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TLR1/TLR2 MyD88-ga bog'liq yo'lni qanday faollashtiradi

TLR1/TLR2 MyD88-ga bog'liq yo'lni qanday faollashtiradi


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Yaqinda men MyD88-ga bog'liq signalizatsiya yo'li haqida o'qidim, xususan uning makrofaglarda va immun tizimining boshqa hujayralarida patogenni tanib olishda faollashishi haqida. Men tushunamanki, PAMP (patogen bilan bog'liq molekulyar naqsh) PRR (naqshni aniqlash retseptorlari) bilan bog'langanda, retseptor turli xil oqsillar o'rtasida kimyoviy reaktsiyalar kaskadini keltirib chiqaradigan konformatsion o'zgarishlarga uchraydi, bu esa oxir-oqibat translokatsiyaga olib keladi. NF-kB ning yadroga, bu esa sitokinlarning ishlab chiqarilishiga olib keladi. Mening savolim, ayniqsa, TLR1 va TLR2 ga nisbatan nozik jihatlar haqida.

Mening o'qishim shuni ko'rsatadiki, ular odatda heterodimer hosil qiladi - bu har doim to'g'rimi? Patogenni tanib olish va immunitetni shakllantirish uchun ikkalasi ham kerakmi?

Patogen TLR1 bilan bog'langanda (TLR2 bilan kompleksdami yoki yo'qmi) retseptor bilan nima sodir bo'ladi. MyD88 oxir-oqibat yollanganligini tushunaman, lekin nima bilan?

Men MyD88 ishga qabul qilingandan keyin sodir bo'layotgan hamma narsani tushunaman deb o'ylayman, lekin men bundan oldin hamma narsaga unchalik aniq emasman. Men MyD88-ni ishga olish darhol keyingi qadam ekanligini tushundim, ammo boshqa manbalar men bilmagan ikkita oqsilga ishora qiladi: TOLLIP va TIRAP. Agar biror narsa bo'lsa, ularning roli nima?

Men hali stackexchange uchun biroz yangiman, shuning uchun agar bu savol standartga mos kelmasa, uzr so'rayman, bu holda savol oddiy bo'lsin: "TLR1/TLR2 MyD88-ga bog'liq yo'lni qanday faollashtiradi?"


TLR odatda homodimerlar (Toll-o'xshash retseptorlar) sifatida ishlaydi, lekin TLR2 TLR1 yoki 6 bilan hamkorlik qilishi mumkin. Uning ligandlari bog'langanda (bu patogen bilan bog'liq molekulyar naqsh deb ataladi) retseptorlari o'zlarining konformatsiyasini o'zgartiradilar va TIRAP (TIR adapteri) bilan bog'lanishiga imkon beradi. oqsil) ularning Toll-interleykin 1 retseptorlari (TIR) ​​domeniga. Keyin TIRAP MYD88-ni ishga oladi, u IRAK1 yoki 4-ni ishga oladi. Keyin signal quyidagi rasmda ko'rsatilganidek (TLR haqidagi Vikipediya maqolasidan olingan) yo'l bo'ylab uzatiladi.


Toll-like retseptorlarining MyD88-ga bog'liq va mustaqil yo'llari ligand-stimulyatsiya qilingan makrofaglarda Triptolidning biologik faolligi bilan shug'ullanadi.

Triptolid - bu Xitoy dorivor o'simlikidan olingan diterpen triepoksidi Tripterygium wilfordii Yallig'lanishga qarshi, immunosupressiv va saratonga qarshi xususiyatlarga ega ilgak F..

Natijalar

Bu erda biz LPS induktsiyalangan sichqoncha makrofaglarida triptolid tomonidan modulyatsiyalangan immun signalizatsiya genlarining ekspression profilini xabar qilamiz. Triptolid bilan davolash massiv tadqiqotida tollga o'xshash retseptorlarni (TLR) o'z ichiga olgan bir yuz to'qson beshta immun signalizatsiya genining 22,5% ifodasini modulyatsiya qildi. TLRlar hujayra ichidagi adapter molekulalari MyD88 va TRIF bilan bog'lanish orqali immun javoblarini keltirib chiqaradi. Triptolid NFKB faollashuvini va TLRlarning quyi oqimidagi boshqa signalizatsiya yo'llarini inhibe qilishi ma'lum bo'lsa-da, triptolid faolligida TLR kaskadining ishtiroki haqida xabar berilmagan. Ushbu tadqiqotda biz triptolidning turli xil TLR agonistlari tomonidan induktsiya qilingan proinflamatuar quyi oqim effektorlarining ifodasini bostirishini ko'rsatamiz. Shuningdek, MyD88 yoki TRIF nokaut qilinganida, TLR tomonidan induktsiya qilingan NFKB faollashuviga triptolidning bostiruvchi ta'siri kuzatildi, bu MyD88 va TRIF vositachiligida NFKB faollashuvi triptolid tomonidan inhibe qilinishi mumkinligini tasdiqladi. TLR kaskadida triptolid TLR4 va TRIF oqsillarini pasaytiradi.

Xulosa

Ushbu tadqiqot triptolid faolligida TLR signalizatsiyasining ishtirokini ochib beradi va yallig'lanish sharoitida triptolid faolligi NFKB faollashuvini qanday kamaytirishi mumkinligini tushunishni yanada oshiradi.


