The potential role of Akkermansia muciniphila in liver health

ABSTRACT

Akkermansia muciniphila (A. muciniphila) is a ‘star strain’ that has attracted much attention in recent years. A. muciniphila can effectively regulate host metabolism, significantly affect host immune function, and play an important role in balancing host health and disease. As one of the organs most closely related to the gut (the two can communicate through the hepatic portal vein and bile duct system), liver is widely affected by intestinal microorganisms. A growing body of evidence suggests that A. muciniphila may alleviate liver-related diseases by improving the intestinal barrier, energy metabolism and regulating inflammation through its protein components and metabolites. This paper systematically reviews the key roles of A. muciniphila and its derivatives in maintaining liver health and improving liver disease.

Plain language summary

Executive summary.

• Gut microbes are closely linked to the liver, and A. muciniphila, as an important gut microbe, has been shown to have the potential to improve liver health in many ways.

Correlation between A. muciniphila & different liver diseases

NAFLD

• NAFLD, along with its common risk factors such as obesity and type 2 diabetes, is associated with decreased A. muciniphila abundance.

• The increased abundance of A. muciniphila is closely related to the effects of drugs currently used to treat NAFLD, such as metformin, Bofutsushosan, or bioactive molecules that have been shown to improve NAFLD.

Alcoholic liver disease

• Alcohol intake causes changes in the composition of the gut microbiota, including A. muciniphila, which breaks down the intestinal barrier.

• Supplementation of live A. muciniphila can reverse the ethanol-induced increase in intestinal permeability and reduce the release of LPS and peptidoglycans from intestinal bacteria into the circulatory system and to the liver tissue, thereby alleviating liver damage caused by inflammation and oxidative stress.

Liver cirrhosis

• Patients at the stage of cirrhosis also showed reduced Akkermansia abundance.

• Live and pasteurized A. muciniphila and its EVs prevented HSCs activation and reduced the relative expression of fibrosis and inflammatory biomarkers.

Liver cancer

• Compared with healthy controls, the abundance of A. muciniphila in liver cancer patients or mice continued to decline.

• A. muciniphila is an important factor influencing anti-tumor immune surveillance, and it is also beneficial for the immunotherapy of liver cancer.

Mechanisms of A. muciniphila in affecting liver health

Improving intestinal barrier

• A. muciniphila can repair the intestinal barrier in many ways, including stimulating the goblet cells to produce new mucins, accelerating the proliferation of intestinal epithelium, increasing the expression of tight-junction related proteins, and remodeling the intestinal microbial community.

Improving energy metabolism

• A. muciniphila ileal colonization results in changes in the expression of genes associated with metabolism and signaling pathways, most of which are involved in lipid metabolism, small molecule biochemistry, and metabolic homeostasis.

• A. muciniphila treatment decreased nutrient absorption and increased energy excretion, increased energy consumption and increased activity of mice.

Regulating immune response

• A. muciniphila treatment can reduce the levels of inflammatory cytokines, increase the levels of anti-inflammatory cytokines, relieve the body's inflammatory signs.

• A. muciniphila can up-regulate the levels of inflammatory cytokines including IFN-γ, IL-6 and TNF-α, and down-regulate the levels of IL-10 to increase the effect of anti-cancer immunotherapy.

• A. muciniphila colonization caused changes in the expression of genes involved in membrane metabolism, signaling and antigen delivery pathways, and upregulation of genes associated with leukocyte antigen presentation.

Conclusion

• A. muciniphila has a close relationship with liver health, and it is a promising research topic to explore the probiotic effects of A. muciniphila in liver disease and its potential in disease treatment.

Future perspective

• The exact causal relationship between A. muciniphila and liver disease and the specific mechanism of action are still unclear and need to be further explored.

• The different bioactive functions of A. muciniphila were related to its strain specificity and intestinal microecological environment.

The gut microbiome is considered a ‘hidden organ’ that plays an important role not only in nutrient absorption and energy metabolism, but also in maintaining normal immune defense functions and regulating inflammatory responses to pathogens and other injuries [1,2]. Anatomically, the liver is connected to the gut by the portal vein, so it is more closely connected to the gut than any other organ. During the body's growth and development, the liver is constantly immersed to products from digestion and absorption in the gut and continuously exposed to enterogenous factors, including bacteria and their products such as lipopolysaccharide (LPS) [3]. When the intestinal barrier is compromised and permeability increases, a large number of toxic factors in the gut converge from the mesenteric vein into the portal vein and flow into the liver, leading to chronic inflammation and related liver disease [4,5]. Therefore, the stable and functional intestinal mucosa that separates the internal environment from food antigens, microorganisms and their metabolites is an important biological barrier for maintaining liver health. The liver, meanwhile, influences gut microbes by secreting bile acids and IgA antibodies (Figure 1). As early as 1967, the critical role of the gut microbiota in liver disease was demonstrated in a germ-free mouse model [6]. Dysregulation of gut microbiota can promote the occurrence of liver diseases to a certain extent, such as non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease (ALD), cirrhosis and liver cancer, etc [7–9].

Figure 1.

The schematic diagram of gut-liver axis.

① The portal vein of the liver collects blood from the small and large intestines, and is also the signature anatomical structure of the gastro-liver communication. In healthy conditions, the portal blood may contain small amounts of potentially pathogenic or toxic compounds, such as dietary components or co-organisms (and their metabolites), and a healthy liver and its immune system are sufficient to deal with such stressors. ② On the contrary, bile acid secreted by the liver enters the intestine through the bile duct and is metabolized by intestinal microorganisms, and the antimicrobial peptides contained in bile also participate in controlling the symbiotic balance of intestinal microorganisms.

