Vitiligo is a widespread condition affecting skin pigmentation, impacting around 0.5 to 2% of people worldwide (Bergqvist and Ezzedine, 2020). Presently, it is classified as an autoimmune disorder, with its development linked to the excessive activation of CD8+ T cells, which are self-reactive cells located in the skin (Perez-Bootello et al., 2023). Overactivation of CD8+ T cells results in the release of IFN-γ. IFN-γ at the diseased skin can increase the expression of keratinocyte chemokines such as CXCL9 and CXCL10, which subsequently attract effector T cells that attack melanocytes (Perez-Bootello et al., 2023; Lee et al., 2024). Recent studies indicate that gut bacteria are essential in forming and sustaining the human innate immune system (Zheng et al., 2020; Donald and Finlay, 2023). The gut microbiome plays a crucial role in immune-related diseases like rheumatoid arthritis, systemic lupus erythematosus, and autoimmune pancreatitis, and is also closely linked to several skin conditions (Ruff et al., 2020; Colucci and Moretti, 2021).

The complex interplay among the gut microbiota-immune-skin axis has driven research into skin conditions linked to the gut microbiota (Mahmud et al., 2022). Besides prevalent conditions such as eczema and psoriasis, research on the connection between gut bacteria and vitiligo has underscored the crucial influence of gut microbiota on immune-related skin diseases (Chen et al., 2021b). Dellacecca et al.’s (2020) animal studies have shown that an imbalance in the gut microbiota, induced by antibiotics, can trigger the development of vitiligo in mice. Research involving humans has shown that people with vitiligo exhibit reduced gut microbiota richness and diversity compared to those without the condition. Additionally, there are marked alterations in species composition, including a notable rise in the Firmicutes/Bacteroidetes ratio among vitiligo sufferers (Bzioueche et al., 2021; Wu et al., 2023). Furthermore, Ni et al. (2020) combined microbiome sequencing with serum untargeted metabolomics analysis to establish a strong correlation between specific gut microbiota genera, the progression of vitiligo, and serum IL-1β levels. Although these discoveries hint at a possible connection between gut bacteria and vitiligo, the scarcity of current research makes the matter complex, particularly due to the lack of studies considering age-related aspects. Therefore, further research is essential to clarify the impact of age on gut microbiota in relation to vitiligo.

This research thoroughly examined the species makeup and possible roles of gut microbiota in individuals with vitiligo and healthy subjects through 16S rRNA high-throughput sequencing. To evaluate how age affects the gut microbiota composition in individuals with vitiligo, the participants were categorized by age. The results not only expand our understanding of the gut microbiome’s connection to vitiligo but also reveal, for the first time, age-specific differences in gut microbiome markers among vitiligo sufferers. These findings enhance our comprehension of how gut microbiota may influence vitiligo development and provide valuable insights for therapeutic approaches that involve altering gut microbiota.

Vitiligo is a chronic dermatological condition characterized by the specific depletion of melanocytes, with potential contributing factors including metabolic irregularities, oxidative stress, inflammation, and autoimmunity (Diotallevi et al., 2023). Recent research has underscored the significant influence of gut microbiota on host metabolism, inflammatory processes, and immune responses (Zheng et al., 2020; Fan and Pedersen, 2021). The developing idea of the gut-skin axis has linked different skin conditions to the composition of gut microbiota, yet research on the connection between gut microbiota and vitiligo is still scarce (Chen et al., 2021b; De Pessemier et al., 2021; Mahmud et al., 2022). We employed 16S rRNA sequencing to analyze the gut microbiota composition in people with stable non-segmental vitiligo, and investigated the influence of age on the condition by dividing participants into different age categories.

