Gut Microbiome And Organ Fibrosis

Abstract: Fibrosis is a pathological process associated with most chronic inflammatory diseases. It is defined as an excessive deposition of extracellular matrix proteins and can affect nearly every tissue and organ system in the body. Fibroproliferative diseases, such as intestinal fibrosis, liver cirrhosis, progressive kidney disease, and cardiovascular disease, often lead to severe organ damage and are a leading cause of morbidity and mortality worldwide, for which there are currently no effective therapies available. In the past decade, a growing body of evidence has highlighted the gut microbiome as a major player in the regulation of the innate and adaptive immune system, with severe implications in the pathogenesis of multiple immune-mediated disorders. Gut microbiota dysbiosis has been associated with the development and progression of fibrotic processes in various organs and is predicted to be a potential therapeutic target for fibrosis management. In this review we summarize the state of the art concerning the crosstalk between intestinal microbiota and organ fibrosis, address the relevance of diet in different fibrotic diseases, and discuss gut microbiome-targeted therapeutic approaches that are currently being explored.

Keywords: gut microbiome; intestinal fibrosis; liver fibrosis; kidney fibrosis; lung fibrosis; heart fibrosis; diet; therapeutic strategies

NEW HERBAL FORMULATION FOR KIDNEY FIBROSIS 

1. Introduction 

Fibrosis is a complex pathological process that results from excessive deposition of extracellular matrix (ECM) components in response to tissue injury [1]. It is the final pathological outcome of most chronic inflammatory diseases and a major contributor to organ malfunction and failure [2]. Fibrotic tissue responses can affect nearly every tissue and organ system and are responsible for up to 45% of all deaths in developed countries [3]. Despite being recognized as a major health problem worldwide, very few treatments are currently available for the treatment of fibrotic disorders and they have limited efficacy [3]. Upon tissue injury, an intricate cascade of events aiming at the repair of tissue architecture and function takes place. Cell damage leads to the immediate release of a myriad of inflammatory mediators such as growth factors, cytokines, and chemokines, which promote leukocyte infiltration, the activation of fibroblasts into myofibroblasts (collagen secreting, α-smooth muscle actin (SMA)-expressing fibroblasts) and the synthesis of ECM components. In cases of minor and non–non-repetitive injury, the increased deposition of ECM components is transient and once the wound is repaired, myofibroblasts undergo apoptosis and the reparative response ceases. When the injury is severe or enduring, however, fibroblast activation persists as a chronic, uncontrolled process and ECM components tend to accumulate continuously, leading to the formation of a permanent fibrotic scar, organ malfunction, and ultimately organ failure [1]. Myofibroblasts are the key cellular mediators of the repair process and the major factor responsible for the secretion of most ECM proteins. These cells can result from the activation of resident fibroblasts and mesenchymal cells but may also originate from a large variety of different cell types such as cells of the vascular wall, endothelial cells, epithelial cells, fibrocytes (circulating fibroblast-like cells derived from bone marrow stem cells) and bone-marrow-derived progenitors such as adipogenic progenitors [4]. Myofibroblasts can be activated through several different stimuli, such as paracrine signals from immune cells, autocrine factors secreted by myofibroblasts, and pathogen-associated molecular patterns (PAMPS) produced by pathogenic microorganisms that interact with pattern recognition receptors (PPRs, such as toll-like receptors (TLRs)) on fibroblasts [5].

The breaking point at which the reparative process is no longer able to cope with the insult and becomes dysregulated, therefore entering a fibrotic path, is not known. Still, it is broadly accepted that once fibrinogenic pathways are activated, the process enters a vicious cycle where even the structural changes of fibrotic tissues themselves are feeders of a further fibrotic response by regulating the differentiation, recruitment, proliferation, and activation of ECM-producing myofibroblasts [6].

The breaking point at which the reparative process is no longer able to cope with the insult and becomes dysregulated, therefore entering a fibrotic path, is not known. Still, it is broadly accepted that once fibrinogenic pathways are activated, the process enters a vicious cycle where even the structural changes of fibrotic tissues themselves are feeders of a further fibrotic response by regulating the differentiation, recruitment, proliferation, and activation of ECM-producing myofibroblasts [6].

