Retinoic Acid and Retinoic Acid Receptors as Pleiotropic Modulators of the Immune System.

Vitamin A is a multifunctional vitamin implicated in a wide range of biological processes. Its control over the immune system and functions are perhaps the most pleiotropic not only for development but also for the functional fate of almost every cell involved in protective or regulatory adaptive or innate immunity. This is especially key at the intestinal border, where dietary vitamin A is first absorbed. Most effects of vitamin A are exerted by its metabolite, retinoic acid (RA), which through ligation of nuclear receptors controls transcriptional expression of RA target genes. In addition to this canonical function, RA and RA receptors (RARs), either as ligand-receptor or separately, play extranuclear, nongenomic roles that greatly expand the multiple mechanisms employed for their numerous and paradoxical functions that ultimately link environmental sensing with immune cell fate. This review discusses RA and RARs and their complex roles in innate and adaptive immunity.

Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.

sLRP1'in up retinol keeps the gut SAAfe.

Retinol is shuttled to myeloid cells for conversion to retinoic acid, but the receptor facilitating uptake of SAA:retinol complexes on myeloid cells is unknown. In a recent issue of Science, Bang et al. (2021) use genetic and biochemical approaches to reveal this critical receptor to be LRP1 and show that this axis is essential for intestinal innate and adaptive immune responses.

Induction of IL-10-producing type 2 innate lymphoid cells by allergen immunotherapy is associated with clinical response.

The role of innate immune cells in allergen immunotherapy that confers immune tolerance to the sensitizing allergen is unclear. Here, we report a role of interleukin-10-producing type 2 innate lymphoid cells (IL-10+ ILC2s) in modulating grass-pollen allergy. We demonstrate that KLRG1+ but not KLRG1- ILC2 produced IL-10 upon activation with IL-33 and retinoic acid. These cells attenuated Th responses and maintained epithelial cell integrity. IL-10+ KLRG1+ ILC2s were lower in patients with grass-pollen allergy when compared to healthy subjects. In a prospective, double-blind, placebo-controlled trial, we demonstrated that the competence of ILC2 to produce IL-10 was restored in patients who received grass-pollen sublingual immunotherapy. The underpinning mechanisms were associated with the modification of retinol metabolic pathway, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathways in the ILCs. Altogether, our findings underscore the contribution of IL-10+ ILC2s in the disease-modifying effect by allergen immunotherapy.

Airway hillocks are injury-resistant reservoirs of unique plastic stem cells.

Airway hillocks are stratified epithelial structures of unknown function1. Hillocks persist for months and have a unique population of basal stem cells that express genes associated with barrier function and cell adhesion. Hillock basal stem cells continually replenish overlying squamous barrier cells. They exhibit dramatically higher turnover than the abundant, largely quiescent classic pseudostratified airway epithelium. Hillocks resist a remarkably broad spectrum of injuries, including toxins, infection, acid and physical injury because hillock squamous cells shield underlying hillock basal stem cells from injury. Hillock basal stem cells are capable of massive clonal expansion that is sufficient to resurface denuded airway, and eventually regenerate normal airway epithelium with each of its six component cell types. Hillock basal stem cells preferentially stratify and keratinize in the setting of retinoic acid signalling inhibition, a known cause of squamous metaplasia2,3. Here we show that mouse hillock expansion is the cause of vitamin A deficiency-induced squamous metaplasia. Finally, we identify human hillocks whose basal stem cells generate functional squamous barrier structures in culture. The existence of hillocks reframes our understanding of airway epithelial regeneration. Furthermore, we show that hillocks are one origin of 'squamous metaplasia', which is long thought to be a precursor of lung cancer.

A randomized phase 2 trial of oral vitamin A for graft-versus-host disease in children and young adults.