Abstrakt

Toll kabi retseptorlari (TLR) tug'ma immunitet reaktsiyasini boshlash uchun patogen va xavf bilan bog'liq bo'lgan molekulyar naqshlarni aniqlay oladigan muhim naqshni aniqlash retseptorlari. TLR1 va 2 bakterial hujayra devorlaridan triatsillangan lipopeptidlar yoki sintetik Pam3CSK4 ligandlari bilan bog'langandan so'ng plazma membranasida heterodimerlanadi. TLR1 / 2 dimerlari TIRAP va MyD88 adapter molekulalari bilan o'zaro ta'sir qiladi, bu esa asosiy transkripsiya omillarini, shu jumladan NF-kB ni faollashtirishga olib keladigan signal kaskadini boshlaydi. So'nggi yigirma yil ichida TLRlar keng qamrovli o'rganilganiga qaramay, ligandlarni bog'lash va retseptorlarni faollashtirishning real vaqt kinetikasi asosan o'rganilmagan. Biz TLR1/2 dimerning MyD88 va TIRAP adapterlari bilan o'zaro ta'siridan foydalanib, TLR faollashuvi va adaptorlarni jalb qilish kinetikasini o'rganishni maqsad qildik. Bioluminesans rezonans energiya uzatish (BRET) tirik hujayralardagi real vaqt rejimida oqsil-oqsil o'zaro ta'sirini aniqlash imkonini beradi va TLRlarga adapterlarni jalb qilishni o'rganish uchun qo'llaniladi. Energiya uzatish TLR2 va TIRAP va TLR2 va MyD88 o'rtasidagi o'zaro ta'sirlarni faqat TIRAP ishtirokida ko'rsatdi. Miqdoriy BRET va konfokal mikroskopiya TIRAP MyD88 ning TLR2 bilan o'zaro ta'siri uchun zarur ekanligini tasdiqladi. Bundan tashqari, Pam3CSK4 stimulyatsiyasi bo'lmaganda oqsillar o'rtasidagi konstitutsiyaviy yaqinlik BRET bilan kuzatilgan va protein ifodasining pasayishi, oqsillarni belgilash strategiyalarining o'zgarishi yoki yorqinroq NanoLuc lusiferazasidan foydalanish bilan bekor qilinmagan. Shu bilan birga, immunopresipitatsiya tadqiqotlari ushbu oqsillar o'rtasidagi konstitutsiyaviy o'zaro ta'sirni ko'rsatmadi, bu BRET bilan kuzatilgan o'zaro ta'sir, ehtimol, oqsilning haddan tashqari ekspressiyasining artefaktlarini anglatadi. Shunday qilib, BRET tadqiqotlarida va TLR yo'lini tekshirishda proteinning haddan tashqari ko'payishidan foydalanishda ehtiyot bo'lish kerak.

Iqtibos: Sampaio NG, Kocan M, Schofield L, Pfleger KDG, Eriksson EM (2018) Bioluminesans rezonans energiyasini uzatish orqali TLR2, MyD88 va TIRAP o'rtasidagi o'zaro ta'sirlarni o'rganish oqsilni haddan tashqari ifodalash artefaktlari bilan to'sqinlik qiladi. PLoS ONE 13(8): e0202408. https://doi.org/10.1371/journal.pone.0202408

Muharrir: Irving Coy Allen, Virjiniya Politexnika Instituti va Davlat Universiteti, Qo'shma Shtatlar

Qabul qildi: 2017 yil 20 dekabr Qabul qilingan: 2 avgust, 2018 yil Nashr etilgan: 2018 yil 23 avgust

Mualliflik huquqi: © 2018 Sampaio va boshqalar. Bu Creative Commons Attribution License shartlari asosida tarqatiladigan ochiq kirish maqolasi boʻlib, asl muallif va manba hisoblangan holda har qanday vositada cheksiz foydalanish, tarqatish va koʻpaytirishga ruxsat beradi.

Ma'lumotlar mavjudligi: Barcha tegishli ma'lumotlar qog'ozda.

Moliyalash: Ushbu ish Viktoriya shtati hukumati operatsion infratuzilmasini qo'llab-quvvatlash va Avstraliya hukumati Milliy sog'liqni saqlash va tibbiy tadqiqotlar kengashi (NHMRC) Mustaqil tadqiqot instituti infratuzilmasini qo'llab-quvvatlash sxemasi NHMRC Dora Lush stipendiyasi, Grant/mukofot raqami: APP1038030 (NGS) NHMRC Grant/mukofot raqamlari: APP10627 va APP1126395 (EME) NHMRC RD Rayt biotibbiyot tadqiqotlari stipendiyasi granti/mukofot raqami 1085842 (KDGP). KDGP laboratoriyasida BRET ishi qisman Avstraliya tadqiqot kengashi Linkage Grant LP160100857 tomonidan moliyalashtiriladi, Promega, BMG Labtech va Dimerix sanoat hamkorlari sifatida. Tadqiqot loyihasi, ma'lumotlarni to'plash va tahlil qilish, nashr qilish qarori yoki qo'lyozmani tayyorlashda moliyachilarning qo'shimcha roli yo'q edi. Ushbu mualliflarning o'ziga xos rollari "muallif hissalari" bo'limida ifodalangan.

Raqobatli manfaatlar: Mualliflar jurnal siyosati bilan tanishib chiqdilar va quyidagi qarama-qarshiliklarga duch kelishdi: KDGP Dimerix Limited kompaniyasining bosh ilmiy maslahatchisi va kompaniyada ulushga ega. KDGP Promega, BMG Labtech va Dimerixdan Avstraliya Tadqiqot Kengashining bog'lanish Grant LP160100857 hamkor tashkilotlari sifatida mablag' oladi. Bu mualliflarning ma'lumotlar va materiallarni almashish bo'yicha barcha PLOS ONE siyosatlariga rioya qilishini o'zgartirmaydi. NGS, MK, LS va EME hech qanday manfaatlar to'qnashuviga ega emas.


Kirish

Birlamchi Sjögren's sindromi (pSS) ekzokrin bezlarning disfunktsiyasi va immunitetning giperaktivligi bilan tavsiflangan otoimmün kasallikdir. pSS bilan og'rigan bemorlarda odatda tupurik va ko'z yoshi ishlab chiqarishni yo'qotish, shuningdek, o'pka va buyrak patologiyalari va B hujayrali limfoma kabi jiddiy tizimli oqibatlar kuzatiladi (1). Tuprik bezining yallig'lanishi kasallikning o'ziga xos belgisi hisoblanadi (2), ammo bu to'qimalarda surunkali yallig'lanishni qo'zg'atuvchi kasallik hodisalari va yo'llari noma'lumligicha qolmoqda. Bir qator tadqiqotlar shuni ko'rsatadiki, tug'ma immunitetning faollashishi pSS (3𠄸) da adaptiv javobdan oldin sodir bo'ladi. Biroq, kasallikning boshlanishi va rivojlanishiga olib keladigan o'ziga xos yo'llar yaxshi tushunilmagan.

