In the article titled “Nutritional and Lifestyle Interventions for Age-Related Macular Degeneration: A Review” , there wAge-related macular degeneration (AMD) is the leading cause of blindness in the developed world. In this narrative review, we will summarize the nutritional interventions evaluated in numerous observational studies and a few randomized clinical trials. The AREDS and AREDS2 studies demonstrated that supplements including vitamins C and E, beta-carotene, and zinc may reduce the progression to advanced AMD in some patients by 25% over five years. This is one of the few nutritional supplements known to have a beneficial effect in any eye disease. Lutein/zeaxanthin supplementation may have beneficial effects in some individuals, whereas omega-3 fatty acids supplementation needs to be further investigated and supported by more evidence. Genetic factors may explain the different patterns of response and explain differences found among individuals. More importantly, a combination of lifestyle behaviors, such as the avoidance of smoking, physical activity, and the adoption of a healthy dietary pattern like the Mediterranean diet, was associated with a lower prevalence of AMD. The adoption of these lifestyles may reduce the prevalence of early-stage AMD and decrease the number of individuals who develop advanced AMD, consequently lowering the onerous and climbing costs associated with treatment of this disease.Age-related macular degeneration (AMD) is the leading cause of blindness in Western countries according to the World Health Organization [1] and this disease can significantly reduce quality of life [2]. As central vision is lost in advanced AMD, aging patients suffer limitations in their ability to function independently, because they have a diminished ability to recognize faces and to read the small print in newspapers, on food packages, and on medication labels [3].The early stages of AMD are characterized by the presence of pigmentary abnormalities and drusen near the fovea [1]. Late-stage AMD has two forms: (a) geographic atrophy, with major loss of the retinal pigment epithelium (RPE) and choriocapillaris; (b) neovascular AMD, with newly formed blood vessels in the macular region that lead to leakage of blood and serum, causing irreversible damage and progressive vision loss [4].The prevalence of AMD increases sharply with age, but despite major geographical and lifestyle differences it appears to be similar in European ancestry populations from the United States, Australia, and Europe . In a meta-analysis performed in 2004 by Friedman and collaborators, the prevalence rates for the late forms of the disease increase from less than 0.5% in subjects aged 50–60 years to 12–16% in individuals aged 80 years and above. Concerning early-stage AMD, an increase from about 1.5% in those with European ancestry aged 40–49 years to more than 25% in individuals aged 80 years and over was found [5].In the recent past, AMD was an ignored and untreated disease of the elderly. Now, hundreds of millions of dollars from the healthcare budgets of Western countries are spent on new treatments for the neovascular form. According to a 2013 United Nations report concerning the aging of the world population, the percentage of people aged 60 years or older is growing faster than any other age group due to fertility declines and increasing longevity. With these longer lifespans, the prevalence of AMD is also rising in the rest of the world outside Western countries. In Asia, the number of people with AMD increased in the last two decades, doubling the number of cases reported globally . For example, in Japan the prevalence of neovascular AMD has changed drastically. AMD was not registered in 1994 and now is the fourth-leading cause of visual disability [8], although it remains relatively low compared with Western countries [8].There are therapies for neovascular AMD, but no effective treatment exists for early-stage AMD and geographic atrophy, which is dramatic in terms of public health over the next two decades [9]. Therefore, there is considerable interest in preventing and delaying the onset of AMD by identifying and modifying risk factors [10]. Disease progression is slow and randomized multicenter clinical trials are extended in time, expensive, and hard to perform and analyze. However, even a modest protective effect on the prevention and/or progression of AMD would have a significant impact on patient welfare and on the burden to society.AMD is a complex disease with numerous inherited risk factors, including one major genetic risk locus on chromosome 1 in the complement factor H region and a second locus in the HTRA/ARMS2 region on chromosome 10, in parallel with minor genetic factors identified through genome-wide association studies [10–12].Similar to genetic polymorphisms and a family history of AMD, increasing age is another very important, consistent, and non-modifiable trait. Aging is clearly associated with an exponential rise in the incidence and prevalence of AMD [13]. Other non-modifiable traits associated with an increased risk of AMD in the literature are light skin color, light iris color , and perhaps being female . These risk factors guide the development of new therapies and novel treatment strategies, but the present situation demands efforts to identify and alter modifiable risk factors.Modifiable risk factors were found using epidemiological studies. It is important to stress that single epidemiological studies cannot be interpreted when isolated from other evidence, unless they can be replicated in independent cohorts. Biological plausibility based on the pathophysiology of the disease added to the associations found in several epidemiological studies adds confidence in the results, but does not prove the existence of causality [10].Epidemiological studies all around the world provided important information concerning the distribution of the different patterns of the disease in several countries and identified several environmental and modifiable risk factors. The most recognized environmental risk factor for AMD is smoking, which has a dose-dependent relationship. The risk of late-stage AMD is multiplied by 2.5 to 4.5 and appears to be cumulative over time . On the other hand, smoking and other environmental factors were not clearly associated with early-stage AMD .Curiously, as AMD shares several characteristics with Alzheimer's disease (AD), such as intra- and extracellular deposits, it was called the “Alzheimer's of the eye” . Additionally, AMD closely mirrors AD in terms of lifestyle and many risk factors . However, the similarity is relative, as the genetic background is completely different .As there are no effective means of primary prevention other than smoking cessation, other possible changes in lifestyle including nutritional changes acquire enormous importance. Multiple studies revealed oxidative stress as one of the mechanisms implicated in the pathogenesis of AMD and diet is generally the main source of antioxidants [24]. Concerning eye tissues, their biological integrity is dependent on the balance between the production of free radicals and their catabolism .The production of free radicals increases with age, but some of the endogenous defense mechanisms decrease, creating an imbalance that leads to progressive damage of cellular structures [25]. The vast antioxidative network includes vitamins , enzymes , carotenoids , and many other compounds  [26]. They act as a protective chain, and the different antioxidants have a synergistic effect and protect each other from direct destruction in the process of neutralizing free radicals [25]. The generation of singlet oxygen free radicals will extract hydrogen atoms from molecules with available six double bonds, such as the omega-3 fatty acid docosahexaenoic acid (DHA), leading to lipid peroxidation [24]. The lipid peroxides cross-link with proteins, nucleic acids, and other compounds, adversely affecting tissue structure and function [27].The eye presents an antioxidant system, with multiple components intervening from the aqueous humor to the retina. The retina possesses an antioxidant system with several components, but vitamins C and E and the carotenoids lutein and zeaxanthin are the most important [28]. Both vitamins cooperate to decrease the retinal epoxide adducts and participate in protection from blue light-induced damage [29]. The carotenoids are concentrated in the plexiform area of the macula, but zeaxanthin predominates in the central fovea whereas lutein is mostly present in the periphery. Zeaxanthin is therefore a more effective antioxidant in the area where the risk of oxidative damage is higher [30]. In addition, both carotenoids are found in the outer segments of rods and probably of cones, which are particularly rich in DHA and therefore potentially vulnerable to lipid oxidation .DHA comprises 40% of the polyunsaturated fatty acids (PUFAs) in the brain and 60% of the lipid constituents of retinal photoreceptor membranes; in humans, DHA is obtained from the diet . Also, DHA may be converted from eicosapentaenoic acid (EPA), but this synthesis involves several steps including elongation and peroxisomal beta-oxidation [33]. DHA and EPA have pleiotropic effects through signal transduction, gene regulation, and plasma membrane dynamics [34]. Health benefits arise from the ability of DHA and EPA to reduce the production of inflammatory eicosanoids, cytokines, and reactive oxygen species [35] and modulation of the expression of numerous genes involved in inflammatory pathways [36]. DHA is present in small quantities in most tissues, but is a major structural lipid of the retina presenting in particularly high levels in this neural tissue . DHA may be involved in the permeability, thickness, fluidity, and other properties of the membrane of photoreceptors [38] and its insufficiency is linked to changes in the function of the retina . The anti-inflammatory actions may inhibit the formation of new choroidal vessels that appear in exudative AMD . EPA and other major dietary omega-3 fatty acids appear to have a similar action [38]. The renewal of retinal membranes demands a steady supply of these omega-3 fatty acids by RPE cells. If there is an imbalance in the retinal lipids, photoreceptor degradation and accumulation may lead to the formation of drusen, formed mainly by the debris of lipids and lipoproteins in the RPE layer and sub-RPE space [34].Although the exact pathogenesis of AMD is not known oxidative stress is considered to be involved, because the human eye, and particularly the retina, is exposed to the production of free radicals leading to a pro-oxidative environment [28]. This vulnerability is related to several factors: (a) high vascularization, which results in high oxygen tension; (b) light-induced stress in the fovea due to increased cellular density and metabolism; (c) retinal tissues that have a high quantity of unsaturated fatty acids and photosensitizing compounds, which are highly susceptible to oxidation; (d) the exposure of the retina to cumulative radiation .The putative mechanisms are light-initiated oxidative damage and a reduction of the macular pigment . Long exposure to bright light, oxidative damage and the presence of oxidized metabolites in the outer segment of photoreceptors of the RPE may contribute to the formation of the drusen and pigment disturbances in the macula .It is therefore conceivable that dietary antioxidants and/or supplements may be beneficial to preventing and/or delaying the progression of AMD due to the decrease of oxidative stress and reduction of inflammatory events [28].In Western countries, the dietary intake of vitamins, carotenoids, essential fatty acids, and other oligoelements is generally enough to supply the needs of healthy individuals. The major dietary sources of the 18-carbon fatty acids alpha-linolenic acid (ALA) and linoleic acid (LA) are vegetable oils [26]. The ingestion of these fatty acids is essential as humans do not possess the enzyme systems necessary to insert double bonds at the omega-3 or omega-6 positions . Diets rich in fish, meat, and