Human Urinary Exosomes As Innate Immune Effectors

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Human Urinary Exosomes as Innate Immune Effectors

† † ‡ Thomas F. Hiemstra,* Philip D. Charles, Tannia Gracia, Svenja S. Hester,§ † ‡ | Laurent Gatto, Raﬁa Al-Lamki,* R. Andres Floto,* Ya Su, Jeremy N. Skepper, † ‡ Kathryn S. Lilley, and Fiona E. Karet Frankl

*Department of Medicine, †Cambridge Centre for Proteome Research and Cambridge Systems Biology Centre, Department of Biochemistry, ‡Department of Medical Genetics, and |Multi-Imaging Centre, Department of Anatomy, University of Cambridge, Cambridge, United Kingdom; and §Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

ABSTRACT Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein. Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are signiﬁcantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm–Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract.

J Am Soc Nephrol 25: ccc–ccc, 2014. doi: 10.1681/ASN.2013101066

Exosomes form as intraluminal vesicles of multi- every cell type along the nephron.13,14 The array of vesicular bodies (MVBs), contain membrane and functions ascribed to exosomes in other tissues has cytoplasmic proteins, have a cytoplasmic-side in- kindled recent interest in the functional signiﬁcance ward membrane orientation, and are released intact of urinary exosomes. Hogan et al.13 suggested inter- into the extracellular space (Figure 1A). First de- action of exosome-like vesicles with primary cilia of scribed in maturing ovine reticulocytes,1 exosomes renal epithelial cells, and Street and coworkers15 are released by many cell types2 and have been con- showed in vitro uptake of exosomes by a renal cortical ventionally regarded as a vehicle for shedding obsolete protein. However, emerging evidence has revealed a variety of exosomal functions, including Received October 11, 2013. Accepted January 6, 2014. 3 the intercellular transfer of membrane receptors Published online ahead of print. Publication date available at 4–6 7 and RNA, induction of immunity, antigen pre- www.jasn.org. sentation,8 modulation of bone mineralization,9 Correspondence: Prof. Fiona E. Karet Frankl, Department of 10 and antiapoptotic responses. Medical Genetics, University of Cambridge, Cambridge Institute Nanovesicles were ﬁrst shown in human urine by for Medical Research, Cambridge Biomedical Campus Box 139, Kanno and colleagues11 and subsequently, were Hills Road, Cambridge CB2 0XY, UK, or Prof. Kathryn S. Lilley, Cambridge Centre for Proteomics and Cambridge Systems Bi- 12 shown to represent exosomes. Consistent with a ology Centre, Department of Biochemistry, University of Cambridge, renal tubular epithelial origin, renal tubular epithelial Cambridge CB2 1QR, UK. Email: [email protected] or fek1000@ cells contain MVBs at the apical surface, and urine cam.ac.uk exosomes contain apical membrane proteins from Copyright © 2014 by the American Society of Nephrology

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Published reports of the urinary exosomal proteome have limited value in illumi- nating the potential functions of urinary exosomes for several reasons. First, protein identification by mass spectrometry (MS) has, until recently, yielded results with unacceptably low reproducibility and high false-positive rates,22,23 and previous reports are not free of these limitations. Sec- ond, studies have often aimed to maximize the number of protein identifications and hence, biomarker candidates rather than applying or reporting rigorous protein identification thresholds. Third, most have relied on pooled samples from up to six donors and have not reported interin- dividual variability or reproducibility. Here, we sought, for the first time, to ascribe functionality to human urinary exosomes. We initially performed rigorous, conservative tandem MS analysis of separate human urinary exosomal samples to allow enrichment scoring24 to elucidation of urine exosomal function.

