Integration of proteomic and metabolomic analysis reveal distinct metabolic alterations of prostate cancer-associated fibroblasts compared to normal fibroblasts from patient's stroma samples.

The prostate gland is a complex and heterogeneous organ composed of epithelium and stroma. Whilst many studies into prostate cancer focus on epithelium, the stroma is known to play a key role in disease with the emergence of a cancer-associated fibroblasts (CAF) phenotype associated upon disease progression. In this work, we studied the metabolic rewiring of stromal fibroblasts following differentiation to a cancer-associated, myofibroblast-like, phenotype. We determined that CAFs were metabolically more active compared to normal fibroblasts. This corresponded with a heightened lipogenic metabolism, as both reservoir species and building block compounds. Interestingly, lipid metabolism affects mitochondria functioning yet the mechanisms of lipid-mediated functions are unclear. Data showing oxidised fatty acids and glutathione system are elevated in CAFs, compared to normal fibroblasts, strengthens the hypothesis that increased metabolic activity is related to mitochondrial activity. This manuscript describes mechanisms responsible for the altered metabolic flux and shows that prostate cancer-derived extracellular vesicles can increase basal respiration in normal fibroblasts, mirroring that of the disease-like phenotype. This indicates that extracellular vesicles derived from prostate cancer cells may drive an altered oxygen-dependent metabolism associated to mitochondria in CAFs.

Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, are able to invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis and here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the ß1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. Spatially, sEV were secreted via filopodia-like cellular protrusions at the leading edge of migrating cells. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI. In vitro assays identified collagen VI as playing the key role in cell adhesion and invasion. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate migration and invasion by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction forces to enable VSMC movement within the complex vascular ECM network.

Extracellular vesicles derived from umbilical cord mesenchymal stromal cells show enhanced anti-inflammatory properties via upregulation of miRNAs after pro-inflammatory priming.

Autoimmune conditions, such as rheumatoid arthritis, are characterised by a loss of immune tolerance, whereby the immune cells attack self-antigens causing pain and inflammation. These conditions can be brought into remission using pharmaceutical treatments, but often have adverse side effects and some patients do not respond favourably to them. Human umbilical cord mesenchymal stromal cells (UCMSCs) present a promising alternative therapeutic due to their innate anti-inflammatory properties which can be strengthened using pro-inflammatory conditions. Their therapeutic mechanism of action has been attributed to paracrine signalling, by which nanosized acellular particles called 'extracellular vesicles' (EVs) are one of the essential components. Therefore, this research analysed the anti-inflammatory properties of UCMSC-EVs 'primed' with pro-inflammatory cytokines and at baseline with no inflammatory cytokines (control). Both control and primed EVs were co-cultured with un-pooled peripheral blood mononuclear cells (PBMCs; n = 6) from healthy donors. Neither control nor primed EVs exerted a pro-inflammatory effect on PBMCs. Instead, the primed EVs showed the immunosuppressive potential by increasing the expression of the anti-inflammatory protein FoxP3 in PBMCs. This may be attributed to the upregulated miRNAs identified in primed EVs in comparison to control EVs (miR-139-5p, miR-140-5p, miR-214-5p). These findings aid in understanding how UCMSC-EVs mediate immunosuppression and support their potential use in treating autoimmune conditions.

An enhanced tilted-angle acoustic tweezer for mechanical phenotyping of cancer cells.

Acoustofluidic devices becomes one of the emerging and versatile tools for many biomedical applications. Most of the previous acoustofluidic devices are used for cells manipulation, and the few devices for cell phenotyping with a limitation in throughput. In this study, an enhanced tilted-angle (ETA) acoustofluidic device is developed and applied for mechanophenotyping of live cells. The ETA Device consists of an interdigital transducer which is positioned along a microfluidic channel. An inclination angle of 5° is introduced between the interdigital transducer and the liquid flow direction. The pressure nodes formed inside the acoustofluidic field in the channel deflect the biological cells from their original course in accordance with their mechanical properties, including volume, compressibility, and density. The threshold power for fully converging the cells to the pressure node is used to calculate the acoustic contrast factor. To demonstrate the ETA device in cell mechanophenotyping, and distinguishing between different cell types, further experimentation is carried out by using A549 (lung cancer cells), MDB-MA-231 (breast cancer cells), and leukocytes. The resulting acoustic contrast factors for the lung and breast cancer cells are different from that of the leukocytes by 27.9% and 21.5%, respectively. These results suggest this methodology can successfully distinguish and phenotype different cell types based on the acoustic contrast factor.

