Background

Pulmonary hypertension (PH) is characterized by remodeling of the pulmonary artery, resulting in irreversible right heart failure and progressive symptoms that often lead to fatality. The extracellular matrix remodeling and fibrosis in the pulmonary vessels, which correlate with loss of compliance, have been linked to chronic perivascular inflammation and immune dysregulation [1,2,3]. Among the five subtypes of PH, group 1 is known as pulmonary arterial hypertension (PAH) [4]. It was 73.5% for patients with PAH to survive 5 years without lung transplants [5]. Patients with other subgroups of PH can receive treatment for their underlying conditions [6,7,8]. However, PAH lacks alternative therapeutic options and can only be managed through drugs that target the carbon monoxide pathway, the endothelin pathway, and the prostacyclin pathway. Despite efforts, no new therapeutic pathways have been proven effective for the treatment of PAH since 2005 [9, 10]. Moreover, treatments for PAH have proven limited in effectiveness to date, and no cure is available.

Emerging evidences suggest that the migration of gut-derived microbes and microbial products to the lungs plays a pivotal role in the pathogenesis of numerous diseases [11, 12], including PAH [13, 14]. Patients with PAH exhibit a greater prevalence of various bacteria that typically promote inflammation, as well as a decreased prevalence of certain species with anti-inflammatory properties, compared to healthy control subjects [13,14,15]. This phenomenon was similarly observed in animal experiments, and the α diversity of the gut microbiota in different ways induced a decrease in animal models compared to the normal groups [16]. Additionally, the ratio of Firmicutes to Bacteroides increased [16,17,18], which served as a sensitive biomarker of gut dysbiosis. The aforementioned findings offer substantiation for the notion that the interplay between the gut microbiota and their metabolites serves as the mediator of the intestinal lung axis.

Nevertheless, despite the distinct alterations observed in the intestinal flora of individuals with PAH and animal models in previous studies, the underlying causal connection between intestinal dysbiosis and PAH remains unresolved. Investigating this causality is of significant clinical importance, as it may contribute to understanding the lung-gut axis in PAH development and facilitate the identification of potential therapeutic targets.

In this context, Mendelian randomization (MR) studies offer an approach to address these limitations by genetically evaluating the genuine causal association between exposure and outcome [19]. This methodology effectively mitigates the influence of unobserved confounding variables, as genetic variants are randomly allocated during conception and thus independent of adaptive lifestyle factors and behaviours. This study employed the genome-wide association study (GWAS) summary statistics obtained from the MiBioGen and NHGRI-EBI GWAS Catalog to conduct a two-sample MR analysis, aiming to assess the causal relationship between gut microbiota and PAH.

Method

Study design

A two-sample MR study was performed to assess the causal relationship between the gut microbiota and PAH. An overview of the study description is presented in the figure below (Fig. 1). Simultaneously, adherence to three fundamental assumptions of MR design is crucial to ensure the validity of instrumental variables: 1) instrumental variables (IV), represented by genetic variations, should exhibit a significant correlation with the gut microbiota (exposure); 2) genetic variations must be independent of both known and unknown confounding factors; and 3) there should be no direct correlation between IV and PAH (outcome).

Fig. 1
figure 1

The flow chart of the study. GWAS = genome-wide association study; LD = linkage disequilibrium; MR = Mendelian randomization; MAF = minor allele frequency; MR-PRESSO = Mendelian randomization pleiotropy residual sum and outlier; SNP = single nucleotide polymorphism

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GWAS data sources

The gut microbiota data utilized in this study were acquired from the international consortium MiBioGen (http://mibiogen.gcc.nl), to our knowledge, which is recognized as the largest publicly accessible sample size Genome-Wide Association Study (GWAS) of the gut microbiome. The datasets encompass the most recent comprehensive meta-analysis of genome-wide proportions, involving 18,340 individuals from 24 population-based cohorts, mostly derived from European populations (N = 13,266). Due to the diverse characteristics of age, sex ratio, and diet among cohorts, the researchers employed per-cohort and whole-study filtering methods to determine the taxa included in GWAS analyses.

