Deficiency of CD73 activity promotes protective cardiac immunity against Trypanosoma cruzi infection but permissive environment in visceral adipose tissue.

Damaged cells release the pro-inflammatory signal ATP, which is degraded by the ectonucleotidases CD39 and CD73 to the anti-inflammatory mediator adenosine (ADO). The balance between ATP/ADO is known to determine the outcome of inflammation/infection. However, modulation of the local immune response in different tissues due to changes in the balance of purinergic metabolites has yet to be investigated. Here, we explored the contribution of CD73-derived ADO on the acute immune response against Trypanosoma cruzi parasite, which invades and proliferates within different target tissues. Deficiency of CD73 activity led to an enhanced cardiac microbicidal immune response with an augmented frequency of macrophages with inflammatory phenotype and increased CD8+ T cell effector functions. The increment of local inducible nitric oxide (NO) synthase (iNOS)+ macrophages and the consequent rise of myocardial NO production in association with reduced ADO levels induced protection against T. cruzi infection as observed by the diminished cardiac parasite burden compared to their wild-type (WT) counterpart. Unexpectedly, parasitemia was substantially raised in CD73KO mice in comparison with WT mice, suggesting the existence of tissue reservoir/s outside myocardium. Indeed, CD73KO liver and visceral adipose tissue (VAT) showed increased parasite burden associated with a reduced ATP/ADO ratio and the lack of substantial microbicidal immune response. These data reveal that the purinergic system has a tissue-dependent impact on the host immune response against T. cruzi infection.

Storage of Carotenoids in Crustaceans as an Adaptation to Modulate Immunopathology and Optimize Immunological and Life-History Strategies.

Why do some invertebrates store so much carotenoids in their tissues? Storage of carotenoids may not simply be passive and dependent on their environmental availability, as storage variation exists at various taxonomic scales, including among individuals within species. While the strong antioxidant and sometimes immune-stimulating properties of carotenoids may be beneficial enough to cause the evolution of features improving their assimilation and storage, they may also have fitness downsides explaining why massive carotenoid storage is not universal. Here, the functional and ecological implications of carotenoid storage for the evolution of invertebrate innate immune defenses are examined, especially in crustaceans, which massively store carotenoids for unclear reasons. Three testable hypotheses about the role of carotenoid storage in immunological (resistance and tolerance) and life-history strategies (with a focus on aging) are proposed, which may ultimately explain the storage of large amounts of these pigments in a context of host-pathogen interactions.

4,4'-Diaponeurosporene-Producing Bacillus subtilis Promotes the Development of the Mucosal Immune System of the Piglet Gut.

Gut mucosal immune responses are known to act as the first line of defense against invasion of pathogenic microorganisms. Piglets have an incompletely developed gut mucosal immune system, making them sensitive to intestinal infections. Promoting the development of the mucosal immune system will increase the pathogen resistance of piglets. The aim of the present study was to investigate the effect of carotenoid (4,4'-diaponeurosporene)-producing Bacillus subtilis (B.s-Dia) on intestinal mucosal immunity in piglets. We showed that oral administration to piglets of B.s-Dia remarkably improved the development of Peyer's patches (PPs) (P < 0.01), and increased villus height (P < 0.01) and colon crypt depth (P < 0.01). In addition, B.s-Dia also increased the number of intraepithelial lymphocytes (P < 0.01), while Bacillus subtilis (B.s) had no significant influence on it (P > 0.05). Moreover, B.s-Dia also increased the number of SIgA+ cells (P < 0.01). Oral administration of either B.s or B.s-Dia increased the number of CD4+ and CD8+ cells in ileum lamina propria (P < 0.01). These results indicate that B.s-Dia contributes to a higher extent to porcine mucosal immune system development than B.s, and might serve as an immunopotentiator candidate. Anat Rec, 302:1800-1807, 2019. © 2019 American Association for Anatomy.

Fate of carotenoid-producing Bacillus aquimaris SH6 colour spores in shrimp gut and their dose-dependent probiotic activities.

