4.2 Forward MR Analysis
The normal balance of gut microbiota protects the intestinal mucosal barrier; hence, the disturbance of gut microbiota ecological balance is a key factor in the development of IBD26. Imbalances in microbiota abundance can alter gut metabolites, increase pro-inflammatory mediators, impair cell barrier function, thereby contributing to the development of inflammatory diseases. In this study, the reduction in the risk of CD and UC by gut microbiota is primarily achieved through the production of short-chain fatty acids (SCFAs). SCFAs inhibit histone deacetylase (HDAC), reduce NF-κB-induced pro-inflammatory mediator expression, while increasing anti-inflammatory mediators, enhancing intestinal epithelial barrier function, promoting antimicrobial peptide (AMP) production, facilitating cell proliferation, and mitigating inflammation27. Acetic acid, propionic acid, and butyric acid are the major components of short-chain fatty acids in the human intestine. Both Family XIII AD3011 group28 and Clostridiales vadin BB60 belong to Clostridiales and can produce butyrate to reduce the risk of IBD. Currently, research on Family XIII AD3011 group and Clostridiales vadin BB60 is limited. Some studies have shown a positive correlation between Family XIII AD3011 group and leukocyte telomere length, reflecting aging, but it is uncertain whether this effect is related to inflammation29. Additionally, the Family XIII AD3011 group is positively correlated with ACOD1 expression levels29. In contrast, ACOD1 is highly expressed in the colon tissues of UC patients, and its expression is positively correlated with the severity of intestinal inflammation. This is in contrast to our findings, as this study demonstrates that Family XIII AD3011 group can reduce the risk of both UC and CD. This requires further in-depth research to clarify. The family Clostridiales vadin BB60 group is a unique unnamed lineage within the order Clostridiales. Studies have found that the abundance of Clostridiales vadin BB60 group increases after drug administration in patients with multiple sclerosis (MS)30. The application of exogenous lipase alters the composition of the gut microbiota in APP/PS1 mice (increased abundance of Clostridiales vadin BB60), improving Alzheimer's disease31, which may suggest that this bacterium can reduce inflammatory responses. Our study confirms that Clostridiales vadin BB60 group can reduce the risk of UC.
Both Lactobacillaceae and Bifidobacteriaceae are bacteria that produce butyrate. Furthermore, Lactobacillus acidophilus in Lactobacillus and Bifidobacterium animalis subsp. lactis in Bifidobacteria may exert effective anti-inflammatory effects by regulating the TLR2-mediated NF-κB and MAPK signaling pathways in intestinal epithelial cells (IECs). These two strains, especially their combination, may be novel adjuvants for IBD therapy32. In normal intestinal epithelium, butyrate is the preferred fuel for IECs, and it maintains intestinal integrity and enhances the epithelial barrier function of the intestine and potential diseases through various pathways. In cancerous colonic epithelium, butyrate has been shown to have anti-tumor effects on colorectal cancer cells. In immune cells, butyrate acts as an immunomodulator on immune cells, including T cells, antigen-presenting cells, monocytes, and neutrophils. It mainly promotes the activation and proliferation of Treg cells, induces IL-10 production, and thereby enhances the immune barrier function of the intestinal mucosa. It also inhibits the release of pro-inflammatory factors such as IL-2, IL-8, and TNF-α, exerting effective anti-inflammatory effects33,34. In other mechanisms, the Bifidobacterium Longum: has antioxidative effects, reduces the expression of intraintestinal cytokines, enhances intestinal barrier function, inhibits NF-κB signaling, and regulates carbohydrate transport and metabolism (e.g., tolerance to bile salts and acids), and modulates the gut microbiota35,36. Moreover, the extracellular vesicles released by Lactobacillus rhamnosus GG (LGG-EVs) improve intestinal inflammation through inhibiting the TLR4-NF-κB-NLRP3 axis activation, reducing pro-inflammatory cytokine levels (TNF-α, IL-1β, IL-6, IL-2), and ameliorating gut dysbiosis37. SCFA butyrate promotes epithelial barrier function, serves as a primary stabilizing mechanism for hypoxia-inducible factor-1 (HIF-1, a transcription factor coordinating barrier protection), serves as the main energy source for colonic cells, and also exerts anti-inflammatory effects in the colonic mucosa by inhibiting and activating G protein-coupled receptors (GPCRs) present in IECs and immune cells through histone deacetylase (HDAC) inhibition27.
