8.1  The anti-inflammatory properties of n-3 PUFAs
As mentioned earlier, an exacerbated immune system response and uncontrolled inflammation are fundamental mechanisms in the development of cardiovascular impairment in patients with COVID-19. Accordingly, a plethora of experimental studies and clinical trials demonstrate that targeting different inflammatory components may be considered promising strategies to control cardiovascular impairment during the acute and remission phases of COVID-19 (Fig. 3 ).
Fig. 3 A summary of the anti-inflammatory mechanisms of n-3 PUFAs. (A) N-3 PUFAs can regulate expression of inflammatory cytokines, chemokines and adhesion molecules, inhibit NLRP3 inflammasomes, activate anti-inflammatory transcription factors (PPARα/γ) and activate GPR120 receptors which inhibit TLR4-mediated activation of NF-κB. (B) N-3 PUFAs are metabolized by COX/5-LOX into 5-series LTs which exert anti-inflammatory effects. (C) N-3 PUFAs can replace n-6 PUFAs, such as AA, altering the inflammatory response. N-3 PUFA will alter cell membrane composition, fluidity and mediated signaling. (D) N-3 PUFAs, DHA and EPA, are metabolized by CYP epoxygenases into bioactive epoxylipids with anti-inflammatory properties. (E) N-3 PUFAs are metabolized by COX/LOX into SPMs which act as potent anti-inflammatory modulators. AA, Arachidonic acid; CCL, Chemokine ligand; COX, Cyclooxygenase; CYP, Cytochrome P450; DHA, Docosahexaenoic acid; EDP, Epoxydocosapentaenoic acid; EEQ, Epoxyeicosatetraenoic acid; EPA, Eicosapentaenoic acid; GRP, G-protein coupled receptor; IL, Interleukin; LOX, Lipoxygenase; LT, Leukotriene; PUFA, Poly unsaturated fatty acid; NFκB, Nuclear factor kappa-light-chain enhancer activated B-cells; NLRP3, NACHT, LRR and PYD domains-containing protein 3; PLA2, Phospholipase A2; PMN, Polymorphonuclear neutrophils; PPAR, Peroxisome proliferator-activated receptor; ROS, Reactive oxygen species; SPMs, Specialized pro-resolving mediators; TLR, Toll like receptor; TNF-α, Tumor necrosis factor-α.

8.1.1  N-3 PUFAs regulate the expression of several proinflammatory innate immune components and modulate macrophage response
A ‘cytokine storm’ and activation of the central innate immune pathway linking the NLRP3 inflammasome, IL-1β, TNF-α and IL-6 response is a primary cause of excessive inflammation reported in COVID-19 that negatively impacts cardiovascular system. Therefore, targeting the different components is a promising approach to ameliorate cardiac complications secondary to COVID-19 (Huang et al., 2020). While there is no direct clinical evidence related to the use of n-3 PUFAs in COVID-19 patients, the application of n-3 PUFAs in several inflammatory settings, including cardiovascular disorders, has been demonstrated to ameliorate detrimental immune reactions by several mechanisms (Rogero et al., 2020). The anti-inflammatory effect of n-3 PUFAs seems to be consistent across several previous clinical findings (Calder, Carr, Gombart, & Eggersdorfer, 2020; Fritsche, 2006; Kiecolt-Glaser et al., 2012; Vedin et al., 2008). Intriguingly, Tan et al. recently demonstrated in a randomized controlled study that high-dose n-3 PUFA supplementation (1.5 g/day EPA and 1.0 g/day DHA) markedly reduces plasma levels of IL-6, IL-1β and TNF-α after 4 weeks of therapy in middle or late-aged patients with chronic venous leg ulcers suggesting n-3 PUFAs as an effective low-risk dietary intervention to modulate inflammation (Tan, Sullenbarger, Prakash, & McDaniel, 2018). This study indicates that n-3 PUFAs could have direct modulatory effects on the main components of the cytokine storm IL-6, IL-1β and TNF-α.
