Lipid‐based regulators of immunity

Abstract Lipids constitute a diverse class of molecular regulators with ubiquitous physiological roles in sustaining life. These carbon‐rich compounds are primarily sourced from exogenous sources and may be used directly as structural cellular building blocks or as a substrate for generating signaling mediators to regulate cell behavior. In both of these roles, lipids play a key role in both immune activation and suppression, leading to inflammation and resolution, respectively. The simple yet elegant structural properties of lipids encompassing size, hydrophobicity, and molecular weight enable unique biodistribution profiles that facilitate preferential accumulation in target tissues to modulate relevant immune cell subsets. Thus, the structural and functional properties of lipids can be leveraged to generate new materials as pharmacological agents for potently modulating the immune system. Here, we discuss the properties of three classes of lipids: polyunsaturated fatty acids, short‐chain fatty acids, and lipid adjuvants. We describe their immunoregulatory functions in modulating disease pathogenesis in preclinical models and in human clinical trials. We conclude with an outlook on harnessing the diverse and potent immune modulating properties of lipids for immunoregulation.


| INTRODUCTION
Lipids, colloquially called fats, are nonpolar hydrocarbons that have a pleiotropic role in modulating physiological functions, including in energy storage, maintaining cell membrane integrity, and cell signaling. 1 Fatty acids (FAs) are a major class of lipids characterized by a hydrocarbon chain functionalized with a carboxylic acid. FAs are primarily sourced from exogenous sources, such as through dietary essential fatty acids (EFAs) and have been demonstrated to influence the immune response through cell signaling that directly and indirectly activate or suppress cells of both the innate (e.g., neutrophils) and adaptive (e.g., T cells and B cells) immune system. Some FAs are associated with inflammatory diseases and may serve as biomarkers for assessing progression, such as the pro-inflammatory metabolites of FA called eicosanoids, for chronic conditions such as rheumatoid arthritis, 2 asthma, 3 and inflammatory bowel disease (IBD). 4 In humans, the two principal immunoregulatory FAs are polyunsaturated fatty acids (PUFAs) and short-chain fatty acids (SCFAs), which together constitute about 15% of the total FAs content, with the remainder comprised of monounsaturated and saturated FAs. 5 PUFAs are characterized by multiple unsaturations (double bonds) in the lipid tail and follow a "ω-x" nomenclature, where "x" refers to the carbon atom with the first unsaturation on the aliphatic chain from the methyl terminus. The key structural difference between ω-3 and ω-6 PUFAs is in the location of a carbon double bond. This seemingly innocuous change has a significant effect on their metabolic derivatives (eicosanoids) which directly modulate immune cells. SCFAs are small molecule metabolites, comprising less than six carbon atoms with no unsaturations and are derived from the breakdown of complex carbohydrates by gut microbiota. 6 These products of bacterial fermentation follow the standard IUPAC naming system for carboxylic acids (e.g., propionate [three carbon atoms], butyrate [four carbon atoms]). By activating G-protein-coupled cell membrane receptors (GPCRs) and inhibiting histone deacetylase (HDAC), SCFAs can modulate the activity of regulatory immune cells.
Outside their structural role in maintaining cell membrane fluidity, extracellular PUFAs are found in tissue microenvironments and are relatively enriched as a fraction of total FA in the brain, heart, and blood plasma. 5 SCFAs diffuse from the gut lumen, which is the primary site for their biosynthesis, via a strong biological gradient and can partition in tissues throughout the body. At homeostasis, SCFA concentration in tissues outside the intestine is negligible, suggesting that SCFAs primarily affect cells associated with the lower gastrointestinal tract. 6 In this review, we will first describe the structural properties of PUFAs, their derivatives, and target signaling pathways, which leads to either activation of pro-inflammatory immune cells and inflammatory mediators or an anti-inflammatory effect that seeks to resolve inflammation. Next, we will describe the structure of SCFA and their dual role as signaling molecules and as epigenetic modulators of immune function, which have been harnessed in treating inflammatory diseases. We will then review lipids used as adjuvants, the immunostimulatory component that enhances the protective immune response to vaccines. We conclude with an outlook on harnessing the immunoregulatory properties of lipids to develop the next generation of immunomodulating medicines ( Figure 1).

