Advancing Gastrointestinal Microbiota Research in Systemic Sclerosis: Lessons Learned from Prior Research and Opportunities to Accelerate Discovery

Introduction

A wealth of evidence supports an association between alterations in gastrointestinal (GI) microbiota and human health and disease. Between 10–100 trillion microorganisms comprise the human microbiota, among which bacteria are the most abundant with a density of 10¹¹-10¹² cells/ml (1). Within the GI tract, over 1000 bacterial species play a critical role in host homeostasis, from nutrition, immune function, and metabolism to defense against pathogenic organisms. Major shifts in the GI microbiota membership and function, which are linked to disease states (i.e., dysbiosis), are commonly observed in patients with autoimmune diseases (2). For example, numerous studies have demonstrated that patients with various autoimmune diseases exhibit reduced diversity of GI microbiota and enrichment of pathobiont species (3).

Dysbiosis is a recognized feature of systemic sclerosis (SSc) (4,5), an autoimmune disease with diverse clinical manifestations and high morbidity and mortality. For example, studies have demonstrated that patients with early SSc exhibit alterations in GI microbiota compared with unaffected controls (6). Furthermore, the abundance of certain bacterial species is associated with the severity of organ involvement in SSc (7). While dysbiosis is common among patients with SSc, the significance of these changes remains unclear. For instance, it is unknown whether dysbiosis is a cause or consequence of immune dysregulation in SSc, or both. Moreover, while the GI microbiota is fairly stable through adulthood, and the most common time period for developing SSc is between 30–50 years of age, a number of external factors can perturb the balance of the GI microbiota, including dietary alterations, environmental shifts, medications, lifestyle changes, and infections (1).

Unlike other autoimmune rheumatic diseases, SSc has a profound predilection for the GI tract. Over 90% of patients with SSc have signs and symptoms of GI involvement (8). Since there are currently no disease modifying therapies for the GI manifestations of SSc, this clinical dimension of SSc typically progresses (worsens) throughout the disease course, representing a major source of morbidity. Patients with SSc-GI manifestations report worse health-related quality of life over time than those without SSc-GI manifestations (9,10), and severe malabsorption is an independent predictor of mortality in patients with SSc (11,12). Interestingly, manipulation of the GI microbiota with broad-spectrum antibiotic therapy offers many patients temporary relief from certain GI symptoms, including distension and bloating (13). This fact is what led our group to embark on the first exploration of the GI microbiota in SSc.

Since the first study of the GI microbiota in SSc was published in 2016 (7), our understanding of the role of GI microbiota in this disease state has evolved. Considering the considerable phenotypic heterogeneity of SSc, it seems increasingly less plausible that a single microbiota signature will define all patients with SSc. Instead, new research points to species-specific organ system associations. This review provides an in-depth coverage of original research conducted over the last 6 years on the GI microbiota in SSc. By comparing and contrasting the methodological approaches to characterize the GI microbiota in SSc, this review offers expert insight into sequence analytic techniques and their capacity to provide sufficient species level resolution for describing inter-individual variations among patients living with SSc. This review also describes emerging research on functional analyses and the application of machine-learning platforms that may propel discovery in this area. Finally, this review will summarize the theoretical basis for personalized approaches to modify the GI microbiota in SSc with the hope of identifying preventative treatment strategies for this condition.

Microbiota Analysis

Sequencing Approaches

Since the majority of bacterial species cannot be readily cultured, high-throughput sequencing facilitates culture-independent analysis. Initial research on the GI microbiota in SSc relied on low-resolution 16S rRNA amplicon sequence analytics (1). In this method, a 16S rRNA region undergoes amplification by PCR with primers that recognize the highly conserved regions of the gene. While this approach provides important compositional information on microbial community diversity, its output is limited to phyla or genus level data. This is due to the fact that this method is based on putative associations between the 16S rRNA gene with taxa defined as an operational taxonomic unit (OTU). Due to the existence of numerous bacterial strains, OTUs are less precise at the species level.