Bacillus aquimaris SH6 spores produce carotenoids that are beneficial to white-leg shrimp (Litopenaeus vannamei) health. However, the optimal dose and mechanisms behind these effects are not well understood. We investigated the fate of SH6 spores in the gut of L. vannamei. Shrimp were divided into six groups administrated with either feed only (negative control) or SH6 spores at 5 × 106 CFU/g pellet (high dose, SH6 spore-H group), 1 × 106 CFU/g pellet (medium dose, SH6 spore-M group), 2 × 105 CFU/g pellet (low dose, SH6 spore-L group), astaxanthin at 0.5 mg/g pellet (Carophyll group), or carotenoids from SH6 vegetative cells at 5 µg/g pellet (SH6 carotenoid group). The growth rate was highest in SH6 spore-H (3.38%/day), followed by SH6 spore-M (2.84%/day) and SH6 spore-L (2.25%/day), which was significantly higher than the control (1.45%/day), Carophyll (1.53%/day) or SH6 carotenoid (1.57%/day) groups. The astaxanthin levels (1.9-2.0 µg/g shrimp) and red-colour scores (21-22) in SH6 spore-H/M were higher than the control (astaxanthin: 1.2 µg/g shrimp; red score: 20) or SH6 spore-L, but lower than the Carophyll and SH6 carotenoids. Feeding with medium and high doses of SH6 spores after 28 days resulted in respective 1.3-2-fold increases in phenol oxidase activity and 8-9 fold increases in Rho mRNA expression compared to the control and low dose group. The live-counts of SH6 in the gut gradually increased during the 28-day feeding period with SH6 spores at different concentrations, starting from 4.1, 8.2, and 5.4 × 104 CFU/g gut at day 1 and reaching 5.3, 5.1, and 4.4 × 105 CFU/g gut in the SH6-H/M/L groups, respectively, at day 28. Gut microbiota became more diversified, resulting in a 2-8-fold increase in total bacterial live-counts compared to the controls. SH6 spore germination was detected by measuring the mRNA expression of a specific sequence coding for SH6 amylase at 4 h, reaching saturation at 24 h. Our results confirm that SH6 spores colonize and germinate in the gut to improve the microbial diversity and boost the immune system of shrimp, exhibiting beneficial effects at >1 × 106 CFU/g pellet.

Structural Organization of 6B9 Molecule, a Monoclonal Antibody Against Lycopene.

Full cDNA and corresponding amino acid (AA) sequences of 6B9 monoclonal antibody (mAb) against lycopene was obtained using Step-Out RACE technology. Variable (V) and constant (C) regions were identified. The light chain of 6B9 contained 238 AA IgM with the highest level of identity (0.93) to both the anti-VEGF receptor antibody and anti-collagen type II FAb CIIC1. The heavy chain was composed of 634 AA with a high identity (0.9) to the Ig mu chain C region. Potential posttranslational modification regions in both chains were identified alongside with disulfide bond sites. The obtained information can be used for making chimeric constructs containing 6B9 mAb (or its fragments) and lycopene, a powerful carotenoid with antioxidant as well as antiproliferating properties, which can be implemented in the treatment of an aggressive form of prostate cancer and possibly other malignancies.

Dietary Intake of Antioxidant Vitamins and Carotenoids and Risk of Developing Active Tuberculosis in a Prospective Population-Based Cohort Study.

Antioxidants may protect against oxidative stress, which is associated with tuberculosis (TB) disease. However, direct evidence for a protective association between dietary antioxidants and TB incidence in humans has been lacking. The relationship between intake of antioxidant vitamins (vitamins A, C, D, and E) and individual carotenoids (α-carotene, ß-carotene, ß-cryptoxanthin, lycopene, and lutein) and TB incidence was examined in the Singapore Chinese Health Study, a prospective cohort study of 63,257 adults aged 45-74 years enrolled during 1993-1998. Baseline intake of these antioxidants was estimated using a validated semiquantitative food frequency questionnaire including questions on use of dietary supplements. After an average of 16.9 years of follow-up, 1,186 incident active TB cases were identified among cohort participants. Compared with the lowest quartile, reduced risk of active TB was observed for the highest quartile of vitamin A intake (hazard ratio = 0.71, 95% confidence interval: 0.59, 0.85; P-trend < 0.01) and ß-carotene intake (hazard ratio = 0.76, 95% confidence interval: 0.63, 0.91; P-trend < 0.01), regardless of smoking status. Lower TB risk was seen for vitamin C intake among current smokers only. Other vitamins and carotenoids were not associated with TB risk. These results suggest that vitamin C may reduce TB risk among current smokers by ameliorating oxidative stress, while vitamin A and ß-carotene may have additional antimycobacterial properties.

