Celiac disease (CD) is an autoimmune inflammatory disease affecting the small intestine, induced by the ingestion of gluten in genetically susceptible individuals (Ludvigsson et al., 2013; Lebwohl et al., 2018). The pooled global prevalence of biopsy-confirmed CD is 0.7%, and the pooled global prevalence of positive results from serologic testing is 1.4% (Singh et al., 2018). This disease can develop at any age, including geriatric populations (Collin et al., 2018). In recent years, the incidence rate of CD has been increasing, partly owing to improved understanding and detection of the disease and to the effective increase in the prevalence of immune disease (Lebwohl and Rubio-Tapia, 2021). The occurrence and development of CD is the result of the interaction of genetic, immune, and environmental factors (D’Avino et al., 2021). Gluten is recognized as an external trigger for CD (Serena et al., 2019b). Gluten is a protein complex mainly existing in wheat, barley, and rye (Wu et al., 2021). Due to its richness in glutamine and proline, it is highly resistant to protease hydrolysis. As a result, it cannot be fully digested by human digestive enzymes, resulting in large peptides (such as 33-mer) that can accumulate on the lamina propria of the small intestine; this can trigger abnormal immune responses in CD susceptible individuals (carrying HLA-DQ2/DQ8 genes), damage the intestinal mucosa, and lead to the occurrence of CD (Shan et al., 2002; Molberg et al., 2005; Zhu et al., 2022). Approximately 30% of the general population carries the HLA-DQ2/8 susceptibility gene, but only 1% of these individuals will develop CD. This indicates that there are other factors involved in the development of the disease, such as infant delivery style, feeding type, timing and amount of gluten intake, and infection and early antibiotic exposure, which indirectly play a pathogenic role in regulating the gut microbiota (Akobeng et al., 2020).

Gut microbiota play an important role in host nutrition metabolism, maintaining the structural integrity of the intestinal mucosal barrier, immune regulation, and resistance to pathogens (Thursby and Juge, 2017; Rinninella et al., 2019). A growing body of evidence indicates that the occurrence of CD is closely related to the ecological imbalance of gut microbiota (Valitutti et al., 2019). Many host-related factors—such as age, sex, genetic susceptibility, and health status—and environmental factors influence the composition of gut microbiota (Olivares et al., 2015; Rehman et al., 2016; Valitutti et al., 2019; Trakman et al., 2022). Garcia-Mazcorro et al. believed that the gut microbiota distribution pattern and metabolic activities differ significantly from person to person, and the impact of food composition, dietary habits and economic level on health varies greatly in between and within countries (Garcia-Mazcorro et al., 2018a; Rinninella et al., 2019). It is suggested that more studies based on local populations should be conducted to draw useful conclusions in biology and medicine, which will pave the way for discovering better treatment protocols for patients with CD.

The gut microbiota also acts as a metabolic organ, interacting with host cells and providing functional support needed to maintain homeostasis (Olshan et al., 2020). Metabolomics is an important part of systems biology and is one of the key technologies for understanding the function of these microbial metabolites (Bauermeister et al., 2022). The fecal metabolome provides functional readings of microbial activity and can serve as an intermediate phenotype to mediate these interactions (Zierer et al., 2018). The metabolic capacity of the gut microbiota has been studied in the CD population, and there are differences in short-chain fatty acid metabolism between untreated and treated CD patients and healthy controls (HC) (Nistal et al., 2012b), as well as differences in specific microbiota that metabolize gluten in CD patients (Caminero et al., 2015). Thus, differences in microbial metabolites in CD patients as compared to the normal population indicate the functional role of the microbiota in the pathogenesis of CD. However, the specific mode of action of gut microbiota disorders involved in the occurrence of CD is not clear; it is likely to cause changes in relevant terminal metabolites in the feces of patients. At present, there is still a lack of consensus on specific bacteria and metabolites in patients with CD, and the identification of specific CD metabolome phenotypes could help identify additional diagnostic tools and therapeutic interventions.