RESULTS

Exosomal samples were obtained from 10 healthy volunteers (five men and five women) ages 23–36 years. Nine subjects were Caucasian, and one man was Mauri- Figure 1. Vesicles isolated from human urine are consistent with exosomes. (A) tian. Samples prepared from 455628 ml sec- – Exosomes are derived from the endocytic pathway (1 4) forming through invagination ond morning void contained 0.5460.18 mg of the limiting membrane of the MVB (3). They are released into the urinary space from protein/ml urine. Exosomal integrity was renal tubular epithelial cells through fusion of the MVB with the apical plasma mem- fi fi fi con rmed by the electron microscopy brane (4). (B and C) Exosomal isolation was con rmed by the identi cation by negative 6 stain EM of nanovesicles (black arrows; characteristic mean 50-nm size distribution. (D) (EM) demonstration of 54.5 14-nm ves- fi Uromodulin (white streaks in B and dark in C; open arrows in B and C) cofractionated icles (Figure 1, B and D), the identi ca- with exosomes but was confirmed to be extraexosomal (5 nm gold-labeled; white tion of known exosomal markers TSG101, arrows in C). (E) Western blot confirmed the presence of known exosomal constituents enolase-1, CD63, podocin, and aquaporin-2 in vesicle preparations but did not confirm them in precipitated protein from exosome- by Western blot and immunogold EM depleted urine. (F) Immuno-EM with 5 (TSG101 and CD63) or 15 nm (AQP2) gold (Figure 1, E and F), and the confirmation particle-labeled antibodies showed vesicular residency of known exosomal con- of a cytoplasmic side inward membrane ori- stituents (arrows). Vesicles were nonpermeabilized; thus, positive staining with an anti- entation (Figure 1F). CD63 antibody directed against an extracellular epitope indicated the cytoplasmic We applied a number of methodological side inward membrane orientation characteristic of exosomes. EDUP, exosome-depleted approaches to overcome limitations of pre- urine protein; HKM, human kidney membrane; MW, molecular weight; TSG101, tumour susceptibility gene 101. vious studies. (1) Samples were not pooled but analyzed separately. (2)MSwasper- formed without and with a uromodulin ex- collecting duct cell line, leading to speculation that exosomes clusion list25 on a high-sensitivity instrument. (3) Data were may provide intrarenal proximal-to-distal transapical renal tu- analyzed using stringent and novel bioinformatics ap- bular epithelial signaling through RNA transfer. However, most proaches26 (Supplemental Material). (4) All ambiguous pep- studies on urine exosomes to date have focused on biomarker tides were excluded unless matched only to products of a discovery, resulting in the publication of several urine exosome single gene. From 50 mg exosomal protein per subject, we protein compendia.12,13,16–21 identified 601 unique proteins by MS, with a median

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2 2 (interquartile range [IQR]) P value of 1.17310 13 (4.08310 4) suggesting a potential role in the targeting of RNA to exo- (Supplemental Table 1). Importantly, the use of a fixed uromodulin somes,4 or involved in innate immunity and the response to exclusion list25 and a posterior error-derived protein type I infection. Uromodulin was present in all 10 samples, consis- error estimator termed espresso (Supplemental Material) un- tent with previous reports, but confirmed as extraexosomal by masked proteins that would not otherwise have been evident immunogold EM (Figure 1D). All MS data have been depos- (Figure 2A). The complete proteome included the known exo- ited with the ProteomeXchange consortium repository somal markers TSG101, CD14, and CD59. However, 307 (51%) (http://proteomecentral.proteomexchange.org; reference had not previously been identified in exosomes from any source PXD000117). compared with the EXOCARTAdatabase.27 There was only min- Using enrichment scoring (ES),24 we found, in addition to imal overlap with the soluble urinary proteome (Supplemental the expected enrichment for cytoskeletal proteins (ES=10.45) Figure 1). and proteins involved in the endocytic pathway and vesicle As previously reported for exosomes,12 the cellular origin of formation (ES=8.06), a very significant enrichment for pro- the majority of identified proteins was in vesicles or the endo- teins involved in immunity and host defense (ES=3.27, cytic vesicular pathway, cytoplasm, plasma membrane, and P,0.001). The MS-derived type I error estimates for proteins nucleus (Figure 2B). Apical membrane proteins from all neph- falling into this grouping were consistently small (Figure 3A), ron segments were detected, including aminopeptidase N, car- indicating high significance. These 29 proteins (Figure 3B, bonic anhydrase II and IV, chloride intracellular channel 1, Table 1) included archetypal antimicrobial proteins and pep- Cubilin, Dipeptidase 1, calbindin-D28k,andVacuolarATPase tides and fell into two categories: those proteins with known 6V0 subunit C. Most proteins were identified from fewer than bacteriostatic (such as mucin-1, fibronectin, and CD14) or one half of samples; 47 (8%) proteins were identified in all 10 bactericidal (e.g., lysozyme C, calprotectin [S100A8/A9], and samples (Figure 2, C and D), and 97 (16%) proteins were dermcidin) roles and those proteins that function as microbial identified in at least 8 of 10 samples. Each of the shared pro- receptors or bind to bacterial surface molecules.28–33 Impor- teins was identified from 6 (IQR=2–24) peptides, indicating a tantly, 28 of 29 immune proteins have known expression in set of consistently identifiable urinary exosomal constituents kidney (Table 1). We confirmed the presence and exosomal (Figure 2E). This core exosomal proteome included a large residency of a representative group of these innate immune number of proteins involved in the endocytic pathway, MVB exosomal proteins, including lysozyme C, dermcidin, mucin- formation, and exosomal biogenesis and cytoskeletal proteins 1, calprotectin, and myeloperoxidase, by Western blot and required for exosomal structural integrity. A considerable immunogold EM (Figure 3C, Supplemental Figure 9). The number was involved in transcription and RNA processing, EM appearances differ markedly from the considerably larger

