Extracellular vesicles from airway secretions: New insights in lung diseases

In e na ional Jou nal o Molecula Sciences Re iew Ex acellula Vesicles om Ai way Sec e ions: New Insigh s in Lung Diseases Lau a Pas o 1, Elisabe h Ve a 1,2, Jose M. Ma in 1,2,3 and Da id Sanz-Rubio 1,*   Ci a ion: Pas o , L.; Ve a, E.; Ma in, J.M.; Sanz-Rubio, D. Ex acellula Vesicles om Ai way Sec e ions: New Insigh s in Lung Diseases. In . J. Mol. Sci. 2021,22, 583. h ps://doi.o g/10.3390/ ijms22020583 Recei ed: 9 Decembe 2020 Accep ed: 29 Decembe 2020 Published: 8 Janua y 2021 Publishe ’s No e: MDPI s ays neu- al wi h ega d o ju isdic ional clai- ms in published maps and ins i u io- nal a ilia ions. Copy igh : © 2021 by he au ho s. Li- censee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and con- di ions o he C ea i e Commons A - ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1T ansla ional Resea ch Uni , Ins i u o de In es igación Sani a ia de A agón (IISA agón), Hospi al Uni e si a io Miguel Se e , 50009 Za agoza, Spain; [email p o ec ed] (L.P.); [email p o ec ed] (E.V.); jmma in @uniza .es (J.M.M.) 2Respi a o y Se ice, Hospi al Uni e si a io Miguel Se e , Uni e si y o Za agoza, 50009 Za agoza, Spain 3Cen o de In es igación Biomédica en Red de En e medades Respi a o ias (CIBERes), 28029 Mad id, Spain *Co espondence: da idsanz [email p o ec ed] Abs ac : Lung diseases (LD) a e one o he mos common causes o dea h wo ldwide. Al hough i is known ha ch onic ai way in lamma ion and excessi e issue epai a e p ocesses associa ed wi h LD such as as hma, ch onic obs uc i e pulmona y disease (COPD) o idiopa hic pulmona y ib osis (IPF), hei speci ic pa hways emain unclea . Ex acellula esicles (EVs) a e he e ogeneous nanoscale memb ane esicles wi h an impo an ole in cell- o-cell communica ion. EVs a e p esen in gene al bio luids as plasma o u ine bu also in sec e ions o he ai way as b onchoal eola la age luid (BALF), induced spu um (IS), nasal la age (NL) o pha yngeal la age. Al e a ions o ai way EV ca go could be c ucial o unde s anding LD. Ai way EVs ha e shown a ole in he pa hogenesis o some LD such as eosinophil inc ease in as hma, he p omo ion o lung cance in i o models in COPD and as bioma ke s o dis inguishing IPF in pa ien s wi h di use lung diseases. In addi ion, hey also ha e a p omising u u e as he apeu ics o LD. In his e iew, we ocus on he impo ance o ai way sec e ions in LD, he pi o al ole o EVs om hose sec e ions on hei pa hophysiology and hei po en ial o bioma ke disco e y. Keywo ds: ex acellula esicle; COPD; as hma; ib osis; induced spu um; nasal la age; o al la age; b onchoal eola la age; exosome 1. In oduc ion Lung diseases (LD) a e among he mos common causes o dea h and disabili y wo ld- wide [ 1 ]. Acco ding o he las upda es o he Fo um o In e na ional Respi a o y Socie ies (FIRS), he high incidence o hose pa hologies is mainly due o global exposu e o pollu ed ai , pa icles, chemicals and in ec ious mic oo ganisms [ 1 ]. Repea ed exposu es o hese agen s induce cell damage and igge immune esponses in he ai way. Ai way epi helium, which is he i s line o de ense agains en i onmen al agen s, is damaged by his con ac . In e ac ions and homeos asis be ween ai way epi helium and mesenchymal, endo helial and immune cells in he ai way and lungs a e also modi ied [ 2 , 3 ]. Ch onic ai way in lamma- ion and excessi e issue epai p ocesses esul in he de elopmen o LD such as as hma, ch onic obs uc i e pulmona y disease (COPD), idiopa hic pulmona y ib osis (IPF) o lung cance , as well as in sys emic in lamma ion and associa ed co-mo bidi ies [3–5]. Ex acellula esicles (EVs) a e he e ogeneous nanoscale memb ane esicles, including exosomes, mic o esicles and apop o ic bodies, ha a e sec e ed by mos cell ypes and hey ha e been mainly