Whole genome shotgun sequencing (WGS) is an alternative approach to 16S rRNA amplicon sequencing (14,15) (Table 1). This method uses sequencing with random primers to sequence overlapping regions of a genome, as opposed to just one specific region of DNA. A major advantage of WGS method is that taxa can be more precisely defined at the species level. While WGS is more costly than 16S sequencing, studies have demonstrated that WGS identifies significantly more bacterial species per read than the 16S method (16). Moreover, unlike 16S sequencing, WGS can identify non-bacterial organisms, including viruses, fungi and protozoa (16), which may contribute to the SSc disease state. As described further below, most of literature on the GI microbiota in SSc employed the 16S method; however, new research uses the WGS approach.

Table 1.

Key differences in 16S rRNA amplicon sequence method and whole genome method of characterizing the GI microbiota

	16S Sequencing	Whole genome sequencing
Methodological Differences	Sequences only a single region of the bacterial genome	Sequences broad regions of the genome
Taxa resolution	Poor species level resolution; Increased risk of species misclassifications	Excellent species level resolution with lower misclassifications
Organisms identified	Bacteria	Bacteria, Viruses, Protozoa, Fungi
Functional information	Not provided	Provided
Archived data	More archived data for reference	Less archived data for reference since this is a newer method
Cost	Less costly	More costly

Study designs

Study design is an important consideration in SSc-GI microbiota research. Early studies investigating the GI microbiota in SSc were insufficiently powered to perform subgroup analyses or create multivariable models that adjusted for confounders (Figure 1). The majority of studies were also cross-sectional, with the exception of one small study that described changes in microbiota over the course of one year in patients with SSc (17). These design issues have delayed our progress in understanding the role of the GI microbiota in the pathogenesis of SSc in contrast to other disease states, such as inflammatory bowel disease (IBD). For example, a study of patients with ulcerative colitis with ileal pouch-anal anastomosis who underwent serial endoscopy and sampling of host mucosa and pouch microbiomes identified pathobionts that were observed prior to endoscopic changes or clinical symptoms (18,19). Longitudinal studies that characterize changes in species abundance relative to the evolution of organ involvement/progression may transform our understanding of the role of the GI microbiota in SSc.

Figure 1. Potential confounders to consider in multivariable models linking the GI microbiota to SSc features.

While a multitude of factors can affect GI microbial composition in SSc, factors of major importance include age, body mass index (BMI), medications, disease duration, geographic location, diet, presence of SIBO, presence of dysmotility. Other factors to consider include sleep and stress, which are often more challenging to quantify.

Studies aiming to understand alterations in the GI microbiota between SSc and unaffected controls have largely matched groups by age and gender. However, given the complexity of external factors that shape the GI microbiota from the time of birth (e.g., birth order, mode of delivery, breastfeeding, antibiotics predelivery) to the time of sampling, it may be impossible to identify the perfect control population for an SSc study. Some have proposed to use household members as controls to help adjust for environmental impacts (e.g., pets, diet); however, studies have demonstrated that healthy first-degree relatives of patients with IBD exhibit dysbiosis that may predispose to disease or subclinical inflammation (20). Thus, selecting the ideal control population is an area of ongoing controversary in SSc microbiota research.

Sampling: Endoscopic vs fecal samples

Most of the SSc GI microbiota studies have utilized fecal samples, which represent a composite of distal GI microbiota and are not representative of region-specific GI microbiota. Endoscopic evaluations can facilitate the acquisition of region-specific samples. Our first study demonstrated microbial community differences between the cecum and sigmoid colon of patients with SSc who underwent colonoscopy with lavage sampling (7). However, unlike IBD, where colonoscopies are routinely done to evaluate disease activity, they are not performed as standard of care in SSc, rendering it a costly research procedure. Furthermore, patients with SSc are often reluctant to undergo colonoscopy, particularly when they suffer from chronic constipation. For these reasons, collecting fecal samples represents a more cost-effective and less invasive approach for sampling the GI microbiota with the caveat that region-specific information is lost.

