Marine-Derived Compounds for the Potential Treatment of Glucocorticoid Resistance in Severe Asthma

One of the challenges to the management of severe asthma is the poor therapeutic response to treatment with glucocorticosteroids. Compounds derived from marine sources have received increasing interest in recent years due to their prominent biologically active properties for biomedical applications, as well as their sustainability and safety for drug development. Based on the pathobiological features associated with glucocorticoid resistance in severe asthma, many studies have already described many glucocorticoid resistance mechanisms as potential therapeutic targets. On the other hand, in the last decade, many studies described the potentially anti-inflammatory effects of marine-derived biologically active compounds. Analyzing the underlying anti-inflammatory mechanisms of action for these marine-derived biologically active compounds, we observed some of the targeted pathogenic molecular mechanisms similar to those described in glucocorticoid (GC) resistant asthma. This article gathers the marine-derived compounds targeting pathogenic molecular mechanism involved in GC resistant asthma and provides a basis for the development of effective marine-derived drugs.


Introduction
Asthma is a chronic inflammatory disease of the lower airways characterized by airway hyperresponsiveness and remodeling, leading to wheeze, cough, chest tightness, and difficulty in breathing. The prevalence of asthma is still increasing, while the potential risk factors for asthma seems to make equal contributions [1]. Although among the population of adults with asthma only 3% to 10% are classified as suffering from severe asthma [2,3], the costs of healthcare per patient are higher than those for stroke, type 2 diabetes, or chronic obstructive pulmonary disease (COPD) [4]. According to the current guidelines [5], difficult-to-control asthma is asthma that is uncontrolled despite treatment with high-dose inhaled glucocorticoids (ICS) combined with long-acting β 2 -agonists or other controllers, or that requires such treatment to maintain good symptom control and reduce exacerbation; severe asthma is considered a subset of difficult-to-control asthma that is uncontrolled despite adherence to maximal optimized Step 4 or Step 5 therapy and treatment of contributory factors, or that worsens when high-dose treatment is reduced.

Mechanisms of Action of Glucocorticoids
Although the topic has been extensively reviewed by Keenan et al. [20] and many others [7,[21][22][23], before delving into the altered cellular and molecular basis of signaling that leads to GCs resistance, it is important to review the heterogenous mechanisms of action by which GCs exert their downstream effect.
GCs have been extensively used in many diseases for a long time, but their molecular mechanisms of action are still not completely understood. GCs bind on the intracellular glucocorticoid receptors (GRs) of the target cell. There are two major variants of GRs with different C-terminal domains: GR-α, and GR-β. GR-α isoform-bind to GCs and affect GR signaling pathways through various post-translational modifications, such as phosphorylation, acetylation, and other modifications [24], while GR-β is unable to bind to GCs and cannot affect GC-induced modification. GR-β probably regulates GC activity, antagonizes GR-α isoform, and regulates GR-α/β heterodimers [25,26].
Genomic mechanisms are mediated by binding to GRs in the cytoplasm and further translocation of the GC/GR complex into the nucleus, while non-genomic mechanisms are mediated through specific interaction with GRs, or nonspecific interactions with the cell membrane [27]. Intracytoplasmic GRs present in inactive forms, in a protein complex, and attached to a chaperone protein. The dissociation of GR and the dissociation of chaperone protein upon activation allow the translocation of GR into the nucleus [28]. Inside the nucleus, the GC/GR complex regulates up to 20% of genes expressed by immune cells by trans-repressing inflammatory genes and stimulating the transcription of anti-inflammatory genes, leading to the reduced activation, recruitment, and survival of inflammatory and epithelial cells [29][30][31]; it also regulates mRNA stability [32] and the immunomodulatory function of smooth muscle cells and airway remodeling in asthma [33].
High concentrations of GCs exert non-genomic actions; inhibit the degranulation of mast cells through the stabilization of the plasma membrane or through a reduction in [Ca2+] elevation [34]; and promote anti-inflammatory effects through negative interference with MAPK signaling pathways [35].