desc ibed in plasma and se um [ 6 – 9 ] o u ine. Howe e , he p esence o EVs has been also epo ed in sec e ions om ai way such as sali a [ 10 ], induced spu um (IS) [ 11 ] o b onchoal eola la age luid (BALF) [ 12 ]. EVs ha e eme ged as a no el and c ucial mechanism o in e cellula communica ion [ 13 , 14 ] wi h an impo an ole in in lamma o y pa hologies, including LD. Ai way inju y modi ies EV ca go in lung epi helium de i ed-EVs p omo ing an immune esponse ia mac ophage ac i a ion [ 15 , 16 ]. In . J. Mol. Sci. 2021,22, 583. h ps://doi.o g/10.3390/ijms22020583 h ps://www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2021,22, 583 2 o 20 The e o e, EVs could con ibu e o cla i y he molecula mechanisms unde lying LD, bu also hey could p o ide new bioma ke s among hei ca go, as well as new EV-based he apeu ic app oaches [17,18]. In his e iew, we aim o summa ize cu en knowledge abou EVs ela ed o LD. We will ocus on he main LD such as COPD, as hma and IPF, and on he po en ial o ai way sec e ions as sou ce o new bioma ke s. Finally, we sugges he po en ial unc ion o EVs om hese sec e ions in he pa hophysiology o LD and hei p omising u u e as o igin o bioma ke s and o LD. 2. Lung Diseases 2.1. As hma As hma is one o he mos p e alen ch onic disease in de eloped coun ies in adul s and child en globally [1], a ec ing mo e han 339 million people [19] and 14% o child en o e he wo ld [ 1 ]. As hma is de ined by he in e na ional Global Ini ia i e o As hma (GINA) as a he e ogeneous disease ela ed o ch onic ai way in lamma ion and cha ac e - ized by in e mi en episodes o wheeze, cough, dyspnea and ches igh ness, as well as e e sible ai way limi a ion [ 19 ]. This disease shows di e en pheno ypes and endo ypes, depending on i s mechanisms and esponses o he apy [20]. In lamma ion in as hma is ex ended om uppe o pe iphe al ai way e ealing di - e en molecula pa e ns. Mos o he pa ien s su e alle gic as hma cha ac e ized by Th2 immune esponse agains en i onmen al s imuli [ 21 ]. A e an igen p esen a ion, which is d i en by dend i ic cells [ 22 ], T-CD4 + lymphocy es sec e e Th2-cy okines (IL-4, IL-13 and IL-5). IL-4 and IL-13 s imula e IgE p oduc ion in B-cells [ 23 ], while IL-5 p o- mo es eosinophil ma u a ion in bone ma ow and u he eosinophil ec ui men in he ai way [ 21 , 24 ]. Non-alle gic T2-eosinophilic esponse is pushed by ala mins and inna e lymphoid cells 2, which a e p oduce s o Th2-cy okines, so hey igge eosinophilic in- lamma ion in ai way [ 25 ]. Finally, non-eosinophilic pheno ype is no mally ela ed o mo e se e e s a us and i is gene ally cha ac e ized by neu ophilic ai way in lamma- ion [ 25 , 26 ]. Al hough i is known ha non-eosinophilic as hma pa hophysiology is d i en by Th17/Th1 cells [ 25 ] and cy okines as IL-17 and IL-22 [ 26 ], i s molecula mechanisms a e poo ly unde s ood. Ai way epi helial inju y p oduced by his in lamma o y si ua ion igge s as hma-cha ac e is ic ai way emodeling, including he inc ease o ai way smoo h muscle bulk and mucus hype sec e ion. Al oge he , as shown in Figu e 1, hese phenomena p oduce ai way na owing and ai way hype esponsi eness o some en i onmen al agen s, which is also a cha ac e is ic ea u e o as hma [27,28]. 2.2. Ch onic Obs uc i e Pulmona y Disease COPD has a no ewo hy impac on global heal h, a ec ing mo e han 200 million peo- ple wo ldwide [ 1 ], leading mo ali y (3 d cause o dea h) and mo bidi y in he wo ld [ 1 , 29 ]. COPD is a LD cha ac e ized by pe sis en ai low limi a ion and espi a o y symp oms due o ai way and/o al eola damage. I is usually caused by signi ican exposu e o i i an s, especially ciga e e smoke, al hough some imes is in luenced by hos ac o s as abno mal lung de elopmen o α1 -an i ipsin gene ic de iciency [ 30 ]. COPD is associa ed o sys emic in lamma