Generation and Application of Monoclonal Antibody Against Lycopene.

A monoclonal antibody (Mab) against lycopene was developed from hybridoma clones obtained from BALB/c mice immunized with trans-isomer of lycopene (t-lycopene, t-LC) conjugated with colloidal gold particles. An alternating immunization schedule which included injection of both formulations of immunogen (without and with Freund's adjuvant) was most effective in the elucidation of a measurable immune response to the t-Lycopene conjugate. Selected hybridoma clones were able to produce an Mab positive in competition assay. In particular, preincubation of 6B9 Mabs with t-LC abolished the ability of 6B9 Mabs to bind LC in the competition assay. Mabs produced by other clones (4F10, 4A3, and 3B12) worked similarly. Analysis of antigen specificity showed that 6B9 Mab raised against t-LC did not recognize other carotenoids such as lutein and carotene. Mab 6B9 was shown to recognize lycopene on a glass surface and in the settings of indirect immunofluorescence experiments performed in cultured hepatocytes and alveolar macrophages incubated with and without lycopene, as well as in sebum and corneocyte specimens from the skin of volunteers supplemented with nutraceutical formulation of lycopene. Newly generated Mabs against lycopene may provide a valuable tool for different analytical assays of lycopene content in various biological, agricultural, and food products.

Immune challenges and visual signalling in tree frogs.

In animals, mate-choice is often based on sexual signals that carry information and help the receiver make the best choice to improve the receiver's fitness. Orange visual sexual signals have been hypothesised to carry immune information because they are often due to carotenoid pigments which are also involved in immunity response. Although many studies have focused on the direct relationships between coloration and immunocompetence, few studies have simultaneously studied immunocompetent response and coloration variation after an immune challenge. We tested this hypothesis on starved and ad libitum-fed males of the European tree frog Hyla arborea. Our results show that male coloration is not a reliable indicator of its immune response capacity in this species. However, after an immune challenge induced by a PHA (Phaseolus vulgaris phytohaemagglutinin) injection, starved males presented a significant coloration loss and this alteration was related to the immune response intensity. Taken together, these results suggest that the brighter (lighter) coloration may be used as a cue by female to exclude males with a recent immune challenge, due to diseases or parasites for example.

Carotenoid-based coloration, condition, and immune responsiveness in the nestlings of a sexually dimorphic bird of prey.

In many birds, nestlings exhibit brightly colored traits that are pigmented by carotenoids. Carotenoids are diet limited and also serve important health-related physiological functions. The proximate mechanisms behind the expression of these carotenoid-pigmented traits are still poorly known, especially in nestlings with sexual size dimorphism. In these nestlings, intrabrood competition levels and growth strategies likely differ between sexes, and this may in turn influence carotenoid allocation rules. We used dietary carotenoid supplementation to test whether wild marsh harrier (Circus aeruginosus) nestlings were carotenoid limited and whether carotenoid allocation strategies varied between sexes, which differ in their size and growth strategies. When supplemented, nestlings used the supplemental carotenoids to increase their coloration independently of their sex. We showed that the condition dependence of the carotenoid level and the response to an immune challenge (phytohemagglutinin test) differed between sexes, possibly because sexual size dimorphism influences growth strategies and/or intrabrood competition levels and access to different types of food. In this species, which often feeds on mammals, a trade-off likely exists between food quantity (energy) and quality (carotenoid content). Finally, carotenoid-based coloration expressed in marsh harrier nestlings appeared to be indicative of immune responsiveness rather than condition, therefore potentially advertising to parents nestling quality or value rather than nutritional need.

Carotenoid-based ornaments of female and male American goldfinches (Spinus tristis) show sex-specific correlations with immune function and metabolic rate.