Laboratoriyamizdagi so'nggi ishlar pSS patogenezida (9) miyeloid differentsiatsiyasining birlamchi javob oqsili 88 (Myd88) adapterining asosiy rolini aniqladi. Myd88 tug'ma va adaptiv immunitet funktsiyasi uchun juda muhimdir, chunki Toll-like retseptorlari (TLR) va Interleykin-1 retseptorlari (IL-1R) oila a'zolarining aksariyati signal uzatish uchun Myd88 ni talab qiladi (10). Yo'q bo'lgan pSS sichqoncha modelidan foydalanish Myd88 (NOD.B10 Myd88−/−), biz Myd88-ga bog'liq yo'llarning o'z-o'zidan faollashishi kasallikning rivojlanishi uchun juda muhim ekanligini aniqladik (9). TLR va IL-1 oilasi a'zolari sichqoncha va inson SS tuprik to'qimalarida ko'paygan bo'lsa-da (3, 11�), nisbatan kam sonli tadqiqotlar bu yo'llarning tuprik va tizimli yallig'lanishga funktsional hissasini o'rgangan va ularning aksariyati Myd88 ga qaratilgan. -mustaqil agonist TLR3 (5, 6, 17�).

Myd88-ga bog'liq bo'lgan TLR yo'llarining pSS patogeneziga qo'shgan hissasini tushunish uchun biz birinchi navbatda NOD.B10Sn-dan taloq to'qimasida RNK ketma-ketligini (RNK-seq) o'tkazdik.H2 b / J (NOD.B10) klinik kasalligi va C57BL / 10 (BL / 10) nazorati bo'lgan ayollar. Biz TLR faollashuvi bilan bog'liq ko'plab genlar o'zgartirilganligini aniqladik, shu jumladan TLR2 va TLR4 vositachiligidagi signal kaskadlarida ishlatilgan. Biz TLR2, TLR2 ko-retseptorlarini (TLR1 va TLR6) topdik va TLR4 darajalari pSS sichqonlaridan olingan taloq va tuprik B hujayralarida o'zgargan. Ushbu topilmalar funktsional ahamiyatga ega edi, chunki klinik kasalligi bo'lgan NOD.B10 ayollaridagi splenotsitlar TLR2 ligatsiyasiga yuqori darajada javob beradi. Bundan tashqari, pSS splenotsitlarini zarar bilan bog'liq molekulyar naqsh (DAMP) Decorin (Dcn) bilan davolash yallig'lanishli sitokin sekretsiyasini TLR4 ga bog'liq ravishda o'zgartirdi. Shunisi e'tiborga loyiqki, Dcn NOD.B10 splenotsitlarida lipopolisakkarid (LPS) bilan solishtirganda aniq yallig'lanish profilini keltirib chiqardi. Bundan tashqari, biz pSS so'lak hujayralarining TLR4 ligatsiyasi natijasida yallig'lanish vositachilari ishlab chiqarilishini aniqladik va bu, ayniqsa, erkaklarnikiga nisbatan urg'ochi hayvonlardan olingan to'qimalarda yaqqol namoyon bo'ldi. Nihoyat, so'lak bezlari to'qimalarida yallig'lanishga qarshi sitokin sekretsiyasi zaiflashdi. Myd88-niqis NOD.B10 sichqonlari. Ushbu ma'lumotlar Myd88-ga bog'liq bo'lgan o'ziga xos TLRlarning pSS-da tartibga solinmaganligini va Dcn ni kasallikdagi yallig'lanishning yangi vositachisi sifatida aniqlaganligini ko'rsatadi. TLR yo'llarining blokadasi mahalliy va tizimli kasallikning namoyon bo'lishini davolash uchun innovatsion terapevtik strategiyani ko'rsatishi mumkin.


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TIR domenini o'z ichiga olgan adapterlarning TLR signalizatsiyasiga qo'shgan hissasi

Shaxsiy TLRlar MyD88, TRIF, TIRAP/MAL yoki TRAM kabi TIR domenini o'z ichiga olgan adapterlar to'plamining a'zolarini farqli ravishda yollaydi. MyD88 barcha TLRlar tomonidan qo'llaniladi va yallig'lanishli sitokin genlarini induktsiya qilish uchun NF-㮫 va MAPKlarni faollashtiradi. TIRAP MyD88 ni TLR2 va TLR4 kabi hujayra yuzasi TLRlariga jalb qiluvchi saralash adapteridir (rasm ​ (1-rasm). 1). Biroq, yaqinda o'tkazilgan tadqiqot shuni ko'rsatdiki, TIRAP TLR9 kabi endosomal TLRlar orqali signalizatsiyada ham ishtirok etadi. TIRAP ning lipid bog'lovchi domeni PI (4,5) P ga bog'lanadi2 plazma membranasida va endosomalarda PI (3)P ga, ularning tegishli joylarida funktsional TLR4 va TLR9 signalizatsiya komplekslarining shakllanishiga vositachilik qiladi. Shunday qilib, TIRAP turli lipidlar bilan bog'lanish orqali ham hujayra yuzasi, ham endosoma TLRlari bilan bog'lanadi (38). Biroq, TLR9 agonistlarining yuqori konsentratsiyasi TIRAP yo'qligida hujayralarni faollashtiradi, bu HSV-1 infektsiyasi kabi tabiiy holatlarda TIRAP TLR9 signalizatsiyasi uchun zarurligini ko'rsatadi (39).

