1.      Introduction

Hyper induction of proinflammatory cytokine production, the ‘cytokine storm’, is correlated directly with tissue injury and with unfavorable prognosis of severe infections [1]. Induced by an influenza infection, various signaling pathways within the infected cell are activated, and this activation is meant to be an adequate biological response [2]. The pathways lead to the activation of our innate immune system among which mast cells, and interferons, proinflammatory cytokines and chemokines levels rise. This innate immune response is activated in a short period of time, minutes to hours after infection, and a cascade of secreted proteins are activated and targeted against the virus, resulting in a state of inflammation. However, excessive and uncontrolled stimulation of the activated innate immune response proved to be quite harmful, especially in influenza and sepsis. ‘Cytokine storm’ modulators, such as IL-10, most promising in the beginning of this century, have not been successful in the clinic [3]. Other modulators, such sphingosine-1-phosphate analog therapy, have been tested in a number of preclinical models, but have not yet been tested in clinical trials [4]. Peroxisome Proliferators-Activated Receptor (PPAR) agonists have also been mentioned as a putative new class of anti-cytokine storm agents [5-6].

Budd et al. point out that for instance gemfibrozil, an old molecule and PPAR-alpha agonist, could reduce mortality in a mouse model of severe influenza due to its ability to inhibit a number of pro-inflammatory cytokines [7]. However, this effect could not replicate in a different model, and fib rates such as gemfibrozil and fenofibrate might have an additional negative impact, for instance impairment of mitochondrial functions [8].

PEA is combined alpha-and gamma-PPAR agonists [9]. Such mixed PPAR-agonists might hold a promise for treating ‘cytokine storm’. A natural PPAR-alpha and PPAR-gamma agonist, biochanin A, has been documented to reduce production of the pro-inflammatory cytokines TNF-α and IL-8 in an LPS-induced inflammation paradigm [10].

PEA is without any documented clinical relevant side-effects and we will review the evidence for PEA as a putative ‘cytokine storm’ modulating agent. Since one of the classical mechanisms of action discovered for PEA is via the modulation of the mast cell, it seems relevant to point out that very recently it was demonstrated that mast cells play a significant role in the pathogenesis of such ‘cytokine storm’ [11]. During the ‘cytokine storm’ synthesis and secretion is stimulated of great number of chemo tactic (RANTES, GM-CSF, MIP-1 alpha, MCP 1, MCP-3, and IP-10), pro-inflammatory (IL-1 beta, IL-6, IL-18, NGF and TNF-alpha), and antiviral (IFN alpha/beta) cytokines. In response to the initial increase of pro-inflammatory proteins, immune competent cells are activated, and recruited to sites of infection. This excess activity contributes further to the pathology induced by influenza viruses, and cause an excessive infiltration of the tissues by immune cells and the micro environment becomes flooded with pro-inflammatory molecules [12]. One of the reasons why the 1918 flu was so aggressive has been postulated to be based on the fact that the 1918 virus may have selectively attenuated the expression of specific innate-response genes [13]. This altered expression lead to extensive damage to the lungs with acute, focal bronchitis and alveoli is, massive pulmonary edema, hemorrhage and rapid destruction of the respiratory epithelium [14]. One of the reasons why people were so susceptible to the 2009 new mutate of H₁N₁ influenza also related to such a lack of appropriate immunity. The influenza H₁N₁ 2009 infection triggered a massive inflammatory response leading to fever, or other tissue damage, eventually leading to organ failure and death [15]. Such pathology is induced by the inflammatory mechanisms initiated by such infections spinning rapidly out of control.

Initial attempts to modulate pathological cytokine triggered immune cascades utilizing corticosteroids were disappointing, with an overall increase in mortality [16]. New immune modulators not compromising the host immunity while modulating the excess reactions of the innate immune system are therefore are greatly needed [11]. PEA can act as such an immune modulator, tempering the overactive inflammatory cascade and thus reducing collateral damage, and his may leads to quicker recovery and less severity of symptoms [17]. We will discuss and review the data related to PEA’s role as an anti-inflammatory agent and its putative relevance in the treatment of severe influenza and other infections, especially there were cytokine storm is to be expected, and in inadequate inflammatory responses.