A meta-analysis showed that the seroprevalence of CD in the general Chinese population is 0.27%, and varies with the geographical origin of the patients, being higher in northern China than in southern China (Zhou et al., 2021). Xinjiang, located in the Northwest of China, has a population of 25.85 million people divided between 55 ethnic groups. Some major ethnic groups are Uyghur (45%), Han (42%), and Kazakh (7%) (Statistics Bureau of Xinjiang Uygur Autonomous Region, 2021). Wheat is the primary food crop for this population. Furthermore, historically, they are characterized by different degrees of genetic exchange with western Europeans (Caucasians). Zhou et al. (2020) reported that the detection rate of CD in Xinjiang is approximately 1.27%, which is higher than the reported national average. These factors may partially account for this high prevalence, although additional external factors should also be taken into consideration. Xinjiang has a unique geographical environment, genetic background, and population composition, which results in the special living and eating habits of its residents. It is well known that differences in dietary habits and genetic background may lead to differences in gut microbiota. Therefore, the current research results of other countries and regions cannot be extrapolated to the characteristics of CD in Xinjiang. The purpose of this study is to analyze the fecal microbial composition and metabolome characteristics in patients diagnosed with CD in Northwest China, and to screen potential biomarkers which could be used for diagnosing the disease. To the best of our knowledge, this study is the first in China to investigate gut microbiota in this clinically important condition.

Advances in the field of microbial research have established a clear link between the gut microbiome and human health (Weir et al., 2021). There is increasing evidence that gut microbiota and their metabolites play an important role in the development of CD, since they are closely related to dietary habits, genetic background, and geographical factors. The incidence of CD in China, for instance, has ethnic and regional differences, but there is a lack of research on gut microbiota related to CD. Xinjiang is located in the Northwest of China, which is a multi-ethnic region with unique geographical environment, genetic background and population composition. Therefore, the conclusions drawn by relevant studies carried out in other countries and regions may not be completely applicable to the population in this region. The purpose of this study is to analyze the fecal microbial composition and metabolome characteristics in patients diagnosed with CD in Northwest China. The results emphasize that the imbalance of gut microbiota resulting in metabolic disorders is an important factor in the pathogenesis of CD, providing some new clues the use of gut microbiota and fecal metabolites as biomarkers to screen or diagnose CD, and providing intervention targets its future treatment.

In the past, CD was considered rare in China; however, a growing number of patients have been diagnosed in recent years. Previous research showed that the seroprevalence of CD in the general Chinese population was 0.27% (Zhou et al., 2021), while the prevalence of positive results from serologic testing in Xinjiang is 1.27% (Zhou et al., 2020); this is higher than the reported national average and closer to the pooled global average (Singh et al., 2018). Gluten is recognized as the main external environmental trigger of CD (Serena et al., 2019b); andhe consumption of gluten-containing cereals in Xinjiang is very high. According to the Xinjiang Statistic Year Book data (2021), the yearly cereal intake per person was 151.81 kg in total: 129.27 kg/person/year in urban Xinjiang and 171.89 kg/person/year in rural Xinjiang. Therefore, high wheat consumption is a critical environmental risk factor for the occurrence of CD in this area. Gut microbiota have host genetic relevance. Some studies have shown that HLA-DQ genotype may affect the first generation of gut colonizers, and this change in gut microbiota increases the risk of CD (Olivares et al., 2015; Valitutti et al., 2019). Data from a meta-analysis data indicate that the frequency of the DQB1*0201 allele frequency is particularly high (22.04%) in the populations (Yuan et al., 2013). Zhou et al. (2020) described the frequency of the HLA-DQ2 and-DQ8 haplotypes among four major ethnic groups in Xinjiang: it was highest in the Uyghur (52.1%), followed by the Hui (44.4%), the Kazakh (40.0%) and the Han (39.4%). Therefore, the genetic susceptibility for CD is highly prevalent in the Xinjiang region. These factors also lead to the special dietary habits of its residents, diet plays an important role in regulating the composition of human gut microbiota and metabolic activities, and dietary factors in early life may largely determine the risk of future health or disease (Chen et al., 2018; Valitutti et al., 2019; Trakman et al., 2022). The intake of carbohydrates and fat in the dietary structure of Xinjiang is high, while the intake of micronutrients, dietary fiber and high-quality protein is low (Yin et al., 2020; Tao et al., 2022). Because of its unique geographical environment, genetic background, population composition, and dietary culture, the fecal microbial composition and metabolome characteristics of patients with CD was unique in this region.