N-3 PUFAs can modulate the transcription and expression of inflammatory genes including cytokines, chemokines and adhesion molecules in cardiomyocytes, fibroblasts, endothelial cells, monocytes and macrophages (Collie-Duguid & Wahle, 1996; De Caterina, Cybulsky, Clinton, Gimbrone, & Libby, 1994; Hughes, Southon, & Pinder, 1996; Miles, Wallace, & Calder, 2000; Sanderson & Calder, 1998). This is primarily achieved through the regulation of key transcription factors, such as inhibiting NF-κB (Kumar, Takada, Boriek, & Aggarwal, 2004; Lo, Chiu, Fu, Lo, & Helton, 1999; Novak, Babcock, Jho, Helton, & Espat, 2003; Zhao, Joshi-Barve, Barve, & Chen, 2004) or activating peroxisome proliferator-activated receptors-α/γ (PPARα/γ) (Gani & Sylte, 2008; Zapata-Gonzalez et al., 2008). Activation of PPARα/γ can directly interfere with the activation of NF-κB and prevent its shuttling to the nucleus reducing the inflammatory burst (Matsumoto et al., 2008; Mishra, Chaudhary, & Sethi, 2004; Poynter & Daynes, 1998; Ricote, Huang, Welch, & Glass, 1999; Vanden Berghe et al., 2003). Interestingly, direct activation of PPAR, using PPAR agonists, was proposed as a therapeutic target for blunting and regulating cytokine storm in COVID-19 patients suggesting n-3 PUFAs could have a promising effect (Ciavarella, Motta, Valente, & Pasquinelli, 2020). Another important immunomodulatory mechanism induced by n-3 PUFAs involves activation of G protein-coupled receptor 120 (GPR120), which mediates strong and wide-ranging anti-inflammatory effects. Research from Oh et al. indicates n-3 PUFAs stimulate GPR120 in both monocytic RAW 264.7 cells and primary intraperitoneal macrophages inhibiting TLR4-mediated inflammatory responses. Knockdown of GPR120 attenuates the protective effects attributed to n-3 PUFA consumption (Oh et al., 2010). These studies together provide evidence that n-3 PUFAs mediate anti-inflammatory effects through different mechanistic pathways.
Cardiac macrophages are primarily derived and replenished from inflammatory monocytes in response to an infection with resident macrophages also having a role. Briefly, macrophages will differentiate into classical M1 inflammatory cells to clean cellular and matrix debris (Epelman et al., 2014). Subsequently, M1 macrophages may undergo polarization and transformation to the alternatively activated or reparatory M2 stage which secrete IL-10 to promote resolution and contribute to wound healing and tissue repair (Murray, 2017). Controlling the migration and the polarization of macrophages to the myocardium in the context of COVID-19 is a tentative approach to limit cardiac injury (Frantz & Nahrendorf, 2014; Fujiu, Wang, & Nagai, 2014; Leblond et al., 2015; van Amerongen, Harmsen, van Rooijen, Petersen, & van Luyn, 2007). In COVID-19, an excessive cardiac recruitment and accumulation of pro-inflammatory M1 macrophages potentially aggravates cardiovascular injury. Notably, as M1 macrophages secrete a large variety of chemokines and cytokines such as TNF-α and IL-1β to recruit and activate other immune cells from both the innate and the adaptive immune system. The effect will impede the reparative phase mediated by M2 macrophages and thus aggravates adverse cardiac remodeling (Dewald et al., 2005; Gordon, Pluddemann, & Martinez Estrada, 2014; Murray & Wynn, 2011; ter Horst et al., 2015).