Glucocorticoids Resistance: Cellular and Molecular Basis
Decreased GC responsiveness can be inherited or acquired. In the case of inherited decreased GC responsiveness, GC insensitivity most probably is not caused by a singular genetic mutation and involves a range of genetic variations. Some of the involved genes have already been determined [36][37][38] and are not the aim of our study.
The research into specific studies dedicated to GC resistance revealed the following responsible mechanisms: • Deficient binding between the GC and the GR or between the GR complex and DNA may be a cause [39]. • Increased antagonism is determined either by increased GR-β expression [40] or by diminished GR-α expression [41]. This can be explained by the IL-2/IL-4-induced suppression of GR-α (and not GR-β) expression in peripheral blood mononuclear cells (PBMCs) [42]. Additionally, IL-2 and IL-4 can synergistically reduce (via the p38MAPK pathway) nuclear translocation and binding affinity in T-cells (reversible by a p38 inhibitor) [43]. Furthermore, IL-17 and IL-23 cytokines were reported to significantly upregulate GR-β [42]. • Inflammation or oxidative stress has the potential to negatively affect GC signaling [22].

•
The expression of various anti-inflammatory genes induced by GCs can be reduced through GR phosphorylation by, for example, p38 mitogen-activated protein kinase (MAPK) and by the reduced activity of histone deacetylase 2 (HDAC2) [43,44].

•
The upregulation of certain cytokines, such as IL-2, IL-4, and IL-13, was detected in the lungs of patients with GC unresponsiveness [45][46][47]; in vitro, the overexpression of these cytokines was associated with the phosphorylation of GR and a decrease in nuclear translocation in inflammatory cells through the activation of p38 mitogenactivated protein kinase [48]. p38MAPK activity was demonstrated to be higher in alveolar macrophages from patients with impaired response to GCs compared to 'responders'. Furthermore, the expression of MKP-1 (DUSP1 gene), an endogenous inhibitor of the MAPK pathway, was significantly diminished in alveolar macrophages after GCS exposure, leading to an increase in p38MAPK activity [49]. Furthermore, p38MAPK inhibitors, such as AZD7624 or SB203580, have recently been investigated in corticosteroid-resistant asthmatic populations [50,51].
• Increased HDAC activity using theophylline, PI3K, and p38 MAPK inhibitors demonstrated beneficial effects [52][53][54], especially in glucocorticoid-resistant asthmatic smokers, where increased antagonism of the GR-α resulted from a reduced ratio of GR-α to GR-β isoforms [55]. Moreover, reduced total HDAC activity in PBMCs isolated from prednisone-dependent asthmatics compared to ICS-maintained moderate asthmatics and healthy volunteers was reported [56]. • GC resistance has been associated with Haemophilus influenzae, Chlamydia pneumoniae, Influenza A virus (IAV), rhinovirus, and Respiratory syncytial virus (RSV) infections [57][58][59][60][61]. The molecular mechanism proposed for glucocorticoid insensitivity in rhinovirusinfected primary human bronchial epithelial cells is the activation of NF-κB and c-Jun N-terminal kinase, which leads to a decrease in GR-α nuclear translocation [62]. The influence of NF-κB activity on GC resistance has also been confirmed by research on the blockade of this pathway [63,64]

•
In a study of human fetal airway smooth muscle cells, TNF-α and IFN-γ cytokines were shown to sustain GC resistance by promoting the Nuclear factor-κB (NF-κB) pathway and Stat1 phosphorylation [67]. TNF-α also demonstrated the potential to activate the c-Jun N-terminal kinase (JNK), which directly phosphorylated GR-α at Ser226 and inhibited GRE-binding [68].

•
The nitrosylation of the glucocorticoid receptor at the HSP90 (chaperone) binding site can be caused by high levels of nitric oxide generated in situ as a result of eosinophilic inflammation. This can decrease its affinity with chaperone proteins that protect it from cytoplasmic degradation. The binding affinity to GCs (ligand) in structural cells, such as fibroblasts, can also be reduced by nitrosylation [69]. In conclusion, asthmatics with persistent airway eosinophilia with increased localized nitric oxide production and possibly increased remodeling may develop GC resistance through the repeated nitrosylation of GR. • Increased NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome/IL-1β activation contributed to glucocorticoid resistance in murine models of steroid-resistant allergic airway disease [70].