The gut microbiota plays a key role in immune system homeostasis by regulating the differentiation and function of immune cells, such as regulatory T cells (Tregs), maintaining immune tolerance and suppressing autoimmune responses (Round and Mazmanian, 2010). However, in patients with vitiligo, dysbiosis of the gut flora may lead to abnormal activation of immune cells, resulting in a reduced or suppressed number of Tregs and dysregulation of effector T cells (e.g., CD8+ T cells), which are more prone to attack their own tissues. Deficiencies in some gut microbiota may over-activate Th1 and Th17 cells, with Th1 secreting cytokines such as IFN-γ and Th17 secreting IL-17, which activate cytotoxic T-lymphocytes (CTLs), including CD8+ T cells, and further promote the attack of melanocytes by self-activated CD8+ T cells (Duan et al., 2012). Dysbiosis of the gut flora can also affect the function of antigen-presenting cells (APCs), causing changes in the way they present otherwise tolerated self-antigens. For example, certain gut bacteria can regulate the maturation and function of dendritic cells (DCs) (Macpherson and Uhr, 2004; Yang et al., 2022). Normally, APCs present antigens to T cells in a precise manner, avoiding overreaction to self-antigens. However, dysregulation of the gut microbiota may alter the expression pattern of molecules on the surface of APCs, leading to false presentation of melanocyte-specific antigens by APCs, which activates self-reactive CD8+ T cells and triggers immune attack against melanocytes. An intact gut barrier serves to prevent the penetration of harmful substances, including antigens and toxins, into the circulation. In contrast, disturbances in the gut microbiota can disrupt the gut barrier, a phenomenon known as ‘leaky gut’. This allows substances that would otherwise be confined to the gut, such as antigens, to enter the bloodstream and activate the systemic immune system (Mishra et al., 2023). These antigens may cross-react with melanocyte-derived proteins, or the activation of the immune system may indirectly lead to the activation of auto-reactive CD8+ T cells, which attack melanocytes.

Metabolites of intestinal flora have an important role in autoimmune initiation. Short-chain fatty acids (SCFAs) (acetic acid, propionic acid, butyric acid) produced by their fermented dietary fibre are critical for immune regulation, e.g., butyric acid inhibits histone deacetylase (HDAC) regulatory gene expression and affects immune cell function and differentiation (Arpaia et al., 2013). Altered production and utilization of SCFAs occurs when gut flora is dysbiotic, e.g., reduced butyrate-producing bacteria and decreased butyric acid levels in patients with inflammatory bowel disease, and this change may lead to abnormal immune cell function, making CD8+ T cells more susceptible to activation to attack melanocytes. Bile acid metabolism is closely related to intestinal flora and autoimmunity. Intestinal flora can influence the metabolic conversion of bile acids to modulate the immune response (Sayin et al., 2013; Rao et al., 2021). Disturbances in bile acid metabolism may additionally impact the immune system’s recognition of and response to self-antigens, thereby triggering autoimmunity. For instance, alterations in the intestinal flora result in aberrant bile acid metabolism, which facilitates Th17 differentiation. Th17 secretes cytokines, such as IL-17, which may activate and recruit CD8+ T cells (Duan et al., 2012), enhance the immune response to melanocyte-specific antigens, and contribute to the pathogenesis of vitiligo. The concept of the gut-skin axis emphasizes the existence of a strong immune link between the gut and the skin. Autoimmune responses initiated by intestinal dysbiosis result in the activation of immune cells and the release of cytokines, which can traverse the bloodstream from the gut to the skin. In the case of psoriasis, for instance, related inflammatory cytokines (e.g., TNF-α and IL-6) can reach the skin and trigger an inflammatory response. In the case of vitiligo, this may be evidenced by an increased migration of autoreactive CD8+ T cells to the skin, where they attack melanocytes and result in depigmentation. Additionally, intestinal flora may influence the skin immune environment through the neuro-endocrine pathway, regulating the production and release of neurotransmitters such as serotonin and dopamine. Serotonin is a crucial neurotransmitter for both skin function and immune regulation, and abnormal levels of serotonin may affect the response of immune cells to melanocytes.

