Chronic inflammation is a defining feature of autoimmune diseases, a group of conditions in which immunological self-tolerance is disturbed due to the recognition of autoantigens by immune cells. Rheumatoid arthritis (RA), the most common chronic inflammatory arthropathy ^[1][2][1,2], is a systemic autoimmune disorder affecting the synovial joints, with a higher incidence in women [3]. RA displays a complex pathophysiology involving the upregulation of pro-inflammatory mediators (interleukins, ILs) and enhanced production of reactive oxygen species (ROS) ^[4][5][4,5]. Both genetic and modifiable lifestyle factors contribute to the risk of RA predisposition ^[6][7][6,7], with diet highly influencing disease activity ^[8][9][8,9]. In particular, a high antioxidant intake is known to reduce onset risk and to ameliorate the clinical course of the disease [10], therefore, the identification of new sources of antioxidant and anti-inflammatory molecules is of high clinical relevance.

Microalgae are photosynthetic prokaryotes and eukaryotes adapted to diverse environments, including extreme habitats ^[11][12][11,12], which are consumed in human nutrition as sources of proteins and other bioactive compounds ^{[13][14][15][16][17]}[13,14,15,16,17]. Several species are non-toxic producers of essential vitamins, lipids, and pigments of therapeutic value ^{[18][19][20][21][22]}[18,19,20,21,22], which could be employed as complementary agents in the management of chronic inflammatory diseases. Moreover, the fast life cycle and light-powered autotrophic metabolism of microalgae allows for large-scale cultivation with lower inputs compared with heterotrophic microorganisms [23].

Pathogenesis and Mediators of Rheumatoid Arthritis

Although the exact etiology of RA remains unknown, the balance between immune cells and the production of inflammatory ILs in the connective tissue that lines the joint capsule (synovium) is altered in the disease onset and progression ^[4][5][4,5]. The healthy synovium consists of a thin lining layer of fibroblasts covering a connective tissue surrounded by blood vessels and enriched in fibroblasts, and innate and adaptive immune cells: the sub-lining layer [24] (Figure 1A). In RA, the lining layer is hyperplastic while the sub-lining layer is infiltrated with B-cells, monocyte-derived macrophages, autoantibody-secreting plasma cells, and differentiated cytotoxic CD4⁺ T-cells involved in the breakdown of tissue tolerance ^[25][26][27][25,26,27]. The release of pro-inflammatory ILs by monocyte-derived M1 macrophages, and osteoclast activation cause progressive bone resorption [28], and autoantibodies produced by differentiated plasma cells further contribute to joint damage [29]. At the intracellular level, three main pro-inflammatory signaling kinase cascades responding to soluble mediators are involved in RA, all being influenced by microalgal metabolites: the Nuclear Factor Kappa-Β (NF-kB)-mediated pathway [30], the Janus kinase 2/Signal Transducers and Activators of Transcription 3 (JAK2/STAT3) pathway [31], and the Jun N-terminal kinase (JNK)/p38 Mitogen-Activated Protein Kinase (p38 MAPK) pathway [32], the latter being predominant in the lining layer and endothelial cells. Pro-inflammatory ILs (Figure 1D) [33] stimulate the production of the soluble cytokine Mediator Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL) by immune cells. RANKL binds its receptor RANK on monocytes and macrophages causing their differentiation into bone-resorbing osteoclasts [34]. Other ILs produced by anti-inflammatory M2 macrophages positively influence the osteoprotegerin (OPG)–RANKL ratio, promoting bone homeostasis [35] (Figure 1D), while the IL-10 family (Figure 1D) appears to play dual roles. Some members stimulate osteogenesis and suppress the synthesis of pro-inflammatory mediators like IL-6, Tumor Necrosis Factor-α (TNF-α), and Vascular endothelial growth factor (VEGF) [36], while others activate the NF-kB and MAPK pathways, stimulating TNF-α, IL-1β, and RANKL production by synovial fibroblasts, promoting osteoclastogenesis.