Statistical analyses

A central goal of SSc microbiota analyses is to identify key species that explain differences between groups of samples (e.g., Diffuse cutaneous disease vs limited cutaneous disease). The risk of type I error is high in these analyses given the high number of bacterial species or genera under investigation. Most studies employ specific methods to correct for multiple hypothesis testing. The use of the traditional Bonferroni method is considered too conservative and may lead to missed findings. Various other multiple-comparison procedures have been utilized to control the false discovery rate (FDR), including a widely used procedure introduced by Benjamini and Hochberg (21). This procedure identifies the expected fraction of non-differentially abundant taxa among taxa deemed significant (the discoveries) and adjusts individual p-values.

Another important analysis issue in SSc microbiota research is sample filtering. Typically, samples are filtered to retain species with at least 10–25% non-zero counts. This is particularly important in cross-cohort integrative analyses that are aiming to identify consistent associations between species and disease features within different cohorts. Collaborating with experienced microbiome statisticians can improve the rigor of this research.

Microbiota Alterations in Early SSc

Due to the abundance of residing lymphocytes within the GI tract, the GI tract itself is considered an immunologic organ (22,23). While few studies have investigated the direct relationship between the immune system and the GI microbiota, a number of studies have demonstrated that patients with SSc exhibit alterations in GI microbial composition compared with healthy controls (HCs) (Figure 2). In the first microbiome study performed in SSc, Volkmann and colleagues demonstrated decreased commensal genera and increased pathobiont genera in the lower GI tract of SSc compared with healthy controls (HCs) using colonic lavage specimens (7). Soon after this original study was published, Andreasson and colleagues demonstrated that severe dysbiosis was observed in patients with SSc, particularly in patients with esophageal dysmotility (24). Several other investigations have confirmed that dysbiosis occurs in SSc (24–27); however, these early studies did not investigate confounding factors that can modify the microbial composition such as medications, disease duration, SSc subsets, or the geographic region of the study patients.

Figure 2. Differences in GI bacterial taxa and GI metabolites in patients with SSc compared with HCs.

Included taxa were found to be differentially abundance in SSc vs controls in at least two different cohorts of patients with SSc. Since very few studies have evaluated GI metabolites in patients with SSc, all metabolites with significant differences between SSc vs controls are listed.

Early studies on the GI microbiome in SSc aimed to understand whether dysbiosis is associated with GI symptoms. Using the UCLA GIT 2.0 questionnaire as a tool to quantify the severity of GI dysfunction, low abundance of Bacteroides fragilis was associated with worse GIT scores (more GI symptoms) (7,25). Another study found that SSc patients with GI symptoms had alterations in the number and distribution of taxa with an increased relative abundance of Desulfovibrio (27). These formative studies helped shaped our initial understanding of how the GI microbiome was altered in SSc and whether it could be linked with clinical manifestations of SSc.

A fundamental question that arises from all of these studies is if dysbiosis is a consequence of autoimmunity and chronic inflammation of SSc or, whether dysbiosis itself perpetuates inflammation. While a bidirectional relationship between the GI microbiota and immune system likely exists, the study of patients with SSc in very early stages (i.e., preclinical phases) of their disease could help further our insight into this relationship and ultimately improve our understanding of the molecular mechanisms that drive the pathogenesis of SSc.

For the early stages of SSc, we refer to the first phases of SSc with an already definite, but recent SSc diagnosis (<3 years), while a very early diagnosis of SSc (VEDOSS) is based on the 2011 European League Against Rheumatism (EULAR) Scleroderma Trial and Research (EUSTAR) proposed criteria where patients experience Raynaud Phenomenon, the presence of puffy fingers and harbor antinuclear antibodies (ANA), as well as SSc-specific autoantibodies (anti-centromere antibody or anti-topoisomerase I) and SSc pattern on nailfold capillaroscopy as necessary features (28). Based on LeRoy and Medsger criteria, subjects that present with RP, SSc specific antibodies and/or SSc pattern at capillaroscopy without any features of fibrosis (puffy fingers included) are defined as very early/preclinical SSc (29).