Regardless of the initial trigger, the innate and adaptive arms of the immune system have a major role in the onset and progression of the fibrotic response and several different immunoregulatory pathways have been pinpointed [7]. Still, several pieces of evidence point to other immune-independent mechanisms triggering fibrotic processes and that inflammation may even be necessary for the reversion of progressive fibrosis [8,9]. Such evidence sheds some light on the lack of success of anti-fibrotic therapies targeting inflammatory processes and suggests that different paths should be Investigated.

In recent years, the role of the gut microbiota in fibrotic processes has been gaining increasing interest. The intestinal microbiota is composed of around 100 trillion bacteria of about 1000 different species which, in healthy conditions, maintain a symbiotic relationship with the host, exerting important and complex functions in metabolism and immunity [10].

Alterations of the gut bacterial population toward a pathological phenotype-dysbiosis- can result in the accumulation of toxic compounds, namely, uremic toxins, and in the depletion of beneficial products (such as short-chain fatty acids (SCFAs)) [11,12]. The dysbiotic state is often associated with disrupted intestinal barrier integrity, facilitating the translocation of bacteria and bacterial products into circulation, and inducing the systemic activation of immune and inflammatory responses that can directly or indirectly cause tissue damage [13]. In genetically susceptible hosts, a dysregulation of the microbiota–immunity interactions is believed to contribute to the onset and progression of a multitude of immune-mediated disorders [13]. An increasing amount of data has been highlighting gut dysbiosis as a major promotor of disease pathogenesis, representing an intrinsic link with the development of fibrosis in several organs (Figure 1).

This review aims to summarize the role of intestinal microbiota in the development and progression of fibrosis in different organs, such as the intestine, the liver, the kidney, the lungs, and the heart, and provide an update on the current strategies targeting the gut microbiome in the treatment of fibrotic diseases.

Figure 1. Gut dysbiosis and organ fibrosis. TMAO, trimethylamine N-oxide; pCS, p-cresyl sulfate; IS, indoxyl sulfate; SCFAs, short-chain fatty acids; KEGG, Kyoto Encyclopedia of Genes and Genomes. ↑ increased; ↓ decreased; = altered. 

2. Intestinal Fibrosis

Chronic inflammation appears to be the major event triggering gut fibrogenesis, through mesenchymal cell recruitment and activation [14]. Intestinal fibrosis-associated inflammation may occur much more severely than in liver, kidney, or lung fibrosis [15,16], such as that occurring in inflammatory bowel disease (IBD). Although the intestine shows an impressive ability to fully regenerate after short-lived insults (infections, acute peptic ulcer, etc.), under the presence of continuous severe inflammation, the mesenchymal cells (in the form of fibroblasts, myofibroblasts, or smooth muscle cells) are continuously activated, producing excessive ECM, and leading to fistulae or stricture formations with possible intestinal obstruction [15]. To date, there is no pharmacological therapy that is effective in reversing intestinal fibrosis; therefore, a deeper understanding of intestinal fibrosis mechanisms is urgent in the search for innovative strategies. 

The understanding of the role of the gut microbiome in the pathogenesis of intestinal fibrosis started to be unraveled in IBD, the most extensively studied disease that is deeply associated with intestinal fibrosis development. IBD includes ulcerative colitis and Crohn's disease. In Crohn's disease, intestinal fibrosis is a frequent complication that may lead to bowel wall thickening, strictures, and stenosis due to general inflammation, causing the remodeling of the entire intestinal wall, associated with enhanced deposition of ECM components [17,18]. In ulcerative colitis, the triggering of fibrosis was recently recognized and is associated with colon shortening and stiffening due to the accumulation of ECM in the mucosal and submucosal layers, as well as with inflammation and disruption of the epithelial layer due to tight-junction damage [19,20].