ion, which is a isk ac o o de elop co-mo bidi ies such as a he oscle osis and o he ca dio ascula diseases, diabe es, me abolic diseases o cance [31,32]. In . J. Mol. Sci. 2021,22, 583 3 o 20 Figu e 1. O e iew o he main p ocesses and e en s ha occu in di e en egions o lung and ai way in as hma, ch onic obs uc i e pulmona y disease (COPD) and in e s i ial pulmona y ib osis (IPF) pa ien s, compa ed o heal hy condi ion. As hma in ol es mainly he uppe ai way p omo ing ai way emodeling, which includes ai way smoo h muscle hype ophy and hype plasia, and goble cell hype plasia and consequen mucus hype sec e ion. These p ocesses cause e e sible ai way na owing. COPD a ec s lowe ai ways and lung pa enchyma causing i e e sible ai way na owing and al eola dis up ion (emphysema) in he lungs. IPF a ec s only he lungs h ough excessi e ib osis p ocesses wi h subsequen abe an al eola e-epi helisa ion (b oncholisa ion). (Componen s o his igu e ha e been ob ained and modi ied om Se ie Medical A , h ps://sma .se ie .com, wi h pe mission unde he C ea i e Commons A ibu ion 3.0 Unpo ed License.). In con as o as hma, in lamma ion in COPD is limi ed o pe iphe al ai ways and lung pa enchyma, esul ing in ib osis, issue des uc ion, small ai way obs uc ion and emphysema [ 33 ]. These mechanisms a e i e e sible and p oduce p og essi e decline in lung unc ion [ 5 ]. Di e en cells a e in ol ed in COPD in lamma ion p ocesses: adap a i e immune cells, inna e immune cells (mainly neu ophils and mac ophages) and s uc u al cells. In esponse o ciga e e smoke o o he noxious pa icles, ai way epi helial cells and mac ophages a e ac i a ed o p oduce se e al in lamma o y media o s [ 34 ]. The ansc ip ion ac o NF- κβ , ac i a ed in al eola COPD mac ophages [ 35 ], up- egula es he exp ession o in lamma o y molecules [ 36 ]. Neu ophils om pe iphe al ai way e- lease CXCL8, p omo ing au oc ine neu ophil ec ui men and se e al p o einases, which con ibu e o loss o elas ici y and pe iphe al ai way collapse. This p ocesses, oge he wi h p o ib o ic e ec o TGF- β eleased om epi helial cells and mac ophages, esul in al eola des uc ion, ib osis and i e e sible ai way na owing [ 36 ] (Figu e 1). Lungs o COPD pa ien s also su e an accele a ed ageing p oduced by an abno mal accumula ion o senescen s uc u al cells [ 37 ]. Finally, eosinophils ha e been epo ed o be inc eased in In . J. Mol. Sci. 2021,22, 583 4 o 20 ai way o some COPD pa ien s, sugges ing he exis ence o a di e en COPD pheno ype whose molecula mechanisms a e poo ly unde s ood [38]. 2.3. Idiopa hic Pulmona y Fib osis IPF is inc easing o e he ime wo ldwide wi h an es ima ed incidence o 2.8–18 cases/100,000 people pe yea in Eu ope and No h Ame ica. Lowe incidence is de ined in Asia and Sou h Ame ica, which may be pa ially due o unde -diagnosis [ 39 ]. In addi ion, IPF is mo e equen in men [ 40 ]. IPF is a ch onic, p og essi e ib osing in e s i ial pneumonia o unknown e iology ha usually a ec s o olde adul s. Disease p og ession a ies be ween pa ien s and is unp edic able, bu IPF leads o se e e pa hological p ocesses which end in p ema u e dea h [41]. Molecula mechanisms o IPF a e no ully unde s ood al hough ch onic dys egula ion in al eola epi helial cells homeos asis has been desc ibed (Figu e 1). IPF biopsies show loss o ype 1 al eola epi helial cells (AEC1s) and educed abno mal enewal capaci y in ype 2 (AEC2s) [ 42 , 43 ]. Fib oblas agg upa ion a e also obse ed be ween abno mal AECs [ 44 , 45 ]. In esponse o hese mic oinju ies, emaining AECs a e ac i a ed o elease ib ogenic g ow h ac o s and cy okines, esul ing in he ec ui men o myo ib oblas om di e en sou ces [ 45 – 47 ]. A his loca ion, myo ib oblas s inc ease sec e ion o abno mal ex acellula ma ix componen s, whose accumula ion and deposi ion leads o ib osis and gas exchange impai men . I also enhances myo ib oblas ac i a ion, es ablishing a posi i e eedback p ocess [ 48 ]. In addi ion, pe iphe al ai way basal cells ac as s em cells o epai damaged al eola ai way wi h new epi helial cells, known as b oncholisa ion [49–51]. 