is partly dependent on the priming involving NF-𝜅B. However, different forms
of vitamin E have been reported to interfere with both, the NF-𝜅B-mediated priming and the actual activation of the NLRP3
complex.
Triggers for the activation of the inflammasome are di-verse (see Section 1.2.2). Reactive oxygen species activate the
inflammasome complex, followed by cleavage of caspase-1 and secretion of IL-1𝛽 and IL-18.[82] Vitamin E is a well-known
antioxidant and, indeed, blocks ROS-induced activation of the inflammasome and the respective formation of ILs in vita-min C-deficient mice with trauma-induced axon degeneration and vision loss.[83] The 𝛼-TOH derivative
[2-(3,4-dihydro-6- hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)ethyl]tri-phenylphosphonium bromide, known as MitoVit E, has been shown to accumulate in isolated mitochondria and in mito-chondria within isolated cells and organs, due to the covalently linkage to the lipophilic triphenyl phosphonium cation, which results in positively charged 𝛼-TOH molecule.[84] Driven by
the mitochondrial membrane potential, the MitoVit E is accu-mulated solely in mitochondria, which significantly decreased their ROS production. Using this MitoVit E, inflammation in hearts of rats with pneumonia-induced sepsis and in isolated cardiomyocytes was attenuated. Further, the application of 21.5
𝜇moles kg−1MitoVit E reduced mitochondrial ROS-dependent
translocation of the NF-𝜅B p65 subunit into the nucleus and the expression of ASC, followed by attenuated cleavage of caspase-1 and release of IL-1𝛽 in inflamed heart tissue.[85]
Tocotrienols and TOHs regulate NF-𝜅B to different extent, with 𝛿- and 𝛾-T3 being the most active forms. With this in line, extracts of annatto seeds (Bixa orellana L.), which contain T3s and predominantly𝛿-T3 (90%), have been reported to
at-tenuate NF-𝜅B-induced priming of the NLRP3 inflammasome, followed by reduced cleavage of caspase-1 and secretion of IL-1𝛽 in J774 macrophages stimulated with LPS and nigericin.[86]
Furthermore, 𝛾-T3 suppresses activation of caspase-1 in J774
macrophages stimulated with LPS and nigericin in a concen-tration dependent manner with 1–5 µm, followed by down-regulation of the expression of IL-1𝛽, IL-18, and NLRP3 mRNA as well as diminished secretion of respective proteins in bone marrow-derived macrophages and peritoneal macrophages ob-tained from db/db mice.[87] Next, an induction of A20 by𝛾-T3
was observed which consequently inhibits the activation of
NF-𝜅B, more precisely the phosphorylation of I𝜅B𝛼 and thereby the
translocation of NF-𝜅B subunits to the nucleus.[88]A20 is one,
but not the only target for the blocking of the priming of the NLRP3 inflammasome by𝛾-T3.[89]In addition,𝛾-T3 inhibits
IL-1𝛽 independently of A20.[87,89] Kim et al. concluded that𝛾-T3
regulates the NLRP3 inflammasome by inducing A20. Deubiq-uitinase A20 in turn inhibits the TRAF6/NF-𝜅B pathway and the
activation of the adenosine monophosphate activated protein ki-nase (AMPK)/autophagy axis. The regulation of the
TRAF6/NF-𝜅B pathway and the AMPK/autophagy axis finally attenuates
caspase-1 cleavage.[87]The regulatory effects of𝛾-T3 have been
exclusively described for the NOD-like receptor subset of the in-flammasome, and not for the AIM2 scaffold inin-flammasome, the adaptor protein ASC, or other caspases.[87]𝛾-Tocotrienol also
at-tenuates the LPS and palmitate-induced activation of the NLRP3 inflammasome which is followed by a reduced secretion of IL-1𝛽 and arachidonic acid as well as synthesis of the correspond-ing lipid mediators. The reduced release of pro-inflammatory mediators subsequently diminished lipotoxicity in bone marrow-derived macrophages.[90]
E can be expected. The cluster of differentiation (CD) 36, a surface marker of macrophages responsible for the uptake of oxidized LDL (oxLDL), has been described to interact with the NLRP3 inflammasome.[91] Notably, 𝛼-TOH downregulated
CD36,[68]suggesting a CD36-mediated regulation of the NLRP3
by 𝛼-TOH. Similarly, COX-2/microsomal prostaglandin E2
synthase-1 (mPGES-1) and 5-lipoxygenase (LOX) contribute to the monosodium urate induced activation of NLRP3,[92] and
𝛼-TOH interacts directly with 5-LOX by forming an irreversible 𝛼-TOH/5-LOX complex.[93] The 𝛼-TOH/5-LOX complex
in-hibits formation of LOX-derived monohydroxy-eicosatetraenoic acids (HETEs).[94] The interaction of𝛼-TOH with COX-2 and
mPGES-1 similarly effects the signaling molecules formed by this pathway.[95]
2.3. Anti-Inflammatory Effects of Physiologically Formed Metabolites of Vitamin E
Structural similarities of the LCMs to their metabolic precur-sors, the different vitamin E forms, suggest that these two classes of molecules share modes of action. Consequently, the anti-inflammatory effects of vitamin E seem to be complicated by the circulating hepatically formed LCMs of vitamin E, which exert actions on immune cells which are at least in part different from that described for TOHs and T3s.[96] So far, knowledge on the
biological mode of action of these LCMs is scarce.