•
The Th2 cytokines IL-13 and IL-5 each possess the ability to induce diminished GRbinding affinity. The effect of hydrocortisone in suppressing LPS-induced IL-6 production by monocytes was demonstrated to be significantly hindered when the cells were primed by IL-13 [71]. Additionally, IL-5-primed eosinophils were unresponsive to GS-induced apoptosis (via synergistic upregulation of nuclear-factor IL-3 due to a cross-talk between GCS-induced trans-activation signaling and IL-5 antiapoptotic pathway) [72].

•
The adoptive transfer of Th17 cells in mice resulted in the development of steroid insensitivity, and Th17 cells and IL-17A levels are frequently associated with CG resistance in asthmatic patients [73][74][75]. Accordingly, the expression of GR-β has been reported to increase Th17 responses [76]. In the obesity phenotype of asthma, the associated steroid resistance may be induced by IL-17 produced by the pulmonary type 3 innate lymphoid cells [77]. The role of IL-7 in GC resistance has been confirmed by the augmentation of dexamethasone anti-inflammatory action in diesel exhaust particle-induced neutrophilic steroid insensitivity secondary to anti-IL-17 therapy [78]. asthma through the concomitant administration of LPS + IFNγ; consequently, PP2A activity (that induced JNK) was attenuated and led to the phosphorylation of GR-α at Ser226, thereby hindering glucocorticoid receptor nuclear translocation in pulmonary macrophages [66]. • LPS promoted a shift from Th2-derived airway eosinophilic inflammation to Th17-drived neutrophilic inflammation in an ovalbumin-sensitized murine asthma model [79]. • Dysregulated IL-10 production is associated with GC insensitivity. This is probably due to impaired IL-10 production, according to Hawrylowicz et al., who compared in vitro stimulated T lymphocytes from corticosteroid-resistant asthmatic with dexamethasone to T lymphocytes from steroid-sensitive asthmatics [80].

•
The induction of Th2/Th17 responses in fungus-exposed patients has the potential to develop GC resistance [65]. More precisely, in neonatal mice, Aspergillus alternata exposure induced IL-33 dependent GC resistant asthma, mediated by ILC2 and Th2 cells [81]. The suggested mechanism underlying glucocorticoid insensitivity is the activation of p38-MAPK in CD4 + T cells and induction of phosphorylation of GR by IL-33 [82].
GCs also produce pro-inflammatory effects under stress conditions [83]. Table 2 depicts the potential targeted molecular and immunopathogenic mechanisms in glucocorticoidresistant severe asthma. Table 2. Potential targeted mechanisms in glucocorticoid-resistant severe asthma.

