marine drugs

Review Different Macrophage Type Triggering as Target of the Action of Biologically Active Substances from Marine Invertebrates

Lyudmila S. Dolmatova 1,* and Igor Yu. Dolmatov 2 1 V.I. Il‘ichev Pacific Oceanological Institute, Far Eastern Branch, Russian Academy of Sciences, Baltiyskaya 43, 690041 Vladivostok, Russia 2 National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Palchevsky 17, 690041 Vladivostok, Russia; [email protected] * Correspondence: [email protected]



Received: 15 November 2019; Accepted: 30 December 2019; Published: 2 January 2020


Abstract: Macrophages play a fundamental role in the immune system. Depending on the microenvironment stimuli, macrophages can acquire distinct phenotypes characterized with different sets of the markers of their functional activities. Polarization of macrophages towards M1 type (classical activation) is involved in inflammation and the related progression of diseases, while, in contrast, alternatively activated M2 macrophages are associated with the anti-inflammatory mechanisms. Reprogramming macrophages to switch their phenotypes could provide a new therapeutic strategy, and targeting the M1/M2 macrophage balance is a promising current trend in pharmacology. Marine invertebrates are a vast source of the variety of structurally diverse compounds with potent pharmacological activities. For years, a large number of studies concerning the immunomodulatory properties of the marine substances have been run with using some intracellular markers of immune stimulation or suppression irrespective of the possible application of marine compounds in reprogramming of macrophage activation, and only few reports clearly demonstrated the macrophage-polarizing activities of some marine compounds during the last decade. In this review, the data on the immunomodulating effects of the extracts and pure compounds of a variety of chemical structure from species of different classes of marine invertebrates are described with focus on their potential in shifting M1/M2 macrophage balance towards M1 or M2 phenotype.

Keywords: marine compounds; inflammation; immunostimulation; macrophage polarization; cytokines; NO; ROS

1. Introduction Macrophages play a key role in initiating and maintaining the inflammatory response, as well as in tissue repair. Inflammation is a complex mechanism involved in the defense of the organism against pathogens. However, if unchecked, inflammation will result in cellular and tissue damage that can lead to chronic disease [1]. Thus, in response to harmful stimuli, including bacterial lipopolysaccharide (LPS), macrophages produce high levels of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukins (IL)-6, IL-8, IL-1β [2]. This results in activation of the inhibitory subunit of nuclear factor kappa B (NF-κB), inhibitor of kappa B (IκB)- kinase, followed by the phosphorylation of IκB protein bound to NF-κB. Afterwards, NF-κB translocates to the nucleus, where it binds to the promoter region of several genes. NF-κB is involved in regulation of expression of several pro-inflammatory enzymes like cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS). iNOS is responsible for synthesis of nitric oxide (NO), which is found in cells in large amounts during inflammation. In turn, COX-2 and phospholipase A2 (PLA2) induce a production of prostaglandin

Mar. Drugs 2020, 18, 37; doi:10.3390/md18010037 www.mdpi.com/journal/marinedrugs Mar. Drugs 2020, 18, 37 2 of 27

E2 (PGE2) [3]. The significant changes in the amount/activity of those molecules allow to use them for monitoring inflammation, and evaluation of the effects of new compounds on those markers in vitro is usually used for exploring their potential inflammatory properties. Additionally, in recent years, stress that arises from accumulation of unfolded proteins within a cell’s endoplasmic reticulum (ER) was identified, and ER stress was found to participate in LPS-induced immune response and regulate inflammation in some pathological models [4]. Therefore, some markers of ER stress, e.g., a transcription factor C/EBP homologous protein (CHOP) [5], are sometimes evaluated in the screening compounds for immunomodulating activity. About four decades ago, the macrophages were first shown to be a morphotypically heterogeneous cell population while exposed to different cytokines [6,7]. Later, these cells were shown to have ability to undergo activation to pro-inflammatory M1 (classically activated) or anti-inflammatory M2 (alternatively activated) phenotypes in response to different exogenic and endogenic stimuli [8]. Among those stimuli, interferon-gamma (IFN-γ) combined with LPS, and mediators of ER-stress activation, such as pancreatic EIF-2alpha kinase, can be mentioned as drivers of the M1 phenotype [9]. Synthetic glucocorticoid dexamethasone and IL-4, IL-10, and IL-13 [8] are inducers of M2 activation. In turn, M1 macrophages release proinflammatory cytokines, such as IL-1β, IL-6, IL-12, IL-18, IL-23, TNFα, type I IFN, and numerous chemokines. Moreover, these cells produce high levels of NO and reactive oxygen species (ROS) [10]. Phenotypic characteristics for M1 macrophages are CD64 and CD80 [11], and also CD14 but not CD16 expression [8]. M2 cells display a high activity of arginase-1 and secrete high levels of IL-10, IL-13 [10,11], and IL-8 [12]. M2-like macrophage phenotype is identified based on the expression of CD64 and CD209 [9]. Moreover, two types of macrophages use different metabolic pathways for energy generation: glycolysis in M1 type [13], and oxidative metabolism in M2 cells [14]. In addition, M1 type has a higher ratio of reduced-to-oxidized glutathione (GSH/GSSG) compared to that in M2 type, and GSH depletion can switch macrophages from the M1 towards the M2 phenotype [15]. The diversity of the microenvironment results in a spectrum of in vivo macrophage phenotypes and functions so that the classical and alternative ways of polarization of macrophages are considered to lead to the extremes of the spectrum of possible phenotypes [16]. Additionally, a new type of macrophages was recently described—regulatory macrophages (MRs). They play a crucial role in anti-inflammatory restriction of inflammation during innate and adaptive immune responses, and dehydrogenase/reductase 9 (DHRS9) is suggested to be their marker [17]. A high plasticity of macrophages and the fact that mixed features of the two types are sometimes present in macrophage populations [18] put a question whether two clearly distinct types of macrophages are present in vivo. However, Jablonski et al. [17] identified a new set of common and distinct M1 and M2 genes regulating pathways linked to M1 and/or M2 phenotypes. This indicates the presence of really distinct traits of the two macrophage types. Moreover, in invertebrates, the expression of arginase in macrophages after stimulation with different stimuli was shown to occur without participation of characteristic for vertebrates Th2 cytokines IL-4/IL-13, and this indicates that the protein regulation of wound healing in lower animals is produced by innate immunity cells [19]. In addition, recent studies showed the existence of two types of macrophage—like phagocytes (referred to as P1 and P2), separated by the gradient centrifugation, in holothurians (Echinodermata). The P1 type has a high level of NO as a marker, increased levels of GSH, and reactive oxygen species (ROS) compared to those in P2, but P2-type had increased arginase activity compared to that in P1 [20,21]. The two cell types also differ in their surface receptor expression [17], but under some stimuli both types could acquire the features of each other [21]. These data indicate the appearance of distinct types of macrophages already in evolutionally ancient animals like echinoderms. The known intracellular signaling ways and mechanisms of macrophage transformation via classical or alternative ways as well as their involvement in pathogenesis of many diseases are more fully described in several reviews [9,22,23]. It was clearly shown that development of a number of diseases is resulted from the M1/M2 disbalance. However, macrophage polarization may be Mar. Drugs 2020, 18, 37 3 of 27 reprogrammed, and a switching from M1 to M2 type was shown to be one of the mechanisms of action of well-known drugs and is considered to become a target for newly developed pharmacological agents [24,25]. Thereby, a number of natural compounds of plant origin were described to be potent for macrophage reprogramming in the last decade [25], but little is known about such substances from marine hydrobionts, in particular, invertebrates. A vast number of marine bioactive compounds were discovered in the last five decades [26,27], and their number is increasing with the development of new methods and instruments of the ocean research and new discoveries especially due to deep-sea studies during the last few years [28]. For years, a number of crude extracts and pure compounds were tested for bioactivities, first of all, for their presumable anti-inflammatory properties, using the markers of inflammation. Only few reports emphasizing the influence of the marine substances on macrophage phenotypes have appeared in the last decade. Nevertheless, taking into consideration, that some intracellular markers of inflammation characterize also the macrophage phenotypes, the results obtained with using such markers may indicate macrophage-polarizing potential of substances tested. Noteworthy, the most studies were conducted in vitro, and LPS was used usually to induce signaling in primary macrophages or cultured cell lines. Recent studies indicate that effects of LPS are time- and concentration-dependent [29,30]. Thus, in bone marrow-derived macrophages short-time (several hours) incubation with 0.1 µg/mL LPS inhibited M1 polarization, while long-term (24 h) incubation promoted M1 and inhibited M2 polarization [30]. Therefore, the results obtained in different cell lines and under different incubation conditions may be distinct, and attention has to be paid to this fact. In this review, the known data on the effects of the extracts and substances from some groups of marine invertebrates (Cnidaria, Porifera, Crustacea, Mollusca, Echinodermata) on the expression of inflammatory and macrophage phenotypic markers are summarized and analyzed with an accent on the promising compounds for M1–M2 macrophage transition targeting.

2. Natural Compounds with Macrophage Phenotype Modulating Activities from Marine Invertebrates of Different Taxa

2.1. Cnidaria Phylum Cnidaria contains over 11,000 species [31] and is one of the richest in secondary metabolites of various structure phyla [32]. These species remain an untapped source; however, a great variety of chemically unique structures with promising bioactive properties was described for this phylum until now [32].

2.1.1. Soft Corals Soft corals (Alcyonacea) is an order of cnidarians belonging to class Anthozoa, subclass Octocorallia. They have proven to be rich sources of several groups of chemically different compounds with anti-inflammatory properties, with mechanisms of action that are reviewed in a number of reports [32,33]. Anti-inflammatory effects of compounds isolated from Taiwanese corals from 2008 until 2012 were fully described in review of Wei et al. [33], where the authors presented all chemical structures of compounds and described their anti-inflammatory activities studied in different immune cells (neutrophils and macrophages) in vitro and in animal models of inflammation. Presenting here some data reported in the review of Wei et al. [33], we focus only on those concerning the capacity of natural substances from the soft corals to inhibit LPS-induced upregulation of NO- and eicosanoid-generating enzymes iNOS and COX-2, respectively, and also the level of NO and TNF-α which are considered to be markers of both inflammation [6,11] and macrophage classical activation pathway [11,20,34,35] in RAW264.7 cells. Far from being a common character for potential anti-inflammatory compounds, of 9 triquinane-type sesquiterpenoids from soft corals Capnella imbricata described in the work of Wei et al. [33], only three were shown to inhibit expression of both iNOS and COX-2 proteins in LPS-stimulated cells and one more—inhibits only iNOS protein [36]. Of three nardosinane-type sesquiterpenoids from Paralemnalia thyrsoides [37], none influenced these markers; and of four Mar. Drugs 2020, 18, 37 4 of 27 nardosinane-type sesquiterpenoid flavalins from Lemnalia flava only one inhibited expression of iNOS and COX-2 proteins [38]. Of six aromadendrane-type sesquiterpenoid lochmolins from the soft coral Sinularia lochmodes, none were able to inhibit accumulation of iNOS protein, and only four compounds inhibited COX-2 (at concentrations of 10–100 µM) [39]. Only one of four selinane- and oppositane-type sesquiterpenoids from soft coral Sinularia leptoclados inhibited iNOS protein expression [40], and one of three ylangene-type sesquiterpenoids from Lemnalia flava reduced iNOS and COX-2 protein accumulation [38]. Inhibition of iNOS expression was described also for sesquiterpenoids erectathiol from Nephthea erecta [41] and for scabralin A from Sinularia scabra [42]. The most anti-inflammatory active were cembrane-based diterpenoids. Almost all of them, described in the review of Wei et al. [33], were found to have iNOS and COX-2 inhibiting ability: gibberosene B from Sinularia gibberosa [43], durumolides A, C [44], F and K [45], 13S-hydroxylobolide [44], deacetyl-13-hydroxylobolide, (7E,11E)- 13,18-dihydroxy-3,4-epoxy-7,11,15(17)-cembratrien-16,14-olide [43], (3E,7E,11E)- 18-acetoxy-3,7,11,15(17)-cembratetraen-16,14-olide [45], durumhemiketalolides A and C[46] from Lobophytum durum, sinulariol A from Lobophytum crassum [47], sarcocrassolide from Sarcophyton crassocaule [48], thioflexibilolide A from Sinularia flexibilis [49], dihydrosinularin and ( )14-deoxycrassin from Sinularia triangular [50], sarcocrassocolide I from Sarcophyton crassocaule [51], − 11-dehydrosinulariolide and 11-epi-sinulariolide acetate from Sinularia discrepans [52]. At the same time, 13R-hydroxylobolide [44], durumolides B and D [44], G-D, L [45], durumhemiketalolide B [46] from Lobophytum durum and grandilobatin D from Sinularia grandilobata [53], 17-dimethylaminolobohedleolide from Lobophytum crassum [47], sarcocrassocolide A-E, sinularolide, 13-acetoxysarcocrassolide from Sarcophyton crassocaule [48], sinularin from Sinularia triangular [50], sarcocrassocolides F-H, J-L from Sarcophyton crassocaule [51], sarcophytolins A-B, D, but not C from Lobophytum sarcophytoides [54], crassarine H from Sinularia crassa [55] and sarcocrassocolides M-O from Sarcophyton crassocaule [56], querciformolide E from Sinularia querciformis, granosolide D, flexibilisolide A, flexilarin, sinulariolide, but not granosolide C and sinulaflexiolide E from Sinularia granosa, inhibited only iNOS protein expression [57], and crassarine F from Sinularia crassa inhibited only COX-2 protein expression [55]. Interestingly, no anti-inflammatory activity was found in macrophages under treatment with most of crassarines (B-E, G, H) from Sinularia crassa [55] and culobophylin A-C from Lobophytum crassum [58]. Anti-inflammatory activity was also abundant among eunicellin-based diterpenoids: simplexin E[59] and klysimplexin S [60], klysimplexin sulfoxide C [61] from Klyxum simplex, lymollin C-D, F-H [62] from Klyxum molle, which were active in reducing both iNOS and COX-2 protein accumulation, while simplexins A and D [59], klysimplexin J-N, R [60], klysimplexin sulfoxide A and B [61] from Klyxum simplex, hirsutalins C, D and H from Cladiella hirsute [63], lymollin B and E from Klyxum molle [62], krempfielins A-D, litophynol B and (1R*,2R*,3R*,6S*,7S*,9R*,10R*,14R*)3- butanoyloxycladiell-11(17)-en-6,7-diol [64] from Cladiella krempfi inhibited only NO generation. For klysimplexins O-R [60], U-X from Klyxum simplex [65], krempfielin A from Cladiella krempfi [64], and hirsutalin E-G from Cladiella hirsuta [63], the iNOS and COX-2 inhibiting activity was not detected. It was shown that of eight verticillane-based diterpenoid cespitularins from Cespitularia hypotentaculata Cespitularin S possessed both iNOS and COX-2 expression inhibiting abilities, and cesputularins K, I, and F had only iNOS expression inhibiting activity [66]. Only one report examined norditerpenoids [66]. Of 18 compounds isolated from Sinularia gyrosa, gyrosanolides B and F,gyrosanin A, (1S*,5R*,8S*,10R*,11S*)- 11-hydroxyl-1- isopropenyl-8-methyl-3,6-dioxo-5,8- epoxycyclotetradec-12-ene-10,12-carbonlactone, (1S*,5S*,8S*,10R*,11S*)-11-hydroxyl-1-isopropenyl-8-methyl-3,6-dioxo-5,8- epoxycyclotetradec-12-ene- 10,12-carbonlactone, leptocladolide B and scabrolide D were able to inhibit iNOS protein accumulation. In addition, diterpenoids gyrosanols A and B were found to inhibit COX-2 protein expression [67]. Almost all numerous sterols studied inhibited iNOS and COX-2 protein accumulation: stoloniferone T and (25S)-24-methylenecholestane- 3β,5α,6β-triol-26-acetate from Clavularia viridis [68], chabrosterol from Nephthea chabroli [41], nebrosteroids D and F, G from Nephthea chabroli [69], griffinisterones F and G and griffinipregnone from Dendronephthya griffini [70], 1α,3β-dihydroxy-24S-methylcholesta-5-ene and Mar. Drugs 2020, 18, 37 5 of 27