Dual-Wave Acoustofluidic Centrifuge for Ultrafast Concentration of Nanoparticles and Extracellular Vesicles.

Extracellular vesicles (EVs) are secreted nanostructures that play various roles in critical cancer processes. They operate as an intercellular communication system, transferring complex sets of biomolecules from cell to cell. The concentration of EVs is difficult to decipher, and there is an unmet technological need for improved (faster, simpler, and gentler) approaches to isolate EVs from complex matrices. Herein, an acoustofluidic concentration of extracellular vesicles (ACEV) is presented, based on a thin-film printed circuit board with interdigital electrodes mounted on a piezoelectric substrate. An angle of 120° is identified between the electrodes and the reference flat of the piezoelectric substrate for simultaneous generation of Rayleigh and shear horizontal waves. The dual waves create a complex acoustic field in a droplet, resulting in effective concentration of nanoparticles and EVs. The ACEV is able to concentrate 20 nm nanospheres within 105 s and four EV dilutions derived from the human prostate cancer (Du145) cell line in approximately 30 s. Cryo-electron microscopy confirmed the preservation of EV integrity. The ACEV device holds great potential to revolutionize investigations of EVs. Its faster, simpler, and gentler approach to EV isolation and concentration can save time and effort in phenotypic and functional studies of EVs.

Oral Progenitor Cell Line-Derived Small Extracellular Vesicles as a Treatment for Preferential Wound Healing Outcome.

Scar formation during wound repair can be devastating for affected individuals. Our group previously documented the therapeutic potential of novel progenitor cell populations from the non-scarring buccal mucosa. These Oral Mucosa Lamina Propria-Progenitor Cells (OMLP-PCs) are multipotent, immunosuppressive, and antibacterial. Small extracellular vesicles (sEVs) may play important roles in stem cell-mediated repair in varied settings; hence, we investigated sEVs from this source for wound repair. We created an hTERT immortalized OMLP-PC line (OMLP-PCL) and confirmed retention of morphology, lineage plasticity, surface markers, and functional properties. sEVs isolated from OMLP-PCL were analyzed by nanoparticle tracking analysis, Cryo-EM and flow cytometry. Compared to bone marrow-derived mesenchymal stromal cells (BM-MSC) sEVs, OMLP-PCL sEVs were more potent at driving wound healing functions, including cell proliferation and wound repopulation and downregulated myofibroblast formation. A reduced scarring potential was further demonstrated in a preclinical in vivo model. Manipulation of OMLP-PCL sEVs may provide novel options for non-scarring wound healing in clinical settings.

Challenges and directions in studying cell-cell communication by extracellular vesicles.

Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. They have important roles in numerous physiological and pathological processes, and show considerable promise as novel biomarkers of disease, as therapeutic agents and as drug delivery vehicles. Intriguingly, however, understanding of the cellular and molecular mechanisms that govern the many observed functions of EVs remains far from comprehensive, at least partly due to technical challenges in working with these small messengers. Here, we highlight areas of consensus as well as contentious issues in our understanding of the intracellular and intercellular journey of EVs: from biogenesis, release and dynamics in the extracellular space, to interaction with and uptake by recipient cells. We define knowledge gaps, identify key questions and challenges, and make recommendations on how to address these.

Cancer-Associated Fibroblast Heterogeneity, Activation and Function: Implications for Prostate Cancer.