To test the hypothesis that early dysbiosis could directly influence the development of autoimmunity and fibrosis, a preclinical study demonstrated that early life dysbiosis induced by antibiotic administration to mice, exacerbated the development of skin and lung fibrosis (30). However, human studies in early and very early SSc are lacking. One study of 106 patients with early SSc (median disease duration of 2 years) from Sweden study showed that dysbiosis was present in this early stage of disease, with a notable increase in the abundance of the pathobiont genera Desulfovibrio (6). In another study, Natalello and colleagues investigated the GI microbiome in 19 patients SSc with early disease (mean disease duration of 2.3 years) and 29 patients with SSc with longer disease duration (mean disease duration 10.8 years). In this study, patients with early SSc patients exhibited greater richness in bacterial species and differences in beta diversity compared with those patients with longer standing disease. This study also demonstrated a relationship between the GI microbiota and the cutaneous subset of SSc, as well as body mass index (BMI) (31).

To date, only one study, by Russo and colleagues, has evaluated the microbiota in patients meeting VEDOSS criteria. This study also investigated fecal metabolites, and found a decrease of pro-tolerogenic bacterial strains and alterations in short-chain fatty acids in both VEDOSS and established SSc patients when compared to HCs. In contrast to the prior study, which demonstrated microbiota differences between patients with early versus established SSc (31), the study evaluating VEDOSS patients demonstrated no differences in the extent of dysbiosis between patients with very early SSc and those with established disease. (32). While the reason for this difference is unclear, future studies are needed to understand when dysbiosis occurs during the SSc disease course and how it evolves over time. Future studies are also needed to understand how individual microbiota signatures predict progression and distinct clinical phenotypes of SSc.

Microbiota Correlates of SSc Features

Microbiota research in SSc has transitioned from attempting to discover a single microbiota signature that applies to all patients with SSc to understanding how specific taxa and their metabolic products associate with SSc features. There is also a growing appreciation that grouping microbes into categories based on their health-promoting versus disease-promoting facilities may lead to misclassifications of the individual microbes’ capacity to affect the host under different conditions. For example, Bacteroides, which was historically deemed a commensal genus, increased the incidence of colitis in mice whose mothers had received peripartum antibiotics (33). Therefore, individual microbes can transition from commensals to pathobionts in certain conditions that alter their functional capacities.

Gastrointestinal tract involvement

As described above, GI involvement occurs in most patients with SSc. One common GI manifestation of SSc is small intestinal bacterial overgrowth (SIBO). In a systematic review and meta-analysis, the prevalence of SIBO was 10-fold higher in the SSc patients (N=1112) compared with controls (N=335) (34). Treatment for SIBO is largely based on antibiotic therapy, and rifaximin is often the drug of choice (34,35). Despite the fact that modulation of the microbiota through antibiotic therapy ameliorates SSc-GI symptoms, few studies have endeavored to understand the relationship between the microbiota and SSc-GI involvement (7,26). One early study found that SSc patients with moderate/severe total GI symptoms had reduced abundance of Bacteroides fragilis and increased abundance of Fusobacterium compared to those with none/mild symptoms (7). This study measured GI symptoms using the UCLA GIT 2.0, a valid questionnaire for assessing GI symptom burden in SSc (36). Patients in this study underwent serial assessment of their microbiome and GI symptoms every three months for one year, and low abundance of Bacteroides was associated with increased GI symptoms over time (17).

However, in this longitudinal study, GIT 2.0 scores did not change significantly over the course of the one year (17). This finding may reflect stable GI disease in this cohort; however, one cannot exclude the possibility that GIT 2.0 scores cannot distinguish disease activity from disease damage in SSc. Research is underway to define new GI disease activity measures in SSc, which will undoubtedly accelerate our progress in identifying microbial biomarkers of SSc-GI involvement that correlate with disease activity.

Additional studies have identified bacterial genera and species associated with specific GI symptoms, such as constipation (25), bloating/distension (25), fecal incontinence and malnutrition (31). However, the findings of these individual studies require replication in other cohorts. Research is also underway to understand the impact of diet on SSc-GI symptoms and the microbiota (37), as dietary manipulation can cause immediate shifts in GI microbial composition without the long-term safety concerns associated with repeated courses of antibiotic therapy, including antibiotic resistance and worsening dysbiosis (38).

