Carotenoid supplementation and retinoic acid in immunoglobulin A regulation of the gut microbiota dysbiosis

Short abstract

Dysbiosis, a broad spectrum of imbalance of the gut microbiota, may progress to microbiota dysfunction. Dysbiosis is linked to some human diseases, such as inflammation-related disorders and metabolic syndromes. However, the underlying mechanisms of the pathogenesis of dysbiosis remain elusive. Recent findings suggest that the microbiome and gut immune responses, like immunoglobulin A production, play critical roles in the gut homeostasis and function, and the progression of dysbiosis. In the past two decades, much progress has been made in better understanding of production of immunoglobulin A and its association with commensal microbiota. The present minireview summarizes the recent findings in the gut microbiota dysbiosis and dysfunction of immunoglobulin A induced by the imbalance of pathogenic bacteria and commensal microbiota. We also propose the potentials of dietary carotenoids, such as β-carotene and astaxanthin, in the improvement of the gut immune system maturation and immunoglobulin A production, and the consequent promotion of the gut health.

Impact statement

The concept of carotenoid metabolism in the gut health has not been well established in the literature. Here, we review and discuss the roles of retinoic acid and carotenoids, including pro-vitamin A carotenoids and xanthophylls in the maturation of the gut immune system and IgA production. This is the first review article about the carotenoid supplements and the metabolites in the regulation of the gut microbiome. We hope this review would provide a new direction for the management of the gut microbiota dysbiosis by application of bioactive carotenoids and the metabolites.

Introduction

Gut microbiota is the microbes coevolve with the host in the environment. Every individual has a unique composition of the microbiota. The phyla Bacteroidetes and Firmicutes are most abundant bacterial species in the adult human gut, accounting for more than 90% of the bacterial populations.¹ Recently, gut microbiota dysbiosis has been shown to be associated with weaknesses in gut barrier function, dysregulated innate, and adaptive immune responses and also with the increased incidence of the inflammatory bowel disease (IBD) and other metabolic disorders.^2,3 Studies show that antibiotics play vital roles in the progression of dysbiosis especially in the overgrowth of the resistant opportunistic bacterial pathogen (OBP).⁴ Another major factor is a diet which can be directly utilized by microbiota, and in turn causes changes in the composition of microbiota. Dysbiosis is accompanied by increased OBP and decreased commensal microbiota like Lactobacillales and Bifidobacterium.⁵ However, the initiation and progression of dysbiosis still need further studies.

The relationship between gut microbiota and the host is based on the gut immune response and tolerance.⁶ Immunoglobulin A (IgA) is one of the critical regulators in protecting the mucosal balance via recognizing and coating particular microbiota to prevent the microbiota from crossing the gut epithelial cell barrier.⁷ Commensal microbiotas could be beneficial to the host health through regulating the production of reactive oxygen species (ROS) and short chain fatty acid (SCFA).⁸ However, in the dysbiosis condition, inhibition of commensal microbiotas by OBP, like Gammaproteobacteria and the relationship between the OBP and production of IgA in dysbiosis are yet exclusive.

Recently, researchers found some bioactive molecules that would be beneficial to improve microbiota composition, and in turn reduce the risk of diseases caused by microbiota dysbiosis. Carotenoid supplementation shows promising improvements in the immune system maturation and IgA production.⁹

Here, we discuss the roles of OBP and commensal microbiota in the pathogenesis of dysbiosis via regulation of the IgA function and immune maturation, and discuss the potential of carotenoid supplementation in dysbiosis prevention and treatment.