1α,3β-dihydroxy-24-methylenecholesta-5-ene from Sinularia sp. [71], 5,24(28)-ergostadien-3β,23S-diol and (22S)-5,24(28)-ergostadien- 3β,17α,22-triol from Nephthea erecta [72], nebrosteroids I-M from Nephthea chabroli [73], minabeolide-1,2,4,5 from Paraminabea acronocephala [74], and 8αH-3β,11-dihydroxy-5α,6α-expoxy-24-methylene-9,11- secocholestan-9-one from Sinularia granosa [75]. Stoloniferone S from Clavularia viridis [68], griffinisterones A-D from Nephthea griffini [76], nebrosteroids A-C, I from Nephthea chabroli [73], griffinisterone H from Dendronephthya griffini [70], 5,24(28)-ergostadien-3β,23R-diol and ergostanoid from Nephthea erecta [72], 5α,8α-epidioxy-22,23-methylene-24-methylcholest-6-en-3β-ol from Lobophytum sarcophytoides [54], paraminabeolides A-D from Paraminabea acronocephala [74], crassarosterosides A-C from Sinularia crassa [74], and 3β,11-dihydroxy-5β,6β-expoxy-24-methylene-9,11-secocholestan-9-one from Sinularia granosa [75] inhibited only iNOS expression. None of hirsutosterols A-G from Cladiella hirsuta [77] showed any anti-inflammatory activity. Among the ceramides and cerebrosides, despite a lower number of them being studied, some were also found that had iNOS- and COX-2 inhibiting properties (ceramide from Sarcophyton ehrenbergi) or iNOS protein expression inhibiting activity (sarcoehrenoside A and cerebrosides 3-6 from Sarcophyton ehrenbergi [78]. Among other metabolites, capilloquinone and capillobenzopyranol, 2-[(2E,6E)-3,7-dimethyl-8-(4-methylfuran-2-yl)octa-2,6-dienyl]-5-methylcyclohexa-2,5-diene-1,4-dione, 2-[(2E,6E)-3,7-dimethyl-8-(4-methylfuran-2-yl)octa-2,6-dienyl]-5-methylbenzene-1,4-diol from Sinularia capillosa [79] and dihydroaustrasulfone alcohol from Cladiella australis [80] were found to inhibit COX-2 and/or iNOS expression. Of note, all examined compounds studied were used at concentration of 10 µM, except for few reports. Thus, 20 and 50 µM concentrations of compounds were used in studies of Chen et al. [60] and Lu et al. [57]; additionally, concentration-dependent effects of compounds were evaluated in the work of Tseng et al. [39] at a concentration range of 1, 10, and 100 µM, and in the studies of Chen et al. [63] at concentrations of 2.5, 10, and 20 µM. For iNOS protein expression inhibiting capacity of cespitularins S from the Formosan soft coral Cespitularia hypotentaculata, the effective concentration 50(EC50s) was 7.80 0.7 µM[66]. Once examined, no toxic effects of griffinisterones A-E to RAW264.7 cells were ± found at the 10 µM concentration, which is effective for inhibiting the upregulation of inflammatory and macrophage classical way markers [76]. In many laboratories, for inducing inflammation, LPS was used at concentration of 0.01 µg/mL, and time of incubation was 16 h [37,38,40–57,59–76,78,79]. In few cases, higher concentrations of LPS were used: 1 [39] or 10 µg/mL [36]. This indicates that, in most studies, there were no standard conditions of macrophage stimulation (a large enough concentration of LPS, known other activating stimuli and long-time incubation) and, in addition, there was lack of data on the phenotypes of macrophages during the experiment. Nevertheless, these studies were useful for generating basic libraries of anti-inflammatory activities from a broad spectrum of soft corals for further research. In case of using standard conditions (LPS concentration, known other activating stimuli and incubation time) for macrophage activation, more reasoned conclusions may be made. Thus, although briarane-based diterpenoids were not shown to be studied for the iNOS and COX-2 inhibiting activities in the review of Wei et al. [33], briarane-type diterpene xcavatolide B (BrD1, 20 µM), isolated from a Formosan gorgonian coral Briareum excavatum (Nutting, 1911), was shown to inhibit LPS (100 ng/mL)-induced expression of pro-inflammatory cytokines IL-6 and, at lesser extent, TNF-α, in mouse bone marrow-derived dendritic cells after 24 h [81]. Authors explain such differences between TNF-α and IL-6 inhibition by more early and rapid TNF-α expression resulted from LPS-induced TNF-α mRNA splicing [82]. Additionally, comparison of cytokine inhibiting properties of different briarane-based diterpenoids (Figure1) revealed that a presence of 8, 17-epoxides of BrDs is crucial for the effects of compounds on LPS-induced expression of the cytokines, but the presence of an α position of the functional group at C-12 and the steric hindrance of longer acyloxyl groups impaired the capacity of compounds to inhibit LPS-induced Mar. Drugs 2020, 18, 37 6 of 27

Mar. Drugs 20202020,, 1818,, xx FORFOR PEERPEER REVIEWREVIEW 66 ofof 2727 cytokine expression. Analysis of the conditions of the experiment indicates that LPS exposure resulted inresultedresulted increase inin of increaseincrease M1-macrophage ofof M1-macrophageM1-macrophage marker expression, markermarker exprexpr andession, BrD1 and can BrD1 be considered can be considered potential potential to shift the to M1-phenotypeshiftshift thethe M1-phenotypeM1-phenotype towards the towardstowards M2-type. thethe M2-type.M2-type.

FigureFigure 1.1. ChemicalChemical structure structure of of the the briarane-based briarane-based diterpenoids. diterpenoids. (1)) ( 1excavatolideexcavatolide) excavatolide BB (BrD1);(BrD1); B (BrD1); ((2)) (2excavatolide) excavatolide K K(BrD2); (BrD2); (3 ())3 excavatolide)excavatolide excavatolide FF F (BrD3);(BrD3); (BrD3); (( (44))) briaexcavatolidebriaexcavatolide R R (BrD4);(BrD4); (((55))) excavatolideexcavatolideexcavatolide Z ZZ (BrD5);(BrD5);(BrD5); ( 6((6))) briaexcavatolide briaexcavatolidebriaexcavatolide B BB (BrD6); (BrD6);(BrD6); ( 7((7))) briaexcavatolide briaexcavatolidebriaexcavatolide K KK (BrD7); (BrD7);(BrD7); ( 8 (()8) briaexcavatolide) briaexcavatolidebriaexcavatolide H HH (BrD8). (BrD8).(BrD8).

Later,Later, excavatolideexcavatolide BB atat concentrationsconcentrations ofof 1,1, 10,10, 25,25, andand 5050 µμMM waswas shownshown toto inhibitinhibit iNOSiNOS proteinprotein expression,expression, andand atat concentrationconcentration ofof 5050 µμMM inhibited inhibited COX-2 COX-2 expressionexpression comparedcompared toto thosethose inducedinducedinduced by byby LPS LPSLPS alone alonealone in RAWinin RAWRAW 264.7 264.7264.7 cells [83cellscells]. These [83][83].. dataTheseThese confirm datadata theconfirmconfirm suggestion thethe suggestionsuggestion about the promisingaboutabout thethe anti-inflammatorypromising anti-inflammatory properties of properties the compound, of the with compound, a mechanism with involving a mechanism the influence involving on thethe macrophageinfluenceinfluence ononphenotype. thethe macrophagemacrophage phenotype.phenotype. MoreMore recent recent studies studies on on the the cembrane-type cembrane-type diterpenoidsditerpenoids ofof anotheranother TaiwanTaiwan species species of of the the same same familyfamilyfamily Briareidae, Briareidae,Briareidae,Briareum Briareum violaceum violaceum(Quoy (Quoy(Quoy and andand Gaimard, Gaimard,Gaimard, 1883) 1883)1883) were werewere carried carriedcarried out outout in inin RAW RAWRAW 264.7 264.7264.7 cells cellscells stimulatedstimulatedstimulated forforfor 16 1616 h hh with withwith LPS LPSLPS at atat a aa concentration concentrationconcentration of ofof 10 1010µ μM[M 84[84].]. OnlyOnly cembranoidscembranoids 1010––1212 (10(10(10 µμM)M) (Figure(Figure(Figure2 ) 2)2) exerted exertedexerted inhibition inhibitioninhibition e ffects effectseffects on iNOS onon iNOSiNOS release releaserelease from the fromfrom cells, thethe and cells,cells, briaviotriol andand briaviotriolbriaviotriol A (11) and briaviodiolAA ((11)) andand A(briaviodiol10) were shownA (10)) werewere to be shown theshown most toto potent bebe thethe suppressorsmostmost potentpotent ofsuppressorssuppressors iNOS release, ofof iNOSiNOS suggesting release,release, their suggestingsuggesting potential theirtheir in shiftingpotential the in M1 shifting phenotype the M1 towards phenotype the M2towards type ofthe macrophages. M2 type of macrophages.

FigureFigure 2. 2. StructuresStructures of of briaviodiol briaviodiol F F ( (99),),), briaviotriols briaviotriolsbriaviotriols A AA ( 10((10))) and andand B BB ( 11((11),),), and andand briaviodiol briaviodiolbriaviodiol A AA ( 12((12).).).

13 TwoTwo newnew cembranecembrane diterpenoidditerpenoid norcembranoids,norcembranoids, sinumerolidesinumerolide AA ((13))) andandand itsitsits epimer,epimer,epimer, E 14 Sinularia 77E-sinumerolide-sinumerolide-sinumerolide A AA ( ((14),),), were werewere isolated isolatedisolated from fromfrom the thethe ethyl ethylethyl acetate acetacetate extract extract of of the the Taiwan Taiwan soft soft coral coral Sinularia numerosa numerosa(Tixier-Durivault, (Tixier-Durivault,(Tixier-Durivault, 1970). 1970).1970). InInIn LPSLPSLPS (10(10(10 ngng/mL)-inducedng/mL)-induced/mL)-induced forforfor 161616 h hh RAW264.7 RAW264.7RAW264.7 macrophage macrophagemacrophage cells, cells,cells, µ compoundscompounds (Figure(Figure3 )3) 1313 and andand 14 14(10 (10(10 μMM each) each) significantly significantly inhibited inhibited LPS-induced LPS-induced iNOS,iNOS, butbut notnot µ COX-2COX-2 proteinprotein expressionexpression inin macrophagesmacrophages [85[85].]. DexamethasoneDexamethasone (10(10 μM)M) waswas usedused asas aa positivepositive controlcontrol as as it isit knownis known as an as anti-inflammatory an anti-inflammatory agent, and agent, is additionally and is additionally able to modulate able to macrophage modulate polarizationmacrophage via polarization M2-way [ 44via]. M2-way In this experiment, [44]. In this dexamethasone experiment,experiment, dexamethasondexamethason inhibited bothe inhibited iNOS and both COX-2 iNOS proteinand COX-2 expression protein compared expression to compared the effects ofto LPSthe effect alone.ss ofof These LPSLPS data alone.alone. apparently TheseThese dadata indicateta apparentlyapparently that the indicateindicate both compoundsthatthat thethe bothboth are ablecompoundscompounds to exert their areare anti-inflammatoryableable toto exertexert ththeir eff ectsanti-inflammatory via influence on macrophageeffects via influence polarization, on butmacrophage with the mechanisms polarization, which but with are distinctly the mechanisms different fromwhichwhich those areare ofdistinctlydistinctly dexamethasone. differentdifferent fromfrom thosethose ofof dexamethasone.