The summary statistics of PAH were downloaded from the NHGRI-EBI GWAS Catalog (https://www.ebi.ac.uk/gwas) on August 27, 2023 for study GCST007228 [20, 21], which conducted a meta-analysis of four independent studies comprising a total of 11,744 samples (2085 PAH cases). These studies include: 1) the UK National Institute of Health Research Bio-Resource (NIHRBR) for Rare Diseases study, which recruited between January 29, 2003 and January 4, 2017; 2) the US National Biological Sample and Data Repository for Pulmonary Arterial Hypertension/PAH Biobank (PAHB) study, and PAH cases recruited between October 3, 2012 and March 14, 2016; and 3) the Paris Pulmonary Hypertension Allele-Associated Risk cohort (PHAAR) study, all of whom were identified by the French PAH Network from 1 January 2003 to 1 April 2010. Similarly, 4) the British Heart Foundation Pulmonary Arterial Hypertension GWAS (BHFPAH) study was recruited from 3 Dec 1998 to 1 Dec 2011 (Table 1).

Table 1 PAH GWAS samples in the study
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As the present study constitutes a reanalysis of previously published data, the acquisition of supplementary ethical approval was deemed unnecessary.

SNP selection

To establish the causal association between the gut microbiota and PAH, we employed a rigorous instrumental variable selection process: 1) Given that only a limited number of gut microbiota possessed three or more independent SNPs at the genome-wide significance threshold (P < 5 × 10−8), we employed a more lenient threshold (P < 1 × 10−5) to include additional SNPs. This approach was adopted to enhance the availability of SNPs for conducting sensitivity analyses, as in previous studies [22]. 2) The reference panel for calculating the linkage disequilibrium (LD) between SNPs consisted of European sample data from the 1000 Genomes project. Among the SNPs with r2 < 0.001 (using a clum** window size of 10 Mb) [44], thereby inducing inflammation. Intriguingly, comparable findings were also observed in hypertensive patients, with normotensive individuals displaying elevated levels of Eubacterium eligens, while essential hypertensive subjects exhibited high levels of Eubacterium fissicatena [45]. Taken together, some gut microbiota may exert SCFA-related anti-proinflammatory effects by preventing PAH initiation and development.

However, it is worth noting that not all SCFA microbial producers possess beneficial features. The families Lachnospiraceae and Ruminococcaceae possess the capacity to produce butyrate and other SCFAs through distinct biosynthetic pathways [46, 47]. In contrast, the findings from our MR analysis revealed that both the genus Lachnospiraceae UCG004 and the genus Ruminococcaceae UCG002 were suggestively associated with increased risks of inducing PAH. The abundance of the two taxa also exhibits an increase within the intestinal lumen of individuals afflicted with various diseases and elderly individuals [48,49,50,51,52]. However, members of this family have consistently demonstrated their capacity to generate favorable metabolites to the host.

However, this study is subject to certain limitations, which necessitate a more cautious interpretation of the findings. The first limitation is the inability to conduct subgroup analysis, such as assessing the severity of pulmonary arterial hypertension (PAH), due to the unavailability of individual level data. This is significant as alterations in intestinal flora among PAH patients were not found to be associated with changes in right heart function [13]. In addition, the limitation of the exposure dataset to the genus level hinders our ability to investigate the causal relationship between gut microbiota and PAH at the species level. Furthermore, the predominance of participants of European descent in genome-wide association studies restricts the generalizability of our findings to other populations. Finally, while our findings establish a causal association between specific gut microbiota and PAH, further research is needed to elucidate the underlying mechanisms.

Conclusions

The results of our MR study identified a potential causal effect of the gut microbiota and PAH. This valuable finding probably contributes to the identification of specific intestinal bacteria as biomarkers for pulmonary PAH, as well as clinical prevention and intervention of PAH through the implementation of fecal microbiota transplantation. Together, despite the presence of compelling evidence connecting gut dysbiosis to the initial development of PAH, the utilization of intestinal microbiota as a therapeutic intervention in clinical settings still requires significant advancements. It is imperative to conduct meticulous experimental investigations to establish the causative relationship between gut dysbiosis, altered gut microbiome, and the pathogenesis of PAH prior to considering the modulation of gut microbiota as a viable therapeutic approach for treating PAH.