Interestingly, evidence demonstrates n-3 PUFAs and/or their biologically active metabolites have the ability to blunt the expression, production and release of IL-1β, TNF-α, and IL-6 by M1 macrophages (Allam-Ndoul, Guenard, Barbier, & Vohl, 2017; Liu et al., 2014; Mildenberger et al., 2017). Schoeniger et al., showed n-3 PUFAs have the ability to down-regulate inflammatory processes and reduce the production and secretion of pro-inflammatory cytokines from RAW 264.7 macrophages infected with microorganisms, R. equi and P. aeruginosa (Schoeniger, Adolph, Fuhrmann, & Schumann, 2011). Moreover, the inhibitory effects of EPA and DHA on the pro-inflammatory NLRP3 inflammasome pathway has also been well-documented in macrophage cell lines as well as in primary human and mouse macrophages (Iverson et al., 2018; Kumar et al., 2016). Kumar et al., investigated the effects of 15-lipoxygenase (LOX) metabolites of ALA on lipopolysaccharide (LPS) -induced inflammation in RAW 264.7 cells and peritoneal macrophages. The findings revealed the anti-inflammatory effects of these metabolites involve inactivation of the NLRP3 inflammasome complex through the PPAR-γ pathway (Kumar et al., 2016). N-3 PUFAs can increase the phagocytic capacity of macrophages, which has been shown through the engulfment of zymosan particles (Chang, Lee, Kim, & Surh, 2015), Pseudomonas aeruginosa, Rhodococcus equi (Adolph, Fuhrmann, & Schumann, 2012), E.coli (Davidson, Kerr, Guy, & Rotondo, 1998) and apoptotic cells (Chang et al., 2015). It has been suggested the increase in phagocytic capacity of macrophages upon n-3 PUFA treatment could be attributed to changes in the cellular membrane composition and structure caused by the incorporation of the n-3 PUFAs (Hellwing, Tigistu-Sahle, Fuhrmann, Kakela, & Schumann, 2018; Schoeniger, Fuhrmann, & Schumann, 2016). Importantly, n-3 PUFAs have been found to promote M2 polarization in macrophage cell lines and primary mouse macrophages enhancing resolution of inflammation and tissue repair after infection (Chang et al., 2015; Ohue-Kitano et al., 2018). Collectively, the modulatory properties of n-3 PUFAs on the immune system could impart a promising beneficial effect on the cardiovascular system in the context of COVID-19, an effect which needs further exploration and confirmation in larger clinical trials.

8.1.2  Shifting to the anti-inflammatory COX- and LOX-derived metabolites of n-3 PUFAs
Accumulating literature demonstrates potent immunomodulatory properties of metabolites generated from n-3 PUFAs and consequently their impact on cardiovascular health (Jamieson, Endo, Darwesh, Samokhvalov, & Seubert, 2017; Schunck, Konkel, Fischer, & Weylandt, 2018). The metabolism of n-3 and n-6 PUFAs is closely interconnected as parent compounds compete for the same metabolic enzymes but result in the production of a wide array of either pro- or anti-inflammatory metabolites. For example, cyclooxygenase (COX) converts the n-6 PUFA arachidonic acid (AA) to the 2-series of prostaglandins (PGs) and the 2-series of thromboxanes (TX), while lipoxygenase (LOX) enzymes metabolize AA to the 4-series leukotrienes (LTs) and the hydroxyicosatetraenoic acids. These lipid mediators are considered pro-inflammatory and are involved in various pathological processes including cardiovascular disorders (Innes & Calder, 2018; Kalinski, 2012; Lewis, Austen, & Soberman, 1990). The synthesis and production of PGE2 occurs in several cells, including dendritic cells, macrophages, fibroblasts and endothelial cells. PGE2 not only mediates vasodilation, endothelial permeability and increase of pain (Ricciotti & FitzGerald, 2011) but also contributes to the tissue influx of neutrophils, mast cells and macrophages and can affect the differentiation of these cells (Kalinski, 2012).
N-3 PUFAs can also act as a substrate for COX and 5-LOX enzymes resulting in production of the 3-series of PGs and TxAs as well as 5-series LTs, which are a set of less inflammatory or even anti-inflammatory metabolites in comparison to the metabolite family derived from AA (Corey, Shih, & Cashman, 1983; Lee et al., 1984; Surette, 2008). These eicosanoids are responsible for producing several physiological responses related to inflammation, and their imbalance has been observed in several diseases (Calder, 2006; Falck et al., 2011). For example, the production of PGE2 and LTB4 by human inflammatory cells was significantly decreased in a diet rich in fish oil (Caughey, Mantzioris, Gibson, Cleland, & James, 1996; Lee et al., 1985; Prescott, 1984; von Schacky, Kiefl, Jendraschak, & Kaminski, 1993). Therefore, the metabolism of n-3 PUFAs by COX and LOX enzymes not only reduce the AA-derived pro-inflammatory metabolites but also alter the metabolic profile towards more biologically active anti-inflammatory mediators (Goldman, Pickett, & Goetzl, 1983; Lee et al., 1984; Lee, Mencia-Huerta, et al., 1984). This may represent one of the central anti-inflammatory and consequently cardioprotective mechanisms of n-3 PUFAs against cardiac complications associated with COVID-19.