In our investigation, a notable decline in the alpha diversity of gut microbiota was observed in individuals with vitiligo in comparison to healthy controls. This pattern remained consistent across analyses conducted on the entire sample and in subgroup analyses based on age. The results indicate that individuals with vitiligo demonstrate reduced diversity in their gut microbiota, which is in line with recent studies reported by Bzioueche et al. (2021) and Luan et al. (2023). However, Ni et al. (2020) reported an increase in gut microbiota diversity in vitiligo patients, but Principal Component Analysis (PCA) contributed the most with 24.53%, also suggesting differences in gut community structure in vitiligo patients. While there are a limited number of conflicting reports, this may be attributed to the scarcity of studies conducted on the subject. The prevailing body of current research consistently upholds the notion of gut microecological dysregulation and reduced diversity in various disease conditions (De Pessemier et al., 2021; Afzaal et al., 2022; Hou et al., 2022). This phenomenon is observed not only in skin disorders like acne vulgaris, atopic dermatitis, and psoriasis but also in other systemic illnesses. Consistent with previous research, the β-diversity outcomes derived from Principal Coordinate Analysis (PCoA) effectively differentiate between individuals and healthy controls, emphasizing the spatial heterogeneity in gut microbiota among vitiligo patients and healthy individuals (Ni et al., 2020; Wu et al., 2023). Nevertheless, no distinction was evident between the DI_m and DI_a groups stratified by age. Generally, the gut microbiota diversity in healthy children differs from that in adults (Radjabzadeh et al., 2020; Korpela et al., 2021). Research on healthy children in northwestern China revealed notable alterations in their gut microbiome as they aged. At the phylum level, the abundances of Actinobacteria notably decreased as children aged. At the genus level, the abundances of Eggerthella, Erysipelatoclostridium, Tyzzerella, Flavonifractor, and Bididobacterium also significantly declined with age, whereas Coprobacer, Lactobacillus, Oscillibacter, Odoribacter and Christensenella showed a marked increase as children grew older (Gao et al., 2023). In our results, there was also a decrease in Actinobacteriota in the HC_a group compared to the HC_m group at the phylum level, but we did not find a significant change at the genus level. Whether the disease state weakens the influence of age remains unknown, particularly in immune system-related skin disorders like vitiligo, necessitating further investigation.

In the microbiome composition, Firmicutes and Bacteroidota are identified as crucial components at the phylum level in both diseased and healthy control groups, consistent with previous research (Bzioueche et al., 2021; Luan et al., 2023; Wu et al., 2023). Notably, our analysis unveiled a marked decrease in the Firmicutes/Bacteroidota ratio within the vitiligo group, contrasting the findings of Bzioueche et al. (2021) and Wu et al. (2023). Additionally, recent research by Luan et al. (2023) did not detect significant variations in this ratio between the groups under comparison. These contradictory findings underscore the necessity for further exploration into assessing the F/B ratio in the gut microbiota of vitiligo patients. Remarkably, genera linked to butyrate production, such as Bifidobacterium, Blautia, Subdoligranulum, and Agathobacter, exhibited substantial decreases in the vitiligo cohort. Butyrate, a crucial SCFA, not in reducing systemic inflammatory responses by preserving the intestinal epithelial barrier but also participates in the citric acid cycle to lower mitochondrial reactive oxygen species production, thereby alleviating autoimmune responses triggered by oxidative stress (Donohoe et al., 2011; Xiao et al., 2022; Xie et al., 2016). Furthermore, SCFA have the potential to impact the activity and quantity of Tregs, which could play a role in the pathogenesis and advancement of vitiligo (Smith et al., 2013; Jin et al., 2024). The genus Bacteroides is significantly enriched in the disease group and is also an important genus that produces short-chain fatty acids (Cheng et al., 2022). Nevertheless, this genus influences the host in multiple ways, impacting not just autoimmune diseases but also affecting the onset and development of cancer via intricate biological processes (Zafar and Saier, 2021; Shin et al., 2024). Contradictory to previous studies, Bzioueche et al. (2021) have reported a decrease in the prevalence of Bacteroides in individuals with vitiligo. This variation may be attributed to the specificity of different Bacteroides species, as there are significant differences among them. For instance, while Bacteroides fragilis, Bacteroides plebeius, and Bacteroides uniformis are notably reduced in individuals with chronic urticaria, Bacteroides ovatus and Bacteroides uniformis are elevated in patients with atopic dermatitis (Lu et al., 2019; Ye et al., 2021). Similarly, the most recent results from Luan et al. (2023), based on metagenomic sequencing, indicate that Bacteroides fragilis is enriched in the vitiligo patient group, while Bacteroides bouchesdurhonensis is reduced. In light of these findings, we postulate that this may be the underlying reason for the discrepancies observed in Bacteroides results across several recent studies on vitiligo-related intestinal microbiota. Consequently, future studies should prioritize metagenomic analysis strategies that are accurate to the species or even strain level.