Gut microbiota dysbiosis causes diseases

Dysbiosis can be understood with the loss of beneficial microbiota, expansion of pathogenic or potentially harmful microbiota, and a decrease of overall microbiota diversity.¹⁰ There are multiple ways to influence the composition of the microbiota, including the genetics of the host, diet, infection and medical interventions, like antibiotics.¹¹ Dysbiosis is associated with several pathological processes, including obesity, non-alcoholic fatty liver disease (NAFLD), and IBD (Table 1).¹² The overgrowth of OBP can cause pathoprogression of some diseases. For example, increased abundance of Escherichia (alcohol-producing bacteria) was correlated with the elevated blood alcohol concentration in patients with non-alcoholic steatohepatitis (NASH), suggestive of involvement of imbalanced gut microbiome in the pathogenesis of NASH.¹³ The bacterial microbiota in IBD has also been investigated. An increase in bacteria in the Proteobacteria phylum, such as Escherichia coli was observed in the IBD patients.¹⁴ Also, fungal microbiota dysbiosis was also found in patients with IBD.¹⁵ Another study demonstrated that the gut microbiota dysbiosis determined the development of NAFLD.¹⁶ Dysbiosis causes the decrease in commensal microbiota, like Faecalibacterium prausnitzii, a dominant butyrate-producing bacteria in the intestine. Butyrate is a SCFA which could be used as an energy source for intestinal epithelial cells and microbiomes. It helps protect the intestinal epithelial barrier integrity and make them resistant to potential pathogens.¹⁷ Similar to IBD, a specific individual microbiota signature seems to be related to the development of obesity. The overall intestinal bacterial diversity was decreased in obese individuals.¹⁸ Lean germ-free (GF) mice would be obese after receiving the transplant of microbiota from obese mice.¹⁹ It seems apparent that changes in microbiota are closely associated with some human diseases. However, whether microbiota dysbiosis is a causal factor or an effect of the disease is yet to be proved. A list of examples of microbiota dysbiosis-associated diseases is provided in Table 1.

Table 1.

Examples of microbiota dysbiosis and the bacteria associated with diseases.

Disease	Dysbiosis	Protective or pathogenic microbiotas, effect on host	References
Obesity	1. Ratio of Firmicutes: Bacteroidetes ↑ 2. Proteobacteria ↑ 3. Compositional microbial diversity ↓	Bifidobacteria treatment reduced the intestinal lipopolysaccharide levels, and suppressed expression of inflammatory and oxidative stress markers in the liver of ob/ob mice fed a high fat diet.	20–24
Diabetes	Ratio of Firmicutes: Bacteroidetes ↓	BB-diabetes-resistant rats had higher levels of Bifidobacterium and Lactobacillus compared to BioBreeding diabetes prone rats (a type 1 diabetic model)	25–27
Inflammatory bowel disease	Proteobacteria ↑ Bifidobacterium ↓	Increased Proteobacteria, and in particular the family of Enterobacteriaceae, containing members such as Escherichia coli, Shigella and Klebsiella.	10,28,29
Nonalcoholic fatty liver disease	Proteobacteria ↑ Bacteroidetes ↓	Proteobacteria/Enterobacteriaceae/Escherichia were similarly represented between microbiomes in healthy and obese mice, but were significantly elevated in nonalcoholic steatohepatitis. Oral administration of Lactobacillus rhamnosus PL60 improved liver steatosis in diet-induced obesity mice.	13,30–32

Gut microbiota dysbiosis leads to IgA dysfunction

There are at least two points of view of the mechanisms in the maintenance of the balance between the host and microbiota via manipulation of the gut immune system. The first is the immune tolerance. The commensal (beneficial) microbiota can induce the mucosal immune cells to recognize them and decrease the inflammatory response. Forkhead box protein P3 (Foxp3) is a protein in regulation of the host immune system response. Induction of Foxp3 triggers regulatory T (Treg) cells, which limit excessive pro-inflammatory responses.³³ A study showed that Bifidobacteria infantis 35624 induced Foxp3⁺ Treg cell activation and increased secretion of toll-like receptor 2 (TLR-2) dependent IL-10, production of dendritic cell-specific intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), and activation of retinoic acid signaling pathways.³³

