Inflammation seems to be the common denominator among the above described seemingly unrelated biological entities, i.e., the gut microbiome, the immune system, and n-3 PUFAs. Inflammation is currently accepted to play a key role in the progression of several chronic diseases, such as atherosclerosis, inflammatory bowel disease, cancer, diabetes, neurodegenerative syndromes, etc. [ 9 ]. In addition, as above described several evidences support the role of both the microbiota and the n-3 PUFAs in regulating inflammation and the immune system [ 45 74 ]. Moreover, dietary n-3 PUFAs, affecting gut integrity, have been shown to reduce clinical colitis and colonic immunopathology by improving epithelial barrier function in animal models [ 84 ]. Indeed, in clinical studies n-3 PUFAs have demonstrated the ability of: (i) Decreasing theratio; (ii) decreasing the levels ofand; (iii) increasing the abundance of butyrate-producing bacterial genera, i.e.,and 87 ]. These data were in line with those obtained in a subsequent study where the authors also found a significant correlation between n-3 PUFAs plasma levels and SCFA-producing bacteria, i.e.,family [ 85 ]. In addition, a diet supplemented with n-3 PUFAs was able to prevent neuropsychiatric disorders and dysbiosis induced by social instability stress during adolescence, and these effects were maintained through adulthood [ 88 89 ] supporting the concept that a healthy diet may have long-lasting beneficial effects and help fight off neurodegenerative diseases. Altogether these data allow hypothesizing a link among n-3 PUFAs intake, gut microbiome shaping and immune system modulation with the final common aim of hampering inflammatory-based disease ( Figure 3 ). Accordingly, this review will particularly focus on the recent studies regarding the therapeutic potential of the combination between fish n-3 PUFAs and probiotic/prebiotic in the IBD and retinal disease.

4.1. Inflammatory Bowel Disease

IBD, specifically Crohn’s disease (CD) and ulcerative colitis (UC), are relapsing and remitting inflammatory diseases of the GI tract without a clear etiology. The symptoms include abdominal pain, diarrhea, weight loss, ulceration, perforation, and bowel obstruction. Although the all picture of the pathogenesis of IBD remains unclear, aberrations in genetics, imbalances in the gut microbiome, dietary and lifestyle factors such as cigarette smoking, medications, and environmental triggers (i.e., geographical location and social status) are all believed to play a role in the disease’s development ( Figure 4 ).

92, Genome-wide association studies (GWAS) have identified 242 loci associated to the presence of IBD [ 5 90 ]. For example, variants at the NOD2 and CDH1 loci confer the largest increase in relative risk of IBD and have been associated to UC, respectively [ 91 93 ]. The NOD2 gene encodes a protein that is activated within the cytoplasm of macrophages and dendritic cells by bacterial ligands [ 94 ], whereas CDH1 gene has a critical role in cell-cell adhesion and is also required for epithelial cell tight junction formation [ 95 ]. Moreover, a meta-analysis revealed that genetic variations in TLR4 gene, the receptor for LPS, conferred a statistically significant risk of developing CD and UC [ 96 97 ]. In addition, polymorphisms in TLR2, the main receptor for gram-positive bacteria, have been associated with IBD in humans and there is an inflammation-dependent induction of TLR2 expression in intestinal macrophages [ 98 ].

Saccharomyces cerevisiae , and anti- Pseudomonas fluorescens -associated sequence 12 [100, The hyper-responsiveness of T cells toward non-pathogenic antigens could represent one of the possible etiologies for IBD. The presence of antibodies against commensal microbial antigens and autoantigens, such as anti-, and anti--associated sequence 12 [ 99 101 ], has been associated to dysbiosis and loss of microbiota responsible for the gut mucus barrier integrity. Since it has been shown that colitis-prone genetically predisposed GF mice colonized by IBD-associated-microbiota developed severe colitis compared to those that were colonized by healthy human microbiota, it can be hypothesized that gut dysbiosis contributes to IBD pathogenesis [ 102 ]. Together, these findings strongly indicate a bidirectional relationship between such diseases and gut dysbiosis, in which dysbiosis potentially contributes to the onset of IBD and also serves as a secondary consequence of gut inflammation [ 103 104 ].