| Eicosanoids and inflammation
Eicosanoids are important regulators of inflammation and comprise prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs), and lipoxins (LXs). [11][12][13] These molecules are converted from PUFAs by cyclooxygenase (COX), lipoxygenase (LOX), and Cytochrome P450 enzymes. Eicosanoids released by ARA metabolism are mostly proinflammatory and are the target of nonsteroidal anti-inflammatory drugs used in suppressing inflammation. 7 Prior to the synthesis of eicosanoids, ARA is released from the sn-2 position of the membrane phospholipids by the action of phospholipase A 2 enzymes, activated by inflammatory stimuli. 14 ARA metabolism results in two-series (two double bonds) PGs and TXs as well as four-series (four double bonds) LTs and LXs. COX-2 is induced in cells by inflammatory stimuli, resulting in a large increase in PG production. PGE 2 , other two-series PGs, and the four-series LTs are among the best-known activators of inflammation 11,12,15 and usually act through GPCRs. 16 ω-3 PUFAs such as EPAs are similarly metabolized by COX, LOX, and Cytochrome P450. However, EPA metabolism yields three-series (three double F I G U R E 1 Overview of discussed immunomodulatory lipids. Immunomodulatory lipids either activate or suppress immune activation. Certain eicosanoids (metabolic products of ω-3 and ω-6 PUFA: prostaglandins, thromboxanes, and leukotrienes) and lipid adjuvants (virosomes, MPLA, and MF59) are pro-inflammatory. Other eicosanoids such as lipoxins, the specialized pro-resolving lipid mediators (metabolic products of ω-3 PUFA; resolvins, protectins, and maresins), and SCFAs (acetate, propionate, and butyrate) are anti-inflammatory. Natural lipids and their derivatives are shaded in blue, synthetic lipids are shaded in violet. MPLA, monophosphoryl lipid A; PUFA, polyunsaturated fatty acid; SCFAs, short-chain fatty acids bonds) PGs and TXs, and five-series (five double bonds) LTs. 14 By incorporating more ω-3 PUFAs into the available pool of PUFAs, the amount of pro-inflammatory eicosanoids is reduced by competition between ARA and the ω-3 PUFAs. 17 However, the metabolism of the ω-3 PUFAs is not regulated solely by supply and demand. EPA itself can negatively regulate COX-2 gene expression and inhibit ARA metabolism. 18 The eicosanoids produced from metabolism of ω-3 PUFAs such as EPAs are structurally distinct from those produced by ARA 14 and are less biologically potent, 19,20 potentially due to a reduced receptor affinity. 21 An important class of eicosanoids produced by the metabolism of both ω-3 and ω-6 PUFAs (overwhelmingly ω-3 PUFA) are the specialized pro-resolving lipid mediators (SPMs). This family of mediators include resolvins produced by EPA (E-series) and DHA (D-series) as well as protectins and maresins produced by DHA. Two series of resolvins and protectins have been identified. One series includes those derived from EPA and DHA via lipoxygenase metabolism, referred to as the Sresolvins, S-protectins, and S-maresins. The second series includes those derived from aspirin-triggered cyclooxygenase (COX-2) or Cytochrome P450 metabolism of EPA and DHA. These lipid mediators are R-resolvins and R-protectins also known as aspirin-triggered resolvins/ protectins. This specialized pathway is transcellular, in which different cells start and end the metabolic pathway. [22][23][24] For example, lipoxin A 4 is a SPM that determines the extent of granulocyte accumulation and activation during inflammation. Lipoxin A 4 formation is achieved by the transcellular biosynthesis of two sequential oxygenation reactions of ARA catalyzed by LOX in interacting cell types. One of these cells is typically a neutrophil, eosinophil, or macrophage and the other is an endothelial, epithelial, parenchymal cell, or platelet. 