The other possible mechanism is the immune ignorance. The lousy microbiota can induce the intestine immune system to recognize them specifically and induce inflammatory responses, while the mucosal immune antibodies ignore other microbiota. IgA production is the primary immune response in the gut.³⁴ IgA can recognize and coat particular microbiota, and consequently prevent the microbiota translocation across the intestinal epithelial barrier, and the destruction of the tight junction in the intestine. Besides the defense and cooperation of microbiota, gut mucosa also can recover from the colitis.³⁵ Research showed that intestinal interferon regulatory transcription factor 3 (IRF3) could be activated by microbiota-derived nucleic acids and subsequently stimulated the expression of thymic stromal lymphopoietin (TSLP), a cytokine mediated in protection of the large intestine.³⁶ These gut immune responses to a variety of microbiota depend on the maturation of host immune organs and the recognition ability of IgA. However, the maturation process of the host immune system induced by microbiota remains uncertain. Moreover, how the individual microbiota induces IgA to recognize them specifically is also not clear.

OBP against the commensal microbiota to induce the production of IgA

IgA is the predominant antibody and is thought to provide the first line of the immune protection at mucosal surfaces in the intestine, through coating the microbiota and neutralizing toxins.³⁷ In addition to the intestine tissue, antigen-specific IgA-secreting cells also distribute in the liver.³⁸ Additionally, IgA is associated with down-regulation of pro-inflammatory epitopes on commensal bacteria.³⁹

Recently, researchers are focusing on the function of the specific IgA-regulated microbiota. Mice with deficiency of or blocked production of IgA contain a higher amount and abundance of Proteobacteria especially Gammaproteobacteria compared with regular wild-type mice.⁴⁰ Interestingly, Proteobacteria is thought to exhibit the immaturity status of microbiota in adult mice. Proteobacteria is mostly associated with the host inflammation with the elevated expression of interferon gamma (IFN-γ), macrophage inflammatory protein 2 (MIP-2), and tumor necrosis factor alpha (TNF-α).⁴¹ The Gammaproteobacteria abundance was increased in the common variable immunodeficiency (CVID) with inhibition of IgA expression.⁴² Dysfunction of IgA can lead to the imbalance of the gut microbiota composition, such as increases in the Gammaproteobacteria and decreases in the Bifidobacterium and Lactobacilli in the gut microbiota, which led to the increased production of the pro-inflammatory cytokines.

Dysbiosis is accompanied by a decreased production of IgA or suppression of the ability of IgA to coat the microbiota. A lower fecal IgA amount more likely reflects initiation of dysbiosis in the gut in the alcoholic steatohepatitis mice, compared with healthy mice,⁴³ though the mechanism by which IgA is inactivated by microbiota needs to be further investigated. Recent research demonstrated that the antibiotic treatment-induced gut dysbiosis led to modification of the immune recognition to the intestinal bacteria.⁴⁴ The antibiotic treatments promote the growth of specific pathogens (for example Neisseria gonorrhoeae and/or Corynebacterium diphtheriae) by a mechanism to which the pathogens can avoid IgA opsonization,⁴⁵ but suppress the colonization of beneficial Lactobacillales and Clostridium cluster IV cells.⁴⁴

The beneficial microbiota is proposed to induce Myd88.⁴⁶ Lack of Myd88 resulted in no significant variation in the composition of the adult microbiota, indicative that the microbiota effects on Myd88 are mono-directional without dysbiosis happening.⁴⁷ Moreover, this commensal microbiota critically affects the IgA production. Deficiency of IgA led to less abundance of Lactobacillales in the gut of mice. When fed the immunocompetent mothers’ milk, the IgA deficient mice accepted milk IgA from the foster mothers, and in turn showed an increased population of Lactobacillales and then decreased Proteobacteria in the gut.⁴⁰

The dysbiosis especially caused by antibiotic treatments is established by the growth of antibiotic-resistant microbiota which is more compatible than commensal microbiota. Bacteriocin generated by microbiota can efficiently influence a strain’s ability to compete with other microbiota in the intestine lumen.⁴⁸

Collectively, dysbiosis causes the competition between OBP and commensal microbiota. OBP can inhibit the activity of commensal microbiota by producing bacteriocin and avoid the recognition of IgA through inhibiting the commensal microbiota-induced, Myd88-regulated IgA production.