Akkermansia muciniphila (A. muciniphila), a gram-negative anaerobic bacterium first identified by Derrien et al., in 2004, is a very popular intestinal microbe in recent years. As the only representative of the verrucomicrobia in the human gut, A. muciniphila accounts for 1–5% of the human gut microbial community [10]. A. muciniphila colonization reaches adult levels within the first year of life. The colonization of A. muciniphila in the intestine was not uniform. Even in the colon, A. muciniphila was less distributed in the ascending colon, but abundant in the transverse and descending colon [11]. A. muciniphila is widely colonized in intestinal mucous layer and is considered as a key species of mucous layer. More and more studies have found that reduced abundance of A. muciniphila is significantly correlated with the occurrence and development of various diseases, including the liver, one of the organs most closely related to the gut [12–14]. A. muciniphila has also been shown to improve intestinal barrier, energy metabolism, and inflammatory responses through its components and metabolites, thereby affecting the progression of liver disease, and is considered a promising candidate for treating microbial-related diseases [4,15]. In the following chapters, we will summarize the existing reports on the effect or improvement of A. muciniphila on liver health, so as to provide references for further understanding of the pathogenesis of chronic liver diseases and development of safe and efficient treatment strategies.

Correlation between A. muciniphila & different liver diseases

NAFLD

With the global prevalence of obesity and high metabolic syndrome, the incidence of NAFLD is increasing year by year. The prevalence of NAFLD is about 20–40% globally, and as high as 75–100% in obese people [16–18]. NAFLD refers to a clinicopathological syndrome characterized by excessive deposition of fat in hepatocytes caused by factors other than alcohol and other definite liver lesions, including simple steatosis, non-alcoholic steatohepatitis, fibrosis and cirrhosis, and even liver cancer. Approximately 22–29% of adults worldwide suffer from NAFLD, which has become one of the most common chronic liver diseases and is seriously endangering people's lives and health [19]. A total of 109 NAFLD patients were selected as the experimental group (77 males, 32 females), and 352 healthy volunteers were selected as the control group (243 males, 109 females) to explore the effect of A. muciniphila on liver function in NAFLD patients and its correlation. The results showed that the mean number of A. muciniphila in the control group was significantly higher than that in the NAFLD group, and the difference between the two groups was statistically significant (p < 0.001) [20]. Although the etiology of NAFLD remains unclear, according to current studies, in addition to genetic factors, it is also significantly correlated with obesity and type 2 diabetes (T2D), which are also the most common risk factors for NAFLD. Moreover, T2D is a metabolic abnormality frequently found in NAFLD patients and is considered to be one of the major determinants of the pathogenesis of NAFLD and the progression of liver disease. A study of 6896 Chinese participants aged 18 to 97 from the Guangdong Gut Microbiome Project found that obesity and high blood sugar were strongly associated with A. muciniphila [21]. Another study in humans also found a negative association between A. muciniphila abundance and obesity, and untreated T2D [22]. To explore the relationship between glucose metabolism disorder and A. muciniphila, Zhang et al. recruited 121 subjects and divided them into three groups according to their glucose intolerance status: normal glucose tolerance (n = 44), pre-diabetes (n = 64), or newly diagnosed T2D (n = 13). The abundance of A. muciniphila was found to be higher in the normal glucose tolerance group than that in the pre-diabetes group, and the relative abundance of Verrucomicrobia was also found to be significantly lower in the pre-diabetic group at the genus level, suggesting that intestinal microbiome disorders may occur before the progression of diabetes [23]. Meanwhile, a study of obese or thin people with and without T2D found that the abundance of A. muciniphila in T2D thin individuals was significantly lower than that in thin individuals without T2D, while this phenomenon was not found in the comparative of obese individuals with and without T2D. Given the close relationship between obesity and T2D, and the key role of the gut microbiome in the pathogenesis of both diseases, the authors speculate that there may be a transitional stage from obesity without T2D to obesity with T2D [24]. A. muciniphila was also positively correlated with indicators related to insulin secretion, which further supported the potential relationship between A. muciniphila in the gut and the disorder of glucose metabolism.

In preclinical and clinical studies, supplementation with live or pasteurized A. muciniphila has been shown to improve systemic metabolic status, including decreased high-fat diet (HFD)-induced weight gain in mice, decreased liver, epididymal, and visceral fat weight, decreased lipid accumulation, increased glucose clearance and decreased serum insulin levels in HFD-fed mice [25–28]. Although we do not yet know the causal relationship between NAFLD and A. muciniphila, increasing evidence suggests that A. muciniphila plays a indispensable role in metabolic balance in the body by regulating energy balance, glucose metabolism, and chronic inflammatory states associated with obesity. In fact, the increased abundance of A. muciniphila is closely associated with the effects of drugs currently used to treat NAFLD, such as metformin, Bofutsushosan or bioactive molecules that have been shown to improve NAFLD, such as cranberry extract, rhubarb supplements, betaine, and quercetin [29–34]. A. muciniphila has been identified as a promising candidate for the prevention or treatment of metabolism-related diseases (Figure 2).

Figure 2.

The schematic diagram describing the close relationship between the occurrence and progression of liver disease and the abundance of A. muciniphila, and supplementing A. muciniphila improves symptoms of liver disease and delays its progression.