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Mar.Mar. Drugs 20202020,, 1818,, 37x FOR PEER REVIEW 77 ofof 2727 Mar. Drugs 2020, 18, x FOR PEER REVIEW 7 of 27

Figure 3. Structure of sinumerolide A (13) and its epimer, 7E-sinumerolide A (14). Figure 3. Structure of sinumerolide A (13) and its epimer, 7E-sinumerolide A (14). Chlorinated compounds are rare among soft coral metabolites [86]. Studies on antiinflammatoryFigure activities 3. Structure of chlorinatedof sinumerolide norcembr A (13) andanoids its epimer, (Figure 7E-sinumerolide 4) from the Indonesian A (14). soft coral ChlorinatedFigure compounds 3. StructureStructure of of sinumerolide sinumerolideare rare amongA ( 13)) and soft its epimer,coral 7E-sinumerolidemetabolites AA[86]. ((14).). Studies on antiinflammatorySinularia sp. [86] were activities conducted of chlorinated in J774 cellsnorcembr stimulatedanoids with (Figure LPS 4) (1 from mg/mL) the Indonesian for 24 h. Almost soft coral all ChlorinatedChlorinated compoundscompounds are rareare amongrare softamong coral soft metabolites coral [86metabolites]. Studies on[86]. antiinflammatory Studies on SinulariacompoundsChlorinated sp. were[86] were inactivecompounds conducted in relationare in J774rare to cells inhibitiamong stimulatedon ofsoft LPS-stimulated withcoral LPS metabolites (1 mg/mL) NO production, for[86]. 24 h.Studies Almost and only onall activitiesantiinflammatory of chlorinated activities norcembranoids of chlorinated (Figure norcembr4) fromanoids the (Figure Indonesian 4) from soft the coral IndonesianSinularia softsp. coral [86] antiinflammatorycompoundsscabrolide D wereslightly activitiesinactive decreased ofin chlorinatedrelation this marker to norcembrinhibiti expresonsion.anoids of Obviously,LPS-stimulated (Figure 4) chlorinated from NO the production, Indonesian norcembranoids andsoft coralonly are wereSinularia conducted sp. [86]in were J774 conducted cells stimulated in J774 with cells LPS stimulated (1 mg/mL) with for LPS 24 h.(1 Almostmg/mL) all for compounds 24 h. Almost were all Sinulariascabrolidenot promising sp. D [86]slightly agents were decreasedto conducted regulate this macrophage in markerJ774 cells expres balance, stimulatedsion. presumably Obviously, with LPS duechlorinated (1 mg/mL)to a chlorine norcembranoidsfor 24 atom. h. Almost areall inactivecompounds in relation were inactive to inhibition in relation of LPS-stimulated to inhibition NO of production,LPS-stimulated and NO only production, scabrolide Dand slightly only compoundsnot promising were agents inactive to regulate in relation macrophage to inhibiti balance,on of presumably LPS-stimulated due to NO a chlorine production, atom. and only scabrolide D slightly decreased this marker expression. Obviously, chlorinated norcembranoids are decreasedscabrolide thisD slightly marker decreased expression. this Obviously, marker expres chlorinatedsion. Obviously, norcembranoids chlorinated are not norcembranoids promising agents are to not promising agents to regulate macrophage balance, presumably due to a chlorine atom. regulatenot promising macrophage agents balance,to regulate presumably macrophage due balance, to a chlorine presumably atom. due to a chlorine atom.

Figure 4. Chemical structure of macrocyclicditerpenoids scabrolide D (15), chloroscabrolides A (16) and B (17), and prescabrolide (18). Figure 4. Chemical structure of macrocyclicditerpenoids scabrolide D (15), chloroscabrolides A (16) Figureand B (17 4. ),Chemical and prescabrolide structure of(18 macrocyclicditerpenoids). scabrolide D (15), chloroscabrolides A (16) OctocoralsFigure 4. Chemical from structurethe genus of macrocyclicditerpenoidsEunicea have to be scabrolidea prolific D source (15), chloroscabrolides of chemically A complex(16) andFigure B ( 174. ),Chemical and prescabrolide structure (of18 macrocyclicditerpenoids). scabrolide D (15), chloroscabrolides A (16) diterpenoids,and B (17 ),including and prescabrolide recently (18 ).isolated group of highly functionalized α-methylene-γ-lactone andOctocorals B (17), and from prescabrolide the genus (18 ).Eunicea have to be a prolific source of chemically complex diterpenoids,cembranolides,Octocorals including fromcharacterized the genusrecently byEunicea the isolated presencehave group to of be a a Δ prolificof6 olefinhighly source known functionalized of as chemically the uprolides α complex-methylene- [87]. diterpenoids, Inγ LPS-lactone (100 Octocorals from the genus Eunicea have to be a prolific source of chemically complex includingcembranolides,μg/mL,Octocorals 6 h recently incubation)-stimulated characterizedfrom isolated the genus group by the Eunicea ofprimary presence highly have murine functionalizedof a toΔ6 macrophagesbeolefin a prolificknownα-methylene- asfromsource the C57B1/6 uprolidesγ -lactoneof chemically mice, [87]. cembranolides, compoundsIn LPScomplex (100 diterpenoids, including recently isolated group of highly functionalized α-methylene-γ-lactone characterizedditerpenoids,μ19g/mL,–21, but 6 hnot incubation)-stimulated byincluding22, the(Figure presence recently5) inhibited of aisolated ∆primary 6the olefin production group murine known of ofmacrophages ashighly TNF theα uprolides functionalizedand IL-6 from [in87 C57B1/6a]. dose-dependent Inα-methylene- LPS mice, (100 compoundsµgγ /manner,mL,-lactone 6 h cembranolides, characterized by the presence of a Δ6 olefin known as the uprolides [87]. In LPS (100 incubation)-stimulatedcembranolides,19the– 21compounds, but not 22 characterized ,were (Figure unable primary 5) inhibited by to murine the induce presence the macrophages productionthe ofproduc a Δ6 oftionolefin from TNF of C57B1knownα TNF and/ 6IL-6and as mice, the inIL-6 auprolides compounds dose-dependent in the absence[87].19 –In21 LPS ,manner,of but LPS.(100 not μg/mL, 6 h incubation)-stimulated primary murine macrophages from C57B1/6 mice, compounds 22μtheApparently,g/mL,, (Figurecompounds 6 h5 ) incubation)-stimulateduprolides inhibited were the19unable– production21 may to inducebe primary considered of TNF the α murineproducand promising IL-6 macrophagestion in of a todose-dependent TNFstimulate andfrom macrophageIL-6 C57B1/6 manner,in the mice, absence polarization the compounds compounds of LPS. via 19–21, but not 22, (Figure 5) inhibited the production of TNFα and IL-6 in a dose-dependent manner, were19Apparently,M2-type–21 unable, but way. not uprolides to 22 induce, (Figure 19 the –5)21 production inhibited may be considered the of TNFproduction and promising IL-6 of inTNF the toα absencestimulateand IL-6 of in macrophageLPS. a dose-dependent Apparently, polarization uprolides manner, via the compounds were unable to induce the production of TNF and IL-6 in the absence of LPS. 19theM2-type– 21compoundsmay way. be considered were unable promising to induce to stimulate the produc macrophagetion of TNF polarization and IL-6 via in M2-type the absence way. of LPS. Apparently, uprolides 19–21 may be considered promising to stimulate macrophage polarization via Apparently, uprolides 19–21 may be considered promising to stimulate macrophage polarization via M2-type way. M2-type way.

Figure 5. Structure of diterpene uprolide N (19), uprolide O (20), uprolide P (21), and dolabellane Figure(22). 5. 19 20 21 22 Figure 5. StructureStructure ofof diterpene diterpene uprolide uprolide N N ( (19), uprolide), uprolide O (O ),(20 uprolide), uprolide P ( P), ( and21), dolabellaneand dolabellane ( ). (22). InFigure addition, 5. Structure three newof diterpene cembranolides uprolide (N23 –(1925),) (Figureuprolide6 6))O which which(20), uprolide werewere isolated isolated P (21), fromandfrom dolabellane an an OkinawanOkinawan Figure 5. Structure of diterpene uprolide N (19), uprolide O (20), uprolide P (21), and dolabellane soft coral,(coral,22). Lobophytum Lobophytumsp., weresp., evaluatedwere evaluated for anti-inflammatory for anti-inflammatory effect in LPS-stimulated effect in LPS-stimulated inflammatory (In22 addition,). three new cembranolides (23–25) (Figure 6) which were isolated from an Okinawan RAWsoftinflammatory coral, 264.7 macrophageLobophytum RAW 264.7 cellssp., macrophage [88were]. All evaluated compounds, cells [88].for especially anti-inflammatoryAll compounds, compound especially23effect, significantly in LPS-stimulatedcompound decreased 23, In addition, three new cembranolides (23–25) (Figure 6) which were isolated from an Okinawan NOinflammatorysignificantly productionIn addition, decreased RAW in three LPS-stimulated 264.7new NO cembranolides productionmacrophage cells. in LPS-stimulated(cells23–25 [88].) (Figure All cells.6)compounds, which were isolatedespecially from compound an Okinawan 23, soft coral, Lobophytum sp., were evaluated for anti-inflammatory effect in LPS-stimulated softsignificantly coral, Lobophytumdecreased NO sp., production were evaluated in LPS-stimulated for anti-inflammatory cells. effect in LPS-stimulated inflammatory RAW 264.7 macrophage cells [88]. All compounds, especially compound 23, inflammatory RAW 264.7 macrophage cells [88]. All compounds, especially compound 23, significantly decreased NO production in LPS-stimulated cells. significantly decreased NO production in LPS-stimulated cells.

Figure 6. Structure of new cembranolides (23)–(25) from an Okinawan soft coral, Lobophytum sp.

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Mar. DrugsFigure2020, 6.18 ,Structure 37 of new cembranolides (23)–(25) from an Okinawan soft coral, Lobophytum sp. 8 of 27

Studies on five new eunicellin-based diterpenoids krempfielins E–I (26–30) along with the knownStudies compounds on five 6-methyl new eunicellin-based ether of litophynol diterpenoids B (31), sclerophytin krempfielins A ( E–I32), (sclerophytin26–30) along B with(33), thelitophynin known compoundsI monoacetate 6-methyl (34), ether 6-acetoxy of litophynol litophynin B (31 ),E sclerophytin(35), (1R*,2 AR* (,332R*),,6 sclerophytinS*,9R*,10R*, B(14R*33)-3-acetoxycladiell-7(16),11(17)-dien-6-ol), litophynin I monoacetate (34), 6-acetoxy (36), and litophynin litophynin E ( 35F), ( (137R*) ,2(FigureR*,3R* ,67)S* ,9fromR*,10 theR*, 14TaiwaneseR*)-3-acetoxycladiell-7(16),11(17)-dien-6-ol soft coral Cladiella krempfi (Hickson, (36), 1919) and litophynin have shown F (37 that) (Figure new 7compounds) from the Taiwanese were not softactive, coral andCladiella only 6-methyl krempfi (Hickson, ether of litophynol 1919) have B shown (31) could that newinhibit compounds both the wereaccumulation not active, of andthe onlypro-inflammatory 6-methyl ether iNOS of litophynol and the B (31expression) could inhibit of COX-2 both the protein accumulation in LPS- ofstimulated the pro-inflammatory RAW264.7 iNOSmacrophages and the expression[89]. 6-acetoxy of COX-2 litophynin protein E in ( LPS-stimulated35) was able to RAW264.7 inhibit iNOS, macrophages and litophynin [89]. 6-acetoxy F (37) litophynininhibited the E ( 35COX-2) was ableprotein to inhibit expression. iNOS, andThese litophynin findings Fdemonstrate (37) inhibited that the the COX-2 compounds protein expression. influence Thesethe different findings stages demonstrate of the thatcell theresponse, compounds and, influenceapparently, the compounds different stages 31 and of the 35 cell may response, be used and, as apparently,potent inhibitors compounds of classi31caland macrophage35 may be usedactivation. as potent inhibitors of classical macrophage activation.

FigureFigure 7.7. Structure of eunicellin-based diterpenoids krempfielinskrempfielins E–I (26)–()–(30),), 6-methyl6-methyl etherether ofof litophynollitophynol BB ((3131),), sclerophytinsclerophytin AA ((3232),), sclerophytinsclerophytin BB ((3333),), litophyninlitophynin II monoacetatemonoacetate ((3434),), 6-acetoxy6-acetoxy litophyninlitophynin EE ((3535),), (1(1R*R*,2,2R*R*,3,3R*R*,6,6S*S*,9,9R*R*,10,10R*R*,14,14R*R*)-3-acetoxycladiell-7(16),11(17)-dien-6-ol)-3-acetoxycladiell-7(16),11(17)-dien-6-ol (36), andand litophyninlitophynin FF ((3737).).

ImmunomodulatingImmunomodulating activityactivity of of polysaccharides polysaccharides of softof soft corals corals is lesser is lesser studied studied compared compared to other to metabolitesother metabolites [90]. Polysaccharides [90]. Polysaccharides isolated isolated from Pseudopterogorgia from Pseudopterogorgia americana americana(Gmelin, (Gmelin, 1791) [90 1791)] induced [90] α theinduced expression the expression of TNF- ,of IL-6, TNF- andα, COX-2IL-6, and in mouseCOX-2 macrophages,in mouse macropha but hadges, no but effect had on no the effect expression on the α ofexpression iNOS protein of iNOS and NOprotein production. and NO However, production. polysaccharides However, polysaccharides reduced expression reduced of TNF- expressionand IL-6 of inTNF- LPS-activatedα and IL-6 macrophages in LPS-activated through macrophages the downregulation through of interleukin-1the downregulation receptor-associated of interleukin-1 kinase κ (IRAK)2receptor-associated expression, mitogen-activatedkinase (IRAK)2 proteinexpression kinase, (MAPK)mitogen-activated phosphorylation, protein and NF-kinaseB activation.(MAPK) Apparently,phosphorylation, polysaccharides and NF- aloneκB activation. do not activate Apparently, polarization polysacc macrophagesharides inalone concentrations do not studied,activate aspolarization the main marker macrophages of M1 macrophages, in concentrations NO level, studied, was notas changed;the main however,marker of they M1 decrease macrophages, expression NO oflevel, proinflammatory was not changed; cytokines however, in LPS-activated they decrease macrophages expression that mayof proinflammatory indicate that these cytokines compounds in mayLPS-activated be potent macrophages in reprograming that the may polarization indicate that of macrophagesthese compounds via the may M1-type be potent way. in reprograming the polarization of macrophages via the M1-type way. 2.1.2. Sea Anemones

2.1.2.Sea Sea anemonesAnemones (Actiniaria) is an order of cnidarians belonging to class Anthozoa, subclass Hexacorallia. They remain poorly studied regarding their potential anti-inflammatory effects [91]. Sea anemones (Actiniaria) is an order of cnidarians belonging to class Anthozoa, subclass The presumable activities of water extracts from two species, Actinia equina (Linnaeus, 1767) and Hexacorallia. They remain poorly studied regarding their potential anti-inflammatory effects [91]. Anemonia sulcata (Pennant, 1777) from Portugal, were examined in RAW264.7 cells. Both preparations The presumable activities of water extracts from two species, Actinia equina (Linnaeus, 1767) and were able to decrease the NO levels in LPS (1 µg/mL)-treated cells (22 h of incubation), but at different Anemonia sulcata (Pennant, 1777) from Portugal, were examined in RAW264.7 cells. Both concentrations. A. sulcata inhibited it in a dose-dependent way with the half maximal inhibitory preparations were able to decrease the NO levels in LPS (1 μg/mL)-treated cells (22 h of incubation), concentration (IC ) of 0.374 mg/mL, and A. equina—at concentrations of 0.125 mg/mL, 0.25 and but at different concentrations.50 A. sulcata inhibited it in a dose-dependent way with the half maximal 0.5 mg/mL (IC50 was not determined). The purine alkaloid homarine (38) (Figure8), the major inhibitory concentration (IC50) of 0.374 mg/mL, and A. equina—at concentrations of 0.125 mg/mL, metabolite found in both extracts, also reduced NO levels. The enhanced level of ROS, another

Mar. Drugs 2020, 18, x FOR PEER REVIEW 9 of 27

0.25 and 0.5 mg/mL (IC50 was not determined). The purine alkaloid homarine (38) (Figure 8), the Mar. Drugs 2020, 18, 37 9 of 27 major metabolite found in both extracts, also reduced NO levels. The enhanced level of ROS, another marker of macrophage activity, was decreased by both extracts, but not homarine, at a 0.0625 mg/mL concentration.marker of macrophage Both extracts activity, and was homarine decreased also by si bothgnificantly extracts, inhibited but not homarine, activity of at PLA2, a 0.0625 which mg/mL is responsibleconcentration. for production Both extracts of prostaglandin and homarine E2 also [6], significantly the extract from inhibited A. equina activity and ofhomarine PLA2, which(each at is concentrationsresponsible for of production 0.5 and 1 ofmg/mL) prostaglandin being more E2 [ 6potent], the extractcompared from toA. the equina extractand of homarine A. sulcata. (each These at factsconcentrations indicate that of 0.5 the and extracts 1 mg/mL) and being homarine more may potent inhibit compared an early to the stage extract of oftheA. signaling sulcata. These pathway facts leadingindicate to that M1-type the extracts macrophages. and homarine In relation may inhibit to such an early parameters stage of studied, the signaling as NO pathway production leading and to PLA2M1-type inhibition, macrophages. homarine In relation displayed to such significant parameters activity studied, that apparently as NO production could be and responsible PLA2 inhibition, for the macrophagehomarine displayed polarization, significant but not activity antioxidant that apparently activity of could the beextracts. responsible Hence, for the the extracts macrophage and homarinepolarization, have but a notpotential antioxidant in modulating activity ofmacropha the extracts.ge polarization Hence, the and extracts redirecting and homarine it towards have the a M2potential phenotype. in modulating macrophage polarization and redirecting it towards the M2 phenotype.