The link between the gut microbiome and intestinal fibrosis was unveiled in IBD, on the one hand, because these patients showed a dysbiotic and pro-inflammatory gut microbiota [21] and, on the other hand, in germ-free animal models of colitis or in Crohn's disease patients undergoing antibiotherapy, where the gut inflammation was found to be absent or improved [22]. Moreover, adherent-invasive Escherichia coli (AIEC), a pathotype of E. coli, and Salmonella enterica serovar Typhimurium (S. Typhimurium) have been shown to induce inflammation (through elevated T helper (TH) 1 and TH17 immune responses) in IBD animal models, leading to subsequent fibrosis development [23–26]. In addition, and similarly to what is found in Crohn's disease patients, extensive ECM deposition was observed in AIEC-infected mice, along with higher expression levels of collagen types I/III, and the enhanced expression of profibrotic mediators such as transforming growth factor-β1 (TGF-β1), connective-tissue growth factor, and insulin-like growth factor I (IGF-I) [26]. In agreement with this, in patients with Crohn's disease, AIEC strains were associated specifically with ileal mucosa and were suggested to locally trigger the initiation or perpetuation of the inflammatory disease [27].

Currently, some mechanisms and molecules associated with the gut microbiome were already recognized to be involved in the pathogenesis of intestinal fibrosis. The bacterial lipopolysaccharides (LPSs), also known as endotoxins, present in the external structure of the gram-negative bacterial cell wall, are known to promote the profibrotic activation of intestinal fibroblasts, with increased nuclear factor-κB (NF-κB)-light-chain-enhancer of activated B cell promoter activity and collagen contraction [28]. Flagellin, the structural protein from the bacterial flagellum, induced the expression of the interleukin (IL)-33 receptor ST2 in the intestinal epithelium of mice co-colonized with AIEC and an attenuated strain of S. Typhimurium, which in turn augmented IL-33 signaling and promoted the development of intestinal fibrosis [24,29].

More recently, the nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECHassociated protein 1 (Keap1) axis was suggested by Piotrowska and colleagues [19] as a promising candidate for the prevention of IBD and its severe complications, such as intestinal fibrosis, given that the Nrf2/Keap1 axis was shown to influence the production of ECM components including collagen and TGF-β1 in the gut. Notably, gut bacteria, their components (such as LPS), or their metabolites (such as urolithin A) were found to activate the Nrf2 pathway [19,30–33]. 

Another interesting mechanism associating intestinal fibrosis and the gut microbiome was described by Jacob and colleagues [34], who found that the intestinal fibrosis and fibroblast activation mediated by the tumor necrosis factor-like cytokine 1A (TNF-L1A) and the tumor necrosis factor ligand superfamily member 15 (TNF-SF15) are dependent on specific microbial populations and are independent of inflammation. First, they demonstrated that the profibrotic and inflammatory phenotype resulting from TNF-L1A-overexpression is abolished in the absence of resident microbiota. Then, germ-free wild-type and TNFL1A-transgenic mice fecal transplanted (by gavage) with stools from specific pathogen-free mice and a healthy human donor showed that reconstitution with specific pathogen-free mice, but not healthy human donor microbiota, resulted in increased intestinal collagen deposition and fibroblast activation in TNF-L1A-transgenic mice. The fibrosis-triggering microbial populations were identified in the cecum as mucolytic bacteria such as the species Mucispirillum schaedleri, the genus Ruminococcus, and the genus Anaeroplasma, and in the ileum, such as the genera Streptococcus and Lactobacillus. In contrast, members of the genera Oscillospira and Coprococcus in the cecum, as well as Faecalibacterium prausnitzii and members of the genus Bacteroides in the ileum, were negatively correlated with fibrosis. Moreover, in vitro, some bacterial strains that were positively correlated with the degree of fibrosis promoted fibroblast migration and collagen expression, whereas other strains that were negatively correlated with fibrosis onset did not. Interestingly, no histologically significant cecal inflammation accompanied the increased cecal collagen deposition under specific pathogen-free microbial conditions, highlighting the importance of TNF-L1A as a pro-fibrotic mediator that can act independently of its pro-inflammatory effects. In sum, this relevant study points to the existence of unique profibrotic mediators, which are either cytokine- or microbiome-driven (or both).