25 Cell culture and animal studies have shown these metabolites to be anti-inflammatory and inflammation resolving. For example, resolvin E1, resolvin D1, and protectin D1 all inhibited transendothelial migration of neutrophils, preventing the infiltration of neutrophils into sites of inflammation; resolvin D1 inhibited interleukin-1β (IL-1β) production; and protectin D1 inhibited tumor necrosis factor (TNF) and IL-1β production. [22][23][24] SPMs have been shown to influence the adaptive immune system as well. For example, D-series resolvins and maresin 1 reduced cytokine production by activated CD8 + T cells and CD4 + T helper (Th)1 and Th17 cells while F I G U R E 2 Overview of the key immunomodulatory effects of PUFAs. Free ω-3 fatty acids (EPA and DHA) compete with free ω-6 fatty acids (e.g. ARA) for cell membrane insertion and metabolism by COX, LOX, and Cytochrome P450 enzymes. Metabolism of ω-3 and ω-6 PUFA results in inflammatory or anti-inflammatory eicosanoids respectively while metabolism of primarily ω-3 PUFA can result in SPMs (resolvins, protectins, and maresins). ω-3 PUFA in the cell membrane can disrupt lipid rafts housing receptors such as TLR-4 and prevent inflammatory stimulation. ω-3 PUFA, SPMs, and eicosanoids of ω-3 PUFA can bind to and regulate PPAR-γ, which subsequently binds and interferes with the translocation of NFκβ to the nucleus. ω-3 PUFA can also act in an anti-inflammatory manner by agonizing GPR120, which causes signal interference with the NFκβ pathway. The figure was created with BioRender.com. ARA, arachidonic acid; COX, cyclooxygenase; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; LOX, lipoxygenase; NFκβ, nuclear factor κβ; PPAR-γ, peroxisome proliferator activated receptor gamma; PUFAs, polyunsaturated fatty acids; SPMs, short-chain fatty acids simultaneously preventing naïve CD4 + T-cell differentiation into Th1 and Th17. 26 SPMs also influence the intracellular production and extracellular release of interferon-γ (IFN-γ) from Th1 cells and IL-17 from Th17 cells. Splenic T cells from mice deficient for elongase 2, the key enzyme involved in the synthesis of DHA from EPA, produced higher amounts of IFN-γ and IL-17 compared to cells from wild-type control mice. 26  generation and function of FoxP3 + regulatory T cells (T reg ) in the presence of D-series resolvins and maresin 1 is enhanced. 26 T reg cultured with SPMs showed significantly enhanced FoxP3 expression, as well as increased expression of the immune checkpoint molecule CTLA-4 and higher production of the anti-inflammatory IL-10 cytokine. Diet enrichment of EPA in mice, 27 as well as humans, 28 increased the concentration of resolvins in the blood and serum. Furthermore, resolvins reduced inflammation in a rat arthritis model. 29 Frequent administration of the precursor of aspirin-triggered resolvin D1 prevented joint stiffness but did not modify paw and joint edema in rats. 29 Transgenic mice expressing fat-1, a gene encoding an enzyme that converts ω-6 to ω-3 PUFA, showed significant improvement in a collagen induced arthritis mouse model. 30 Clinical arthritis score, inflammatory cell infiltration, and inflammatory cytokine expression in the spleen and ankle were attenuated while T reg expansion and differentiation was enhanced in fat-1 mice compared to wild-type mice.