FigureFigure 8. 8. StructureStructure of of homarine. homarine. 2.2. Porifera 2.2. Porifera The phylum Porifera contains about 5500 species, which are considered the most prolific regarding The phylum Porifera contains about 5500 species, which are considered the most prolific their pharmacological potential. They are a rich source of a variety of secondary metabolites including regarding their pharmacological potential. They are a rich source of a variety of secondary terpenoids, macrolides, and sterols [92]. Nevertheless, studies on their anti-inflammatory activities are metabolites including terpenoids, macrolides, and sterols [92]. Nevertheless, studies on their mostly at the early stage. anti-inflammatory activities are mostly at the early stage. Methanol extracts of Hyrtios and Haliclona species (Demospongiae), collected near Kosrae Island, Methanol extracts of Hyrtios and Haliclona species (Demospongiae), collected near Kosrae Micronesia, Central Pacific, were examined for their activities in the LPS (1 µg/mL)-treated for 24 h Raw Island, Micronesia, Central Pacific, were examined for their activities in the LPS (1 μg/mL)-treated 264.7 cells [93]. Both extracts (25, 50, and 100 µg/mL each) exhibited dose-dependent ability to reduce for 24 h Raw 264.7 cells [93]. Both extracts (25, 50, and 100 μg/mL each) exhibited dose-dependent NO generation, and at concentration of 10 µg/mL, the extracts inhibited the proinflammatory cytokine ability to reduce NO generation, and at concentration of 10 μg/mL, the extracts inhibited the IL-1β production, whose level characterizes M1 macrophage phenotype [12]. This research shows the proinflammatory cytokine IL-1β production, whose level characterizes M1 macrophage phenotype potential of the extracts as anti-inflammatory drug candidates with macrophage polarizing properties. [12]. This research shows the potential of the extracts as anti-inflammatory drug candidates with Similarly, ethanol extracts from the species of two other sponge genera, Mycale (Oxymycale) acerata macrophage polarizing properties. (Kirkpatrick, 1907), Isodictya erinacea (Topsent, 1916), and Isodictya toxophila (Burton, 1932) collected Similarly, ethanol extracts from the species of two other sponge genera, Mycale (Oxymycale) from the Eastern Weddell Sea and the South Shetland Islands were used for screening their impact on acerata (Kirkpatrick, 1907), Isodictya erinacea (Topsent, 1916), and Isodictya toxophila (Burton, 1932) inflammatory mediators at non-toxic concentrations (50, 125, and 250 µg/mL for each species) in the LPS collected from the Eastern Weddell Sea and the South Shetland Islands were used for screening their (1 µg/mL)-stimulated Raw 264.7 cells [94]. The effects of the extracts were distinct regarding different impact on inflammatory mediators at non-toxic concentrations (50, 125, and 250 μg/mL for each markers. Thus, the extract of Isodictya erinacea inhibited significantly and dose-dependently the release species) in the LPS (1 μg/mL)-stimulated Raw 264.7 cells [94]. The effects of the extracts were distinct of IL-1β and PGE , although not affecting LTB The extracts of the sponge Isodictya toxophila decreased regarding different2 markers. Thus, the extract4. of Isodictya erinacea inhibited significantly and the release of IL-1β and LTB4 at two non-cytotoxic concentrations (50 and 125 µg/mL) and also dose-dependently the release of IL-1β and PGE2, although not affecting LTB4. The extracts of the diminished PGE2 release at the intermediate concentration (125 µg/mL). Mycale (Oxymycale) acerata’s sponge Isodictya toxophila decreased the release of IL-1β and LTB4 at two non-cytotoxic extract decreased the release of all inflammatory mediators studied IL-1β, PGE2, and LTB4 at the lower concentrations (50 and 125 μg/mL) and also diminished PGE2 release at the intermediate concentration of 50 µg/mL. Taking into consideration, the role of these mediators in the mechanisms concentration (125 μg/mL). Mycale (Oxymycale) acerata’s extract decreased the release of all of polarization to M1-type macrophages [12], the extracts, especially that from Mycale (Oxymycale) inflammatory mediators studied IL-1β, PGE2, and LTB4 at the lower concentration of 50 μg/mL. aceratas, seem to have potential for pharmaceutical application as macrophage targeting agents. Taking into consideration, the role of these mediators in the mechanisms of polarization to M1-type The extract [dichloromethane/methanol (1:1)] (100 µg/mL) of another species of the Isodictya genus, macrophages [12], the extracts, especially that from Mycale (Oxymycale) aceratas, seem to have Isodictya palmata (Ellis & Solander, 1786), inhabiting the North Atlantic, was also shown to reduce the potential for pharmaceutical application as macrophage targeting agents. release of pro-inflammatory cytokines (IL-12 and IL-10) in human monocyte-derived dendritic cells [95]. The extract [dichloromethane/methanol (1:1)] (100 μg/mL) of another species of the Isodictya Moreover, this study revealed that the extract reduced CD86 expression. CD86 is one of the cell genus, Isodictya palmata (Ellis & Solander, 1786), inhabiting the North Atlantic, was also shown to surface markers for M1-type macrophages [96], and the findings clearly demonstrate the involvement reduce the release of pro-inflammatory cytokines (IL-12 and IL-10) in human monocyte-derived of polarization of macrophages to an M2 type in the mechanisms of the influence of the extract on dendritic cells [95]. Moreover, this study revealed that the extract reduced CD86 expression. CD86 is macrophages and indicate that the extract has potential in application as a macrophage-targeted anti-inflammatory drug. Mar. Drugs 2020, 18, x FOR PEER REVIEW 10 of 27 Mar. Drugs 2020, 18, x FOR PEER REVIEW 10 of 27 one of the cell surface markers for M1-type macrophages [96], and the findings clearly demonstrate theone involvement of the cell surface of polarization markers forof macrophages M1-type macrop to anhages M2 type [96], in and th ethe mechanisms findings clearly of the demonstrateinfluence of the involvement of polarization of macrophages to an M2 type in the mechanisms of the influence of theMar. Drugsextract2020 on, 18 , 37macrophages and indicate that the extract has potential in application 10as of 27a macrophage-targetedthe extract on macrophages anti-inflammatory and indicate drug. that the extract has potential in application as a macrophage-targetedThe identified compound anti-inflammatory girolline drug. (39 ) (Figure 9), isolated from the marine sponge PseudoaxinyssaTheThe identifiedidentified cantharella compound compound (Demospongiae), girolline girolline (39) (Figurewas(39) evaluated9(Figure), isolated 9),for from itsisolated theanti-inflammatory marine from sponge the marine Pseudoaxinyssaactivity sponge using thecantharellaPseudoaxinyssa model of(Demospongiae), stimulation cantharella of(Demospongiae), cell was response evaluated by was forflag itsevaluatedelline-induced anti-inflammatory for its activation anti-inflammatory activity of the using Toll-like activity the modelreceptor using of (TLR),stimulationthe model followed of of stimulation cell by response the activation of by cell flagelline-induced response of the nuclear by flag transcription activationelline-induced of thefactor activation Toll-like NF-κB receptorof and the the Toll-like (TLR), production followed receptor of proinflammatoryby(TLR), the activationfollowed by ofcytokines thethe nuclearactivation [96]. transcriptionGirolline of the nuclear(2 μ factorg/mL) transcription NF-almostκB andcompletely factor the production NF- abolishedκB and of NF-the proinflammatory κproductionB activity inof THP1-derivedcytokinesproinflammatory [96]. macrophages Girolline cytokines (2 µ[96].andg/mL) Girollinedecreased almost (2 completelythe μg/mL) secret almostion abolished of thecompletely pro-inflammatory NF-κB abolished activity in NF-cytokine THP1-derivedκB activity IL-6 inin bothmacrophagesTHP1-derived flagellin-treated and macrophages decreased human the andpe secretionripheral decreased ofblood the the pro-inflammatory mononuclear secretion of thecells cytokine pro-inflammatory and THP1-derived IL-6 in both cytokine flagellin-treated macrophages IL-6 in [97].humanboth Elongationflagellin-treated peripheral factor blood human 2 mononuclearwas pedefinedripheral cellsto bloodbe and a molecularTHP1-derivedmononuclear target cells macrophages of andgirolline. THP1-derived [97 It]. is Elongation noteworthy macrophages factor that 2 flagellinewas[97]. defined Elongation is known to be factor a molecularto be 2 wasthe TLR5 targetdefined ligand, of to girolline. be while a molecular It LPS is noteworthy is TLR4target agonist of that girolline. flagelline [98]. Apparently,It is knownnoteworthy tothe be data that the obtainedTLR5flagelline ligand, with is known whilegirolline LPSto onbe is thisthe TLR4 modelTLR5 agonist ligand,need [98 to ].while be Apparently, proved LPS iswith TLR4 the a datawell-known agonist obtained [98]. inducer withApparently, girolline of macrophage the on data this polarizationmodelobtained need with to begirolline sure proved on on its with this potency amodel well-known as need macrophage to inducerbe proved polarization of macrophagewith a well-known modulator. polarization inducer to of be macrophage sure on its potencypolarization as macrophage to be sure on polarization its potency modulator. as macrophage polarization modulator.

Figure 9. Structure of girolline. FigureFigure 9.9. StructureStructure ofof girolline.girolline. 2.3. Mollusca Mollusca 2.3. Mollusca Phylum Mollusca contains about 50,000 species [99], [99], and only a small proportion of them were Phylum Mollusca contains about 50,000 species [99], and only a small proportion of them were used as as a a source source for for anti-inflammatory anti-inflammatory preparations preparations and/or and /wereor were tested tested for in for order in order to highlight to highlight their used as a source for anti-inflammatory preparations and/or were tested for in order to highlight their immunomodulatorytheir immunomodulatory properti propertieses [100]. [The100 ].muricidae The muricidae molluscs molluscs are known are known for their for production their production of the immunomodulatory properties [100]. The muricidae molluscs are known for their production of the Tyrianof the Tyrianpurple purpledye, and dye, some and of some the dye of the precursors dye precursors,, e.g., tyrindoleninone e.g., tyrindoleninone (Figure 10), (Figure with 10 specific), with Tyrian purple dye, and some of the dye precursors, e.g., tyrindoleninone (Figure 10), with specific anticancerspecific anticancer activity [3]. activity Hypobranchial [3]. Hypobranchial gland and gland egg andchloroform egg chloroform extracts, extracts,and brominated and brominated indoles anticancer activity [3]. Hypobranchial gland and egg chloroform extracts, and brominated indoles fromindoles the from Australian the Australian muricid muricid DicathaisDicathais orbita (Gmelin, orbita (Gmelin, 1791) (Gastropoda, 1791) (Gastropoda, Neogastropoda), Neogastropoda), were from the Australian muricid Dicathais orbita (Gmelin, 1791) (Gastropoda, Neogastropoda), were testedwere tested on LPS-stimulated on LPS-stimulated RAW264 RAW264.7.7 macrophages macrophages for the for capacity the capacity to modulate to modulate expression expression of the of tested on LPS-stimulated RAW264.7 macrophages for the capacity to modulate expression of the markersthe markers of inflammation. of inflammation. Both Both hypobranchial hypobranchial gland gland and and egg egg chloroform chloroform extracts extracts inhibited inhibited the markers of inflammation. Both hypobranchial gland and egg chloroform extracts inhibited the production of NO in LPS-stimulated RAW264.7 macrophages with IC50 of 30.8 μg/mL and 40 μg/mL, production of NO in LPS-stimulated RAW264.7 macrophages with IC50 of 30.8 µg/mL and 40 µg/mL, production of NO in LPS-stimulated RAW264.7 macrophages with IC50 of 30.8 μg/mL and 40 μg/mL, respectively. The hypobranchial gland extract also inhibited the production of TNFα with IC50 of respectively. The hypobranchial gland extract also inhibited the production of TNFα with IC50 of respectively. The hypobranchial gland extract also inhibited the production of TNFα with IC50 of 43.03 µμgg/mL./mL. IsolatedIsolated from from the the extract extract tyrindoleninone tyrindoleninone (40 ()40 and) and tyriverdin tyriverdin (42) ( were42) were tested tested along along with 43.03 μg/mL. Isolated from the extract tyrindoleninone (40) and tyriverdin (42) were tested along witha range a range of synthetic of synthetic brominated brominated indole indole derivatives, derivatives, and mono-brominated and mono-brominated indole indole (41) was (41 shown) was with a range of synthetic brominated indole derivatives, and mono-brominated indole (41) was shownmost active most in active inhibition in inhibition of NO production, of NO production which indicates, which indicates that the that position the position of the bromine of the bromine atom on shown most active in inhibition of NO production, which indicates that the position of the bromine atomthe isatin on the (43 )isatin benzene (43) ring benzene is important ring is forimportant immunomodulating for immunomodulating activity. The activity. chemical The structure chemical of atom on the isatin (43) benzene ring is important for immunomodulating activity. The chemical structurecompounds of iscompounds shown in Figure is shown 10. Apparently,in Figure 10. brominated Apparently, indoles brominated from muricids indoles are from potent muricids for use are in structure of compounds is shown in Figure 10. Apparently, brominated indoles from muricids are potentmacrophage for use reprogramming. in macrophage reprogramming. potent for use in macrophage reprogramming.

