Abstract

Oxysterols can be derived from the diet, physiologically produced via specific enzymes, or are generated by autoxidation. These molecules have physiological properties and can also adversely affect vital organs. Indeed, some of them have pro-oxidant and pro-inflammatory activities and can lead to major pathologies. The present review focuses on oxysterols (7-ketocholesterol, 7β-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 5,6α-epoxycholesterol, 5,6β-epoxycholesterol, and cholestane-3β, 5α, 6β-triol) involved either in cholesterol metabolism, age-related diseases (such as cardiovascular, neurodegenerative, and eye diseases, e.g., sarcopenia), and inflammatory diseases (especially Behcet’s disease and bowel and lung diseases (e.g., sarcoidosis, COVID-19)). Metabolic pathways associated with oxysterol-induced inflammation are discussed considering the cytokinic TLR4 pathway, non-cytokinic pathways, and the contribution of Ca2+ and K+ channels. Therapeutic approaches targeting oxysterol-induced inflammation either by natural or synthetic molecules are also presented.

Keywords: oxysterols, inflammatory diseases, natural molecules, nutrients, edible oils, synthetic molecules

1. Introduction

1.1. Oxysterols: Origins and Biogenesis

In humans, there are several sources of oxysterols which result from cholesterol oxidation. These compounds can be formed endogenously in different tissues, or can enter the body from the diet [1]. Oxysterols can be generated either by the auto-oxidation of cholesterol following a free radical attack [2], or by specific enzymes, usually of the cytochrome P450 type [3]. In general, the oxysterols oxidized on the steroid nucleus are generated by auto-oxidation, while those oxidized on the side chain result from enzymatic attack [4].

1.1.1. Dietary Origin of Oxysterols

Cholesterol is an important component of many foods. During industrial processes, cholesterol can be subjected to oxidation, leading to the formation of oxysterols [5]. Foods rich in cholesterol such as eggs, egg powder commonly used in processed foods, clarified butter, dairy products, red meat, ham (bacon), and dried or tinned fish are the richest in the following oxysterols: 7-ketocholesterol (7KC), 7β-hydroxycholesterol (7β-OHC), 5,6 α-epoxycholesterol, and 5,6 β-epoxycholesterol [6,7]. During food storage and preparation, cholesterol can easily undergo oxidation when exposed to high temperatures, oxygen or ozone, or light (ultraviolet exposure) [8]. Dehydrated products are particularly sensitive to oxidation [9]. The oxysterols found mainly in food are those oxidized at C7 (7KC, 7β-OHC) as well as epoxycholesterols (5, 6α/5, 6β-epoxycholesterol), but also 25-hydroxycholesterol (25-OHC) formed both enzymatically and by autoxidation [7,10].

In food products, the level of oxysterols can reach 10 to 100 μM [11]. After ingestion of a meal rich in oxysterols (salami, parmesan), assays showed that plasma concentrations of oxysterols (7KC, 7β-OHC) were increased, mainly in chylomicrons, after 5 h. Similar results were shown in rats after gastric infusion of oxysterols [12]. This shows that intestinal absorption varies according to the type of sterol. Also, in rats, it was shown that 92% of dietary oxysterols were absorbed [13]. The majority of ingested oxysterols are absorbed in the form of esters in the upper intestinal tract and taken up in plasma by chylomicrons. Oxysterols can be found in all types of lipoproteins, but the majority are present in low-density lipoprotein (LDL) [14,15]. Depending on their esterification, oxysterols can be transported in the plasma either by albumin or LDL [16]. Measurements carried out with 7KC, 20 α-hydroxycholesterol (20 α-OHC), and 25-OHC have shown that albumin preferentially transports 25-OHC [16,17].

1.1.2. Enzymatic Formation of Oxysterols

Several oxysterols can be produced physiologically by enzymatic oxidation during cholesterol metabolism. A large proportion of oxysterols are formed in this way. In terms of the major oxysterols, 4β-hydroxycholesterol (4β-OHC), 27-hydroxycholesterol (27-OHC), 24(S)-hydroxycholesterol (24(S)-OHC), 7α-OHC, and 25-OHC are produced, respectively, by CYP3A4, CYP27A1, CYP46A1, CYP7A1, and cholesterol 25-hydroxylase (CH25H) [4]. Other enzymes are responsible for the formation of oxysterols, which are generally detected in trace amounts in biological fluids or tissues. Most of these are intermediates in cholesterol metabolism reactions, some of which probably have very short lifetimes [18].

The enzymes involved in cholesterol oxidation are most often members of the cytochrome P450 superfamily of enzymes, but they may also belong to the hydroxysteroid dehydrogenase (HSD) family [19]. CH25H is the exception and is not a member of either of these enzyme families [10]. Not all of these enzymes are ubiquitously expressed, leading to cell-type-specific oxysterol profiles.

CYP3A4 enables the conversion of cholesterol to 4β-OHC, the major oxysterol found in plasma, but also the conversion of cholesterol to 25-OHC [20]. This cytochrome enzyme is expressed in the endoplasmic reticulum (ER) of the liver. It is also involved in the metabolism of many xenobiotics and is estimated to be capable of metabolizing almost half of the drugs used [4,21].