ABSTRACT: Vitamin A plays a key role in the maintenance of gastrointestinal homeostasis and promotes a tolerogenic phenotype in tissue resident macrophages. We conducted a prospective randomized double-blinded placebo-controlled clinical trial in which 80 recipients of hematopoietic stem cell transplantation (HSCT) were randomized 1:1 to receive pretransplant high-dose vitamin A or placebo. A single oral dose of vitamin A of 4000 IU/kg, maximum 250 000 IU was given before conditioning. The primary end point was incidence of acute graft-versus-host disease (GVHD) at day +100. In an intent-to-treat analysis, incidence of acute GVHD was 12.5% in the vitamin A arm and 20% in the placebo arm (P = .5). Incidence of acute gastrointestinal (GI) GVHD was 2.5% in the vitamin A arm (P = .09) and 12.5% in the placebo arm at day +180. Incidence of chronic GVHD was 5% in the vitamin A arm and 15% in the placebo arm (P = .02) at 1 year. In an "as treated" analysis, cumulative incidence of acute GI GVHD at day +180 was 0% and 12.5% in recipients of vitamin A and placebo, respectively (P = .02), and cumulative incidence of chronic GVHD was 2.7% and 15% in recipients of vitamin A and placebo, respectively (P = .01). The only possibly attributable toxicity was asymptomatic grade 3 hyperbilirubinemia in 1 recipient of vitamin A at day +30, which self-resolved. Absolute CCR9+ CD8+ effector memory T cells, reflecting gut T-cell trafficking, were lower in the vitamin A arm at day +30 after HSCT (P = .01). Levels of serum amyloid A-1, a vitamin A transport protein with proinflammatory effects, were lower in the vitamin A arm. The vitamin A arm had lower interleukin-6 (IL-6), IL-8, and suppressor of tumorigenicity 2 levels and likely a more favorable gut microbiome and short chain fatty acids. Pre-HSCT oral vitamin A is inexpensive, has low toxicity, and reduces GVHD. This trial was registered at www.ClinicalTrials.gov as NCT03202849.

Interleukin-33-Activated Islet-Resident Innate Lymphoid Cells Promote Insulin Secretion through Myeloid Cell Retinoic Acid Production.

Pancreatic-islet inflammation contributes to the failure of ß cell insulin secretion during obesity and type 2 diabetes. However, little is known about the nature and function of resident immune cells in this context or in homeostasis. Here we show that interleukin (IL)-33 was produced by islet mesenchymal cells and enhanced by a diabetes milieu (glucose, IL-1ß, and palmitate). IL-33 promoted ß cell function through islet-resident group 2 innate lymphoid cells (ILC2s) that elicited retinoic acid (RA)-producing capacities in macrophages and dendritic cells via the secretion of IL-13 and colony-stimulating factor 2. In turn, local RA signaled to the ß cells to increase insulin secretion. This IL-33-ILC2 axis was activated after acute ß cell stress but was defective during chronic obesity. Accordingly, IL-33 injections rescued islet function in obese mice. Our findings provide evidence that an immunometabolic crosstalk between islet-derived IL-33, ILC2s, and myeloid cells fosters insulin secretion.

Adipocyte HSL is required for maintaining circulating vitamin A and RBP4 levels during fasting.

Vitamin A (retinol) is distributed via the blood bound to its specific carrier protein, retinol-binding protein 4 (RBP4). Retinol-loaded RBP4 is secreted into the circulation exclusively from hepatocytes, thereby mobilizing hepatic retinoid stores that represent the major vitamin A reserves in the body. The relevance of extrahepatic retinoid stores for circulating retinol and RBP4 levels that are usually kept within narrow physiological limits is unknown. Here, we show that fasting affects retinoid mobilization in a tissue-specific manner, and that hormone-sensitive lipase (HSL) in adipose tissue is required to maintain serum concentrations of retinol and RBP4 during fasting in mice. We found that extracellular retinol-free apo-RBP4 induces retinol release by adipocytes in an HSL-dependent manner. Consistently, global or adipocyte-specific HSL deficiency leads to an accumulation of retinoids in adipose tissue and a drop of serum retinol and RBP4 during fasting, which affects retinoid-responsive gene expression in eye and kidney and lowers renal retinoid content. These findings establish a novel crosstalk between liver and adipose tissue retinoid stores for the maintenance of systemic vitamin A homeostasis during fasting.

Phenotypic landscape of intestinal organoid regeneration.

The development of intestinal organoids from single adult intestinal stem cells in vitro recapitulates the regenerative capacity of the intestinal epithelium1,2. Here we unravel the mechanisms that orchestrate both organoid formation and the regeneration of intestinal tissue, using an image-based screen to assay an annotated library of compounds. We generate multivariate feature profiles for hundreds of thousands of organoids to quantitatively describe their phenotypic landscape. We then use these phenotypic fingerprints to infer regulatory genetic interactions, establishing a new approach to the mapping of genetic interactions in an emergent system. This allows us to identify genes that regulate cell-fate transitions and maintain the balance between regeneration and homeostasis, unravelling previously unknown roles for several pathways, among them retinoic acid signalling. We then characterize a crucial role for retinoic acid nuclear receptors in controlling exit from the regenerative state and driving enterocyte differentiation. By combining quantitative imaging with RNA sequencing, we show the role of endogenous retinoic acid metabolism in initiating transcriptional programs that guide the cell-fate transitions of intestinal epithelium, and we identify an inhibitor of the retinoid X receptor that improves intestinal regeneration in vivo.