3. Ai way Sec e ions Sampling he uppe and lowe ai ways is a ema kably help ul ool in clinical p ac ice. Di e en ai way sec e ions a e used and has been la ely in es iga ed as a p omising sou ce o bioma ke s, including b onchoal eola la age luid (BALF), induced spu um (IS), nasal la age luid (NL) o pha yngeal la age luid (PHAL). 3.1. B onchoal eola La age Fluid BALF is an es ablished p ocedu e o washing he b onchial ee wi h saline which allows sampling pe iphe al ai ways [ 52 ]. I is an in asi e me hod, as i is pe o med oge he wi h a lexible ibe op ic b onchoscopy. BALF con ains he cells in he al eola space which ha e been in con ac wi h he ins illed luid (al eola mac ophages, CD4 + and CD8 + lymphocy es, neu ophils, eosinophils and mas cells [ 53 ]), ai way epi helial cells (con amina ing BALF i exceed 5% o o al cell coun [ 53 ]), and di e en soluble componen s om he endogenous luid in he al eoli, named he epi helial lining luid (ELF) [ 54 ]. These componen s p oceed mainly om local cell sec e o y p oduc ion [ 55 – 57 ], bu also om he ascula space in con ac wi h he al eola space [ 54 ]. In clinical p ac ice, BALF is used in diagnosis o espi a o y in ec ions [ 58 ] and as a pa o he diagnosis o in cha ac e iza ion o non-in ec i e LD, bo h in adul s and child en [ 59 ]. Howe e , i s in asi e na u e has limi ed i s use as a esea ch ool. 3.2. Induced Spu um Spu um induc ion is a ela i ely non-in asi e me hod o collec a sample o cells and sec e ions om lowe ai ways as shown in Figu e 2[ 60 ]. Spu um is induced by inhala ion o hype onic o iso onic s e ile saline solu ion and ob ained by u he di ec expec o a ion [ 61 ]. IS is a sa e and less in asi e me hod o sample ELF han BALF [ 62 , 63 ]. Howe e , IS con ains also sali a and squamous cells om uppe ai ways, which a e conside ed con aminan componen s and ep esen di e en p opo ions o IS [64,65]. In . J. Mol. Sci. 2021,22, 583 5 o 20 Figu e 2. Ana omical o igin o minimally in asi e and non-in asi e ai way sec e ions. Induced spu um comes om lowe ai way while nasal la age and pha yngeal la age ha e hei o igin in di e en pa s o he uppe ai way. (Componen s o his igu e ha e been ob ained and modi ied om Se ie Medical A , h ps://sma .se ie .com, wi h pe mission unde he C ea i e Commons A ibu ion 3.0 Unpo ed License.). Mos o IS esea ch has been dedica ed o as hma, al hough mul i ude o s udies ha e in es iga ed he u ili y o IS in o he LD, especially COPD and ch onic cough [ 66 ]. Di e en ial cell coun in IS is used in clinical p ac ice o assess as hma pheno ypes [ 67 ], al hough e e ence pa ame e s a y among di e en s udies and popula ions [ 68 – 70 ]. In bo h as hma [ 71 ] and COPD [ 72 ], cul u es om IS a e pe o med o iden i y he p esence o pa hogens and manage an ibio ic he apy. Ex ended bioma ke s esea ch has been done in IS. Fo example, assessmen o in lamma o y pa e n o exace ba ions in as hma and COPD by IS cy ology allows he apy adjus men and p edic ion o new exace ba ion episodes [ 73 ]. IS can wo k as a minimally in asi e BALF su oga e in assessmen o low ai way s a us in LD wi h low ai way in ol emen , al hough u he esea ch is needed in o de o op imize p o ocols and s anda dize e e ence pa ame e s. In his way, IS has been p oposed and s udied as a non-in asi e su oga e o BALF in in e s i ial LD diagnosis [74]. 