2.3.1. Interactions of LCMs and NF-𝜅B
Compared to TOHs, T3s, and𝛿-T3 in particular, are potent in-hibitors of NF-𝜅B and phosphorylation of the I𝜅B𝛼 subunit.[78]
Indeed, an LCM formed from𝛼-TOH, namely 𝛼-13′-COOH, did not affect translocation of the p65 subunit in murine RAW264.7 macrophages.[97]However, direct effects of the LCMs of vitamin
E on NF-𝜅B activity cannot be excluded. Furthermore, effects of
𝛿-T3-13′-COOH on NF-𝜅B translocation and activity could be
ex-pected due to the structural similarity with𝛿-T3 but this needs further investigations.
2.3.2. Interactions of LCMs and the NLRP3 Inflammasome
The LCMs of vitamin E are anti-inflammatory molecules, al-though the molecular principles of the inhibitory action on NF-𝜅B-mediated inflammation have not yet been shown. As reported earlier, the NLRP3 inflammasome is activated by ROS. The LCMs𝛼-13′-COOH and 𝛿-13′-COOH increased intracellular and intramitochondrial ROS formation in HepG2 cells,[98]thus
assuming an activation of NLRP3. In agreement with this, the LCMs of𝛼-TOH increased the expression of CD36,[68]which has
been described to interact with the NLRP3 inflammasome.[91]
The reported interaction of the arachidonic acid signaling path-way with the NLRP3 inflammasome[92]is a conceivable target for
the LCMs, since inhibitory effects of 9′-COOH, 13′-COOH,[99]
and 𝛼-13′-COOH,[97] 𝛿-T3-13′-COOH[100] on COX-2-dependent
production of PGE2 in IL-𝛽-stimulated A549 cells and in
LPS-stimulated RAW264.7 macrophages have been described. In addition,𝛼-13′-COOH[101]and𝛿-T3-13′-COOH[102]are potent
selective inhibitors of the mPGES1, as tested in IL-1𝛽-stimulated A549 cells. With this in line, 𝛿-13′-COOH[103] and
𝛼-13′-COOH[97] selectively inhibit 5-LOX activity and the respective
biosynthesis of 5-LOX-derived lipid mediators.[101] Although an
effect of the LCMs on NLRP3 seems to be evident, further studies are required to evaluate the role of LCMs of vitamin E on the initiation and activation of NLRP3 and other inflammasomes.