eggs will provide EPA, DHA, and arachidonic acid, but they can also be synthesized from ALA and LA . However, the conversion rate of ALA to omega-3 PUFAs is relatively low in humans and involves several steps [48]. Due to this, oils rich in ALA do not significantly increase DHA and EPA levels .In contrast, lutein and zeaxanthin are not synthesized by humans. They are found in plants such as fruits and spices (lutein) and lettuce, broccoli, and spinach (zeaxanthin) [10].The role of nutrition and nutritional supplements has been raised by a number of observational and randomized clinical trials. Three types of nutritional factors have mainly been investigated for their potential protection against age-related eye diseases: antioxidants , zinc, the carotenoids lutein and zeaxanthin, and omega-3 PUFAs. The results of the most important randomized clinical trials concerning the effects of these nutritional factors on AMD are presented and discussed below [28].AREDS was a complex randomized clinical trial designed to evaluate the effect of vitamins C (500 mg), E (400 IU) and beta-carotene (15 mg), with or without zinc (80 mg), and copper (2 mg), on the progression of AMD . Most of the 4757 patients, aged 55 to 80 years, were taking nutritional supplements at the time of study enrollment and, to standardize this a daily dose of a multivitamin and minerals tablet (Centrum®) was provided and 66% of the participants opted to use this supplement. The supplements were provided in much higher concentrations than the recommended daily intake [50]. Therefore, the effects found were attributed to the AREDS supplements [50]. The patients were followed up for a mean period of 6.3 years [28].The form of vitamin E used in AREDS was alpha-tocopherol, the form that is mainly present in tissues and blood [51]. Vitamin E exists as eight fat-soluble compounds of tocopherols and tocotrienols, and each subgroup has several subtypes . Nuts and seeds, whole grains, and dark leafy vegetables  are rich sources of alpha-tocopherol, which is absorbed and accumulated . The dose used in the AREDS trial was 400 UI daily, whereas the recommended daily intake is only 22.4 UI [50].Beta-carotene is a carotenoid often referred to as provitamin A [51]. Due to its antioxidative properties, beta-carotene was included in the multivitamin supplements, but a solid role in the prevention of AMD was not strongly supported [51]. Also, due to its effective antioxidant activity, vitamin C (ascorbate) was included in the AREDS formulation. However, there was no association between vitamin C intake and AMD in the Pathologies Oculaires Liées à l'Age (POLA) study or in the Eye Disease Case-Control Study Group [51–53].At the beginning of the study, 1117 patients had few if any drusen (Category 1) and 1063 patients had extensive small drusen, pigment abnormalities, or at least 1 intermediate size drusen and were classified as Category 2. Category 3 included 1621 participants possessing extensive intermediate drusen, geographic atrophy not involving the center of the macula, or at least one large drusen. Finally, 956 participants were included in Category 4 as they had advanced AMD or visual acuity less than 20/32 due to AMD in one eye [28]. The results showed that treatment with zinc alone or in combination with antioxidants reduced the risk of progression to advanced AMD in patients in Categories 3 and 4 [28]. The reduction of risk at five years for those taking the supplements plus zinc was 25% when compared to placebo controls. The treatment effect persisted following five additional years of follow-up after the end of the clinical trial [54]. During the study, 407 participants without geographic atrophy at baseline developed, at least, moderate geographic atrophy, not necessarily involving the center of the macula [28]. No significant differences among treatments with antioxidants, with zinc, or antioxidants plus zinc on the progression of AMD were found [49].After the publication of the AREDS report in 2001, the AREDS formulation became the standard of care. Due to serious concerns about the safety of beta-carotene, which increased the risk of lung cancer in smokers, this compound was replaced in the randomized clinical trial known as AREDS2 [28]. In addition to this concern, it was found that beta-carotene was not detectable in the human retina contrary to lutein, zeaxanthin, and mesozeaxanthin [10]. Moreover, in the Beta-Carotene and Retinol Efficacy Trial (CARET) study, the combination of retinol (vitamin A) and beta-carotene increased the risk of lung cancer and cardiovascular events [55]. This evidence discourages the use of beta-carotene in the prevention of AMD due not only to the side effects, but also to poor efficacy [51]. In addition, vitamin A alone was found to have no solid results concerning the prevalence of AMD [51] and it was not included in the original AREDS formulation.AREDS2 was a phase 3 study and controlled clinical trial, involving 4203 patients aged 50 to 85 years [56]. Conducted in 2006–2012, the selected participants were at risk of progression to advanced AMD with bilateral large drusen or large drusen in one eye and advanced AMD in the other eye . The main aim of AREDS2 was to improve the original AREDS formulation, making it more effective and safer. The lutein/zeaxanthin daily dose chosen was in the ratio 5 : 1 (10 mg of lutein : 2 mg of zeaxanthin) based on a small-scale study . These daily doses are substantially higher than a typical Western diet (1-2 mg of lutein : 0.2 mg of zeaxanthin). Primary randomization considered four groups of participants: (a) lutein (10 mg) + zeaxanthin (2 mg); (b) fish oil (350 mg of DHA + 650 mg of EPA); (c) lutein + zeaxanthin + DHA + EPA; and (d) placebo [56].The expected 25% incremental improvement over the original AREDS results was not obtained [56]. The addition of lutein + zeaxanthin, DHA + EPA, or both to the AREDS formulation did not further reduce the risk of progression to advanced AMD. In the analyses evaluating the patients assigned to lutein/zeaxanthin (2123) versus the patients not assigned to lutein/zeaxanthin (2080), an added 10% reduction was found in the risk of progression to advanced AMD in patients assigned to lutein/zeaxanthin versus those not assigned to lutein/zeaxanthin. However, the patients who received DHA + EPA did not show this effect. Lutein/zeaxanthin was not related to an increase in lung cancer incidence, contrary to beta-carotene that was associated with a rise in lung cancer in former smokers . Mesozeaxanthin is now available in the market, but there is no randomized clinical trial supporting its superiority to lutein/zeaxanthin [10].In brief, the evidence on beneficial and adverse effects from AREDS2 and other studies suggests that lutein (2 mg) + zeaxanthin (15 mg) could be better than beta-carotene (15 mg) in AREDS-type supplements, particularly for former or current smokers. Other studies also found a correlation between high plasma levels of lutein/zeaxanthin and a low risk of AMD, such as the POLA Study and others .Large-scale epidemiologic observational studies, such as the Eye Disease Case Control Study (USA), agree with the findings of beneficial effects of dietary intake of carotenoids [61] and several other studies demonstrated an increase in macular pigment associated with the intake of carotenoids . There are other epidemiological studies with other conclusions. For instance, the Rotterdam study showed a reduced risk for AMD in subjects with high dietary intakes of beta-carotene, vitamins C and E, and zinc [64], while the Physicians' Health Study did not reveal benefits for vitamins C or E [65].µM/L in AREDS participants, whereas it was 31.6 µM/L in men and 40.5 µM/L in women enrolled in the French POLA study . The plasma levels of vitamin C of this French study were comparable to those of the EUREYE Study implemented in seven European countries [67].Interestingly, data from the United States differs from European data, which could be explained by differences in nutritional patterns or supplement intake. For example, the majority of the participants in the AREDS study used vitamin supplements (Centrum) in addition to the supplementation tested in the study [4]. The mean baseline plasma vitamin C was 62 A systematic review of prospective studies of dietary intake found no evidence that diets high in antioxidant vitamins prevent AMD . More specifically, there is no evidence from randomized clinical trials that healthy people should take vitamin and antioxidant supplements to delay or prevent the onset of AMD [68]. Recently, it was reported that high dietary intake of folate was associated with a decreased risk of progression of geographic atrophy, using a large prospective cohort with a high number of incident cases [70]. This association could be modified by genetic susceptibility, particularly associated with C3 alleles [70]. In this same study, thiamin, riboflavin, niacin, and vitamins B6 and B12 were not significantly related to progression [70]. Folate is provided by green vegetables, fruits, nuts, beans, and peas, the same sources as lutein and zeaxanthin [70].Zinc levels are high in ocular tissues, but the distribution is not uniform [47]. It is the most abundant trace metal in the retina and is preferentially located in the inner nuclear and PR layers, that is, the regions affected by AMD [71]. Total zinc concentration in the RPE is high enough to release reactive zinc in the high micromolar range [71]. In photoreceptors, the loosely bound zinc ions may play a role in regeneration of rhodopsin and in the phototransduction cascade [71]. The US Food and Drug Administration (FDA) recommends 11 mg/day of zinc for men and 8 mg/day for women. A daily replenishment of approximately 1% of total body zinc is obtained from the diet and prolonged periods of zinc depletion cannot be compensated [71]. Oysters and other seafood contain more zinc per serving than any other food [50]. Beef, poultry, and pork generally provide the necessary intake [50]. Although zinc is present in beans, cereals, and nuts, plant-based phytates may inhibit their absorption [50]. Therefore, if meat and seafood are scarce in the diet, the consumption of zinc may not be sufficient as the body does not store zinc .Zinc is a cofactor of many active ocular enzymes, including superoxide dismutase and catalase . Zinc also binds complement factor H, inducing large multimeric forms that lose complement 3b inhibitory activity [73]. This action can help to suppress the chronic inflammatory events in the retina that can lead to AMD [73]. In vitro studies suggested that zinc supplementation attenuates endothelial cell activation and may affect the progression of AMD [74]. In an animal model of light-induced retinal degeneration, zinc supplementation induced changes in gene expression, enhancing antioxidative retinal capacity and the reduction of oxidative damage [75].In AMD, the content of zinc in human RPE/choroid is decreased by 24%. On the other hand, high levels of zinc are present in drusen, probably affecting the oligomineralisation of complement factor H . Also, the expression of some transport, sensory, and trafficking/storage proteins was found to be downregulated in AMD maculas . These changes in retinal zinc homeostasis justify its presence in the AREDS formulation and in other trials. As high doses of zinc may interfere with copper absorption, the AREDS formulation included a copper supplement (2 mg/day) [4].A systematic review of these trials with zinc was published in 2013 [76]. Ten studies analyzing the effect of zinc intake, both from supplements and/or foods, in treatment and primary prevention were included: four randomized clinical trials, four prospective cohorts, and two retrospective cohort studies. The review found that current evidence on zinc intake for the prevention of AMD is inconclusive. However, AREDS revealed that 80 mg of zinc oxide, alone or in combination with antioxidants, significantly reduced the risk of progression to advanced AMD in individuals with moderate AMD. The risk of visual acuity loss was of a similar magnitude, but not statistically significant. The