| Agonist of GPR120
A third proposed mechanism of PUFA regulation of NFκβ involves GPR120, a GPCR expressed on macrophages. 50 ω-3 PUFAs, agonists of GPR120, inhibited the macrophage response to endotoxin, an effect which involved maintenance of cytosolic inhibitor of nuclear factor kappa B (Iκβ) and a decrease in production of TNF and IL-6, suggesting that GPR120 is involved in anti-inflammatory signaling. 50 Iκβ binds to NF-κβ to form an inactive complex where the common pathway for NF-κβ activation is based on phosphorylation-induced, proteasome-mediated degradation of Iκβ. 51 Therefore, a maintenance of cytosolic Iκβ regulates activation of NF-κβ. The effects produced by the synthetic agonist were similar to those produced by DHA and EPA. 50 It was observed that both EPA and DHA, but not ARA enhanced GPR120-mediated gene activation. 50 Moreover, the ability of DHA to inhibit the responsiveness of macrophages to endotoxin was eliminated in GPR120 knockdown cells. These data support that the inhibitory effect of DHA on NFκβ might occur via GPR120 due to signal interference with the pathway that activates NFκβ.

| SHORT-CHAIN FATTY ACIDS
SCFAs are carboxylic acids and are one to six carbon atoms in length.
Acetate (two carbons), propionate (three carbons), and butyrate (four . AAI was not mitigated in GPR41 À/À mice treated with propionate after house dust mite exposure, but the same treatment ameliorated AAI in wild-type and GPR43 À/À mice. 68 Neutrophilassociated GPR43 has been shown to have a role in the recruitment of polymorphonuclear leukocyte to the site of inflammation, likely in a protein kinase p38α-dependent manner. 69 GPR43 activation has also been shown to induce chemotaxis of neutrophils in vitro. 70  inflammasome and enhances the production of IL-18, a critical component for maintaining epithelial integrity and intestinal homeostasis. 71 As with ω-3 PUFA derivatives, SCFA suppress cell adhesion molecules expressed by activated monocytes, neutrophils, and Overview of the mechanisms of SCFA-mediated immune modulation. SCFAs are produced by anaerobic metabolism of dietary fiber in the gut by bacteria. These two-carbon (acetate), three-carbon (propionate), and four-carbon (butyrate) metabolic products influence the immune system by two modes of action, GPCR activation and HDAC inhibition. SCFAs modulate the innate immune system by activating GPR41, GPR43, and GPR109a, which are expressed on cells such as monocytes, neutrophils, and macrophages. SCFAs modulate the adaptive immune system by inhibiting HDAC, leading to modulation of the mTOR pathway, subsequently modifying the ratio of effector to regulatory T cells. The figure was created with BioRender.com. GPCR, G-protein-coupled cell membrane receptor; HDAC, histone deacetylase; mTOR, mammalian target of rapamycin; SCFA, short-chain fatty acids endothelial cells, mitigating the infiltration of inflammatory immune cells. [72][73][74] Acetate inhibits LPS-stimulated TNF production by peripheral blood mononuclear cells in both mice and humans via GPR43. 75 GPR43 activation has been demonstrated to induce the differentiation and enhance the function of FoxP3 + T regs through epigenetic modulation. 76 3.2 | SCFA-mediated adaptive immune cell modulation by histone deacetylase inhibition which can be acetylated at lysine 516 by HDAC inhibitors (e.g., SCFAs) and coactivator p300. 59 S6K phosphorylates ribosomal protein 6 (rS6), which is an important target in the mammalian target of rapamycin (mTOR) pathway and is a key metabolic pathway in T cells. In general, mTOR activity promotes effector T cells at high levels and promotes T reg at low levels. Increased phosphorylation of rS6 was observed under acetate and propionate supplementation. 77 Therefore, SCFAs can increase mTOR activity and support the generation of both effector and T reg .
It has also been demonstrated that SCFA promote the function of T regs . 76 SCFAs increase the activity of FoxP3 + T cells and IL-10 production via HDAC inhibition, which regulates gene expression of the (TCR)β À/À Tcrδ À/À mice, and NOD-scid IL2Rgamma null (NSG) mice grafted with purified B cells. 83 These effects were extended to autoantibody responses in lupus-prone MRL/Fas lpr/lpr and NZB/W F1 mice, 83 supporting a potential role of SCFA in systemic lupus erythematosus therapy.