Figure 2. The human urinary exosomal proteome. (A) Using conventional methods (Mascot scoring and no uromodulin exclusion), only 237 proteins would have been evident (green). The application of a uromodulin exclusion list (purple) to conventional methods and the use of espresso (cyan) greatly increased protein identifications, which was further amplified by their combination. (B) Cellular locali- zation of 601 proteins identified from exosomal pellets by tandem MS. A significant proportion consisted of cytoplasmic and membrane proteins or constituents of the endocytic pathway or vesicles. (C) Heat map showing the overlap between subjects for all proteins identified from two or more peptides (yellow) or one peptide (red) or not seen (black). (D) Most proteins were identified in the minority of subjects, consistent with the stochastic nature of MS. The x axis represents the number of subjects in whom each protein was observed; 47 proteins were observed in all 10 subjects, showing the value of analyzing multiple samples separately. (E) Principal protein groupings were consistent with previous reports of exosomes but also included those exosomes with an innate immune role.

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been proposed as an antimicrobial urinary defense protein38 and it cofractionates with exosomes during ultracentrifugation.25,39 Initial colony counting40 showed significant reduction in bacteria after incubation with exosomes (with or without uromodulin) but not uromodulin alone. However, this method is unable to assess real-time growth. To achieve this result, we transformed BL21 with the luxCDABE op- eron41 (BL21-lux) to constitutively express luciferase; transfected organisms sponta- neously omit 490-nm light. Compared with buffer alone, exosomes from each of four healthy volunteers significantly inhibited growth of BL21-lux,whereas growth was not altered by uromodulin (Figure 4). There was only minor interin- dividual variation (Supplemental Figure 2). The effect of exosomes on bacterial growth occurred rapidly, becoming appar- ent within 30 minutes of coincubation (Supplemental Figure 3). Complete inhibition of bacterial growth was dependent on exosomal structural integrity (Figure 4A, Supplemental Figure 4). To assess clinical relevance, we first transformed a uropathogenic E. coli (UPEC) strain, obtained from an infected patient, to express the luxCDABE cluster (UPEC-lux). Despite more vigorous growth, UPEC-lux growth was still almost arrested by exosomes from healthy volunteers (Figure 4A). We repeated these experiments with both BL21-lux and UPEC-lux using exosome-depleted urine Figure 3. Exosomes are enriched for innate immune proteins. (A) The exosomal as growth medium. Exosomes were equally proteome was significantly enriched for proteins with a known role in host defense. Proteins falling into this category (red) were identified with a high degree of statistical effective against both organisms, although confidence compared with all nonimmune proteins called. (B) The immune group as expected, their overall growth was slower included bactericidal and bacteriostatic proteins as well as those proteins known to in urine. We next sought to determine function as bacterial or viral receptors. (C) Western blot confirmed the presence of whether exosomes were effective against a representative group of proteins identified by MS, including myeloperoxidase (MPO), well characterized, luxCDABE-transformed, mucin-1 (MUC1), dermcidin (DCD), calprotectin (S100A8/A9 heterodimer), and lysozyme commensal (Nissle) or standard uropatho- C (LYZ). Positive controls represent human kidney membrane, except DCD (purified genic (UTI89 and CFT073) model strains of DCD) and calprotectin (neutrophil lysate). Immunogold EM shows the decoration of E. coli. In both exosome-depleted donor vesicles with 5-nm gold particles. urine and Luria-Bertani (LB) media, we observed consistent, significant growth inhi- and more heterogeneous neutrophil granule,34 where some of bition of all three organisms by exosomes but not by uromodulin these proteins are also found. alone (Figure 4A). Colony counting at peak bacterial growth Because of this enrichment, we next tested the hypothesis confirmed that the observed differences in luminescence were that exosomes may inhibit growth of or kill Escherichia coli,the attributable to significant differences in the number of viable organism responsible for up to 90% of urinary tract infections organisms (Figure 4B). (UTIs) in humans.35–37 First, BL21, a laboratory strain of The effect of coincubation with exosomes on bacterial E. coli, was incubated with exosomes, suspension buffer alone, or integrity was next evaluated by scanning EM. UPEC-lux in- purified human uromodulin, because uromodulin has itself cubated with exosomes or control and freeze-dried after 5 or

4 Journal of the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc,2014 mScNephrol Soc Am J Table 1. Identiﬁed exosomal proteins with a known innate immune role No. of Known Present in P Peptide IPI Identiﬁer Gene Symbol Protein Putative Immune Role Subjects Disambiguated Expression Exocarta Value Count with Protein in Kidney IPI00221224 ANPEP Aminopeptidase-N E. coli receptor viral sensing Yes 0 10 292 Yes 25: IPI00022463 TF Transferrin Bactericidal Yes 0 10 23 ✔ Yes ccc IPI00023673 LGALS3BP Galectin-3-binding protein Possibly bacteriostatic; Yes 0 10 31 ✔ Yes – ccc secreted in breast milk—

