Immunosuppressive drugs are used to mitigate the immune response in solid organ transplantation. The major classes of approved maintenance immunosuppressive drugs are calcineurin inhibitors (cyclosporine and tacrolimus), glucocorticoids (primarily prednisolone), the co-signal inhibitor belatacept, mammalian target of rapamycin (mTOR) inhibitors (sirolimus and everolimus) and antiproliferative agents (azathioprine and mycophenolic acid) ( Figure 4 ) (Wiesner and Fung, 2011). All of them inhibit T cell proliferation and activation. Indeed, proliferation of T cells depend on a three-step cascade that precedes cell cycle entry. The first step starts with the respective interaction of T cell receptor (TCR) and B7 on the surface of T cell with MHC presenting antigen and CD80/CD86 expressed by APC; the induced signaling cascade in T cell involves the calcium-dependent phosphatase calcineurin. Belatacept, a fusion protein, mimics the inhibitory receptor CTLA4 for B7, and thus blocks the co-stimulation signal (Rangel, 2010). Calcineurin inhibitors block the calcineurin-dependent NFAT signaling pathway (Wang et al., 2022). The second step is the transcription of different cytokine genes including that of the autocrine factor IL-2; glucocorticoids also block this step. IL-2 is secreted and binds to its receptor CD25 expressed by activated T cells and thus provokes the third step, i.e., a mTOR-dependent signaling cascade leading to cell cycle entry. Basiliximab, a monoclonal antibody against CD25 inhibits IL2 binding to its receptor, and the mTOR inhibitors blocks the third step of T cell activation (Zaza et al., 2014). Antiproliferative agents inhibit cell cycle by blocking a T and B cell specific pathway of nucleotide synthesis required for the S phase of cell cycle. Thus, antiproliferative agents are also inhibitors of B cell proliferation. In addition to their role on T cell activation and proliferation, glucocorticoids are known to affect B cell, neutrophil, macrophage, DC and NK cell activation and/or proliferation (Allison and Eugui, 2000; Muscari et al., 2022).

To prevent graft rejection and promote graft tolerance in the long term, immunosuppressive drugs are commonly used in different combinations of calcineurin inhibitors, anti-proliferative agents or mTOR inhibitors, with or without glucocorticoids (Wiesner and Fung, 2011; Hartono et al., 2013; Enderby and Keller, 2015; Claeys and Vermeire, 2019; Fuehner et al., 2019). The treatment of established graft cellular rejection usually consists in the use of a pulse therapy of corticosteroids and increased doses of calcineurin inhibitors, and/or of anti-thymocyte antibodies in case of severe or resistant rejection (Cooper, 2020). The most common treatment of ABMR consists in plasmapheresis to deplete circulating alloreactive antibodies, and either steroid pulse or, less frequently, rituximab to limit B-cell proliferation and activation (Alasfar et al., 2023; Rostaing et al., 2023).

The use of powerful antirejection agents may lead to opportunistic infections, reactivation of latent organisms, and post-surgery complications. The pattern of common infections after solid organ transplantation varies according to the immunosuppressive therapy and environmental exposure (Patel and Paya, 1997; Singh and Limaye, 2015; Fishman, 2017). These infections include bacterial (e.g., Clostridium spp., Enterobacteriaceae, Streptococcus pneumoniae), viral (e.g., cytomegalovirus, Epstein-Barr virus, respiratory viruses), and fungal (e.g., Aspergillus species, Candida species, Pneumocystis spp.) species (Karuthu and Blumberg, 2012; van Delden et al., 2020). Whenever necessary, transplant patients receive antimicrobial drugs in addition to their immunosuppressive regimen.