CYP27A1 or 27-hydroxylase converts cholesterol into 27-OHC and is involved in the ‘acidic’ pathway of bile acid formation. This mitochondrial enzyme is expressed in a wide variety of tissues but mainly in the liver, endothelial cells, and monocytes/macrophages [4,21].

CYP46A1 or 24-hydroxylase converts cholesterol to 24(S)-OHC. It is expressed in the ER, mainly in the neurons of the central nervous system, but also in small quantities in the testes and ovaries. It enables the excretion of cerebral cholesterol, as cholesterol cannot cross the blood–brain barrier (BBB), whereas 24(S)-OHC can [4,21].

CYP7A1 or 7α-hydroxylase is the first enzyme involved in the ‘classic’ bile acid formation pathway. It converts cholesterol into 7α-OHC. It is highly expressed in the liver and more specifically in within the ER of hepatocytes. Its expression is regulated by bile acid levels [21]. It can also produce 7KC from 7-dehydrocholesterol [4].

CH25H or 25-hydroxylase catalyzes the conversion of cholesterol into 25-OHC. It is expressed in the ER of most tissues. It is not a member of the cytochrome enzyme family and is a non-heme iron-containing protein [21,22].

1.1.3. Formation of Oxysterols by Autoxidation

The carbons in rings A and B of the sterane nucleus of cholesterol are the most sensitive to free radical attack, particularly carbons 5, 6, and 7. There are two types of autoxidation, type I and II.

Type I autoxidation involves oxidation by free radicals (superoxide anion (O2•−), hydrogen peroxide (H2O2), hydroxyl radical (HO•), nitric oxide (NO), and peroxynitrites ONOO−). These can be generated by cellular metabolism, or by their subsequent decomposition into hydroxyl radicals, by the dismutation of two superoxide anions into hydrogen peroxide (2 O2•− → H2O2) or by the Fenton reaction (H2O2 + Men+ → HO• + Me(n+1)+) where Me is a transition metal such as copper, iron, or aluminum [8]. Most often, oxidation by ROS or reactive nitrogen species (RNS) result in the loss of a hydrogen on carbon 7, due to the weak bond between carbon and hydrogen. The dissociation energy of this bond is 88 kcal/mol [23]. As this local oxidation at C7 is fairly stable, it can then easily react with molecular oxygen to form a peroxyl radical (COO•). Subsequently, by reacting with another hydrogen lost by another molecule, the peroxyl radical will form a cholesterol hydroperoxide (7α- or 7β-OOHC). As the hydroperoxide function is very unstable, hydroperoxide cholesterol breaks down into 7α-OHC, 7β-OHC, or 7KC. These three oxidized C7 oxysterols are the ones formed mainly by auto-oxidation [24]. 7KC is the major oxysterol in OxLDL, accounting for around 30% of total sterols [25,26].

Type II cholesterol auto-oxidation involves non-radical attack by oxygen singletons (1ΔgO2, hypoclorous acid (HOCl) or ozone (O3)). The involvement of ozone is interesting given that it is linked to atmospheric pollution. Oxygen singletons can be formed by the reaction of hydrogen peroxide (H2O2) with HOCl, both of which are produced during inflammatory reactions in the presence of myeloperoxidase. According to Iuliano [8] C5, C6, C7 hydroperoxides, 5,6-epoxides (α/β), as well as secosterols, are formed as primary compounds in these reactions [23]. The 5,6α/β-epoxycholesterols can be taken up by the enzyme cholesterol epoxide hydrolase to form cholestane-3β, 5α, 6β-triol. It has been shown that aminolysis of α-epoxycholesterols can give alkylamino-oxysterols. These include dendrogenin A, which can be formed when a 5,6α-epoxycholesterol reacts with a histamine through the action of an enzyme that has yet to be identified at the molecular level, dendrogenin A synthase [27,28,29].

2. Involvement of Oxysterols in Inflammatory Human Diseases

There is substantial evidence that oxysterols can adversely affect certain major and vital organs such as the heart, brain, blood vessels, bones, pancreas, and eyes [30]. These oxysterols might play a role in the development and course of chronic illness [30,31] and also of infectious diseases, especially COVID-19 [32,33] (Figure 1). Actually, a number of clinical studies have shown that people with type 2 diabetes, obesity, hypercholesterolemia, and atherosclerosis have higher levels of various oxysterols [34,35]. These can be used as useful indicators to diagnose certain pathologies, or to forecast the occurrence and progression of diseases such as cardiovascular diseases, Alzheimer’s disease, diabetes mellitus, multiple sclerosis, osteoporosis, lung cancer, breast cancer, and infertility [36]. In addition, some oxysterols could be also used as biomarkers of some rare diseases such as X-linked adrenoleukodystrophy (X-ALD) [37] and Niemann–Pick disease [38,39]. It is noteworthy that oxysterols involved in inflammatory diseases have often simultaneously pro-oxidant and pro-inflammatory properties contributing to the aggravation of symptoms via an overproduction of pro-inflammatory cytokines at the system level or in the tissues affected by the disease [40,41,42].

Figure 1.