When analyzed using LEfSe, it was found that Oscillospiraceae was enriched in the DI_a group. Oscillospiraceae, a family of microbes commonly found in the intestines of both humans and animals, have their Oscillospira levels affected by numerous elements such as age, dietary habits, prebiotics, probiotics, and exposure to heavy metals. An increase in Oscillospira abundance was linked to a high-fat diet, obesity, type 2 diabetes, constipation, and depression, while the trembling spirochete showed a direct connection to gallstones and could serve as a biomarker for symptomatic gallstone formation. The presence of Oscillospira was notably lower in individuals suffering from inflammatory bowel disease and showed a negative correlation with the intensity of enteritis and enteropathy (Yang et al., 2021). Oscillospira abundance was notably lower in the gut of overweight children (Chen et al., 2021c). Aging involves a persistent, mild inflammation marked by increased levels of inflammatory substances in the bloodstream. Circulating concentrations of many inflammatory markers and mediators are higher in older adults than in young adults. Increasing research indicates that age-related dysregulation of the gut microbiota contributes to the overall inflammatory state in older adults (Shintouo et al., 2020). Among these, Oscillospira is strongly negatively correlated with pro-inflammatory monocyte chemotactic protein 1 (Conley et al., 2016). Oscillospira levels rise as people get older, but they decline in some age-related illnesses or conditions linked to poor aging, such as cardiometabolic diseases (Taniguchi et al., 2018). Variations in the microbiome due to aging are significantly diverse and shaped by personal and external environmental influences. Ageing is accompanied by changes in the microbiome, which in turn affects the rate of age-related decline (Ghosh et al., 2022). Microbial biomarkers in the DI_m group were more diverse and included Veillonella, Burkholderiales, Veillonellaceae and Sutterellaceae. Veillonella are bacteria that significantly contribute to various bodily regions, such as the oral cavity, gut, and respiratory system. The impact of microorganisms of the genus Veillonella varies significantly in different populations, especially in children and the elderly. In children, Veillonella parvula might play a protective role in the initial stages of immune system development, potentially aiding in the prevention of asthma, bronchitis, and autism (Arrieta et al., 2015; Djais et al., 2019). Research in epidemiology indicates that the abundance of Veillonella parvula, commonly found in the gut, is markedly reduced in children who are susceptible to asthma. In addition, Veillonella plays an important role in children’s oral health, acting as an early settling species of oral biofilms, forming aggregates with a wide range of bacteria, providing nutrients for periodontal pathogens, and influencing oral ecology (Djais et al., 2019). In adults, Veillonella’s impact is then associated with a variety of diseases. For instance, the abundance of Veillonella is notably higher in conditions like primary sclerosing cholangitis, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis (Kummen et al., 2017; Loomba et al., 2020). In addition, Veillonella atypica was more enriched in athletes because of its ability to utilize lactic acid as a carbon source to convert lactic acid into propionic acid, improving athletic performance and endurance (Scheiman et al., 2019). Burkholderiales is an order containing a wide variety of bacteria, some species of which are directly related to human health (respiratory infections, chronic lung diseases). Sutterellaceae is a group of bacteria found in the human gut that includes the genera Parasutterella and Sutterella. Parabacteroides distasonis is a bacterium in the family Sutterellaceae and is considered one of the core flora of the human body. There was a strong inverse relationship with conditions like obesity, non-alcoholic fatty liver disease, and diabetes, indicating it might positively influence glycolipid metabolism. This bacterium has the potential to alleviate obesity symptoms, insulin resistance, lipid metabolism issues, and NAFLD by producing succinic acid and secondary bile acids, thereby activating various signaling pathways and serving as a multi-targeted regulator. It is considered a promising probiotic for combating metabolic syndrome (Wang et al., 2019). Research discovered that Sutterella produced excessive IgA protease, breaking down IgA and thus reducing its levels in the gut lining, which weakened the intestinal antimicrobial immune defense (Kaakoush, 2020), and could be linked to immune irregularities in vitiligo. In summary, microorganisms of the genus Oscillospiraceae and Veillonella have diverse roles and influences in different populations, and their protective effects in children differ from the potential risks associated with disease in adults. Future studies need to further explore the specific mechanisms of action and health effects of microorganisms of the genera Oscillospiraceae and Veillonella in different populations. Based on the LEfSe analysis, it was elucidated that the characteristic microbiota in vitiligo patients is predominantly characterized by Bacteroides, whereas the characteristic microbiota in the healthy control group is primarily dominated by Bifidobacterium. Bifidobacterium, a renowned probiotic, produces short-chain fatty acids like butyrate and influences tryptophan metabolism, thereby improving atopic dermatitis via the gut-skin connection (Fang et al., 2022). Intriguingly, Bzioueche et al. (2021) observed a depletion of both Bifidobacterium and Bacteroides in the lesional skin microbiota of vitiligo patients. Consequently, further investigation is needed to explore the relationship between gut microbiota and skin microbiota. Moreover, subgroup analysis conducted on disease-healthy controls also revealed similar distinct outcomes. In addition to the aforementioned major differential genera, Parabacteroides, another distinct genus, was identified in the disease group through subgroup analysis. Analogous to Bacteroides, this genus not only generates short-chain fatty acids like acetic acid but also serves as an important bacterial source of sphingolipids, which can modulate immunity and inflammation by stimulating natural killer T cells and triggering rapid cytokine release (Heaver et al., 2018; Lei et al., 2021a; Cui et al., 2022). Consistent with our study, Liu et al. (2024) recently observed an enrichment of both genera in infants with severe atopic dermatitis. However, in a vitiligo-related study by Wu et al. (2023), contradictory results were reported. As mentioned earlier, these discrepant results may be limited not only by species specificity but also by limited studies.