Fecalibacterium prausnitzii , Clostridium clusters IV and XIVa, some Bacteroides species, Bifidobacterium ) and an increase of harmful bacteria (i.e., adherent-invasive Escherichia coli , Fusobacterium , Campylobacter concisus , Enterohepatic Helicobacter , Clostridium difficile , Veillonella ) in IBD patients [ It is well known that the microbiota and the gut have a symbiotic relationship: The human host supplies the nutrients needed for the survival of the microbes, and these latter protect the host against pathogens, and act as regulatory factors of the immune responses [ 56 ]. An aberrant mucosal immune system and irregular mucosal epithelium with increased intestinal permeability (IP) may permit the translocation of bacterial-derived toxins causing gut inflammation. The communication between the gut microbiota system and all the organs of the human body is regulated by the IP [ 105 106 ]. Indeed, IP degree is very changeable and results from the interconnection between several factors: Type of diet; gene expression; intestinal/liver pathology; surface mucus; integrity of tight junctions; and production of immunoglobulins [ 107 ]. Several investigators detected a decrease of several protective bacteria (i.e.,clusters IV and XIVa, somespecies,) and an increase of harmful bacteria (i.e., adherent-invasive) in IBD patients [ 108 109 ].

113, Ruminococcus torques , Bacteroides , Prevotella ), increased IP and inflammation [ Lactobacillus and Akkermansia muciniphila blooming, and reduced gut inflammation [ Akkermansia muciniphila could be a paradox because this is a mucin-user bacterium and at the same time, it is crucial for the maintenance of the mucus layer integrity [ Akkermansia muciniphila has been associated to a mucus layer thickening and a reduction of IP [ Several mechanisms are responsible for the intestinal inflammation caused by the consumption of high-fat diets (HFDs), including both changes in the intestinal barrier and composition of the intestinal microbiota. Experimental and clinical data indicated a direct correlation between plasma endotoxin levels and dietary fat intake, which suggested an increase of the IP [ 110 111 ] due to the decrease of epithelial tight junction proteins, such as occludin. Indeed, several experimental studies showed a dramatic decrease of intestinal occludin expression associated to the administration of a HFD [ 112 114 ]. On the other hand, high consumption of carbohydrates, such as glucose, sucrose, lactose, or fructose, overwhelms absorptive mechanisms of the intestine, resulting in high luminal sugar concentrations used by the microbiota as an energy source [ 115 ]. In support of this hypothesis, consumption of a high sugar diet was demonstrated to promote intestinal dysbiosis, the expansion of harmful bacteria (such as,), increased IP and inflammation [ 116 117 ]. In contrast, a fish-oil based diet has been associated withandblooming, and reduced gut inflammation [ 118 119 ]. The observed increase in the abundance ofcould be a paradox because this is a mucin-user bacterium and at the same time, it is crucial for the maintenance of the mucus layer integrity [ 120 ]. However, this latest effect appeared to be more significant as proved by the fact that increased intestinal levels ofhas been associated to a mucus layer thickening and a reduction of IP [ 121 ].

Recently, the treatment for IBD has made progress from simply controlling symptoms to modifying the course of the disease by achieving and maintaining remission which is defined as complete mucosal healing and normalization of blood markers, as well as disappearance of symptoms [ 122 ]. However, the development of a safer and more effective novel treatment for IBD is in great need.