other two randomized clinical trials showed a statistically significant increase in visual acuity in early-stage AMD patients and the other one revealed no effect of zinc treatment on visual acuity in advanced AMD patients [76]. Furthermore, results from the remaining six cohort studies on associations between zinc intake and incidence of AMD were inconsistent. The authors of the systematic review concluded, based on the strength of AREDS, that zinc treatment may be effective in preventing progression to advanced AMD, but zinc supplementation alone may not be sufficient to produce clinically meaningful changes in visual acuity [76].Due to possible secondary effects of high doses of zinc in the stomach, AREDS2 evaluated the effects of a lower dose of zinc (25 mg versus 80 mg), but no significant difference in AMD progression was reported [56]. Note that, as stated previously, most of the participants in both AREDS trials simultaneously took Centrum that included the recommended daily intake of zinc, increasing the daily dose of zinc [4]. As a trend favoring the higher dose of zinc was found [77], the present AREDS2 formulation includes 80 mg of zinc. It should be noted that the doses of daily zinc are much higher than recommended by the FDA, but the secondary effects described were minimal.More recently, it was reported that response to AREDS supplements is affected by genetic factors and the effectiveness of antioxidant/zinc supplementation is influenced by genotype [78]. Particularly, in AREDS patients with the high-risk CFH/low-risk ARMS2 genotype, zinc-containing treatments may worsen outcomes for patients who have moderate to severe AMD [77].As already mentioned, the carotenoids, lutein/zeaxanthin, are concentrated at the central fovea composing the macular pigment . By scavenging reactive oxygen species and filtering potentially damaging blue light, they protect the macular region from photooxidative injury and can decrease a toxic byproduct of the visual cycle (A2E), stimulated by this blue light . Lutein/zeaxanthin supplements may offer protection to decrease the number of lipofuscin granules and increase the stability of lysosomes [42]. Using animal models, lutein/zeaxanthin preserved macular health and improved functional abnormalities [14]. The absorption of poorly focused short wavelengths by the macular pigment decreases chromatic aberration and improves visual resolution .Lutein/zeaxanthin supplementation from foods can increase pigment macular density, but this capacity varies among individuals [79]. Physical activity may directly contribute to a denser macular pigment due to the reduction of inflammation and oxidative stress, or indirectly by reducing obesity. In fact, obesity is related to a lower density of a macular pigment in this study and others and may increase oxidative stress [13]. The status of the macular pigment may be improved by a healthy diet and physical exercise .Despite strong biological plausibility, the epidemiological studies have not found consistent results [80]. More importantly, as stated above, the AREDS2 trial failed to confidently demonstrate protective effects of lutein/zeaxanthin in the primary analyses [56]. However, there was a 26% risk reduction when the analysis was restricted to a subgroup of participants at the bottom 20% of the dietary intake of lutein/zeaxanthin . This secondary analysis supports the hypothesis that supplements are effective only when the background dietary intake is below a sufficient threshold [81].This hypothesis is supported by a long-running (two decades of follow-up), very large prospective cohort from the Nurses' Health Study and Health Professionals Follow-up Study that found an association between a higher intake of lutein/zeaxanthin and a 40% lower risk of advanced AMD [80]. The intake of carotenoids was not associated with intermediate AMD. This finding was interpreted as an effect on the progression of AMD but not on the initiation of the disease [80]. Although there is no causal inference, this is probably the best available evidence in the absence in the near future of a well-designed large-scale randomized trial like AREDS2 [80].Some other studies found an association between carotenoids and intermediate AMD. However, only one of three prospective cohort studies reported a significant inverse association between the intake of lutein/zeaxanthin and intermediate AMD [42]. In late-stage AMD the dysfunction or loss of macular photoreceptors may not be rehabilitated [82]. A meta-analysis of eight high-quality randomized clinical trials involving 1176 AMD patients in which a comparison of lutein/zeaxanthin intervention with placebo was performed [42], with the outcomes of visual acuity, contrast sensitivity, glare recovery time, and subjective perception of visual quality, found that lutein/zeaxanthin improved visual acuity and contrast sensitivity of AMD patients and, more importantly, there was a dose-response relationship [42].The intake of xanthophyll carotenoids is far below the recommended level and there is a tendency for a decrease in Western countries [83]. A recommendation for an increased intake of lutein/zeaxanthin from food sources or supplements should be advised, especially for AMD patients. The results of the Taurine, Omega-3 Fatty Acids, Zinc, Antioxidant, Lutein (TOZAL) study showed that AMD patients require at least six months of supplementation to have a positive outcome, such as changes in visual acuity and other visual parameters [84].In AREDS2, the addition of DHA + EPA to the original AREDS formulation  did not further reduce the risk of AMD. This finding was unexpected as epidemiological studies have for more than 10 years pointed to a beneficial effect of dietary omega-3 in the prophylaxis of AMD [85]. There is consistent evidence that the intake of high doses of DHA present in oily fish is associated with a decreased risk of neovascular AMD . Concerning the dietary consumption of n-3 fatty acids, the Eye Disease Case Control Study in the United States demonstrated an association between a higher intake of n-3 fatty acids and a lower risk of AMD among individuals on a diet low in LA . The Blue Mountains Eye Study in Australia demonstrated a protective effect of n-3 fatty acids in late-stage AMD in individuals in the highest quintile of intake [43]. Subjects in the AREDS trial who reported the highest consumption of n-3 fatty acids were also less likely to have neovascular AMD at baseline [44]. Other studies have reported an inverse association between the dietary consumption of n-3 fatty acids and neovascular AMD . Some researchers criticize AREDS2, stating that the design, setting, and selection of patients in the AREDS2 trial did not allow for ascertaining the potential of omega-3 supplements [85], due to an inadequate duration of treatment, inadequate dose, or both . Another possible explanation is that the effect may be modified by underlying genetic risk factors . It is also possible that there is an optimal level of supplementation with omega-3 fatty acids and if the level is not optimal it could be ineffective or detrimental. A more appropriate conclusion is that in a well-nourished population AREDS2 was not more effective than the original formulation [22].A prospective randomized prospective study, the Nutritional AMD Treatment-2 (NAT-2) trial, had a true placebo group and demonstrated that patients who achieved high red blood cell membrane EPA/DHA levels were significantly protected against AMD versus those patients with low levels of EPA/DHA . However, in the same NAT-2 study, with supplementation with 840 mg/day DHA  or placebo  for three years, dynamic drusen remodeling was not affected by DHA supplementation [34]. This drusen remodeling showed a tendency to be influenced by CFH genotype [34].In an open-label experiment, it was reported that high doses of EPA (3.4 g) and DHA (1.6 g) on a daily basis for six months improved the visual acuity of patients with dry AMD [88]. Also regarding the intake of omega-3 PUFAs, a Cochrane meta-analysis published in 2012 and updated in 2015 concluded that there is currently no evidence to support the idea that increasing levels of omega-3 PUFAs in the diet prevents or slows the progression of AMD, in accordance with the AREDS2 trial .In short, the majority of evidence suggesting a positive effect of dietary omega-3 intake on the development and progression of AMD comes from observational studies and this remains to be demonstrated in randomized clinical trials [39]. In a recent paper, the systemic confounders that may affect the serum measurements of omega-3 and omega-6 PUFAs in patients with retinal disease were discussed [91]. In that pilot study, they demonstrate that serum lipid profiles are significantly altered by variables like fasting and medication. Patient fasting status may affect the serum levels of unsaturated and saturated fatty acid levels, influencing the results and conclusions of the studies [91].Summing up the current evidence, it is reasonable to advise AMD patients to consume dietary fatty fish  or fish oil supplements. It is prudent to inform the patients of the doubts concerning their efficacy and the lack of support from large randomized clinical trials.Dietary patterns where nutrients in the food can interact synergistically is a recent paradigmatic shift in nutritional sciences. Numerous studies show that a healthy diet, the maintenance of an adequate body weight, and an active lifestyle are important to maintaining health and avoiding the physical and cognitive degeneration associated with aging .The Mediterranean diet is one of the most studied healthy dietary patterns. This diet is rich in vegetables and fruits, thereby providing high amounts of bioactive antioxidant compounds. Furthermore, olive oil and fatty fish rich in omega-3 fatty acids are present in the Mediterranean dietary pattern .From the scientific point of view, there are major differences between the Mediterranean diet and the so-called Western diet. In fact, the INTERHEART and INTERSTROKE studies  considered the existence of three main dietary patterns, evaluated by a dietary risk score: (a) Western diet with high intake of fried foods, salty snacks, eggs, and red meat; (b) oriental diet, high in tofu and sauces like soy and others; (c) prudent diet with a high intake of fruits and vegetables with some characteristics of the Mediterranean diet. It was found that the Western diet increased the population risk for acute myocardial infarction and stroke by approximately 30%. In contrast, the prudent diet decreased the same risk by 30%. The oriental diet was neutral, probably because the high intake of fruits, fish, and vegetables was offset by the high salt intake and other factors .Defining a Mediterranean diet is difficult considering that the geographical region includes more than 17 countries [26]. According to De Lorgeril, the Mediterranean dietary pattern is inspired by the traditional diet found in Greece and Southern Italy [26]. It has the following characteristics: (a) high consumption of fruits, legumes, and other vegetables, bread and other cereals, potatoes, beans, nuts, and seeds; (b) olive oil is the main source of monounsaturated fat; (c) dairy products, fish, and poultry are consumed in low to moderate amounts; only low quantities of red and processed meat are present; (d) wine is consumed during meals in low to moderate amounts .Summarizing the prospective observational studies, a meta-analysis was published by Sofi and collaborators in 2008 and updated in 2010 . The studies prospectively analyzed the association between adherence to a Mediterranean diet, mortality, and incidence of diseases, with a total of more than 2 million subjects followed from 3 to 18 years. Greater adherence to a Mediterranean diet was associated with an improvement in health status, namely, a major reduction in overall mortality (9%), mortality from cardiovascular diseases (CVD) (9%), and incidence of mortality from cancer (6%). The reduced incidence of Parkinson's and Alzheimer's diseases (13%) demonstrated that the adherence to the Mediterranean diet prevented these