| Therapeutic role of SCFAs in autoimmune diseases
SCFAs have been demonstrated to be immune modulating in a range of autoimmune diseases, particularly those that affect the gut. In active IBDs including Crohn's disease (CD), and ulcerative colitis (UC), SCFAproducing bacteria (particularly those of phylum Firmicutes) are reduced, leading to dysbiosis. In particular, a decrease in Firmicutes prausnitzii, a butyrate producing bacteria from the Clostridium cluster IV, is an indicator of IBD. 84,85 Dysbiosis results in a decreased amount of SCFAs in the fecal matter of patients with IBD. 77 One study found that acetate and propionate, but not butyrate, are reduced in UC patients 86 while a separate study found both butyrate and propionate to be reduced in IBD patients. 87 The apparent differences might be attributed to dietary differences and the method of analysis. 86 In a rat sepsis model, butyrate prevented liver, kidney, and lung damage, thereby improving the survival rates. 90 The increase in survival rate was shown to be caused by downregulation of high-mobility group box protein 1 (HMGB1) by butyrate supplementation, which is a pro-inflammatory cytokine that activates multiple membrane receptors, including receptors for advanced glycation end products 91  SCFAs have the potential to exacerbate inflammation. For example, it has been documented that chronic elevation of SCFA higher than physiological levels can cause T-cell-induced inflammatory responses in the renal system. 80 In addition, in mouse models of colonic inflammation, the therapeutic effect of SCFA has been inconsistent and may be context dependent. For example, SCFA enemas did not prevent or reduce intestinal damage in 2,4,6-trinitrobenzene sulfonic-acid (TNBS)induced colitis in rats 95 but reduced colonic mucosal damage and serum inflammatory cytokines (IL-6, TNF, and IL-1β) in dextran sodium sulfate (DSS)-treated mice. 96 In contrast, butyrate did not prevent DSSinduced intestinal damage in mice exposed to antibiotics. 97  preparations. Therefore, subunit vaccine development often relies on adjuvants, which are molecular components that can safely boost the immunogenicity of the vaccine. Since its initial use in the 1920s, insoluble aluminum salts (alum) remained the only adjuvant in licensed vaccines for several decades. 99 As the overall effectiveness of an immunization regimen can be improved by adjuvanting the vaccine formulation, the development of new adjuvants is an important focus in vaccines formulations. 100 In particular, an evolving understanding of the relationship between lipids and antigen presenting cells has made it possible to design safe lipid adjuvants with strong and robust immune stimulating effects (Figure 4).

| Free lipids as vaccine adjuvants
It is known that free cationic lipids, such as those with quaternary ammonium head groups, disrupt cell monolayers and can cause hemo-   115 Despite its low viral content, Inflexal ® imparts a robust immune response compared to conventional influenza vaccines. 116,117 Virosomes are also being further explored for malaria and respiratory syncytial virus vaccines. 118

| Oil-in-water nanoemulsions
MF59 is a safe and effective vaccine adjuvant, formulated as an oil-inwater nanoemulsion of squalene that produces approximately 160-nm-sized droplets, 125  showed that the cellular influx observed in CCR2 +/+ mice was significantly different than the influx found with CCR2 À/À mice, supporting the theory that cellular recruitment is the mode of action of MF59. 129 Another study showed that mice deficient in ICAM-1 showed significantly lower antibody titers against a Plasmodium falciparum vaccine than did wild type controls. 130 This ICAM-1 dependent immune response was found to be a specific mechanism for emulsion adjuvants such as MF59 but not for immune potentiator adjuvants such as MPLA. In vitro studies with MF59 found that rather than DCs being the target of this adjuvant, monocytes, macrophages, and granulocytes were targeted by MF59. 131 Accordingly, it was postulated that a key component of the mechanism of MF59 was chemokine-driven immune cell recruitment and chemokine-release that would create a positive feedback loop, strongly enhancing the numbers of immune cells at the injection site. These cells could then further participate in antigen uptake and transport to the draining lymph nodes. AS03 ® is another squalene, oil-in-water emulsion adjuvant that was approved for use as an emergency pandemic adjuvant for influenza by the European Medicines Agency. AS03 ® was more effective than alum in inducing a high antigen-specific antibody response and induced higher levels of cytokines and stimulated more monocyte and granulocyte recruitment to the draining lymph nodes than aluminum hydroxide. 132

| CONCLUSIONS
Lipids are important mediators of immune homeostasis and may be leveraged for treating a range of disorders. Their full potential as stand-alone immunomodulators or adjuvants is only now beginning to be realized. In this review, we focused on three classes of lipids-two  The immunomodulatory effect of PUFA has been assessed solely based on diet supplementation. 17,134,135 Unlike SCFA, the immune modulating properties of PUFA are not due to the PUFA themselves, but rather from the eicosanoids derived from PUFA metabolism. [11][12][13] Therefore, an additional consideration is the metabolism of PUFA in target tissues. Reflecting this complexity, clinical trials using ω-3 PUFA have garnered mixed results. For example, dietary supplementation of ω-3 PUFA has been extensively studied in the context of cardiovascular disease. 136 (6)