Alcoholic liver disease

ALD is another very common chronic liver disease and one of the main causes of death from chronic liver disease. Some of the milder forms of alcoholic liver damage can be reversed with abstinence, while some of the more severe forms, such as cirrhosis, can be irreversible and life-threatening. Alcohol produces harmful reactive oxygen species (ROS) at every step of human metabolism, inducing oxidative stress. Superoxide anions and hydroxyethyl radicals produced during cytochrome P4502E1 (CYP2E1) metabolism also induce mitochondrial dysfunction, depleting antioxidant and recruiting inflammatory cells, increasing the sensitivity of hepatocytes to free radical damage. The pathogenic factor of ALD is single, but the mechanism is complex, involving multiple links of steatosis, inflammation, fibrosis and cancerosis. It is the result of the joint action of many factors such as susceptibility genes, intestinal microecology, oxygen stress injury, immune injury and programmed cell death. In addition to the direct effects of ethanol and its metabolites, the progression of ALD is also closely related to intestinal microorganisms. Clinical studies have shown that alcohol intake can lead to changes in the composition of gut microbiota, resulting in an imbalance of intestinal microorganism and disruption of the intestinal barrier. For example, Lowe et al. conducted a special dietary intervention for 10 days in wild-type mice to construct a model of early alcoholic steatohepatitis and analyze changes in intestinal microbial composition caused by alcohol intake [35]. It was found that acute and chronic alcohol intake caused changes in various bacterias in the cecum, and the loss of Akkermansia was observed to be an early marker of alcohol-induced intestinal ecological disorder. Patients with alcohol use disorder showed intestinal microbiome characteristics with low Akkermansia abundance [36]. In addition, decreased abundance of A. muciniphila was associated with the severity of ALD [37]. Intervention with synthetic human α-defensin 5 in mice effectively reversed alcohol-induced deleterious effects, which were associated with changes in the microbe composition of the cecum, especially the increase of A. muciniphila [38]. Besides, ethanol metabolites induce a redistribution of tight junction associated proteins and adhesion junction associated proteins [39,40], resulting in disruption of these junctions and an increase in intestinal permeability, which in turn leads to intestinal barrier disruption and bacterial endotoxin translocation. Endotoxin promotes liver inflammation, metabolic disorders, and further progression of ALD by activating toll-like receptor 4 (TLR4) signaling pathway. Modern medicine has also verified that ALD patients often have elevated serum endotoxin, which is correlated with the severity of the disease, suggesting a close relationship between intestinal function and ALD. In a mouse model, live A. muciniphila supplementation can reverse the ethanol-induced increase in intestinal permeability, increase mucus thickness and the expression of tight junction protein, and then reduce LPS and peptidoglycan released by intestinal bacteria into the circulation system and reach the liver tissue, thereby alleviating liver injury caused by inflammation and oxidative stress [37]; At the same time, A. muciniphila can also directly affect the chronic inflammatory response of the body and alleviate liver damage [41]. The intestinal vascular barrier controls the translocation of antigens and prevents bacteria from entering the blood circulation, which plays an important role in ALD. In an experimental ALD model, ethanol feeding increased the expression of the integral membrane protein Pv-1 associated with the endothelial fenegration diaphragm, which can be used as a marker of endothelial barrier dysfunction, indicating changes in endothelial barrier function. A. muciniphila administration tended to restore the intestinal vascular barrier and protect mice from ethanol-induced liver injury [42]. A. muciniphila has also been reported to down-regulate the expression of CCAAT/enhancer binding protein α (C/EBPα), and C/EBPα acetylation has been shown to promote the progression of ALD through down-regulation of miR-233 with anti-inflammatory and anti-fibrotic functions [43,44]. These reports suggest that A. muciniphila may play an important role in regulating susceptibility to liver disease associated with acute alcohol consumption. In addition, some plant-derived compounds have preventive and therapeutic effects on ALD, and the mechanisms behind this phenomenon have also been shown to be related to intestinal A. muciniphila abundance. For example, rhubarb extract supplementation improved alcohol-induced liver injury and down-regulated key markers of inflammation and oxidative stress in liver tissue, which showed the same trend as increased A. muciniphila abundance [45]. Berberine has been reported to have beneficial effects in improving acute and chronic alcoholic liver injury. This beneficial effect was accompanied by IL6/signal transduction and transcriptional activator 3 (STAT3) signaling pathway dependent activation of immunosuppressive cell populations, an increased number of blood and liver immunosuppressive functional cells, and a decreased number of cytotoxic T cells. It is worth noting that berberine changed the entire intestinal microbial community, among which the abundance of A. muciniphila was most significantly increased by berberine administration. After excluding the influence of intestinal microorganisms, berberine's function on the immunosuppressive cell population was also eliminated, and its protective effect against alcohol-induced liver injury in mice was also lost [46]. These studies also further provide credible evidence for the positive role of A. muciniphila in protecting the liver from alcohol-related damage.