major chronic neurodegenerative diseases associated with aging . Prevención con Dieta Mediterránea (PREDIMED) trial, a multicenter randomized clinical trial in Spain [99], showed that relatively small changes in diet have beneficial effects [99]. The clinical trial included 7447 subjects at risk of CVD. The conclusions demonstrated that the adoption of a Mediterranean-style diet reduced the risk of cardiovascular complications by 30%, including a reduction of the risk of stroke by 40% over a follow-up of approximately five years [99]. In 2013, the Cochrane Heart Group published a review analyzing the Mediterranean dietary pattern for the primary prevention of CVD [100], concluding that the Mediterranean diet may reduce some cardiovascular risk factors  [100]. Additional randomized clinical trials are necessary, but there is sufficient scientific evidence to recommend the Mediterranean dietary pattern to individuals who want to age in good health while still appreciating food .TheAssessing dietary patterns with respect to AMD is relatively recent. Association of a particular nutrient to AMD is difficult to dissociate from other aspects of the diet. More importantly, there are synergistic relationships between the food components .In the Carotenoids Age-Related Eye Disease Study (CAREDS), high adherence to a Mediterranean diet was associated with a lower prevalence of early AMD [86]. Previously, it was reported that advanced AMD was related to overall diet quality using the Health Eating Index (HEI) [102]. Data from the Melbourne Collaborative Study showed that a diet rich in fruits, vegetables, nuts, and chicken was associated with a lower prevalence of AMD [103]. Conversely, the Western diet was associated with increased odds of AMD [104]. The oriental pattern of diet was linked to decreased odds of AMD [104].Merle and collaborators used the data from 2525 individuals from the AREDS trial and dietary information collected from food-frequency questionnaires and the alternate Mediterranean diet score [86]. This score was constructed from individual intakes of vegetables, fruit, legumes, whole grain, nuts, fish, red and processed meats, alcohol, and the ratio of monounsaturated to saturated fats. They reported that a high score was associated with a 26% reduced risk of progression to advanced AMD after adjustments for demographic, behavioral, ocular, and genetic factors. The consumption of fish and vegetables was associated with a lower risk of progression to advanced AMD [86]. The administration of AREDS supplements did not change the protective effect of the Mediterranean diet on the risk of progression [86]. In addition, they reported that the Mediterranean score was associated with a lower risk of advanced AMD among individuals carrying the CFH Y402H non-risk (T) allele. CFH is one of the main genes implicated in AMD and the risk allele leads to complement activation, increasing AMD risk. The authors hypothesize that the effect of the Mediterranean diet is stronger among subjects with the non-risk allele [86]. The biological plausibility of the benefits of the Mediterranean diet involves a decrease in oxidative stress and inflammation. Accordingly, individuals with higher adherence to the Mediterranean diet have higher plasma concentrations of some important and beneficial biomarkers [105].In a population in the central region of Portugal, it was reported that higher adherence to a Mediterranean diet score was associated with a lower AMD risk [6]. In this Coimbra study, the consumption of fruit, beta-carotene, and vitamin E was particularly beneficial [6]. In an update of the study presented at the American Academy of Ophthalmology meeting , it was reported for the first time that coffee, also rich in antioxidants, was protective. Of the participants in this study who consumed caffeine , 54.4% did not have AMD (data not published).As one of the key components of the Mediterranean diet, olive oil was studied in the ALIENOR  study, a population-based study aimed at verifying the associations of nutritional factors such as antioxidants, macular pigment, and fatty acids [106]. After adjustments for the multiple potential confounders, there was a decreased risk of late-stage AMD among olive oil users in 654 subjects (1269 eyes) with a mean age of 72.7 years, with complete data suggesting a protective role of olive oil consumption for late-stage AMD in an elderly French population [106]. On the other hand, no association was found between olive oil consumption and early-stage AMD, and the use of other types of fats was not associated with any stage of AMD. Similar results were found in an Australian cohort , where an inverse association between the intake of olive oil and the prevalence of late-stage AMD was found [107].Olive oil is a mixture of lipids and antioxidant compounds and it contains monounsaturated fatty acids (MUFAs) and polyphenols that have strong antioxidant and anti-inflammatory properties [86]. A laboratory study in ARPE-19 human retinal pigment epithelial cells reported that the major polyphenol of olive oil, hydroxytyrosol, prevented the degeneration of retinal pigment epithelial cells induced by oxidative stress [108]. The protection probably does not rely on oleic acid, the main MUFA component, as the associations between MUFA intake and AMD are inconsistent in the literature. It is suggested that the protection derives from phenolic compounds, including oleocanthal, hydroxytyrosol, and oleuropein [106]. However, more studies are needed to find the mechanism by which olive oil has beneficial effects on late-stage AMD [106].No association was found between oils rich in omega-3 and any stage of AMD. The ALIENOR study also found no association between olive oil consumption and early-stage AMD [106]. This lack of association suggests that the risk factors are dissimilar at different stages of the disease. Some risk factors may favor the accumulation of drusen and the pigmentary disturbances. Others may have an influence on neoangiogenesis and/or apoptosis. Another explanation, as suggested by some studies, is that early-stage AMD may represent a very heterogeneous group of patients, and some of these patients present a low risk of developing late-stage AMD [109–111].In epidemiological studies a healthy diet, avoiding food high in sugar, fat, alcohol, refined starch, and oils, the absence of smoking, and physical activity  were associated with a reduced occurrence of early-stage or advanced AMD, or both [3]. This risk reduction was greater when multiple lifestyles were considered together, as shown by CAREDS [3]. In this study, women aged 50–74 years who displayed a combination of healthy lifestyle factors such as healthy diet, physical exercise, and not smoking had threefold lower odds for early-stage AMD when compared to women who displayed unhealthy lifestyles. The several mechanisms of the healthy lifestyle that lead to protection are difficult to disentangle from one another [3]. The energy expenditure of the physical exercise leads to an increase in daily nutrient intake. Both physical activity and diet may contribute to better vitamin D status, also associated with lower risk of AMD [112].Insufficient physical activity increases the occurrence of numerous metabolic and vascular diseases [113] and may facilitate the progression of some cases of AMD . Sedentarism may contribute to increasing inflammatory events and dysfunction of the endothelium [113]. This hypothesis is supported by the finding that elevated C-reactive protein in patients with neovascular AMD is partly explained by physical inactivity [115]. On the contrary, physical activity can upregulate the enzymatic systems related to antioxidant protection, reducing oxidative events [113].There is another important factor that can modify the benefits of the healthy lifestyles: genetic risk [116]. In fact, in the Unites States, the Y402H (rs1061170) variant within the complement factor H (CFH) gene and the A69S (rs10490924) variant within the age-related maculopathy susceptibility 2 (ARMS2) locus increased the risk (1.5- to 3-fold) for both early-stage and late-stage AMD in individuals of European ancestry . Wang and collaborators reported that the ingestion of lutein only reduces the incidence of AMD among persons with two or more alleles from common CFH and ARMS2 variants [118]. Meyers and collaborators studied the joint associations of diet, lifestyle, and genes in AMD and concluded that physicians should recommend the adoption of healthy lifestyles at early ages in individuals who have a family history of AMD and also they should motivate AMD patients to follow the same advice [116].Stressing that the benefits and safety of the use of high-dose antioxidants for long periods in the prevention or delay of the progression of AMD in early stages has not been established [119], the adoption of healthy lifestyles at early ages may ultimately significantly benefit the patients and families at genetic risk. Public health interventions to consume plant-rich, high lutein diets, physical activity, and cessation of smoking are recommended strategies for AMD prevention. These recommendations are particularly important in individuals at genetic risk, with a family history of AMD or both. The combination of healthy lifestyles practices may be more important in the reduction of AMD than a focus on one. Collectively, these changes may reduce oxidative stress, blood pressure, and blood lipids. In short, they may reduce the systemic inflammation that contributes to the pathogenesis of AMD and other chronic diseases, including neurodegenerative diseases, although there is a lack of clinical trials to give solid evidence for these benefits [116]. These recommendations can lead to an improvement of outcomes through genotype-directed therapy [12]. The knowledge of modifiable risk factors along with information concerning genetic risk variants for AMD has greatly improved the management of patients and the ability to predict which patients will develop or progress to advanced forms of AMD [87]. Individualized prevention and treatment strategies and personalized medicine are becoming a reality [87].Changes in lifestyle, such as eating a healthy diet like the Mediterranean diet, and if necessary complementing dietary patterns of selected individuals with appropriate supplements, may play an important role and avoid progression to advanced stages of AMD [86]. There are good rates of compliance with dietary advice; that is, patients change their diet, but patient education by health professionals can be improved [120].The nutritional advice of clinicians to their patients must be balanced and they should discuss what is supported by scientific evidence and the current doubts. This advice should be supported by high levels of evidence, preferably from randomized clinical trials [119].Current evidence shows that all AMD patients should be advised to increase their consumption of green leafy vegetables and to eat oily fish, at least twice a week. A Mediterranean type of diet may provide additional benefits in other age-related diseases beyond AMD [26].Patients with moderate or advanced AMD should be advised to use AREDS-based supplements. Current smokers or ex-smokers are advised to avoid formulations with beta-carotene [120].If patients are unsure of their dietary lutein intake, they should be advised to use lutein/zeaxanthin AREDS-based supplements, given the lack of side effects found in most trials [120]. If they have a normal or high dietary lutein intake they can consume the modified beta-carotene free AREDS formulation [120].Dietary modifications may not only delay the onset of AMD, but can also slow the progression of the disease. Supplementation with AREDS-based supplement slows the progression of AMD, but does not prevent its development.More importantly, the adoption of a Mediterranean diet and physical activity and avoiding smoking and sedentary behavior may reduce the prevalence of the early stages of AMD, decrease the number of individuals who develop advanced AMD, and consequently reduce the burden associated with the treatment of this disease.