2. Anti-Inflammatory and Immunomodulatory Metabolites from Microalgae

2.1. Carotenes and Xanthophylls

Like bacteria, fungi, and plants, microalgae synthetize C40 lipophilic pigments consisting of a polyene chain of conjugated double bonds (Figure 2) with terminally linked ionone rings known as carotenoids ^[37][38][37,38]. β-carotene (Figure 2B)—a structural element of photosystems [39]—and the antioxidant lycopene (Figure 2A) are anti-inflammatory carotenes ^[40][41][40,41] usually introduced in the diet with carrots (Daucus carota) and tomatoes (Solanum lycopersicum), respectively, although present also in microalgae [42]. Xanthophylls are oxygenated carotenoids containing hydroxyl and ketone groups in the ionone rings, which serve different functions in phototrophs. The non-ketolated xanthophyll lutein (Figure 2C) participates in light-harvesting and photoprotection, while the ketocarotenoid astaxanthin (ASTX, Figure 2D) scavenges harmful ROS generated by photosynthetic electron transport under excess light [43]. Lutein is an anti-inflammatory carotenoid [44] abundantly found in green leafy vegetables and egg yolk, while ASTX is a potent antioxidant uniquely synthetized by a few microalgal species. Abiotic stresses induce a hypercarotenogenic response in several chlorophytes, including the halophile Dunaliella salina (Chlorophyceae), which overaccumulates β-carotene in lipid bodies (plastoglobules) inside the chloroplast [45], and in the freshwater species Haematococcus pluvialis (Haematococcaceae), which forms haematocysts filled with ASTX-rich cytoplasmic lipid droplets [46]. Other microalgal xanthophylls with anti-inflammatory and immunomodulatory properties are fucoxanthin and diatoxanthin produced by several diatoms (stramenopiles) and by the haptophyte Tisochrysis lutea (Coccolithophyceae) (Figure 2E,F) ^[47][48][47,48]. As discussed in the following paragraphs, carotenoids appear to interfere in all major pro-inflammatory pathways implicated in the onset and progression of RA.

Astaxanthin: The Red Gold of Algae

With recognized safety for human consumption [49], approved Novel Food status [50], and an established role in promoting bone homeostasis in degenerative skeletal diseases [51], ASTX is the microalgal pigment of highest biopharmaceutical value. Clinical studies have shown that ASTX intake reduces the levels of systemic inflammatory biomarkers ^[52][53][52,53] and potentiates the pain-relieving effect of anti-inflammatory therapies [54]. The pharmacological effects of ASTX derive from its strong antioxidant-activity mediated via ROS quenching [55] (Figure 2G, top panel) and direct free radical scavenging ^[56][57][56,57] (Figure 2G, bottom panel). This amphipathic molecule is symmetrically arranged within the lipid bilayer [58], thus exerting antioxidant activity on both intra- and extracellular environments. Notably, the higher number of hydroxyl groups compared with other carotenoids confers to ASTX superior ROS-detoxifying capacity [59]. Early studies showed that ASTX suppressed ROS production ^{[60][61][62][63]}[60,61,62,63] and secretion of pro-inflammatory ILs by cultured human-activated monocytes [64]. Moreover ASTX stimulated the expression of ROS-scavenging enzymes in chondrocytes challenged with IL-1β [65], and inhibited pro-inflammatory and osteoclastogenic gene expression in macrophages challenged with RANKL [66]. Lastly, the administration of ASTX promoted cartilage health in animal models of arthritis and osteoasthritis ^[67][68][69][67,68,69]. Notably, the esterified biological form of ASTX displays higher bioavailability compared with synthetic ester-free derivatives ^{[70][71][72][73]}[70,71,72,73], suggesting the need to improve its production from natural sources. However, a major limitation to the clinical use of ASTX lies in its low solubility in gastrointestinal fluids [74], requiring encapsulation in polysaccharide, lipid, and protein nanoparticles to enhance its delivery and release ^{[75][76][77][78][79][80]}[75,76,77,78,79,80].