2.      PEA’s Activity in Various Inflammatory Paradigms: Initial Findings

After several investigators had indicated that extracts of egg yolk, peanut oil and soybean lecithin have inflammatory activity, purification of these substances was achieved and identified as N (2-hydroxyethyl) palmitamide, now called Palmitoyl Ethanol Amide (PEA) [18]. In 1957 Kuhl et al tested this PEA in a passive joint anaphylaxis assay in the guinea pig [18]. This passively sensitizing the guinea pig knee joints leads to inflammation and swelling, and PEA could counteract the swelling. This was the reason for the authors to state that PEA is a natural occurring anti-inflammatory agent [18]. In 1959, more specific details regarding the anti inflammatory action of PEA were discussed by Ganley et al from the Merck Institute for Therapeutic Research [19]. They assessed the mortality in mice after an anaphylaxis test with intra peritoneally injected killed smooth Bordetella pertussis cells, and found PEA, even in low dose, to increase survival from 20% (untreated) to 70% in the high dose group. The effect was comparable to the rescue data with high dose hydrocortisone [19].                                                

In the beginning of the 70s the modifying effects of PEA on immunological reactions were well established [20]. Perlik et al. summarized: "It has been shown that N-(2-hydroxyethyl)-palmitamide (PEA) can decrease the intensity of several inflammatory and immunological processes." [21]. This clearly was a premonition, as ‘cytokine storm’ was only first described in 1981 [22].

In the period between 1972 and 1977 in total 3627 patients and volunteers completed 6 different placebo-controlled double-blind trials of which 1937 received PEA up to 1800 mg/day. Relevant side effects were not reported and especially the trials conducted during the flu season demonstrated a treatment, as well as a prophylactic effect. The last study in children was not significant due to the fact that during the study period no influenza epidemic occurred. The results can be seen in Table 1 [24].

However, the mechanism of action of PEA remained unknown, and in spite of much speculations, it remained an enigma till in the 90s of last century Nobel laureate Rita Levi-Montalcini and her co-workers proved PEA to be an anti-inflammatory agent. This was based on its properties to inhibit overactive mast cells and reduce the secretion of pro-inflammatory proteins such as NGF and TNF-alpha [24,25].

3.     PEA: A Pleitropic Agent, Multiple Mechanisms Relevant for Treating Infections?

Following the initial discovery by Levi-Montalcini that PEA acted as an autacoids mast cell modulator in 1993 [26], many experiments were conducted and her findings were duplicated in a variety of in vitro and in vivo paradigms [27-31]. Furthermore, activated mast cells have been described since as important pathogenetical factors in a number of inflammatory and auto-immune disorders [32]. Mast cells amplify immune responses via a number of different mechanisms [33]. Mast cells also seem to contribute to lung pathology during infections [34]. Moreover, in nearly all infection these potent immuno modulatory cells play an important pathogenetical role [32]. This also holds true for viral lung infections: human H₁N₁, H₃N₂, and influenza B virus isolates activate mast cells in vitro [35]. Mast cells enhance lung injury that results from H₅N₁ infection by releasing proinflammatory mediators, including histamine, tryptase, and gamma interferon (IFN-γ) [36].

Already in 1996 it was demonstrated that PEA can inhibit excitotoxic neuronal injury, and such injury is intimately connected to enhanced markers of inflammation [25]. Levi-Montalcini and her group published the results of a series of experiments with pure PEA and concluded that by providing the cells with exogenous PEA: “one might be making available quantities of its physiological modulator sufficient to restore cellular homeostasis in the face of an excitotoxic challenge [37].”Since the discovery of PEA as a mast cell modulator and an inhibitor of injury-induced damage, many new mechanisms have been discovered (Figure 1).

; IKK/NF-kappa B: I kappa B kinase/NF-kappa B signaling pathways; IL-1b: Interleukin-1 beta; iNOS: inducible Nitric Oxide Syntheses; NOS: Nitric Oxide Syntheses; PPAR: Peroxisome Proliferators Activated Receptor; S100B: S100 calcium binding protein B; TLR4: Toll-Like Receptor 4; TNF-𝛼: Tumor Necrosis Factor-𝛼.

We will discuss briefly some of these, in relation to influenza and cytokine storm and to the mechanisms of action of PEA: the Toll like receptors and the PPAR receptors.