3. Inflammatory Diseases
3.1. The Importance of C-Reactive ProteinOne of the acute phase proteins, the C-reactive protein (CRP), more precisely its pentameric form, is predominantly synthe-sized by the liver. The systemic concentration of CRP in healthy humans is below 1 mg L−1 and rises up to 100- to 1000-fold following inflammatory stimuli and is accompanied by an increase of IL-6, IL-1𝛽, or TNF𝛼.[104,105] C-reactive protein and
IL-6 regulate each other in a self-reinforcing manner. Evidence is emerging that the circulating pentameric form of CRP (pCRP) is functionally inert and has no intrinsic pro-inflammatory properties.[106] The dissociation of pCRP to the monomeric
form (mCRP) finally activates the CRP system, which further activates platelets, leukocytes, endothelial cells, and the com-plement system.[106] Therefore, CRP is considered rather an
unspecific marker for inflammation than a specific biomarker due to the lack of specificity in differentiating infection from other inflammatory processes.[107]Notably, CRP serves as both a
marker of inflammation and an active regulator of inflammatory processes.[107]As defined by Yeh et al., CRP levels of more than
10 mg L−1 could be an indicator for infections, autoimmune diseases, or cancers, whereas CRP concentrations between 1 and 10 mg L−1can be a predictor for chronic inflammatory diseases such as atherosclerosis and related cardiovascular complications in primary and secondary prevention,[107]and for the early
diag-nosis of non-alcoholic steatohepatitis (NASH)[108]and DMT2.[109]
3.2. Atherosclerosis
With 31%, CVDs are still the leading cause of global mortality.[110]
Figure 5. Pathogenesis of atherosclerosis. Atherosclerosis is a chronic inflammatory disease of the inner wall of arteries. The initial pathological pro-cess is the endothelial dysfunction enabling monocytes to immigrate though the endothelial layer into the subintimal tissue and to differentiate into macrophages. Oxidative stress modifies LDL to oxLDL. The excessive uptake of oxLDL by macrophages results in foam cell formation. An increased secretion of chemokines, cytokines and CRP is the consequence. A) With increasing grade of inflammation, the lesion area growths, smooth muscle cells immigrate and a more or less stable plaque forms, which is characterized by a necrotic lipid core and stabilizing fibrotic cap. In advanced disease stages, bloodflow is affected and atherosclerotic plaques may get unstable, finally resulting in plaque rupture or erosion of the endothelium. Consequently, a thrombus is formed that may occlude arteries at the site of its formation or more downstream in smaller vessels, causing life-threatening events such as MI or stroke. B) The Glagovian phenomenon describes an adaptive widening of the arterial diameter while the diameter of the vascular lumen re-mains unchanged but may also decrease with more progressed plaque formation. CRP, C-reactive protein; CV, cardiovascular events; LDL, low-density lipoproteins; oxLDL, oxidized low density lipoprotein.
various oxidative, inflammatory and lipid-related triggers cause the secretion of cytokines and chemokines by macrophages, further augment the inflammatory processes within the arterial wall. Over decades, processes which are likely irreversible cause, i) loss of elasticity, ii) reduction of the vascular lumen, and iii) adaptive widening of the arterial diameter (Glagovian phe-nomenon), while the diameter of the vascular lumen remains unchanged or decreases. In the following, plaque rupture or plaque erosion cause thrombus formation and in turn occlu-sion of the artery, resulting in acute events such as myocardial infarction (MI) and stroke (Figure 5).
3.2.1. Role of Inflammation in Atherosclerosis
Recent human trials focusing on the contribution of the IL-1𝛽/IL-6/CRP pathway to atherogenesis revealed the importance of targeting inflammatory processes during effective treatment of atherosclerosis. Notably, CRP concentrations between 1 and 5 mg L−1are a strong predictor of peripheral vascular disease, coronary artery disease, MI, and stroke even after adjustment for traditional risk factors, such as LDL cholesterol.[104]In brief,
CRP plasma concentrations are increased in patients with stable
coronary artery disease by about twofold compared to healthy in-dividuals and even further after acute events. Concentrations of CRP above 3 mg L−1are associated with increased risk of coro-nary events (about twofold) in stable and unstable angina pectoris and are predictive for future events. Patients with CRP concen-trations of more than 200 mg L−1after an acute MI are more prone to develop myocardial rupture and are at increased risk of recurrent events.[107]Results of the Canakinumab