Figure 10. 40 41 42 43 Figure 10. TheThe structure structure of of tyrindoleninone tyrindoleninone ( 40),), 6-bromoistain 6-bromoistain ( (41), tyriverdin ( 42), and isatin (43). Figure 10. The structure of tyrindoleninone (40), 6-bromoistain (41), tyriverdin (42), and isatin (43). Aplysia depilans (Gmelin, 1791) (Gastropoda, Aplysiida) is distributed in the Mediterranean Sea and in the Atlantic Ocean. In the methanol extract from A. depilans twenty-two fatty acids, cis-5,8,11,14-eicosatetraenoic (arachidonic acid), being the major one, and eight carotenoids (zeaxanthin Mar. Drugs 2020, 18, 37 11 of 27 as the major xanthophyll, fucoxanthin and two isomers, neoxanthin, lutein, zeaxanthin, and α- and β-carotene) were identified [101]. The extract of the A. depilans digestive gland was able to decrease the NO level in a dose-dependent way (IC50 value of 0.663 mg/mL) in LPS (1 µg/mL) -exposed RAW 264.7 cells, incubated for 18 h. This suggests that the anti-inflammatory capacity of the extract may be due to the inhibition of iNOS expression and, possibly, involves inhibition of macrophage polarization through the M1-way. The anti-inflammatory activity of the A. depilans digestive gland obviously may be partially resulted from the activity of the metabolites identified, mainly with unsaturated fatty acids and carotenoids. Cis-5,8,11,14-eicosatetraenoic, being n-6 polyunsaturated fatty acid (PUFA), is considered a pro-inflammatory fatty acid, once it is a precursor of the synthesis of eicosanoids, increasing the inflammatory response [102]. On the other hand, long-chain n-3 PUFAs, such as cis-5,8,11,14,17-eicosapentaenoic (EPA) and cis-4,7,10,13,16,19-docosahexaenoic (DHA) acids, are known also to inhibit iNOS expression [103], decrease the production of inflammatory mediators (eicosanoids, cytokines and NO) [102], and shift M1/M2 macrophage balance towards the M2 phenotype [32]. Carotenoids identified in this work were shown earlier to influence the expression of the markers of inflammation and macrophage polarization. Thus, zeaxanthin and fucoxanthin inhibited the NO production, as well as expression of iNOS, COX-2, and pro-inflammatory cytokines [104]. Hence, the extract may be considered a potent M1–M2 modulator due to its constituents’ fatty acids and carotenoids identified. The anti-inflammatory preparation Lyprinol® is an example of practical application of fatty acid-rich extract from mollusks. Lyprinol® is prepared from New Zealand green-lipped mussel Perna canaliculus (Gmelin, 1791) (Bivalvia, Mytilida) and contains five main lipid classes including sterol esters, triglycerides, sterols, polar lipids and free fatty acids, mainly EPA and DHA, and also carotenoids. Lyprinol subfractions inhibit PGE2 production by activated macrophages [105]. Moreover, in human THP-1 monocytes activated by a combination of IFN-γ (10 ng/mL) and LPS (1 µg/mL) during 16 h incubation, the extract reduced the production of pro-inflammatory cytokins TNF-α and IL-12 [106]. As the standard combination for activation of M1 stage was used in this study, the ability of the extract to decrease the level of M1-macrophage markers demonstrates that the preparation can promote switching macrophage polarization towards M2-type macrophages. The influence of the ratio of n-3 PUFA and n-6 PUFA for anti-inflammatory activity of the preparation was demonstrated in the studies on the Mediterranean molluscs belonging to different genera: Armina maculata (Rafinesque, 1814) and Armina tigrina (Rafinesque, 1814) (Gastropoda, Nudibranchia) on the one side and Aglaja tricolorata (Renier, 1807) (Gastropoda, Cephalaspidea) on the another [107]. In addition, 23, 26, and 18 fatty acids (FA) were identified in the extracts of A. maculata, A. tigrina, and A. tricolorata, respectively. A. tricolorata’s extract exhibited higher proportion of n-3 PUFA (n-6/n-3 < 1) in contrast with the A. maculata and A. tigrina with a n-6/n-3 ratio higher than 1.75. The significantly higher reduction of NO levels was shown in LPS-challenged RAW 264.7 macrophages exposed to A. tricolorata’s extract compared to the effects of the extracts of two other species. Since n-3 PUFA have been reported as anti-inflammatory agents influencing cell signaling and macrophage polarization [103,104], the higher proportion of those FA in A. tricolorata’s extract is a possible reason of such different effects of extracts studied. A non-standard approach to evaluate the compound bioactivity was suggested in the case of bone remodeling when two opposite physiological processes are executed by the two types of cells. Osteoclasts and osteoblasts play opposite roles in this process and osteoclasts are considered a primary target for the treatment of bone resorption [108]. To test the capacity of fermented extract (FO) of the Pacific oyster Crassostrea gigas (Thunberg, 1793) (Bivalvia, Ostreida) to prevent osteoclast differentiation, induced by the receptor activator of NF-κB ligand (RANKL), the RAW 264.7 cells were used as osteoclast precursors. ROS are known to promote the transcription of osteoclast-specific genes in RANKL-induced osteoclasts [109]; therefore, the ROS level is an important marker of an osteoclast functional phenotype. FO was shown to decrease RANKL-stimulated ROS production and to inhibit RANKL-induced nuclear translocation of NF-κB in RAW 264.7 cells. Taking into consideration that Mar. Drugs 2020, 18, x FOR PEER REVIEW 12 of 27 Mar. Drugs 2020, 18, 37 12 of 27 RANKL-stimulated ROS production and to inhibit RANKL-induced nuclear translocation of NF-κB in RAW 264.7 cells. Taking into consideration that FO inhibited the rise of markers which FOcharacterize inhibited the the polarization rise of markers of macrophages which characterize via M1-type the polarization (ROS, nuclear of macrophagestranslocation viaof NF- M1-typeκB), it κ (ROS,can be nuclear suggested translocation that FO is ofable NF- alsoB), to itpreven can bet classical suggested macrophage that FO is polarization able also to preventinduced classicalthrough κ macrophagethe NF-κB-associated polarization ways. induced However, through this the suggestion NF- B-associated must be ways. verified However, in the thisstandard suggestion conditions must bewith verified using inthe the known standard activators conditions of macrop withhage using polarization the known activatorstowards the of M1-type. macrophage polarization towardsRecently, the M1-type. the most fundamental studies of the anti-inflammatory mechanisms of mollusk-derivedRecently, the mostpreparations fundamental were studies reported of the by anti-inflammatory Dong et al. [110]. mechanisms The water of extract mollusk-derived of widely preparationsdistributed in were the reportedworld ocean by Dong scallop et al.Atrina [110 ].pectinata The water (Glenn, extract 1904) of widely (Bivalvia, distributed Ostreida) in was the worldtested oceanfor anti-inflammatory scallop Atrina pectinata activity (Glenn,in LPS (250 1904) ng/mL (Bivalvia,)-stimulated Ostreida) RAW was 264.7 tested cells. for The anti-inflammatory scallop extract activityeffectively in LPSreduced (250 ngthe/mL)-stimulated synthesis of NO, RAW ROS 264.7generation cells. Theand scallopthe expression extract eofff ectivelyIL-6 and reduced TNF-α. The the α synthesismechanisms of NO,of such ROS reduction generation were and theexamined expression and ofhighlighted IL-6 and TNF-as related. The to mechanisms downregulation of such of reductionMAPK (JNK, were p38 examined and ERK) and and highlighted NF-κB signaling. as related toAccording downregulation to these of findings, MAPK (JNK,mechanisms p38 and of ERK) the κ andanti-inflammatory NF- B signaling. effect According of the toextract these findings,were real mechanismsized via the of same the anti-inflammatory ways that are described effect of thefor extractpolarization were realizedof macropha via theges same towards ways the that M2-type. are described for polarization of macrophages towards the M2-type.In addition to PUFA, the digestive glands of some mollusk species contain a large amount of etherIn lipids addition 1-O to-alkyl-2,3-diacyl-sn-glycerols PUFA, the digestive glands of which some molluskare formed species by containfatty acids a large and amount fatty alcohols of ether lipids(compounds 1-O-alkyl-2,3-diacyl-sn-glycerols 44, 45 in Figure 11). The which1-O-alkyl-sn-glycerols are formed by fatty were acids isolat anded fatty from alcohols the digestive (compounds gland 44of, 45thein squid Figure Berryteuthis 11). The 1- magisterO-alkyl-sn-glycerols (Berry, 1913) were (Cephalopoda, isolated from Oegopsida). the digestive This gland compound of the squid was Berryteuthisdescribed to magister cause an(Berry, increase 1913) in (Cephalopoda,the level of ROS Oegopsida). and the synthesis This compound of NO and was IL-6 described expression to causein the anRAW264.7 increase incell the line level at of non-to ROS andxic concentrations the synthesis of of NO 0.1–5 and IL-6μg/mL expression [111]. This in the effect RAW264.7 was comparable cell line at µ non-toxicwith that concentrations produced by ofLPS 0.1–5 (1 μgg/mL)./mL [111 This]. This implies effect wasthat comparablethe compound with thatmay produced be a promising by LPS µ (1immunostimulantg/mL). This implies agent. that However, the compound further mayresearch be a has promising to be performed immunostimulant for understanding agent. However, whether furthermacrophage research polarization has to be performed mechanisms for are understanding involved in whetherthis effect. macrophage polarization mechanisms are involved in this effect.

Figure 11. Structures of the alkyl glycerols: (44) chimyl alcohol; (45) batyl alcohol. Figure 11. Structures of the alkyl glycerols: (44) chimyl alcohol; (45) batyl alcohol. 2.4. Crustacea 2.4. CrustaceaThe sub-phylum Crustacea (Arthropoda) holds a massive biodiversity as it includes about 68,000 species [99]. Lipids of crustaceans are mostly studied for their bioactive properties [112]. The sub-phylum Crustacea (Arthropoda) holds a massive biodiversity as it includes about However, classes of Crustacea are distinctly different from each other regarding the main constitutive 68,000 species [99]. Lipids of crustaceans are mostly studied for their bioactive properties [112]. compounds. Thus, more than 80% of marine copepod Calanus oil consists of wax esters, i.e., long-chain However, classes of Crustacea are distinctly different from each other regarding the main fatty alcohols linked to long-chain fatty acids [113]. Lipids of shrimps consist of free fatty acids, constitutive compounds. Thus, more than 80% of marine copepod Calanus oil consists of wax esters, triglycerides, carotenoids and other lipids [114]. Carotenoids include astaxhantin (3,3’-dihydroxy-β, i.e., long-chain fatty alcohols linked to long-chain fatty acids [113]. Lipids of shrimps consist of free β’-carotene-4,4’-dione), astaxanthin esters, β-criptoxanthin, α-carotene, β-carotene, meso-zeaxanthin, fatty acids, triglycerides, carotenoids and other lipids [114]. Carotenoids include astaxhantin canthaxanthin, lutein, zeaxanthin and crustacyanin, and some of these compounds were reported to (3,3’-dihydroxy-β, β’-carotene-4,4’-dione), astaxanthin esters, β-criptoxanthin, α-carotene, have anti-inflammatory and macrophage activity modulating properties [115]. Crabs (Decapoda) are β-carotene, meso-zeaxanthin, canthaxanthin, lutein, zeaxanthin and crustacyanin, and some of these essentially constituted by xanthophylls, astaxanthin esters being the main compounds [116]. compounds were reported to have anti-inflammatory and macrophage activity modulating Thus, lipids of the Antarctic krill Euphausia superba (Malacostraca) are rich in n-3 PUFA EPA (12%) properties [115]. Crabs (Decapoda) are essentially constituted by xanthophylls, astaxanthin esters and DHA (7%) [117]. The high content of the two biologically active components, EPA and DHA, are being the main compounds [116]. responsible for the majority of physiological e ects of krill oil [118]. Thus, lipids of the Antarctic krill Euphausiaff superba (Malacostraca) are rich in n-3 PUFA EPA ® (12%)The and anti-inflammatory DHA (7%) [117]. mechanisms The high content of FlexPro of the MD two (FP-MD),biologically a novel active multi-ingredient components, EPA dietary and supplement,DHA, are responsible consisting for of the a mixturemajority of of krillphysiological oil, astaxanthin effects andof krill hyaluronic oil [118]. acid were studied in RAW264.7 macrophage cells challenged with l µg/mL LPS [119]. FP-MD significantly inhibited the The anti-inflammatory mechanisms of FlexPro MD® (FP-MD), a novel multi-ingredient dietary α β mRNAsupplement, levels consisting of pro-inflammatory of a mixture cytokines of krill oil, IL-6, astaxanthin TNF- and and IL-1 hyaluronic, and, in acid contrast, were itstudied elevated in theRAW264.7 mRNA macrophage levels of anti-inflammatory cells challenged cytokine with l μg/mL IL-10. LPS In addition,[119]. FP-MD FP-MD significantly reduced LPS-inducedinhibited the