4. PEA, Toll-Like Receptors and PPAR Receptors

Toll Like Receptors (TLRs) are key molecules that alert the immune system to the presence of microbial infections [38]. TLRs are single, membrane-spanning receptors expressed in immune cell such as macrophages, dendritic cells, mast cells and T-cells, that recognize structurally conserved molecules derived from micro-organisms. The acute lung injury caused by infections is secondary to the generation of host-derived, oxidized phospholipids such as oxidized 1-palmitoyl-2-arachidonoyl-phosphaticylcholine potently stimulating Toll-Like Receptor 4 (TLR4)-dependent inflammation [39]. Furthermore, TLR4−/−mice are highly refractory to influenza-induced lethality [40]. The Toll Like Receptor 4 (TLR4) ligand fimbriae H protein (Fim H) has been associated with the induction of the innate antiviral responses in the lung leading to protection against lethal influenza infection in mice [1]. New chemical entities and TLR-4 antagonists Eritoran (E5564) (Eisai, Inc.), an extremely potent TLR-4 antagonist, is highly protective when administered therapeutically to mice infected with a lethal dose of influenza [41].

In a model for chronic colon inflammation PEA treatment dose-dependently via the TLR4/PPAR-alpha mechanism improved all macroscopic signs of colitis ulcerous and decreases significantly the expression and release of all the proinflammatory markers tested: iNOS, COX2, S100B and GFAP protein expression [42]. In that study PEA, dose-dependently, ameliorated colitis in wild-type mice and PEA decreases enteric activation and inflammatory markers expression and release in both the colitis model in mice as well as in human colitis samples. PEA also reduces macrophage and neutrophil infiltration in both experimental mouse colitis model as well as human colitis samples. The authors defined PEA as a new pharmaceutical tool in inflammation, due to fact that PEA could counteract mucosal immune cells infiltration, enteric abnormal activation and inhibit the release of proinflammatory mediators during colitis. As PEA is a pleitropic molecule, its anti-viral and immune-modulating properties might be related to other mechanisms of action [43]. For instance influenza virus activates the cellular IKK/NF-kappa B [44] signaling pathway for replication, and recently it has been suggested that this pathway may be a suitable target for antiviral intervention. Ptors cross-talk and PEA as a PPAR-alpha agonist can amelioration oxidative/nitrosative stress induced by NF-kappa B [45] and inhibit many proinflammatory genes [46].

5. Conclusion

Over 600 papers have been referenced in Pub Med in the last 50 years describing PEA’s anti-inflammatory profile. PEA has been tested in a variety of animal models for infections and inflammation since 1957, and the results are concordant. PEA was also tested positive for the treatment and the prophylaxis of influenza and common cold in 5 clinical placebo controlled clinical trials. PEA is an endogenous compound, available as a food supplement and has a dual action in infections. It may modulate hyperactive immune reactions and mitigates ‘cytokine storm’ most probably via its role as a PPAR-alpha agonist.

Figure 1: Main and downstream targets of Palmitoyl ethanol amide: CCL4: Chemokines (C-C motif) Ligand 4; COX2: Cyclo OXygenase-2; GFAP: Glial Fibrillary Acidic Protein; GPR: G Protein-coupled Receptor 

