In this pilot randomized controlled study investigating the effects of repeated oral FMT on systemic inflammation in PWH on ART, we observed significant reductions across a broad array of inflammatory proteins. Notably, these effects persisted until the final visit, 16 weeks post-intervention, suggesting a sustainable modulation of systemic inflammation. Unlike previous interventions that targeted the gut microbiome with prebiotics, probiotics, and synbiotics (reviewed in [11]), our study directly measured changes in inflammation by assessing a comprehensive panel of inflammatory proteins. Additionally, whereas previous pilot studies noted only limited engraftment of donor’s microbiota following three different modalities of FMT [17, 46, 47], our current research identifies potential key microbial species whose changes correlate significantly with long-lasting variations in inflammatory marker levels, thereby highlighting their potential for targeted interventions in the field of microbiome therapeutics.
So far, no interventions targeting the microbiota of PWH on ART, including prebiotics [48, 49], probiotics [14, 50], synbiotics [13], or rifaximin [15] have convincingly proved to temper inflammation or enhance boost immune recovery. Although some studies have reported mixed effects on diverse inflammatory cytokines, the evaluation of inflammation has typically been restricted to a limited set of molecules [17, 46]. Our previously reported study indicated that repeated oral FMT induces modest yet enduring changes in gut microbiota structure. This was particularly notable in the Ruminococcaceae and Lachnospiraceae families, commonly depleted in PWH [10] and are major butyrate producers. Concurrently, there was a reduction in IFABP, a biomarker of intestinal barrier integrity and an independent mortality predictor in PWH [17]. Here, we have further elucidated the effects of FMT on inflammation, achieving a broader resolution.
Among the 46 DEIPs analyzed following FMT, 45 exhibited downregulation across proinflammatory and regulatory domains. Notably, enhanced signaling via cytokine-cytokine receptor interactions and the IL-10 pathway suggests a shift toward an anti-inflammatory profile in patients undergoing FMT. However, this extensive modulation suggests a broad systemic impact beyond mere anti-inflammatory effects, challenging simplistic interpretations of immune responses. Such widespread downregulation might reflect a resetting of the immune system, which often remains in a state of heightened activation in chronic HIV infection [1, 2, 18] despite effective viral suppression through ART. While the generalized reduction in inflammatory markers indicates that FMT can efficiently modulate immune responses—potentially reducing the risk of inflammation-related comorbidities—a reduction in both pro-inflammatory and regulatory proteins may reflect a move toward homeostasis in an activated immune system. Thus, these findings need to be replicated in larger intervention studies. In contrast to this general trend, Persephin (PSPN) level increased post-FMT. Given PSPN’s critical role in neuronal survival and differentiation, its upregulation post-FMT raises intriguing possibilities regarding the gut microbiota’s impact on the gut-brain axis, hinting at specific pathways of gut-immune-neural axis restoration or a unique compensatory response to microbiome modification [51].
To further investigate mechanistic insights of these proteins, we performed an enrichment analysis of biological functions with the list of 46 DEIPs. Most of the identified proteins related to functions that include pro-inflammatory cytokines and chemokines, such as TNF, a central mediator of acute inflammation; IL1B and IL6, cytokines involved in fever and acute phase reactions; and CCL20 and CCL22, chemokines responsible for immune cell chemotaxis. In addition, some proteins were grouped in a second cluster, suggesting their role in modulation of adaptive immune responses, as they are involved in lymphoid tissue organization and B cell function. For example, TNFRSF13C is essential for B cell development; TNFSF11 (RANKL) is involved in T cell and dendritic cell regulation and bone metabolism; and LTBR is crucial for lymphoid tissue development. Thus, these 46 DEIPs play critical roles in the immune response and clinical progression of HIV, affecting both proinflammatory and anti-inflammatory pathways. For instance, IL-6 is frequently reported to be elevated in PWH on ART and serves as an independent predictor of mortality in this population [1, 52]. TNF is associated with inflammation and HIV persistence during ART, partly through signaling [53, 54]. CCL20 (macrophage Inflammatory Protein-3 alpha or MIP-3 alpha), a protein involved in recruiting cells to sites of inflammation, is typically elevated in HIV [55]. Conversely, key proteins in the IL-1 pathway, like IL10RA, the IL-10 receptor, and IL1RN, the IL-1 receptor antagonist, could be particularly relevant in the pathogenesis of inflammation-related cardiovascular events [56, 57]. Interestingly, microbiome-derived signals can influence these proteins’ expression in the epithelia [58–60].
The network analysis revealed a nuanced landscape of protein expression changes following FMT, suggesting that not all DEIPs are uniformly influenced by alterations in the microbiome. We identified a subset of proteins, including FLT3LG, CCL20, IL17A, CLEC7A, ADAM23, OSCAR, SIRPB1, LAIR1, AMBN, and CKMT1A_CKMT1B, that showed significant correlations with at least 20 distinct bacterial species each. This suggests targeted modulation by the microbiome, likely through specific microbial metabolic activities or immunomodulatory mechanisms. For example, in PWH, gut dendritic cells are activated by Prevotella sp., positively correlated with IL17-A changes in our study, promoting maturation and activation of dendritic cells, thereby enhancing their ability to present antigens and activate T cells [61, 62].
