Host-Microbiota Interplay in IBD: The Emerging Role of Extracellular Vesicles, Perinatal Immune Priming, and Gut-Resident Immune Cells

Abstract

The human gut is populated by innumerable microorganisms which govern equilibrium and well-being. Fluctuations in the composition and function of intestinal microbiota have been shown to result in persistent ailments such as inflammatory bowel disease (IBD). Yet, conclusive cause-effect studies must be formulated in this context. This chapter features current advancements in the field of host-microbiota interactions and their association with IBD. The role of bacterial extracellular vesicles (BEVs) and modification of intestinal EV proteomes with distinctive host-microbiota interactions in IBD, perinatal immune priming in offspring from maternal IBD and the function of gut-resident immune cells in IBD have been discussed here. These compelling developments would be crucial in expanding our understanding of IBD pathogenesis, detection of novel diagnostic repertoire and therapeutic targets for this disease.

1. Introduction

A plethora of assorted microorganisms inhabits the human gastrointestinal tract. The flexibility of the hefty genome of this community allows it to adapt well within the intestinal environment and complement the host [1]. The depth of association of the microbiome with human biology is accurately demonstrated by the spectrum of tasks delegated to the microbiome including pathogen defence [2], nutrient metabolism [3], assisting immune maturation [4] and maintaining metabolic homeostasis [5]. Humans and their gut microbiota are thus known to be co-evolved in a symbiotic manner. The composition of the gut microbiota varies notably among individuals [6, 7] and determines the susceptibility of the host to several diseases including inflammatory bowel disease (IBD) [8, 9, 10]. IBD has emerged as a global health challenge in the last decade [11].

IBD is a chronic and relapsing inflammatory disorder of the intestine and has two subtypes, Crohn’s disease (CD) and ulcerative colitis (UC) [12]. Although sharing some clinical features and being studied together in the past, these two diseases represent discrete pathophysiological entities. Crohn’s disease is characterized by segmental inflammation with clear distinctions between affected and unaffected bowel segments. The earliest mucosal lesions appear over Peyer’s patches and the terminal ileum is affected the most [13]. On the contrary, ulcerative colitis is characterized by continuous inflammation extending proximally from the rectum to the colon. Inflammation is restricted to the mucosal layer, with neutrophils permeating the lamina propria and the intestinal crypts and forming cryptic abscesses [13, 14].

Compositional and metabolic changes in the intestinal microbiota have been extensively associated with chronic inflammation; however, several aspects of our understanding of IBD pathogenesis remained unclear. This chapter highlights the significant updates in the research related to the host-microbiota interactions as well as the role of the immune system in IBD, which might provide new avenues for disease prevention and treatment.

2. Extracellular vesicles and IBD

Extracellular vesicles (EVs) have gained recognition recently as novel mediators for cell-to-cell as well as interspecies and even interkingdom interaction [15]. EVs are submicron entities found circulating in all bodily fluids and in all species, including bacteria. EVs of the eukaryotic cells emerge either from the budding of the plasma membrane or the fusion of multivesicular endosomes with the plasma membrane. EVs derived from Gram-positive and Gram-negative bacteria may disperse in extracellular space by outward budding of the prokaryotic membrane [16, 17]. EVs contain a bioactive cargo of nucleic acids (DNA, mRNA, microRNA, and other noncoding RNAs), proteins (receptors, transcription factors, enzymes, and extracellular matrix proteins), small molecular metabolites, and lipids, which can govern the functions of the recipient cell [18, 19, 20]. Based on their biogenesis and size, EVs have been categorized into microvesicles, exosomes, ectosomes, oncosomes, and outer membrane vesicles (Table 1) [21].

Ev type	Diameter (nm)	Density (g/ml)	Origin	Morphology	Composition
Exosomes	40–150	1.13–1.19	Derived from the plasma membrane by multivesicular endosome pathway	Cup-shaped	Surrounded by a phospholipid membrane containing relatively high levels of cholesterol, sphingomyelin, and ceramide and containing detergent-resistant membrane domains
Microvesicles	100–1000	Unknown	Released from the plasma membrane during cell stress	Cup-shaped	Insufficiently known
Membrane particles	50–80, 600	1.032–1.068	The plasma membrane of epithelial cells	Cup-shaped	CD133
Apoptotic vesicles	>2000	1.16–1.28	Plasma membrane, endoplasmic reticulum	Heterogeneous	Histones, DNA, immature glycoepitopes

Table 1.