Antiinflamma-tory Thrombosis Outcome Study (CANTOS), in which IL-1𝛽 was targeted by a monoclonal antibody, revealed a decrease of CRP concentrations by 26–41% in patients after application of 150 or 300 mg anti-IL-1𝛽 antibody every 3 months, respectively. In addi-tion, the hazard ratios for the primary endpoint (first occurrence of nonfatal MI, any nonfatal stroke, or cardiovascular death) as compared with placebo were as follows: 0.85 (95% CI, 0.74–0.98;
p= 0.021) in the 150 mg group and 0.86 (95% CI, 0.75–0.99; p =
0.031) in the 300 mg group.[111]
In contrast, the Cardiovascular Inflammation Reduction Trial (CIRT), in which the anti-inflammatory compound methotrexate was used, revealed neither a reduction of the cardiovascular event rate nor of IL-6, IL-1𝛽, and CRP.[112]However,
A systemic meta-analysis of trials using methotrexate in weekly doses of 13–15 mg for at least 3 months showed a 21% (95% CI, 0.73–0.87) risk reduction of sever cardiovascular events .[115]
The most pronounced effect of methotrexate has been reported by Choi et al., who reported a hazard ratio of 0.4 (95% Cl, 0.2– 0.8) for total mortality, 0.3 (95% Cl, 0.2–0.7) for cardiovascular and 0.6 (95% Cl, 0.2–1.2) for non-cardiovascular deaths when comparing patients with rheumatoid arthritis treated with and without 13–25 mg methotrexate per week.[113]Comparable doses
of 20 mg methotrexate per week were applied in CIRT in patients with previous MI or multivessel coronary disease and additional DMT2 or metabolic syndrome. The investigators explained the negative outcome of CIRT with the target of methotrexate, namely adenosine signaling, which impacts less pronounced on atherothrombosis.[116] Notably, inflammation in patients
suffering from arthritis, characterized by a constant low-grade inflammation and additional acute inflammatory burst, is much more pronounced than in patients with chronic low-grade inflammation such as patients with DMT2 or metabolic syn-drome. Furthermore, the grade of inflammation significantly impacts atherogenesis and cardiovascular events.[111] Next, it
must be acknowledged that different types of inflammatory diseases display their own characteristics although they involve similar or even general signaling pathways. This means that an anti-inflammatory therapy can be effective for some conditions but is not necessarily effective for all inflammatory diseases. Methotrexate, for example, is effective in arthritis but its effects on atherosclerosis and cardiovascular events are still under discussion.
It was a crucial prerequisite that the patients of the CANTOS trial had elevated CRP concentrations of more than 2 mg L−1 (median baseline 4.2± 2.80 to 7.10 mg L−1, SD), whereas the median baseline concentration of CRP in CIRT was 1.6 mg L−1 (± 0.76–3.50 mg L−1, SD), which is in the normal range.[116]It has
been therefore discussed that anti-inflammation therapies are beneficial only for patients with persistently elevated inflamma-tory state. So far, the inhibition of the IL-1𝛽/IL-6/CRP pathway is the only effective and trail-validated anti-inflammatory ap-proach that reduces cardiovascular event rates.[116] Since the
reported effect of the IL-1𝛽-targeting antibody canakinumab is independent of lipid and blood pressure lowering,[111]combined
therapies could be considered as an effective future therapeutic concept against atherosclerosis and respective cardiovascular events.[116]
3.2.2. Vitamin E and Atherosclerosis—Plaque Stability versus Cardiovascular Events
The role of vitamin E in the prevention of CVD has been in-tensively studied. Although higher plasma levels of vitamin E are associated with a lower risk for severe coronary disease (Nurses’ Health Study, NHS),[117] supplementation of vitamin
E in randomized human trials failed to have valid effects on the risk of cardiovascular events.[118] Neither supplementation
of vitamin E as a single compound (e.g., Heart Outcomes Pre-vention Evaluation (HOPE) study[119] or in combination with
other micronutrients (e.g., Supplémentation en Vitamines et
Minéraux Antioxydants SU.VI.MAX study),[120]in primary (e.g.,
Physicians’ Health Study)[121] or secondary prevention (e.g.,
Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico (GISSI) study)[122] revealed significant
cardiopro-tective effects of vitamin E. Based on the current state of research, supplementation of vitamin E is not recommended for preventing CVD anymore.