2014 , prevents diarrheal illness IPI00394972 IPI00394975 TRIM5 Tripartite motif Viral sensing; antiviral responses No ,0.001 10 10 ✔ Yes IPI00852949 IPI00939323 containing protein 5 IPI00909886 CARD9 Caspase recruitment Antifungal responses; deﬁciency No ,0.001 10 3 Yes domain-containing 9 results in mucosal and systemic candidiasis IPI00024254 IFIT3 IFN-induced protein Viral sensing No ,0.001 10 2 Yes with tetratricopeptide repeats 3 IPI00004573 PIGR Polymeric Ig receptor Expression of IgA and IgM at Yes 0 10 135 ✔ Yes apical surface IPI00247063 MME Neprilysin Unknown Yes 0 10 86 ✔ Yes IPI00797452 KRT10 Keratin 10 Cell surface receptor for Yes 0 10 416 ✔ Yes Staphylococcus aureus IPI00027462 S100A9 S100A8/A9 (Calprotectin; Bactericidal Yes 0 (A9) 9 11 Yes IPI00007047 S100A8 occur as heterodiner) Yes ,0.001 (A8) 6 8 Yes IPI00019038 LYZ Lysozyme C Bactericidal Yes 0 8 102 Yes IPI00294713, IPI00306378 MASP2 Mannan-binding lectin Bactericidal enterobacterial Yes 0 8 35 ✔ Yes serine peptidase 2 receptor IPI00013955, IPI00218163, MUC1 Mucin-1 Unknown bacterial receptor Yes ,0.001 7 8 ✔ Yes IPI00218164, IPI00218165, IPI00218166, IPI00218168, IPI00218169, IPI00607673, IPI00902840, IPI00978078 IPI00007244, IPI00236554, MPO Myeloperoxidase Bactericidal fungicidal Yes ,0.001 4 5 ✔ Yes mueRl o rn Exosomes Urine for Role Immune

IPI00236556 www.jasn.org IPI00029260 CD14 Monocyte differentiation Binds bacterial cell walls Yes ,0.001 4 5 Yes antigen CD14 IPI00219806 S100A7 Psoriasin Bactericidal No ,0.001 3 19 ✔ Yes IPI00218733 SOD1 Superoxide dismutase 1 Protects against schistosomiasis; Yes ,0.001 3 4 ✔ Yes mechanism unknown AI RESEARCH BASIC IPI00027547 DCD Dermcidin Bactericidal Yes ,0.001 2 177 ✔ Yes IPI00032328, IPI00215894 KNG1 Kininogen-1 Bactericidal Yes ,0.001 2 95 ✔ Yes IPI00009276 PROCR Protein C receptor Unknown; protective against No ,0.001 2 4 Yes E. coli bacteremia 5 BASIC RESEARCH www.jasn.org

15 minutes showed clear evidence of exosome-induced bacterial lysis (Figure 5). Organisms incubated with exo- No Yes Yes Yes No Yes Yes Yes

Known somes for 5 minutes showed an increased proportion of in Kidney Expression lysed phenotypes (12%) compared with control samples (1.8%, P=0.003), increasing to 56% versus 2% after 15 minutes (Figure 5, A and C), consistent with the rapid in- ✔ ✔ ✔ ✔ ✔ hibition of BL21-lux growth observed earlier (Supplemen- tal Figure 3).

Disambiguated All these bacterial growth experiments were performed at pH 5.5–6.0, typical of omnivorous human urinary pH; exosomes were, however, also effective at pH 6.5 and pH Count Peptide 7.0 (Supplemental Figures 5 and 6). Furthermore, we noted that 0.5 mg/ml exosomes were effective against a 2 4 2 4 2 4 1 2 1111 1 1 1 starting 1 UPEC-lux concentration (representing 500 rela-

No. of .

Subjects tive light units) of 100 CFU/ml (Supplemental Figure

with Protein 8), conﬁrming our observations to be highly physiologically relevant. P Value 0.001 0.001 0.001 0.001 0.001 0.001 0.001 , , , , , , , DISCUSSION

Urinary exosomes released by renal tubular epithelia are No 0 No Yes No No Yes Exocarta