In this study, Firmicutes and Bacteroidetes were the dominant genera between the two groups, similar to other human gut microbiota studies (Verdu et al., 2015; Kumar et al., 2018; Valitutti et al., 2019), while Proteobacteria community abundance in the CD group was significantly higher than in the HC group, which may contribute to the development of the disease. In rat models, the combined action of gliadin and Proteobacteria member Shigella has been proved to enhance the destruction of the intestinal epithelial barrier (Cinova et al., 2011), possibly because Proteobacteria dominated bacteria and toxins are more likely to penetrate the intestinal mucus layer (Jakobsson et al., 2015). Wacklin et al. (2013) found that increased Proteobacteria colonization was associated with the common gastrointestinal manifestations of CD. In addition, Proteobacteria with adhesive and invasive properties are thought to promote inflammatory responses, leading to inflammatory bowel disease in genetically or immunologically susceptible populations (Mukhopadhya et al., 2012). Therefore, this confirms that most individuals share a common microbiome with inter-individual variability, which can be explained by factors such as diet, geography, and ethnicity.

Current studies show that the change of microbiota in CD patients is mainly manifested by the increase of Gram-negative bacteria and the decrease of Gram-positive bacteria (Girbovan et al., 2017). At the genus level, fecal samples of CD in the region showed a significant decrease in the numbers of Firmicutes (Ruminococcus, Faecalibacterium, Blautia, Gemmiger, and Anaerostipes), which are all Gram-positive bacteria. There was also a higher abundance of Proteobacteria (Holdemanella, Haemophilus, Sutterella), Bacteroidetes (Veillonella, Allisonella), and Firmicutes (Streptococcus, Lactobacillus), which are mainly Gram-negative bacteria, indicating the imbalance of CD fecal microbiota. Among them, Streptococcus, Haemophilus and Veronococcus are all opportunistic pathogens, which mainly colonize and infect human beings and cause many diseases (Nørskov-Lauritsen, 2014; Andam and Hanage, 2015; Diricks et al., 2022). Moreover, several cohort studies have shown that different gastrointestinal infections increase the risk of CD, with viral and bacterial factors being involved in the pathophysiology of the disease (Caminero and Verdu, 2019). Previous studies have found that the incidence of Streptococcus significantly increased in the saliva and tissues of CD patients (Nistal et al., 2012a, 2016; Iaffaldano et al., 2018; Serena et al., 2019a; Zhan et al., 2022), and similar results were found in the stool samples of this study. Multiple studies (Ludvigsson et al., 2008; Thomas et al., 2008; Tjernberg et al., 2020) have shown an increased risk of invasive pneumococcal disease in CD, and the European CD guidelines recommend that CD patients are vaccinated with the Streptococcus pneumoniae vaccine (Al-Toma et al., 2019). Furthermore, Veroniella is a proinflammatory taxon that increases in patients with inflammatory bowel disease and irritable bowel syndrome (Shukla et al., 2015; Ruigrok et al., 2021) and promotes the expression of several inflammatory cytokines (TNF-α, IL-6, and IL-1β) in the colon (Shukla et al., 2015). It is also increased in stool samples from patients with CD or refractory CD (Garcia-Mazcorro et al., 2018b; Panelli et al., 2020). In general, the main features are increased abundance of potentially pathogenic Gram-negative bacteria, especially highly pathogenic gut bacteria, the activation of proinflammatory pathways, and the induced secretion of Th1 type proinflammatory cytokines (De Palma et al., 2010). In addition, most reports show that compared with healthy people, patients with CD have an imbalance of Lactobacillus, which shows that the diversity and abundance of probiotics are reduced (Girbovan et al., 2017; Valitutti et al., 2019; Serena et al., 2019a; Francavilla et al., 2020). Interestingly, we did not find these changes, and Nistal et al., 2012b also found similar results, which may be because the presence of Lactobacillus in the intestines is affected by dietary intake (Graf et al., 2015). People in Xinjiang have a high intake of fermented dairy products in their diet, and CD patients may have spontaneous excessive intake due to gastrointestinal symptoms. Therefore, the people in Northwest China have unique gut microbiota characteristics, and Lactobacillus as a supplement may not improve CD in this region.