The continuous remodeling of the tumor microenvironment (TME) during prostate tumorigenesis is emerging as a critical event that facilitates cancer growth, progression and drug-resistance. Recent advances have identified extensive communication networks that enable tumor-stroma cross-talk, and emphasized the functional importance of diverse, heterogeneous stromal fibroblast populations during malignant growth. Cancer-associated fibroblasts (CAFs) are a vital component of the TME, which mediate key oncogenic events including angiogenesis, immunosuppression, metastatic progression and therapeutic resistance, thus presenting an attractive therapeutic target. Nevertheless, how fibroblast heterogeneity, recruitment, cell-of-origin and differential functions contribute to prostate cancer remains to be fully delineated. Developing our molecular understanding of these processes is fundamental to developing new therapies and biomarkers that can ultimately improve clinical outcomes. In this review, we explore the current challenges surrounding fibroblast identification, discuss new mechanistic insights into fibroblast functions during normal prostate tissue homeostasis and tumorigenesis, and illustrate the diverse nature of fibroblast recruitment and CAF generation. We also highlight the promise of CAF-targeted therapies for the treatment of prostate cancer.

Stroma-derived extracellular vesicle mRNA signatures inform histological nature of prostate cancer.

Histological assessment of prostate cancer is the key diagnostic test and can predict disease outcome. This is however an invasive procedure that carries associated risks, hence non-invasive assays to support the diagnostic pathway are much needed. A key feature of disease progression, and subsequent poor prognosis, is the presence of an altered stroma. Here we explored the utility of prostate stromal cell-derived vesicles as indicators of an altered tumour environment. We compared vesicles from six donor-matched pairs of adjacent-normal versus disease-associated primary stromal cultures. We identified 19 differentially expressed transcripts that discriminate disease from normal stromal extracellular vesicles (EVs). EVs isolated from patient serum were investigated for these putative disease-discriminating mRNA. A set of transcripts including Caveolin-1 (CAV1), TMP2, THBS1, and CTGF were found to be successful in discriminating clinically insignificant (Gleason = 6) disease from clinically significant (Gleason > 8) prostate cancer. Furthermore, correlation between transcript expression and progression-free survival suggests that levels of these mRNA may predict disease outcome. Informed by a machine learning approach, combining measures of the five most informative EV-associated mRNAs with PSA was shown to significantly improve assay sensitivity and specificity. An in-silico model was produced, showcasing the superiority of this multi-modal liquid biopsy compared to needle biopsy for predicting disease progression. This proof of concept highlights the utility of serum EV analytics as a companion diagnostic test with prognostic utility, which may obviate the need for biopsy.

The role of bacterial extracellular vesicles in chronic wound infections: Current knowledge and future challenges.

Chronic wounds are a significant global problem with an increasing economic and patient welfare impact. How wounds move from an acute to chronic, non-healing, state is not well understood although it is likely that it is driven by a poorly regulated local inflammatory state. Opportunistic pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa are well known to stimulate a pro-inflammatory response and so their presence may further drive chronicity. Studies have demonstrated that host cell extracellular vesicles (hEVs), in particular exosomes, have multiple roles in both increasing and decreasing chronicity within wounds; however, the role of bacterial extracellular vesicles (bEVs) is still poorly understood. The aim of this review is to evaluate bEV biogenesis and function within chronic wound relevant bacterial species to determine what, if any, role bEVs may have in driving wound chronicity. We determine that bEVs drive chronicity by both increasing persistence of key pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa and stimulating a pro-inflammatory response by the host. Data also suggest that both bEVs and hEVs show therapeutic promise, providing vaccine candidates, decoy targets for bacterial toxins or modulating the bacterial species within chronic wound biofilms. Caution should, however, be used when interpreting findings to date as the bEV field is still in its infancy and as such lacks consistency in bEV isolation and characterization. It is of primary importance that this is addressed, allowing meaningful conclusions to be drawn and increasing reproducibility within the field.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

Prostate Cancer Cell Extracellular Vesicles Increase Mineralisation of Bone Osteoblast Precursor Cells in an In Vitro Model.