2.2. Anti-Inflammatory Mechanisms of Action of Astaxanthin and Other Carotenoids

2.2.1. NF-κB Pathway

ASTX and β-carotene interfere with the NF-κB pathway blocking the translocation of the NF-κB transcription factor to the nucleus, thereby suppressing ROS and pro-inflammatory gene expression. This effect is likely mediated through targeting the Inhibitor of the NF-κB γ subunit (IKK-γ) of the IkB kinase complex ^{[81][82][83][84]}[81,82,83,84]. This prevents the phosphorylation and subsequent proteasome-mediated degradation of the IkBα binding factor, which abolishes the release of NF-κB [30]. A similar inhibitory effect has been proposed for fucoxanthin and diatoxanthin ^[85][86][85,86]. The Mitogen- and Stress-activated protein Kinase-1 (MSK1) is a nucleus-localized factor, which activates the NF-κB pathway [87] and the transcriptional regulator cAMP-responsive Element-Binding Protein (CREB) [88]. Phosphorylated CREB binds CREB-Responsive Elements (CRE) promoting pro-inflammatory gene expression [89]. These events are suppressed by ASTX, which inhibits MSK1 autophosphorylation [90]. Lastly, in silico simulations suggested that ASTX and β-carotene extracellularly interact with IL-6 and TNF-α, preventing their binding to membrane receptors [91]. ASTX may also interact with the NF-κB-Inducing Kinase (NIK) and block the phosphorylation of the IKK-α subunit of the IkB α kinase complex, suppressing the NF-κB pathway [92].

2.2.2. JAK2/STAT3 and JNK/p38 MAPK Pathways

β-carotene and ASTX further modulate the pro-inflammatory pathways mediated by the JNK/p38 MAPK [93] and JAK2/STAT3 kinases ^[84][94][84,94], the latter responding to IL-6 in the pathogenesis of RA and osteoarthritis ^[31][95][31,95]. Phosphorylation of STAT3 dimers by JAK2 induces nuclear translocation and the differentiation of CD4⁺ T cells into the highly reactive T helper 17 (Th17) cell type [96]. TNFs and IL-1 activate the JNK/p38 MAPK pathway starting a phosphorylation cascade ending with JNK/p38 MAPK nuclear translocation [97], and phosphorylation of pro-inflammatory transcription factors (ELK1, MEF2, ATF2, and STAT1) [98] and of the MAPK-activated kinase 2 (MK2), which, in turn, targets the tristetrapolin (TTP) factor, promoting stabilization of IL mRNAs [99]. These oxidant-sensitive inflammatory pathways are also modulated by lutein [100], as reported using extracts enriched in this xanthophyll from different species of the chlorophyte genus Tetraselmis (Chlorodendrophyceae) [101].

2.2.3. Other Pro-Inflammatory Pathways Targeted by Microalgal Carotenoids

Mitochondrial disfunction is a key pathogenic driver in RA [102], and ASTX was reported to attenuate organellar ROS production in human chondrocytes treated with IL-1β [69]. In addition to suppressing pro-inflammatory pathways, ASTX is also suggested to promote cartilage homeostasis via the transcriptional regulator nuclear factor-erythroid 2-related factor 2 (Nrf2) ^[68][93][68,93]. ASTX is suggested to stabilize and promote the nuclear translocation of Nfr2, which binds so-called antioxidant response elements (AREs), enhancing the expression of anti-inflammatory and ROS-detoxifying genes ^[103][104][103,104].

2.3. Lipids and Their Derivatives

A hallmark of RA is the altered fatty acid profile of the synovium ^[105][106][105,106], while the intake of polyunsaturated fatty acids (PUFAs) correlates with joint health and mitigates the risk of RA onset [107]. Microalgal mass cultivation is a more sustainable way to derive functional lipids compared with cold water fish ^{[108][109][110][111][112]}[108,109,110,111,112]. Phytoplankton occupies the lowest trophic level in oceans and freshwater basins, representing the primary PUFAs producer in aquatic food webs [113]. Global warning and ocean acidification are predicted to affect phytoplankton ecology ^[114][115][114,115], thus reducing PUFAs availability to higher trophic levels and, eventually, putting at risk the supply for human nutrition [116]. Moreover, upon stress acclimation, microalgae synthetise a wider range of anti-inflammatory and immunomodulatory lipids compared to animals ^{[117][118][119][120][121]}[117,118,119,120,121].