Despite inflammation in CeD being localized to the small intestine, studies in both pediatric and adult populations have frequently employed fecal analysis to investigate the microbiota due to ease of sampling. However, functional heterogeneity of each gastrointestinal tract segment gives rise to regional differences in gut microbial populations (Martinez-Guryn et al., 2019). The regional tropism of gastrointestinal tract microbiota can be a determinant of specific diseases. A recent study found that (Constante et al., 2022) microbiota clustering based on Marsh grades within the duodenal mucosa, but not within aspirate or fecal samples, indicated the duodenal microbiota is altered in association with intestinal damage, also suggests that the result based only on fecal analysis must be interpreted with caution. Our study also found that the fecal microbiota and metabolome were not associated with intestinal injury. Therefore, the severity of intestinal damage in CD may mainly affect the change of the duodenal mucosal microbiota. Although this is a fair argument to propose, it is possible that events in the large bowel influence disease pathogenesis upstream along the gastrointestinal tract. Irrespective of their primary role in CD pathogenesis, the disease-specific microbial signature identified here might be used as another adjutant, noninvasive biomarker to screen for CD.

Gut microbiota regulate a series of key metabolic functions in the body, and its imbalance is an important factor in regulating host metabolic disorders (Pascale et al., 2018). In a recent study, aspirates from the small intestine of CD donors were poured into the stomachs of mice. The intestinal contents from mice colonized with CD microbiota exhibited altered gluten metabolism, suggesting that proteolytic function is transmitted by the microbiota associated with an inflamed celiac duodenum, and further supporting the role of microbial metabolism in CD (Constante et al., 2022). The fecal metabolome can serve as an intermediate phenotype mediating host-microbiome interactions. It could potentially be a new tool for exploring the links between microbiome composition, host phenotype, and heritable complex traits (Zierer et al., 2018). To the best of our knowledge, there are few studies using LC–MS/MS untargeted metabolomics to investigate fecal metabolome changes in adult CD subjects. There is also a lack of joint analysis of relevant microorganisms and metabolites in patients with CD. In this study, we used 16S rDNA and metabolomics sequencing to reveal changes in gut microbiome composition and underlying mechanisms of gut microbiome mediated metabolic dysfunction in CD patients in Northwest China. The significant difference in fecal metabolite levels between CD and HC groups suggest that CD can cause changes in fecal related terminal metabolites. In our study, 222 fecal metabolites that were differentially regulated between sample sets were identified. Finally, 13 metabolites with significant differences between the CD and HC groups were screened using VIP >2.0 and p < 0.05 as screening conditions. Mainly, the levels of amino acids and their derivatives (metabolites, steroids, lipids, choline, oxidized derivatives of unsaturated fatty acids, esters, and niacin metabolites) were decreased in the CD group. According to pathway enrichment analysis, they are mainly involved in the metabolic pathways of biosynthesis and metabolism of amino acids, biosynthesis of unsaturated fatty acids, nicotinate and nicotinamide metabolism, and in aminoacyl tRNA biosynthesis. The decreased levels of choline (Acetylcholine), methionine (S-methyl-L-methionine, a derivative of methionine) and lipids (especially lysoPA (16_0_0_0), docosapentaenoic acid (22 N-3)) indicated the deficiency of one-carbon metabolism (OCM) in CD patients. Martín-Masot et al. (2020) were the first to describe metabolic deficiencies in OCM in patients with CD. OCM is a network of biochemical reactions in which one-carbon units are transferred to various biosynthetic pathways; folic acid and methionine cycles are two important components of this network (Newman and Maddocks, 2017; Pan et al., 2021; Lionaki et al., 2022). They are functionally used for biosynthesis of phospholipids, amino acids and DNA, as well as methylation of proteins, RNA, and DNA. CD mainly causes progressive villous atrophy, crypt hyperplasia and proliferation of epithelial lymphocytes in small intestinal mucosa, affecting the absorption of trace elements such as folic acid and vitamin B₁₂. This results in malnutrition (Balaban et al., 2019) and damage to energy generation pathways (Sharma et al., 2015; Upadhyay et al., 2020), while a close dependence between the regulatory pathways of energy and OCM, as well as the balance between input and output, remains (Luciano-Mateo et al., 2017). Therefore, the deficiency of OCM in CD patients may be related to energy deficiency caused by malabsorption. These findings will provide new translational opportunities for CD dietary interventions, drug development, and biomarker identification.