Dysregulated cysteine metabolism leads to worsened liver pathology in diabetes-tuberculosis comorbid condition.

Diabetes mellitus (DM) is a risk factor for developing active tuberculosis (TB) with a 3-fold increase in susceptibility and a 4-fold higher relapse rate. With increasing DM prevalence in TB endemic regions, understanding pathophysiological changes associated with DM-TB comorbidity is imperative. In this study, streptozotocin (STZ)-induced DM C57BL/6 mice were aerosol infected with low dose (100-120 CFU) Mycobacterium tuberculosis H37Rv. At 3 weeks post infection (w.p.i.), multiple tissue mycobacterial load and metabolites were profiled. The liver proteome of DM-TB and controls were analyzed using quantitative proteomics, and multi-omics data were integrated. DM-TB mice showed dysregulated multi-tissue (lungs, liver, brain, kidney and thigh muscle) metabolism. In contrast, the mycobacterial burden in the lung, spleen and liver was similar at 3 w.p.i. in DM-TB and TB groups. Enrichment analysis of deregulated liver metabolites (n = 20; log2DM-TB/TB>±1.0) showed significant perturbation in cysteine-methionine, glycine-serine, BCAA and fatty acid metabolism. 60 out of 1660 identified liver proteins showed deregulation (log2DM-TB/TB>±1.0) and contributed from perturbed cysteine-methionine metabolism corroborating metabolomics data. In addition, amino acid biosynthesis, retinol metabolism and polyol biosynthetic process were also differentially enriched in the livers of DM-TB groups. Global correlation analysis of liver metabolome and proteome data showed a strong association between aspartic acid, pyruvic acid, leucine and isoleucine with CYP450 enzymes involved in retinol metabolism, while iminodiacetic acid, isoleucine and γ-aminobutyric acid (GABA) strong positive correlation involved in cysteine metabolism. Targeting perturbed cysteine metabolism using micro molecules, like DL-Propargylglycine, might help prevent liver damage in DM-TB comorbid conditions.

Vitamin A enhanced periosteal osteoclastogenesis is associated with increased number of tissue-derived macrophages/osteoclast progenitors.

A deleterious effect of elevated levels of vitamin A on bone health has been reported in clinical studies. Mechanistic studies in rodents have shown that numbers of periosteal osteoclasts are increased, while endocortical osteoclasts are simultaneously decreased by vitamin A treatment. The present study investigated the in vitro and in vivo effect of all-trans retinoic acid (ATRA), the active metabolite of vitamin A, on periosteal osteoclast progenitors. Mouse calvarial bone cells were cultured in media containing ATRA, with or without the osteoclastogenic cytokine receptor activator of nuclear factor kappa B-ligand (RANKL), on plastic dishes or bone discs. Whereas ATRA did not stimulate osteoclast formation alone, the compound robustly potentiated the formation of RANKL-induced bone resorbing osteoclasts. This effect was due to stimulation by ATRA (half-maximal stimulation ∼3 nM) on the numbers of macrophages/osteoclast progenitors in the bone cell cultures, as assessed by mRNA and protein expression of several macrophage and osteoclast progenitor cell markers, such as macrophage colony-stimulating factor receptor, receptor activator of nuclear factor kappa B, F4/80, and CD11b, as well as by flow cytometry (FACS) analysis of CD11b+/F480+/Gr1- cells. The stimulation of macrophage numbers in the periosteal cell cultures was not mediated by increased macrophage colony-stimulating factor or interleukin-34. In contrast, ATRA did not enhance macrophages in bone marrow cell cultures. Importantly, ATRA treatment upregulated the mRNA expression of several macrophage-related genes in the periosteum of tibia in adult mice. These observations demonstrate a novel mechanism by which vitamin A enhances osteoclast formation specifically on periosteal surfaces.

Verbascum species as a new source of saffron apocarotenoids and molecular tools for the biotechnological production of crocins and picrocrocin.

Crocins are glucosylated apocarotenoids present in flowers and fruits of a few plant species, including saffron, gardenia, and Buddleja. The biosynthesis of crocins in these plants has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems. Mullein (Verbascum sp.) has been identified as a new source of crocins and picrocrocin. In this work, we have identified eight enzymes involved in the cleavage of carotenoids in two Verbascum species, V. giganteum and V. sinuatum. Four of them were homologous to the previously identified BdCCD4.1 and BdCCD4.3 from Buddleja, involved in the biosynthesis of crocins. These enzymes were analyzed for apocarotenogenic activity in bacteria and Nicotiana benthamiana plants using a virus-driven system. Metabolic analyses of bacterial extracts and N. benthamiana leaves showed the efficient activity of these enzymes to produce crocins using ß-carotene and zeaxanthin as substrates. Accumulations of 0.17% of crocins in N. benthamiana dry leaves were reached in only 2 weeks using a recombinant virus expressing VgCCD4.1, similar to the amounts previously produced using the canonical saffron CsCCD2L. The identification of these enzymes, which display a particularly broad substrate spectrum, opens new avenues for apocarotenoid biotechnological production.