Interstitial lung disease

The gut-lung axis refers to the ability of the GI microbiota to influence the course and outcome of underlying lung disease, and vice versa (39). Gut dysbiosis is associated with pulmonary diseases, including asthma and chronic obstructive pulmonary disease (40,41). Moreover, accumulating evidence links the Western diet with shifts in the microbiota and predisposition to inflammatory respiratory diseases (42).

The role of the GI microbiota in interstitial lung disease (ILD) is an evolving area of research (43) (Figure 3). In a murine model of hypersensitivity pneumonitis, Bacteroidetes phylum was enriched in streptomycin-treated mice during the perinatal period (44). In SSc, patients with ILD were found to have higher fecal calprotectin levels than patients without ILD (45). In another study, which included two geographically-distinct SSc cohorts (USA and Sweden), patients with ILD had marked differences in beta diversity compared with those without ILD (6). Beta diversity represents the degree of difference in community membership or structure between two samples.

Figure 3. Perturbations of the gut lung axis in dysbiosis.

Changes in the GI microbiota causes intestinal barrier disruption leading to translocation of bacteria and their components into the circulation triggering systemic inflammation. In dysbiosis, the production of metabolites, such as short chain fatty acids, is altered, affecting immune function and defense against respiratory pathogens. In ILD, it is plausible that gut dysbiosis worsens inflammation leading to increased production of pulmonary cytokines, chemokines and growth factors that perpetuate fibrosis. It is also possible that pulmonary pathogens could enter the GI tract directly during coughing episodes related to underlying esophageal dysfunction in SSc.

In the first study to employ WGS to characterize the GI microbiota in SSc, a multicenter study of SSc patients from 5 continents demonstrated significant differences in specific species abundance in patients with and without ILD (46). This study also found that specific species were correlated with the severity of ILD as measured by the quantitative radiological extent on high-resolution computed tomography of the chest. For example, the abundance of specific species categorized as commensal in other disease states (e.g., Bifidobacterium adolescentis (47)) was lower in patients with increased QILD scores. Ongoing research on the GI microbiota and ILD may reveal important pathogenic participants, particularly as this research evolves to consider the effects of GI metabolites on ILD course in SSc.

Understanding the GI Metabolome

The metabolome collectively refers to metabolites produced from biochemical processes within a given system (48). In the GI tract, bacteria participate in numerous metabolic processes, including digestion of carbohydrates, which results in the production of specific metabolites (49). The dynamic interplay between the GI microbiota and its metabolic products contributes to host homeostasis, and alterations in the GI metabolome has been linked with specific disease states (50,51).

The primary techniques for analyzing fecal metabolites include chromatography (gas chromatography (GS) or liquid chromatography (LC)), mass spectrometry (MS), and nuclear magnetic resonance. The metabolite coverage of each method varies. Consequently, using multiple platforms to measure metabolite levels achieves the broadest coverage. MS analysis can be performed directly or in conjunction with preliminary separation techniques such as GC-MS, LC-MS, or capillary electrophoresis (52,53).

To date, a Human Metabolome Database (54) is available with more than 100,000 compounds, of which approximately 7,000 derive from studies using fecal samples (55). The most common human fecal metabolites are short chain fatty acids (SCFAs) such as acetic, propionic and butyric, while lipids are the least common. SCFAs can induce immune tolerance via their interactions with T regulatory lymphocytes (5,56).

Bellocchi and colleagues performed the first study to examine the GI microbiome in relation to serum metabolites using an untargeted approach (high-performance LC coupled to MS) (27). In this study comparing 59 patients with SSc and 28 HCs, 17 metabolite intermediates distinguished SSc from HCs, including glycerophospholipid metabolites and benzene derivatives. In this study, an association was observed between the pathobiont genera, Desulfovibrio, and alpha-N-phenylacetyl-l-glutamine and 2,4-dinitrobenzenesulfonic acid (27).

A more recent study analyzed fecal metabolites using a targeted approach with LC-MS (57). In this study comparing HCs (N=79) and patients with early SSc (disease duration <3 years) (N=115), levels of several fecal metabolites were significantly different in the SSc patients, including methioninesulfoxide. Another recent study found differences in the levels of fecal SCFA in patients meting VEDOSS criteria and those with established SSc (32). Future studies are needed to understand the role that fecal metabolites play in driving inflammation in SSc, both within the GI tract and systemically. Such metabolites may emerge as important markers of GI disease activity and improve our ability to objectively monitor progression of GI involvement in SSc.