Liver cirrhosis

Cirrhosis is the result of the continuous development of various chronic liver diseases. Unsurprisingly, patients at the stage of cirrhosis also showed reduced Akkermansia abundance [47]. Various etiologies lead to inflammation, necrosis and other changes in hepatocytes, among which the imbalance between synthesis and degradation of extracellular matrix (ECM) in the liver is the key to abnormal deposition of fibrous connective tissue in the liver and subsequent development of liver fibrosis and even cirrhosis. Hepatic stellate cells (HSCs) were first described by von Kupffer in 1876, and activated HSCs play a key role in liver fibrosis by producing an excess of ECM. Activation of HSCs is usually the result of chronic inflammation in the liver, leading to transdifferentiation of quiescent cells into activated spindle-like cells [48]. Activated HSCs shows significant phenotypic changes, including up-regulation of type I collagen expression and increased release of inflammatory, proliferative and fibrotic factors [49]. A study found that A. muciniphila treated with heat inactivation could significantly down-regulate the expression of fibrosis markers and promote the transition of LPS-activated HSCs to quiescent state [50]. Raftar et al. also studied the role of live and pasteurized A. muciniphila and its EVs in the prevention of liver fibrosis, and found that all studied A. muciniphila preparations prevented HSCs activation and reduced the relative expression of fibrosis and inflammatory biomarkers [51]. A. muciniphila EVs had the most significant effect on inhibiting HSCs activation. A. muciniphila treatment also ameliorates endoplasmic reticulum stress in liver and muscle, reduces plasma levels of LPS binding protein (LBP) and leptin, and inactivates LPS/LBP downstream signaling (e.g., decreased phosphorylated c-Jun N-terminal kinase (JNK) and increased nuclear factor-κB inhibitor alpha (IKBA) expression) in liver and muscle, as well as increases plasma anti-inflammatory factors (such as α-tocopherol, β-sitosterol) and decrease biochemical and inflammatory cytokines levels to improve metabolic inflammation in the body [51,52]. These studies all show that A. muciniphila plays an important role in alleviating liver inflammation and liver fibrosis. Besides, Kang-Xian (KX) pills is a Chinese patent medicine for the treatment of chronic hepatitis and cirrhosis. A study on the mechanism of KX in alleviating liver injury found that KX treatment significantly improved the liver function of chronic hepatic injury model mice, and KX also increased the level of colonic tight-linking protein and decreased the expression of liver pro-inflammatory cytokines. Further 16S rRNA sequencing analysis showed that KX treatment increased the relative abundance of Akkermansia, and after eliminating the influence of intestinal microorganisms, KX would no longer have the effect of alleviating liver disease, and its effect on liver inflammation would disappear [53]. These studies also further support the pivotal role of A. muciniphila in the improvement of liver fibrosis.

Liver cancer

The final stage in the development of fatty liver, hepatitis, cirrhosis and other liver diseases is liver cancer. Despite significant differences in the number and distribution of intestinal microorganisms at different stages of liver disease progression, A. muciniphila abundance consistently decreased, even in patients with liver cancer or in mice compared with healthy controls [54]. It can be seen that the imbalance of A. muciniphila bacteria is a key factor in the progression of liver disease. In addition, A. muciniphila is also an important factor influencing anti-tumor immune surveillance. One study found that A. muciniphila isolated with breast milk improved the severity of non-alcoholic steatohepatitis and inhibited its progression to liver cancer. This was accompanied by an increase in liver CXCR6⁺ natural killer T (NKT) cells and a decrease in macrophage infiltration. Further studies showed that the antitumor ability of A. muciniphila was not obvious in NKT cell-deficient mice. In vitro, A. muciniphila promoted the killing effect of NKT cells on HepG2 cell [54]. Intestinal microorganism imbalance can induce TLR dependent amplification of liver monocyte bone marrow derived suppressor cells (mMDSC) and inhibition of T cell abundance, while supplementation of single A. muciniphila can reduce the infiltration of MDSCs, reduce liver inflammation and fibrosis, and delay the progression of liver disease to liver cancer [55]. A. muciniphila benefits have also been shown in the immunotherapy of liver cancer. By investigating the dynamic change characteristics of intestinal microflora, it was found that fecal samples of liver cancer patients who responded to anti-PD-1 immunotherapy showed higher taxonomic richness and more gene counts than those of patients who did not respond, and the former generally had a higher abundance of A. muciniphila [56,57]. More importantly, antibiotic treatment can counteract the effect of PD-1 antibody on tumor growth. Further studies have found that changes in intestinal microorganism can lead to changes in glycerophospholipid metabolism, which may affect the expression of immunorelated cytokines IFN-γ and IL-2 in the tumor microenvironment, resulting in differences in the efficacy of PD-1 antibodies [58].

Although there is still no intuitive evidence showing the correlation between A. muciniphila and liver cancer treatment, a considerable amount of treatment has been conducted on A. muciniphila and other cancer models. This may help us to speculate the role of A. muciniphila in the treatment of liver cancer to some extent. For instance, oral supplementation of A. muciniphila enhanced the antitumor efficacy of IL-2, and macrophages treated with A. muciniphila extracellular vesicles (EVs) showed significant inhibitory effect on tumor cell growth [59,60]. In addition, the anti-tumor effects of chemotherapy drugs also show a certain correlation with intestinal microbes. For example, gemcitabine has been reported to significantly reduce the proportion of gram-positive firmicutes and gram-negative bacteroides in the gut of tumor-bearing mice, while the abundance of A. muciniphila increased from 5% to 33%, and activation of the typical nuclear factor-κB (NF-κB) pathway was found in mouse tumor tissues [61]. 16S rDNA gene sequencing analysis showed that the abundance of A. muciniphila was significantly increased in individuals treated with FOLFOX (oxaliplatin, fluorouracil and calcium leucovorin), and was positively correlated with the therapeutic effect. A. muciniphila colonization significantly enhanced the anti-cancer efficacy of FOLFOX [62]. A. muciniphila combined with cisplatin also improved the efficiency of tumor inhibition. Pathological analysis showed that A. muciniphila combined with cisplatin down-regulated the levels of Ki-67, p53 and factor-related suicide ligand proteins, increased the levels of IFN-γ, IL-6 and TNF-α, and inhibited the expression of CD4⁺ CD⁺ Foxp3⁺ Treg in peripheral blood and spleen of mice [63]. These evidences all suggest that A. muciniphila plays an important role in a variety of anti-tumor therapeutic strategies. Therefore, in-depth study on the mechanism of A. muciniphila promoting tumor treatment effect and development of A. muciniphila-based treatment strategies may become one of the promising directions of tumor treatment.