Corroborating the results of Jacob and colleagues [34], a cohort of children with Crohn's disease from the RISK cohort (Risk Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease Progression in Children with Crohn's disease) showed that the bacteria from the genus Ruminococcus are implicated in structuring complications. Moreover, taxa belonging to the Veillonella genus were also found to be increased in penetrating complications, suggesting differential microbial populations in different disease phenotypes [35]. 

Beyond IBD, there is evidence as to the role of the gut microbiota in fibrosis onset in radiation-induced intestinal injury [36]. Zhao and colleagues [36] showed that antibiotic pre-treatment regimens improved the reconstitution ability of the gut microbiota in mice after radiation. This antibiotic pre-treatment in mice effectively reduced the content of LPS, inhibited the TLR4/MyD88/NF-κB signaling pathway and regulated macrophage cell polarization in the ileum, downregulated TGF-β1 phosphorylated Smad-3 and SMA protein levels and upregulated E-cadherin protein expression. In sum, Zhao and Colleagues [36] suggest that antibiotic pre-treatment may significantly improve the survival rate and attenuate intestinal injury after radiation by reducing inflammation and preventing intestinal fibrosis. In brief, there are several arguments implicating the role of the gut microbiome in the pathogenesis of intestinal fibrosis, directly or through inflammation. For that reason, modulation of the gut microbiome (as discussed in Chapter 8) may constitute a valuable therapeutic tool in managing intestinal fibrosis. 

The gut–liver axis comprises a bidirectional interaction/communication pathway between the gastrointestinal tract and the liver, through the biliary tract, portal vein, and systemic circulation, enabling the transport of gut-derived products directly to the liver, where they influence several liver functions, and the liver feedback route to the intestine, where it controls metabolic functions and influences gut barrier integrity and microbiota composition [38]. This interdependence explains the influence of the altered gut microbiome (gut dysbiosis) and disturbances in the intestinal barrier in the increased portal influx of bacteria, bacterial fragments, and their products to the liver [39]. The translocated microbes and molecules then activate PPRs on liver cells, stimulating the production of inflammatory cytokines and the synthesis of ECM by hepatic stellate cells, which contribute to chronic inflammation and progressive fibrosis [40]. 

Recent evidence has demonstrated an association between intestinal dysbiosis and non-alcoholic fatty liver disease (NAFLD), although causality is yet to be established [41]. A change in the gut microbiota composition according to the fibrosis stage has been observed in NAFLD patients. Using 16S rRNA sequencing, Boursier et al. [42] found a higher abundance of the class Bacteroidetes and a lower abundance of the genus Prevotella in patients with NASH, the aggressive form of NAFLD that comprises inflammation, hepatocellular damage, steatosis, and fibrosis. Among these patients (i.e., NASH) those with higher fibrosis (stage 2 or higher) also showed a higher abundance of the genus Ruminococcus [42]. Loomba et al. [43], using whole-genome metagenomics, identified an increased abundance of the species Escherichia coli and Bacteroides vulgatus in patients with NAFLD with advanced fibrosis. Similarly, a greater abundance of the genus Escherichia has been observed in obese children with NASH, compared with children with obesity without NASH [44]. However, recently, Schwimmer et al. [45] showed that a high abundance of Prevotella copri was associated with more severe fibrosis in children with NAFLD. There is a lack of accordance across studies, which can be explained by the great variability in study design and population selection, as well as by different geographies and dietary patterns [41]. Moreover, the different NAFLD phenotypes may result from different microbiome signatures on the host according to its genetic predisposition or environmental factors [39]. 

Still, in addition to changes in the microbiome composition, alterations in the functional capacity of the gut microbiome have been demonstrated in NAFLD. When the functional profile of the gut microbiota was predicted through bioinformatics, a significant shift in the metabolic function of gut microbiota was revealed in more serious NAFLD lesions (NASH and significant fibrosis), mainly impacting Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to carbohydrate, lipid, and amino acid metabolism [42]. Moreover, in pediatric NASH patients, elevated serum ethanol concentrations have been observed, most likely from a gut-microbiota source that is enriched in alcohol-producing bacteria (e.g., E. coli) [44]. This may be a risk factor driving disease progression, since the role of alcohol metabolism in the generation of reactive oxygen species is well established, which then influences liver inflammation [44].