Involvement of cholesterol-oxidized products in inflammatory diseases, with a focus on cardiovascular, neurodegenerative, eye, osteoporosis, sarcopenia, bowel, lung and Behcet’s diseases.

2.1. Cardiovascular Diseases

Cardiovascular disease, often associated with atherosclerosis, is the main cause of morbidity and death. Atherosclerosis is a lipid metabolic issue in addition to a chronic inflammatory illness [43]. Oxysterols are the main component that can aid in the development of atherosclerosis and have been demonstrated in numerous empirical investigations to have a role at different stages of the atherosclerotic process [30,44]. Patients with atherosclerotic lesions or cardiovascular illnesses have higher plasma levels of oxysterols [36]. The oxysterols found mainly in atheromatous plaques are mainly 7KC, 7β-OHC, 7α-OHC, 5,6α-epoxycholesterol, 5, 6β-epoxycholesterol, and cholestane-3β, 5α, 6β-triol; they can reach levels up to 100 times higher than normal plasma levels [45,46]. 7α- and 7β-OHC comprise 75–85% of oxysterols present in the plaques at various locations. They are also among the most important oxysterols that are present in an atherosclerotic lesion. Their amount increases with the degree of atherosclerosis and is almost directly correlated with cholesterol levels [30]. Atherosclerotic patients’ plasma and atheromatous plaques have been found to include certain oxysterols (7β-OHC, 7KC, and 25-OHC). These oxysterols are strong in vitro inducers of MCP-1, MIP-1β, TNF-α, and/or IL-8 secretion, the latter of which involves the MEK/ERK1/2 cell signaling pathway [41].

An increase in 7β-OHC appears to be a biomarker of cardiovascular risk [47]. Björkhem showed that there was a considerable rise in 7β-OHC levels in patients exhibiting rapid progression of carotid atherosclerosis [48].

Due to its pro-oxidant and pro-inflammatory properties, it is now well established that 7KC contributes to the development of atherosclerosis [31]. 7KC has been shown to contribute to the pathophysiology of atherosclerotic plaques. It induces an increase in the expression of cell adhesion molecules (Inter Cellular Adhesion Molecule-1 (ICAM-1), Vascular Cell Adhesion Protein-1 (VCAM-1) and E-selectin due to the elevation of ROS at vascular endothelial cells level, which promotes the recruitment of macrophages to plaques in the early stages of the pathology [26].

A recent study investigated in-depth proteomic profiling and showed the effects of 7KC on the macrophage proteome. Atherogenic/M1 indicators, cholesterol metabolism, biosynthesis and transport, and nutrient transport in general were among the dynamic alterations that 7KC independently mediated. These effects prime the macrophage, increasing the release of important pro-inflammatory factors, including TNFα release triggered by LPS [49].

In human atherosclerotic plaque-derived monocytes and macrophages, 25-OHC contributes to the production of foam cells and stimulates the release of pro-inflammatory cytokines and chemokines, including IL-1, IL-6, IL-8, CCL5, and M-CSF. However, in certain situations, 25-OHC can block the Akt/NFB signaling pathway, induce IFN, repress SREBP, and antagonize inflammasomes, all of which can reduce the activity of inflammasomes [50].

Other oxysterols aid in the rupture and erosion of plaque in advanced atherosclerotic lesions. For instance, it was noted that α-triol promotes smooth muscle cell calcification, which damages the artery wall [51].

2.2. Neurodegenerative Diseases

Among the neurodegenerative diseases where the increase of the oxysterols amount was noticed are Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Huntington’s disease, multiple sclerosis, and X-linked adrenoleukodystrophy (X-ALD). The most commonly accepted forms of oxysterols that may contribute to the pathophysiology of neurodegenerative disease phases are 24(S)-OHC and 27-OHC, 7KC, 7β-OHC, 7α-OHC, 25-OHC, 27-OHC, 5,6α-epoxycholesterol, 5,6 β-epoxycholesterol, 4β-OHC, and 4α-OHC [52].

Variations in the levels of 24(S)-OHC and 27-OHC in the blood and/or cerebrospinal fluid have been linked to a number of neurological illnesses. To remove extra cholesterol from the brain, CYP46A1 almost exclusively produces 24(S)-OHC in neurons; this oxysterol, unlike cholesterol, can pass through the BBB [53] and is linked to an activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPH-oxidase or NOX), whose activity is itself modulated by oxysterols [54].

7KC influences neurodegeneration by acting on the interactions of the Aβ 1-42 peptide with the plasma membrane, promoting its incorporation into fibrillar deposits [55]. These results on the relationships between 7KC and Aβ 1–42 on synthetic lipid membranes, or cells in culture, are in agreement with in vivo observations of patient brains [56]. In addition, the effects of 7KC on microglia, particularly at the lysosomal level, could lead to a reduction in the efficiency of phagocytosis of the Aβ 1–42 peptide and promote the appearance of amyloid plaques [57]. Izumi, et al. [58] demonstrated the impact of 25-OHC on hippocampal plasticity and learning involve NLRP3 inflammasome and cellular stress responses in multiple neuropsychiatric illnesses.

Apart from these general findings, we will indicate the impact of oxysterols in the most pronounced neurodegenerative diseases separately and in a more detailed manner.