The analysis of predicted functional pathways highlights the significance of glycan biosynthesis and metabolism, as well as the metabolism of cofactors and vitamins pathways in disease groups, and the Hypoxia-inducible factor 1 (HIF-1) pathway. Glycans play a crucial role in cellular biology, and any changes in their biosynthesis and metabolism can have implications for disease mechanisms such as autoimmunity and chronic inflammation (Alves et al., 2022; Pinho et al., 2023). In a recent study, Ali et al. (2022) discovered a notably higher presence of advanced glycation end-products (AGEs) in the affected skin of individuals with vitiligo when contrasted with healthy subjects. Additionally, vitamins are essential for maintaining intestinal immune homeostasis, which involves antibody production and the activation of immune cells (Hosomi and Kunisawa, 2017). For instance, the active variant of 1α,25-dihydroxyvitamin D3 plays a role in regulating immune reactions by influencing the function of immune cells such as T cells and B cells (Mann et al., 2015; Hosomi and Kunisawa, 2017). A recent study by Agarwal et al. (2015) indicated that the average serum levels of vitamin B12 in vitiligo patients were notably lower than those in the control group. Nonetheless, it is important to highlight that vitamin C, recognized for its antioxidant and immune-regulating properties, is not recommended for the treatment of vitiligo, as it may disrupt melanin production pathways (Chen et al., 2021a; Dutta et al., 2022). In addition, Metabolic processes, such as the fermentation of dietary fibre by intestinal flora to produce SCFAs, consume oxygen, thereby reducing the local oxygen concentration in the intestine and creating a hypoxic microenvironment. This hypoxic state will activate the intracellular hypoxia-sensing mechanism, which will stabilize and activate HIF-1, a key transcription factor that regulates a series of genes related to hypoxia adaptation. These include genes promoting angiogenesis, altered glucose metabolism and cell proliferation and survival, which enable the cells to adapt to the hypoxic environment. The activation of the HIF-1 pathway will consequently impact the metabolism and function of intestinal epithelial cells, thereby modifying the intestinal microenvironment, including pH and redox status. These modifications will influence the survival and reproduction of the intestinal flora, resulting in an increase or decrease in the number of specific flora, which in turn affects the composition of the intestinal flora. Additionally, the HIF-1 pathway can regulate the absorption and transport of certain nutrients by the intestinal epithelial cells, which may indirectly affect the metabolic activities of the intestinal flora. To illustrate, the fermentation and utilization of substances such as dietary fibre by the intestinal flora may be affected, resulting in alterations to the production of metabolites by the intestinal flora (Pral et al., 2021). Modifications to the structure of the flora and its metabolites may influence the local or systemic immune response of the body, thereby affecting the development of vitiligo.