Lachnospiraceae, a family of bacteria, is a major producer of butyrate34. Our study demonstrates that Lachnospiraceae UCG008 is associated with a reduced risk of CD. A cross-sectional study identified a decrease in Lachnospiraceae abundance as a potential target microbiota for IBD development38. In clinical studies, gut dysbiosis is associated with CD39 and may play a crucial role in triggering disease recurrence after surgery. Features of the mucosa-associated microbiota in CD patients and postoperative disease recurrence patients include a reduction in butyrate-producing taxa, such as Lachnospiraceae40. Anti-tumor necrosis factor monoclonal antibodies, such as adalimumab (ADA), significantly improve clinical remission and induce clinical response in patients with mild to moderate active CD by increasing protective microbial taxa at the genus level, such as Lachnospiraceae_unclassified41. Furthermore, changes in gut microbiota can be used to predict the efficacy of ADA. Siberian ginseng increases the abundance of Lachnospiraceae to reverse the proportion of splenic and peripheral blood lymphocyte subpopulations, stimulate intestinal CD4 + T cells, and IgA-secreting cells, thereby ameliorating immune dysregulation42. Fecal microbiota transplantation (FMT) in clinical trials may alleviate UC in patients by increasing the abundance of Lachnospiraceae43. In basic research, Lachnospiraceae and Ruminococcaceae in the Clostridiales order primarily reside in the outer mucus layer and come into contact with the intestinal lumen, which helps prevent bacteria and other microorganisms from entering the intestinal wall44. Lachnospiraceae UCG008 and Lachnospiraceae UCG010 are two distinct genera within the family Lachnospiraceae that have not yet been classified. Our study demonstrates that Lachnospiraceae UCG008 can reduce the risk of CD, while Lachnospiraceae UCG010 promotes the development of UC. Limited research has shown that the intake of modern wheat may exert anti-inflammatory effects by increasing the abundance of Lachnospiraceae_UCG-008 spp., promoting butyrate production45. Smoking in AS patients increases the abundance of g_Lachnospiraceae_UCG-008, possibly as a manifestation of compensatory anti-inflammatory effects46. On the other hand, Lachnospiraceae_UCG-010 has been found to be involved in mediating hepatic inflammation, leading to lipid metabolism disorders47. The Chinese herbal formula Erwu Lv Xue Pill may alleviate rheumatoid arthritis by reducing the abundance of Lachnospiraceae_UCG-01048. The water extract of Cymbopogon citratus (DC.) Stapf (CCAE) may improve loperamide-induced constipation in mice by reducing the abundance of Lachnospiraceae UCG-01049. The bacteria of these two genera in the Lachnospiraceae family may be the focus of further research on the etiology of IBD.
Paraprevotella is a genus within the family Prevotellaceae. Mendelian randomization studies have shown that this bacterium is a protective factor for CD. Basic research has shown that, compared to the low-fat diet group, the exercise group with a higher abundance of Paraprevotallaceae reduces inflammation and promotes intestinal health50. Additionally, supplementation with flaxseed increases the abundance of Paraprevotella in the intestinal mucosa of CD mice and elevates butyrate levels, thus exerting beneficial effects on the CD mouse model51. Some studies have shown that Paraprevotella is positively correlated with the concentrations of propionate, butyrate, and valerate in the intestines52. There is also evidence that antigen B (AgB) can prevent IBD by increasing the abundance of Paraprevotella and Ruminococcaceae in the intestines of DSS-induced mice, as the increase of beneficial gut microbiota can ameliorate the inflammatory state26. This also corroborates our findings.
Turicibacter is an obligate anaerobic Gram-positive bacterium that produces lactic acid through sugar fermentation and is commonly detected in the gastrointestinal tracts and feces of humans and animals. It has been shown that Turicibacter is positively correlated with pro-inflammatory cytokines such as IL-6, IL-1β, TNF-α, and IFN-γ53.
Intestinimonas is a genus within the family Oscillospiraceae. Currently, research on Intestinimonas is limited. One study has shown a positive correlation between triglycerides (TG) and Intestinimonas, where high TG levels lead to hyperlipidemia and subsequently promote inflammation. TNF-α can be produced by adipocytes and promotes the production of IL-6 by activating the MAPK and NF-κB signaling pathways. IL-6 induces inflammation by affecting the secretion function of intestinal epithelial cells54.
The family Oxalobacteraceae belongs to the order Burkholderiales and is composed of anaerobic Gram-negative bacteria. The genus Oxalobacter is one of its genera. A two-sample Mendelian randomization analysis revealed a positive correlation between Oxalobacter and IBD, which is consistent with our research findings. Studies have shown that a fiber-rich diet is associated with reduced levels of Oxalobacteraceae55, and such a diet can promote the production of anti-inflammatory substances by probiotics56. Gene set enrichment analysis has also identified a correlation between Oxalobacteraceae and IBD57. However, other studies have reported a lower frequency of Oxalobacter colonization in individuals with inflammatory bowel disease Further research is needed to explore its underlying mechanisms.