These discrepancies in cardioprotective effects of vitamin E may be the result of crucial differences in study designs. As dis-cussed earlier, the impact of doses and duration of vitamin E sup-plementation and proposed interactions between the different vitamin E forms or with other food ingredients or medications cannot be excluded.[123] In addition, the application of𝛼-TOH
or non-𝛼-TOH seems to be crucial for the evaluation of cardio-protective effects. In observational studies all forms of vitamin E,𝛼-TOH as well as non-𝛼-TOH forms, depending on the di-etary intake, contribute to the described cardioprotective effects, whereas in clinical studies preferably𝛼-TOH was supplemented. 𝛼-TOH is the most active form of vitamin E, at least in
prevent-ing fetus resorption, and is the most abundant form of vitamin E in healthy humans. However,𝛼-TOH and non-𝛼-TOH forms accumulate in different tissues,[124] and mediate at least in part
different biological functions.[125]Mechanistic studies described
unique antioxidant and anti-inflammatory properties for non- 𝛼-TOH forms, which consequently suggested beneficial effects in the prevention and therapy of chronic diseases.[125]Consequently,
the application of𝛼-TOH only may impact the outcome of
clin-ical studies. In addition, metabolites of vitamin E, formed dur-ing hepatic catabolism, may have valuable contributions to the biological activity of vitamin E. The protein responsible for reg-ulating the formation of the metabolites of the different vitamin E forms is𝛼-TTP (see Section 2.1). Research on these metabo-lites provide evidence for their physiological relevance and effec-tive interactions of the long-chain metabolites of tocopherols with lipid metabolism[68]and inflammation.[67,97]Taking this into
ac-count, we are confident that the role of vitamin E in cardiovascu-lar disease is not fully understood.
An additional parameter that is crucial for the outcome of hu-man studies investigating cardio-protective effects of vitamin E is a proper cohort collection. The data by Levy and colleagues may shed new light on the story by demonstrating that the hap-toglobin (Hp) genotype of the study participants may be relevant for cardioprotective effects of vitamin E. In their study, high-dose vitamin E supplementation decreased the risk of cardiovascular events in patients with DMT2 and Hp genotype 2-2 (OR 0.58, 95% Cl, 0.4–0.86; p= 0.006).[126,127]The homozygous Hp 2-2
pheno-type is present in 36% of humans and is considered as an in-dependent predictor of cardiovascular outcomes in people with DMT2 who have a five times higher odds ratio compared to in-dividuals with the Hp 1-1 genotype.[128] A meta-analysis of the
HOPE trial and the Israel Cardiovascular Events Reduction with Vitamin E study revealed a significant reduction of cardiovascular death, MI and stroke (OR 0.58, 95% CI, 0.40–0.86; p= 0.006) in diabetic individuals with Hp 2-2 genotype compared to diabetics with Hp 1-1 or Hp 2-1 genotype.[126]The results of these studies
Figure 6. Pathogenesis of non-alcoholic liver diseases. Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease globally and may develop into more serious disease stages. Non-alcoholic fatty liver disease is hallmarked by the pathological accumulation lipids in hepatocytes. The progressing inflammation and accumulation of lipids results in non-alcoholic steatohepatitis (NASH), which can transform to the more severe stages liver fibrosis and cirrhosis. The histological changes during fibrosis affect blood flow through the liver, which is partly reversible, whereas cirrhosis causes an irreversible disruption of normal liver architecture and results in liver dysfunction. With NASH and the more severe stages of liver disease the risk of hepatocellular carcinoma increases. The global prevalence for patients with NALFD who develop inflammatory NASH is between 5% and 20%. About 10–20% of NASH patients develop liver fibroses (stage IV) and less than 5% are faced with cirrhosis.
DMT2 and Hp genotype 2-2 (OR: 0.66, 95% CI, 0.45–0.95; p= 0.025) by high-dose vitamin E supplementation.[129]
Notably, these trials tested the capacity of vitamin E to pre-vent cardiovascular epre-vents and therewith its ability to stabilize atherosclerotic plaques. As outlined above, recent studies provide clear evidence that the progression of atherosclerosis is triggered by chronic low-grade inflammation. As vitamin E alters acute ox-idative and inflammatory stress, it likely dampens also peak levels of oxidative and inflammatory stress during acute cardiovascular events such as MI. Within the first 48 h following MI, plasma concentrations of antioxidants, such as vitamin E, drop,[130]
sup-porting the concept that vitamin E deficiency during acute events may contribute to the severity of the outcome. Indeed, a recent study of our group demonstrated the potential of𝛼-TOH to pre-serve cardiac function after acute MI in a mouse model of my-ocardial ischemia/reperfusion.[131]However, human trials are on
demand to confirm the relevance of this finding.