2.2.1. Alzheimer’s Disease

The most prevalent kind of dementia, Alzheimer’s disease (AD), causes cognitive impairment and progressive cognitive loss. Numerous studies, with varying methodologies and findings, have been conducted and are currently underway to examine the significance of circulating oxidized cholesterols in AD [59].

The identification of the apolipoprotein E (ApoE) ε4 allele’s genetic link with both familial and sporadic late-onset AD raised the prospect of lipid metabolism and transport abnormalities in AD patients’ brains [60].

In early-onset Alzheimer’s disease, there is an elevation of 27-OHC in the blood plasma and CSF fluid, which is thought to be a risk factor. In addition to decreasing brain glucose uptake, GLUT4 expression, and spatial memory, the release of 27-OHC from circulation into the brain can also activate the renin–angiotensin system (RAS), which can result in ischemic brain injury, oxidative stress, and compromised cognitive performance. Furthermore, hypertension and insulin resistance, two risk factors for Alzheimer’s disease, may be exacerbated by RAS activation [61].

One of the primary ways that excess cholesterol is eliminated from the brain is by the neuron-specific enzyme CYP46A1 converting it to 24(S)-OHC [62]. When comparing to control in AD, some studies indicated elevated amounts of 24(S)-OHC in the blood [63,64] but others discovered tiny quantities [65,66].

According to An et al. [67] who used random forest machine learning and 5-fold cross-validation, a panel of five oxysterols were identified that could distinguish patients with mild cognitive impairment (MCI) from controls with good performance. So, given their role in controlling neuronal death, neuroinflammation, oxidative stress, Aβ accumulation, and other molecular pathways underlying AD pathology, the intracerebral and extracerebral equilibrium of two side-chain enzymatical oxysterols—specifically, the flux of 24(S)-OHC from the brain into the circulation and the reverse flux of 27-OHC—has been identified as a critical factor in AD pathogenesis. As a result, these oxysterol patterns in biological fluids and organs might be better markers of disease than specific individual components.

In rats, cognitive performance and cholesterol metabolism were adversely affected when 26-OHC was administered via the tail vein [68]. High plasma levels of 26-OHC were found to be strongly associated with moderate cognitive impairment (MCI) [69]. In comparison to controls, Popp, Meichsner, Kölsch, Lewczuk, Maier, Kornhuber, Jessen and Lütjohann [63] and Zarrouk, et al. [70] reported high amounts of 26-OHC in the bloodstream while Mateos, et al. [71] and Hughes, Kuller, Lopez, Becker, Evans, Sutton-Tyrrell and Rosano [64] reported low levels, and Costa, Joaquim, Nunes, Kerr, Ferreira, Forlenza, Gattaz and Talib [65] found no difference. Otherwise, there were reports of elevated levels of 7-oxycholesterol in AD [72].

When 24(S)-OHC and 27-OHC are present, β-amyloidogenesis—which is connected to Alzheimer’s disease—increases and proteins linked to Parkinson’s disease fluctuate in expression [73].

Radhakrishnan, et al. [74] noticed increased 7KC levels in the brains of 3xTg mouse model of AD. Microglia activation and elevated oxidative stress in astrocytes were the outcomes of applying 7KC to a microglia cell line alone or to mixed astrocyte and microglia cultures.

According to Choi, et al. [75], 25-OHC in AD, when administered in vivo, mechanistically increase the esterification of cholesterol, alters the dynamics of cell membranes, further decreases phagocytosis, raises the synthesis of pro-inflammatory cytokines, and hinders microglial surveillance. Furthermore, it has been demonstrated that amyloid-beta (Aβ) increases 25-OHC levels and CH25H expression in microglia, worsening these functional deficits.

Papotti et al. [76] measured and correlated particular lipid markers in patients with varying levels of cognitive decline, such as those with AD and those with MCI related to AD (MCI-AD) carriers or non-carriers of the APOE4 genotype. They concluded that compared to non-carriers, AD APOE4 carriers displayed increased PCSK9 and 24(S)-OHC in CSF. CSF PCSK9 and 27-HOC were negatively correlated in AD, and CSF PCSK9 and 24(S)-OHC were negatively correlated only in AD APOE4 carriers. In AD, APOE4 carriers displayed a positive association between CSF and serum PCSK9, indicating PCSK9 exchange between the brain and the periphery. So, ApoE4-associated lipid changes in AD may be specifically indicated by PCSK9 and 24(S)-OHC, which may aid in the clinical development of the AD spectrum.

A total of 142 people between the ages of 49 and 88 were included in an AD study. Subjects with AD pathology had greater levels of cerebrospinal fluid (CSF) 24S-OHC and a higher 24(S)-OHC/27-OHC ratio. Aβ1–42 levels were associated with CSF desmosterol [77].

Postmortem frozen brain tissue CSF from patients with late-stage AD (Braak stages III–IV) and early-stage AD (Braak stages I–II) were examined for lipids. Brain tissue and mitochondria isolated from late-stage AD brain tissue had increased amounts of oxysterols namely 26-OHC, 25-OHC, and 7-oxycholesterol, with the exception of 24(S)-OHC, which was lower in late AD [78].