Mechanisms of A. muciniphila in affecting liver health

Improving intestinal barrier

The intestinal barrier mainly includes intestinal mucus layer, epithelial cells and intercellular tight junction, bacteriostatic components produced by intestinal microorganisms, and immunoactive substances secreted by intestinal lymphocytes and intestinal microorganisms. The damage of any part will have adverse effects on the body and threaten the life and health of the body. Intestinal epithelial cells mainly consist of intestinal cells, Paneth cells, goblets, tufted cells, intestinal endocrine cells and micropleated cells, which together form a continuous polarized monolayer, leading to the separation of lumen and lamina lamina. These cells play an important role in substance absorption, hormone secretion, antimicrobial peptide secretion, mucus production, and antigenic response. The mucus barrier is a giant network of highly glycosylated mucins secreted by goblet cells. According to its function, the mucus layer can be divided into two layers. The outer layer can be adhered by bacteria, which is conducive to wrapping bacteria and promoting the discharge of pathogenic bacteria through intestinal peristalsis. The inner layer is closely adhered to the surface of small intestinal epithelial cells to prevent the invasion of pathogenic bacteria. Tight-junction proteins between intestinal epithelial cells seal the intercellular space, forming a strong intestinal barrier to limit the passage of bacteria, toxins, inflammatory mediators, and other harmful substances through the intestinal mucosa into surrounding tissues and blood circulation.

A. muciniphila is well known as mucin-degrading bacteria. However, it has been reported that A. muciniphila can not only degrade mucin, but also stimulate goblet cells to produce more new mucin [64]. At the same time, the new mucin can provide nutrients for A. muciniphila and stimulate its proliferation, forming a feedback loop to continuously renew the intestinal mucus layer. It has a good protective effect on intestinal epithelial cells. A. muciniphila has also been reported to accelerate the proliferation of intestinal epithelium through Wnt/β-catenin signaling pathway after colonizing intestines, thereby repairing the damaged intestinal mucosa and preventing the invasion of pathogenic microorganisms [65]. A. muciniphila treatment can also promote the proliferation of small intestinal stem cells and the differentiation of small intestinal epithelial cells, thus accelerating the regeneration of intestinal epithelial cells and maintaining intestinal homeostasis [66]. Besides, A. muciniphila can also increase the expression of RegIIIγ in the colon of mice, which is known to have a direct bactericide effect on gram-positive bacteria in the intestine, suggesting that A. muciniphila may manipulate host immunity by increasing the expression of RegIIIγ, thereby promoting its own survival, remodeling intestinal microbial community, and improving intestinal barrier function [67].

In addition, oral administration of A. muciniphila increased mRNA levels of a series of tight junction related proteins in mice, including occludin, jam3 and claudin, and intestinal permeability experiments showed that A. muciniphila reduced the entry of oral FITC-dextran into the blood circulation [68]. Cruz-Lebrón et al. found that the medium used by A. muciniphila also stimulated the increase of transepithelial electrical resistance (TEER) value (an indicator of the integrity of the monolayer epithelium) and up-regulated the expression of claudin 4 in epithelial monolayers. However, there are differences in the time points of the two indicators indicating the improvement of intestinal barrier function. To be specific, TEER value was significantly increased at 3 h and 6 h after A. muciniphila-medium treatment, while claudin 4 expression was up-regulated at 24 h after A. muciniphila-medium treatment. Therefore, A. muciniphila-medium may regulate intestinal barrier by two different mechanisms. In the process of verifying this phenomenon, the authors also found that the effect of A. muciniphila-medium on improving intestinal barrier was independent of EVs [69]. In contrast, Chelakkot et al. found that feeding A. muciniphila EVs reversed HFD-induced intestinal damage and improved intestinal permeability, and mechanism studies showed that EVs significantly up-regulated the expression of occludin, zonal occludens and claudin-5, the main tight junction proteins responsible for maintaining intestinal barrier integrity [70]. The authors further treated Caco-2 cells with A. muciniphila EVs to further explore the mechanism by which they regulate intestinal epithelial tight junctions and found that EVs induced phosphorylation of adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK), a kinase responsible for tight junction reorganization and stability, in a dose-dependent manner. Ashrafian et al. also demonstrated the efficacy of EVs in improving intestinal barrier permeability [21]. It is not clear why there are two diametrically opposed views. Through further literature investigation, we found that in the two literatures that mentioned the positive role of A. muciniphila EVs in improving intestinal barrier, A. muciniphila‘s medium was both supplemented with mucin [70,71], while only brian heart infusion was used for A. muciniphila culture in Cruz-Lebrón et al.'s work. According to the literature, the expression of specific proteins in A. muciniphila can respond to the changes of intestinal mucosal environment. For example, Lee et al. found that mucin-containing media induced upregulation of A. muciniphila's putative mucin-degradation related enzymes, including Amuc_1220, Amuc_1120 and Amuc_0052, etc [72]. The absence of mucin in the medium results in the upregulation of outer membrane protein Amuc_1100 and other 79 genes encoding secretory protein candidates of A. muciniphila, among which Amuc_1100 has been shown to enhance TEER and protect epithelial integrity through TLR2-mediated signaling, and A. muciniphila grown in mucin-deficient medium showed more effective effects in reducing obesity and improving intestinal barrier than that in mucin-containing medium [73–75]. Although this phenomenon still supports the role of mucin deficiency in promoting the intestinal barrier repair function of A. muciniphila, it is confirmed that cultural environment differences affect protein expression in A. muciniphila. At the same time, bacteria can sense the nutritional environment and induce the expression of specific protein components in EVs [76]. It can be speculated that the biological activity of A. muciniphila EVs depends largely on the bacterial culture environment. This provides direction for further research to determine the function of EVs in intestinal barrier repair (Figure 3). Although there is some contradiction in the determination of the composition of A. muciniphila in intestinal barrier repair, the function of A. muciniphila itself in intestinal barrier repair is widely recognized. Increased intestinal mucosal permeability is an important cause of chronic inflammation, and improving the intestinal barrier may be the basic core competitiveness of many health effects of A. muciniphila.