Microbiota regulates IgA dysfunction via inhibition of the immune organ maturation

Besides competition between the commensal microbiota and pathogens, dysbiosis is also associated with the immune immaturity. The gut microbiota homeostasis is essential for maturation of the immune system. Studies confirmed that lymphoid follicles are immature and IgA production is insufficient in GF mice. However, colonization with commensal bacteria could reverse the above deficiency by induction of the intestinal IgA response and maturation of lymphoid follicles in those mice.^49,50

The gut microbiota controls the peripheral lymphoid volume expansion and maintenance. In the GF mice, the secondary lymphoid organs are undeveloped. Under normal condition, CD45⁺/CD103⁺/RALDH⁺ (retinoic acid dehydrogenases⁺) immune cells move to the peripheral lymph nodes in healthy mouse pups after birth, driven by commensal microbiome.⁵¹ On the other hand, an unstable structure of gut microbiota composition with high abundance of Proteobacteria is also observed in non-disease states in the neonatal period, while the neonatal immune system is still under development to maturation and lacks IgA in the gut mucosa.⁵² Given that OPB inhibits the commensal microbiota activity, it is likely that OPB suppresses the maturation of the host immune system, especially in the production of IgA.

Micronutrients in dysbiosis management

The management of dysbiosis should base on global approaches, not only to consider the diet quality and quantity control, but also to include strategies focused on increasing of intakes of foods rich in bioactive components, such as prebiotics, probiotics, and others which could modulate either composition or metabolic/immunological activity of the gut microbiota.⁵³

Bioactive compounds to increase the commensal microbiota

Prebiotics exert the beneficial role in the gut via increasing the population of good microbiota like Lactobacillus and Bifidobacterium. The prebiotics such as fructooligosaccharides, fibers, and polyphenols can be metabolized by particular microbiota and in turn confer health benefits to the host.⁵⁴ For instance, polysaccharides can be used to produce SCFA which is beneficial to the gut integrity, host metabolism, and immunological responses. The study showed that SCFA produced by commensal microbiota promotes the peripheral regulatory T cell production and activation, and induces IL-10 production, which could mitigate inflammation in infection tissues.⁵⁵ Another study revealed that consumption of the blackcurrant extract powder containing polyphenols increased the amount of Bifidobacterium and Lactobacillus but decreased the enzymatic activity of β-glucuronidase, a bacterial enzyme considered as a risk factor for colorectal cancer.⁵⁶ The IgA production was also improved via prebiotics supplementations. Administration of fructooligosaccharides increased the concentration of IgA and expression of polymeric immunoglobulin receptor (pIgR) in the intestine in mice.⁵⁷ It would be interesting to explore the underlying mechanism by which that commensal microbiota metabolizes the fructooligosaccharides and subsequently increases the production of IgA.

Supplementation of carotenoids increases the IgA production and maturation of gut-associated immune tissues

Carotenoid metabolism

Carotenoids are pigments abundant in many fruits, vegetables, and some animal products. Human beings are unable to synthesize carotenoids from endogenous precursors and must acquire them from the diet. The absorption of dietary carotenoids in the intestine is controlled, at least partially by the scavenger receptor class B type 1 (SRB1), which is regulated by beta-carotene oxygenase 1 (BCO1) activity via homeobox transcription factor intestine-specific homeobox (ISX).⁵⁸ However, the regulation of SRB1 in the large intestine has not been investigated.