Mar. Drugs 2020, 18, 37 13 of 27 phosphorylation levels of NF-κB p65 and inhibitor of κB-α (IκB-α). The changes in the functional markers indicate that FP-MD could shift the macrophage balance by increasing M2 type activity and inhibiting the M1-type in RAW264.7 macrophage cells, and, most possibly, carotenoids and n-3 PUFA contributed to this effect. The expression levels of pro-inflammatory cytokines and inflammatory markers also reduced in mice with LPS-induced inflammatory arthritis after treatment with FP-MD that suggests possibility of involvement of the mechanisms including inhibiting NF-κB in the anti-inflammatory effects of FP-MD. Similarly, krill oil from Euphausia superba (80 mg/kg/day for one month) [117] effectively inhibited the expression of iNOS and cyclooxygenase-2 COX-2 induced by the administration of LPS (250 µg/kg, 7 days) and decreased ROS and malondialdehyde levels in mice brain. Studies of the major components of krill oil EPA and DHA in microglia (CNS macrophage)-like BV-2 cell line revealed that these PUFAs dose-dependently decreased LPS (1 µg/mL)-induced nitric oxide and ROS generation, COX-2 and iNOS expression as well as NF-κB activity in model macrophages after 24 h of incubation. These results confirm the data on the macrophage-polarization modulating capacity of n-3 PUFA [102] and suggest that anti-inflammatory effect of krill oil in mice may result from its capacity to negatively influence classical activation of microglial cells. However, further studies in vivo are necessary to clarify the mechanisms of the anti-inflammatory effects of krill oil, as the direct inhibition of Th1 activity, possibly, through induction of apoptosis, but not increase in polarization of T-cells towards Th2 phenotype, associated with macrophage polarization, was reported at least ex vivo [120]. A focus on carotenoid ingredients of shrimps was made in the studies of anti-inflammatory properties of a shrimp waste extract [121]. Comparison of shrimp extract and commercial astaxanthin showed a suppressive effect of those preparations on the generation of O2(-), NO and TNF-α secretion in LPS-induced rat alveolar macrophages, while purified shrimp astaxanthin did not suppress O2(-) production, and the effect of shrimp extract on TNF-α secretion was more pronounced. These findings support the suggestion about the ability of the shrimp extracts to influence macrophage functional phenotypes through an alternative way, and indicate that it may be partly caused by the presence of astaxanthin. Crustaceans of the order Decapoda (crab, shrimp, prawn, and lobster) are also a valuable source of chitin [122]. Being isolated from tissues, purified chitin is a plain polysaccharide. There is a large body of knowledge on the use of chitosans as biomaterials, and, apparently most important, as drug carriers. However, of special interest is the fact that some of the structural constituents of chitosan may modulate the effects of other molecules, which can basically promote the drug delivery targeting M1 or M2 macrophages dependent on the aim of application. Thus, treatment of RAW264.7 macrophages with a combination of chitosan hydrolysate/chitooligosaccharide mixture and IFN-γ significantly induced NO production in a dose-dependent manner, while a low-molecular weight chitosan inhibited NO production in combination with IFN-γ [123]. Both hydrolysate and chitooligosaccharide mixture through binding to the receptors of CD14, TLR4, which are the markers of M1 macrophages [8,96], promoted the migration of NF-κB into the nucleus, and a specific inhibitor of NF-κB blocked NO production. Apparently, different chitosan derivatives may modulate the effects of inflammation mediators in a different manner, may be, through distinct capacities in binding the cell surface receptors/changing the cell phenotype. Taking into consideration that tumor-associated macrophages (TAMs) have the protumoral functions, and their polarization via M2-type way is important for tumor progression [124], the capacity of the compounds to induce proinflammatory way of macrophage activation indicates a potential of these compounds in treatment of cancer.

2.5. Echinodermata Some of the most familiar seashore animals are members of the phylum Echinodermata. The phylum contains about 7000 living species, including the crinoids, asteroids (sea stars), ophiuroids (brittle stars), echinoids (sea urchins), and holothurians (sea cucumbers) [99]. Mar. Drugs 2020, 18, x FOR PEER REVIEW 14 of 27 Mar. Drugs 2020, 18, x FOR PEER REVIEW 14 of 27

2.5.2.5. EchinodermataEchinodermata SomeSome ofof thethe mostmost familiarfamiliar seashoreseashore animalsanimals areare membersmembers ofof thethe phylumphylum Echinodermata.Echinodermata. TheThe phylumphylum containscontains aboutabout 70007000 livingliving species,species, includinincludingg thethe crinoids,crinoids, asteroidsasteroids (sea(sea stars),stars), ophiuroidsophiuroids Mar. Drugs 2020, 18, 37 14 of 27 (brittle(brittle stars),stars), echinoidsechinoids (sea(sea urchins),urchins), andand holothuriansholothurians (sea(sea cucumbers)cucumbers) [99].[99].

2.5.1. Crinoidea 2.5.1.2.5.1. Crinoidea To date, there are about 600 species of crinoids [125]. Their small number and mostly deep-sea ToTo date,date, therethere areare aboutabout 600600 speciesspecies ofof crinoidscrinoids [[125].125]. TheirTheir smallsmall numbernumber andand mostlymostly deep-seadeep-sea habitat apparently result in a lack of studies on their anti-inflammatory capacities. Attention was habitathabitat apparentlyapparently resultresult inin aa lacklack ofof studiesstudies onon theirtheir anti-inflammatoryanti-inflammatory capacities.capacities. AttentionAttention waswas mostly paid to naphthopyrone compounds. The two naphthopyrones 6-methoxycomaparvin (46) mostlymostly paidpaid toto naphthopyrone naphthopyrone compounds. compounds. The Th twoe two naphthopyrones naphthopyrones 6-methoxycomaparvin 6-methoxycomaparvin (46) and(46) and 6-methoxycomaparvin 5-methyl ether (47) were isolated from a bioactive methanol-soluble 6-methoxycomaparvinand 6-methoxycomaparvin 5-methyl 5-methyl ether ether (47) were (47) isolatedwere isolated from a from bioactive a bioactive methanol-soluble methanol-soluble extract extract of the Fijian crinoid Comanthus parvicirrus (Müller, 1841) (Figure 12) [126]. It was shown that ofextract the Fijian of the crinoid Fijian crinoidComanthus Comanthus parvicirrus parvicirrus(Müller, (Müller, 1841) (Figure 1841) (Figure 12)[126 12)]. It[126]. was It shown was shown that these that these compounds completely inhibit TNF-alpha-induced NF-κB activation in the leukemia cell line compoundsthese compounds completely completely inhibit inhibit TNF-alpha-induced TNF-alpha-induced NF-κB activationNF-κB activation in the leukemia in the leukemia cell line cell K562 line at K562 at a relatively high concentration of 300 μM. This indicates possible slight anti-inflammatory aK562 relatively at a relatively high concentration high concentration of 300 µ M.of 300 This μ indicatesM. This indicates possible slightpossible anti-inflammatory slight anti-inflammatory effect of effect of those substances. thoseeffect substances.of those substances.

Figure 12. Structure of naphthopyrones of the Comanthus parvicirrus. (46) 6-methoxycomaparvin; (47) FigureFigure 12. StructureStructure of of naphthopyrones naphthopyrones of of the the ComanthusComanthus parvicirrus parvicirrus. (46.() 6-methoxycomaparvin;46) 6-methoxycomaparvin; (47) 6-methoxycomaparvin 5-methyl ether. (6-methoxycomaparvin47) 6-methoxycomaparvin 5-methyl 5-methyl ether. ether.

Further studies on the bioactivity of the species of the same genus Comanthus bennetti FurtherFurther studiesstudies onon thethe bioactivitybioactivity ofof thethe speciesspecies ofof thethe samesame genusgenus ComanthusComanthus bennettibennetti revealedrevealed revealed that the crude extract significantly inhibited the expression of pro-inflammatory thatthat thethe crudecrude extractextract significantlysignificantly inhibitedinhibited ththee expressionexpression ofof pro-inflammatorypro-inflammatory proteinsproteins inin proteins in LPS-stimulated RAW 264.7 cells [127]. Isolated from the extract comaparvin LPS-stimulatedLPS-stimulated RAWRAW 264.7264.7 cellscells [127].[127]. IsolatedIsolated fromfrom thethe extractextract comaparvincomaparvin (5,8-dihydroxy-10-methoxy-2-propylbenzo[h]chromen-4-one) (compound 48 in Figure 13) at (5,8-dihydroxy-10-methoxy-2-propyl(5,8-dihydroxy-10-methoxy-2-propylbenzo[h]chromen-4-one)benzo[h]chromen-4-one) (compound(compound 4848 inin FigureFigure 13)13) atat concentrations of 1, 10, 25, and 50 µM dose-dependently decreased the expression of iNOS protein and concentrationsconcentrations ofof 1,1, 10,10, 25,25, andand 5050 μμMM dose-dependentlydose-dependently decreaseddecreased thethe expressionexpression ofof iNOSiNOS proteinprotein mRNA in LPS (0.01 µg/mL)-stimulated for 16 h macrophage cells, but did not inhibit cyclooxygenase-2 andand mRNAmRNA inin LPSLPS (0.01(0.01 μμg/mL)-stimulatedg/mL)-stimulated forfor 1616 hh macropmacrophagehage cells,cells, butbut diddid notnot inhibitinhibit (COX-2) expression. Apparently, the comaparvin-induced anti-inflammatory effects observed show cyclooxygenase-2cyclooxygenase-2 (COX-2)(COX-2) expression.expression. ApparentlyApparently,, thethe comaparvin-inducedcomaparvin-induced anti-inflammatoryanti-inflammatory downstream COX-2 expression. However, changes in the marker of M1 macrophage suggest that the effectseffects observedobserved showshow downstreamdownstream COX-2COX-2 expression.expression. However,However, changeschanges inin thethe markermarker ofof M1M1 mechanisms of the anti-inflammatory e ect of the compound may involve the inhibition of classical macrophagemacrophage suggestsuggest thatthat thethe mechanismsmechanismsff ofof thethe anti-inflammatoryanti-inflammatory effecteffect ofof thethe compoundcompound maymay activationinvolveinvolve thethe of inhibitioninhibition macrophages. ofof classiclassicalcal activationactivation ofof macrophages.macrophages.

FigureFigure 13.13. StructureStructure ofof comaparvin.comaparvin. Figure 13. Structure of comaparvin. 2.5.2. Asteroidea 2.5.2.2.5.2. AsteroideaAsteroidea Sea stars contribute to Echinoderm variety with about 2000 species [128]. A lot of its structurally Sea stars contribute to Echinoderm variety with about 2000 species [128]. A lot of its structurally variableSea secondarystars contribute metabolites to Echinoderm attract the variety attention with of ab researchers;out 2000 species however, [128]. little A islot known of its structurally about their variable secondary metabolites attract the attention of researchers; however, little is known about immunomodulatoryvariable secondary metabolites capacities. attract the attention of researchers; however, little is known about their immunomodulatory capacities. theirStudies immunomodulatory on the lipidomic capacities. profiling of the sea star Marthasterias glacialis (Linnaeus, 1758), Studies on the lipidomic profiling of the sea star Marthasterias glacialis (Linnaeus, 1758), collectedStudies in weston the Portugal, lipidomic revealed profiling two majorof the classes sea star of compounds: Marthasterias fatty glacialis acids (Linnaeus, and sterols 1758), [129]. collected in west Portugal, revealed two major classes of compounds: fatty acids and sterols [129]. Amongcollected those, in west the Portugal, predominant revealed compounds two major were cl cis-11-eicosenoicasses of compounds: and cis-11,14fatty acids eicosadienoic and sterols acids,[129]. Among those, the predominant compounds were cis-11-eicosenoic and cis-11,14 eicosadienoic acids, and,Among additionally, those, the predominant the unsaturated compounds sterol ergosta-7,22-dien-3-olas. were cis-11-eicosenoic and These cis-11,14 compounds eicosadienoic as well acids, as and, additionally, the unsaturated sterol ergosta-7,22-dien-3-olas. These compounds as well as lipophilicand, additionally, extract were the evaluated unsaturated for their sterol anti-inflammatory ergosta-7,22-dien-3-olas. effects in theThese LPS (1compoundsµg/mL)-induced as well RAW as

264.7 macrophages model of inflammation for 24 h. In these studies, the mechanisms involved in the immunomodulating effects of metabolites were studied in more detail compared to those of any other marine compounds reported above. The extract significantly diminished the levels of LPS-induced NO as a result of downregulation of iNOS, an effect for which all compounds tested contributed, Mar. Drugs 2020, 18, 37 15 of 27 notably ergosta-7,22-dien-3-ol. However, only the extract, but not single compounds, prevented an LPS-induced increase of the expression of COX-2, IL-6 level, and ROS generation. The extract and some of its constituents also inhibited the activation of the NF-kB pathway, which is the trigger of the inflammatory response, with subsequent ROS and ER-stress marker elevation [3]. In this study, ER-stress status was also evaluated by the expression of the endpoint protein of the CHOP, an ER-stress marker [5]. It is noteworthy that CHOP damages the RAW 264.7 cells largely dependent on pro-inflammatory stimuli used [130]. In this study, LPS increased CHOP expression levels, and this effect was partly reverted by the extract and its components, indicating the compounds could attenuate NF-kB activation and subsequent expression of some inflammatory markers. Apparently, the mechanisms of action of the compounds are distinct as they modulated different levels of the inflammation pathway. However, the fact that all of them were active in NO level inhibition indicates that the extract could inhibit the classical activation of macrophages, and its constituents contribute to this effect. Steroidal glycosides are the predominant metabolites of starfish, and are responsible for their general toxicity [131]. Steroidal glycosides, protolinckiosides A-D (1-4, respectively) were isolated from the MeOH/EtOH extract of the tropical starfish Protoreaster lincki (Blainville, 1834). The structures of 1-4 were elucidated as (3β,4β,5α,6β,7α,15α,16β,25S)-4,6,7,8,15,16,26-heptahydroxycholestan-3-yl 2-O-methyl-β-d-xylopyranoside (1), (3β,5α,6β,15α,24S)-3,5,6,8,15-pentahydroxycholestan-24-yl α-l-arabinofuranoside, sodium (3β,6β,15α,16β,24R)-29-(β-d-galactofuranosyloxy)-6,8,16-trihydroxy-3- [(2-O-methyl-β-d-xylopyranosyl)oxy]stigmast-4-en-15-yl sulfate and sodium (3β,6β,15α, 16β,22E,24R)-28-(β-d-galactofuranosyloxy)-6,8,16-trihydroxy-3-[(2-O-methyl- β-d-xylopyranosyl)oxy] ergosta-4,22-dien-15-yl sulfate. All compounds significantly decreased the ROS content in LPS-challenged RAW 264.7 macrophages. These results are too limited to discuss the participation of the compounds in macrophage –polarization mechanisms. In addition, unique steroid glycosides, polyhydroxysteroids capelloside A, and oscinasteroside (24S)-24-O-(3-O-methyl-β-d-xylopyranosyl)-5a- cholestane-3β,6β,8,15α,24-pentoI 15-O-sulfate(24S)- 24-O-(3-O-methyl-β-d-xylopyranosyl)-5a- cholestane-3β,6β,8,15α,24-pentoI 15-O-sulfate(24S)- 24-O-(3-O-methyl-β-d-xylopyranosyl)-5a- cholestane-3β,6β,8,15α,24-pentoI 15-O-sulfate coscinasteroside B coscinasteroside B coscinasteroside B (24S)-24-O-(3-O-methyl- β-d-xylopyranosyl)-5a- cholestane-3β,6β,8,15α,24-pentoI 15-O-sulfate B, isolated from the ethanolic extract of the starfish Ogmaster capella (Müller & Troschel, 1842) (Valvatida) inhabiting the Western Pacific [132], and granulatoside D (compound 49, Figure 14), isolated from the ethanolic extract of the starfish Choriaster granulatus (Lutken, 1869) occurring in the South Pacific Ocean [133] were studied for bioactivities at 0.1 µM concentration. Both steroid glycosides from O. capella at non-toxic concentrations effectively inhibited the ROS level in LPS (1.0 µg/mL)-induced for 24 h RAW 264.7 cells, but not in un-stimulated cells. Neither of the compounds affected the NO levels in macrophages elevated by LPS treatment. Moreover, granulatoside D at a dose of 0.1 µM dually affected ROS, increasing its level in peritoneal murine macrophages and decreasing in LPS—pretreated peritoneal macrophages, which suggests that the effect of this compound on ROS generation depends on the initial environment factors. These findings indicate that the steroid glycosides from the species studied may have oxidant-antioxidant balance modulating capacities, but their possible immunomodulating ability needs to be further studied. Mar. Drugs 2020, 18, x FOR PEER REVIEW 16 of 27 Mar. Drugs 2020 18 Mar. Drugs 2020, 18,, x FOR, 37 PEER REVIEW 16 of16 27 of 27 Mar. Drugs 2020, 18, x FOR PEER REVIEW 16 of 27