Present in present in human urine, where their function is unknown. Here, we examined the normal human urinary exosomal proteome through in-depth MS and showed significant enrichment for known innate immune proteins. EM studies confirmed the exosomal residency of antibacterial proteins and peptides. Urinary exosomes from healthy volunteers inhibited the growth of different uropathogenic and commensal E. coli strains and induced bacterial lysis, and these effects were dependent on the structural integrity of urinary exosomes. These findings suggest that urinary receptor exosomes function within the renal tract as innate immune Bactericidal bacteriostatic Bactericidal and bacteriostatic Yes Bactericidal Microbial receptor Bacteriostatic (binds iron) Bacteriostatic (binds iron) Bactericidal fungicidal Bactericidal and bacteriostatic Yes effectors. Although many of the innate immune proteins identi- fiedinouranalysis had beenpreviously identified inurinary exosomal analyses (Supplemental Table 1),12,13,16–21 enrichment for this protein grouping has not previously been reported. However, enrichment analysis relies on the reliability of the background proteome. Our analysis epithelium associated protein 1

-Defensin 9 of separate urinary exosomal preparations, along with key b methodological advances, has revealed a number of proteins identified from urinary exosomes for the first time. Furthermore, rather than place emphasis on maximizing the number of protein identifications, we made every at- FN1 Fibronectin DEFB109 PLUNC Palate lung and nasal LCN1LCN2 Lipocalin 1 Lipocalin 2 PGLYRP1 Peptidoglycan recognition HTN1 Histatin-1 LTF Lactoferrin tempt to ensure the robustness of those proteins ultimately included in this proteome. These key aspects of our study enabled the clear identification of enrichment for innate immune proteins (confirmed by direct methods) as well as er Gene Symbol Protein Putative Immune Role

ﬁ the expected enrichment for those proteins with a cytoskeletal role, involved in MVB or exosomal biogenesis, or Continued resident on the luminal surface of the nephron. IPI Identi UTIs represent the most common bacterial infection in humans, with an estimated 150 million UTIs per annum IPI00339224, IPI00339225, IPI00339226, IPI00339227, IPI00339228, IPI00339319, IPI00411462, IPI00414283, IPI00479723, IPI00556632, IPI00855777, IPI00855785, IPI00867588 42,43 35,36,44 IPI00935308 IPI00009856 Table 1. IPI00021085 IPI00022418, IPI00339223, IPI00012024 Group: LCN1 Group: LCN2 IPI00298860 globally. Most of these UTIs are caused by E. coli.

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The anatomic location of the distal urethra results in its continuous exposure to large numbers of bacteria. Nevertheless, the urinary tract is usually sterile above the ure- thral meatus, indicating the existence of highlyeffectiveinnateimmunemecha- nisms within the renal tract. To date, these mechanisms remain incompletely under- stood. For infection to occur, uropathogenic organisms must overcome a variety of physical and immune obstacles and as- cend the urinary tract. First, organisms must adhere to uroplakin-covered urothe- lium, which is limited by the shear flow of urine and the glycoprotein uromodulin, which impairs the ability of bacteria to adhere and facilitates their expulsion during voiding.45 Second, invading organisms are exposed to free radicals and soluble antimicrobial proteins and peptides present within urine, although the soluble concentrations of these proteins and peptides seem too low to be directly bactericidal.46,47 To evade these defenses, organisms typically invade epithelial cells, where they proliferate intracellularly before egressing back into the urinary space, a process that may, in turn, trigger acute in- flammatory responses through Toll-like receptor activation. Our findings indicate that, in addition, this defensive array in- cludes exosomes, which must be evaded or overwhelmed for UTI to occur. Consti- tutively released by renal tubular epithelia, a continuous stream of exosomes acts as an

(P=0.04), bacterial growth did not significantly differ with uromodulin compared with control in any experiment. Intact exosomes inhibited growth of BL21 compared with lysed exosomes (shown in green; P,0.001); lysed exosomes induced some growth inhibition compared with buffer only (P,0.001). As expected, given the protein- and carbohydrate-rich nature of LB medium compared with urine, all organisms grew Figure 4. Exosomes inhibit growth of E. coli. (A) All exosomal additions were made at more vigorously in LB. For Nissle, UTI89, and 25 mg/ml. All curves represent mean6SEM; y axes show luminescence (relative light E. coli O6:H1 (CFT073) grown in urine, the num- units3106), and x axes show time in hours. Growth curves are left-censored at the ber of viable organisms at peak bacterial growth onset of detectable bacterial growth and arranged by growth medium (columns) and (dashed lines labeled A–C) was additionally as- organism (rows). All data represent at least three replicates per condition. Data for sessed by conventional colony counting (corre- BL21 represent triplicates for each of four biologic replicates. For all strains of E. coli sponding bar graphs in B). Addition of exosomes tested, highly significant inhibition of growth in either medium occurred with the to donor urine resulted in significant reductions addition of exosomes compared with controls (P values shown). With the exception of in the number of CFU for Nissle (P=0.009), UTI89 UPEC in LB, where uromodulin achieved some growth inhibition compared with control (P=0.05), and CFT073 (P=0.003). Colony counts are shown as CFU per milliliter3106.