Carotenoids are characterized into two subgroups, e.g. pro-vitamin A carotenoids and non-pro-vitamin A carotenoids, or xanthophylls. Pro-vitamin A carotenoids can be cleaved by BCO1 (at the 15, 15′ double bound) resulting in a production of vitamin A, which can be further converted into retinoic acid in host cells, such as dendritic cells and B cells. All carotenoids can be asymmetrically cleaved by beta-carotene oxygenase 2 (BCO2) to generate apocarotenals.^59,60 As fat-soluble bioactive compounds, the blood carotenoid bioavailability is low 10–40%. Likely, carotenoids may reach the colon and be fermented in the gut by microbiota.⁶¹ However, the utilization of carotenoids by microbiota has not been evidenced, and their biological functions in the gut microbiome are yet well studied.⁶²

Carotenoid function

Low levels of carotenoids are considered antioxidants. However, the clinical trials have demonstrated that over-dosed carotenoids are toxic.⁶³ Functionally, carotenoids play essential roles in all aspects of cell functions, such as in regulation of the host immune responses by activation of macrophages, natural killer cells, T cells, and/or Treg cells (for some recent review articles please visit literature^64–70). Carotenoids are beneficial for protecting humans against age-related eye diseases,⁷¹ metabolic syndromes,⁷² stroke,⁷³ heart and cardiovascular diseases,^74,75 diabetes,⁷⁶ inflammation,⁷⁷ and even body composition changes.⁷⁸ Unlike retinoic acid (RA), carotenoid metabolism in the gut and their impacts on the gut microbiome remain unknown.

Retinoic acid

RA is a critical regulator for the intestinal immune response. Lack of vitamin A in the mouse diet suppresses pro-inflammatory Th17 cell generation in the gut via reduction of commensal microbes.⁷⁹ The primary function of RA in B cells is to induce the IgA production and class switching.⁸⁰ Inhibition of the RA pathway in B cells significantly decreases the IgA secretion but increases the Lachnospiraceae (Erec482+) and Lactobacillus/Streptococcus in the fecal samples, compared with the control group, which are thought to be associated with colorectal adenomas. Supplementation of β-carotene increased IgA production and transfer to neonatal mice through maternal milk.⁸¹ Moreover, Bifidobacteria infantis 35624 can attenuate the colitis by inducing the CD103+ retinoid acid secreting dendritic cells.⁸² However, the direct effect of RA on the microbiota is poorly understood.

Astaxanthin supplementation

Recently, some nutrition studies have been focused on the health benefits of xanthophylls, such as zeaxanthin, lutein, and astaxanthin. Astaxanthin is an oxycarotenoid found in high amounts largely in some microalgae and marine animals, such as salmon and shrimp.⁸³ The biological functions of astaxanthin have been reported in protection against inflammation and the associated diseases, including but not limited to ulcers, cancer, neurodegeneration, diabetes, and cardiovascular disease.⁸⁴ However, how astaxanthin alters the gut immune system remains exclusive. The treatment with astaxanthin could significantly reduce bacterial loads and attenuate gastric inflammation in mice infected with H. pylori.⁸⁵ Mice administrated with astaxanthin showed significantly lowered colonization levels and inflammation scores in the stressed rats.⁸⁶ Supplementation of astaxanthin increased the production of IgA antibody-secreting cells (ASC) in the small intestine of mice at weanling age.⁸⁷

Astaxanthin also induces the host immune responses, such as activation of T cells, B cells, and NK cells, and production of IFN-γ and IL-6.⁸⁸ Astaxanthin activates T cells and NK cells to produce IFN-γ, and in turn mediates B cell differentiation and maturation, resulting in IgA production. Recently, a published human study indicated that the supplementation of astaxanthin increased sIgA levels which may protect against the exercise-induced mucosal immunity dysfunction.⁸⁹ Further studies suggested that astaxanthin enhanced the antibody production through the release of IL-1α, which is one of the significant regulating factors in B cell differentiation.⁹⁰ Thus, astaxanthin confers its beneficial effects by regulation of the maturation of B cell and production of IgA. Astaxanthin could have a potential in the prevention or treatment of dysbiosis and its associated diseases.