Figure 14. Structure of the granulatoside D. Figure 14. Structure of the granulatoside D. Figure 14. Structure of the granulatoside D. In contrast, another group of steroid glycosides of a rare structure, containing carbohydrate In contrast,In contrast, another another group group of steroid of steroid glycosides glycosides of a of rare a rare structure, structure, containing containing carbohydrate carbohydrate moietiesIn contrast, incorporated another into group a macrocycle, of steroid luzonico glycosidessides of A-E, a rare and structure, a related containingopen carbohydrate carbohydrate chain moietiesmoieties incorporated incorporated into into a macrocycle, a macrocycle, luzonico luzonicosidessides A-E, A-E, and and a related a related open open carbohydrate carbohydrate chain chain moietiessteroid glycosideincorporated luzonicoside into a macrocycle, F from the luzonico tropicalsides and A-E, sub-tropical and a related western open Indo-Pacificcarbohydrate starfish chain steroidsteroid glycoside glycoside luzonicoside luzonicoside F from F from the thetropical tropical and and sub-tropical sub-tropical western western Indo-Pacific Indo-Pacific starfish starfish steroidEchinaster glycoside luzonicus luzonicoside (Gray, 1840) F (Spinulosida)from the tropical increased and sub-tropicalthe ROS level western and NO Indo-Pacific synthesis instarfish RAW EchinasterEchinaster luzonicus luzonicus (Gray,(Gray, 1840) 1840) (Spinulosida) (Spinulosida) increased increased the theROS ROS level level and and NO NO synthesis synthesis in RAW in RAW Echinaster264.7 cells luzonicus [134]. As (Gray, these compounds 1840) (Spinulosida) were earlier increased shown the to inhibitROS level also andcancer NO cells synthesis [7], these in resultsRAW 264.7264.7 cells cells [134]. [134 As]. Asthese these compounds compounds were were earlier earlier shown shown to inhibit to inhibit also also cancer cancer cells cells [7], [these7], these results results 264.7demonstrate cells [134]. a potentialAs these compoundsefficacy of luzonicosides,were earlier sh ownmainly to inhibitA (50) also and cancer D (51 cells) (Figure [7], these 15), resultsagainst demonstratedemonstrate a potential a potential efficacy efficacy of luzonicosides, of luzonicosides, mainly mainly A ( A50) ( 50and) and D ( D51 ()51 (Figure) (Figure 15), 15 against), against demonstratecancer development, a potential and efficacy also of ofstimulating luzonicosides, the macrophage mainly A (to50 )transform and D (51 phenotype) (Figure 15),towards against the cancercancer development, development, and and also also of ofstimulating stimulating th thee macrophage macrophage to to transform transform phenotype towardstowards thethe M1 cancerM1 (“anti-cancer”) development, type. and also of stimulating the macrophage to transform phenotype towards the M1(“anti-cancer”) (“anti-cancer”) type. type. M1 (“anti-cancer”) type.

Figure 15. Structures of luzonicoside A (50) and luzonicoside D (51). FigureFigure 15. Structures 15. Structures of luzonicoside of luzonicoside A (50 A) ( 50and) and luzonicoside luzonicoside D (51 D) (.51 ). Figure 15. Structures of luzonicoside A (50) and luzonicoside D (51). Pyrrole oligoglycosides are also rarely found compounds. New plancipyrrosides (Figure 16) A PyrrolePyrrole oligoglycosides oligoglycosides are arealso also rarely rarely found found compounds. compounds. New Newplancipyrrosides plancipyrrosides (Figure (Figure 16) A 16) (52) Pyrroleand B ( oligoglycosides53) from the methanol are also extractrarely foundof the compounds.Vietnamese starfishNew plancipyrrosides Acanthaster planci (Figure (Linnaeus, 16) A (52A() and52) B and (53 B) from (53) from the methanol the methanol extract extract of the of theVietnamese Vietnamese starfish starfish AcanthasterAcanthaster planci planci (Linnaeus,(Linnaeus, (1758)52) and (Valvatida) B (53) from were the testedmethanol in RAW264.7extract of thecells Vietnamese and shown starfish to have Acanthaster different planci activity (Linnaeus, against 1758)1758) (Valvatida) (Valvatida) were were tested tested in RAW264.7RAW264.7 cells cells and and shown shown to haveto have different different activity activity against against increased 1758)increased (Valvatida) NO production were tested in inLPS RAW264.7 (1 μg/mL)-stimulated cells and shown cells to haveafter different24 h incubation activity against [135]. increasedNO production NO production in LPS (1 µing /mL)-stimulatedLPS (1 μg/mL)-stimulated cells after 24 hcells incubation after [24135 ].h Plancipyrrosideincubation [135]. B ( 53) Plancipyrroside B (53) exhibited a higher inhibitory effect with IC50 of 5.94 ± 0.34 μM and may be exhibitedincreased aNO higher production inhibitory in eff ectLPS with (1 ICμg/mL)-stimulatedof 5.94 0.34 µ Mcells and after may be24 consideredh incubation potent [135]. for Plancipyrroside B (53) exhibited a higher inhibitory50 effect with IC50 of 5.94 ± 0.34 μM and may be Plancipyrrosideconsidered potent B (for53) furtherexhibited studies a higher of their inhibitory immunomodulating effect± with IC properties.50 of 5.94 ± 0.34 μM and may be consideredfurther studies potent offor their further immunomodulating studies of their immunomodulating properties. properties. considered potent for further studies of their immunomodulating properties.

FigureFigure 16. 16.Structure Structure ofof thetheplancipyrrosides plancipyrrosides A A ( 52(52)) and and B B ( 53(53).). Figure 16. Structure of the plancipyrrosides A (52) and B (53). 2.5.3. Echinoidea Figure 16. Structure of the plancipyrrosides A (52) and B (53). 2.5.3. Echinoidea 2.5.3. EchinoideaSea urchins contain unique compounds like polyhydroxynaphthoquinones involved in the sea 2.5.3. SeaEchinoidea urchins contain unique compounds like polyhydroxynaphthoquinones involved in the sea urchin’sSea urchins pigmentation contain (spinochromesunique compounds or echinochromes) like polyhydroxynaphthoquinones [136]. J774 macrophages involved were used in inthe research sea urchin’sSea urchinspigmentation contain (spinochrounique compoundsmes or echinochromes) like polyhydroxynaphthoquinones [136]. J774 macrophages involved were in usedthe sea in urchin’son the pigmentation bioactivity of spinochromes(spinochromes (compounds or echinochromes)54–57 in Figure[136]. 17J774) isolated macrophages from four were common used regular in urchin’sresearch pigmentationon the bioactivity (spinochro of spinochromesmes or echinochromes) (compounds [136].54–57 J 774in Figure macrophages 17) isolated were from used four in researchsea urchins on the collected bioactivity in theof spinochromes Indian Ocean: (compoundsEchinometra mathaei54–57 in(Blainville, Figure 17) 1825), isolatedDiadema from savignyifour researchcommon on regular the bioactivity sea urchins of collectedspinochromes in the (compounds Indian Ocean: 54 –Echinometra57 in Figure mathaei 17) isolated (Blainville, from 1825),four common(Audouin, regular 1809) seaTripneustes urchins collected gratilla (Linnaeus, in the Indian 1758) Ocean: and Toxopneustes Echinometra pileolus mathaei(Lamarck, (Blainville, 1816). 1825), In LPS commonDiadema regularsavignyi sea(Audouin, urchins collected1809) Tripneustes in the Indian gratilla Ocean: (Linnaeus, Echinometra 1758) mathaeiand Toxopneustes (Blainville, pileolus1825), Diadema(10 ng /savignyimL)-induced (Audouin, macrophages, 1809) Tripneustes each of the gratilla spinochromes (Linnaeus, caused 1758) an increaseand Toxopneustes in TNF-α productionpileolus Diadema(Lamarck, savignyi 1816). (Audouin,In LPS (10 1809)ng/mL)-induced Tripneustes macrophages,gratilla (Linnaeus, each 1758)of the and spinochromes Toxopneustes caused pileolus an (Lamarck, 1816). In LPS (10 ng/mL)-induced macrophages, each of the spinochromes caused an (Lamarck, 1816). In LPS (10 ng/mL)-induced macrophages, each of the spinochromes caused an

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increase in TNF-α production after 6 h incubation while no effect was found without LPS afterstimulation. 6 h incubation One of while the nopossible effect wasexplanations found without of such LPS synergistic stimulation. effect One ofof thespinochromes possible explanations and LPS ofmay such be synergisticrelated to a eshortffect ofperiod spinochromes of cell incubation and LPS, maywhen be LPS related alone to or a each short of period the compounds of cell incubation, are not whenable to LPS induce alone the or each massive of the signaling, compounds and are triggering not able to the induce process the massive of TNF- signaling,α production and triggering after the themultiplying process ofsignaling TNF-α productionwith spinochromes. after the In multiplying any case, spinochromes signaling with may spinochromes. be perspective In to any be case,used spinochromesfor activation macrophages may be perspective towards to the be M1-type, used for activationin accordance macrophages with data towardson the pro-inflammatory the M1-type, in accordancerole of naphthoquino with datanes on thein mice pro-inflammatory [137]. role of naphthoquinones in mice [137].

Figure 17. Structure of spinochromes A ((5454),), BB ((5555),), CC ((5656),), andand EE ((5757).).

InIn addition,addition, macrophage-stimulating macrophage-stimulating activity activi wasty found was for found SEP-2, afor water-soluble SEP-2, a polysaccharide,water-soluble isolatedpolysaccharide, from Strongylocentrotus isolated from Strongylocentrotus nudus (A. Agassiz, nudus 1864), (A. inhabiting Agassiz, 1864), the Yellow inhabiting Sea, and the preliminaryYellow Sea, characterized as d-glucan containing a (1 4)-linked d-Glcp backbone→ with (1 3)-linked d-Glcp side and preliminary characterized as d-glucan→ containing a (1 4)-linked d-Glcp→ backbone with chains.(1→3)-linked SEP-2 d-Glcp was shown side tochains. enhance SEP-2 significantly was shown ROS to level, enhance NO production,significantly and ROS inflammatory level, NO cytokinesproduction, secretion and inflammatory (IL-1β, IL-6 cytokines and TNF- αsecretion) in RAW264.7 (IL-1β, cell IL-6 line and [138 TNF-]. Theseα) in resultsRAW264.7 suggest cell thatline SEP-2[138]. mightTheseinduce results macrophage suggest that polarization SEP-2 might toward induce the pro-inflammatory macrophage polarization M1 state. toward the pro-inflammatoryThe sea urchins M1 are state. also a rich source for obtaining antimicrobic peptides. One of them, centrocin 1, was isolatedThe sea from urchinsS. droebachiensis are also a(O.F. rich Müller,source 1776)for obtaining (Echinoida) antimicrobic and is characterized peptides. as One a heterodimer of them, formedcentrocin by 1, monomers was isolated of from 30 (heavy S. droebachiensis chain) and (O.F. 12 amino Müller, acid 1776) residues (Echinoida) (light and chain) is characterized connected by as a singlea heterodimer disulphide formed bridge. by In monomers this study of [139 30], (heavy macrophage-like chain) and THP-1 12 amino cells wereacid residues stimulated (light by adding chain) 0.1connected ng/mL LPS,by a concentrations single disulphide of which bridge. were In preliminarily this study [139], selected macrophage-like based on in vitro THP-1experiments cells were to givestimulated a close by to maximumadding 0.1 release ng/mL of LPS, cytokines concentrations over a period of which of 6 h.were The preliminarily centrocin 1-peptide selected derivatives based on atin nontoxicvitro experiments concentrations to give significantly a close to reducedmaximum the release level of of the cytoki pro-inflammatorynes over a period cytokine of 6 TNF-h. Theα incentrocin LPS-stimulated 1-peptide cells, derivatives and some at of nontoxic the peptides concen reducedtrations the significantly production reduced of another the inflammation level of the marker,pro-inflammatory IL-6, in LPS-stimulated cytokine TNF- cells.α in Thus, LPS-stimulated these peptides cells, may and have some a potential of the forpeptides using inreduced promoting the macrophageproduction of phenotype another inflammation towards M2 type.marker, IL-6, in LPS-stimulated cells. Thus, these peptides may have a potential for using in promoting macrophage phenotype towards M2 type. 2.5.4. Holothuroidea 2.5.4.There Holothuroidea are about 1500 species of sea cucumbers [140]. Many species are of commercial valueThere [141] are and about are the 1500 main species objects of ofsea studies cucumbers for prospective [140]. Many using species in pharmaceuticalare of commercial industry. value The[141] immunostimulating and are the main substancesobjects of studies from holothurians for prospective of the using Far-East in pharmaceutical Pacific were the industry. most studied. The Triterpeneimmunostimulating glycosidecucumarioside substances from A2-2 holothurians (A2-2) (compound of the58 Far-East, Figure 18Pacific), isolated were from the themost Far studied. Eastern sea cucumber Cucumaria japonica (Dendrochirotida), is known to exert significant immunomodulatory Triterpene glycoside cucumarioside A2-2 (A2-2) (compound 58, Figure 18), isolated from the Far eEasternffects in sea vivo cucumber[142]. The workCucumaria of Pislyagin japonica et al.(Dendrochirotida), [143] is, apparently, is oneknown of the to first exert of those significant clearly indicatingimmunomodulatory that immunomodulatory effects in vivo [142]. activity The of work marine of triterpenePislyagin et glycosides al. [143] is, is associatedapparently, with one e offfects the onfirst macrophage of those clearly polarization indicatingin vivo thatand immunomodulatoin vitro.A2-2 (1.5ry mgactivity/kg) stimulates of marine NO triterpene and ROS glycosides production is in the spleen macrophage in comparison with LPS (1.5 mg/kg) activity that indicates an activation associated with effects on macrophage polarization in vivo and in vitro. A2-2 (1.5 mg/kg) stimulates ofNO macrophages and ROS production towards classicalin the spleen phenotype. macrophage Studies in comparison on RAW 264.7 with macrophages LPS (1.5 mg/kg) treated activity with LPSthat µ γ (1indicatesg/mL) andan activation IFN- (20 ngof/ mL)macrophages for M1 polarization towards orclassical IL-4 (20 phenotype. ng/mL) for M2Studies polarization on RAW showed 264.7 thatmacrophages A2-2 stimulated treated polarization with LPS (1 through μg/mL) the and M1-type IFN-γway. (20 ng/mL) for M1 polarization or IL-4 (20 ng/mL) for M2 polarization showed that A2-2 stimulated polarization through the M1-type way.