Figure 3.

The regulation of intestinal gene expression by different parts of A. muciniphila colonization and the mechanism of A. muciniphila improvement in liver disease.

Improving energy metabolism

As the metabolic center and the second energy storage, liver is the most prone to damage due to metabolic stress. Metabolic syndrome, characterized by central obesity, insulin resistance, hypertension, and dyslipidemia, is a major global health problem and a significant risk factor for the most common non-alcoholic liver disease and continues to affect the progression of chronic liver disease. Gut microbes control food processing and metabolism of energy and nutrients, which have a significant impact on human health. In order to study the interaction mechanism between A. muciniphila and host, Derrien et al. inoculated A. muciniphila into germ-free mice by gavage, and observed that A. muciniphila could colonize different intestinal regions to different degrees, and what was more interesting was that A. muciniphila colonized in different intestinal segments could participate in regulating different signaling pathways [77]. For example, A. muciniphila ileal colonization results in changes in metabolic and signaling pathway-related gene expression, primarily by regulating PPARα-dependent pathways. Most of these genes and pathways are involved in lipid metabolism, small molecule biochemistry and metabolic homeostasis, and their colonization in the colon is involved in the regulation of gene expression involved in vitamin and mineral metabolism, which also reflects the regulation of host biological metabolism by A. muciniphila (Figure 3).

Depommier et al. studied the influence of A. muciniphila on energy consumption of mice by indirect calorimetry, and found that compared with mice in the same litter, HFD-fed mice had lower energy consumption, and A. muciniphila treatment completely eliminated the influence of HFD, so that the energy consumption of HFD-fed mice was the same as that of mice fed a normal diet (ND) [78]. The mice treated with A. muciniphila showed a significant increase in motor activity. In addition, A. muciniphila treatment significantly increased the excretion of fecal energy. Further analysis of markers related to lipid, peptide and carbohydrate absorption in jejunum showed that A. muciniphila treatment significantly decreased the transcription levels of major glucose transporters glucose transporter 2 (GLUT2) and sodium/glucose cotransporter 1 (SGLT1), and to some extent the transcription levels of the fructose transporter, glucose transporter 5 (GLUT5). This suggests that the increased energy loss observed in faeces may be related to reduced carbohydrate absorption. In addition, supplementing A. muciniphila regulates serum 3β-chenodeoxycholic acid levels and stimulates insulin secretion and fibroblast growth factor 15/19 (FGF15/19) expression, as well as increases intestinal 2-oleoylglycerol levels, which have been shown to stimulate the release of glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) from L cells in the intestine involved in glucose homeostasis, thus achieving lower blood glucose levels and improved glucose homeostasis [24,67]. A. muciniphila treatment can also reduce the overexpression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, a key enzyme in liver gluconeogenesis, and alleviate insulin resistance [79]. Other than that, A. muciniphila treatment caused specific loss of interscapular brown adipose tissue (iBAT), but had no significant effect on white adipocyte (WAT) diameter or fat mass of mice epididymis. Transcriptional analysis of thermogenic genes showed that A. muciniphila significantly induced the transcription of mitochondrial specific genes encoding uncoupling protein 1 and related thermogenic differentiation markers in iBAT, while no changes in the expression of related genes were observed in groin WAT (igWAT) of mice. A. muciniphila treatment also increased rectal temperature and skin temperature at iBAT, suggesting that the anti-obesity effect of A. muciniphila is mediated by iBAT rather than igWAT [25]. In order to further explore the mechanism by which A. muciniphila improves obesity, Lee et al. treated 3T3-L1 cells with A. muciniphila cell lysates and found that A. muciniphila cell lysates reduced lipid accumulation in 3T3-L1 cells and inhibited adipocyte differentiation [43]; besides, A. muciniphila treatment down-regulatedthe expression of genes involved in adipocyte differentiation and lipid synthesis in 3T3-L1 cells, and the expression levels of fatty acid synthase and acetyl-CoA car-boxylase were also decreased to some extent. A. muciniphila treatment also significantly reduced the expressions of sterol regulatory element-binding proteins and fatty acid translocase involved in lipogenesis and fatty acid transport in liver and muscle [43,51,80]. It is of great significance to inhibit the occurrence and development of obesity. In addition, A. muciniphila treatment can induce the expression of low density lipoprotein receptor and apolipoprotein E in the liver cells of cyclic-AMP-responsive-element-binding protein H (CREBH) deficient mice, thereby increasing the uptake of apolipoprotein B100 and apolipoprotein E-mediated medium density lipoprotein, thus accelerating the clearance of circulating triglyceride-rich lipoproteins and chylomicron [52], which is important to reverse the metabolic disorder caused by high-fat diet and prevent the progression of the disease.

Besides, compared with live A. muciniphila, pasteurized counterpart showed better effects in reducing fat mass, alleviating insulin resistance and dyslipidemia in mice. Amuc_1100 is one of the most abundant outer membrane proteins of A. muciniphila with a molecular weight of 32 kDa. Plovier et al. constructed recombinant protein Amuc_1100 and found that it appears to be thermally stable at the temperature required for pasteurization [81], and Amuc_1100 partially replicated the active functions of A. muciniphila, including its role in improving obesity, insulin resistance, and intestinal barrier. Based on the fact that Amuc_1100 remains stable at 70°C, it is speculated that it can still signal in pasteurized cells, and that pasteurization enhances the effect of A. muciniphila by increasing the protein's accessibility to the host. In addition, Yoon et al. found that a protein called P9 secreted by A. muciniphila could significantly reduce HFD-induced weight gain and food intake in mice, as well as reduce adipose tissue volume and glucose intolerance during the exploration of the mechanism by which A. muciniphila improves metabolic diseases [25]. GLP-1 regulates BAT thermogenesis. Studies have shown that P9 can bind to intercellular adhesion molecule 2 (ICAM-2) receptors and induce GLP-1 secretion in a dose-dependent manner, with a much higher effect than Amuc_1100. In addition, P9 regulates glucose homeostasis and body weight gain by activating GLP-1 receptor pathway, and this process is highly dependent on IL-6 participation. These reports prove that A. muciniphila is a promising candidate strain for the prevention or treatment of metabolic related diseases. With the deepening of the research on A. muciniphila, the reason and debate become more and more clear, and the mechanism of A. muciniphila becomes clear. It is worth further exploring the probiotic mechanism of A. muciniphila to realize the regulation of body function by manipulating A. muciniphila as soon as possible.