Classification of extracellular vesicles.

EVs produced by commensal bacteria in the gastrointestinal tract are distributed throughout the gut lumen and carry a variety of compounds with a potential role in bacterial survival and host interaction [22]. EVs have been studied in many pathological and non-pathological conditions, including colorectal cancer and IBD. The role of extracellular products from commensal bacteria in immunomodulation and maintaining the homeostasis of the intestinal tract has gained attention since 1967 [23]. A recent study of bacterial extracellular vesicles (BEVs)-host interactions by Gul et al. investigated the effect of BEVs derived from the gut commensal bacterium Bacteroides thetaiotaomicron on host immune cells. Dendritic cells, macrophages and monocytes were of particular interest as they play key roles in regulating the immune response in IBD [24].

Genes expressed in each of the immune cell-types were identified by single-cell RNA sequencing and were assumed to be all translated into functional proteins to establish the host-microbe protein-protein interaction (PPI) networks. Even though there were a large number of BEV-human PPIs, most of the bacterial proteins were hubs with the potential to interact with thousands of host proteins. It was found that a total of 48 BEV proteins comprising of hydrolases, proteases, and other catabolic enzymes without a specific cleavage site, communicate with the host immune cells (Figure 1). Toll-like receptor (TLR) pathway analysis revealed that targets for BEVs differ among different cells and between the same cells in healthy versus disease (ulcerative colitis) conditions [25]. These findings thus, suggest the role of cell-type as well as health status in influencing BEV-host interaction.

Figure 1.

Interactions of BEV proteins with immune cells in (i) Healthy state (ii) Ulcerative colitis (No. of expressed genes/No. of interacting proteins presented for each cell-type).

Zhang et al. [26] elucidated the association of microbiome and intestinal EV proteins in pediatric IBD. Mucosal-luminal interface samples collected from a pediatric IBD inception cohort were subjected to metaproteomic characterization for both the human and microbiota proteins. Microbial proteins related to oxidative stress responses were found to be upregulated in IBD cases compared to controls. Human proteins related to oxidative antimicrobial activities were found to be abundant in isolated free EVs and their expression was elevated in IBD cases, corresponding with the alteration of microbial functions [26]. Hence, EVs could serve as promising biomarkers with diagnostic and/or therapeutic potential in IBD.

3. Mother to child transfer of IBD

Pre- as well as post-natal bacterial colonisation plays a significant role in sculpting the immune system. Microbes transmitted from mother to infant presumably adapt to and persist in the infant gut than non-maternally acquired strains. Human trials have demonstrated the influence of maternal health status and microbiology on the development of the neonatal microbiome and immune system [27, 28]. The role of IBD in the maternal microbiome composition during pregnancy and its impact on the offspring’s microbiome was investigated by Torres et al. by sampling pregnant women with and without IBD for their stool and saliva at each trimester, combined with their clinical and obstetric records. Post-delivery, the neonates were pursued with serial stool samples at time points of 7, 14, 30, 60, and 90 days, respectively, along with thorough health and exposure metadata. Stool samples from mother–baby pairs were then gavaged into 6–8 weeks old germ-free mice (GFM) for their immune phenotyping. 16S rRNA sequencing and microbiome analysis of the samples revealed that women with IBD maintained altered gut bacterial diversity throughout the pregnancy, with an enrichment of Gammaproteobacteria and a reduction in Bacteroidetes, compared with healthy controls. Offsprings to the IBD mothers demonstrated similarity to the bacterial diversity and composition trends of the mothers, to at least 3 months after birth compared with the offsprings to control mothers [29]. GFM inoculated with the stools from the third trimester IBD mother and 90-days infant showed a considerable reduction in the microbial diversity and fewer class-switched memory B cells and regulatory T cells in the colon, indicating the possible role of microbial factors from maternal IBD in influencing the immune system of the offspring [30].