cDC, makrofaglar va MEFlarda TLR signalizatsiyasi. TLR4 hujayra yuzasida, TLR3 esa endosoma bo'linmasida joylashadi. Gomo- yoki geterodimer hosil bo'lishi ikkita asosiy quyi oqim adapter oqsillari, MyD88 va TRIF uchun signalni boshlaydi. TIRAP signalni TLR4 dan MyD88 ga uzatadi, TRAM esa TLR4 dan TRIF ga signalni vositachilik qiladi. TLR ulanishi MyD88-ga asoslangan va shuningdek, IRAK1 va IRAK4 ni o'z ichiga olgan Myddosome shakllanishiga olib keladi. IRAK1 faollashuvi TRAF6 va TAK1 ning o'zida K63 bilan bog'langan poliubiquitinatsiyadan keyin TRAF6 faollashuvini keltirib chiqaradi. TAK1 faollashuvi IKK kompleksi-NF-㮫 va MAPKlarning faollashishiga olib keladi. MAPK faollashuvi AP1s transkripsiya faktorining faollashuviga olib keladi. TRAF6 ECSIT ubiquitinatsiyasini rag'batlantiradi, buning natijasida mitoxondriyal va hujayrali ROS hosil bo'ladi. TLR ulanishi, shuningdek, TRAF6 va TRAF3 ishga qabul qilingandan keyin TRIF faollashuvini keltirib chiqaradi. TRAF6 RIP-1ni ishga oladi, u MAPK faollashtirilgandan so'ng TAK1 kompleksini faollashtiradi. RIP-1 faollashuvi Pellino-1 tomonidan ubiquitinatsiyani tartibga soladi. Pellino-1 DEAF-1 bilan bog'lanish orqali IRF3 faollashuvini tartibga soladi. TRAF3 IRF3 fosforillanishi uchun TBK1 va IKKi ni ishga oladi. PIKfyve-dan PtdIns5P TBK1 va IRF3 o'rtasida murakkab shakllanishni osonlashtiradi. Bir nechta salbiy regulyatorlar signal kompleksining shakllanishini yoki ubiquitinatsiyani inhibe qilish orqali TLR signalizatsiyasini modulyatsiya qiladi. MyD88 ST2825, NRDP-1, SOCS1 va Cbl-b TRIF SARM va TAG TRAF3 SOCS3 va DUBA tomonidan bostiriladi va TRAF6 A20, USP4, CYLD, TANK, TRIMP38 tomonidan bostiriladi. NF-㮫 Bcl-3, I㮫NS, Nurr1, ATF3 va PDLIM2 tomonidan bostiriladi, IRF3 faollashuvi esa Pin1 va RAUL tomonidan salbiy tartibga solinadi.

TRIF TLR3 va TLR4 ga jalb qilinadi va I toifadagi IFN va yallig'lanishli sitokin genlarini induktsiya qilish uchun IRF3, NF-㮫 va MAPKlarni faollashtirishga olib keladigan muqobil yo'lni targ'ib qiladi. TRAM tanlab TLR4 ga jalb qilinadi, lekin TRIF va TLR4 o'rtasidagi bog'lanish uchun TLR3 emas. TLR3 to'g'ridan-to'g'ri TRIF bilan o'zaro ta'sir qiladi va bu o'zaro ta'sir epidermal o'sish omili ErbB1 va Btk (40, 41) tomonidan TLR3 sitoplazmatik domenidagi ikkita tirozin qoldiqlarini fosforlanishini talab qiladi. Birgalikda, adapterdan foydalanishga qarab, TLR signalizatsiyasi asosan ikkita yo'lga bo'linadi: MyD88-ga bog'liq va TRIF-ga bog'liq yo'llar.


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C57BL/6 sichqonlari va TRIF lps2/lps2 mutant sichqonlari Jekson laboratoriyasidan xarid qilingan. MyD88 −/− sichqonlari Shizuo Akiradan sovg'a bo'ldi (Ross Kedl, Kolorado universiteti sog'liqni saqlash fanlari markazi orqali). C57BL/6 × 129Sv sichqonlarining F2 avlodidan olingan SHIP heterozigotlari SHIP +/+ va SHIP −/− littermates hosil qilish uchun yetishtirildi. Barcha sichqonlar Luisvill universitetida maxsus patogensiz hayvonlar muassasasida saqlangan va eksperimentlar uning Hayvonlarni parvarish qilish va foydalanish bo'yicha institutsional qo'mitasi nazorati ostida o'tkazilgan. ning lipid A tuzilmalaridan olingan sMLA va sDLA E. coli LPS mos ravishda Invivogen va Peptides Internationaldan sotib olindi. Ikkala birikma ham har birining to'liq eritish zahiralari har doim bir vaqtning o'zida reagent tayyorlashga ta'sir qilishi mumkin bo'lgan faollikdagi farqlarni minimallashtirish uchun tayyorlanmaguncha yumshoq vortekslash orqali DMSOda eritildi. Eritilgan birikmalar bir martalik foydalanish uchun kichik hajmlarda ajratilgan va �ଌ da saqlanadi. M-CSF and GM-CSF were purchased from Rɭ Systems. Single-strand cDNA synthesis enzymes were obtained from Invitrogen. Enzyme mix 2X, containing SYBR Green dye for quantitative real-time PCR (QRT-PCR) was purchased from Applied Biosystems. All phospho-specific Abs and TLR4 Ab used for immunoblotting experiments in this study were purchased from Cell Signaling Technology, and all nonphospho-specific and β-actin Abs were from Santa Cruz Biotechnology, except for the MyD88-specific Ab, which was from eBioscience. HRP-conjugated anti-rabbit and anti-goat secondary Abs were from Jackson ImmunoResearch.

Generation of bone marrow-derived DCs and macrophages

Bone marrow-derived DCs (BMDCs) were prepared according to a protocol modified from that of Lutz et al. (22). In brief, femurs and tibiae from 8- to 12-wk-old mice were collected and flushed with sterile HBSS twice. The resulting bone morrow cells were resuspended in R10F (RPMI 1640 medium containing 10% heat-inactivated FBS, 2 mM l -glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin) plus 50 µM 2-ME and 5 ng/ml GM-CSF. A total of 2 × 10 6 cells per bacteriological culture plate were cultured for 10 d, with feeding on days 3 and 8 by adding 10 ml fresh medium, and on day 6 by replacing half of the culture medium. Nonadherent cells were collected on day 10 and verified to be at least 85�% CD11b + /CD11c + /MHC-II + /CD86 low /Gr1 − /CD4 − /CD8 − /B220 − /CD19 − by flow cytometry before use in experiments.

Bone marrow-derived macrophages (BMDMs) were prepared according to a protocol modified from Sag et al. (23). In brief, bone marrow cells obtained as described earlier were cultured overnight in standard tissue culture plates in the presence of 10 ng/ml M-CSF. Nonadherent cells from this initial culture were then transferred to low-attachment six-well plates (Corning Life Sciences) in 4 ml R5F containing 30% L929 conditioned medium and 10 ng/ml M-CSF for 7 d, adding 1.5 ml fresh medium on days 3 and 5. Cells were verified to be at least 90�% CD11b + /CD11c − /MHC-II − /CD80 − /CD86 − /Gr1 − /CD4 − /CD8 − /B220 − /CD19 − /F4/80 + by flow cytometry before use in experiments.