3.3. Nasal La age Fluid NL is a non-in asi e p ocedu e o ob ain cy ology om nasal ca i ies [ 75 ] (Figu e 2). Di e en nasal la age echniques ha e been desc ibed [ 76 , 77 ] consis ing on ins ill s e ile saline solu ion. NL is a mix u e o saline and nasal sec e ions, con aining epi helial cells, neu ophils, eosinophils, lymphocy es and hei sec e ed media o s [78]. NL has been applied o assess he in lamma o y s a us o a speci ic pa o uppe ai ways, he nasal ca i ies. Mos NL s udies ha e been ocused in hini is, as i is a disease cha ac e ized by nasal mucosa in lamma ion and nasal hype sec e ion [ 75 , 78 , 79 ]. Recen s udies ha e shown ha NL cy ology is a bes subs i u e o IS cy ology han blood cell coun in iden i ica ion o in lamma o y pheno ypes o as hma [ 80 , 81 ]. In addi ion, despi e he weake ela ionship be ween nasal mucosa in lamma ion and COPD, ano he wo k e ealed ha IL-8 concen a ion in NL ha e posi i e co ela ion wi h disease se e i y and ciga e e pack-yea s in COPD pa ien s [ 82 ]. NL has a g ea po en ial as a non-in asi e subs i u e o IS in hose pa ien s o si ua ions whe e pe o m IS could be isked. 3.4. Pha yngeal La age Fluid PHAL is a ecen ly de eloped non-in asi e echnique o collec cells om he o opha- yngeal ca i y o assess mucosal in lamma ion o he uppe ai way [ 83 ]. Figu e 2shows ha PHAL con ains cells om he o opha yngeal mucosa (a majo p opo ion o squamous In . J. Mol. Sci. 2021,22, 583 6 o 20 epi helial cells, ollowed by a high pe cen age o neu ophils and o he in lamma o y cells [83]) and soluble ac o s and p o eins [84]. Highe PHAL lymphocy es numbe ha e been ound in he pa ien s wi h obs uc i e sleep apnea (OSA) [ 85 , 86 ]. Ou g oup epo ed also ha T-CD4 + cells numbe in PHAL co ela ed wi h mo e se e e OSA s a us, as well as PHAL concen a ion o IL-6 and IL-8 [ 84 ]. Thus, PHAL is eme ging as a new non-in asi e ool o assess uppe ai way in lamma ion, which could be pa icula ly help ul in some LD wi h concomi an uppe ai way in lamma ion such as as hma. 4. Ex acellula Vesicles EVs a e a he e ogeneous popula ion o small memb anous s uc u es eleased in o he ex acellula en i onmen by mos cells and pa icipa e in in e cellula communica- ion [ 87 ]. EVs ha e been classi ied acco ding o di e en c i e ia as hei size o biogenesis, di e en ia ing be ween exosomes, mic o esicles (MVs), and apop o ic bodies (ABs) [ 88 – 90 ]. Exosomes (30–150 nm) esul om a complex biogenesis which in ol es he endosomal memb ane in agina ion, gene a ing in aluminal esicles (ILVs) inside he la e endosome (MVB), and he usion be ween he MVB memb ane and he plasma memb ane eleases exosomes [ 91 , 92 ]. MVs (100 nm o 1000 nm), also named ec osomes o mic opa icles, a e gene a ed di ec ly by ou wa d budding o he plasma memb ane [ 93 , 94 ]. ABs (1 o 5 µ m) a e eleased unde going apop osis, ia plasma memb ane bubbling o agmen a ion due o cell disassembly [95,96]. EVs con ain biomolecules om hei o igin cell, including p o eins, lipids, me aboli es and di e en nucleic acids (DNA, mRNA, miRNA and o he noncoding RNA) [89,97,98]. Thei molecula ca go can be uploaded in o he esicles selec i ely, mos ly in exosomes. This p ocess depends on cell s a us and ype [ 99 ]. EV elease has been epo ed in pla ele s [ 93 ], umo cells [ 100 ], dend i ic cells [ 101 ], T [ 102 ] and B [ 103 ] cells, eosinophils [ 104 ], epi helial cells [ 15 , 16 ], endo helial cells [ 105 ] o mesenchymal s em cells (MSCs) [ 106 ]. Thei accessi- bili y in body luids (plasma [ 6 , 8 ], se um [ 9 ], u ine [ 9 ], sali a [ 10 ], BALF [ 12 ] o IS [ 11 ]) and he speci ici y o hei ca go associa ed wi h pa hophysiological condi ions [ 107 , 108 ] con e hem g ea po en ial as bioma ke s. EVs ha e been b oadly s udied because o hei associa ion wi h pa hologies, including in- lamma o y diseases [ 109 , 110 ] ca dio ascula diseases [ 111 , 112 ], me abolic diseases [113,114] , cance [ 115 , 116 ] o LD [ 107 , 117 , 118 ]. They ha e been linked o p o-in lamma o y p ocesses, pa icipa