Another study by Kim et al. made use of fecal calprotectin (FC) to monitor intestinal inflammation in pregnant women and their offsprings. FC is a non-glycosylated, calcium- and zinc-binding protein with antimicrobial, antiproliferative, and immunomodulatory properties, and it is used as a surrogate marker of intestinal inflammation [31]. FC levels decreased gradually in mothers with IBD during the 3 trimesters of pregnancy, contrary to the control mothers in which small gradual increase in FC levels was reported [32]. The rising levels of FC in healthy pregnancy correlated with the increase in pro-inflammatory phylum Proteobacteria and a decrease in anti-inflammatory Faecalibacterium [33]. Babies born to mothers with IBD presented significantly higher FC levels compared with control babies starting at 2 months of life and throughout 36 months. FC levels in both pregnant women with IBD and their babies were positively correlated with Streptococcus abundance and negatively correlated with that of Alistipes [32]. Consequently, maternal IBD has the potential to adversely affect the offspring’s intestinal milieu during early life after birth, which can have significant health-related consequences later.

4. Gut-resident macrophages and microbial dysbiosis in IBD

Intestinal epithelium mononuclear phagocytes (MPs) have been designated as the ‘sensors’ and ‘responders’ to the intestinal environment by virtue of their location and function. They are represented by heterogeneous dendritic cell (DC) and macrophage subsets which are vital for the induction of immune response and regulation of inflammation [34]. Mononuclear phagocytes keep the intestinal inflammation in check either through direct regulation of microbiota or through the release of local anti-inflammatory molecules. Mononuclear phagocytes expressing the fractalkine receptor CX3CR1 and displaying a macrophage phenotype, play a key role in the uptake and sampling of bacterial and fungal antigens from the intestinal lumen [35, 36, 37, 38, 39].

Gut microbiota has a crucial role in maintaining tolerogenic function i.e., immunological tolerance of intestinal macrophages and bacterial dysbiosis has strongly been associated with intestinal inflammation and IBD [40, 41, 42]. Intestinal epithelium-adhering bacteria can interact with CX3CR1 MPs to regulate the immune balance in health and diseases. The enrichment of adherent-invasive Escherichia coli in ileal mucosa has been described in active Crohn’s disease [43, 44]. This bacterium stimulates the production of IL-10 by CX3CR MPs and suppresses the Th17 immune responses [44, 45]. Klebsiella species derived from the oral cavity have been found to inhabit the intestine of IBD patients and induce severe colitis by the activation of Th1 proinflammatory immune response [46].

Koscsó et al. [47] performed extensive phenotypical, transcriptional, and functional analyses of intestinal inflammatory MPs in Salmonella colitis model. CX3CR1⁺MPs were identified as the predominating inflammatory cell type and were further divided into monocyte-derived Nos2⁺ CX3CR1^lo, lymph migratory Ccr7⁺CX3CR1^int and mucosa resident Cxcl13⁺CX3CR1^hi subsets. An increase in MPs in the inflamed bowel was found to be directly related to the increase in CX3CR1^lo, CX3CR1^int and CX3CR1^hi macrophage populations and thus, have an apparent role in the induction of pathogen-specific mucosal IgA response [34, 47]. These studies suggest that CX3CR1 MPs are crucial in maintaining immune homeostatic conditions and controlling intestinal disease development.

5. Disease-specific signatures of Crohn’s disease and ulcerative colitis

Inflammatory bowel disease (IBD) involves chronic intestinal inflammation linked with critical ailment and has two subtypes- ulcerative colitis (UC), which directly affects the colon and Crohn’s disease (CD) which can affect any part of the gastrointestinal (GI) tract. Macroscopic patterns of inflammation can at times distinguish between UC and CD but an insight of mucosal and peripheral immunological as well as microbial signatures differentiating these two subtypes becomes necessary for the diagnosis, prevention of recurrence or complications, and effective treatment [48].