The human monocytic cell line THP-1 was purchased from American Type Culture Collection (TIB 202) and cultured in RPMI 1640 medium (Life Technologies A10491) containing 4.5 g/L d -glucose, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, 10 mM HEPES, 300 µg/L l -glutamine, with added 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-ME, and 10% FBS. The cells were cultured at a density of 2𠄶 × 10 5 /ml at 37° in a 5% CO2 incubator. Cells were used between weeks 6 and 11 of culture.

QRT-PCR

BMDCs (1 × 10 6 ) were rested for 2 h in polystyrene tubes (12 × 75 mm) at 37ଌ and then treated with 100 ng/ml sMLA or sDLA (diluted in R10F). In all experiments, DMSO was used as vehicle control. Cells were lysed 1, 2, or 3 h after activation in guanidine thiocyanate buffer. Total RNA was isolated using the RNeasy Mini Kit (Qiagen), and cDNA was synthesized using the SuperScript III Platinum Two-Step QRT-PCR kit (Invitrogen Life Technologies). QRT-PCR was performed using the Applied Biosystems 7500 Fast system or CFX96 Real-Time PCR Detection System and Power SYBR Green RT-PCR mastermix. QuantiTect Primers (Qiagen) were used for all QRT-PCR assays except for primers used to measure β-actin mRNA (forward: 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ reverse: 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′), which were purchased from Sigma-Genosys. Expression of each target gene was normalized to β-actin, and fold expression over vehicle control was calculated using the 2 −Δ㥌τ method (24).

Immunoblotting and immunoprecipitation

BMDCs (2𠄳 × 10 6 ) were rested for 2 h in 12 × 75 mm polystyrene tubes at 37ଌ and then exposed to sMLA or sDLA. In all experiments, DMSO was used as vehicle control. After the indicated time points, cells were centrifuged in ice-cold HBSS containing 50 µM NaF and then lysed in radioimmunoprecipitation assay lysis buffer containing Complete Mini protease inhibitor mixture tablets (Roche), phosphatase inhibitor mixture (Sigma-Aldrich), and 250 nM okadaic acid (Sigma-Aldrich). The resulting lysates were tested for their protein concentrations using the BCA assay (Pierce Biotechnology) and then mixed with 5X SDS sample buffer (1× final concentration). Lysates containing equal amounts of protein were loaded onto 10% SDS-PAGE gels for electrophoresis, after which resolved proteins were transferred onto nitrocellulose membranes (GE Healthcare) and blocked with 5% nonfat dry milk (NFDM) for 1 h. Primary Abs were dissolved in 5% BSA except for β-actin and IRF3 total Abs, which were dissolved in 5% NFDM and incubated with the blocked membranes overnight at 4ଌ. After exposure to HRP-conjugated anti-rabbit or anti-goat secondary Abs for 1 h in NFDM, bands were visualized using the ECL detection system (GE healthcare), and band intensities were analyzed with Quantity One Software (Bio-Rad version 4.6.6).

THP-1 cells (1.5𠄳 × 10 6 ) were rested for 2 h at 37ଌ in 12 × 75 mm polystyrene tubes. The cells were then exposed to 100 ng/ml sMLA, sDLA, or the vehicle control DMSO. At the indicated times, the cells were washed in 4ଌ HBSS containing 50 µM NaF and lysed in radioimmunoprecipitation assay buffer containing Complete Mini protease inhibitor mixture tablets (Roche), phosphatase inhibitor mixture (Sigma-Aldrich), and 250 nM okadaic acid (Sigma-Aldrich). Equal amounts of protein, as determined by the Pierce Biotechnology BCA assay, were loaded on 8% SDS-PAGE gels and subsequently immunoblotted for pSHIP1 (Cell Signaling 3941), SHIP1 (Cell Signaling 2728), and β-actin (Santa Cruz Biotechnology sc1616). Bands were visualized by autoradiography or the FUJI LAS 4000. Measurements of band intensities were analyzed with either Quantity One Software (Bio-Rad version 4.6.6) or Multi Gauge software (FUJIFILM).

Immunoprecipitation of MyD88 was performed as described previously (25). In brief, 3𠄶 × 10 6 BMDCs were lysed in 1% digitonin lysis buffer. After incubating with primary Abs overnight, beads conjugated to anti-rabbit secondary Abs were added to the lysates and incubated for another 2𠄴 h at 4ଌ. Beads were collected by a brief centrifugation, and immunoprecipitated proteins were released by suspension in 2X sample buffer before resolving by SDS-PAGE and immunoblotting.

Statistical Analysis and sample normalization

Two-factor (time versus treatment) ANOVA and post hoc Tukey’s tests were performed to determine the significance of the differences between sDLA- versus sMLA-induced cellular effects on gene expression and signaling. A p value π.05 was considered to indicate statistically significant differences between treatment groups. All QRT-PCR data were normalized to β-actin, and phosphoprotein band intensities were normalized to total protein levels for IRF3 and I㮫 kinase (IKK)α/β, and to β-actin for SHIP1. Lysates from DMSO alone-treated cells were used as reference point for calculation of fold expression and intensities.


Natijalar

Mal is essential for TLR2/6, but not TLR2/1-dependent activation of NFκB

Earlier studies have shown that TLR2 and TLR4 are the sole TLRs that engage both MyD88 and Mal for signal transduction ( Fitzgerald va boshqalar, 2001 Horng va boshqalar, 2001 Yamamoto va boshqalar, 2002 ). Here, we analysed whether the nature of the TLR2 heteromer influences the contribution of Mal to the signalling response. In full accordance with the proposed model that Mal and MyD88 are both required for TLR2-dependent cytokine formation, the formation of TNF-α was severely impaired in MyD88-, Mal- and TLR2-deficient mouse macrophages on stimulation with the diacylated lipopeptide MALP2 (Figure 1A). In contrast, the TNF-α response to the triacylated TLR2 ligand Pam3CSK4 was largely independent of Mal, although the response was somewhat reduced in Mal-deficient cells at low Pam3CSK4 concentrations (Figure 1B). Notably, MyD88- and Mal-deficient cells exhibited an impaired response to LPS from Escherichia coli, but responded normally to PolyI:C stimulation (Figure 1C and data not shown), in full accordance with the published phenotype ( Yamamoto va boshqalar, 2002 Kenzel va boshqalar, 2009). These data indicate that, in contrast to the universal role of MyD88, Mal has a more subtle function in the TNF-α production depending on the degree of acylation of the lipopeptide ligand and probably the nature of the TLR2-containing heteromer.