ing in an igen p esen a ion [ 101 , 103 ] o mac ophage ac i a ion [ 15 ], as well as o immunomodula o y p ocesses [ 106 ]. Addi ionally, EVs could ha e a he apeu ic ap- p oach as ehicles o d ug deli e y, gi en hei in insic biocompa ibili y and speci ic a ge ac i i y [ 119 ]. P omising examples o i a e no el exosome-based modali y o cance he apy [120] o nanopa icle-guided as hma he apy [121]. In he las decade, he esea ch on EVs has inc eased. Howe e , he op imal isola ion me hod emains unclea and mos imes depends on he downs eam analyses [ 13 , 122 – 124 ]. Thei cha ac e iza ion equi es ollow se e al poin s ha include p o ein ma ke s, size o mo phology. Those a e desc ibed in he MISEV2018 [ 87 ] upda ing he MISEV2014 [ 125 ], showing ha he EV ield is con inuously in de elopmen and equi es u he s u-dies. 5. EVs in Lung Pa hophysiology Mos lung cells elease EVs unde physiological and pa hological condi ions. B onchial epi helial cells (BECs) and al eola mac ophages a e he majo sou ces o pulmona y EVs [ 126 ], bu also ascula endo helial cells, ib oblas s, MSCs and dend i ic cells. All oge he a e in ol ed in lung homeos asis, as well as in LD de elopmen . T ans e o EVs ca go be ween ai way epi helial cells can al e he sec e ions o hei a ge cells, including mucins [ 127 ]. Memb ane associa ed mucins a e p esen in acheo- b onchial epi helial cells exosomes su ace, sugges ing hei con ibu ion o inna e mucosal de ense o ai way [ 128 ]. Simila ly, al eola mac ophages elease EVs ha con ain he sup- p esso o cy okines signaling (SOCS) 1 and 3. These EVs ha e immunomodula o y e ec s In . J. Mol. Sci. 2021,22, 583 7 o 20 in he al eola epi helial cells and hei sec e ion is diminished by ciga e e smoking [ 129 ]. Table 1shows some o he p o eins, lipids and miRNAs con ained in EVs ha ha e been ela ed wi h as hma, COPD and IPF. Table 1. P o eins, lipids and miRNAs con ained in ex acellula esicles (EVs) ha ha e been associa ed wi h as hma, ch onic obs uc i e pulmona y disease (COPD) and idiopa hic pulmona y ib osis (IPF). EV Ca go As hma COPD IPF P o eins ADAM10 [11], MHC-II [101,103,130], HLA-DR (IS) [11] α1-an i ypsin [131], CD66b [132,133], CD31, NE [132]Tissue ac o [134], WNT5A [135] Lipids Leuko iene B4 [136] PGE2 [137] miRNAs miR-223 and miR-142a [ 138 ], le -7, miR-200 amilies, miR-21 [139] miR-210 [140], miR-21 [141], Le -7e, le -7g, miR-26b [142] miR-21 [143], miR-328 [144], miR-33a, miR-142 and le -7d [ 145 ] 5.1. EVs in As hma EVs a e in ol ed in se e al p ocesses o as hma immuni y and in lamma ion. One o he mos impo an is an igen p esen a ion abili y o EVs eleased om dend i ic cells [ 101 ] and B cells [ 103 , 130 ]. Figu e 3shows ha MHC-II and co-s imula o y molecules in he su aces o hese EVs allow in e ac ion wi h T cells and subsequen di e en ia ion o T cells, p oli e a ion o T-CD4+Th2 cells and sec e ion o Th2 cy okines [101,130]. As hma-like in lamma ion d i en by IL-13 enhances exosomes sec e ion in BECs in- ducing p oli e a ion and chemo axis o undi e en ia ed mac ophages in he lungs [ 126 ]. B onchocons ic ion mechanical s ess also s imula es exosomes sec e ion om BECs, p o- ducing ypical subepi helial angiogenesis [ 146 ]. Fu he mo e, mechanical signals egula e miRNA ca go o EVs eleased om BECs [ 147 ]. In con as , EV immunomodula o y e ec s has been epo ed o be diminished in as hma, such as EV-media ed anspo o SOCS3 om al eola mac ophages o BECs [148]. Eosinophil exosomes o as hma ic pa ien s di e hei capaci ies om non-as hma ic subjec s. They p omo e eosinophil chemo axis inc easing ROS and ni ic oxide (NO) le els, and adhesion, ia ICAM-1 and in eg in- α2 up egula ion [ 149 ] (Figu e 3). These exosomes also p omo e apop osis and educe wound healing capaci y in BECs ia TNF o CCL26 up egula ion [ 150 ]. In addi