124, Lactococcus lactis expressing IL-10, had demonstrated a significant remission of disease activity in CD patients. Furthermore, oligofructose-enriched inulin (OF-IN) administration was able to induce an improvement in CD associated to a reduction of the Ruminococcus gnavus . In addition, it has been recorded that the intake of this prebiotic is able to increase the abundance of the Bifidobacterium longum . This bacteria neutralizes reactive oxygen species and exerts anti-inflammatory effects at the site of inflammation, reducing gastrointestinal discomfort and tissue injury [ Bifidobacterium longum or a multistrain mix of Bifidobacterium longum , Lactobacillus acidophilus , and Streptococcus faecalis , increased expression of tight junction proteins in IBD [®, a mixture of 4 species of Lactobacillus, 3 species of Bifidobacterium, and 1 species of Streptococcus, has been shown to: (i) Improve epithelial barrier damage; (ii) induce remission in active UC; and (iii) decrease pro-inflammatory mucosal cytokine expression [ The role of gut microbiota in colitis development was confirmed by using animal models. GF mice displayed minimal inflammation or delayed onset of chemically and genetically induced colitis compared to the conventionally raised (CONV-R) animals [ 123 125 ]. However, higher mortality was seen in GF than CONV-R mice after giving dextran sulfate sodium (DSS), due to massive gut epithelial injury [ 126 ]. The seemingly paradoxical phenomenon could be explained by the lack of immune maturation and/or tolerance as well as the impairment of epithelial turnover (which is dependent on commensal colonization) in GF mouse intestine [ 127 128 ]. Gut microbiota metabolites act as important signals for the monitoring of the correct function of the epithelial barrier and the immune cells. Similarly, immune-driven signals central to gut homeostasis can also modulate the metabolism of immune cells [ 129 ]. It is well known that several hematopoietic cells produce IL-10 and its importance for maintaining tolerance within the intestinal microbiota come from experimental observations using IL-10- or IL-10R-deficient mice (both these mouse models develop spontaneous colitis) (see below, [ 130 ]), and clinical data (IBD patients are characterized by decreased levels of the anti-inflammatory cytokine IL-10). In line with these results, administration of genetically modified probiotic, i.e.,expressing IL-10, had demonstrated a significant remission of disease activity in CD patients. Furthermore, oligofructose-enriched inulin (OF-IN) administration was able to induce an improvement in CD associated to a reduction of the. In addition, it has been recorded that the intake of this prebiotic is able to increase the abundance of the. This bacteria neutralizes reactive oxygen species and exerts anti-inflammatory effects at the site of inflammation, reducing gastrointestinal discomfort and tissue injury [ 131 ]. Previous studies also showed that different probiotic combinations, especiallyor a multistrain mix of, and, increased expression of tight junction proteins in IBD [ 132 ]. Furthermore, VSL#3, a mixture of 4 species of Lactobacillus, 3 species of Bifidobacterium, and 1 species of Streptococcus, has been shown to: (i) Improve epithelial barrier damage; (ii) induce remission in active UC; and (iii) decrease pro-inflammatory mucosal cytokine expression [ 133 ].

Akkermansia genus) compared to wild-type [ Meta-analyses revealed a low incidence of IBD in eskimos, whose diet is particularly rich in n-3 PUFAs [ 134 ]. Starting from these clinical evidences, the impact of dietary n-3 PUFAs has been evaluated in different models of colitis. All the results obtained in these experiments are coherent and indicate that n-3 PUFAs decrease chemically-induced intestinal tissue damage and inflammation [ 135 ]. Similarly, in fat-1 transgenic mice, characterized by high levels of endogenous n-3 PUFAs, the intestinal tissue damage was significantly reduced compared to wild type mice, together with decreased expression of TNF-α, IL-1β, and increased synthesis of SPMs [ 136 137 ]. In addition, the fat-1 mice displayed higher gut microbiota diversity and more abundance of Verrucomicrobiota plylum (genus) compared to wild-type [ 138 ].