Mal is not sufficient to drive NFκB activation in the absence of MyD88

To further resolve whether Mal and MyD88 occupy discrete positions in NFκB activation, we transfected wild-type (WT), MyD88 and Mal knock-out macrophages with plasmids encoding for these adaptor proteins or parental plasmid. As shown in Figure 2A, expression of the adaptors MyD88 or Mal in WT macrophages led to a strong, ligand-independent induction of an NFκB-dependent luciferase reporter gene, which was comparable to that obtained by MALP2 stimulation. Similarly, expression of both adaptors potently induced NFκB activation in Mal-deficient macrophages (Figure 2C). This indicated that MyD88 is located downstream of Mal, as it does not require Mal for transcriptional activation. In contrast, expression of Mal in MyD88 knock-out macrophages did not activate the NFκB-dependent reporter (Figure 2B). These observations were fully compatible with a model, in which Mal is located upstream of MyD88 and requires the presence of MyD88 for the induction of NFκB. The relative contribution of Mal and MyD88 was not cell-type specific, as embryonic fibroblasts from WT-, MyD88- and Mal-deficient mice exhibited the same phenotype (data not shown).

The PI3K activity in response to TLR2/6 agonists is delayed in MyD88-deficient cells

Several studies have underlined the importance of PI3K function in NFκB activation downstream of IL-1R and various TLRs ( O'Neill va boshqalar, 1997 Arbibe va boshqalar, 2000 Ojaniemi va boshqalar, 2003). However, although TLR2 was the first TLR, which was reported to activate PI3K, the relationship of PI3K to the TLR2-adaptor proteins had not been clarified. First, we analysed, whether PI3K is located up- or downstream of Mal and MyD88. We used Akt phosphorylation as a read out of PI3K activity. Two principal pathways, which connect PI3K to phosphorylation of Akt on residues Thr308 and Ser473, have been described. First, PI3K catalyses the phosphorylation of phosphatidylinositol(4,5)P2 (PIP2) to PIP3, which, in turn, binds to and phosphorylates Akt through the phosphatidylinositol-dependent kinases (PDK1 and PDK2) ( Cantley, 2002 ). More recently, it has been shown that mammalian TOR (mTOR) directly phosphorylates Akt on Ser473 in vitro and facilitates Thr308 phosphorylation by PDK1 ( Sarbassov va boshqalar, 2005 ).

As depicted in Figure 3A, MALP2 induced phosphorylation of Akt in WT macrophages with a maximum between 10 and 20 min after stimulation. MALP2-induced Akt phosphorylation was PI3K –dependent, as it was abrogated by preincubation of the cells with the PI3K inhibitor LY294002. As compared with WT cells, Akt phosphorylation by MALP2 was severely impaired in Mal-deficient macrophages (Figure 3C), as well as Mal-deficient mouse embryonic fribroblasts (MEF) (Figure 3D). In contrast, Akt phosphorylation was induced by MALP2 in MyD88-deficient macrophages (Figure 3E). Notably, the peak of Akt phosphorylation was retarded when compared with WT cells (maximal phosphorylation at 60 min after stimulation). These findings indicated that Mal, but not MyD88, essentially contributed to TLR2/6-induced PI3K activation, although MyD88 accelerated the kinetics of this process. Intriguingly, the TLR2/1 agonist Pam3CSK4 induced PI3K activity independently of Mal and of MyD88 (Figure 3B, C and E). Accordingly, TLR2/6 and TLR2/1 induced PI3K activation through distinct pathways, which differ in the usage of Mal and MyD88.

Mal requires PI3K for efficient NFκB activation

We have shown that Mal is essential for PI3K activation. As PI3K has been implicated in the transactivation of the NFκB subunit p65 in response to TLR2 stimulation ( Strassheim va boshqalar, 2004 ), we wondered whether PI3K was involved in ligand-independent NFκB activation on heterologous expression of Mal. To clarify this subject, we transfected HEK293 cells with TLR2 and an NFκB-dependent ELAM-luciferase reporter plasmid. As shown in Figure 4A, NFκB activation by the diacylated lipopeptide FSL-1 was inhibited by increasing concentrations of LY294002 in a dose-dependent fashion. This finding confirmed earlier reports on an important role of PI3K in the response to different TLR2 agonists ( Arbibe va boshqalar, 2000 Henneke va boshqalar, 2005). Next, we transfected HEK293 cells with Mal and ELAM-luciferase reporter plasmids and inhibited PI3K with LY294002. As shown in Figure 4B, Mal transfection in the presence of endogenous MyD88 in these cells was sufficient to potently induce the ELAM-luciferase reporter and this activation was inhibited by LY294002 in a dose-dependent fashion. NFκB transcriptional activity relies on two mechanisms: degradation of the repressor IκB allows its translocation to the nucleus and phosphorylation of the p65 subunit on residue Ser536 further enhances gene expression ( Perkins, 1997 Jefferies va boshqalar, 2001). To better understand the specific role of PI3K in TLR2-dependent NFκB activation, we made use of a p65 reporter system, which is activated independently of IκB degradation ( Vanden Berghe va boshqalar, 1998). As shown in Figure 4C, incubation of the cells with increasing concentrations of LY294002 decreased the p65-Gal4/Gal-luciferase reporter response to heterologous Mal expression. Hence, PI3K activation downstream of Mal is essential for the transactivation of the NFκB subunit p65.