Strategies to Restore Homeostasis of the GI microbiome

Targeted therapies aimed at restoring homeostasis of the GI microbiome are an area of major interest in SSc and other autoimmune disease. While some factors that shape GI microbial composition are unmodifiable (e.g., genetics, delivery mode, birth order), many factors are modifiable, and these include diet, exercise, mind-body approaches, as described further below.

Dietary manipulation

Dietary changes can induce rapid effects on the GI microbiota. In animal models, diet changes account for the majority (57%) of the total structural variation in GI microbiota; whereas genetic mutations explain only 12% of changes (58). The GI microbiota acts on digested nutrients, which in turn shape its composition and metabolic activities. For example, in a cross-over study of healthy individuals living in Denmark, the relative abundance of 14 bacterial species was altered during the 8-week low-gluten diet intervention compared with the high-gluten diet intervention (59). Moreover, the low-gluten diet was associated with a reduction in both fasting and postprandial hydrogen exhalation and improved postprandial well-being after a standardized test meal in this study.

The consumption of a Mediterranean-type diet is also associated with alterations in the GI microbiota compared with a Western-type dietary pattern and confers a myriad of health benefits to the host (60). To our knowledge, there has been one study investigating the relationship between diet and the microbiome in SSc (37). In this single-center, cross-sectional, observational study, we found that patients who consumed a diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) did not have significant differences in their GI symptom severity compared with patients who did consume a low FODMAP diet; however, Entercococcus (a purported pathobiont in other disease states) was less abundance in the low FODMAP group (37).

Nutrition-centered interventional studies are lacking in SSc, and many patients with SSc struggle to discover what type of diet is right for them. Patients are inundated with information about diets for patients with autoimmune diseases on the internet and social media, which can make it overwhelming for them to know what dietary modifications to make. Patients with SSc may also have limitations in what they can eat due to difficulty swallowing, decreased oral aperture, poor dentition and/or slow GI motility. Given the phenotypic heterogeneity of SSc, it is unlikely that a single dietary intervention will confer the same benefit to all patients with SSc. However, we encourage patients to work with licensed nutritionists/dieticians, keep food diaries, undergo monitoring of their vitamin levels and continually monitor how individual foods make them feel. Figure 4 describes an approach to advising patients on their nutritional journey with SSc.

Figure 4. Proposed guide for helping patients on their nutritional journey with SSc.

In Step 1, when patients learn how to eat mindfully, they develop a heightened awareness of how a particular food or combination of foods makes them feel, which better prepares them for Step 2 (creating a food and symptom diary). Recording symptoms and food intake helps patients identify patterns and make connections between food intake and their physical sensations and emotional feelings. In Step 3, a patient will form their health care team to guide them on their nutritional journey. This team generally consists of their scleroderma rheumatologist, gastroenterologist, dietician/nutritionist and other providers, such as psychotherapists or acupuncturists. In Step 4, patients are advised to make gentle changes to their diet and not drastic changes that are unsustainable in the long-term. Humility is important as it will help the patient to understand that even when they eat all of the “right” foods for them, they may still have bad days in terms of symptoms, which could be related to non-food factors, such as stress or medication changes. Finally, in Step 5, the patient learns to let their symptoms guide their daily food intake. For example, if they awaken feeling worse reflux symptoms, they may opt to avoid all reflux-provoking food that day.

Medications and Supplements

Antibiotics

Rifaximin is an oral antibiotic with broad-spectrum activity and is an FDA-approved for the treatment of irritable bowel syndrome (IBS) (61). Treatment with rifaximin is associated with symptomatic improvements in patients with SIBO in association with IBS (2), as well as SSc (34,62). Exactly how rifaximin favorably modifies the GI microbiota remains unclear. It is also uncertain whether repeated course of rifaximin can perpetuate dysbiosis in autoimmune diseases such as SSc. Antibiotic resistance is also of concern, and for these reasons, antibiotics, including rifaximin, should be used sparingly in SSc.