The immunosuppressive drugs may also induce malignancies by reducing the vigilance of immune cells towards potentially carcinogenic cells. This have been pointed out in primary or secondary immunodeficiency disorders (Ilham et al., 2023). A population-based study of transplant recipients observed a 2-fold overall increased risk of cancer, ranging from non-Hodgkin lymphoma to transplant-related cancer (kidney, liver, and lung) (Engels et al., 2011). In addition, a recent study showed that kidney transplant recipients with ultra-long-term survival of more than 20 years have an increased risk of developing post-transplant lymphoproliferative disorder and renal cell carcinoma (Fuhrmann et al., 2022).

Other adverse effects (see European Medicines Agency Summary of Product Characteristics) are also observed with immunosuppressive drugs. Indeed, cyclosporine and tacrolimus are nephrotoxic (Griffin and Nelson, 2016) and favor post-transplant diabetes mellitus, hypertension as well as a large spectrum of cardiovascular diseases including heart failure, and coronary artery disease. Corticosteroids, calcineurin and mTOR inhibitors also favor post-transplant diabetes mellitus. Mycophenolic acid entails gastro-intestinal disorders such as nausea, vomiting and diarrhea. It also induces cell apoptosis and architectural remodelling of the lower gastro-intestinal tract, exhibiting colitis-like or inflammatory bowel disease-like patterns (Selbst et al., 2009).

In addition, immunosuppressive drugs as well as antimicrobial agents, frequently given to transplant patients, induce gut microbiota modifications due to their antimicrobial and immunosuppressive properties. The comparison of gut microbiome from kidney transplant patients and healthy controls revealed that age, body mass index (BMI), estimated glomerular filtration rate (eGFR) and medications (mycophenolate mofetil, antibiotics and proton-pump inhibitors) were the main co-variables explaining variations in the gut microbiota of kidney transplant patients (Swarte et al., 2020). However, it is quite difficult to identify a typical alteration induced by each type of immunosuppressive drug in transplant patients, as they are treated with combined therapy. However, efforts have been made to characterize the contribution of each type of drug, mainly using preclinical models (Gabarre et al., 2022). Indeed, administration of glucocorticoids, tacrolimus or mycophenolic acid to preclinical models result in alteration of the composition of gut microbiota and the fecal concentration of bacterial metabolites (Wu et al., 2018; Zhang et al., 2018; Taylor et al., 2019; Jardou et al., 2021). The most relevant clinical contribution has been reported in the paper by Swarte and colleagues, which supports that mycophenolic acid is one of the main drivers of changes occurring in the gut microbiome of kidney transplant recipients. In this work, they found that the gut microbiome of kidney transplant patients more than one-year post-transplantation, is significantly different from that of healthy population, containing more Pseudomonadota (ex Proteobacteria), less Actinomycetota (ex Actinobacteria), with a functional loss of butyrate-producing bacteria. The use of mycophenolic acid and antibiotics was associated with variation in the gut microbiome of kidney transplant patients and correlated with lower diversity (Swarte et al., 2020). Liver transplant recipients experiencing TCMR presented a lower diversity index, which represents microbial diversity, in the post-transplant as compared to the pre-transplantation period (Kato et al., 2017). At the phylum level, an increase in Pseudomonadota and Actinomycetota and a decrease in Bacillota (ex Firmicutes) were observed (Kato et al., 2017). Moreover, the paper by Swarte and colleagues provide some evidence that age and BMI are variables that are associated with alteration in the microbiota of kidney transplant patients (Swarte et al., 2020).

The gut microbiota is composed of a diverse and complex microbial community, which contributes to human health. This community of microorganisms includes bacteria, archaea, viruses and fungi that are distributed throughout the gastro-intestinal tract (Kho and Lal, 2018). Among the bacterial species, four main phyla represent 98% of the gut microbiota in healthy adults, of which Bacillota (60-80%) and Bacteroidota (15-25%) are the dominant bacterial phyla, followed by Pseudomonadota and Actinomycetota (Jandhyala et al., 2015; Zhang et al., 2019). The diversity of the gut microbiota depends on host age, genetic parameters, physiological status and health condition, and on exposure to various environmental factors including diet and medication (Falony et al., 2016; Zhernakova et al., 2016; The Milieu Intérieur Consortium et al., 2019). The gut microbiota exerts important effects on host homeostasis, including digestion of dietary fibers, synthesis of essential vitamins, maintenance of the intestinal barrier integrity as well as education and homeostasis of the immune system (Macfarlane and Macfarlane, 2012; Yu et al., 2012; Sommer and Bäckhed, 2013; Takiishi et al., 2017; Okumura and Takeda, 2018). The gut microbiota is also involved in drug metabolism, potentially leading to the activation, inactivation or toxicity of medicines (Li et al., 2016; Guo et al., 2019; Zimmermann et al., 2019).