Previous studies have found higher levels of short-chain fatty acids (SCFAs) in both untreated and treated patients with CD than in healthy controls (Nistal et al., 2012b; Tjellström et al., 2013; Caminero et al., 2015)—these findings usually involve pediatric patients. However, in this study, there was no significant difference in the content and composition of SCFAs between adult CD patients and HCs, which is consistent with the results of Niccolai et al.’s (2019) study. They considered that age may affect the composition of SCFAs, and the disease-specific mechanism involving gut microbiota is still unclear. In the gastrointestinal tract, tryptophan can be metabolized by the gut microbiota into ligands of the aryl hydrocarbon receptor (AhR), which contributes to intestinal homeostasis (Roager and Licht, 2018). Lamas et al. (2020) identified a potential pathogenic mechanism related to the impaired production of AhR ligands by the gut microbiota in CD. A recent study has been found that tryptophan metabolism in patients with potential CD is lower than in both CD and HC groups. The altered metabolic profile of patients with potential CD suggested that gluten intolerance was evident at the metabolic level before intestinal damage (Upadhyay et al., 2022). This study found that tryptophan derivative N-Acetyl-D-tryptophan in stool metabolites of CD patients was downregulated. Therefore, based on the aforementioned evidence, a tryptophan-enriched diet may be an effective adjuvant treatment for CD.

We further explored the potential use of gut microbiota related metabolites in the noninvasive diagnosis of CD. The gut microbiota of four genera (Clostridium_IV, Veillonella, Ruminococcus, and Gemmiger) can distinguish CD from HC, with an AUC of 0.85. The model based on six metabolites (D-alanyl-D-serine, acetylcholine, coronopilin, formiminoglutamic acid, N(6)-methyllysine, and lysoPA) performed better than microbial characteristics, with an AUC of 0.98. The current diagnosis of CD is based on a combination of features, including CD specific serum antibodies (anti-tTG antibody, anti-endomysial antibody, or anti-DGP antibody) and the presence of villous atrophy assessed by intestinal mucosal biopsy (Bai and Ciacci, 2017; Al-Toma et al., 2019). However, poor or inadequate biopsy specimens and patchy mucosal damage may increase the risk of false positive or false negative results (Pais et al., 2008; Ravelli et al., 2010; Mubarak et al., 2011). Therefore, we need more accurate noninvasive biomarkers to complement traditional serological tests to diagnose CD.

In this study, the gut microbiota and metabolites were linked with a multi-omics integration method. Correlation analysis showed that the decrease of Gram-positive bacteria, such as Firmicutes (Clostridium_IV, Ruminococcus Christensenella), was closely related to amino acid metabolism, esters, alcohols and other organic compounds. Overall, these results suggest a significant interaction between gut microbiota and metabolites that may influence the occurrence of CD. By studying the correlation between some metabolites and microbial communities, it may eventually help to manipulate the function of microbial communities in CD therapeutically (McHardy et al., 2013; Gundamaraju et al., 2017).

This study has some limitations. Most importantly, it has a relatively small sample size, and different diagnostic models based on microbial composition and metabolites may limit the generalization of the results, requiring external validation through larger samples and multi-center trials. Nevertheless, confounding factors can be effectively controlled by recruiting age-, sex-and ethnic-matched healthy subjects. Moreover, this was a case–control study. Although our data proposed the functional association between microbiome, metabolome, and disease, cause and effect could not be distinguished, and the mechanism of functional association needs to be further explored.

In conclusion, studying the relationship between human gut microbiota and CD in Northwest China has highlighted some unique microbiota and metabolic characteristics and opened a new way for researchers to explore the potential beneficial effects of supplementing specific nutrients. This study provides an experimental basis for formulating potential diagnostic and therapeutic targets in the future. These results should encourage more research on CD in China.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI-PRJNA890948.

The studies involving human participants were reviewed and approved by the consent of the ethics committee of the people’s Hospital of Xinjiang Uygur Autonomous Region (approval number: KY202203110 67). The patients/participants provided their written informed consent to participate in this study.

TS and GF: conceptualization. TS: writing original draft. YF and AA: data curation. WL and TL: software. HL and MW: formal analysis. ZL and JL: methodology. AM and HJ: project administration. GF: supervision. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (82260116), the Natural Science Foundation of Xinjiang Province (2021D01C149), and the Hospital Project of People’s Hospital of Xinjiang Uygur Autonomous Region (20200406).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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