8.1.3  Anti-inflammatory features of the n-3 PUFAs-derived specialized pro-resolving mediators (SPMs)
Metabolism of n-3 PUFAs also generates another group of highly specialized pro-resolving mediators (SPMs) which include resolvins ‘resolution phase interaction products’ produced from both EPA (E-series, RvE1-2) and DHA (D-series, RvD1-6) as well as protectins and maresins produced from DHA (Serhan et al., 2002; Serhan, Chiang, & Van Dyke, 2008). Both the COX and LOX pathways are involved in the synthesis of these metabolites with distinct epimers being produced in the presence and absence of aspirin (Mas, Croft, Zahra, Barden, & Mori, 2012).
SPMs possess potent anti-inflammatory and inflammation resolving properties which is essential to terminate ongoing inflammatory processes, accelerate the cleaning process and aid in tissue regeneration and wound healing allowing tissue homeostasis to return (Serhan et al., 2000; Serhan et al., 2002; Spite et al., 2009; Titos et al., 2011). Several mechanistic pathways contribute to the anti-inflammatory effects of resolvins, protectins and maresins. This includes preventing the migration of neutrophils and monocytes across epithelial cells and promoting clearance of polymorphonuclear (PMNs) leukocytes, apoptotic cells and debris from the site of inflammation (Campbell et al., 2007; Serhan et al., 2002). Krishnamoorthy et al. showed resolvins inhibit tissue migration of neutrophils by lowering the expression of surface adhesion receptors on neutrophils, such as CD11b or CD18, and reducing the production of the chemokine IL-8 (Krishnamoorthy et al., 2010). Additionally, the partial agonist/antagonist activity of RvE1 toward LTB4 receptors on PMNs will inhibit NF-κB activation, abolish pro-inflammatory cytokine production and reduce PMN leukocyte infiltration (Arita et al., 2007; Serhan et al., 2002; Serhan et al., 2008). Resolvins can blunt reactive oxygen species (ROS) production from neutrophils, induce neutrophil apoptosis and clearance by macrophages, as well contribute to inhibiting chemokine signaling (Ariel et al., 2006; Schwab, Chiang, Arita, & Serhan, 2007; Serhan & Chiang, 2004). Furthermore, Morin et al. demonstrated a diet enriched with DHA and monoglycerides can significantly increase the levels of RvD2 and RvD3, which correlate with reduced levels of proinflammatory mediators CRP, IL-6, TNF-α, and IL-1β in a rat model of hypertension (Morin, Rousseau, Blier, & Fortin, 2015). Additionally, there is growing evidence for a role of SPMs in regulating the humoral immune response. A study conducted by Ramon et al., showed 17-hydroxydocosahexaenoic acid (17-HDHA), the precursor of the D-series SPMs (RvD1, 17R-RvD1, RvD2), can reduce IL-6 secretion in human B cells, increase B cell antibody production and promote B cell differentiation to an antibody secreting cell (Ramon, Gao, Serhan, & Phipps, 2012). These new findings highlight the potential applications of SPMs as non-toxic, supportive adjuvants and as anti-inflammatory therapeutic molecules particularly during infection as in the case of COVID-19.
Resolvins, protectins and maresins play a pivotal role regulating the function of macrophages. Sulciner et al. demonstrates RvD1, RvD2 or RvE1 can inhibit debris-stimulated cancer progression by enhancing clearance of debris via macrophage phagocytosis in multiple tumors. These resolvins suppressed the release of the proinflammatory cytokines/chemokines, including TNFα, IL-6, IL-8, chemokine ligand 4, and chemokine ligand 5, by human macrophages cocultured with tumor cell debris (Sulciner et al., 2018). Maresins are conjugates of sulfides synthetized by macrophages, which are also participants in acute inflammation resolution and seem to promote tissue regeneration (Serhan et al., 2009). Maresin-1 biosynthesis involves an active intermediate (13S,14S-epoxi-DHA) that stimulates macrophage conversion from M1 (pro-inflammatory) to M2 (anti-inflammatory) phenotype (Dalli, Ramon, Norris, Colas, & Serhan, 2015). It is noteworthy that M2 macrophages secrete resolvins, protectins and maresins to dampen inflammation and restore homeostasis (Bouchery & Harris, 2017; Ramon et al., 2016) and at the same time augment phagocytic capacity of macrophages and other cells to remove debris from the site(s) of infection and injury and enhance microbial clearance (Dalli et al., 2013; Norris et al., 2018; Poorani, Bhatt, Dwarakanath, & Das, 2016).