Divergence from the ‘normal’ composition and function of the gut microbiota, called dysbiosis, can occur in pathological conditions such as chronic gastro-intestinal diseases, cardiovascular diseases, metabolic disorders, neurological disorders, carcinogenesis and others (Guinane and Cotter, 2013; Petersen and Round, 2014; Lloyd-Price et al., 2016; Schroeder and Bäckhed, 2016). Changes in the composition of microbiota and alterations of the production of microbial metabolites are referred to as structural and functional dysbiosis, respectively. Increasing data shows that dysbiosis is not only associated with disease occurrence but also partly responsible for disease development.

The gut microbiota produces a large amount of metabolites that influence host homeostasis (Fan and Pedersen, 2021; Wang et al., 2023). There are short-chain fatty acids (SCFA), represented by acetate, propionate, and butyrate, produced from undigested dietary fiber. Bacterial members of the gut microbiota generate carbon and energy from fermentable carbohydrates that resist digestion by host metabolic enzymes. This process is termed “prebiosis” (Hugenholtz et al., 2013). These fermentable carbohydrates are metabolized in the colon by the microbiota via the glycolytic and pentose phosphate pathways that generate deoxy-hexoses and hexoses from starch, cellulose and fructans, and pentoses from xylans and pectins, respectively (Cummings, 1981; Macfarlane and Macfarlane, 2003; Deleu et al., 2021). The main SCFA produced by the gut microbiota are acetate (C2), propionate (C3) and butyrate (C4), in approximate proportions of 60:20:20 (Chambers et al., 2018). Formate (C1), valerate (C5), caproate (C6) and branched-chain fatty acids (e.g., isobutyrate, 2-methyl-butyrate, and isovalerate) can also be produced by the gut microbiota (Macfarlane and Macfarlane, 2003; Heaney, 2020).

The most abundant phyla in the gastro-intestinal tract, i.e., Bacteroidota mainly produce acetate and propionate, whereas Bacillota mostly release acetate, propionate and butyrate (Macfarlane and Macfarlane, 2003; He et al., 2020). Pathways for the production of acetate are commonly spread among bacteria species and this SFCA achieves the highest concentrations in the intestine (Louis and Flint, 2017), whereas more specific pathways and substrates have been described for propionate and butyrate production. Bacteroidota and Negativicutes (from Bacillota phylum) use the succinate pathway for propionate biosynthesis while Lachnospiraceae (from Bacillota phylum) use the propanediol pathway (Reichardt et al., 2014). Moreover, there are two pathways for butyrate synthesis mediated by specific enzymes: butyrate kinase for Coprococcus eutactus and Coprococcus comes species; or butyryl CoA and acetate CoA transferase for Faecalibacterium prausnitzii, Eubacterium rectale and Roseburia intestinalis species (Reichardt et al., 2014; Deleu et al., 2021).

SCFA can passively penetrate and pass through the apical membrane of digestive epithelial cells (Rechkemmer and von Engelhardt, 1988). SCFA can also be actively transported by sodium-coupled monocarboxylate transporter 1 (SMCT1) or monocarboxylate transporter 1 (MCT1) located on the apical side of digestive epithelial cells (Cuff et al., 2002; Miyauchi et al., 2004; Gill et al., 2005; Gupta et al., 2006). SCFA are used by colonocytes as a source of energy that provides 6 to 10% of their daily caloric requirements (Bergman, 1990). The remainder is transported through the basolateral membrane into the bloodstream through MCT4 and MCT5, located there (Gill et al., 2005; Bik et al., 2018) ( Figure 5 ).