Skeletal metastases are the most common form of secondary tumour associated with prostate cancer (PCa). The aberrant function of bone cells neighbouring these tumours leads to the devel-opment of osteoblastic lesions. Communication between PCa cells and bone cells in bone envi-ronments governs both the formation/development of the associated lesion, and growth of the secondary tumour. Using osteoblasts as a model system, we observed that PCa cells and their conditioned medium could stimulate and increase mineralisation and osteoblasts' differentiation. Secreted factors within PCa-conditioned medium responsible for osteoblastic changes included small extracellular vesicles (sEVs), which were sufficient to drive osteoblastogenesis. Using MiR-seq, we profiled the miRNA content of PCa sEVs, showing that miR-16-5p was highly ex-pressed. MiR-16 was subsequently higher in EV-treated 7F2 cells and a miR-16 mimic could also stimulate mineralisation. Next, using RNA-seq of extracellular vesicle (EV)-treated 7F2 cells, we observed a large degree of gene downregulation and an increased mineralisation. Ingenuity® Pathway Analysis (IPA®) revealed that miR-16-5p (and other miRs) was a likely upstream effec-tor. MiR-16-5p targets in 7F2 cells, possibly involved in osteoblastogenesis, were included for val-idation, namely AXIN2, PLSCR4, ADRB2 and DLL1. We then confirmed the targeting and dow-regulation of these genes by sEV miR-16-5p using luciferase UTR (untranslated region) reporters. Conversely, the overexpression of PLSCR4, ADRB2 and DLL1 lead to decreased osteoblastogene-sis. These results indicate that miR-16 is an inducer of osteoblastogenesis and is transmitted through prostate cancer-derived sEVs. The mechanism is a likely contributor towards the for-mation of osteoblastic lesions in metastatic PCa.

Macrophage Plasticity and Function in the Lung Tumour Microenvironment Revealed in 3D Heterotypic Spheroid and Explant Models.

In non-small cell lung cancer (NSCLC), stroma-resident and tumour-infiltrating macrophages may facilitate an immunosuppressive tumour microenvironment (TME) and hamper immunotherapeutic responses. Analysis of tumour-associated macrophage (TAM) plasticity in NSCLC is largely lacking. We established a novel, multi-marker, dual analysis approach for assessing monocyte-derived macrophage (Mφ) polarisation and M1/M2 phenotypic plasticity. We developed a flow cytometry-based, two-marker analysis (CD64 and CD206) of CD14+ cells. The phenotype and immune function of in vitro-induced TAMs was studied in a heterotypic spheroid and tumour-derived explant model of NSCLC. Heterotypic spheroids and NSCLC explants skewed Mφs from an M1- (CD206loCD64hi) to M2-like (CD206hiCD64lo) phenotype. Lipopolysaccharide (LPS) and IFNγ treatment reversed M2-like Mφ polarisation, indicating the plasticity of Mφs. Importantly, antigen-specific CD8+ T cell responses were reduced in the presence of tumour explant-conditioned Mφs, but not spheroid-conditioned Mφs, suggesting explants are likely a more relevant model of the immune TME than cell line-derived spheroids. Our data indicates the importance of multi-marker, functional analyses within Mφ subsets and the advantages of the ex vivo NSCLC explant model in immunomodulation studies. We highlight the plasticity of the M1/M2 phenotype using the explant model and provide a tool for studying therapeutic interventions designed to reprogram M2-like Mφ-induced immunosuppression.

Correction: Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB.

Correction for 'Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB' by Roman Mikhaylov et al., Lab Chip, 2020, 20, 1807-1814, DOI: 10.1039/C9LC01192G.

Analysis of Neat Biofluids Obtained During Cardiac Surgery Using Nanoparticle Tracking Analysis: Methodological Considerations.

Small extracellular vesicles (sEVs) are those nanovesicles 30-150 nm in size with a role in cell signalling and potential as biomarkers of disease. Nanoparticle tracking analysis (NTA) techniques are commonly used to measure sEV concentration in biofluids. However, this quantification technique can be susceptible to sample handing and machine settings. Moreover, some classes of lipoproteins are of similar sizes and could therefore confound sEV quantification, particularly in blood-derived preparations, such serum and plasma. Here we have provided methodological information on NTA measurements and systematically investigated potential factors that could interfere with the reliability and repeatability of results obtained when looking at neat biofluids (i.e., human serum and pericardial fluid) obtained from patients undergoing cardiac surgery and from healthy controls. Data suggest that variables that can affect vesicle quantification include the level of contamination from lipoproteins, number of sample freeze/thaw cycles, sample filtration, using saline-based diluents, video length and keeping the number of particles per frame within defined limits. Those parameters that are of less concern include focus, the "Maximum Jump" setting and the number of videos recorded. However, if these settings are clearly inappropriate the results obtained will be spurious. Similarly, good experimental practice suggests that multiple videos should be recorded. In conclusion, NTA is a perfectible, but still commonly used system for sEVs analyses. Provided users handle their samples with a highly robust and consistent protocol, and accurately report these aspects, they can obtain data that could potentially translate into new clinical biomarkers for diagnosis and monitoring of cardiovascular disease.

Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB.

Acoustofluidics has been increasingly applied in biology, medicine and chemistry due to its versatility in manipulating fluids, cells and nano-/micro-particles. In this paper, we develop a novel and simple technology to fabricate a surface acoustic wave (SAW)-based acoustofluidic device by clamping electrodes made using a printed circuit board (PCB) with a piezoelectric substrate. The PCB-based SAW (PCB-SAW) device is systematically characterised and benchmarked with a SAW device made using the conventional photolithography process with the same specifications. Microparticle manipulations such as streaming in droplets and patterning in microchannels were demonstrated in the PCB-SAW device. In addition, the PCB-SAW device was applied as an acoustic tweezer to pattern lung cancer cells to form three or four traces inside the microchannel in a controllable manner. Cell viability of ∼97% was achieved after acoustic manipulation using the PCB-SAW device, which proved its ability as a suitable tool for acoustophoretic applications.

Pro-Inflammatory Priming of Umbilical Cord Mesenchymal Stromal Cells Alters the Protein Cargo of Their Extracellular Vesicles.

Umbilical cord mesenchymal stromal cells (UCMSCs) have shown an ability to modulate the immune system through the secretion of paracrine mediators, such as extracellular vesicles (EVs). However, the culture conditions that UCMSCs are grown in can alter their secretome and thereby affect their immunomodulatory potential. UCMSCs are commonly cultured at 21% O2 in vitro, but recent research is exploring their growth at lower oxygen conditions to emulate circulating oxygen levels in vivo. Additionally, a pro-inflammatory culture environment is known to enhance UCMSC anti-inflammatory potential. Therefore, this paper examined EVs from UCMSCs grown in normal oxygen (21% O2), low oxygen (5% O2) and pro-inflammatory conditions to see the impact of culture conditions on the EV profile. EVs were isolated from UCMSC conditioned media and characterised based on size, morphology and surface marker expression. EV protein cargo was analysed using a proximity-based extension assay. Results showed that EVs had a similar size and morphology. Differences were found in EV protein cargo, with pro-inflammatory primed EVs showing an increase in proteins associated with chemotaxis and angiogenesis. This showed that the UCMSC culture environment could alter the EV protein profile and might have downstream implications for their functions in immunomodulation.

Targeting Antigen to the Surface of EVs Improves the In Vivo Immunogenicity of Human and Non-human Adenoviral Vaccines in Mice.

Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5.

Considerations towards a roadmap for collection, handling and storage of blood extracellular vesicles.

There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids ("liquid biopsies"). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non-EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single-centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence-based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood.

Neutrophil-derived miR-223 as local biomarker of bacterial peritonitis.

Infection remains a major cause of morbidity, mortality and technique failure in patients with end stage kidney failure who receive peritoneal dialysis (PD). Recent research suggests that the early inflammatory response at the site of infection carries diagnostically relevant information, suggesting that organ and pathogen-specific "immune fingerprints" may guide targeted treatment decisions and allow patient stratification and risk prediction at the point of care. Here, we recorded microRNA profiles in the PD effluent of patients presenting with symptoms of acute peritonitis and show that elevated peritoneal miR-223 and reduced miR-31 levels were useful predictors of bacterial infection. Cell culture experiments indicated that miR-223 was predominantly produced by infiltrating immune cells (neutrophils, monocytes), while miR-31 was mainly derived from the local tissue (mesothelial cells, fibroblasts). miR-223 was found to be functionally stabilised in PD effluent from peritonitis patients, with a proportion likely to be incorporated into neutrophil-derived exosomes. Our study demonstrates that microRNAs are useful biomarkers of bacterial infection in PD-related peritonitis and have the potential to contribute to disease-specific immune fingerprints. Exosome-encapsulated microRNAs may have a functional role in intercellular communication between immune cells responding to the infection and the local tissue, to help clear the infection, resolve the inflammation and restore homeostasis.