Recognition of microbial pathogens and dead cells and their phagocytic uptake by specialized immune cells are essential to maintain host homeostasis. Phagosomes undergo fusion and fission events with endosomal and lysosomal compartments, a process called ‘phagosome maturation’, which leads to the degradation of the phagosomal content. However, many phagocytic cells also act as antigen-presenting cells and must balance degradation and peptide preservation. Emerging evidence indicates that receptor engagement by phagosomal cargo, as well as inflammatory mediators and cellular activation affect many aspects of phagosome maturation. Unsurprisingly, pathogens have developed strategies to hijack this machinery, thereby interfering with host immunity. Here, we highlight progress in this field, summarize findings on the impact of immune signals, and discuss consequences for pathogen elimination. Self and non-self immune signals are able to delay or accelerate phagosome maturation, and their effects are dependent on the phagocytic cell type, duration of stimulation, and whether the stimulus is particle bound or present in the cellular environment.Acceleration of phagosome maturation enhances pathogen killing, while a delay in phagosome maturation preserves antigenic peptides for presentation to T cells and to initiate adaptive immune responses.Besides its functions in pathogen killing and antigen presentation, the phagosome also functions as a signaling platform and interacts with other cell organelles.Some pathogens are able to arrest phagosome maturation to enhance their intraphagosomal survival and replication or to promote phagosomal escape.The latex bead phagocytosis model system combined with mass spectrometry is a powerful technique to analyze changes in the phagosomal proteome. Dictyostelium discoideum, phagocytosis serves mainly in the uptake of nutrients Phagocytosis appeared during the evolution of unicellular eukaryotic organisms and describes the ingestion of large particles ≥0.5 μm)  μm DictyBox 1During phagosome maturation, the phagosome interacts with endosomes and lysosomes , which cSoon after engulfment, the formed phagosome fuses with early endosomes and acquires the small GTPase Rab5. The Rab5 effector Rabaptin-5 recruits the class III phosphoinositide 3-kinase vacuolar protein-sorting 34 (Vps34) The transition from an early phagosome to a late phagosome is marked by the conversion from Rab5 to Rab7, which induces the CORVET-to-homotypic fusion and vacuole-sorting (HOPS) switch Finally, the phagosome interacts with lysosomes to develop into a phagolysosome, which is mediated by different soluble NSF attachment protein receptors (SNAREs) trans-Golgi Network, mediated by different Rab proteins  and the retromer complex, respectively During phagosome maturation, intraluminal vesicles are also formed for the degradation of transmembrane proteins Alt-text: Box 1The phagocytic process is initiated by the recognition of a particle ligand by cell surface receptors. To achieve selective uptake of baits, professional phagocytes express specific but partially redundant arrays of receptors. Receptor recognition launches signaling pathways that induce remodeling of the actin cytoskeleton and extension of membrane protrusions that surround the particle to form a phagocytic cup. Once the phagocytic cup is sealed, a phagosome is formed that gradually matures via fusion and fission events with vesicles of the endocytic compartment. Initially, the formed phagosome interacts with different types of endosomes to gradually mature from an early phagosome into a late phagosome . Endosomvacuolar ATPase , which produces reactive oxygen species (ROS). During phagolysosomal fusion, the organelle acquires hydrolytic enzymes, including glycosidases, lipases, DNAses, and proteases, such as cathepsins, which require a low pH for optimal activity Small GTPases of the Rab family represent an important group of proteins involved in phagosome maturation. The binding of specific Rab proteins to intracellular organelles enables targeting specificity and facilitates interactions of phagosomes with different compartments over time . Rab proWhile phagosome maturation is generally conserved, its extent and outcome are dependent on the phagocyte type. Phagosomes in neutrophils and in most MΦ populations rapidly destroy phagocytic cargo. By contrast, DC phagosomes often only partially degrade their cargo to preserve antigenic peptides for presentation to T lymphocytes to initiate adaptive immune responses. Hence, phagosome maturation in phagocytes is adapted to their specific function in immunity Recently, increasing evidence indicates that the phagosome is more than a degradation machinery and also functions as a signaling platform + T cell responses As mentioned above, kinetics of phagosome maturation display remarkable differences between MΦs and DCs. Phagosome maturation proceeds quickly in most MΦs, as illustrated by the rapid acquisition of hydrolytic enzymes and the v-ATPase + T cells or on MHC I molecules to CD8+ T cells In contrast to most MΦs, DCs are believed to only partially degrade phagocytosed pathogens to preserve peptides for presentation to T cells. Consequently, acidification and antigen degradation kinetics of DC phagosomes are slower compared with MΦs pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and cytokines, and can be sensed at the cell membrane, or are present on the phagocytosed particles themselves, such as PAMPs, DAMPs, and opsonins. The effect of a specific stimulus, such as the one of lipopolysaccharide (LPS) of Gram-negative bacteria, may differ depending on whether it is present in the phagocyte environment or at the phagocytosed particle. Immune signals may accelerate or delay phagosome maturation to enhance microbial killing or antigen presentation, respectively. Below, we first discuss the impact of different immune signals present in the cellular or tissue environment, and then focus on the signals present at phagocytosed particles and their effects on phagosome maturation . One family of PRRs are Toll-like receptors (TLRs), which recognize both intra- and extracellular microbial patterns and activate innate immune cells Escherichia coli but not those containing apoptotic cells was impaired in MyD88- and TLR2/TLR4-deficient MΦs compared with wild-type (WT) MΦs E. coli during phagocytosis of apoptotic cells did not influence phagolysosomal fusion kinetics of phagosomes containing apoptotic cells. The authors also demonstrated that, in DCs, the presence of TLR ligands within E. coli or apoptotic cell phagosomes promoted phagosomal antigen presentation to CD4+ T cells in a phagosome-autonomous way PAMPs are conserved molecular motifs associated with certain groups of pathogens that are recognized by polyuridylic acid (polyU) also decreased phagosomal degradation and acidification in vitro, as measured by flow cytometry after incubation of cells with beads coupled to ovalbumin (OVA) or a pH-sensitive dye, respectively. Moreover, immunization of mice with polyU enhanced cross-presentation and promoted more efficient responses of cytotoxic T lymphocytes in vivoCpG only caused a minor delay in antigen degradation, yet increased phagosomal acidification and resulted in less efficient cross-presentation of phagosomal antigen in DCs In addition to their presence in phagosomes, TLR ligands can also be present in the extracellular environment , where tinterferon gamma (IFN-γ) induced delayed phagosomal proteolysis, phagolysosomal fusion, and acquisition of maturation markers Mycobacterium tuberculosis (Mtb) 19-kDa lipoprotein also delayed phagosome maturation immunoglobulin G (IgG)-opsonized zymosanCD40 ligand did not induce major differences in phagosomal degradation In addition to TLR agonists, cellular activation by cytokines can also directly influence phagosome maturation. Stimulation of MΦs with In conclusion, although the effect of TLR signaling on phagosome maturation remains a controversial topic, under specific conditions TLR activation by stimuli in the cell environment can delay phagosome maturation to promote efficient antigen presentation. The extent of these effects depends on the specific TLR involved and the duration of stimulation. Similarly, M1-like MΦ polarization negatively regulates phagosome maturation kinetics, whereas M2-like MΦ polarization accelerates phagosome maturation. Yet, more research is needed to unravel the mechanistic details and to examine the effect of other immune stimuli, such as TGF-β, on phagosome maturation.Box 2Acanthamoeba castellaniiDuring the 1960s, Wetzel and Korn developed an ingenious method using polystyrene particles to isolate latex bead-containing phagosomes (LBPs) from The use of this model system is sometimes more advantageous than the use of other phagocytic cargo, especially if one aims to isolate pure and homogeneous phagosome preparations for mass spectrometry. Due to the buoyancy of the latex beads, the isolation of LBPs requires only one to two centrifugation steps to separate the floating fraction containing the LBPs from contaminants trapped in other sucrose layers. By contrast, the isolation of bacteria-containing phagosomes is more laborious and yields less pure phagosome preparations In addition to the mentioned advantages, functional assays in combination with flow cytometry are also available for accurately quantifying phagosome maturation and phagocytic uptake at a single organelle level Alt-text: Box 2Not only immune stimuli present in the phagocyte environment, but also immune signals present at the phagocytosed particle itself can impact phagosomal maturation. Upon