SCFA are selective agonists of three different G-protein coupled receptors (GPCR) in intestinal cells, namely, GPR41 (free fatty acid receptor 3 or FFAR3), GPR43 (free fatty acid receptor 2 or FFAR2) and, only for butyrate, GPR109A (hydroxycarboxylic acid receptor 2 or HCAR2) (Brown et al., 2003; Thangaraju et al., 2009; Zhao et al., 2018). These receptors are also expressed on various other cell types such as immune cells (e.g., neutrophils, B and T cells, macrophages and DC) and endothelial cells, and in different organs and tissues such as the brain, adipose tissue, spleen and muscles (Le Poul et al., 2003; Vinolo et al., 2011; Li et al., 2017; van der Hee and Wells, 2021) ( Figure 5 ). Of note, SCFA receptors have not been detected so far on NK cells.

In addition, some SCFA, particularly butyrate, have histone deacetylase (HDAC) inhibitory activity leading to epigenetic modification of the genes, allowing their differential expression (Licciardi et al., 2011). A well-known example of this pathway is the acetylation of the Foxp3 locus by butyrate in T cells leading to Foxp3 transcription factor expression and differentiation of regulatory T lymphocyte (Treg) (Arpaia et al., 2013).

Neutrophils are the first immune cells to be attracted to an inflammatory site. They can be activated by pathogens to produce cytokines. These cytokines initiate the recruitment and activation of other immune cells, coordinating the overall immune response (Mayadas et al., 2014). Neutrophil express receptors for immunoglobulins and are activated by antibodies bound to antigens, leading to the production of cytokines and ROS and the secretion of granules. ROS production is known to be toxic to tissues while granule secretion may cause tissue damage or help tissue repair (Kruger et al., 2015; Wang, 2018). In the context of solid organ transplantation, neutrophils are typically the first type of leukocytes to infiltrate transplanted organs and to orchestrate local inflammation and possibly provoke tissue damage (Schofield et al., 2013; Scozzi et al., 2017).

In vitro, propionate and butyrate significantly reduce the production of pro-inflammatory molecules, such as TNF-α and cytokine-induced neutrophil chemoattractant-2 (CINC-2αβ) induced by the bacterial product lipopolysaccharide (Vinolo et al., 2011). Butyrate blocks microbial product-induced ROS production, whereas acetate favors the basal production of ROS (Vinolo et al., 2009). In addition, propionate and butyrate provoke caspase-8 and 9-dependent apoptosis of non-activated neutrophils and facilitate activated neutrophil apoptosis by inhibiting HDAC activity (Aoyama et al., 2010). Moreover, in a mouse model, acetate was protective against ischemia- and reperfusion-induced injuries, which was associated with a decreased number of activated neutrophils found in kidney tissue and a reduced level of oxidative stress in kidney cells (Andrade-Oliveira et al., 2015).

B lymphocytes play a key role in the humoral immune response and are involved in organ rejection by producing DSA. Moreover, B cells can affect the transplanted organs by interacting with and regulating T cells (Schmitz et al., 2020). Propionate and butyrate treatment inhibit immunoglobulin class-switch recombination and somatic hypermutation in B cells and diminish plasma cell differentiation (Sanchez et al., 2020). Moreover, acetate promotes B10 lymphocytes, a subpopulation of regulatory B cells that produce the potent Th1 inhibitory and anti-inflammatory cytokine IL10 (Kalampokis et al., 2013; Matsumoto et al., 2014; Daïen et al., 2021). In a mouse model, butyrate has been shown to induce B10 cell differentiation (Kim et al., 2021).