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Figure 18. Structure of cucumarioside A2-2.

A number of studies concernedFigure th 18.e Structurebioactivity of cucumariosideof sulfated fucans A2-2. of sea cucumbers. The water soluble protein-sulfated fucan (PSF) complex isolated from the body wall of Apostichopus japonicus Figure 18. Structure of cucumarioside A22-2. (Selenka,A number 1867) (old of studies species concerned name is Stichopus the bioactivity japonicus of) (Synallactida)sulfated fucans and of seafour cucumbers. its purified The fractions water soluble protein-sulfated fucan (PSF) complex isolated from the body wall of Apostichopus japonicus (F1, F2A, F number3 and F 4 of) mostly studies consisted concerned of thethneutrale bioactivity sugars, of proteins, sulfatedsulfated and fucans sulfates of seasea in cucumbers.cucumbers. various proportions, The water (Selenka, 1867) (old species name is Stichopus japonicus) (Synallactida) and four its purified fractions andsoluble fucose protein-sulfated was their major fucan monosaccharide. (PSF) complexcomplex isolatedA different from concentration the body wallwall of ofof samples ApostichopusApostichopus (2, 5, japonicus and 10 (F1, F2, F3 and F4) mostly consisted of neutral sugars, proteins, and sulfates in various proportions, μ(Selenka,g/mL) induced 1867) (old significant speciesspecies nameexpression isis Stichopus of NO, japonicus COX-2) and (Synallactida) release of andpro-inflammatory four its purifiedpurified cytokines fractionsfractions and fucose was their major monosaccharide. A different concentration of samples (2, 5, and 10 IL-1(F1,,F βF,2 IL-6,,,F F33 andand TNF- F F44α)) ,mostly mostlyand IL-12 consisted consisted compared of of neutral neutral to LPS sugars, sugars,(1–2 μg/mL), proteins, proteins, which and and was sulfates sulfates used in inas various a positive proportions, control in μg/mL) induced significant expression of NO, COX-2 and release of pro-inflammatory cytokines RAW264.7and fucosefucose wascellswas their aftertheir major major18 h monosaccharide. ofmonosaccharide. incubation A[144]. diAff erent diffTheerent concentrationenhanced concentration expression of samples of samples of (2, markers 5, and(2, 105, ofµandg /mL)M1 10 IL-1β, IL-6, TNF-α, and IL-12 compared to LPS (1–2 μg/mL), which was used as a positive control in macrophagesinducedμg/mL) induced significant in cells significant expression exposed expression to of fractions NO, COX-2 of displays NO, and COX-2 releasethat fractionsand of pro-inflammatoryrelease could of influencepro-inflammatory cytokines classical IL-1activation cytokinesβ, IL-6, RAW264.7 cells after 18 h of incubation [144]. The enhanced expression of markers of M1 ofTNF-IL-1 macrophages.βα, IL-6,, and TNF- IL-12 αIn compared, andthese IL-12 studies, to compared LPS (1–2the mainµ gto/mL), LPS back which(1–2bone μ wasg/mL), of highly used which as stimulating a positivewas used control markersas a positive in RAW264.7expression control cells Fin3 macrophages in cells exposed to fractions displays that fractions could influence classical activation fractionafterRAW264.7 18 h(59 of ) incubationcellswas after(1→3)- [14418α -l-linked].h Theof enhancedincubation fucosyl expression residue[144]. Thewith of markers enhancedsulfation of M1atexpression C-2 macrophages and/or of C-4markers in cells(Figure exposedof 19).M1 of macrophages. In these studies, the main backbone of highly stimulating markers expression F3 Earlier,tomacrophages fractions a pivotal displays in cells role that exposed of fractions sulfates to fractions could and influenceM. displays w. of classicalcompounds that fractions activation in couldactivating of macrophages. influence RAW classical 264.7 In these cellsactivation studies, was fraction (59) was (1→3)-α-l-linked fucosyl residue with sulfation at C-2 and/or C-4 (Figureα 19). establishedtheof macrophages. main backbone [145]. In ofthese highly studies, stimulating the main markers backbone expression of highly F3 stimulatingfraction (59 )markers was (1 expression3)- -l-linked F3 Earlier, a pivotal role of sulfates and M. w. of compounds in activating RAW 264.7→ cells was fucosylfraction residue(59) was with (1→ sulfation3)-α-l-linked at C-2 andfucosyl/or C-4 residue (Figure with 19). sulfation Earlier, a pivotalat C-2 roleand/or of sulfatesC-4 (Figure and M. 19). w. established [145]. ofEarlier, compounds a pivotal in activating role of sulfates RAW 264.7 and cellsM. w. was of established compounds [145 in]. activating RAW 264.7 cells was established [145].

Figure 19. The proposed structure of the SF3 fraction from A. japonicus.

Figure 19. The proposed structure of the SF3 fraction from A. japonicus. In addition, theFigure immunostimulating 19. The proposed structureeffects of of glycosaminoglycan the SF3 fraction from (AHG)A. japonicus. (compound 60) from ApostichopusIn addition, japonicus the immunostimulating(Selenka, 1867) (Synallactida) effects of glycosaminoglycan(Figure 20) in cyclophosphamide-induced (AHG) (compound 60) In addition, theFigure immunostimulating 19. The proposed structure effects ofof glycosaminoglycanthe SF3 fraction from A. (AHG) japonicus. (compound 60) from immunosuppressionfrom Apostichopus japonicus was studied(Selenka, with 1867) using (Synallactida) a broad set of (Figure markers 20 of) in mice cyclophosphamide-induced macrophage activation Apostichopus japonicus (Selenka, 1867) (Synallactida) (Figure 20) in cyclophosphamide-induced [146].immunosuppressionIn It addition,was revealed the wasimmunostimulating that studied AHG with (10 usingmg/kg) effect a broadenhanceds of setglycosaminoglycan of the markers expression of mice (AHG)of macrophage NO, (compoundTNF-α activation, IL-6, 60 IL-1) [from146β,]. immunosuppression was studied with using a broad set of markers of mice macrophage activation IL-18,ItApostichopus was and revealed related japonicus that mRNA, AHG (Selenka, (10 which mg/kg) are1867) enhanced the (Synallactida)markers the expression of macrophage (Fig ofure NO, 20) TNF-polarizati inα ,cyclophosphamide-induced IL-6,on IL-1 towardsβ, IL-18, the and M1-type related [146]. It was revealed that AHG (10 mg/kg) enhanced the expression of NO, TNF-α, IL-6, IL-1β, [11].mRNA,immunosuppression The which mechanisms are the was markers studiedof immunostimulation of macrophage with using polarizationa broad were set of towardsshown markers theto of M1-type micebe realized macrophage [11]. The through mechanisms activation the IL-18, and related mRNA, which are the markers of macrophage polarization towards the M1-type phosphorylationof[146]. immunostimulation It was revealed of MAPK werethat and AHG shown NF- (10κ toB. mg/kg) be Apparently, realized enhanced through glycosaminoglycan the the expression phosphorylation stimulatesof NO, of TNF- MAPK theα classical, IL-6, and NF-IL-1 wayκβB. , [11]. The mechanisms of immunostimulation were shown to be realized through the ofApparently,IL-18, macrophage and related glycosaminoglycan activation. mRNA, which stimulates are the markers the classical of macrophage way of macrophage polarizati activation.on towards the M1-type phosphorylation of MAPK and NF-κB. Apparently, glycosaminoglycan stimulates the classical way [11]. The mechanisms of immunostimulation were shown to be realized through the of macrophage activation. phosphorylation of MAPK and NF-κB. Apparently, glycosaminoglycan stimulates the classical way of macrophage activation.

FigureFigure 20. 20. HypotheticalHypothetical structure structure for fucose branchesbranches stretchingstretching fromfrom a a core core chondroitin chondroitin sulfate sulfate unit unit of

ofintact intact glycosaminoglycan glycosaminoglycan from fromA .Ajaponicus. japonicusAHG-E. AHG-E. Figure 20. Hypothetical structure for fucose branches stretching from a core chondroitin sulfate unit Inof intact contrast, glycos ethylaminoglycan acetate solventfrom A. japonicus fraction AHG-E. (phenol content of about 20.4 mg/g) of A. japonicus Figure 20. Hypothetical structure for fucose branches stretching from a core chondroitin sulfate unit at concentrations of 10, 50, and 100 µg/mL significantly inhibited the production of NO and PGE2 by inhibitingof intact glycos iNOSaminoglycan and COX-2 from at their A. japonicus protein AHG-E. and gene levels, as well as production and the gene

Mar. Drugs 2020, 18, 37 19 of 27 transcription of proinflammatory cytokines IL-1β, TNF-α in LPS (1 µg/mL) stimulated RAW264.7 cells [147]. The responsible molecular signaling for these effects was found to be through suppression of the phosphorylation of MAPK molecules; ERK and p38 MAPK [148]. These findings, apparently, indicate that phenols of the sea cucumber are able to reverse the M1-type polarization of macrophages. For screening new metabolites, as well for understanding some mechanisms of macrophage polarization, more simple models may be useful. Previously, P1 and P2 types of phagocytes of the Far-Eastern sea cucumber Eupentacta fraudatrix (Dendrochirotida) were shown to be phenotypically distinct and similar to M1 and M2 macrophages, respectively [21]. Using phagocytes as a model for studying a new extract of Far-Eastern holothurians [149], rich with carotenoids and n-3 PUFA as the main biocompounds, both the extract (0.01, 0.1 and 1 µg/mL) and dexamethasone as positive control (0.1, 1 and 100 µM) were shown to downregulate the level of IL-1α-like substances in P2 phagocytes after 24 h incubation in a concentration-dependent manner. After 48 h, the changes in the levels of pro-inflammatory cytokine-like substances induced by both preparations in P1 cells were opposite to those induced in P2 type phagocytes. This fact indicates that inhibition of pro-inflammatory cytokines is involved in the mechanism of action of the extract and corresponds to known wound-healing activity of the extract [150]. Further studies are needed for confirmation of the potent immunomodulating activity of the extract and the mechanisms involved.

3. Conclusions The new paradigm of polarization of macrophages via two distinct ways has founded the understanding of impaired switching between M1 and M2 phenotypes of macrophages as triggering mechanism of development of pathological states and diseases. As a result, correction of M1/M2 balance has become a new therapeutic target, as well as the modern pharmacological trend. Based predominantly on the evaluation of changes in NO and COX-2 protein levels, analysis of the numerous data on the effects of marine extracts and compounds of different chemical structure in LPS-stimulated macrophages highlighted the fact that soft corals are rich in potentially active substances modulating macrophage polarization towards M2 (anti-inflammatory) type. The most frequent potent activity was found for cembrane-based and eunicellin-based diterpenoids, sterols, ceramides, and cerebrosides. Studies on pure compounds from Porifera were rarer, but the recently studied extracts were shown to promote M2-way of macrophage polarization with the use of a broad set of the markers, including interleukins and cell surface markers. In addition, several extracts from Molluscs of different orders (Aplysiida, Mytilida, Cephalaspidea, Ostreida) were argued to be promising for promotion of M2-type polarization. These extracts were rich with carotenoids as well as with n-3 PUFA and n-6 PUFA, which contributed to the extract’s activities. However, compound 1-O-alkyl-sn-glycerols from the Oegopsida was shown to stimulate macrophages towards M1 phenotype. Carotenoid, EPA, and DHA-rich lipids from Crustaceans (krills and shrimps) have shown the promising activity regarding M2-polarization promotion. Different derivatives of polysaccharide chitin have been revealed to activate macrophage polarization through both M1 and M2 ways. Compounds from tissues of Echinoderms (Holothuroidea, Echinoidea, Asteroidea, Crinoidea) were more often studied with a focus on their macrophage-polarizing properties. The larger proportion of those compounds studied had M1 macrophage promoting abilities (triterpene glycoside cucumarioside A2-2, protein-sulfated fucan complex, glycosaminoglycans from sea cucumbers; polyhydroxynaphthoquinones (echinochromes), water-soluble polysaccharide SEP-2 from sea urchins; steroid glycosides luzonicosides from sea stars). Obviously, the chemical composition and structure of marine substances influence the way and intensity of their effects on macrophage polarization, but exact mechanisms of such dependence are little studied. However, the predominant capacity of more hydrophilic compounds, mostly from echinoderm tissues, and also from squid tissues, compared to that of the hydrophobic molecules produced by Mar. Drugs 2020, 18, 37 20 of 27 other invertebrates, to stimulate classical way of macrophage activation suggests that hydrophilic properties are important for the compound binding to different surface receptors of macrophages and for subsequent signaling cascade. This may have an implication in further constructing new drugs. On the other hand, most of the reports cover the compounds that promote the reduction in classic polarization of macrophages. This apparently results from the screening character of the studies aimed to establish the anti-inflammatory properties of molecules and using the corresponding stimuli to promote the M1-way macrophage activation in control cells. Nevertheless, the rare reports on the investigation of marine molecule effects in macrophages stimulated toward the M2 phenotype extends the understanding of the mechanisms of macrophage polarization shift, and indicate the importance of the evaluation of the effects of compounds on both types of macrophages. On the whole, the species of different phyla of marine invertebrates are a promising source to obtain drugs that affect the polarization of macrophages towards both the M2 and the M1 types. Further fundamental research of the mechanisms of the effects of marine compounds on macrophage polarization switching is particularly important for creating new medicines with strong anti-inflammatory or immunostimulating properties.

Author Contributions: L.D. conseptualized the manuscript, carried out the review of literature, drafted and corrected the manuscript. I.D. contributed to drafting, editing and correcting the manuscript. Both authors read and approved the submitted version of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest.

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