Regulating immune response

At present, although the pathogenesis of chronic liver disease has not been fully elucidated, it can be confirmed that inflammation plays a key role throughout the entire pathological process of chronic liver disease. Intestinal immunity is the most important component of mucosal immune system, and immune cells in intestinal mucosal tissues account for 80% of all immune cells [82]. Intestinal mucosal immunity is closely related to extra-mucosal immunity, and they can interact with each other through immune cells, various cytokines or other molecules. Although bacteria are officially regarded as non-self components by the host immune system, bacterial colonization in the gut is essential for maintaining the non-inflammatory balance of immunity and immune tolerance in mammals [83]. The imbalance of intestinal microorganism may break immune homeostasis in the intestine and affect the progression of many diseases.

A. muciniphila abundance has been shown to be inversely associated with a variety of immune disorders-related diseases, and A. muciniphila has also been reported to have the ability to regulate the host immune response and may play a role in immune tolerance to symbiotic microorganisms [28,77]. Various animal models were constructed to investigate the role of A. muciniphila in inflammation-related diseases. A. muciniphila treatment was found to reduce the levels of inflammatory cytokines including IL-2, IL-6, IL-1b, IL-17, and TNF-α in inflammatory animals, and increase the levels of anti-inflammatory cytokines including IL-10, IL-35, and TGF-β, as well as relieve the body's signs of disease [41,80,84–88]. It was also found that A. muciniphila can improve the immune function, increase the chemotaxis and phagocytosis function of macrophages, the toxic effect of NK cells [84]. In LPS-induced LX-2 inflammatory cell model, A. muciniphila significantly inhibited the expression of TLR-2 and TLR-4 genes, and also significantly improved the serum inflammatory factor levels in HFD/CCl₄-induced liver injury mice [41]. Moreover, A. muciniphila can reverse HFD-induced liver inflammation, inhibit the expression of macrophage markers F4/80 and Fpr2, and reduce the number of Treg cells in spleen and mesenteric lymph node in line with that in the blank control group [80]. At the same time, A. muciniphila can regulate the proportion of M1/M2 type macrophages by regulating the production of inflammatory chemokines at the lesion site, so as to relieve the level of lesion inflammation [86]. These reports all provide evidence for the positive role of A. muciniphila in regulating inflammatory response. On the other hand, studies in some tumor models have shown that A. muciniphila can up-regulate the levels of inflammatory cytokines including IFN-γ, IL-6 and TNF-α, and down-regulate the levels of IL-10 to increase the effect of anti-cancer immunotherapy [63]. A. muciniphila can also induce antigen-specific T cell responses to regulate host immune function and restore the efficacy of PD-1 blocking in an IL-12-dependent manner by increasing recruitment of CCR9⁺ CXCR3⁺ CD4⁺ T lymphocytes in mouse tumor lesions [89]. Thus, A. muciniphila seems to regulate the body's immune balance through a complex mechanism.

Through the study of A. muciniphila-host interaction, it was found that A. muciniphila colonization resulted in the highest gene expression difference in the colon, followed by ileum and cecum (Figure 3). Analysis of biological functions in the transcriptome showed that A. muciniphila colonization caused changes in the expression of genes involved in membrane metabolism, signaling and antigen delivery pathways, and upregulation of genes associated with leukocyte antigen presentation [77]. It is important to note that the balance between pathways and processes involved in A. muciniphila varies from intestinal region to intestinal region; Its colonization modulated the immunomodulatory process most significantly in the colon, followed by the cecum and ileum. A. muciniphila supplementation down-regulates the expression of immunoglobulin and its receptors in the colon, and down-regulates several genes encoding chemokines, such as Cxcl13 and Ccl12, as well as cytokines IL5 and complement factors C1ra and C5ar1. A. muciniphila supplementation also down-regulates immune-related genes in the colon, including Blk, Cd4, Cd72, Tlr7 and Tlr12. In addition, A. muciniphila colonization is also involved in down-regulating the expression of immune-related pathways in the colon, such as “IgA producing intestinal immune network”, “cytokine-cytokine receptor interaction”, and “inflammatory response pathway” [27]. Analysis of local immune cell populations of MLNs by flow cytometry showed that A. muciniphila had no effect on the percentage of dendritic cells (DCs), but increased total B cell populations and significantly reduced total neutrophils and total T cell populations. Studies on the composition of various subsets of DC, B cells and T cells in MLNs showed that A. muciniphila had no effect on the percentage of various subsets of DC, but tended to reduce the activity of DC subsets by decreasing the expression of antigen presenting molecule MHCII on DCS [90].