Mal interacts with p85a independently of TLR2 and MyD88

As outlined above, PI3K activation occurred downstream of Mal. Next, we analysed whether Mal and p85α, the regulatory subunit of PI3K, interacted physically with each other. First, RAW264.7 macrophages were stimulated with MALP2 for the indicated time periods (Figure 5A). Then cells were lysed and cell lysates were subjected to immunoprecipitation and western blot analysis. Under these conditions, we found that endogenous p85α and Mal interacted in an inducible fashion with a maximal effect 5–10 min after stimulation. The same result was obtained in immortalized bone-marrow macrophages from C57BI/6 mice (data not shown). In the case of another TLR, namely TLR5, it has been postulated that the adaptor MyD88 bridges the interaction between the TIR domain of the receptor and PI3K ( Rhee va boshqalar, 2006). Therefore, we wondered whether MyD88 was involved in the association of Mal with p85α. To address this question, we created the cell line HEK-MyD88si, in which MyD88 expression is effectively silenced by shRNA as confirmed by immunoblot and functional analysis (abrogated NFκB response to IL-1β, Figure 5B and C). Importantly, the response to polyI:C, which stimulates NFκB in an MyD88-independent fashion, was preserved in these cells. HEK293 and HEK293-MyD88si cells were co-transfected with Mal and p85α, respectively. Immunoprecipitation and western blot analysis of these constructs revealed that MyD88 expression was not a prerequisite for the interaction of Mal and p85α (Figure 5B). Furthermore, the only established transmembrane partners of Mal, TLR2 and 4 were not essential in this context, as the used HEK293 cells did not express either of these TLRs (data not shown). In full accordance with this finding, endogenous Mal and p85α interacted in response to TLR2 stimulation in an inducible fashion in MyD88-deficient macrophages (Figure 5D). Notably, the colocalization of both proteins was retarded in MyD88 deficient as compared with WT macrophages. Accordingly, although MyD88 is not required for TLR2-dependent PI3Kinase activation, it does influence the kinetics of both the physical Mal–p85α interaction and Akt phosphorylation (Figures 3E and 5D).

Next, we assessed the spatial distribution of the Mal–p85α complex. To this end, we exploited the observation that heterologous expression leads to ligand-independent interaction of both proteins (Figure 5B). Therefore, we stably expressed GFP-p85α in HEK293 cells and transiently transfected the resulting clonal HEK-GFP-p85 cell line with the fluorescent fusion construct CFP-Mal. As shown in Figure 5E, CFP-Mal is predominantly localized at the plasma membrane and in internal vesicle-like structures, whereas p85α seemed equally distributed throughout the cytoplasm in resting cells, in accordance with earlier reports ( Gillham va boshqalar, 1999 Kagan and Medzhitov, 2006 ). When both proteins were co-expressed in HEK-GFP-p85 cells, GFP-p85α co-localized with CFP-Mal at the plasma membrane.

The p85α regulatory subunit contains an amino-terminal and a carboxi-terminal Src homology domain 2 (SH2), both of which mediate the association with proteins containing phosphotyrosines in the context of YxxM/W sequence motifs. The so-called inter-SH2 domain binds to p110, the catalytic subunit of PI3K. Furthermore, an amino-terminal SH3 domain mediates the interaction of p85α with proteins containing PxxP motives. As the amino-terminus of Mal comprises various PxxP motives, we analysed whether the SH3 domain of p85α was critical for the interaction with Mal. We engineered a vector encoding for GFP-SH3 and expressed it together with Mal in HEK293 cells. In contrast to full-length p85α, the GFP-tagged SH3 domain of p85α did not colocalize with Mal (Figure 5E). The confocal-microscopy data were confirmed by immunoprecipitation analysis, in which GFP-SH3 was found not to be associated with Mal under conditions in which full-length p85α constitutively interacted with Mal (Figure 5F). Hence, the SH3 domain of p85α was structurally not sufficient for the interaction with Mal. Next we assessed, whether a change in the residue Pro125 of Mal disrupted the Mal–p85α association. Mal/Pro125 is an essential residue involved in Mal-Btk ( B ruton's t yrosine k inase) interaction and subsequent Mal phosphorylation on Tyr residues ( Piao va boshqalar, 2008). However, exchange of this residue by histidine (MalP125H Fitzgerald va boshqalar, 2001 ) did not affect the interaction of Mal with p85α. Hence, the SH3 domain of p85α was not sufficient and the Pro125 in Mal was not necessary for the formation of the Mal–p85α complex.

Mal mediates PIP3 generation and membrane ruffling at the leading edge

The presented data show that Mal is required for proper PI3K activation on TLR2 stimulation. PI3K has earlier been shown to mediate PIP3 generation and membrane ruffling at the leading edge of macrophages ( Evans va boshqalar, 2006 Lasunskaia va boshqalar, 2006). Accordingly, we wondered whether TLR2 ligands induced these morphological changes and whether Mal was a critical intermediate in this process.

First, we used a quantitative HPLC analysis to evaluate PIP3 accumulation. WT-, Mal- and MyD88-deficient macrophages were chased with 32 P and stimulated with FSL-1 for 90 min in the presence or absence of LY294002. Subsequently, the cells were lysed and, after lipid extraction and HPLC separation (Figure 6A and B), the levels of PIP3 were quantified. We found, in full accordance with the data presented above, that engagement of TLR2/6 induced the specific formation of PIP3 as compared with PIP2, and that both PI3K and Mal were essential in this process. However, as opposed to our data on Akt phosphorylation under these conditions, MyD88 was necessary for PIP3 shakllanishi. Hence, although Mal, but not MyD88, is required for PI3K activation by diacylated lipoproteins, both are necessary for PIP3 shakllanishi.