Potentially Therapeutic Effect of Marine-Derived Biologically Active Compounds in Severe Asthma
Experimental studies with marine compounds demonstrating their effectiveness in in vitro or in vivo models of bronchial asthma are scarce [84][85][86][87][88]. On the other hand, recent studies revealed the significant potential of marine compounds to interfere with molecular mechanisms similar to those involved in GC-resistant asthma. Therefore, a standardized inclusion-exclusion criterion was implemented, aiming to justify the current review. The literature search queries were performed until September, 2021. We included and analyzed all the original articles from PubMed and Scopus databases, with marine compounds that potentially target these molecular mechanisms; we excluded reviews and generalized or irrelevant studies (results illustrated in Table 3). Some of these compounds from different marine sources are well characterized and have well-defined structures (Table 4), while others are extracts with complex compositions.
Mojabanchromanol (MC), a chromanol isolated from the brown algae Sargassum horneri, demonstrated anti-oxidant effects through the attenuation of particulate matterinduced oxidative stress, the reduction of the ROS-mediated phosphorylation of MAPK extracellular signal-regulated kinase 1/2 (Erk1/2) and of c-JNK, and the inhibition of the secretion of pro-inflammatory cytokines (IL-6, IL-1β and IL-33). The authors proposed that mojabanchromanol be developed as a therapeutic agent against particulate matter-induced airway inflammatory responses [84].
Sargachromenol, isolated from Sargassum horneri, demonstrated anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, by reducing the nitric oxide (NO); and in intracellular reactive oxygen species (ROS), by decreasing the mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and by inhibiting the activation of NFκB and MAPK signaling [90].      Some of these compounds from different marine sources are well characterized and have well-defined structures (Table 4), while others are extracts with complex compositions. Some of these compounds from different marine sources are well characterized and have well-defined structures (Table 4), while others are extracts with complex compositions. Some of these compounds from different marine sources are well characterized and have well-defined structures (Table 4), while others are extracts with complex compositions.
Mojabanchromanol (MC), a chromanol isolated from the brown algae Sargassum horneri, demonstrated anti-oxidant effects through the attenuation of particulate matterinduced oxidative stress, the reduction of the ROS-mediated phosphorylation of MAPK extracellular signal-regulated kinase 1/2 (Erk1/2) and of c-JNK, and the inhibition of the secretion of pro-inflammatory cytokines (IL-6, IL-1β and IL-33). The authors proposed that mojabanchromanol be developed as a therapeutic agent against particulate matterinduced airway inflammatory responses [84].
Sargachromenol, isolated from Sargassum horneri, demonstrated anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, by reducing the nitric oxide (NO); and in intracellular reactive oxygen species (ROS), by decreasing the mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and by inhibiting the activation of NFκB and MAPK signaling [90].
Fucoidan, purified from Saccharina japonica, reduced the production of NO, and downregulated the expression of the MAPK (including p38, ENK and JNK) and NF-κB (including p65 and IKKα/IKKβ) signaling pathways in a zebrafish experiment [91].
An extract from Korean marine alga Ulva pertusa, 4-hydroxy-2,3-dimethyl-2-nonen-4-olide, moderately inhibited the release of the pro-inflammatory cytokines IL-12 p40 and IL-6 from bone marrow-derived dendritic cells, as well as signal transduction by inhibiting phosphorylation of NF-kB, and, thus, warranted further study to evaluate its potential as a "therapeutic agent for inflammation-associated maladies" [94].