3.3. Non-Alcoholic Fatty Liver
Non-alcoholic fatty liver (NAFL) is globally the most common chronic liver disease,[132]with steadily increasing prevalence.[133]
Worldwide, between 6% and 33% of the population suffer from fatty liver,[134,135] with the highest prevalence of 46%
in Asia and the United States.[136] The National Health and
Nutrition Examination Surveys reported a significant asso-ciation of the prevalence of fat liver and the global burden
of metabolic comorbidities, such as obesity, DMT2, dyslipi-demia, and metabolic syndrome.[133] Furthermore, ethnic
background,[132,137] gender,[138] and age[139] significantly affect
the prevalence of NAFLD.[136]Fatty liver development is a silent
epidemic until a certain stage of the disease so that it remains often undiagnosed and may therefore be under-reported in the general population and especially in children.[140]
Non-alcoholic fatty liver disease is a complex disease with many facets but is hallmarked by chronic inflammation and excessive triglyceride deposition in hepatocytes. During its development, several stages and severities of the disease can be defined (Figure 6). Non-alcoholic fatty liver is characterized by the accumulation of lipids to amounts of 5–10% of the liver mass without signs of inflammation. if not treated, inflammatory pro-cesses trigger the formation of NASH which is accompanied by the so-called ballooning (swelling and rounding of hepatocytes cytoplasm[141]) as well as apoptosis and necrosis of hepatocytes,
with or without fibrosis. Patients with NASH have an increased risk of liver cirrhosis and liver cancer.[140] To date, the gold
standard for diagnosing NASH is liver biopsy. However, there are emerging non-invasive biomarkers that may replace liver biopsy.[142]
3.3.1. Role of Inflammation in Fatty Liver Disease
of energy and nutrients such as saturated fatty acids, fructose, and others.[143] Besides, obesity alters the gut microbial
popu-lations as reported from high-fat diet fed mice.[144] The
interac-tion of the gut-liver-adipose tissue axis triggers pro-inflammatory signaling cascades involving NF-𝜅B, STAT3, and the inflamma-some, resulting in increased production of pro-inflammatory cytokines.[145,146] Both obesity and NAFLD are characterized by
increased expression of TNF𝛼 and IL-6 in adipose tissue and liver, with increasing severity of the diseases. Further, the gut micro-biome is triggering TLR signaling. Toll-like receptors 2, 4, and 9 are triggered by saturated fatty acids, such as palmitic acid, as well as bacterial endotoxins and bacterial DNA, respectively, and drive in interaction with NLRP3 the progression of NAFLD, with TLR4 most likely playing a key role.[147]
Diet-induced excessive uptake and accumulation of lipids in the liver, such as free fatty acids and saturated fatty acids in particular,[144]further activate Kupffer cells. Kupffer cells express
all types of inflammasomes, except for NLRP1.[148]Indeed,
Kupf-fer cells are the main source of IL-1𝛽 in the liver via the activa-tion of the NLRP3 inflammasome. For instance, free fatty acids act as so-called DAMPs and increase ROS generation[149]
result-ing in the activation of the NLRP3 inflammasome.[146,150]There
is a mutual influence of the TLR and inflammasome signaling in NAFLD,[147] and the contribution of the NLRP3
inflamma-some to inflammation and fibrosis in NAFLD has been shown in NAFLD patients, which exhibit increased production of pro-IL-18 and pro-IL-1𝛽, as well as in experimental NASH mouse models
using MCC950, an inhibitor of the NLRP3 inflammasome,[151]
and in murine NLRP3-deficient mice.[152]
Data on the role of CRP as an independent marker in NAFLD is contradictory. Often, obesity seems to account for most of the production of inflammatory mediators, including CRP,[153]
which makes it difficult to use CRP as an independent clinical marker for the evaluation of NASH.[154] However, in a Chinese
study with 25 843 volunteers, CRP was an independent risk fac-tor for NAFLD.[108] Compared to the group with CRP levels of
<1 mg L−1, elevated levels of 1–3 and>3 mg L−1CRP correlated
with an increased prevalence of NAFLD of 1.09 (95% CI, 1.01– 1.17) and 1.24 (95% CI, 1.13–1.35), respectively.[108]Even in a