As in NAFLD, intestinal dysbiosis has been demonstrated to be an important hallmark of alcoholic liver disease (ALD). During the development and progression of ALD, there are changes in the structure, composition, and function of the intestinal microbiome [40]. Chronic alcohol intake causes changes in the taxonomic composition of the intestinal microbiome and increases the circulating levels of LPS, likely due to an increase in gut permeability, which then accumulates in the liver and activates PPRs, resulting, as already emphasized, in the production of inflammatory cytokines and the activation of hepatic stellate cells, which would then increase the expression of ECM [40,46]. Moreover, the severity of ALD was shown to be associated with the degree of intestinal dysbiosis [47]. Patients with severe alcoholic hepatitis harbored larger amounts of Bifidobacteria, Streptococci, and Enterobacteria and less of the genus Atopobium compared with patients with a heavy intake of alcohol but no hepatitis [47]. Additionally, the severe phenotype was transmiscible from patients to mice through fecal microbiota transplantation [47]. These animals showed an increased intestinal permeability, which led to increased bacterial translocation, along with a decrease in bile acid derivatives, which in turn could affect the efficiency of alcohol metabolism [47].

Cirrhosis, late-stage fibrosis and an extreme manifestation of chronic liver injury is associated with marked gut barrier impairment matching the disease progression that occurs with microbial translocation. Translocated bacteria, dominated by the phylum Proteobacteria, are abundant in the portal vein and the hepatic and peripheral blood of decompensated cirrhotic patients and are associated with increased systemic inflammation [48]. The physiopathological mechanism involved in complications such as hepatic encephalopathy and spontaneous bacterial peritonitis is strictly associated with the translocation of enteric bacteria or their products into the systemic circulation [49]. Regarding microbiome composition, recently, metagenomic techniques have been used to characterize the fecal microbiome in cirrhosis, showing reduced diversity and overgrowth of potentially pathogenic taxa belonging to the Enterococcaceae, Staphylococcaceae, and Enterobacteriaceae families, and a decreased abundance of potentially beneficial auautochthonous taxa, namely, those belonging to the Lachnospiraceae and Ruminococcaceae families [50,51]. The reduced secretion of bile acid reported in cirrhosis could favor the overgrowth of these pathogenic bacteria [52]. A distinctive feature of cirrhosis is the invasion of the lower intestinal tract by microorganisms of oral origin, such as bacteria from the genera Veillonella and Streptococcus [50].

As recent evidence indicates, independently of the underlying etiology, liver fibrosis itself is typically accompanied by gut dysbiosis [53]. Together with the severely compromised gut barrier, gut dysbiosis, with the overgrowth of potentially pathogenic bacteria, drives hepatic inflammatory immune responses through portal delivery of PAMPs. These PAMPs are recognized by PPRs, such as TLRs and nucleotide-binding oligomerization domain-like receptors (NOD-NLRs), on the surface of hepatic stellate cells, hepatocytes, or immune cells [54]. Therefore, hepatic stellate cell fibrogenesis can be triggered either directly or indirectly via inflammatory signals produced by neighboring cells [54]. Furthermore, the altered microbiome also results in the intestinal deconjugation of bile acids and the production of secondary bile acids that suppress farnesoid-X receptor signaling [39]. Farnesoid-X receptor signaling exerts protective effects on intestinal epithelial barrier properties, and therefore its suppression promotes the disruption of the intestinal barrier [55], which can contribute to the perpetuation of the insult on the liver and the persistent activation of stellate cells, which would then lead to disruption of the balance between ECM deposition and dissolution, triggering progressive liver fibrosis. 

In conclusion, although it is now accepted that liver damage and fibrosis can result from the interplay between the gut microbiota and the host liver and immune cells, further studies are needed to better understand this interaction for future microbiome target strategies.