Mojabanchromanol (MC), a chromanol isolated from the brown algae Sargassum horneri, demonstrated anti-oxidant effects through the attenuation of particulate matterinduced oxidative stress, the reduction of the ROS-mediated phosphorylation of MAPK extracellular signal-regulated kinase 1/2 (Erk1/2) and of c-JNK, and the inhibition of the secretion of pro-inflammatory cytokines (IL-6, IL-1β and IL-33). The authors proposed that mojabanchromanol be developed as a therapeutic agent against particulate matterinduced airway inflammatory responses [84].
Sargachromenol, isolated from Sargassum horneri, demonstrated anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, by reducing the nitric oxide (NO); and in intracellular reactive oxygen species (ROS), by decreasing the mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and by inhibiting the activation of NFκB and MAPK signaling [90].
Fucoidan, purified from Saccharina japonica, reduced the production of NO, and downregulated the expression of the MAPK (including p38, ENK and JNK) and NF-κB (including p65 and IKKα/IKKβ) signaling pathways in a zebrafish experiment [91].
An extract from Korean marine alga Ulva pertusa, 4-hydroxy-2,3-dimethyl-2-nonen-4-olide, moderately inhibited the release of the pro-inflammatory cytokines IL-12 p40 and IL-6 from bone marrow-derived dendritic cells, as well as signal transduction by inhibiting phosphorylation of NF-kB, and, thus, warranted further study to evaluate its potential as a "therapeutic agent for inflammation-associated maladies" [94].
Fucoidan, purified from Saccharina japonica, reduced the production of NO, and downregulated the expression of the MAPK (including p38, ENK and JNK) and NF-κB (including p65 and IKKα/IKKβ) signaling pathways in a zebrafish experiment [91].
An extract from Korean marine alga Ulva pertusa, 4-hydroxy-2,3-dimethyl-2-nonen-4olide, moderately inhibited the release of the pro-inflammatory cytokines IL-12 p40 and IL-6 from bone marrow-derived dendritic cells, as well as signal transduction by inhibiting phosphorylation of NF-kB, and, thus, warranted further study to evaluate its potential as a "therapeutic agent for inflammation-associated maladies" [94].
Pyrenocine A, produced from the marine-derived fungus Penicillium paxilli, produces immunosuppressive effects through the inhibition of pro-inflammatory mediators (TNF-α and PGE2) and inhibits the expression of genes related to NFκB activation in macrophages stimulated with LPS [108].
Lobocrassin B, an extract from the soft coral Lobophytum crissum, inhibited the production of TNF-α and NF-κB, an important transcription factor responsible for cytokine production, in mouse dendritic cells [116].
Didemnin B (Depsipeptides) was extracted and isolated from Trididemnum solidum; it exhibited strong anti-inflammatory and immunosuppressive activity through the inhibition of iNOS and NF-kB [122,123].
Another ethanol extract with a commercial grade of 70%, separated from Sargassum horneri, demonstrated similar effects: it significantly repressed the secretions of inflammatory cytokines and reduced protein expression in PGE2, TNF-α, IL-6, IL-1β, NF-κB, and MAPKs from PM-activated macrophages [128].
A sulfated polysaccharide with a sulfate content of 9.07% from Saccharina japonica showed significant inhibition of NO and PGE2 production via the downregulation of iNOS and COX-2 expression. The polysaccharide also suppressed TNF-α and (IL)-1β production via the NF-κB and MAPK signal pathways in LPS-induced RAW cells [131].
A sulfated polysaccharide isolated from Sargassum fulvellum demonstrated a significant and concentration-dependent decrease in the production levels of NO, TNF-α, PGE2, IL-6, and IL-1β in LPS-treated RAW 264.7 macrophages [132].
The fatty acids from the Arctoscopus japonicus egg of a cold-water marine fish presented anti-inflammatory effects through the suppression of the expression of iNOS, COX-2, IL-1β, IL-6, and TNF-α, and a reduction in the phosphorylation levels of NF-κB p-65, p38, ERK1/2, and JNK, key components of the NF-κB and MAPK pathways [139].