2.2.2. Parkinson’s Disease

Recognition of Parkinson disease (PD) is primarily based on clinical findings that link numerous nonmotor characteristics, including hyposmia, sleep disorders, behavioral or mental health issues, and dysautonomia, with complex motor impairment, or Parkinsonism, which includes rigidity, akinesia, rest tremor, and gait disturbance [79]. Oxysterols can control proteins implicated in Parkinson’s disease progression. LXR and estrogen receptors are two types of nuclear receptors that were engaged in the mechanisms of this regulation [80].

By enhancing LXR-mediated transcription of alpha-synuclein and decreasing estrogen receptor-mediated transcription of tyrosine hydroxylase, 27-OHC favorably regulates the expression of this protein [80]. It has also been demonstrated that the LXR pathway, namely 27-OHC, regulates alpha-synuclein expression in SK-N-SH neuroblastoma cells and MO3.13 oligodendrocyte cell lines [81].

The severity of the disease was associated with 24(S)-OHC levels in CSF, and some patients have higher 27-OHC levels in their CSF [48]. Feeding of rabbits with a diet supplemented with 2% cholesterol for 12 weeks revealed that long-term ingestion of this kind of food caused an increase in alpha-synuclein in the substantia nigra of these animals [82].

In Parkinson’s disease, elevated levels of 24(S)-OHC, 27-OHC, 7β-OHC, and 7KC have been measured in the visual cortex [45,83].

2.2.3. Multiple Sclerosis

Immune-mediated demyelination and axon loss in the central nervous system are hallmarks of multiple sclerosis (MS). Based on an array of investigations, oxysterols in MS patients may serve as indicators of particular disease stages [84]. In MS, 24(S)-OHC may be an appropriate biomarker of brain damage and neuronal metabolism [85].

One well-established indicator of neuroaxonal damage in MS is serum neurofilament light chain (sNfL). At follow-up, there was a positive correlation between 7KC and 7β-HOC and sNfL levels. After controlling for LDL-C or HDL-C, the relationships between 7KC or 7β-HOC and sNfL were still significant [86].

Research revealed that individuals with MS experienced a considerable drop in 24(S)-OHC levels [87]. Furthermore, 24(S)-OHC diffuses into the cerebrospinal fluid in cases of neuronal degeneration and may have lipotoxic effects on many central nervous system cells, including oligodendrocytes [88].

Serum 24(S)-OHC levels show a negative correlation with normalized brain volume measurements in patients with MS who have relapses [89]. This discovery also indicates a potential involvement of these oxysterols in MS, since elevated levels of 24(S)-OHC and 27-OHC have been documented in patients with a comparable illness [90]. Patients with MS in the progressive phase also had higher circulating levels of 15-OHC and 15-KC [87].

Higher levels of total hydroxy-octadecanoic acid (total HODEs), including 9-HODE and 12-HODE, were also found in patients with MS. This oxidative stress biomarker (total HODEs) was linked to an elevated level of 7KC and 7β-OHC, which are primarily produced by auto-oxidation, and additionally to an increase in 24(S)-OHC, which may be a sign of neuronal death [91].

During times when the disease is active, patients with MS have higher fluxes of 24(S)-OHC, which can lead to elevated plasma levels of this oxysterol. Furthermore, there is a correlation between the amount of brain atrophy and the plasma levels of this oxysterol in the blood. An elevated flux of 24(S)-OHC from the brain into the cerebrospinal fluid (CSF) in patients who have neuronal injury and/or demyelination was also recorded. Otherwise, increased 27-OHC flow from the circulation into the CSF is caused by a BBB deficiency [92].

2.2.4. Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset non-demyelinating neurodegenerative illness, leading to motor impairments. There are two types: sporadic (90% of cases) and familial (caused by mutations in over 20 genes). Oxysterols may be involved in ALS because of their partial capacity to bind the LXR receptors, as well as other receptors like oxysterol binding proteins (OSBP) [93].

Compared to the control (persons without ALS) and treatment groups (ALS patients treated with riluzole), an untreated ALS group had greater amounts of 24-OHC and 25-OHC in their CSF. 25-OHC may also be directly involved in the pathophysiology of ALS through GSK3-ß activation and neuronal apoptosis [94]. It has been shown that 25-OHC can restore the membrane and functional characteristics of neuromuscular junctions in the early stages of the disease [95]. The CYP27A1 enzyme is shown to be less active in ALS patients, which makes it more difficult for the central nervous system to eliminate excess cholesterol that could be harmful to neuronal cells. This is further exacerbated by a decrease in the neuroprotective LXR ligands such as 3,7-diHCA [96]. On the other hand, in spite of the fact that levels of 24(S)-OHC, 27-OHC, and 25-OHC are typically higher in ALS patients, there was no statistically significant association between the presence of ALS and the levels of these oxysterols in the plasma of ALS patients [97].

2.2.5. X-Linked Adrenoleukodystrophy

Adrenoleukodystrophy (ALD) is caused by an ABCD1 mutation, a genetic condition that often follows an X-linked inheritance pattern (X-ALD). The buildup of very long-chain fatty acids (VLCFA) is linked to this progressive neurodegenerative disorder. Severe cerebral inflammatory demyelination and milder spinal cord axonopathy are the primary manifestations of X-ALD [98]. The primary impacted areas of X-ALD are the adrenal cortex and inflammatory demyelinating lesions, where lipid peroxidation, particularly that generating oxysterols, primarily develops [99].