Additionally, we identified that Lachnospiraceae UCG-010, Paraprevotella, Eubacterium ruminantium, Eubacterium coprostanoligenes, and Ruminococcaceae UCG005 promote the development of UC or CD.
Probiotics are living organisms that regulate the gut microbiota in various ways to improve gut health. When probiotics are administered in appropriate proportions, they exert positive effects on the host organism27. Oral administration of infant Bifidobacterium reduces levels of C-reactive protein (CRP) and TNF-α in gastrointestinal and non-gastrointestinal inflammatory diseases58. Long Bifidobacterium significantly reduces disease activity index and rectal bleeding, leading to clinical remission59. A probiotic mixture (Streptococcus salivarius, Lactobacillus acidophilus, and Bifidobacterium bifidum strains) demonstrates shortened recovery time, reduced disease activity, and improved endoscopic images in UC patients60. However, research on Family XIII AD3011 group and Clostridiales vadin BB60 group as probiotics is still in its preliminary stages. This study aims to provide new insights for future IBD treatment strategies.
4.3 Reverse MR Analysis
We also conducted reverse Mendelian randomization (MR) analysis and identified 15 bacteria, including Butyrivibrio and Eubacterium eligens group, whose abundance changes are associated with CD or UC. Although these bacteria have been shown to be associated with CD or UC in this study, they are not the causes of UC or CD but rather the consequences of disease development.
The most representative bacterial phyla (90%) in the gut species are mainly composed of four classes: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria2. Compared to healthy individuals, the gut mucosa of IBD patients in either active or quiescent states undergo various changes, one of which is alterations in the composition and functionality of the microbiota, commonly referred to as dysbiosis61. Dysbiosis is defined as a decrease in gut microbiota diversity due to changes in the balance between symbiotic and potentially pathogenic microorganisms5. CD and UC gut microbiomes exhibit a generally decreased classification diversity compared to the healthy gut microbiome. For example, many studies have reported decreased bacterial diversity and richness in IBD patients, characterized by a reduction in the Firmicutes/Bacteroidetes ratio, mainly due to a decrease in Firmicutes and an increase in Bacteroidetes62, as well as an increase in Actinobacteria and Proteobacteria.
IBD patients also show a reduction in species that produce butyrate2. The genera Butyrivibrio63,64, Oscillibacter63, Anaerotruncus63, Lachnospiraceae, and Ruminococcaceae all belong to the phylum Firmicutes and include Holdemanella65, and are all butyrate-producing bacteria. Furthermore, research has found that at the genus/species level, Lachnospiraceae and Ruminococcaceae, both belonging to the family Ruminococcaceae, are decreased in inflamed mucosa of IBD compared to healthy controls66.
Oscillibacter bacteria are often increased in IBD and are associated with its progression and development. Mucosal biopsies from inflamed and non-inflamed regions of the gut of IBD patients and healthy individuals showed a decrease in Firmicutes in IBD samples, an increase in Oscillibacter abundance62, and the genus Barnesiella belongs to the phylum Bacteroidetes. In a study, colitis-induced mice exhibited higher abundance of Ruminiclostridium 6 compared to the healthy control group67. Related studies have indicated that the abundance of Erysipelatoclostridium, a type of clostridial spore-forming bacteria, was significantly reduced in AOM/DSS-induced colitis-associated colorectal cancer mice68. However, multiple studies have found a lower abundance of Roseburia in UC patients, and this species is negatively correlated with disease activity69.
4.4 Strengths and Limitations
Our study has several strengths. Firstly, we employed the MR analysis method to explore the relationship between different gut microbiota and the risk of IBD, thereby controlling for potential confounding factors. The large sample size provided sufficient power to estimate the causal effects of gut microbiota on IBD. Secondly, we investigated 150 different families and genera of gut microbiota. Due to substantial differences among phyla and classes of gut microbiota, we only utilized data at the family and genus levels to obtain more specific conclusions.
However, this study also has several limitations. Firstly, some batches of SNP data remained pleiotropic even after MR-PRESSO, and the conclusions obtained from MR-Egger may not be sufficiently robust. Additionally, our study population is predominantly composed of Europeans, and limited data suggest that the impact of environmental exposures on IBD risk may vary by ethnicity or gender. Therefore, the conclusions cannot be generalized to larger populations at this time, and further research is needed to explore the impact of dietary intake on IBD incidence in different ethnic groups. As we used aggregated data instead of individual-level data, we were unable to conduct stratified analyses for variables such as gender. The statistical power of some correlation conclusions in this study is low, which may increase the risk of Type II errors. As the method of ratio estimation assumes linear causal relationships, this study cannot exclude the possibility of non-linear associations between diet and IBD incidence. Finally, the presence of individual canalization is inevitable in MR studies. However, it is important to note that as long as the assumptions of instrumental variables are met in the application of SNPs in this study, the MR conclusions remain valid.