5.1 Microbial signatures of IBD subtypes

Gut microbiota dysbiosis has been associated with disease phenotypes in IBD and may be a causative or synergistic factor in prolonged or chronic inflammation. Microbial dysbiosis in IBD is characterized by a significant reduction in bacterial diversity and alterations in some specific taxa, including enrichment of the phyla Proteobacteria and Bacteroidetes, and a reduction in Firmicutes [49, 50, 51, 52]. CD has been presented with a decrease in the proportion of Firmicutes and a slight increase in Enterobacteriaceae when compared with controls and UC patients [53]. Bacteroides, Eubacterium, Faecalibacterium, and Ruminococcus are the main bacterial genera reduced in the fecal samples of CD patients [54, 55]. A reduction in Faecalibacterium prausnitzii has been implicated in the etiology of CD, suggesting a critical role for the organism as an integral component of the anti-inflammatory balance in health and in CD pathogenesis. The phylum Proteobacteria is highly abundant in patients with active UC and decreased significantly in patients in remission, where as vice-versa for Firmicutes. Patients with active UC show an enrichment of Klebsiella, Enterococcus, and Haemophilus, while those in remission have higher numbers of Roseburia, Lachnospira, Blautia, and Faecalibacterium [56].

5.2 Immune cell signatures of IBD subtypes

An elaborated knowledge of the inflammatory landscape and immune markers of IBD in circulation and tissues become essential for the effective disease management in IBD subtypes. In this view, Mitsialis et al. carried out multidimensional immunophenotyping of colonic mucosa and peripheral blood of IBD (UC & CD) and non-IBD subjects to provide a deep understanding of the disease-specific immunophenotypes in UC and CD (Figure 2) [57]. Active ulcerative colitis (UCa) mucosa had relatively more B cells and fewer T cells and cytokine-producing effector memory (EM)-T cell subsets- IFNG⁺TNF⁺ were reduced whereas IL17A⁺⁺CD161⁺ subsets were enriched. CXCR3⁺ plasmablasts were found to be expanded in UCa. HLA-DR⁺CD38⁺ memory regulatory T cells (mTregs) were also abundant in UCa and co-expressed various chemokine receptors implying an activated memory phenotype. UCa mucosa was enriched with granulocytes expressing chemokine receptors (CXCR3, CCR6) and unconventional granulocyte markers (HLA-DR, CD38, and CD56) described to be up-regulated on granulocytes in other human diseases (Table 2).

Figure 2.

Disease-specific immunosignatures of Crohn’s disease (CD) and Ulcerative colitis (UC) mucosa and periphery.

Ulcerative colitis (UC) specific immunophenotypes		Crohn’s disease (CD) specific immunophenotypes
B:T cell ratio	+	HLA-DR+CD38+ T cells; IL17A^+ HLA-DR^+ CD38^+ CD161^+ DN Effector Memory T cells; IL1B^+ HLA-DR^+ CD38^+ T cells	-; +; +
Cytokine-producingeffector memory (EM)-T cell subsets: IFNG+TNF+ IL17A++CD161+	-; +	IL1B+ IFNG+ TNF+ naïve B-cell clusters	+
CXCR3+ plasmablasts	+	CD14+ and IL1B+ macrophages/monocytes clusters	+
HLA-DR+CD38+ mTregs	+	Innate lymphoid cells (ILCs): ILC1 and ILC1-like clusters ILC3	+; -
Chemokine receptors CXCR3, CCR6	+

Table 2.

Disease specific alterations of immune cells in IBD subtypes.

“+”= enriched; and “-” = reduced.

In case of active Crohn’s disease mucosa (CDa), HLA-DR⁺CD38⁺ T cells co-expressing IFNG⁺TNF⁺ were diminished whereasIL17A⁺ HLA-DR⁺ CD38⁺ CD161⁺ DN EM T cells and IL1B⁺ HLA-DR⁺ CD38⁺ T cells demonstrated expansion. IL1B⁺ IFNG⁺ TNF⁺ naïve B-cell clusters were augmented in CDa mucosa and included CD44⁺⁺ (marker of activated B cells), CCR7⁺, AHR⁺, HLA-DR⁺, CD38⁺ and CD11C⁺, a marker expressed in B cells and proficient in antigen presentation linked with autoimmunity [57]. Total CD14⁺ as well as IL1B+ macrophages/monocytes clusters were increased in peripheral CDa. Innate lymphoid cells (ILCs) signatures could differentiate Crohn’s disease from ulcerative colitis. ILC1 and ILC1-like clusters were increased more in the mucosa in case of CDa than UCa whereas ILC3 were specifically reduced in UCa mucosa (Table 2). These findings could be explored for targeted therapeutics and possibly harnessed for personalized approaches to IBD therapy in the future.