phagocytosis of microbes, their PAMPs can influence the kinetics of phagosome maturation. Since most microbes express a diverse variety of PAMPs, inert particles coated with a particular PAMP can be used to study the specific effects of this PAMP on phagosome maturation . This moPam3CSK4, which is recognized by the TLR2/TLR1 heterodimer, enhanced phagosomal acidification Box 3LC3-associated phagocytosis (LAP) is a form of phagocytosis that is characterized by the receptor-mediated recruitment of autophagy proteins, such as LC3, to the phagosome. Although LAP uses parts of the autophagy machinery, LC3-positive phagosomes are distinct from autophagosomes. The former contain extracellular instead of intracellular material and are surrounded by a single lipid membrane structure instead of a double lipid membrane LAP is triggered by phagocytic baits that contain PRR ligands, such as bacteria or beads coupled to zymosan, LPS, or Pam3CSK4 Aspergillus fumigatus. However, another study showed that Atg5 and Atg7 were dispensable for FcγR-induced LAP in murine cells LAP is at the interface between phagocytosis and autophagy, using both autophagy proteins, such as the class III PI3-kinase Vps34, and endosomal, LAP-specific proteins, such as Rubicon. Rubicon promotes PI3P production by Vps34 and ROS production by NOX2, which are both essential for LAP In most publications, LAP is induced by TLR-activating particles and pathogens, which induce MΦ activation and, thus, significantly alter phagosome functions. This might explain apparently contradicting results in murine cells, where LC3-positive phagosomes, formed after LAP, show accelerated phagosome maturation and cargo degradation Strong evidence points toward an important role of LAP in immunity because it is involved in the regulation of IFN production Alt-text: Box 3In MΦs, coating of beads with the lipopeptide fibronectin, calreticulin, or phosphatidylserineE. coli with IgG increased phagosome maturation kinetics and phagosomal killing E. coli with C1q negatively influenced bacterial killing DAMPs, which are associated with tissue damage and cell death, do not appear to influence phagosome maturation kinetics in MΦs, as demonstrated for In conclusion, only certain PAMPs and opsonins signal from within the phagosome to positively or negatively modulate phagosome maturation. Why other PAMPs and opsonins fail to influence phagosome maturation kinetics remains to be elucidated. Surprisingly, the tested DAMPs did not modulate phagosome maturation when coupled to beads. Therefore, in future studies, the use of more physiological cargo will be needed to study in detail the influence of DAMPs on phagosome maturation.Initially, the role of phagosomes in innate immunity was seen as a function of a degradation machinery modulated by signaling events at the cell surface and within the cytosol. Over the past few years, more evidence has emerged demonstrating that the phagosome is a signaling platform that integrates intraphagosomal, intracellular, and extracellular signals. There is also evidence that signaling from the plasma membrane and the phagosome might be different, because plasma membrane-localized TLRs induce specific signaling from endocytic compartments Box 4LegionellaMycobacteriumThrough optimization, latex bead phagosomes et al., unpublished data, 2017). Phagosomal proteomes are relatively small (∼3000–4000 proteins) compared with the cellular proteome, but have wide ranges in protein abundance, with an estimated 40 proteins comprising approximately 50% of the protein mass Since phagosomes are dynamic intracellular organelles, their composition changes significantly during maturation and in response to external stimuli and the cellular activation status Alt-text: Box 4Additional insight came from the mass spectrometric analysis of phagosomal proteomes , which aHowever, although phagosome maturation is modulated by cellular signaling, it is also able to affect signaling in return. One example comprises nucleic acid-sensing TLRs that are confined to endocytic compartments and that only become fully functional after proteolytic processing and cleavage of their ectodomains by endolysosomal proteases acquired by phagosomes during maturation. For example, in addition to different cathepsins, asparaginyl endopeptidase (AEP) has been identified as the key protease that controls the proteolytic maturation of TLR9 Intracellular segregation of nucleic acid-sensing TLRs to endosomes is also thought to provide one level of protection from autoimmunity, which might occur when cell surface TLRs bind to self nucleic acids that are released during necrotic and apoptotic cell death. Defects in the engulfment and digestion of apoptotic cells lead to the release of intracellular molecules, such as DNA and histones, which induce inflammation and autoimmunity. Alternatively, autoimmune responses may be provoked after microbial infections. Recently, a new mechanism was identified whereby phagocytosis of infected apoptotic cells by DCs leads to the presentation of both pathogenic peptides and self peptides, inducing autoreactive Th17 cells and autoimmunity Since phagosome maturation is essential for host defense, several pathogens have developed mechanisms to interfere with phagosome maturation, to escape the phagosomal lumen, or to survive within phagosomes. For example, some fungal pathogens have developed strategies to interfere with Rab dynamics to subvert phagosome maturation Legionella pneumophila (Lpn) is the causative agent of Legionnaires disease, which infects alveolar MΦs upon inhalation of contaminated aerosols Another pathogen that directly interferes with phagosome maturation is Mtb. Mtb causes 1.8 million deaths and over 10 million new cases of TB annually. It is transmitted via contaminated aerosols and infects mainly alveolar MΦs, but also DCs and epithelial cells. After its engulfment, Mtb arrests phagosome maturation at an early stage, preventing acidification, phagolysosomal fusion, and acquisition of lysosomal hydrolases The dynamic and profound changes that phagosomes undergo during their maturation enable immune cells to maintain homeostasis and to respond rapidly to microbial threats. Therefore, phagosome maturation has a direct impact on the outcome of immune responses and is regulated not only at the cellular level, but also at the organelle level. In recent years, an increasing body of evidence has demonstrated several links between the phagosome maturation machinery and different signaling pathways. In addition, polarization of phagocyte cell populations enables the innate immune system to initiate and shape immune responses. For example, the M1-like and M2-like phenotypes during MΦ polarization are able to induce different features of phagosome maturation, even when they are located in the same tissue. Interestingly, these characteristics are not restricted to MΦs, as nicely demonstrated by the emerging concept of neutrophil polarization The kinetics of phagosomal maturation are susceptible to various stimuli ranging from cytokines and PAMPs to DAMPs and opsonins. Other factors, such as the duration of stimulation and the engaged phagocyte type, also influence phagosomal fate. Therefore, phagosome maturation can be stimulated to enhance pathogen killing or to prevent presentation of self peptides and autoimmunity. By contrast, phagosome maturation can be delayed to preserve pathogenic peptides for presentation to T cells to efficiently induce adaptive immunity. More work is needed to identify the signaling pathways to and from the phagosome and to better understand how different phagocytes regulate these aspects at the molecular level. It is currently not known whether immune signals modulate phagosome maturation upon triggering of a single signal transduction pathway or if multiple pathways are involved. In addition, the effects of other types of environmental stimuli  have not yet been studied (see Outstanding Questions).Outstanding QuestionsWhat are the molecular signaling pathways to and from the phagosome and how are they regulated?Are immune signals able to modulate phagosome maturation upon triggering of a single signal transduction pathway or are multiple pathways involved? Is it possible to identify common key molecules?Which Rab proteins have key roles in the modulation of phagosome maturation?Are phagosomes only involved in intracellular communication or do they also participate in intercellular communication?What is the effect of other types of environmental stimulus, such as allergens, on phagosome maturation?How can the phagosome as a signaling platform be modulated by new therapeutics to enhance antigen presentation and pathogen killing?Based on current progress, the idea emerges that phagosomes function as signaling platforms that integrate multiple intraphagosomal, intracellular, and extracellular signals, which each can modulate phagosome maturation. Proteomic studies revealed that many proteins known to be involved in signaling, such as receptors and kinases, are present in phagosomes. The phosphorylation status of these proteins is only one aspect that influences phagosome maturation. Finally, the various interactions between phagosomes, the inflammasome, and surrounding organelles, such as autophagosomes and mitochondria, suggest that the phagosome is not a solitary organelle and can communicate to other organelles. Future research could focus on these interactions and the impact of immune signals, especially when phagosome maturation is impaired by pathogens to evade host immunity. This might help to develop new therapeutics that enhance pathogen killing and adaptive immunity.