Prebiotics/probiotics

Prebiotics are substrates selectively consumed by host micro-organisms, conferring a health benefit (63). The main prebiotics are inulin, fructo-oligosaccharides, galacto-oligosaccharides, lactulose, xylo-oligosaccharides, and mannan-oligosaccharides (64). These substances are found in foods such as chicory root, artichoke, garlic, onions, leeks, asparagus, beans, lentils and bananas (65). In animal models of colitis, prebiotics have shown beneficial effects (66). However, human studies, show mixed outcomes in patients with IBD (67–69). To the best of our knowledge, no studies have investigated prebiotic supplementation in patients with SSc. Some patients with SSc have a difficult time tolerating foods rich in prebiotics, particularly patients with slow GI motility. Thus, lower consumption of these foods may contribute to dysbiosis in SSc.

Probiotics supplements contain live microorganisms that may also confer health benefits (63). Probiotics supplements commonly consist of one or more of the following: Bifidobacterium, Lactobacillus, Saccharomyces boulardii. However, cultured food products (e.g., yogurt, Kefir) and fermented foods (sauerkraut, pickled vegetables) naturally contain probiotics (70). Administration of oral Bifidobacterium and Lactobacillus is associated with improvements in colitis in mouse models (71–73). However, similar to prebiotics, human studies employing probiotic interventions have yielded conflicting findings (74–76). These inconsistencies may be attributable to variability in the types and dosages of probiotics, as well as the viability of probiotic strains in acidic environments of the upper gastrointestinal tract (5). In SSc patients, two relatively small, randomized controlled trials found that treatment with probiotic supplementation was not associated with an improvement in GI symptoms compared with placebo (77,78). The lack of efficacy could be due to the fact that future research is needed to personalize probiotics for specific SSc patient subgroups.

Fecal Microbial Transplant

Fecal microbial transplant (FMT) involves the transfer of the entire fecal microbial community present in the feces from healthy donors (79). Stools from a healthy donor are homogenized, filtered and then used for fresh fecal preparation, frozen fecal preparation, fecal capsules or washed microbiota preparation (80). FMT has been studied most extensively in IBD, but with variable results (81). The first study of FMT in SSc patients was performed in 2020, where commercially available anaerobic cultivated human intestinal microbiota (ACHIM) was administered via gastroduodenoscopy in SSc patients (N=5) with GI symptoms such as diarrhea, distention/bloating and/or fecal incontinence (82). This small study demonstrated improvements in GI symptoms; however, serious complications, including laryngospasms and duodenal perforation occurred during endoscopy, highlighting the need to employ safer delivery methods (e.g., oral capsule) (80).

A subsequent study of 67 SSc patients randomly assigned to either placebo (n=34) or ACHIM treatment (n=33) did not meet its primary endpoints (diarrhea and bloating) (83). However, in a subgroup of patients with severe fecal incontinence, an improvement was noted in this symptom (84). Numerous questions remain regarding the role of FMT in SSc, including how to personalize FMT for individual patients with unique manifestations of SSc and for patients residing in different geographic regions, and whether serial FMT is necessary generate a sustained treatment response, given the resiliency of the host microbiome.

Mind-Body Approaches

The brain-gut axis refers to the bidirectional communication between the central and the enteric systems, essentially linking intestinal functions with emotional and cognitive areas of the brain (85). The microbiota plays an integral role in modulating the brain-gut axis. For example, in IBS, alterations in the microbiota are associated with enteric nervous system abnormalities (86). Transfer of the microbiota of IBS patients to germ-free rats induces a visceral hypersensitivity phenotype (87), and treatment with rifaximin is associated with improvement in IBS symptoms compared with placebo (88).

Mind-body practices (i.e., any health practice that promotes tranquility and relaxation of the mind and body) have the capacity to modify the GI microbiota via the brain-gut axis (Table 2). These practices include yoga, breathwork, mindfulness, meditation, tai chi and qi gong. For example, a study of 56 Tibetan Buddhist monks demonstrated significant differences in the microbiota of these individuals compared with control subjects living in a neighboring community (89). Individuals consuming a vegan diet and participating in an advanced meditation program experienced an increase in branch short-chain fatty acid over the course of the study compared with household controls (90).