6. Conclusion

Even though there has been a massive upsurge in the research related to host-microbiota interactions as well as the role of genetics, environmental factors, and the immune system in IBD, several facets of IBD pathogenesis remain obscure. This chapter collates the contemporary advancements in host-microbiota investigations which can be pivotal in detecting the hallmarks of IBD leading to upgraded comprehension of its pathogenesis, extension of the diagnostic repertoire and discovery of cutting-edge therapeutic targets for this disease.

EVs have emerged as prominent tools in deciphering the complex host-microbiota interactions in healthy as well as disease states. They not only regulate the gut microbiome communities, but also actively participate in the disharmony between bacteria and their hosts. EVs derived from gut commensal bacteria have been studied to play a crucial role in immunomodulation and regulating gut homeostasis in IBD [22]. The first proteomic characterization of intestinal EVs from children with new-onset IBD illustrated the presence of host defense proteins in the isolated EV samples, especially the reactive oxidant-producing enzymes responsible for increased oxidative stress in the intestine [26]. Increased oxidative stress triggers microbial defense responses and functional alterations leading to gut microbial dysbiosis and mucosal inflammation [58]. This learning is crucial for the thorough analysis of host–microbiome interactions underlying the development of IBD and the potential use of EVs as diagnostic markers and/or therapeutic agents.

Dysbiosis of microbiota in germ-free mice have been demonstrated to cause abnormal imprinting of the intestinal immune system [29]. It provides a potential link between early life exposures, microbiome and future risk of IBD, highlighting the consequences of the abnormal establishment of early life microbiome during the development of the immune system. Maternal IBD negatively impacts the development of a baby’s intestinal ecosystem. Dysbiosis, in pregnant women with IBD or during early infancy can be aimed for promoting the development of a healthy microbiome in the offspring and reducing the potential risk of IBD.

Intestinal resident macrophages are acknowledged as key cellular sensors, integrating signals from the luminal microbiota to regulate intestinal homeostasis. Recent studies affirm their role in promoting anti-inflammatory environment in the healthy gut and switching to a proinflammatory state in response to any alterations in the intestinal microbiota [59]. Follow-up studies should be done to devise tools for identifying patients with compromised resident intestinal macrophages function and evaluating the clinical advantages of targeting the microbiota and immune dysfunctions within this subset of IBD patients. Intestinal macrophage subsets also exhibit peculiar activity in stimulating mucosal IgA responses [47]. This differential activity can be harnessed for designing anti-inflammatory therapies aimed at modulating macrophage function in inflammatory bowel disease.

IBD includes Crohn’s disease and ulcerative colitis which are two distinct pathological conditions macroscopically, but often misinterpreted or difficult to distinguish on a deeper extent. There has been evidence of disease-specific statistical shifts in some bacterial species as well as phyla, peculiar to each subtype of IBD [56]. Single-cell analysis with CyTOF on IBD and non-IBD colonic mucosa and blood to identify disease-specific immune signatures revealed the abundance of HLA-DR⁺CD38⁺ T cells in both active Crohn’s disease (CDa) and ulcerative colitis (UCa) mucosa [57]. CD38 has been involved in colitis in mice [60] whereas CD38⁺ effector T cells in pediatric IBD [61], suggesting that CD38 could be targeted for IBD therapy. Various disease-specific mucosal signatures associated with differential cytokine expression were also reported. IL1B signatures particular to CD involved HLA-DR⁺CD38⁺ T cells, naïve B cells, and DCs. IL1B⁺ macrophages/monocytes were augmented in both CDa and Uca mucosa, along with a specific expansion of IL1B+ monocytes to only peripheral CDa [57, 62]. Thus, exploiting IL1B can be a promising therapeutic strategy for subsets of Crohn’s disease. These extrusive microbial and immunological signatures of IBD can also be of high biological and diagnostic potential. To sum up, the above-discussed studies have a robust potential of heralding state-of-art diagnostic as well as therapeutic avenues in the field of inflammatory bowel disease. Further translational work based upon these findings can lead to the upgradation of our insight and methodology towards gut disorders as critical as IBD with a prospect of personalized therapies soon.

Conflict of interest

The authors declare no conflict of interest.