Genetic tuning of ß-carotene oxygenase-1 activity rescues cone photoreceptor function in STRA6-deficient mice.

Rod and cone photoreceptors in the retina mediate dim light and daylight vision, respectively. Despite their distinctive functions, rod and cone visual pigments utilize the same vitamin A-derived chromophore. To sustain vision, vitamin A precursors must be acquired in the gut, metabolized, and distributed to the eyes. Deficiencies in this pathway in inherited ocular disease states deplete cone photoreceptors from chromophore and eventually lead to cell death, whereas the more abundant rod photoreceptors are less affected. However, pathways that support cone function and survival under such conditions are largely unknown. Using biochemical, histological, and physiological approaches, we herein show that intervention with ß-carotene in STRA6-deficient mice improved chromophore supply to cone photoreceptors. Relieving the inherent negative feedback regulation of ß-carotene oxygenase-1 activity in the intestine by genetic means further bolstered cone photoreceptor functioning in the STRA6-deficient eyes. A vitamin A-rich diet, however, did not improve cone photoreceptor function in STRA6-deficiency. We provide evidence that the beneficial effect of ß-carotene on cones results from favorable serum kinetics of retinyl esters in lipoproteins. The respective alterations in lipoprotein metabolism maintained a steady supply of retinoids to the STRA6-deficient eyes, which ameliorated the competition for chromophore between rod and cone photoreceptors. Together, our study elucidates a cone photoreceptor-survival pathway and unravels an unexpected metabolic connection between the gut and the retina.

Vitamin A-treated natural killer cells reduce interferon-gamma production and support regulatory T-cell differentiation.

Natural killer (NK) cells are innate cytotoxic lymphocytes that contribute to immune responses against stressed, transformed, or infected cells. NK cell effector functions are regulated by microenvironmental factors, including cytokines, metabolites, and nutrients. Vitamin A is an essential micronutrient that plays an indispensable role in embryogenesis and development, but was also reported to regulate immune responses. However, the role of vitamin A in regulating NK cell functions remains poorly understood. Here, we show that the most prevalent vitamin A metabolite, all-trans retinoic acid (atRA), induces transcriptional and functional changes in NK cells leading to altered metabolism and reduced IFN-γ production in response to a wide range of stimuli. atRA-exposed NK cells display a reduced ability to support dendritic cell (DC) maturation and to eliminate immature DCs. Moreover, they support the polarization and proliferation of regulatory T cells. These results imply that in vitamin A-enriched environments, NK cells can acquire functions that might promote tolerogenic immunity and/or immunosuppression.

Preexisting helminth challenge exacerbates infection and reactivation of gammaherpesvirus in tissue resident macrophages.

Even though gammaherpesvirus and parasitic infections are endemic in parts of the world, there is a lack of understanding about the outcome of coinfection. In humans, coinfections usually occur sequentially, with fluctuating order and timing in different hosts. However, experimental studies in mice generally do not address the variables of order and timing of coinfections. We sought to examine the variable of coinfection order in a system of gammaherpesvirus-helminth coinfection. Our previous work demonstrated that infection with the intestinal parasite, Heligmosomoides polygyrus, induced transient reactivation from latency of murine gammaherpesvirus-68 (MHV68). In this report, we reverse the order of coinfection, infecting with H. polygyrus first, followed by MHV68, and examined the effects of preexisting parasite infection on MHV68 acute and latent infection. We found that preexisting parasite infection increased the propensity of MHV68 to reactivate from latency. However, when we examined the mechanism for reactivation, we found that preexisting parasite infection increased the ability of MHV68 to reactivate in a vitamin A dependent manner, a distinct mechanism to what we found previously with parasite-induced reactivation after latency establishment. We determined that H. polygyrus infection increased both acute and latent MHV68 infection in a population of tissue resident macrophages, called large peritoneal macrophages. We demonstrate that this population of macrophages and vitamin A are required for increased acute and latent infection during parasite coinfection.

Fungi Take Control of Lymphocyte Recirculation.