co-hort of obese patients, CRP levels were found to be higher in the sub-cohort of obese patients with diagnosed NAFLD compared to obese patients with normal livers (7.95± 3.63 vs 4.75 ± 1.48 mg L−1, p < 0.05).[155] Therefore, Ziemia´nski et al. concluded that
CRP levels can be a useful marker to identify obese patients at increased risk of NAFLD.[155]In addition, high CRP levels were
associated with extensive liver fibrosis in NASH differentiating different stages of NAFLD even after adjusting for age, sex, and BMI.[156]
3.3.2. Vitamin E in the Therapy of NAFLD/NASH
To date, effective therapies of NAFLD are scarce. However, lifestyle management (e.g., weight reduction) and the treatment with PPAR𝛾 agonists and vitamin E have been shown to be
effec-tive against NAFLD depending on the disease stage. However, pharmacological doses of vitamin E, more precisely 𝛼-TOH,
are needed to treat NAFLD, as outlined below. NAFLD is a
combination of inflammation, oxidative stress, and excessive accumulation of lipids in the liver. Therefore, medications cur-rently used or tested for the therapy of NAFLD/NASH are used to treat comorbidities, such as DMT2, obesity, dyslipidemia, and hypertension. The prevalence of NAFLD/NASH is significantly increased in obese people (BMI>30), and reductions in body weight of 3–5% and 9% positively affect steatosis and necroin-flammation as well as fibrosis, respectively.[134,140,157] Increases
in aerobic and anaerobic exercise also contribute to decreases in hepatic lipid accumulation.[158] However, in patients with
progressing to advanced fibrosis lifestyle interventions alone are not sufficient.[159]
One of the most important randomized human trials inves-tigating pharmacological approaches for treating NAFLD and NASH is the Pioglitazone versus Vitamin E versus Placebo for the Treatment of Nondiabetic Patients with Nonalcoholic Steatohepatitis (PIVENS) study.[160] The dual PPAR𝛼/𝛾 agonist
saroglitazar and the PPAR𝛾 agonist pioglitazone are approved
medications in diabetes and were shown to reduce steatosis, NASH, fibrosis, and inflammation in pre-clinical trials.[161] In
the PIVENS study, 30 mg d−1 pioglitazone failed to improve the primary outcome of the study defined as biopsy-confirmed reduction of NASH (34% vs 19%; pioglitazone vs placebo; p= 0.04).[160]Taking this into account, moderate effects of
pioglita-zone cannot be excluded. Furthermore, pioglitapioglita-zone significantly reduces steatosis, inflammation, and hepatocellular ballooning, improves insulin resistance and liver enzyme levels, and re-solves steatohepatitis.[160]Despite these benefits, pioglitazone is
also known for adverse effects, such as weight gain, increased cardiovascular event rates, increased risk of osteoporosis and bladder cancer. Thus, the U. S. Food and Drug Administration raised concerns on the safety of long-term medications with pioglitazone. However, the European Association for the Study of the Liver and the Guidance Statements of the American Association for the Study of Liver Diseases (AASLD) currently recommend pioglitazone for treating NASH.[140]
Next to pioglitazone, RRR-𝛼-TOH is currently one of the few established therapies for NAFLD.[160] The combined treatment
of 45 patients with biopsy-confirmed NASH with 1000 mg d−1 vitamin C and 1000 IU d−1RRR-𝛼-TOH for 6 months showed significant improvements of the fibrosis score, whereas markers for necroinflammation remained unchanged.[162] As reported
in the PIVENS study, the 2-year intervention with 800 IU d−1
RRR-𝛼-TOH in non-diabetic patients with biopsy-diagnosed
NAFLD significantly reduced the primary outcome (steatohep-atitis) compared to placebo (43% vs 19%; p= 0.001). Additional major improvements were decreased transaminase concentra-tions (p< 0.001), steatosis (p = 0.005) and inflammation (p = 0.02), whereas the degree of fibrosis remained unchanged (p= 0.24).[160]In another pilot study, children diagnosed with
obesity-induced NASH and elevated liver enzyme (aminotransferase) levels were selected. Treatment with daily doses of 400–1200 IU DL-𝛼-TOH for up to 10 months significantly decreased concentrations of liver markers alanine aminotransferase (ALT) (p< 0.001), aspartate aminotransferase (AST) (p < 0.002), and alkaline phosphatase (p< 0.003).[163] The Treatment of