Th2 Cytokines
The whole culture extract of a marine-derived actinomycete strain, culture CNQ431, identified as a Streptomyces sp., demonstrated potent suppression of Th2 cytokines IL-5 and IL-13, but also the production of the dendritic cell-associated cytokines IL-1 and TNF-α, indicating immunosuppressive effects on both the APCs (i.e., dendritic cells) and the Th2 cells in a mouse splenocyte assay [124].
An ethanol extract from Sargassum horneri, obtained from a brown alga, was found to have antioxidant, anti-inflammatory, and anti-allergic effects in a BALB/c mouse model of asthma sensitized with ovalbumin. IL-4, IL-5, and IL-13 were found to be decreased in the lungs of PM-exacerbated asthmatic mice. Concomitantly, the Th17 cell response, the expression of responses of relevant effector cytokines, IL-17a and Th2/Th17, were also decreased [86].
A methanol extract of Sargassum hemiphyllum, a brown seaweed, inhibited the increase of TNF-α-induced NF-kB protein levels, the transcription factor of TNF-α, and IL-8 and TNF-α release, suggesting an inhibitory effect on atopic allergic reactions [130]. Exopolysaccharide EPCP1-2, an extracellular polysaccharide extracted from Crypthecodinium cohnii, has significant potential to inhibit macrophage proliferation, as well as to downregulate the expression of TLR4, TAK1, MAPKs, and NF-κB protein. It acts as an anti-inflammatory agent through macrophage suppression on the RAW 264.7 macrophage cell line and is a potent regulatory MAPK, and NF-κB signaling pathways [133].
Spirulina extract (Immulina ® ), a high-molecular-weight polysaccharide extract from the cyanobacterium Arthrospira platensis (Spirulina), showed anti-inflammatory and inhibitory effects in an induced allergic inflammatory response and on histamine release from RBL-2H3 mast cells. It also has the potential to inhibit the IgE-antigen-complex-induced production of TNF-α, IL-4, leukotrienes, and histamine, and showed promising effects with respect to the relief of allergic rhinitis symptoms [137,138].
A component of this extract, n-hexane, a fatty-acid-rich fraction, ameliorated allergic airway inflammation in a mouse model of ovalbumin-induced asthma: eosinophil infiltration and goblet cell hyperplasia were significantly reduced around the airways, and the concentrations of Th2-related cytokines (IL-4, IL-5, and IL-13) and Th17-related cytokines (IL-17) were significantly decreased in the spleen and bronchoalveolar lavage fluid [88].
Phlorotannins isolated from brown algae, Eckolonia cava, exhibited anti-allergic activities through the inhibition of degranulation: the tested compounds suppressed the binding between IgE and FcεRI receptors [140].
Reticulol, a polyketide isolated from Graphostroma sp. MCCC 3A00421 deep-sea hydrothermal sulfide deposits from the Atlantic Ocean, showed potent inhibition of degranulation with an IC50 value of 13.5 µM [141].
An extract of Apostichopus japonicus, obtained from a sea cucumber, showed antioxidant and anti-inflammation effects in mice with ovalbumin-induced asthma. The hyper-responsiveness of airways was significantly lower, the number of eosinophils in the lungs was decreased, and T regulatory cells significantly increased in the mesenteric lymph nodes [143].
Peridinin and fucoxanthin, carotenoids isolated from Symbiodinium sp., a symbiotic dinoflagellate, and from Petalonia fascia, a brown alga, respectively, were shown to suppress allergic inflammatory responses through the inhibition of delayed-type hypersensitivity in mice, and to reduce the number of eosinophils in both the ear lobe and peripheral blood. The inhibitory effect of peridinin was higher than that of fucoxanthin [144].
Grassystatin A, obtained from the cyanobacterium Lyngbya confervoides, inhibited the upregulation of IL-17 and interferon-γ (INF-γ) in response to antigen presentation, reduced T cell proliferation in a dose-dependent manner, and inhibited the upregulation of IL-17 and IFN-γ in response to antigen presentation [106].
Ogipeptins A-D, obtained from the culture broth of the Japanese marine Gramnegative bacterium Pseudoalteromonas sp. SANK 71903, decreased TNF-α release by human U937 monocytic cells [107].
Carijoside, a steroid glycoside extracted from Carijoa sp., inhibited the superoxide anion generation and elastase release by human neutrophils [117]. On the other hand, rossinones A and B, terpene-derived metabolites of the Aplidium speciesascidian family of Polyclinidae, inhibited only the superoxide production [120].
A brominated indol isolated form the gastropod mollusk Dicathais orbita, 6-bromoisatin, inhibited inflammation in a murine model of LPS-induced acute lung injury by significantly reducing TNF-α and IL-1β production and associated lung damage [121].
Sinulerectol A and Sinulerectol B, as extracts isolated from the marine soft coral Sinularia erecta, showed anti-inflammatory activities in neutrophil pro-inflammatory responses [125].
Sargassum horneri (Turner), a C. Agardh ethanolic extract with 70% ethanol lyophilized at −40 • C, obtained from a brown alga, showed antioxidant and anti-inflammatory effects through a dose-dependent reduction of the mRNA level of cytokines, including IL-1β, and pro-inflammatory genes, such as iNOS and COX-2, in LPS-stimulated macrophage activation. In addition, the anti-inflammatory effects were obtained by inhibiting ERK, p-p38 and NF-κB phosphorylation and by the release of IL-1β in LPS-stimulated macrophages [129].

Conclusions
In recent years, efforts have been made to understand the mechanisms underlying GC resistance. To overcome GC resistance, which is frequently associated with high doses of GC treatment, there is an urgent need for more specific targeted therapies. Natural compounds have been demonstrated to be effective against various pathological mechanisms through a variety of pathways. Some of these mechanisms were also shown to be involved in GC resistance. This paper reviewed the marine compounds potentially acting on the mechanisms involved in GC-resistant severe asthma. The article provided a basis for the development of effective marine-derived drugs as new and safe sources for the potential treatment of glucocorticoid-resistant severe asthma.

Conflicts of Interest:
The authors declare no conflict of interest.