In order to explore the specific molecular mechanism of A. muciniphila's involvement in immune regulation, Wang et al. managed to obtain Amuc_1100, one of the most abundant outer membrane proteins in A. muciniphila, and explored its role in inflammation regulation, taking A. muciniphila as a reference [91]. A. muciniphila or Amuc_1100 was found to improve colitis, reduce infiltrating macrophages and CD8⁺ cytotoxic T lymphocytes in colon, and reduce CD16/32⁺ macrophages in spleen and mesenteric lymph nodes (MLN) in colitis mice, and increase PD-1⁺ cytotoxic T lymphocytes (CTLs) in spleen. Moreover, A. muciniphila and Amuc_1100 can reduce tumorgenesis by amplifying CTL in colon and MLN. It can be seen that Amuc_1100 is an important protein in the immunomodulatory function of A. muciniphila. Shi et al. expressed and purified Amuc (molecular weight ∼40 kDa), also one of the most abundant outer membrane proteins in A. muciniphila, and studied its efficacy in mediating anti-tumor immunotherapy [59]. It was found that oral administration of Amuc showed an anti-tumor effect equivalent to that of A. muciniphila, and pretreatment of A. muciniphila with Amuc-specific antibody greatly impaired the tumor inhibition effect of A. muciniphila, suggesting that Amuc plays an important role in A. muciniphila-induced antitumor immunotherapy. In addition, Amuc increased the proportion of CTLs in the tumor immune microenvironment while decreasing the proportion of Tregs. Studies on the underlying mechanism of the tumor-specific immune response mediated by Amuc have shown that the tumor inhibitory effect of Amuc is partly mediated by the TLR2 signaling pathway. Recently, a team of researchers led by Jon Clardy of Harvard Medical School and Ramnik Xavier of the Broad Institute demonstrated that A. muciniphila can induce the secretion of certain cytokines by human immune cells through cell membrane phospholipids (diacyl phosphatidylethanolamine, a15:0-i15:0 PE) and reset the activation threshold of dendritic cells, regulating subsequent immune stimulation [92]. In dose-response studies, a15:0-i15:0 PE up-regulated the secretion levels of TNF-α and IL-6, but did not affect the secretion levels of IL-10 and IL-12p70. Gene knockout experiments in mice showed that a15:0-i15:0 PE also induced cellular response via TLR2 pathway. Interestingly, researchers found that low doses of a15:0-i15:0 PE (0.15 μmol/L, ~1% of EC50) inhibited the immune activation induced by addition of Pam3CSK4 and LPS 18 h later, and this phenomenon did not occur at high doses of a15:0-i15:0 PE, at short intervals (6 h), or at co-stimulation. This suggests that low doses of a15:0-i15:0 PE can “passivate” the response of cells to other immunogens, on the one hand ignoring low-dose stimulus, on the other hand moderating high-dose stimulus, which may be the potential mechanism of A. muciniphila bacteria on immune regulation. These reports suggest that intestinal colonization of A. muciniphila induces a variety of immune responsive-related pathways, including innate and adaptive immunity, and that its outer membrane proteins and lipid components play important roles in immunomodulatory functions. Immune-related studies based on A. muciniphila provide a promising immunomodulatory strategy.

Conclusion

Based on the above analysis, there is a close relationship between A. muciniphila and liver health, and to explore the probiotic effect of A. muciniphila in liver disease and its potential in disease treatment is a promising topic for future research. More importantly, the safety and tolerability of live A. muciniphila have been demonstrated in the clinic. Besides, pasteurization does not eliminate the bioactive functions of live A. muciniphila bacteria, and even enhances some functions to a certain extent. Moreover, the EVs or membrane protein components produced by A. muciniphila can partially exhibit similar functions to live A. muciniphila, which provides a broad scope for the development of safe and effective treatment agents for A. muciniphila bacteria.

Future perspective

Although great progress has been made in the studies on the relationship between A. muciniphila and liver disease, the causal relationship and specific mechanism of action of the two remain unclear. In addition, many current studies are carried out at the animal level. Considering the differences between animals and human beings, as well as the complexity of naturally formed diseases compared with model diseases, systematic and comprehensive clinical studies are needed to provide exact basis for the application of A. muciniphila. In addition, according to literature reports, the nutritional environment of A. muciniphila has an important impact on the expression of the specific proteins and the function of A. muciniphila. Therefore, in the process of A. muciniphila preparation, the influence of culture environment on the activity of bacteria should be taken into account in order to obtain bacteria with stable biological engineering.

On the other side, in addition to the widely reported probiotic function of A. muciniphila, many studies have also mentioned that supplementing A. muciniphila has the possibility of causing inflammation [93,94]. This phenomenon may be due to the strain specificity of the bacteria. Different bacterial strains have different genome homology and different functions [95,96]. For example, Zhai et al. reported differences in anti-inflammatory function between strain A. musiniphila ATCC BAA-835^T and murine-derived A. musiniphila 139 [97]. In addition, the function of A. musiniphila has also been reported to be related to the intestinal microecological environment [98]. A. musiniphila is a symbiotic intestinal bacteria that is not pathogenic to the host itself. However, the presence of certain pathogenic bacteria, such as S. typhimurium, may make them potentially harmful bacteria. Therefore, more research should be devoted to exploring the specific conditions under which A. musiniphila exert different biological activities, which has important implications for developing the next generation of probiotics for the treatment of human diseases.

Funding Statement

The work was supported by the China Postdoctoral Science Foundation (2021M703354), the Shanghai Post-doctoral Excellence Program (2021430), and the China National Postdoctoral Program for Innovative Talents (BX20220322). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Author contributions

M Zhang conducted writing – review & editing; Y Wang conducted writing – original draft and investigation; Y Gan conducted conceptualization.

Financial disclosure

The work was supported by the China Postdoctoral Science Foundation (2021M703354), the Shanghai Post-doctoral Excellence Program (2021430), and the China National Postdoctoral Program for Innovative Talents (BX20220322). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.