per day for 2 years.[164]None of the intervention groups reached
significance in the primary outcome of sustained reduction in ALT. However, DL-𝛼-TOH supplementation significantly im-proved hepatocellular ballooning (p= 0.006) and NAFLD activity score (p= 0.02). Analyses of the PIVENS and TONIC cohorts, revealed a relationship between CYP4F2 polymorphisms and the pharmacological effectiveness of vitamin E. However, this interaction seems to be complicated by age differences, which cannot be explained yet.[165]Consequently, the AASLD guidelines
recommend vitamin E treatment of non-diabetic adult patients with biopsy-diagnosed NASH. Notably, studies on treatment of patients with diabetes and histologic characterization of cirrhosis are still warranted and vitamin E is therefore not recommended until reliable data exist.[140]
While promising effects of vitamin E for the treatment of NAFLD have been described, the molecular modes of action of vitamin E in NAFLD are still unknown. However, a contribution of hepatically formed𝛼-LCMs can be expected as these
metabo-lites exert anti-inflammatory effects.[97]Due to the key role of the
NLRP3 inflammasome in the progression of NAFLD it will be in-teresting to study whether and how vitamin E or even its metabo-lites interfere with the NLRP3 inflammasome.
4. Conclusions and Perspective
Inflammatory processes regulated by the innate and adaptive immune system are indispensable in the defense against sev-eral non-communicable diseases. However, chronic low-grade or massive acute inflammatory responses vouch a variety of danger, such as the initiation and progression of inflammation-driven diseases. Therefore, investigating underlying signaling pathways is fundamental for the discovery of drug targets that may be used for future preventive and therapeutic approaches. In the basic re-search of inflammation using in vitro studies, animal studies as well as clinical trials in humans the use of reasonable model sys-tems and a proper sample collection is crucial for further data in-terpretation. Recent studies, namely CANTOS and CIRT, showed the diversity of the success rate of anti-inflammatory treatments, depending on the inflammatory status of the patients present-ing and the signalpresent-ing pathways affected by the anti-inflammatory agent applied. Furthermore, the heterogeneity of the patients must be considered for finding personalized therapies. For the evaluation of studies investigating the effectiveness of vitamin E, we have to be aware of the discrepancies between in vitro and in vivo studies. In general, in vitro studies mainly serve as proof-of-principle studies often using non-physiological concentrations of the bioactive molecules of interest to unravel mechanistic signal-ing pathways. Hence, various factors occurrsignal-ing in vivo, such as metabolism, systemic and local accumulation of the compound of interest as well as synergistic effects with other nutritional fac-tors, medications or lifestyle factors cannot be considered. There-fore, the transfer of results between different model systems is limited.
Vitamin E, particularly 𝛼-TOH, is acknowledged as one of the major lipophilic antioxidants and is accounted for its anti-inflammatory capacity. Therefore, vitamin E supplementation may be a reasonable approach for the therapy of certain inflam-matory diseases, such as NAFLD. Based on recent study results,
we propose that the vitamin E-derived LCMs represent a new class of regulatory structures with biological functions and are therefore potential lead structures for drug development, that need further exploration. However, the identification of key reg-ulators by which vitamin E or related LCMs affect inflammatory diseases are missing.
Acknowledgements
M.W. and L.B. contributed equally to this work.
Open access funding enabled and organized by Projekt DEAL.
Conflict of Interest
The authors declare no conflict of interest.
Keywords
atherosclerosis, fatty liver disease, inflammasome, inflammatory dis-eases, nucleotide-binding domain and leucine-rich repeat pyrin domain (NLRP) 3, tocopherol
Received: January 31, 2020 Revised: June 26, 2020 Published online: July 29, 2020
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