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even with contemporary high-sensitivity mass spectrometers, mitigation against the masking effect of abundant proteins is incomplete. Lower abundance proteins may, therefore, have gone undetected or may not have been consistently seen. In contrast to the situation in vivo, where exosomes would be continually replenished, our in vitro experiments used a single exosomal dose; however, the strength of the observed effect further supports its physiologic relevance. Finally, uromodulin cofractionates with urinary exosomes during isolation; therefore, we have not directly assessed the effects of uromodulin- free exosomal preparations, because the exosomal population would be highly compromised. Our findings add to an expanding body of evidence that exosomes are biologically active in a wide variety of tissues.4,10,48–50 They are consistent with a report of protection against viral respiratory pathogens by respiratory epithelium- derived exosomes,51 in which Kesimer and colleagues51 showed inhibition of influenza A virus infection of Madin– Darby canine kidney cells by respiratory epithelium-derived exosomes in vitro. The demonstration that urinary exosomes are able to induce bacterial lysis and inhibit growth at bacterial concentrations that are highly physiologically relevant makes Figure 5. Exosomes induce lysis of E. coli. (A) Incubation of UPEC identifying factors that modulate exosomal release and con- with exosomes (Exos) showed rapid induction of bacterial lysis. stitution key priorities for future work and may reveal poten- An increase in lytic phenotypes was detected as early as 5 mi- tial therapeutic targets for the treatment of UTIs. nutes after incubation (P=0.003); after 15 minutes, more than 50% of UPEC had undergone lysis (P,0.001). (B–D) Intact organisms (B and D, white arrow) and whole organisms that had lost CONCISE METHODS integrity (C and D, black arrows) were counted, but lysed bacterial fragments (D, open arrows) were not. Exosomal Isolation Exosomes were isolated from 10 healthy volunteer urine samples as innate immune sentinel within the urinary tract. Because exo- previously described.25 The five men and five women ages 23–36 somes contain molecules at once attractive and lethal to bac- years were on no regular medication and had not consumed anti- teria, they provide vehicles for the efficient, targeted distal biotics or other medication within the previous 1 month. Current delivery of antimicrobial molecules and confer on renal epithelia UTI was excluded. Briefly, subjects urinated directly into a container the ability to effect distant bacterial killing. Furthermore, they with protease inhibitors, including PMSF (500 ml 0.5 M solution), may function as decoys in limiting interaction of bacterial leupeptin (450 mg), and sodium azide (15 ml 100 mM solution). adhesion molecules with epithelial surfaces. Urine was centrifuged within 30 minutes of collection (Beckman Our study has several strengths. First, this study is the first AVANTI J26-XP centrifuge; JA-17 fixed angle rotor; polyallomer comprehensive report of in-depth proteomic analysis of 50-ml centrifuge bottles) for 20 minutes at 17,0003g. The superna- individual, rather than pooled, urinary exosomal samples. tant was passed through a sterile 0.22-mm filter and ultracentrifuged Second, we report that a urinary exosomal proteome was (Beckman Optim L-100 XP VAC Ultracentrifuge; Ti45 fixed-angle constituted by statistically robust and reproducibly identifiable titanium rotor; Beckman 70-ml polycarbonate ultracentrifuge bot- protein identifications. Third, we show a clinically relevant tles) for 135 minutes at 235,0003g and 4°C. Each ultracentrifugation effect of urinary exosomes on the most common human pellet was suspended in 50 ml suspension buffer (250 mM sucrose and urinary pathogen E. coli using several methodologies. How- 10 mM triethanolamine [pH 7.6]) and pooled with the other pellets ever, our findings should be interpreted within the limitations from the same urine sample. To avoid confounding effects on bacte- of the study. It is not possible to infer from our data which rial growth, urine samples for these experiments were collected with- exosomal proteins represent the key effectors, because targeted out protease inhibitors or sodium azide, triethanolamine was removed deletion of individual exosomal proteins has not yet been pos- from the suspension buffer, and exosomal pellets were washed one sible. More than one third of innate immune proteins were time by resuspension and ultracentrifugation. consistently identified ($8 of 10 samples), and this subgroup may be central. However, we cannot exclude the possibility that Exosome-Depleted Urinary Protein other less consistently identified members of this proteome or Samples were prepared as described for exosomal isolation above. The indeed, unidentified molecules are important. Furthermore, supernatant from the 235,0003g centrifugation was retained after

8 Journal of the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc,2014 www.jasn.org BASIC RESEARCH harvesting exosomal pellets. This supernatant contained the nonexo- scoring was performed using the DAVID bioinformatics tool.24 This somal soluble urinary protein. For MS, protein was precipitated from method provides a measure (by tests of proportions) of whether these supernatants (typically 360 ml per subject) by the addition of functional categories are overrepresented within a gene list compared ammonium acetate and acetone (Supplemental Material). Pellets with what is expected from stochastic sampling of the entire human were resuspended with Laemmli sample buffer. gene set.