Factors regulating leukocyte migration to neonatal lymph nodes are not sufficiently identified. In this issue of Immunity, Zhang et al. (2016) reveal that fungi drive emigration of gut DCs to lymph nodes, where these DCs instruct endothelial cell receptivity to leukocytes.

Peripheral Lymphoid Volume Expansion and Maintenance Are Controlled by Gut Microbiota via RALDH+ Dendritic Cells.

Lymphocyte homing to draining lymph nodes is critical for the initiation of immune responses. Secondary lymphoid organs of germ-free mice are underdeveloped. How gut commensal microbes remotely regulate cellularity and volume of secondary lymphoid organs remains unknown. We report here that, driven by commensal fungi, a wave of CD45(+)CD103(+)RALDH(+) cells migrates to the peripheral lymph nodes after birth. The arrival of these cells introduces high amounts of retinoic acid, mediates the neonatal to adult addressin switch on endothelial cells, and directs the homing of lymphocytes to both gut-associated lymphoid tissues and peripheral lymph nodes. In adult mice, a small number of these RALDH(+) cells might serve to maintain the volume of secondary lymphoid organs. Homing deficiency of these cells was associated with lymph node attrition in vitamin-A-deficient mice, suggesting a perpetual dependence on retinoic acid signaling for structural and functional maintenance of peripheral immune organs.

Harnessing global fisheries to tackle micronutrient deficiencies.

Micronutrient deficiencies account for an estimated one million premature deaths annually, and for some nations can reduce gross domestic product1,2 by up to 11%, highlighting the need for food policies that focus on improving nutrition rather than simply increasing the volume of food produced3. People gain nutrients from a varied diet, although fish-which are a rich source of bioavailable micronutrients that are essential to human health4-are often overlooked. A lack of understanding of the nutrient composition of most fish5 and how nutrient yields vary among fisheries has hindered the policy shifts that are needed to effectively harness the potential of fisheries for food and nutrition security6. Here, using the concentration of 7 nutrients in more than 350 species of marine fish, we estimate how environmental and ecological traits predict nutrient content of marine finfish species. We use this predictive model to quantify the global spatial patterns of the concentrations of nutrients in marine fisheries and compare nutrient yields to the prevalence of micronutrient deficiencies in human populations. We find that species from tropical thermal regimes contain higher concentrations of calcium, iron and zinc; smaller species contain higher concentrations of calcium, iron and omega-3 fatty acids; and species from cold thermal regimes or those with a pelagic feeding pathway contain higher concentrations of omega-3 fatty acids. There is no relationship between nutrient concentrations and total fishery yield, highlighting that the nutrient quality of a fishery is determined by the species composition. For a number of countries in which nutrient intakes are inadequate, nutrients available in marine finfish catches exceed the dietary requirements for populations that live within 100 km of the coast, and a fraction of current landings could be particularly impactful for children under 5 years of age. Our analyses suggest that fish-based food strategies have the potential to substantially contribute to global food and nutrition security.

Vitomics: A novel paradigm for examining the role of vitamins in human biology.

The conventional view of vitamins reflects a diverse group of small molecules that facilitate critical aspects of metabolism and prevent potentially fatal deficiency syndromes. However, vitamins also contribute to the shaping and maintenance of the human phenome over lifecycle and evolutionary timescales, enabling a degree of phenotypic plasticity that operates to allow adaptive responses that are appropriate to key periods of sensitivity (i.e., epigenetic response during prenatal development within the lifecycle or as an evolved response to environmental challenge over a great many lifecycles). Individually, vitamins are important, but their effect is often based on nutrient-nutrient (vitamin-vitamin), nutrient-gene (vitamin-gene), and gene-gene interactions, and the environmental influence of shifting geophysical cycles, as well as evolving cultural practices. These ideas will be explored within what I refer to as the "adaptive vitome (vitomics)" paradigm.

Enhanced stability and clinical absorption of a form of encapsulated vitamin A for food fortification.

Food fortification is an effective strategy to address vitamin A (VitA) deficiency, which is the leading cause of childhood blindness and drastically increases mortality from severe infections. However, VitA food fortification remains challenging due to significant degradation during storage and cooking. We utilized an FDA-approved, thermostable, and pH-responsive basic methacrylate copolymer (BMC) to encapsulate and stabilize VitA in microparticles (MPs). Encapsulation of VitA in VitA-BMC MPs greatly improved stability during simulated cooking conditions and long-term storage. VitA absorption was nine times greater from cooked MPs than from cooked free VitA in rats. In a randomized controlled cross-over study in healthy premenopausal women, VitA was readily released from MPs after consumption and had a similar absorption profile to free VitA. This VitA encapsulation technology will enable global food fortification strategies toward eliminating VitA deficiency.