The role of resolvins in the resolution of inflammation has been demonstrated in several animal models of ALI and ARDS (Gao et al., 2017; Uddin & Levy, 2011; Wang, Yan, Hao, & Jin, 2018; Zhang et al., 2019). These studies carried out using rat and mouse models infected with the E.coli endotoxin, LPS, suggested the pro-resolving effects of these molecules could be attributed, for example, to the suppression of neutrophil infiltration due to reduced expression and release of pro-inflammatory cytokines from alveolar macrophages (Uddin & Levy, 2011; Zhang et al., 2019). Further, it has been demonstrated protectins may reduce the replication of influenza (Morita et al., 2013) and potentially affect the inflammatory manifestations of respiratory viral diseases (Russell & Schwarze, 2014).
Importantly, pro-inflammatory cytokines, TNF-α and IL-6, will inhibit the activities of desaturases, which are essential for the generation of AA, EPA and DHA from their precursors LA and ALA (Das, 2013). Hence, in instances where there is a substantial degree of inflammation due to high levels of IL-6 and TNF-α, such as following COVID-19 infection, a deficiency of EPA and DHA and subsequent decreased generation of resolvins, protectins and maresins can occur (Das, 2018). Thus, administration of PUFAs and/or their metabolites, resolvins, protectins and maresins can suppress inappropriate production of IL-6 and TNF-α to resolve inflammation, enhance recovery and limit cytokine storm (Das, 2019) in COVID-19. Together, the studies imply administration of n-3 PUFA may enhance recovery from infections and further, if present in adequate amounts, may modulate the response to infections.

8.1.4  Role of CYP-mediated metabolites in ameliorating inflammation
CYP2J and CYP2C isoforms, the constitutively expressed cytochrome P450 (CYP) epoxygenases found in the cardiovascular system, metabolize EPA into 5 regioisomeric epoxyeicosatetraenoic acids (5,6-, 8,9-, 11,12-, 14,15-, 17,18-EEQ) and DHA into 6 regioisomeric epoxydocosapentaenoic acids (4,5-, 7,8-, 10,11-, 13,14-, 16,17-, 19,20-EDP) (Arnold et al., 2010; Konkel & Schunck, 2011; Westphal, Konkel, & Schunck, 2015). Recent evidence suggests that 17,18-EEQ and 19,20-EDP mediate several anti-inflammatory effects of n-3 PUFAs in various models of tissue injury (Arnold et al., 2010; Ulu et al., 2014; Wang, Chai, Lu, & Lee, 2011). For example, Fang et al. demonstrated a n-3 PUFA-rich diet attenuates MI injury in mice by producing a protective eicosanoid pattern, which results in shifting the metabolite profile to a more anti-inflammatory state by increasing the levels of the 19,20-EDP and 17,18-EEQ and decreasing the pro-inflammatory PGE2 (Fang et al., 2018). The cardioprotective effects of n-3 PUFAs are also attributed to their ability to attenuate the NLRP3 inflammasome complex cascade (Darwesh, Jamieson, Wang, Samokhvalov, & Seubert, 2019). Importantly, the anti-inflammatory features of CYP-derived epoxy metabolites have been reported in numerous models. For example, in TNFα-induced retinal vascular inflammation, Capozzi et al. demonstrated 19,20-EDP can ameliorate vascular adhesion molecule and intracellular adhesion molecule expression and reduce leukocyte adherence to human retinal microvascular endothelial cell monolayers (Capozzi, Hammer, McCollum, & Penn, 2016). Additionally, evidence demonstrates intraperitoneal infusions of 17,18-EEQ and 19,20-EDP protect against allergic intestinal inflammation and kidney fibrosis in corresponding mouse models (Kunisawa et al., 2015; Sharma et al., 2016). 