Moreover, a diet high in fibers and n-3 PUFAs is protective for the development of IBD, whereas a diet high in refined sugars, complex carbohydrates, and n-6 PUFAs (i.e., red meat), increases the risk of acquiring IBD. A number of studies indicated that homeostasis is crucial to have a good n-3 to n-6 PUFAs ratio, the former being anti-inflammatory and the latter pro-inflammatory molecules [ 1 139 ]. Therefore, dietary n-3 PUFAs supplementation is encouraged as anti-inflammatory adjuvants for IBD [ 140 141 ]. On the contrary, a low-fiber diet may increase the risk of CD, and, if associated to high consumption of sugar and soft drinks, it may also increase UC risk [ 142 ]. However, thus far, the effects of dietary interventions on CD and UC are uncertain [ 143 ]. The cornerstone is that dietary fiber is a plant-based carbohydrate that resists digestion by intestinal and pancreatic enzymes in the human GI tract. Soluble and insoluble dietary fibers are essential for gastrointestinal mucosa health because they serve as important substrates for the gut microbiota. The fermentation products selectively promote the growth of beneficial Bifidobacteria and Lactobacilli and exert anti-inflammatory (such as, inhibition of NFκB transcription) and anti-carcinogenic functions [ 144 ].

Clostridium clusters, and a reduction of Protebacteria and Bacillaceae [ Parabacteroides , and reduced the genus Bacteroides that include mucolytic species [ Two large clinical trials designed at evaluating the effects of n-3 PUFA supplementation on CD provided conflicting results and concluded that supplementation was not effective in preventing CD relapse [ 145 146 ]. Intriguingly, another study suggested that IBD patients that achieved a n-3/n-6 ratio of 1 maintained disease remission at a significantly higher rate compared to those did not reach this goal [ 147 ]. A double-blind, randomized study was carried out in 38 pediatric CD patients, who received, for 12 months, 5-aminosalicylic acid (50 mg/kg/d) + n-3 PUFAs (3 capsule/d, each capsule contained 400 mg/g EPA and 200 mg/g DHA) or 5-5-aminosalicylic acid (50 mg/kg/d) + olive oil placebo capsules [ 148 ]. The results indicated that the addition of enteric-coated capsules to a conventional therapy with 5-aminosalicylic acid delayed the relapse of the disease even though it could not prevent it. Another study used enteric-coated n-3 PUFAs capsules for remission maintenance in adult CD patients at high risk of relapse, and found n-3 PUFAs to be more effective than placebo [ 149 ]. However, two multicentre, randomized, double-blind, placebo-controlled studies, called Epanova Program in Crohn’s 1 and 2, (EPIC1 and EPIC2), found that relapse occurred in 32% with n-3 PUFAs and 36% with placebo in EPIC1, and 48% with n-3 PUFAs and 49% with placebo in EPIC2, indicating that n-3 PUFAs did not reduce the rate of relapse in patients with quiescent CD [ 146 ]. Of interest, two random double-blind placebo crossover studies reported significant improvement of the disease [ 150 ] and of the oxidative stress status in active UC patients receiving sulfasalazine, respectively [ 151 ]. Thus, the efficacy of n-3 PUFA or fish oil against CD or UC is, at best, marginal. However, none of the studies reported any adverse effects associated with n-3 PUFA supplementation. A recent meta-analysis of observational studies showed that fish consumption was inversely associated with the risk of CD. Moreover, there was a strong inverse association between dietary n-3 PUFAs intake and the risk of UC [ 152 ]. Previously published studies revealed that fish consumption and dietary n-3 PUFAs intake might play a role in the etiology of IBD [ 153 154 ]. In line with these findings, one study revealed that the Mediterranean diet, rich in fish and seafood, reduced inflammation [ 151 ] and normalized the gut microbiome in CD patients, i.e., an increase in Bacteroidetes andclusters, and a reduction of Protebacteria and Bacillaceae [ 155 ]. In a pilot study, short-term EPA-supplementation reduced mucosal inflammation favoring an improvement of both endoscopic and histological inflammation in almost all patients, together with a significant up-regulation of IL-10 expression, and a reduction of STAT3 activation [ 156 ]. Moreover, microbiota analysis showed that EPA treatment increased the family Porphyromonadaceae and the genus, and reduced the genusthat include mucolytic species [ 157 ].