FLT3LG, which decreased in our study in the FMT group, plays a crucial role in developing and maturing dendritic cells [63] and correlated in our study with changes in Faecalibacterium prausnitzii, a dominant commensal of the human gut and a major butyrate producer [64]. Other DEIPs highlight the potential role of FMT in shaping pathogen-specific defense mechanisms. These include CCL20, essential for mucosal immunity directly regulated by certain bacteria such as Prevotella sp. [65]; IL17A, which expression modulates the microbiome composition [66]; and CLEC7A, relevant for antifungal immunity and innate immune responses [67]. The association of these proteins with a broad array of bacterial species underscores their potential as biomarkers for evaluating the efficacy and understanding the biological modulatory effects of FMT.
We identified specific bacterial species significantly associated with changes in plasma DEIPs following FMT. Species within the Clostridiales order, including Clostridium sp. and Ruminococcus sp., were frequently correlated with proteins like OSCAR, CLEC7A, IL17A, and FLT3LG. These findings align with our selection of stool donors based on high butyrate and Faecalibacterium content, emphasizing the role of butyrate-producing bacteria in modulating immune responses. Our previous analysis identified pronounced engraftment in the Lachnospiraceae and Ruminococcaceae families, with their abundance remaining elevated after 48 weeks [17]. Butyrate can inhibit NF-κB signaling [32], partly by inhibiting human histone deacetylases [29, 48]. This inhibition facilitates the transcription of genes involved in regulatory T cell function, such as Foxp3 [68]. As a result, butyrate induces a tolerogenic response in human dendritic cells. Our network analysis of the protein-protein interactions identified the NF-kB signaling pathway as significantly regulated post-FMT, suggesting this is a critical pathway influenced by microbiome changes post-FMT.
Among the list of DEIPs associated with at least 15 bacterial species, FLT3LG, IL17A, and OSCAR also correlated with at least 15 bacterial genes, emphasizing the potential influence of the gut microbiome on these proteins. Consequently, these proteins could serve as indicators for microbial-driven inflammatory responses. We also found changes in bacterial functions, which correlated with alterations in the plasma proteome. Key bacterial functions identified included transcriptional regulation (ArsR family), carbohydrate metabolism (basic endochitinase B, N-acetylglucosamine-binding protein A, beta-glucoside kinase, oxalate decarboxylase, polygalacturonase), and protein secretion systems (type VI secretion system secreted protein Hcp). Additionally, there were changes in proteins involved in cellular transport (fructose transport system permease protein, teichoic acid exporter) and metabolic processes (3,4-dihydroxyphenylacetate 2,3-dioxygenase, 3-hydroxypropionyl-coenzyme A dehydratase, serine/threonine-protein phosphatase 6 regulatory ankyrin subunit, GTP cyclohydrolase IV).
These bacterial functions suggest mechanisms by which FMT could reduce inflammation in PWH. The enhancement of carbohydrate metabolism and degradation (e.g., endochitinase, polygalacturonase, oxalate decarboxylase) may lead to increased production of SCFAs like butyrate, known for their anti-inflammatory properties and ability to strengthen the gut barrier [39, 41]. The presence of proteins involved in transcriptional regulation and secretion systems indicates a potential modulation of bacterial virulence and host-microbe interactions, potentially reducing pathogenic bacterial load and associated inflammation. Changes in cellular transport proteins could improve nutrient absorption and microbiota stability, contributing to a healthier gut environment [39, 42]. Lastly, alterations in enzymes such as GTP cyclohydrolase IV and serine/threonine-protein phosphatase may influence host immune responses and signaling pathways, further contributing to the anti-inflammatory effects observed.
Several factors must be taken into consideration when interpreting our results. First, our previous analysis utilized 16S rRNA sequencing across 11 study visits, providing higher temporal resolution but limited taxonomic detail [17]. In contrast, the current analysis employs shotgun metagenomics over four study visits, allowing us to achieve species-level resolution, albeit with less frequent sampling. This difference in methodology provides more detailed taxonomic insights in the current study but less temporal resolution. Here, we used mOTUs2 to profile the microbiome composition at the species level. Given our limited sample size, we chose this method to ensure higher accuracy in role assignment, thereby mitigating the risk of false discoveries despite its lower sensitivity compared to other tools [69]. In this study, we measured microbiome function indirectly by assigning significant bacterial genes to their functions or proteins. However, a direct assessment would have required analyzing higher functional levels of the microbiome, such as its transcriptome, proteome, or metabolome, which should be considered in next studies.
The strengths of our pilot study include (i) the randomized controlled trial design, which allows us to attribute observed changes directly to the FMT intervention rather than to natural microbial variations; (ii) the use of a novel proteomic assay, which allows for a more detailed, efficient and precise measurement of inflammatory biomarkers than in previous studies [17, 46, 47]; (iii) the application of species-level resolution in microbial analysis; (iv) the lack of significant differences in dietary intake between the groups, minimizing confounding variables; (v) the comprehensive longitudinal analysis, which helps in understanding the changes over time; and (vi) the careful selection of donors with microbiota profiles high in Faecalibacterium spp. and butyrate, targeting anti-inflammatory properties.
While randomized controlled trials are essential for establishing causality, our longitudinal correlation analysis between fecal bacteria and plasma proteins should be considered preliminary. The immune system is intricately regulated and often follows a non-linear response pattern to interventions. This motivates further mechanistic studies to elucidate how FMT modulates inflammation. Additionally, future research should directly measure microbiome functions through metatranscriptomics, metaproteomics, or metabolomics. It remains crucial to explore factors that may enhance the effects of FMT, including donor selection, baseline microbiome composition, inflammatory profiles, and the potential need for antibiotic preconditioning regimens.