T lymphocytes are central in the immune response against foreign antigens occurring after solid organ transplantation. T lymphocytes recognize antigens presented by APC and either differentiate into cytotoxic cells, the Tc, or activate effector inflammatory cells that damage foreign tissue (Issa et al., 2010). SCFA modulates T lymphocyte differentiation and function through the inhibition of HDAC activity or via GPR binding (Wang et al., 2023). The most potent effect of SCFA on T cells is the promotion of Treg, a subpopulation with potent inhibitory function on Th cells and considered as the major actor of immune tolerance against self-antigens (Bayati et al., 2021). Of note, the potential use of Treg as an alternative or as a supplement to immunosuppressive therapies in organ transplantation to reduce graft rejection and avoid associated adverse effects has been discussed and investigated (Mathew et al., 2018; Sánchez-Fueyo et al., 2020; Juneja et al., 2022). In vitro, butyrate and to a lesser extent the other SCFA isovalerate and propionate, stimulate, via inhibition of HDAC, the generation of Treg (Arpaia et al., 2013; Furusawa et al., 2013; Kespohl et al., 2017; Verma et al., 2018). The positive regulation of Treg by butyrate was confirmed in a mouse model (Arpaia et al., 2013). In addition, in a mouse model of kidney transplantation, acetate and butyrate supplementation prolonged allograft survival by promoting tolerance towards graft (Wu et al., 2020). By depleting Treg or by using GPR43 deficient mice, the authors showed that the benefit of acetate supplementation resulted from the promotion of Treg differentiation through a GPR43-dependent pathway (Wu et al., 2020).

APC, including DC and macrophages play a significant role in the immune response after transplantation. Macrophages may differentiate into pro-inflammatory cells with high microbicidal activity, called M1, or into anti-inflammatory cells with low microbicidal potential, known as M2 (Yunna et al., 2020). M1 macrophages are involved in acute and chronic inflammation, as well as in graft rejection. In contrast, M2 macrophages have a beneficial role in promoting Treg differentiation and immune tolerance towards the graft (Schmidt et al., 2016). Some authors propose to target DC and to modulate macrophages as a new treatment strategy to improve transplant outcomes (Merad et al., 2007; Panzer, 2022). Butyrate and propionate strongly inhibit human monocyte-derived DC activation by bacterial products (Nastasi et al., 2015). Butyrate has the same effect on murine bone marrow-derived DC: murine DC preincubated with butyrate favors the production of the anti-inflammatory cytokine IL10 by mouse splenocytes (Berndt et al., 2012). More recently, it was shown in vitro and in vivo that butyrate, through its HDAC inhibitory activity, imprints a potent anti-microbial activity during macrophage differentiation (Schulthess et al., 2019). Wang et al. (2020b) showed in vitro that propionate and butyrate suppress the pro-inflammatory M1 phenotype of macrophages while promoting the anti-inflammatory M2 phenotype (Wang et al., 2020b). They also demonstrated in a mouse model that the fermentable fiber inulin-induced reduction of alcoholic liver disease is associated with an increased intestinal content of propionate and butyrate and, in parallel, with the suppression of hepatic M1 macrophages and an increased number of M2 macrophages (Wang et al., 2020b). Another paper showed in a mouse model of kidney injury that acetate decreases the numbers of kidney infiltrating macrophages and kidney cell oxidative stress that may result from ROS production by macrophages (Andrade-Oliveira et al., 2015). In addition, in vitro butyrate reduces the production of inflammatory mediators (TNF-α, IL-6, inducible nitric oxide synthase) by macrophages (Ohira et al., 2013). Using high fiber diet in a mouse model of airways viral infection, Trompette et al. (2018) demonstrated that butyrate diminishes the ability of macrophages to produce the proinflammatory mediator C-X-C motif ligand 1 (CXCL1) and the subsequent airways neutrophil influx (Trompette et al., 2018). Finally, Singh et al. (2014), combining in vitro and in vivo experiments, brought evidence that butyrate activates GPR109A and promotes the production of IL10 by colonic macrophages, boosting Treg differentiation and reducing colon carcinogenesis (Singh et al., 2014).