ion, hei angiogenic e ec on b onchial smoo h muscle cells is media ed by VEGFA [ 150 ]. Simila ly, exosomes om neu ophils enhance neu ophils chemo axis h ough hei leuko iene B4 ca go [ 136 ] and adhesion by ICAM-1 up egula ion in lung endo helial cells [151]. Few s udies desc ibe he e ec o as hma pa hophysiology on EV-miRNAs [ 152 ]. One o hem desc ibed he inc ease o miR-223 and miR-142a on EVs om BALF o alle gen- ea ed mice [ 138 ] while o he , pe o med on as hma ic pa ien s, desc ibed 24 miRNAs di e en ially exp essed including membe s o le -7 and miR-200 amilies and miR-21 [ 139 ]. MiR-21 a ge s he p o-in lamma o y Th1 cy okine, IL-12, and down egula e Th2 esponse, which is cha ac e is ic o as hma in lamma ion [ 153 ]. Acco dingly, miR-21 is inc eased in se um o as hma ic pa ien s and in e sely co ela ed wi h FEV 1 [ 154 ]. Las ly, a se o EV-miRNAs om BALF and miRNAs om lung issue we e al e ed in alle gen-exposed mice and we e in e sely co ela ed be ween bo h o igins [155]. In . J. Mol. Sci. 2021,22, 583 8 o 20 Figu e 3. Summa y o some o he main epo ed oles o ex acellula esicles (EVs) in he pa hophysiological mechanisms o as hma, ch onic obs uc i e pulmona y disease (COPD) and in e s i ial pulmona y ib osis (IPF). The igu e shows he cellula o igin o he EVs as well as hei cellula des iny o hei main ac ion. In he uppe and la ge lowe ai way o as hma pa ien s, EVs con ibu e o an igen p esen a ion, EVs om endo helial cells pa icipa e in mac ophage chemo axis while eosinophil EVs could ac in hei own chemo axis, adhesion and ec ui men . In COPD, EVs could p omo e emphysema de elopmen inc easing MMP1 sec e ion in lung epi helial cells, h ough myo ib oblas di e en ia ion o enhancing he p o eolysis o he ex acellula ma ix. In he al eoli o IPF pa ien s, EVs om mac ophages could inc ease he collagen 1-A exp ession in ib oblas , inc easing he ex acellula ma ix deposi ion, as well as p omo e ib oblas p oli e a ion. (Componen s o his igu e ha e been ob ained and modi ied om Se ie Medical A , h ps://sma .se ie .com, wi h pe mission unde he C ea i e Commons A ibu ion 3.0 Unpo ed License.). 5.2. EVs in COPD EVs may also play a main ole in COPD pa hophysiology. One o he ac o s associa ed wi h COPD, ciga e e smoke (CS), modi ies EV ca go om di e en lung cells. CS induces signi ican elease o CCN1-en iched exosomes om BECs, as well as he clea age o ma ix- associa ed CCN amily p o ein in o a sho e iso o m [156], which p omo es MMP1 sec e- ion in lung epi helial cells con ibu ing o emphysema de elopmen [156–158] (Figu e 3) . Simila ly, mac ophages exposed o CS elease MVs wi h p o eoly ic and collagenoly ic ac i i y in hei su ace as shown in Figu e 3[ 159 ]. In addi ion, he inc eased mac ophage- MVs elease up egula es se e al p o-in lamma o y and chemoa ac an media o s, as ICAM-1, IL-8 and MCP-1, in al eola epi helial cells [ 160 ]. O he s udies ha e ocused on EV non-coding RNAs in smoke s and COPD pa ien s. MiR-210 is o e exp essed in bo h BEC-EVs and lung issue samples a e CS exposu e. miR-210 loaded-EVs om COPD pa ien s showed he induc ion o lung ib oblas s di e en ia ion in o myo ib oblas s by silencing he au ophagy- ela ed ac o ATG7 [ 140 ]. Di e en p o iles o long non-coding RNAs (lncRNAs) we e ound in ci cula ing exosomes om non-smoke s and smoke s o di e en smoking p oduc s. EV-lncRNAs al e a ions ound in smoke s could lead o pulmona y ib osis and mi ophagy, which a e hallma k p ocesses in COPD and IPF. In addi ion, he a ge s o some o he di e en ial lncRNAs a e in ol ed in pa hological p ocesses o COPD, IPF and as hma [161]. In . J. Mol. Sci. 2021,22, 583 9 o 20 O e exp ession o exosomal miR-21 om BECs also p omo es myo ib oblas di e en- ia ion a ge ing hypoxia-inducible ac o 1 α (HIF-1 α ) [ 141 ]. Howe e , dec eased miR-21 