Patients with X-ALD have elevated levels of 7KC in their plasma. According to Nury, et al. [100], 7KC stimulates oxidative stress and peroxisomal dysfunction in microglial cells, which results in microglial cell activation and proliferation, which may be a factor in demyelination and dementia. By promoting brain inflammation through the activation of the NLRP3 inflammasome pathway, which is essential for demyelination and oligodendrocyte loss, 25-OHC was found to be a powerful mediator in the pathophysiology of X-ALD [101]. By triggering mitochondrial ROS, 25-OHC facilitates the assembly and activation of the NLRP3 inflammasome, which in turn causes oligodendrocyte death, IL-1β release, and microglia recruitment, ultimately resulting in severe neuroinflammation and demyelination [102].

2.2.6. Autism Spectrum Disorder (ASD)

The hallmarks of autism spectrum disorder (ASD) include limited-repetitive patterns of behavior, interests, or hobbies, as well as ongoing deficiencies in social communication and engagement. It is now known that neuro-immune disorders and neuro-inflammation play a major role in the development and maintenance of ASD [103]. 24(S)-OHC was high in children and as a potential marker of ASD, although 7α-OHC and 25-OHC were only marginally significant. Age and 24(S)-OHC in patients had an inverse relationship [104]. Variants in the liver X receptor gene and oxysterol dysregulation in ASD were examined and it was found that 27-OHC may be used as an ASD diagnostic indicator [105]. While 27-R-OHC levels were lower in the ASD group than in the control group, 24-OHC and 25-OHC levels were significantly greater. The autistic group had a significantly greater ratio of 24(S)-OHC to 27-OHC. According to the receiver operating characteristic study, this ratio had “acceptable discrimination potential” and was statistically significant in its ability to distinguish between diagnoses of ASD and non-ASD [106].

2.3. Eye Diseases

The quality of life of millions of people is impacted by ocular degeneration, a significant public health concern, that includes cataracts, glaucoma, macular degeneration, and diabetic retinopathy. Oxysterols cause inflammation and cell death pathways and are linked to the pathogenesis of eye degeneration [107].

2.3.1. Cataract

The most common cause of blindness globally is a cataract, which is defined as the loss of transparency in the natural lens of the eye [108]. Oxysterols could have a function in the formation of cataracts. They may change intracellular lipid homeostasis and Na+/K+ ATPase activity, which may be significant risk factors in cataract physiopathology [48]. Cataract development is linked to abnormalities in the metabolism of cholesterol, such as sterol 27-hydroxylase (CYP27A1) or 7-dehydrocholesterol reductase [109].

Studies conducted in clinics revealed that cataractous human lenses had higher levels of cholesterol, 7β-OHC, 7KC, 5α,6-α-epoxycholestanol, 20α-OHC, and 25-OHC than normal lenses [107].

Certain oxysterols, including 7β-OHC, 7KC, 5α, 6α-epoxycholestanol, 20α-OHC, and 25-OHC, were detected by gas chromatography in human cataracts retrieved after conventional eye surgery, but no cholesterol oxides were found in any healthy lens [110]. 7KC has also been detected in the lenses of cataract patients (around 4 nmol/mol of free cholesterol while it is undetectable in controls) [110,111].

2.3.2. Age-Related Macular Degeneration (AMD)

In the retina, the macula is responsible for central vision and color perception. Disorders of the macula can be of genetic origin or are linked to age, such as AMD.

AMD and atherosclerosis may have comparable mechanisms. It is commonly recognized that oxysterols play a part in the development of atherosclerosis. Due to their cytotoxic, pro-inflammatory, and pro-oxidant qualities, oxysterols may be implicated in the retinal pigment epithelium and photoreceptor lesions that occur in AMD, since cholesterol is a component of drusens [112].

During AMD, oxidative stress appears in retinal pigment epithelial cells leading to the formation of oxysterols [26,113]. In primary cultures of pig retinal pigment epithelial cells, 24(S)-OHC, 25-OHC, or 7KC caused minor mitochondrial dysfunctions but a notable 2- to 4-fold increase in reactive oxygen species generation. Additionally, in decreasing order (25-OHC > 24(S)-OHC > 7KC), they increased IL-8 gene expression and IL-8 protein secretion [42].

Studies have shown a correlation between oxysterols and AMD, either at the level of the oxysterols or the genes that produce or bind them. In fact, substantial levels of 7KC have been discovered in the drusen, a type of proteolipidic deposit that is typical in cases of AMD [107].

7KC accumulates in the neural retina fractions of monkeys between 3 and 5 times more than in the pigment epithelium and choriocapillaris fractions. This accumulation occurs mainly at the level of drusen [114]. It has been shown that 7KC has chemoattractant activity towards retinal microglia, which triggers their activation and migration into the subretinal space, probably by a mechanism involving the inflammasome [115]. Additionally, on retinal pigment epithelial cells, 7KC induces the secretion of vascular endothelial growth factor (VEGF) [116].

