**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 [18–20]. Based on their biogenesis and size, EVs have been categorized into microvesicles, exosomes, ectosomes, oncosomes, and outer membrane vesicles (**Table 1**) [21].

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


### **Table 1.**

*Classification of extracellular vesicles.*

### **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).*

(ulcerative colitis) conditions [25]. These findings thus, suggest the role of cell-type as well as health status in influencing BEV-host interaction.

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.