Acute respiratory distress syndrome (ARDS) is a complex clinical syndrome characterized by acute inflammation, microvascular damage, and increased pulmonary vascular and epithelial permeability, frequently resulting in acute respiratory failure and death. Current best practice for ARDS involves “lung-protective ventilation,” which entails low tidal volumes and limiting the plateau pressures in mechanically ventilated patients. Although considerable progress has been made in understanding the pathogenesis of ARDS, little progress has been made in the development of specific therapies to combat injury and inflammation. In recent years, several natural products have been studied in experimental models and have been shown to inhibit multiple inflammatory pathways associated with acute lung injury and ARDS at a molecular level. Because of the pleiotropic effects of these agents, many of them also activate antioxidant pathways through nuclear factor erythroid-related factor 2, thereby targeting multiple pathways. Several of these agents are prescribed for treatment of inflammatory conditions in the Asian subcontinent and have shown to be relatively safe. Here we review natural remedies shown to attenuate lung injury and inflammation in experimental models. Translational human studies in patients with ARDS may facilitate treatment of this devastating disease. Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality rates –3. TheseEndothelial injury is an underlying cause of increased permeability and pulmonary edema in ALI and ARDS, but epithelial injury also plays an important role in their development. Endothelial activation may also lead to obstruction or destruction of the pulmonary vasculature . Injury κB (NF-κB), interferon regulatory factor families, STAT, and AP-1. There is also simultaneous activation of oxidant and antioxidant pathways, particularly in innate immune cells.Contemporary approaches to develop drug therapies have not been productive. In particular, blockade of single cytokines and chemokines have failed to improve outcomes because of the complex pathogenesis and nature of ARDS. Therefore, defining the contribution of proximal signaling pathways that amplify the inflammatory response and developing therapies to specifically block them is an attractive approach, one that may limit injury and inflammation associated with this devastating disease. Intracellular signaling pathways triggered by diverse pattern-recognition receptors converge on signaling hubs, including transcription factors nuclear factor Nuclear factor erythroid 2-related factor 2 (Nrf2), a member of the cap'n'collar family of basic leucine zipper transcription factors, provides a key antioxidant response. Most widely studied experimental models use lipopolysaccharide (LPS), an endotoxin of gram-negative bacteria. More recently, the roles of other molecules , intracytoplasmic receptors , amplifiers , and several others are being recognized. The detailed molecular mechanisms of lung injury and ARDS have been reviewed extensively in several recent publications , 9, 15.Since its initial description in 1967, little progress has been made in the development of novel therapies for ARDS. To date, no pharmacological agents have demonstrated efficacy in preventing ARDS or improving its symptoms, and the morbidity and mortality continue to be significant , 17. HenInflammation is an important component of ALI and ARDS, as inflammation is what damages the respiratory membrane. Most inflammatory cells, including macrophages and neutrophils, release inflammatory cytokines in response to various stimuli. LPS, a main component of the outer membranes of gram-negative bacteria, has been identified as a key risk factor for ALI and ARDS. LPS binds to Toll-like receptor 4 (TLR4), which induces activation of intracellular pathways. Ligand binding to TLR4 induces the recruitment and activation of adaptor proteins through the Toll/interleukin- (IL-) 1 receptor (TIR) domain. Recruitment of the adaptor protein myeloid differentiation primary response gene 88 (MYD88) to the receptor complex will only occur if the TIR domain contains adaptor protein . MYD88 recruits IL-1 receptor-associated kinase 4 (IRAK4), which forms an active complex capable of recruiting the tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6).κB kinase (IKK) enzyme complex and regulatory scaffold proteins .. cis-act in vitro studies , and activator protein 1). It inhibits TREM-1 in bone marrow macrophages. Curcumin also inhibits p300-HAT, a key epigenetic element known to activate transcription of the genes that regulate inflammation. Curcumin exhibits antioxidant effects at the level of the KEAP1–Nrf2 complex, resulting in the dissociation of KEAP1 and Nrf2, followed by nuclear accumulation of Nrf2. Kang et al. [Curcumin (or diferuloylmethane), a natural product found in turmeric, has been shown to inhibit multiple proinflammatory pathways and to activate anti-inflammatory pathways . Curcumig et al.  showed tγ, leading to the suppression of the expression and release of TNF-α [Six different phase 1 human trials found no toxicity from curcumin. Both human and laboratory studies have found evidence of anti-inflammatory properties of curcumin, and it inhibits a bevy of enzymes and mediators of inflammation , 52. Theof TNF-α .Since its first description 50 years ago, there has been an increase in the understanding of molecular pathogenesis and pathophysiology for the development of ARDS. However, to date the best practice involves “lung-protective ventilation” in mechanically ventilated patients with ARDS with no specific therapies directed towards lung inflammation. The inflammatory response in patients with ARDS is initiated, amplified, and modulated by a complex network of proinflammatory signaling pathways and oxidant stress generated by a variety of cell types in the lungs. Here, we reviewed some natural products whose biological effects may be useful in the development of new therapies for ARDS. Many of these agents have pleiotropic effects, such as inhibiting proinflammatory signaling while activating antioxidant defense mechanisms. One of the benefits of these natural products is that they have been consumed in the Asian subcontinent for centuries with no significant toxicity. However, to be developed for therapies, systematic studies (including pharmacokinetics and pharmacodynamics) must be carried out in human trials.Although great strides have been made in the last several decades in defining molecular pathways for ALI and ARDS, these discoveries have not been translated into actual changes in medical treatments for patients with ARDS. To date, supportive strategies and lung-protective ventilation are the only approaches that have been shown to improve outcomes in these patients. A major challenge in generating effective therapeutics has been the ability to develop reliable animal models of critical illness that allow for the generation and testing of novel hypotheses and, ultimately, the translation of these findings to the human condition . Areas o in vitro and in vivo data for many products that have been studied in preclinical models of ARDS.The prognosis of patients with ARDS continues to be abysmal, with mortality rates ranging from 30% to 40%. Therefore, ARDS represents an unmet medical need and there is an urgent need to develop new therapies to treat patients with this condition. To date, treatment of the inciting event, lung-protective ventilation with lower tidal volumes, and optimal management of fluids remain the key therapeutic strategies for ARDS, but no specific therapies yet exist. Because of the complex nature of the disease , neither blocking individual proinflammatory cytokines with antibodies nor the use of antioxidants has been rewarding. An interest in natural therapies as anti-inflammatory and antioxidative agents for systemic conditions has been growing. Given the complexity of the pathogenesis of ARDS, many natural products have been tested as pleiotropic agents that may help combat the inflammation and promote healing of the lung. We have reviewed theThe challenge lies in conducting translational studies to prove the efficacy and safety of these compounds in clinical trials. Although many of the aforementioned agents are widely consumed as herbal supplements or food additives in Asian countries, further study is needed before they can be adopted as therapies for ARDS. The pharmacokinetics and pharmacodynamics of these compounds in the setting of ARDS need to be established. The systemic administration of these compounds can also be challenging, so novel approaches to administer these compounds as nanomedicine or through aerosolization are other potential avenues for future study. Translational studies using these agents in patients with ARDS will provide potential opportunity to develop much-needed novel therapies for this devastating disease.


Neurogenesis is the process through which neural stem and progenitor cells generate neurons. During the development of the mouse neocortex, stem and progenitor cells sequentially give rise to neurons destined to different cortical layers and then switch to gliogenesis resulting in the generation of astrocytes and oligodendrocytes. Precise spatial and temporal regulation of neural progenitor differentiation is key for the proper formation of the complex structure of the neocortex. Dynamic changes in gene expression underlie the coordinated differentiation program, which enables the cells to generate the RNAs and proteins required at different stages of neurogenesis and across different cell types. Here, we review the contribution of epigenetic mechanisms, with a focus on Polycomb proteins, to the regulation of gene expression programs during mouse neocortical development. Moreover, we discuss the recent emerging concept of epigenetic and transcriptional pre-patterning in neocortical progenitor cells as well as post-transcriptional mechanisms for the fine-tuning of mRNA abundance. The generation of neocortical neurons during mouse development is the result of balanced proliferative and differentiative divisions of neural stem and progenitor cells  and Polycomb (PcG) groups were identified as part of an evolutionary conserved epigenetic memory system that acts antagonistically to maintain active and repressed gene expression states, important during stem cell differentiation and embryonic development  and the brain lipid-binding protein (Fabp7/Blbp) acquire H3K4me3  are H3K27me3-positive during the expansion phase of NPCs  has shown that H3K4me3 and H3K27me3 marks are highly dynamic during neocortical lineage progression , a key proneural transcription factor that can suppress astrocytic differentiation  family proteins, part of PRC1, has been reported to modulate the balance between self-renewal and lineage commitment  miR-92 and miR-92b, and both miRNAs regulate bIP specification in the developing neocortex  (reviewed in Zhao et al., 6A during neocortex development leads to a prolonged cell cycle of aRGs and extends neuron production to postnatal stages, suggesting that m6A regulates cortical neurogenesis (Yoon et al., 6A, several encode transcription factors regulating NPC fate, such as Pax6, Sox2, Neurog2, and Tbr2. The presence of m6A on these transcripts promotes their rapid turnover, and in absence of the m6A methyltransferase complex component Mettl14, several neuronal lineage proteins, like Neurod1 and Tbr2, are precociously expressed in aRG. This observation led to the proposal of the novel concept of transcriptional pre-patterning during cortical neurogenesis, whereby a subset of neuronal lineage genes is already expressed in aRG but their levels actively suppressed post-transcriptionally by m6A-dependent decay (Yoon et al., 6A during neurogenesis found that Mettl14 deletion results in decreased radial glia proliferation and premature differentiation (Wang et al., 6A in specific neural subpopulations, the two studies (Yoon et al., In addition, recently a new epitranscriptomic mechanism has been identified that regulates the metabolism and translation of mRNAs, which involves the post-transcriptional modification of mRNAs by Whereas, transcriptome analysis provides a snapshot of a cell's gene expression pattern at a specific point in time, the corresponding epigenetic information captures gene regulatory mechanisms, developmental origins, and potential future responses to developmental stimuli (Mo et al., That said, the transition from “closed” to “open” chromatin, and vice versa, is determined by regulatory proteins, most prominently a special class of transcription factors, called pioneer factors (Shlyueva et al., cis-regulatory elements, among which enhancers are the most abundant (Spitz and Furlong, Differential gene expression in specific cell types is mainly controlled by distal in vitro and in the mouse developing neocortex, chromatin interactions change dynamically, frequently