was obse ed in BEC exosomes exposed o CS. This compensa o y mechanism in esponse o CS educed M2 mac ophage pola iza ion and, consequen ly, epi helial-mesenchymal ansi ion in ol ed in ai way emodeling [ 162 ]. Finally, CS induce on endo helial cells he p oduc ion o MVs en iched in le -7d, miR-191, miR-126 and miR-125a, whose majo e ec in ecipien mac ophages was he impai men o e e ocy osis [163]. Su ace p o eins iden i ied in ci cula ing endo helial MVs sugges ha endo helial MVs ha e an impo an egula o y ole in he coagula ion and in lamma ion p ocesses in COPD [ 164 ]. I has been also epo ed ha EV-media ed anspo o α1 -an i ypsin om endo helial cells o al eola epi helial cells is impai ed by CS exposu e [ 131 ]. In addi ion, exosomes om ac i a ed neu ophils con ain neu ophil elas ase (NE) in hei su aces, p o ec ing NE om α1 -an i ypsin p o eolysis, and enabling he deg ada ion o he ex acellula ma ix ia NE + exosomes [ 132 ] (Figu e 3). These esul s sugges ha endo helial and neu ophil de i ed-EVs con ibu e o emphysema de elopmen in COPD. 5.3. EVs in IPF EVs implica ion in IPF emains unclea . Up o now, mos ly EV s udies ela ed o mechanisms in ol ed in ib osis de elopmen used in i o o mu ine models and ocused hei miRNA ca go. MiR-21 is up egula ed in EVs om se um o a lung ib osis mice model and om human IPF pa ien s and i is co ela ed wi h IPF p og ession and mo ali y [ 143 ]. Ano he s udy showed ha miR-21 up egula ion is p omo ed ia TGF- β 1 and i has p o- ib o ic e ec s including myo ib oblas di e en ia ion as desc ibed in Figu e 3[ 165 ]. Kidney epi helial cells ha e been epo ed o elease an inc eased amoun o exosomes in esponse o inju y, loaded wi h speci ic p o- ib o ic ca go such as TGF- β 1 mRNA, which p oduce ib oblas s p oli e a ion, ac i a ion and epai p ocesses [166]. On an IPF a model, miR-328 was o e exp essed in M2 mac ophage-de i ed exo- somes wi h associa ed p oli e a i e e ec s in ib oblas s ia silencing FAM13A and o e - exp ession o ce ain p o eins as collagen 1A and α -SMA (Figu e 3) [ 144 ]. Th ee mo e exosomal mic oRNAs ha e been epo ed o be al e ed in IS om IPF pa ien s, miR-33a, miR-142 and le -7d [145]. Las ly, a ecen s udy showed inc ease amoun o EVs ca ying WNT5A in BALF om IPF pa ien s. These EVs o igina ing om lung ib oblas s ha e au oc ine e ec s in i o s imula ing ib oblas p oli e a ion [ 135 ]. Impai men o block o EV-WNT5A supp esses he ib ogenic e ec s o EVs imp o ing he IPF pa hology. 6. EVs in Ai way Sec e ions and Po en ial U ili y in Clinical P ac ice P esence o EVs in ai way sec e ions was i s desc ibed on BALF o heal hy sub- jec s [ 12 ]. By con as , he isola ion o EVs om spu um was epo ed 13 yea s la e om spon aneous spu um o cys ic ib osis pa ien s [ 167 ]. Following we p esen he eme ging ele ance o EVs om ai ways sec e ion on LD. 6.1. Ai way EVs in As hma The as majo i y o EV s udies in pa ien s wi h as hma ha e been done using BALF. Inc eased amoun o exosomes wi h speci ic p o ein su ace ma ke s [ 168 ], lipid [ 168 ] and miRNA [ 139 ] p o iles we e ound in BALF o as hma ic pa ien s. Exosome concen a ion and exp ession o al e ed EV-miRNAs we e co ela ed wi h as hma se e i y bioma ke s such as se um eosinophil and IgE le els [ 168 ] and FEV 1 [ 139 ]. Up o now, only one wo k has eco e ed EVs om IS o as hma ic pa ien s [ 11 ]. Exosomes om IS o mild alle gic as hma pa ien s con ain sho RNA species and su ace ma ke s as e aspanins and HLA-DR, sugges ing he easibili y o IS as an accessible minimally-in asi e sou ce o EV bioma ke s [11]. EVs we e isola ed om NL o he i s ime on 2011 [ 169 ]. Mo e han 600 p o eins ha e been iden i ied among NL-EV ca go, inding p o eins ela ed wi h an imic obial In . J. Mol. Sci. 2021,22, 583 16 o 20 83. 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