Table 2.

Mind-body approaches which may favorably affect GI microbial composition and immune function

Approach	Effect(s) on Microbiome	Effects(s) on Immune System
Meditation	Increased commensal bacteria89,95 Decreased pathobiont bacteria95	Enhanced immune function without activating inflammatory signals98
Yoga	Increase short-chain fatty acid production90	Downregulate proinflammatory markers99
Tai-Chi or Qi Gong	Increased alpha diversity96 Decreased pathobiont bacteria96,97	Increased natural killer cell cytoxicity100 Increased Immunoglobulin G100

For patients with SSc, finding the right mind-body practice is a personal journey and depends on their underlying disease among many other factors. Certain physical limitations (e.g., hand contractures) may affect their ability to practice certain yoga poses, for instance. In a patient with end-stage lung disease secondary to ILD, an intense breathwork practice, such as the Wim Hof Method, may be unadvisable. However, experienced teachers of these health practices will offer modifications to the exercises, and patients can learn how to adapt the practice to suit their needs.

Summary

Since the first study was published on the GI microbiome in SSc in 2016, our understanding of the role of this inherited genome in SSc pathogenesis has evolved. Advances in sequencing techniques have improved our ability to define taxa at a species level and to study the dynamic interaction between specific bacterial species and their metabolites. In addition, evaluating microbial signatures in relation to defined SSc phenotypes has transformed our understanding of how the GI microbiome may interact with the immune system to generate inflammation and fibrosis in distant organ systems, including the lungs.

However, more progress is needed to advance this field of study in SSc. First, to understand whether dysbiosis drives SSc pathogenesis, longitudinal studies of patients with very early SSc are required. Second, to discover microbiome profiles that are generalizable to all patients with SSc, analyses need to account for the critical external factors that shape the microbiome, including geographic environment (91–94). Third, to achieve adequate statistical power to perform these multivariable analyses, collaborative, international research efforts are needed. Finally, interventional studies (e.g., dietary interventions, probiotic or prebiotic supplementation, FMT) should ideally enrich for a patient population most likely to benefit from the intervention to advance precision medicine in SSc. The microbiome represents exciting area of research discovery in autoimmune diseases with great potential to guide the development of preventative treatment strategies for SSc.

Key Points.

• Alterations of the GI microbiota (i.e., dysbiosis) occur relatively early in the SSc disease course

• Specific bacterial species are associated with clinical features of SSc, including GI symptoms and ILD

• An integrative analysis of GI microbiome-metabolome will further our knowledge of disease mechanisms underlying specific SSc phenotypes

• Modulating of the GI microbiome via targeted therapies, supplements and fecal microbial transplant is promising, but these interventions are still under investigation

• Nutritional and mind-body interventions offer hope for improving homeostasis of the GI microbiome in SSc

Synopsis.

Dysbiosis is a feature of patients with systemic sclerosis (SSc). While a causal relationship between the GI microbiota and SSc pathogenesis has not been established, alterations in the GI microbiota are appreciated early in the SSc disease course. Moreover, recent research has illuminated specific microbial signatures that define SSc phenotypes. The present review summarizes new research on the GI microbiome in SSc with a focus on technical advancements and the emerging study of the GI metabolome. This review also addresses diverse modalities for manipulating the GI microbiome with the hope of developing preventative treatment strategies to avert progression of SSc.

Clinics Care Points.

• Alterations of the GI microbiota of SSc are a feature of SSc and are present early in the disease course, prior to the onset of over GI symptoms

• Advancements in sequencing techniques to characterize the GI microbiome has illuminated connections between specific bacterial species and SSc features (e.g., GI symptoms, ILD severity)

• Integrative analyses that examine the GI microbiota and metabolome could reveal specific metabolites that drive disease pathogenesis and/or serve as markers of disease activity in SSc

• Interventions aiming to restore homeostasis of the GI microbiota in SSc include dietary changes, medications, supplements, as well as a host of mind-body interventions