related to neural transcription factors that contribute to chromosome reorganization (Bonev et al., During neural differentiation, both in vivo during neocortex development at the Eomes gene promoter (Albert et al., It is well-established that epigenetic mechanisms contribute to the regulation of gene expression during stem cell differentiation and development. In this review, we have summarized recent advances in our understanding of the role of Polycomb proteins during mouse neocortex development. In particular, recent epigenome profiling has shed further light on the context-dependent functions of Polycomb proteins during the proliferative and neurogenic phase of neocortex development. It remains to be shown on a genome-wide scale how PcG targets change with the transition to the gliogenic phase. Moreover, in the future, it will be interesting to apply the emerging CRISPR/Cas9-based epigenome editing tools (Pulecio et al., The epigenetic landscape provides a framework within which many transcription factors operate, but which, in turn, is modulated by the action of transcription factors and gene expression itself. During development, epigenetic patterning is important for the correct spatio-temporal regulation of gene expression. In addition, the translation of expressed mRNAs is regulated by miRNAs and novel epitranscriptomic modifications, providing a multi-layered mechanism to precisely control the dynamic expression of genes, both at the mRNA and protein level. The challenge for the future will be to integrate the different layers of transcriptional and post-transcriptional gene regulation into a comprehensive framework that allows to link the different mechanisms and to understand the cross-talk between these systems.MA designed the figures. MA and WH wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study is to investigate the Protein-Protein Interaction Network of Celiac Disease.. Understanding the molecular mechanisms and involved pathway may lead to the development of drug target discovery. The protein interaction network is one of the supportive fields to discover the pathogenesis biomarkers for celiac disease.  Celiac disease (CD) is an autoimmune disease with susceptibility of individuals to gluten of wheat, rye and barleyIn the present study, we collected the articles that focused on the proteomic data in celiac disease. According to the gene expression investigations of these articles, 31 candidate proteins were selected for this study. The networks of related differentially expressed protein were explored using Cytoscape 3.3 and the PPI analysis methods such as MCODE and ClueGO. According to the network analysis Ubiquitin C, Heat shock protein 90kDa alpha (cytosolic and Grp94); class A, B and 1 member, Heat shock 70kDa protein, and protein 5 , T-complex, Chaperon in containing TCP1; subunit 7 (beta) and subunit 4 (delta) and subunit 2 (beta), have been introduced as hub-bottlnecks proteins. HSP90AA1, MKKS, EZR, HSPA14, APOB and CAD have been determined as seed proteins. Chaperons have a bold presentation in curtail area in network therefore these key proteins beside the other hub-bottlneck proteins may be a suitable candidates biomarker panel for diagnosis, prognosis and treatment processes in celiac disease.  Celiac disease (CD) is an autoimmune disease in susceptible individuals sensitive to gluten component of wheat, rye and barley , 2, whicThe proteomics study has been used to identify proteins or changes in protein expression of CD to discover a biomarkers using several techniques, such as antibody and tissue arrays, two-dimensional (2D) gel electrophoresis, mass spectrometry. Results of these techniques are suggesting that we may approached new insight in some diagnostic biomarkers by observing body fluids with posttranslational modifications during signaling  which wiPubMed , ISI of web knowledge, and Google scholar were searched for full text articles on the associated keywords "proteomic" and "celiac disease" published until April 2016 , 25. BasThe topological analyses were carried out via algorithms such as centrality measures. Graph centrality measures like degree, betweenness and closeness centrality are so useful in the identification of nodes that are functionally crucial in the network . In the  To identify crutial nodes in PPI networks, two centrality criteria, including degree and betweenness have been used in this study. First, we prepared the list of proteins, which were hubs in PPI network. The results indicate that some nodes have a large number of links to other protein nodes and act as hubs. Then we determine the bottlenecks (highest betweeness nodes) in the network . It was As represented in The results of MCODE analysis were shown in Functional distribution of the biological process of celiac modules was shown in Since proteins act as complex and corporate with each other in cellular events and pathways, their deregulation are resulted in disorders. One way to evaluate and analyze the pathways is molecular mapping . Recentl


Frizzled 6 (FZD6) belongs to a family of proteins that serve as receptors in the WNT signaling pathway. FZD6 plays an important role in the establishment of planar cell polarity in many embryonic processes such as convergent extension during gastrulation, neural tube closure, or hair patterning. Based on its role during hair development, we hypothesized that FZD6 may have similar expression pattern and function in the dental lamina, which is also a distinct epithelial protrusion growing characteristically angled into the mesenchyme. Diphyodont minipig was selected as a model species because its dentition closely resemble human ones with successional generation of teeth initiated from the dental lamina. We revealed asymmetrical expression of FZD6 in the dental lamina of early as well as late stages during its regression with stronger expression located on the labial side of the dental lamina. During lamina regression, FZD6-positive cells were found in its superficial part and the signal coincided with the upregulation of molecules involved in epithelial-mesenchymal transition and increased migratory potential of epithelial cells. FZD6-expression was also turned on during differentiation of cells producing hard tissues, in which mature odontoblasts, ameloblasts, or surrounding osteoblasts were FZD6-positive. On the other hand, the tip of successional lamina and its lingual part, in which progenitor cells are located, exhibited FZD6-negativity. In conclusion, asymmetrical expression of FZD6 correlates with the growth directionality and side-specific morphological differences in the dental lamina of diphyodont species. Based on observed expression pattern, we propose that the dental lamina is other epithelial tissue, where planar cell polarity signaling is involved during its asymmetrical growth. Minipig embryos and fetuses were obtained from Liběchov animal facility . The day after insemination was established as day 1 of gestation. Staged embryos and fetuses were obtained by hysterectomy. All samples were fixed in 4% neutral formaldehyde and decalcified in 12.5% EDTA in 4% PFA until the mandibular bones of embryos were soft enough for further processing. Sections were stained with Haematoxylin-Eosin and alternative slides were used for immunohistochemical labeling. All procedures were conducted following a protocol approved by the Laboratory Animal Science Committee of the Institute of Animal Physiology and Genetics, Academy of Sciences .For detection of FZD6-positivity, we performed immunohistochemical labeling. After deparaffinization and rehydration, antigen retrieval was performed in a water bath (97°C) in citrate buffer (pH = 6) for 20 min. Blocking serum was applied to the sections for 20 min and slides were incubated for 1 h at room temperature with primary FZD6 antibody . The secondary antibody was applied for 30 min. Streptavidin-FITC complex  was used for visualization of FZD6-positive cells (30 min). DAPI  or DRAQ5  were applied for the counterstaining. The photos taken under a fluorescence microscope Leica DM LB2  were merged together in Adobe Photoshop 7.0 (USA). High power images were taken on confocal microscope Leica SP5 using 40x (air) objectives  with Leica Application Suite software.PCP components are often localized asymmetrically in different types of cells. The Frizzled family was found to be required for producing the correct orientation of cuticular bristles and hairs (Guo et al., At the cap stage Figure , distincIt is known that FZD6 is transducing signals in non-canonical WNT pathway and Wnt5a is one of its representative, which signals upstream of PCP pathway in mammals (Moon et al., Wnt3a is expressed in the enamel knot at the cap stage (Millar et al., FZD can also mediate canonical signaling with activation of β-catenin. WNT3a is strongly expressed in the inner enamel epithelium of humans during the bell stage (Wang et al., FZD6 was also distributed in distinct areas of the mesenchyme surrounding epithelial thickenings during the early stages of odontogenesis; however, mesenchymal expression was downregulated and only small spots were visible on the mesenchymal cell surface at older stages. On the other hand, FZD6 was mostly expressed in the epithelium with uniform expression throughout the whole thickness of the oral epithelium. This finding is consistent with observations in FZD6 knockout mice, where numerous genes encoding keratins, keratin-associated proteins and transglutaminases and their substrates were significantly downregulated (Cui et al., During later stages of odontogenesis, the dental lamina protruded deeply into the mesenchyme and morphological changes were obvious in its superficial part Figure . In the While, the most distal tip of the successional dental lamina was FZD6-negative, β-catenin was previously described in the tip and lingual side of the dental lamina in snake and alligator (Wu et al., The minipig similar to human has a diphyodont type of dentition, where only two generation of teeth are initiated. During embryonic period, the dental lamina undergoes major morphological changes and becomes thinner and disconnected from the oral epithelium. Cells are elongated in the superficial area of the dental lamina (Buchtová et al., Later during dental lamina regression, the dental lamina become fragmented, and only occasional epithelial islands remain in the superficial area Figure . Some ofInterestingly, distinct expression was obvious especially in the basal layers of the lamina epithelium facing the tooth where clusters of elongated cells were moving out of the dental lamina. The expression of FZD6 during lamina regression coincides with the upregulation of molecules involved in epithelial-mesenchymal transition of epithelial cells (Buchtová et al., Both canonical and non-canonical WNT pathways were previously shown to be involved in the differentiation of hard-tissue producing cells (Millar et al., In agreement with these findings, FZD6 expression was switched on during the differentiation of odontoblasts, ameloblasts, and osteoblasts in minipigs Figure  at time Wnt5a-deficient mice exhibit delayed odontoblast differentiation (Lin et al., Ror2 mutant mice (Lin et al., Wnt5a-deficient mice were shorter and thicker than in control animals. Similarly in Ror2-deficient mice, odontoblasts were polarized but appeared to be shorter in comparison to littermate control animals (Lin et al., Wnt5a is expressed in the dental mesenchyme, its receptor is also expressed in the epithelium. It was proposed that another receptor must be involved in WNT5a-mediated signaling as defects in tooth development in Ror2 mutants occur much later than observed in WNT5a-deficient mice (Lin et al., While the tooth phenotype of FZD6 mutant mice has not yet been described, Odontoblasts and ameloblasts are highly polarized cells with a characteristic morphology and arrangement of cellular compartments. In odontoblasts, FZD6 was expressed asymmetrically only on the side facing the dentin Figures . In amelIn summary, FZD6 was expressed asymmetrically in the dental lamina at early as well as late stages of diphyodont dentition (Figure FZD6 expression was also switched on during the differentiation of odontoblasts, ameloblasts, and osteoblasts in minipigs at time when the production of hard tissues started. In odontoblasts, FZD6 was expressed asymmetrically only on the side facing the dentin therefore it is possible that FZD6 can be involved in the establishment of cellular polarity in the odontoblasts during their differentiation. However, the exact role of FZD6 in the growth directionality of the lamina and differentiation of hard tissue producing cells has to be proven experimentally in future.MB and VB designed the project. IP, HD, ML, and JS performed and interpreted experiments. IP, MB, and JS wrote manuscript with contribution of VB. All authors approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.