1. Hawiger J, Veach RA, Zienkiewicz J (2015) New paradigms in sepsis: from prevention to protection of failing microcirculation. J Thromb Haemost 13: 1743-1756.
2. Goraya MU, Wang S, Munir M, Chen JL (2015) Induction of innate immunity and its perturbation by influenza viruses. Protein Cell 6: 712-721.
3. Asadullah K, Sterry W, Volk HD (2003) Interleukin-10 therapy, review of a new approach. Pharmacol Rev 55: 241-269.
4. Walsh KB, Teijaro JR, Rosen H, Oldstone MB (2011) Quelling the storm: utilization of sphingosine-1-phosphate receptor signaling to ameliorate influenza virus-induced cytokine storm. Immunol Res 51: 15-25.
5. Kahlich R, Klíma J, Cihla F, Franková V, Masek K, et al. (1979) Studies on prophylactic efficacy of N-2-hydroxyethyl palmitamide (Impulsin) in acute respiratory infections. Serologically controlled field trials. J Hyg Epidemiol Microbiol Immunol 23:11-24.
6. Tisoncik JR, Korth MJ, Simmons CP, Farrar J, Martin TR, et al. (2012) Into the eye of the cytokine storm. Microbiol Mol Biol Rev 76: 16-32.
7. Budd A, Alleva L, Alsharifi M, Koskinen A, Smythe V, et al. (2007) Increased survival after gemfibrozil treatment of severe mouse influenza. Antimicrob Agents Chemother 51: 2965-2968.
8. Brunmair B, Lest A, Staniek K, Gras F, Scharf N, et al. (2004) Fenofibrate impairs rat mitochondrial function by inhibition of respiratory complex I. J Pharmacol Exp Ther 311: 109-114.
9. Park MH, Park JY, Lee HJ, Kim DH, Chung KW, et al. (2013) The novel PPAR α/γ dual agonist MHY 966 modulates UVB-induced skin inflammation by inhibiting NF-κB activity. PLoS One 8: e76820.
10. Sithisarn P, Michaelis M, Schubert-Zsilavecz M, Cinatl J Jr (2013) Differential antiviral and anti-inflammatory mechanisms of the flavonoids biochanin A and baicalein in H5N1 influenza A virus-infected cells. Antiviral Res 97: 41-48.
11. Liu Q, Zhou YH, Yang ZQ (2015) The cytokine storm of severe influenza and development of immunomodulatory therapy. Cell MolImmunol 1-8.
12. Rao KN, Brown MA (2008) Mast cells: multifaceted immune cells with diverse roles in health and disease. Ann NY Acad Sci 1143: 83-104.
13. Kash JC, Tumpey TM, Proll SC, Carter V, Perwitasari O, et al. (2006) Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus. Nature 443: 578-581.
14. Kobasa D, Jones SM, Shinya K, Kash JC, Copps J, et al. (2007) Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature 445: 319-323.
15. Liu Y, Chen H, Sun Y, Chen F (2012) Antiviral role of Toll-like receptors and cytokines against the new 2009 H1N1 virus infection. MolBiol Rep 39: 1163-1172.
16. Cronin L, Cook DJ, Carlet J, Heyland DK, King D, et al. (1995) Corticosteroid treatment for sepsis: a critical appraisal and meta-analysis of the literature. Crit Care Med 23:1430-1439.
17. Keppel Hesselink JM, Kopsky DJ, Witkamp RF (2014) Palmitoylethanolamide (PEA)-‘promiscuous’ anti-inflammatory and analgesic molecule at the interface between nutrition and pharma. Pharma Nutrition 1:19-25.
18. Kuehl FA, Jacob TA, Ganley OH, Ormond RE, Meisinger MAP (1957) The identification of N-(2-hydroxyethyl)-palmitide as a naturally occuring anti-inflammatory agent. J Am Chem Soc 79: 5577-5578.
19. Ganley OH, Robinson HJ (1959) Antianaphylactic and antiserotonin activity of a compound obtained from egg yolk, peanut oil, and soybean lecithin. J Allergy 30: 415-419.
20. Perlik F, Raskova H, Elis J (1971) Anti-inflammatory properties of N(2-hydroxyethyl) palmitamide. Acta Physiol Acad Sci Hung 39:395-400.
21. Perlík F, Krejcí J, Elis J, Pekárek J, Svejcar J (1973) The effect of N-(2-hydroxyethyl)-palmitamide on delayed hypersensitivity in guinea-pig. Experientia 29: 67-68.
22. Clark IA (2007) The advent of the cytokine storm Immunology and Cell Biology 85: 271-273.
23. Keppel Hesselink JM, de Boer T, Witkamp RF (2013) Palmitoylethanolamide: A Natural Body-Own Anti-Inflammatory Agent, Effective and Safe against Influenza and Common Cold. Int J Inflam artikel ID: 151028.
24. Facci L, Dal Toso R, Romanello S, Buriani A, Skaper SD, et al. (1995) Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide. Proc Natl AcadSci USA 11: 3376-3380.