Without any risk alleles in genes coding for complement factor H members, an allele in the cholesterol-24S-hydroxylase (CYP46A1) gene may increase the risk of exudative AMD [117].

2.4. Osteoporosis

Osteoporosis (OP), a systemic bone disease, is characterized by reduced bone strength, microarchitectural alterations, and an elevated risk of fracture. As they are involved in a number of critical biological processes, cholesterol oxidation products are significant substances in the preservation of bone metabolic equilibrium [118]. Certain research indicates that oxysterols such α-triol may have a role in osteoporosis [119]. Comparably, it was demonstrated that a major decrease in trabecular and cortical bone resulted from an increase in 27-OHC, either by injection or by genetically altering the CYP7B1 enzyme [120].

An endogenous selective estrogen receptor modulator (SERM), 27-OHC controls bone homeostasis by competitively binding to the estrogen receptor [121]. When 27-OHC concentration is raised pharmacologically, bone trabeculae and cortical bone are significantly reduced, which ultimately leads to OP [122].

22S-HOC and 20S-HOC, influence bone homeostasis by encouraging mesenchymal stem cells to differentiate osteogenically while preventing their lipogenic development [123]. 22R-OHC, 22S-OHC, and 20S-OHC have been shown to have pro-osteogenic effects on M2-B104 cells, whereas 7KC and cholestane-3beta-5alpha-6beta-triol have been shown to have anti-osteogenic effects on rat bone marrow stromal cells [119,124].

Using alveolar bone healing models and periodontal ligament stem cells, 22S-OHC and 20S-OHC together encourage periodontal regeneration [118,125].

According to Nelson, Wardell and McDonnell [121] and He and Nelson [126], 27-OHC interacts with the estrogen receptor and may also encourage osteoporosis [127]. Osteoblast differentiation is inhibited by 25-OHC [128]. The process of bone damage caused by exposure to combined metals (Fe and Pb) may involve 7KC. This could be connected to variations in inflammatory levels in vivo that promote osteoclast proliferation [129].

2.5. Sarcopenia

Sarcopenia is defined by a progressive loss of skeletal muscle mass and strength that occurs naturally with age [130]. Elevated amounts of pro-inflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), as well as C-reactive protein (CRP), are linked to sarcopenia. These pro-inflammatory cytokines change intercellular communication, raise adiposity, and disrupt insulin-like growth factor 1 (IGF-1) signalling in the skeletal muscles [131]. Skeletal muscle contraction can result in the production and release of cytokines known as myokines, such as apelin, which can impact skeletal muscle function and act on oxidative stress and inflammation [132]. It is likely that the positive effects of apelin on muscle metabolism are achieved by activating AMP-activated protein kinase and Akt, which in turn triggers mitochondriogenesis [133].

There was an increase in 7KC, and particularly 7β-OHC, in the plasma of sarcopenic patients [134]. The same authors established that the cytotoxic effects of 7β-OHC on myoblasts are somewhat greater than those of 7KC. These two oxysterols had less of an adverse effect on differentiated C2C12 cells (myotubes). Furthermore, apelin implicated in sarcopenia and inflammatory biomarkers (CRP, TNF-α, IL-6, IL-8, and LTB4) were also measured in the serum [135,136].

In their clinical investigation, Priyadarsini, Nanda, Devi and Mohapatra [130] verified that oxidative stress and inflammation are present in sarcopenic individuals and found new oxidative stress biomarkers, particularly oxysterols (7KC, 7β-OHC) produced by cholesterol autoxidation, that may influence the skeletal muscle atrophy.

Song, et al. [137] investigated how muscle-specific proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) regulates Nrf2 to modify mitochondrial oxidative stress in elderly sarcopenia. PGC-1α) and nuclear factor erythroid 2-related factor 2 (Nrf2) expression in C2C12 cells were decreased by either PGC-1α silencing or 7β-OHC treatment. Moreover, PGC-1α silence reduced the expression of the Nrf2 protein and elevated damaging ROS in the cells treated with 7β-OHC. Conversely, PGC-1α overexpression raised the production of the Nrf2 protein and reduced the damaging ROS in cells treated with 7β-OHC.

2.6. Bowel Diseases

Inflammatory diseases of the gastrointestinal tract include Crohn’s disease, ulcerative colitis, and inflammatory bowel diseases (IBDs). The first area of the body to be exposed to the effects of oxysterols is the gut, where the dietary sources of oxysterols, primarily foods high in cholesterol, come from. This main interaction may disrupt the balance of the human digestive system and contribute to injury to the intestinal mucosa [138].

Oxysterols could contribute to the evolution and worsening of these pathologies through their pro-inflammatory and pro-oxidant effects. Secretion of pro-inflammatory cytokines has been described on colon epithelial cells (Caco-2) treated with a mixture of 7KC, 5,6α-epoxycholesterol, 5,6β-epoxycholesteol, 7α-OHC, and 7β-OHC [139]. In inflammatory bowel diseases, oxysterols can participate in alteration of the intestinal barrier via activation of matrix metalloproteinases (MMP) which degrade cell junctions. Addition of a mixture of oxysterols composed of 7KC, 7α-OHC, 7β-OHC, and 5,6-epoxycholesterol to colonic epithelial cells induces their death by apoptosis [31,140]. The integrity of the intestinal epithelium and the vascular endothelium barrier can be weakened by 7KC and 25-OHC [141].