17,18-EEQ was able to inhibit TNFα-induced inflammation in human lung tissue obtained from patients undergoing surgery for lung carcinoma via inhibition of NF-κB and activation of the transcription factor PPAR-γ (Morin, Sirois, Echave, Albadine, & Rousseau, 2010). The anti-inflammatory properties of DHA epoxides were also well demonstrated using animal models of inflammatory pain. For example, Morisseau et al. demonstrated that direct injection of the DHA epoxides, EDPs, together with the pro-inflammatory carrageenan into the paw or spinal cord of male Sprague-Dawley rats resulted in significant antihyperalgesic activity. Surprisingly, both the parent free fatty acid DHA and the corresponding diols were inactive, supporting the hypothesis that the epoxylipids mediate many of the beneficial effects of the parent compounds (Morisseau et al., 2010). The bacterial endotoxin, LPS, has a marked role in triggering inflammatory injury which can result in several cardiovascular complications. In a study using HL-1 cardiac cells, 19,20-EDP protected against LPS-stimulated inflammatory injury by activating the histone deacetylase Sirtuin-1 inhibiting the activation the pro-inflammatory transcription factor NF-κB (Samokhvalov, Jamieson, Vriend, Quan, & Seubert, 2015). The accumulating evidence suggests the anti-inflammatory properties of CYP-epoxygenase metabolites of n-3 PUFAs have a substantial role in activating protective responses in models of cardiovascular injury. However, further investigation is required to elucidate whether the protective properties limit cardiovascular injury secondary to COVID-19 infection.

8.1.5  N-3 PUFAs alter cell membrane structure and function - modulation of the lipid raft
Within a cell, n-3 PUFAs can be found incorporated into phospholipid membranes where elevating levels will replace existing n-6 PUFAs thereby altering the composition and properties of lipid rafts (Lordan et al., 2017; Lordan et al., 2020). The increased incorporation of n-3 PUFAs into membrane bilayers can have a role in mediating immunomodulatory effects by altering membrane composition, fluidity and function. These changes will impact membrane-mediated signaling, protein trafficking, generation of bioactive lipids, cytokine secretion and gene activation in both innate and adaptive immune responses. For example, a change in fluidity can interfere with the dimerization and expression of the TLR4 subunits, blocking the downstream inflammatory reaction (Ciesielska & Kwiatkowska, 2015; Takashima et al., 2016). Evidence of these effects by n-3 PUFAs have been demonstrated to impact the maturation of dendritic cells, macrophage function and T and B cell polarization/activation (Katagiri, Kiyokawa, & Fujimoto, 2001; Kim et al., 2010; McMurray, Bonilla, & Chapkin, 2011; Rockett, Salameh, Carraway, Morrison, & Shaikh, 2010; Shaikh and Edidin, 2006, Shaikh and Edidin, 2008). Interestingly, DHA appears to be better than EPA in replacing n-6 PUFAs and cholesterol in plasma membranes of aortic endothelial cells enhancing the fluidity of the phospholipid membrane (Hashimoto, Hossain, Yamasaki, Yazawa, & Masumura, 1999).
In most cell types, AA is the predominant n-6 PUFA in membrane phospholipids (Yaqoob, Pala, Cortina-Borja, Newsholme, & Calder, 2000). Inflammatory immune cells such as monocytes, neutrophils, macrophages and lymphocytes often contain a large amount of AA in their membrane. The high membrane AA composition is important during normal inflammatory responses. Under stress conditions activation of phospholipase A2 liberates AA from the cell membrane leading to metabolism and production of many pro-inflammatory metabolites (Ford, Hazen, Saffitz, & Gross, 1991; Hazen, Ford, & Gross, 1991; Leslie, 2015; Mancuso et al., 2003). Supplementation with n-3 PUFAs leads to the substitution of AA with EPA and DHA in the cell membrane which can alter immune cell reaction in response to stress stimuli by shifting the metabolic profile to less proinflammatory or even anti-inflammatory metabolite predominance (Brouard & Pascaud, 1990; Faber et al., 2011; Gibney & Hunter, 1993; Grando et al., 2009). Therefore, increasing n-3 PUFAs, such as EPA and DHA, in the phospholipids has a potential benefit of ameliorating detrimental effects during uncontrolled inflammatory responses (Lordan et al., 2020).