25. Skaper SD, Buriani A, Dal Toso R, Petrelli L, Romanello S, et al. (1996) The ALIAmidepalmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons. Proc Natl AcadSci USA 93: 3984-3989.
26. Aloe L, Leon A, Levi-Montalcini R (1993) A proposed autacoid mechanism controlling mastocyte behaviour. Agents Actions 39: C145-147.
27. De Filippis D, Negro L, Vaia M, Cinelli MP, Iuvone T (2013) New insights in mast cell modulation by palmitoylethanolamide. CNS NeurolDisord Drug Targets 12: 78-83.
28. De Filippis D, D'Amico A, Luvone T (2008) Cannabinomimetic control of mast cell mediator release: new perspective in chronic inflammation. J Neuroendocrinol 20: 20-25.
29. Skaper SD, Facci L, Romanello S, Leon A (1996) Mast cell activation causes delayed neurodegeneration in mixed hippocampal cultures via the nitric oxide pathway. J Neurochem 66: 1157-1166.
30. D'Agostino G, La Rana G, Russo R, Sasso O, Iacono A, et al. (2007) Acute intracerebroventricular administration of palmitoylethanolamide, an endogenous peroxisome proliferator-activated receptor-alpha agonist, modulates carrageenan-induced paw edema in mice. J Pharmacol Exp Ther 322: 1137-1143.
31. Wang J, Zheng J, Kulkarni A, Wang W, Garg S, et al. (2014) Palmitoylethanolamide Regulates Development of Intestinal Radiation Injury in a Mast Cell-Dependent Manner. Dig Dis Sci 59: 2693-703.
32. Rao KN, Brown MA (2008 ) Mast cells: multifaceted immune cells with diverse roles in health and disease. Ann N Y Acad Sci 1143: 83-104.
33. Brown MA and Hatfield JK (2012) Mast Cells are Important Modifiers of Autoimmune Disease: With so Much Evidence, Why is There Still Controversy? Front Immunol 3: 147.
34. Boogaard van den FE, Brands X, Roelofs JJ, de Beer R, De Boer OJ, et al. (2014) Mast cellsimpair host defenseduringmurine Streptococcus pneumoniae pneumonia. J Infect Dis. 210: 1376-1384.
35. Graham AC, Hilmer KM, Zickovich JM, Obar JJ (2013) Inflammatory response of mast cells during influenza A virus infection is mediated by active infection and RIG-I signaling. J Immunol 190: 4676-4684.
36. Hu Y, Jin Y, Han D, Zhang G, Cao S, et al. (2012) Mast cell-induced lung injury in mice infected with H5N1 influenza virus. J Virol 86: 3347-3356.
37. Hesselink JM (2013) Evolution in pharmacologic thinking around the natural analgesic palmitoylethanolamide: from nonspecific resistance to PPAR-α agonist and effective nutraceutical. J Pain Res 6: 625-634.
38. Hajishengallis G, Lambris JD (2016) More than complementing Tolls: complement-Toll-like receptor synergy and crosstalk in innate immunity and inflammation. Immunol Rev 274: 233-244.
39. Saito T, Gale M Jr. (2007) Principles of intracellular viral recognition. Curr Opin Immunol 19: 17-23.
40. Liu Y, Chen H, Sun Y, Chen F (2012) Antiviral role of Toll-like receptors and cytokines against the new 2009 H1N1 virus infection. MolBiol Rep 39: 1163-1172.
41. Shirey KA, Lai W, Scott AJ, Lipsky M, Mistry P, et al. (2013) The TLR4 antagonist Eritoran protects mice from lethal influenza infection Nature 23: 498-502.
42. Esposito G, Capoccia E, Turco F, Palumbo I, Lu J, et al. (2014) Palmitoylethanolamide improves colon inflammation through an enteric glia/toll like receptor 4-dependent PPAR-α activation Gut 63: 1300-1312.
43. Haasbach E, Reiling SJ, Ehrhardt C, Droebner K, Rückle A, et al. (2013) The NF-kappaB inhibitor SC75741 protects mice against highly pathogenic avian influenza A virus. Antiviral Res 99: 336-344.
44. D'Agostino G, La Rana G, Russo R, Sasso O, Iacono A, et al. (2009) Central administration of palmitoylethanolamide reduces hyperalgesia in mice via inhibition of NF-kappaB nuclear signalling in dorsal root ganglia. Eur J Pharmacol 613: 54-59.
45. Impellizzeri D, Ahmad A, Bruschetta G, Di Paola R, Crupi R, et al. (2015) The anti-inflammatory effects of palmitoylethanolamide (PEA) on endotoxin-induced uveitis in rats. Eur J Pharmacol. Aug 15: 28-35.
46. Nhu QM, Shirey K, Teijaro JR, Farber DL, Netzel-Arnett S, et al. (2010) Novel signaling interactions between proteinase-activated receptor 2 and Toll-like receptors in vitro and in vivo. Mucosal Immunol 3: 29-39.