2.3.1. Long-Chain Polyunsaturated Fatty Acids

Several microalgae accumulate very long-chain PUFAs ^[122][123][122,123], including the omega-3 (ω-3, n-3) PUFAs [124] α-linolenic (18:3), docosahexaenoic (DHA, 22:6, n-3, Figure 3A), docosapentaenoic (DPA, n-3, 22:5, Figure 3B), and eicosapentaenoic (EPA, 20:5, n-3, Figure 3C) acids but also ω-6 PUFAs like arachidonic (ARA, 20:4, n-6, Figure 3H), γ-linolenic (18:3), linoleic (18:2), and dihomo-γ-linolenic (DGLA, 20:3, n-6, Figure 3D) acids. These molecules are biosynthetic precursors of anti-inflammatory signaling molecules and interfere with pro-inflammatory pathways ^[125][126][125,126]. The administration of lipids from the DGLA-hyperproducing freshwater chlorophyte Lobosphaera incisa (Trebouxiophyceae) suppressed the expression of the NF-κB pathway-related genes in an animal model of chronic inflammation [127]. Similarly, lipid extracts from the haptophyte Pavlova lutheri (Prymnesiophyceae), an EPA- and DHA-hyperaccumulating strain, inhibited IL-6 and TNF-α production in activated human macrophages, possibly through suppressing the NF-κB pathway [128]. Several heterotrophic marine microorganisms are strong DHA producers, such as the dinoflagellate Crypthecodinium cohnii (Dinophyceae) and, above all, the thraustochytrids protists Thraustochytrium spp., Aurantiochytrium (formerly Schizochytrium) limacinum (Labyrinthulomycetes) ^{[129][130][131][132][133]}[129,130,131,132,133], and related genera [134]. Although these microorganisms cannot exploit light energy to drive their metabolism, they are capable of fermenting plant biomass hydrolysates, affording cost-effective heterotrophic cultivation using renewable feedstocks ^{[135][136][137][138][139][140][141]}[135,136,137,138,139,140,141]. Arguably, the most valuable heterotrophic sources of DHA are Schizochytrium spp., which recently obtained Novel Food status ^[142][143][142,143]. Notably, a recent human clinical trial investigated the supplementation of an EPA- and DHA-enriched oil from a Schizochytrium sp. in RA patients, reporting beneficial effects on joint health and the blood levels of inflammatory markers [144].

2.3.2. Betaine Lipids

Betaine lipids are anti-inflammatory and immunomodulatory glycerolipids in which the phosphate and carbohydrate moieties attached to the glycerol backbone are replaced with positively charged ether-bond betaine groups. Betaine lipids are widely distributed in all clades of eukaryotic microalgae, where they act as acyl group donors upon membrane lipid remodelling and during the accumulation of storage neutral lipids ^[145][146][145,146]. Betaine lipids derive from the turnover of membrane phospholipids under abiotic stresses, mainly temperature and nutrient starvation ^{[147][148][149]}[147,148,149]. The betaine lipid 1,2-diacylglyceryl-3-O-4’-(N,N,N-trimethyl)-homoserine (DGTS, Figure 3E) is the most abundant betaine lipid in microalgae, followed by 1,2-diacylglyceryl-3-O-carboxy-(hydroxymethyl)-choline (DGCC, Figure 3F), and 1,2-diacylglyceryl-3-O-2’-(hydroxymethyl)-(N,N,N-trimethyl)-β-alanine (DGTA, Figure 3G), with evidence of DGTS-mediated inhibition of the IKK-β kinase causing suppression of the NF-κB pathway and secretion of pro-inflammatory ILs by Th1 and Th2 cells, as recently reported with extracts from the oleaginous chlorophyte Chromochloris zofingiensis (Chlorophyceae) [150].