Sample Preparation for MS Protein Confirmation and Antibodies Protein from resuspended exosomal pellets was concentrated by Proteins identified by MS were confirmed by Western blotting precipitation and quantified using a Bradford protein binding according to standard methods and immuno-EM (described below). colorimetric assay (Bio-Rad). Protein pellets were stored at 280°C For each protein, the same antibodies (Supplemental Material) were until use. used for both techniques. For MS, protein pellets were suspended in Laemmli sample buffer and incubated at 95°C; 50 mg protein from each sample was fragmented Bacterial Transformation on a 4%/12% SDS–polyacrylamide gel. After staining, each gel track Bacteria were transformed by electroporation to express the luxCDABE was separated into 28 equal sections, which were processed indi- operon (Bioware plasmid pXEN13), which results in ampicillin re- vidually for the remainder of the workflow. After destaining, pro- sistance and constitutive expression of luciferase. teins were reduced and alkylated in gel, washed with NH4HCO3,and dehydrated in acetonitrile, and proteins were digested with modified Bacterial Growth Assays trypsin. Exosomal samples were collected from the same 10 healthy volunteers Liquid chromatography–MS/MS was performed using an Eksigent described earlier, but in contrast to samples prepared for MS, these NanoLC-1D Plus (Eksigent Technologies) HPLC system and an LTQ samples were handled without protease inhibitors, triethanolamine, Orbitrap Mass Spectrometer (Thermo Fisher Scientific). Peptides or sodium azide. were separated by reverse-phase chromatography (Dionex). Peptides were loaded onto a 5-cm C18 precolumn (300 mm inner diameter; Bacterial Colony Counting LC Packings) from the autosampler. Peptides were eluted onto the Colony counting was performed essentially as in Miles and Misra.40 analytical column using gradients for solvent A (water+0.1% formic Briefly, serial dilutions of overnight bacterial cultures were spotted acid) and B (acetonitrile+0.1% formic acid) of 5%–50% B over onto ampicillin-impregnated agar plates and incubated at 37°C for 18 40 minutes. A New-Objective nanospray source was used for electro- hours. Colony counts were made from the highest bacterial concen- spray ionization. m/z Values of eluting ions were measured in the tration that yielded distinct colonies. Orbitrap mass analyzer with a mass range of 350–1600, and the res- olution was set at 7500. Bacterial Growth Curves For each growth assay, negative controls comprised suspension buffer MS Data Processing only or purified uromodulin (P135–1; SCIPAC) diluted in suspension Peptides from each gel segment were run two times. All segments were buffer. Overnight bacterial cultures were diluted in LB broth to lu- runwith dynamicexclusion. From these runs, a fixed exclusion list was minescence of 100–500 relative light units. At 4°C, 10 mlbacterial generated for the abundant protein uromodulin and superimposed culture was placed in each well of a 96-well plate; 5 mg exosomal on a dynamic exclusion list as described elsewhere.25 Data from these protein, uromodulin, or an equal volume of suspension buffer was two sets of runs were combined. MS data were processed using the added to each well. Volumes in all wells were standardized to 200 ml SEQUEST Bioworks Browser (version 3.3.1 SP1; Thermo Fisher Sci- with LB or exosome-depleted urine as appropriate (Supplemental entific) to generate MS/MS peak lists. Combined peak list files were Figure 7). All samples were evaluated at least in triplicate. Plates submitted to the MASCOT search algorithm (version 2.2.1; Matrix were incubated at 37°C, and luminescence readings were obtained Science) and searched against the IPI-Human Database, version 4.3. hourly until growth ceased. Exosomes were chemically lysed by se- Spectra were rescored using MASCOT-Percolator, a machine learning quential addition of ammonium acetate in methanol and acetone as tool that minimizes false discoveries and incorporates target decoy described for protein precipitation above. searching.26 Protein identification required two or more unique peptides, with a false discovery rate of 0.1. Single peptide identifications EM were included if the MASCOT-percolator posterior error probability Negative staining transmission EM with neutralized phosphotunstic was ,0.01. In addition, we included proteins based on an in-house acid or uranyl acetate on carbon film grids was performed with an FEI protein type I error estimator termed espresso (Supplemental Mate- Tecnai G2 electron microscope operated at 120 kV using an AMT rial). The cellular location of proteins was evaluated by searching the XR30B digital camera. For immunolabeling, samples were placed Ensembl gene identifier for each protein against the Human Protein on glow-discharged nickel grids for 30 seconds. Primary antibodies Atlas Database (http://www.proteinatlas.org). Renal expression was were applied for 15 minutes at room temperature. Secondary anti- evaluated using the bioGPS Gene Portal System (http://www.biogps. bodies labeled with 5-, 10-, or 15-nm gold particles were applied for org). Comparison of exosomal proteins with previous reports was 15 minutes. Grids were blocked with PBS. For scanning EM, bacterial made using the Exocarta exosomal protein database.27 Enrichment cultures were incubated with exosomes, and 10 mlwerespottedonto