Conspicuous ornamentation has been linked to immunological and physiological condition in males of many species. In species where both sexes are ornamented, it is unclear whether the signal content of ornaments differs between males and females. We examined the immunological and physiological correlates of carotenoid-based bill and plumage ornamentation in American goldfinches Spinus tristis, a species in which bright orange bills are sexually monomorphic but yellow plumage is sexually dimorphic during the breeding season. Because bill color is dynamic over short periods while plumage color is static over longer time frames, we tested whether these signals have the potential to provide temporal information about immunity and condition. In both sexes, bill color (but not plumage color) was negatively related to leukocyte differential, a measure of recent stress, while plumage color (but not bill color) was positively related to resting metabolic rate. In females, bill color also positively correlated with immunoglobulin Y, a component of acquired immunity, while plumage color positively predicted natural antibody levels, a component of innate immunity. In males, neither bill color nor plumage color predicted immune function, suggesting that the mechanisms underlying these signals vary with sex. Our results demonstrate that dynamic signals such as bill coloration do not merely reflect the same information provided by static signals but that these two classes of signal provide information about different temporal aspects of phenotypic quality. Furthermore, our results indicate that a signal expressed in both sexes has the potential to provide different information depending on the sex of the bearer.

Fat stores in a migratory bird: a reservoir of carotenoid pigments for times of need?

Carotenoids are well known for their immune-stimulant function in birds and other vertebrates. Moreover, they have potential antioxidant capacity, scavenging free radicals and protecting cell compartments from oxidation. Most essential carotenoids are fat soluble and could be stored for times of need especially in adipose tissues, built up by migratory birds as the main source of energy on long-distance flights. In an exclusive diet experiment, garden warblers (Sylvia borin) were fed ad libitum with an experimental diet, enriched with two different dose rates of carotenoids, or with control food, during the period of their first autumn migration. Plasma carotenoid content was measured via HPLC and chroma of plasma and fat examined with a spectrophotometer. Birds were infected with Isospora spp. and intensity of infection determined by oocyst counts 3 days post infection. Plasma lutein levels and chroma of subcutaneous fat stores were positively correlated and chroma values of these fat stores increased in the birds that got the higher dose rate, whereas they decreased significantly in the control group after infection with Isospora spp. Chroma of subcutaneous fat deposits in vivo and intensity of Isospora infection were negatively correlated. By measuring the chroma of fat deposits in vivo, we show that fat can be a reservoir for carotenoids. These colorful antioxidants are stored in the fat and taken from there in times of a higher demand, e.g. when mounting an immune response to parasites.

Tomato Lycopene and Inflammatory Cascade: Basic Interactions and Clinical Implications

Lycopene, a natural carotenoid found in tomato, has been reported to possess various health benefits, such as cardiovascular and cancer preventive properties. However, the experimental basis for such health benefits is not fully understood. One of the possible mechanisms for its protective activities is by down-regulation of the inflammatory response. That includes the inhibition of pivotal pro-inflammatory mediators, such as the reduction of reactive oxygen species, the inhibition of synthesis and release of pro-inflammatory cytokines, changes in the expression of cyclooxygenase and lipoxygenase, modifications of eicosanoid synthesis, and modulation of signal transduction pathways, including that of the inducible nitric oxide synthase via its inhibitory effects on Nuclear Factor-kB (NF-kB), Activated protein-1 (AP-1) and mitogen-activated protein kinase (MAPK) signaling. Recent data suggest that lycopene also exhibits anti-inflammatory activity through induction of programmed cell death in activated immune cells. This review will discuss recent data on the control of inflammatory signaling exerted by tomato lycopene in isolated cells, in animal models and in clinical trials, focusing on the dose of the carotenoid and the biological environment in which it acts. A clear understanding of the molecular mechanisms of action of lycopene is crucial in the valuation of this molecule as a potential preventive and therapeutic agent.

Carotenoid-based coloration predicts resistance to oxidative damage during immune challenge.

Many animal ornaments may have evolved as signals advertising the quality of the bearer. The honesty of the information content of these signals would rely on the costs associated with their expression, these being relatively greater for low-quality than for high-quality individuals. Given the physiological functions of carotenoids, carotenoid-based ornaments could indicate individual immunocompetence, and possibly the ability to mount an immune response at a lower cost. We evaluated whether the red carotenoid-based coloration of male red-legged partridges (Alectoris rufa) predicts the capacity of the individual to counteract the oxidative stress generated by a cell-mediated immune response. Individuals were subcutaneously injected with phytohaemagglutinin (PHA) or phosphate buffer solution (PBS) as a control. We found that eye ring pigmentation predicted the change in the amount of peroxidized lipids (TBARS) in blood after the PHA-induced inflammatory challenge. The degree of pigmentation of this carotenoid-based ornament was also negatively related to individual changes in gamma-glutamyl transferase (GGT), another biomarker of oxidative stress involved in antioxidant metabolism (i.e. glutathione recycling). However, changes in circulating carotenoids did not significantly explain changes in lipid peroxidation or GGT levels, suggesting that the higher resistance to oxidative stress of those individuals with more pigmented eye rings was not directly mediated by their greater circulating levels of carotenoids. Our results indicate that carotenoid-based coloration can predict not only immune responsiveness (more coloured males mount greater responses) but also an individual's ability to counter the oxidative stress generated during immune challenge (more coloured males experience less oxidative damage when mounting an immune response).