Finally, we investigated the potential relationship between age and vitiligo characteristics based on microbiota and disease severity. The findings revealed a positive association between Bacteroides and Parabacteroides with the VASI score and disease duration in vitiligo patients. Nevertheless, as the patients aged, a downward trend was observed. Moreover, an upward trend was observed in VASI concerning disease duration, although no significant correlation was found with age. This finding is similar to the conclusions drawn by Demirkan et al. (2018), who observed that VASI in individuals with vitiligo showed a moderate association with the disease duration (r = 0.555; p < 0.001) and a weaker correlation with age (r = 0.349; p = 0.043). Nevertheless, these results contradict those reported by Patvekar et al. (2017). Given the scarcity of pertinent research available at present, additional exploration is required. Furthermore, some studies have indicated a notable positive correlation among VASI, disease duration, lesion location, and quality of life index (Algharably et al., 2019; Awal et al., 2024). However, our study did not incorporate other pertinent indicators, which constitutes a limitation of this research. In addition, we evaluated a number of genera as predictors of vitiligo, among which Parabacteroides, Lachnospira, Bacteroides, and Sutterella had some degree of diagnostic accuracy, which may help in the diagnosis of vitiligo.

Currently, some studies have revealed the efficacy of probiotics in the treatment of certain skin disorders such as atopic dermatitis, acne and psoriasis (Szántó et al., 2019). Researchers are actively exploring the relationship between microbiome changes and the development of vitiligo in order to identify potential therapeutic targets. Touni et al. (2024b) found that oral administration of neomycin affected the gut microbiota, thereby delaying the development of vitiligo in mice. A recent report showed that Bacillus subtilis-derived-exopolysaccharide halts depigmentation and autoimmunity in vitiligo (Touni et al., 2024a). The preceding studies indicate that the utilization of microbiome-targeted therapies has the potential to serve as a valuable strategy for preserving overall skin health and managing various dermatological conditions. While the application of microbiome-targeted therapies for vitiligo remains to be extensively researched, the modification of the microbiome composition through probiotics or analogous medications has the potential to emerge as an effective alternative approach for treating vitiligo in the future. This approach has the potential to mitigate the adverse effects associated with conventional therapies and enhance the therapeutic efficacy of the drugs, thereby reducing symptoms and improving the quality of life of patients with vitiligo.

In conclusion, the research findings not only illustrate variations in the composition of gut microbiota between vitiligo patients and healthy individuals within the same age group but also emphasize the age-related aspect of these variances. The study identifies specific microbial markers in the gut microbiota of vitiligo patients across different age groups. However, further investigation is crucial in the field of age-related disease microbiome studies. Upcoming studies ought to utilize sophisticated methods like metagenomics and metabolomics to deeply investigate the variations in gut microbiota at a finer species and metabolite functional level concerning disease onset and advancement. Additionally, increasing the sample size and conducting multicenter studies are essential to validate the reliability of the results. These endeavors will establish a theoretical basis for advancing the clinical application of gut microbiota-based therapeutic interventions for vitiligo.