J Am Soc Nephrol 25: ccc–ccc, 2014 Immune Role for Urine Exosomes 9 BASIC RESEARCH www.jasn.org glass coverslips, quench-frozen in propane-cooled liquid nitrogen, 8. Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, freeze-dried (Edwards Auto 306 Turbo), and gold-coated. Scanning Melief CJ, Geuze HJ: B lymphocytes secrete antigen-presenting vesi- – EM was carried out with a Philips XL-30 FEG–scanning EM, and cles. JExpMed183: 1161 1172, 1996 9. Boyan BD, Wong KL, Fang M, Schwartz Z: 1alpha,25(OH)2D3 is an images were captured with an accelerating voltage of 5 kV. autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases. JSteroid Statistical Analyses Biochem Mol Biol 103: 467–472, 2007 Data were analyzed with Stata SE, version 12.1. Data are presented as 10. Sirois I, Raymond MA, Brassard N, Cailhier JF, Fedjaev M, Hamelin K, means6SDs or median (IQR) as appropriate. Parametric continuous Londono I, Bendayan M, Pshezhetsky AV, Hébert MJ: Caspase-3- dependent export of TCTP: A novel pathway for antiapoptotic in- variables were compared with the t test, and nonparametric contin- tercellular communication. Cell Death Differ 18: 549–562, 2011 uous variables were compared with the Wilcoxon sign rank test. Bac- 11. Kanno K, Sasaki S, Hirata Y, Ishikawa S, Fushimi K, Nakanishi S, Bichet terial growth was compared using response feature analysis with the DG, Marumo F: Urinary excretion of aquaporin-2 in patients with di- area under the curve as the response feature, and comparisons were abetes insipidus. NEnglJMed332: 1540–1545, 1995 fi made by one-way ANOVA. Proportions were compared with Pearson’s 12. Pisitkun T, Shen RF, Knepper MA: Identi cation and proteomic pro- filing of exosomes in human urine. Proc Natl Acad Sci U S A 101: 13368– chi-squared test of proportions. 13373, 2004 13. Hogan MC, Manganelli L, Woollard JR, Masyuk AI, Masyuk TV, Tammachote R, Huang BQ, Leontovich AA, Beito TG, Madden BJ, Charlesworth MC, ACKNOWLEDGMENTS Torres VE, LaRusso NF, Harris PC, Ward CJ: Characterization of PKD protein- positive exosome-like vesicles. J Am Soc Nephrol 20: 278–288, 2009 14. Dear JW, Street JM, Bailey MA: Urinary exosomes: A reservoir for We thank M. Clatworthy and M. Berry for the generous donation of biomarker discovery and potential mediators of intrarenal signalling. UTI89 E. coli and Ardeypharm GmbH for Nissle E. coli (Mutaflor). Proteomics 13: 1572–1580, 2013 This work was supported by an Action Medical Research Training 15. Street JM, Birkhoff W, Menzies RI, Webb DJ, Bailey MA, Dear JW: Exosomal transmission of functional aquaporin 2 in kidney cortical Fellowship (to T.F.H.), Wellcome Trust Grant 088489 (to F.E.K.F.)and collecting duct cells. J Physiol 589: 6119–6127, 2011 Strategic Award 079895 (to Cambridge Institute for Medical Research), 16. Gonzales PA, Pisitkun T, Hoffert JD, Tchapyjnikov D, Star RA, Kleta R, the National Institute for Health Research Cambridge Biomedical Wang NS, Knepper MA: Large-scale proteomics and phosphoproteomics Research Centre (CBRC), and the Biotechnology and Biological Sci- of urinary exosomes. J Am Soc Nephrol 20: 363–379, 2009 ences Research Council (BBSRC). T.F.H. received a Raymond and 17. Zhou H, Pisitkun T, Aponte A, Yuen PS, Hoffert JD, Yasuda H, Hu X, Chawla L, Shen RF, Knepper MA, Star RA: Exosomal Fetuin-A identified Beverley Sackler Research Studentship and is currently supported by by proteomics: A novel urinary biomarker for detecting acute kidney the CBRC. P.D.C. was supported by BBSRC Research Studentship BB/ injury. Kidney Int 70: 1847–1857, 2006 D526088/1. L.G. is supported by a 7th Framework Programme of the 18. Nilsson J, Skog J, Nordstrand A, Baranov V, Mincheva-Nilsson L, European Union (262067-PRIME-XS). 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