Astaxanthin alters the gut microbiota

Most recently, we conducted a pilot study to determine the potential role of astaxanthin in the gut microbiome. Six-week-old BCO2 knockout (KO) and the genetic background C57BL/6J mice (WT) (with mixed gender) were fed either AIN-93M (control diet) or AIN-93M supplemented with 0.04% astaxanthin (w/w, astaxanthin diet) for eight weeks (Oklahoma State University Institutional Animal Care and Use Committee Protocol # HS-14–4, approved by the chair of the IACUC). The results showed that astaxanthin administration considerably altered the cecal microbiota at the phylum, which mostly differed by gender and BCO2 gene expression (Figure 1). In particular, Proteobacteria and Bacteroides, the OBP, were significantly more abundant in both male and female BCO2 KO mice, which could be diminished by application of astaxanthin in female WT and BCO2 KO only. On the contrast, astaxanthin drastically increased the abundance of Actinobacteria and Bifidobacterium, the commensal microbiota in male WT mice only. There are more than a dozen of commensal microbiota genera with increased abundance at the phylum of Firmicutes in mice after astaxanthin treatment (Figure 1, detailed data not shown). So far, the results suggested that astaxanthin administration and the metabolic enzyme, e.g., BCO2 remarkably impact the gut microbiome in mice, although the vast majority of the microbiota identified in the current study in Figure 1 had not been well characterized in literature.

Figure 1.

The phylum-level changes of the cecal microbiota in BCO2 knockout (KO) and the wild-type (WT) mice after administration of 0.04% astaxanthin (w/w). Six-week-old mice were fed either the control (AIN-93M) or astaxanthin (ASTX, AIM-93M supplemented with 0.04% astaxanthin (w/w)) diet for eight weeks. The cecal DNA samples were subjected to 16s rRNA sequencing (by Novogene, Inc.). The heatmap of the abundance changes is shown at the phylum level only. (A color version of this figure is available in the online journal.)

Conclusion

Dysbiosis is one of the most crucial factors in gut dysfunction that can induce many human diseases including obesity, metabolic syndrome, IBD, and NAFLD. The onset and development of the gut dysbiosis are complex and may involve various mechanisms. A better understanding of the pathophysiological mechanisms would be beneficial for the development of new preventative and therapeutic strategies. Impaired IgA production and function exert a vital role in initiation of the imbalance between OBP and commensal microbiota. Carotenoids, such as β-carotene (in the metabolite form of RA) and astaxanthin may contribute to the gut immune homeostasis by directly regulating IgA production, thereby preventing of and/or delaying the development of dysbiosis (Figure 2). Future investigations are warranted to elucidate the precise mechanisms by which carotenoids are preventive/protective in the gut dysbiosis.

Figure 2.

Regulation of microbiota homeostasis by immunoglobulin A (IgA) and carotenoids. Commensal microbiota can induce Treg cells via Myd88 signaling to up-regulate the T Follicular Helper cells, which in turn stimulates the plasma cells to produce IgA. The increased opportunistic bacterial pathogens (OBP) inhibit the commensal microbiota, leading to suppression of IgA production in the plasma cells. OBP also triggers the enhanced expression of macrophage inflammatory protein 2 (MIP-2), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ), inflammation-associated factors in the gut tissue. Dietary carotenoids (including pro-vitamin A carotenoids and non-pro-vitamin A carotenoids, e.g. xanthophylls) and their supplementation may increase the production of retinoic acid (RA). RA sequentially induces the maturation of gut immune system, e.g. B cell activation, and IgA production. Xanthophylls activate T cells and natural killer cells resulting in the production of IFN-γ. IFN-γ further stimulates the differentiation and maturation of B cells to produce IgA, and in turn promote the gut health. (A color version of this figure is available in the online journal.)

Authors’ contributions

All authors participated in the experimental design, conducting research, data interpretation, literature review, and manuscript preparation. Authors had read and approved the final version of the manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This project was made possible in part by support from the USDA National Institute of Food and Agriculture 2015–67018-23176, and USDA NIFA Hatch Project OKL02992 (to DL), and the Priority academic program development of Jiangsu Higher Education Institutions (PAPD) (to XS).