Inhibitory effect of carotenoids on the degranulation of mast cells via suppression of antigen-induced aggregation of high affinity IgE receptors.

Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is critical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and beta-carotene significantly inhibited the antigen-induced release of beta-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-derived mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (Fc epsilonRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited Fc epsilonRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of Fc epsilonRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of Fc epsilonRI to lipid rafts, which are known as platforms of the aggregation of Fc epsilonRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of Fc epsilonRI to lipid rafts. This is the first report that focused on the aggregation of Fc epsilonRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts.

Testosterone and carotenoids: an integrated view of trade-offs between immunity and sexual signalling.

Allocation tradeoffs with the immune system can enforce honesty on sexual signals that act as indicators of individual quality. Such tradeoffs can be brought about by (1) the dual action of testosterone, which stimulates sexual signals but also suppresses immune functions, and/or (2) competition for carotenoids, which can be deposited as ornamental pigments or used as antioxidants in support of immune functions. Recent studies integrate these two mechanisms by showing that testosterone treatment in male birds upregulates circulating lipoproteins, plasma carriers for carotenoids, and increases bio-availability of carotenoids, which enhances carotenoid-based sexual coloration and immune responsiveness.

Costly carotenoids: a trade-off between predation and infection risk?

Carotenoid reserves in copepods seem costly in terms of predation risk because they make individuals conspicuous. However, carotenoids also seem to play an important role in immune defence as free radical scavengers. To test whether predation risk influences carotenoid levels and whether changes in carotenoid levels are related to changes in immune defence, I examined individual changes in large carotenoid and other lipid droplets upon exposure to predation risk and subsequent exposure to parasites in the copepod Macrocyclops albidus. Copepods reduced carotenoid reserves upon exposure to predators, through which they potentially avoided the costs of being conspicuous under predation risk. Thus, the size of carotenoid reserves is a plastic trait. Such a decrease in carotenoid reserves may also have a negative impact on the copepods' immune system as individuals that decreased their reserves suffered higher parasite prevalence upon exposure to the cestode Schistocephalus solidus. These results suggest that carotenoid reserves may be individually optimized to trade-off each individual's unique costs (predation risk) and benefits (immune defence) of having these reserves.

Lycopene suppresses the lipopolysaccharide-induced phenotypic and functional maturation of murine dendritic cells through inhibition of mitogen-activated protein kinases and nuclear factor-kappaB.

Dendritic cells (DC) are the most potent of antigen-presenting cells. The most important function of DC is to initiate the immune response by presenting antigens to naïve T lymphocytes. Currently, little is known about the basic action of lycopene in murine bone marrow (BM)-derived DC. In the present study, we have revealed that lycopene significantly attenuates the phenotypic and functional maturation of murine BM-DC, especially in lipopolysaccharide-induced DC maturation. We found that lycopene down-regulates the expression of costimulatory molecules (CD80 and CD86) and major histocompatibility complex type II molecules. We also determined that lycopene-treated DC were poor stimulators of naïve allogeneic T-cell proliferation and induced lower levels of interleukin-2 in responding T cells. They also exhibited impaired interleukin-12 production. Additionally, lycopene was able to inhibit mitogen-activated protein kinases, such as ERK1/2, p38 and JNK, and the transcription factor, nuclear factor-kappaB. Assessment of the in vivo effects of lycopene may reveal an inability to induce a normal cell-mediated immune response, despite the ability of the cells to migrate to the spleen. This data provides new insight into the immunopharmacology of lycopene and suggests a novel approach to the manipulation of DC for therapeutic application.