EBI2 (Epstein–Barr virus-Induced gene, or GPR183) is a G-protein coupled receptor that is activated by certain oxysterols, such as 7,25-diOHC [142]. In both steady state and during inflammation, production of colonic lymphoid structures is mediated via the EBI2-7,25-diOHC axis and enhanced oxysterol synthesis. Likewise, oxysterols encourage the downregulation of the CH25H enzyme, which may have multiple roles in the pathophysiology of intestinal fibrosis and inflammatory bowel disorders [143].

Independent of EBI2-mediated cell migration, a large intake of 25-OHC has been demonstrated to modify intestinal immunity, limit plasma cell differentiation, and impair IgA production and the response, through SREBP2 [144].

Inflammatory signals raise 7,25-diOHC, and during colitis, EBI2 regulated the recruitment of inflammatory cells. As a result, studies using EBI2-deficient mice show that in an innate model of intestinal inflammation, these animals were less prone to colitis. Increased synthesis of the EBI2 ligand 7,25-diOHC links colonic inflammation to the oxysterol EBI2 pathway. Furthermore, in patients with ulcerative colitis, there is a strong association between colonic inflammation and the expression of CH25H and CYP7B1 [145].

2.7. Lung Diseases

Three respiratory infections, tuberculosis (TB), severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), and silicosis, have been demonstrated to raise the local synthesis of oxysterol in the lung [146].

2.7.1. Tuberculosis

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), an infectious respiratory disease. A novel finding is that the host immunological response to Mtb infection is regulated by oxysterols and their receptors [146].

Compared to animals that were not infected, mice with Mtb infections had higher lung expression of the oxysterol-producing enzymes CH25H and CYP7B1 and increased levels of 25-OHC which correlated with this [147].

7α,25-OHC is the most powerful endogenous agonist, and both it and 25-OHC are ligands for the oxysterol-sensing receptor EBI2. Dendritic cells, eosinophils, macrophages, innate lymphoid cells (ILCs), T cells, and B cells are among the immune cells that express EBI2/GPR183 [146].

According to Bohrer, et al. [148], oxysterol synthesis triggers the migration of TB EBI2/GPR183+ immune cells to the lung, indicating the potential utility of EBI2/GPR183 and oxysterols as biomarkers for early TB diagnosis and prediction of disease severity. MTb catalyzes the change from 25-OHC to 25-hydroxycholest-4-en-3-one using the 3HSD enzyme. Consequently, MTb interferes with the human immune response by modulating the activity of 25-OHC through its enzymatic system [149]. However, people with chronic obstructive pulmonary disease, another illness linked to persistent infection, have higher than normal amounts of 25-OHC in their lungs [150].

In humans, the enzyme 3-hydroxysteroid dehydrogenase (3HSD) type 7 controls the amount of 7,25-dihydroxycholesterol. A 3HSD homologue found in MTb may functionally resemble 3HSD type 7 enzyme, obstructing immune cell movement driven by oxysterol [149].

When 7α,25-OHC activates EBI2/GPR183 in primary human monocytes, Mtb and Mycobacterium bovis BCG were restricted intracellularly. The addition of an EBI2/GPR183 antagonist eliminated this effect, indicating that this receptor regulates the intracellular growth of mycobacteria and maybe other microorganisms [151].

Investigation on THP-1-derived macrophages and primary human monocyte-derived macrophages showed that Mtb infection causes the production of IL-36, which in turn promotes synthesis of the LXR ligands 25-OHC and 27-OHC. The synthesis of antimicrobial peptides like cathelicidin and defensins, which improve mycobacterial control, is triggered by LXR activation [152].

2.7.2. SARS-CoV-2 and Respiratory Diseases

In COVID-19 and influenza, there is evidence that oxysterols play a role in the immune response to severe viral respiratory infections [153]. Numerous investigations have demonstrated that oxysterol concentrations vary during SARS-CoV-2 infection. According to a study that tracked the kinetics of serum 25-OHC over time in a single female COVID-19 patient, 25-OHC levels significantly increased later in the infection when the patient’s clinical condition significantly deteriorated and peaked two days before the patient’s death [154]. In a different study, serum concentrations of 27-OHC were inversely correlated with disease severity, and 7KC and 7β-OHC were higher than in healthy matched controls [32].

In a different study of COVID-19 patients with various degrees of metabolic comorbidities there was a drop in 7KC and an increase in 25-OHC, 24(S)-OHC, and 27-OHC compared to healthy controls. The concentrations of 4β-OHC and 7α-OHC did not differ [155].

Single-cell sequencing of bronchoalveolar lavage samples from COVID-19 patients with moderate and severe disease revealed that the oxysterol-producing enzymes CH25H and CYP7B1 are increased in lung macrophages and myeloid dendritic cells, compared to those of healthy controls. Increased expression is associated with the severity of the disease. In addition, EBI2/GPR183 expression in macrophages increased with COVID-19 infection [152].

In a murine model of COVID-19 infection using a mouse-adapted