Next, we analysed whether engagement of TLR2 by lipoproteins led to macrophage polarization and assessed the roles of Mal and MyD88 in this context. For that reason, we transfected RAW264.7 macrophages with a plasmid construct, which encodes for the pleckstrin homology (PH) domain of Akt fused to GFP (GFP-PH). This domain recognizes and translocates to PIP3-rich domains in the plasma membrane ( Downward, 1998 ). As shown in Figure 7A, GFP-PH was homogeneously distributed throughout the cytoplasm in resting macrophages. In contrast, stimulation of TLR2 with diacylated lipoproteins (FSL-1, 90 min) led to progressive macrophage polarization, membrane ruffling and localization of GFP-PH at the plasma membrane of the leading edge. Notably, when RAW264.7 or WT macrophages were transfected with Mal (Figure 7A and data not shown), we observed a similar pattern of GFP-PH localization at the leading edge structure, which indicated that ligand-independent activation of Mal drives PIP3 formation and macrophage polarization. Furthermore, treatment of Mal-transfected macrophages with LY29400 inhibited cell polarization, which confirmed the involvement of PI3K in these processes. However, when Mal or MyD88-deficient macrophages were transfected with GFP-PH and were stimulated with FSL-1, accumulation of GFP-PH at the leading edge was abrogated (Figure 7B). Hence, Mal and MyD88 are both required for TLR2/6-mediated PI3K-dependent macrophage polarization and PIP3 formation, whereas only Mal is essential for Akt phosphorylation.


MUHOKAZA

OPN is expressed in chronic inflammatory diseases, which are often caused by Gram-positive bacteria, such as staphylococci, streptococci, mycobacteria, or by Gram-negative bacteria containing LPS variants that do not stimulate TLR4 (Bartonella spp., Xlamidiya spp., Helicobacter) [53, 54]. DCs were previously reported to produce conspicuous levels of OPN and to be present in mycobacterial and B. henselae granulomas, as well as in Helicobacter pylori-infected gastric epithelium [41, 55, 56]. Thus, it is reasonable to infer that DCs represent a source of OPN contributing to the inflammatory response. Here, we show that heat-killed, Gram-positive bacteria, mainly interacting with TLR2, boost OPN release by DC, whereas Gram-negative enterobacteria and other TLR3/TLR4 agonists do not. In line with these findings, the up-regulation of OPN production was also observed in the presence of nonenteric, Gram-negative bacteria, such as Bartonella spp. va Legionella, which contain LPS variants unable to activate TLR4 but mainly recognized by TLR2.

The host-pathogen interaction involves the simultaneous recognition of several microbial products, each one with specific agonist activities, and it is now clear that the combined activation of different receptors may result in complementary, synergistic, or antagonistic effects modulating innate and adaptive immunity. Numerous reports emphasize the synergy between different TLR signaling to enhance the expression of costimulatory molecules and the production of inflammatory cytokines [43, 44, 57, 58]. Nevertheless, growing evidence also demonstrates that simultaneous triggering of selected TLR may result in antagonism and cross-tolerance [59– 63]. Selective down-modulation of specific cytokines and chemokines by TLR cross-talk may be a mechanism evolved to regulate the DC response to pathogens and to prevent potential detrimental effects. Moreover, in certain situations, the activation of particular TLR responses might represent an escape mechanism implemented by some microorganisms.

In the present work, we show that in the presence of simultaneous triggering of TLR2 and TLR3/4, the induction of OPN is abrogated completely. The observation that the same effect is obtained when TLR2 is stimulated in the presence of CM from TLR3/4-stimulated DCs points to the existence of a soluble mediator(s) that may account for this regulation. Preliminary characterization demonstrated that the molecule is heat-labile and has a molecular mass >10 kDa. These results are in line with many reports that envisage autocrine cytokines, such as IFN-β, IL-10, and TGF [60, 61] as regulators of the TLR cross-talk. In our experimental setting, IFN-β appeared a particularly interesting candidate, as it is induced by TLR3 and TLR4 and is known to suppress OPN in human CD4 T cells [64]. Consistent with this report, we show that exogenous IFN-β inhibits TLR2-mediated OPN production in a dose-dependent manner. However, when DCs were treated with combinations of TLR2 and TLR3/TLR4 agonists, neither the neutralization of endogenous type I IFNs nor the blockade of IFNAR2 could rescue OPN secretion, demonstrating that type I IFN is not the response element accounting for TLR interference in our system. In addition, we also excluded a role for TGF-β, IL-10 the IL-10-related cytokine IL-29 (IFNλ1), and factors preferentially induced by TLR3 and/or TLR4, such as IFN-inducible protein 10 (IP-10), a proliferation-inducing ligand (APRIL), and B cell activating factor (BAFF) (data not shown). Further efforts are being dedicated to the purification and identification of this putative inhibitory molecule(s).

To gain insight into the mechanism leading to the release of the inhibitory factor(s), we investigated the importance of some steps in the TLR3/4 pathways. Our results indicate that OPN is preferentially induced by those TLR that signal exclusively through the MyD88 pathway. In keeping with this, OPN is up-regulated by IL-1β, which also signals via MyD88. Importantly, MyD88 silencing led to a significant decrease in OPN up-regulation in Mo-DCs following S. aureus stimulation, implying that MyD88 is a crucial component of this response.

On the contrary, TLR4 or TLR3 stimulation sharply limits the MyD88-dependent OPN production. Among TLR, TLR4 is unique for its ability to make use of two adaptor molecules, i.e., MyD88 and TRIF, whereas TLR3 uses TRIF exclusively. As dynasore and chloroquine, respectively, prevented the inhibitory effect of LPS and Poly I:C on TLR2-induced OPN, we propose that the signaling through the TRIF pathway blocks the MyD88-dependent OPN up-regulation. In line with these evidences, the inhibitory effect of LPS on TLR2-induced OPN was partially prevented by TRIF knockdown.

However, this is independent from TBK activation, as the TBK inhibitor BX795 did not rescue OPN production. These results suggest that the TLR3/4- and TRIF-dependent pathways leading to IFN-β induction and to the inhibition of MyD88-mediated OPN production bifurcate upstream of TBK-1, further excluding a role of an IFN-mediated, negative feedback loop. In general, there are very little data in the literature describing the suppressive role of TRIF on TLR/PRR signaling. Recently, Seregin et al. [63] showed that in vivo TRIF acts as a negative regulator of the TLR/MyD88 signal in multiple cell types (macrophages NK, B, and T cells and DCs), but the molecular mechanism remains unexplored, as well as the key question of whether TRIF-negative regulation is dependent on ligand binding. Another group showed that TRIF can suppress TLR5-mediated proinflammatory responses in intestinal epithelial cells by inducing the degradation of this receptor as well as that of TLR3, -6, -7, -8, -9, and -10 [5]. However, this is unlikely in our experimental system, as TLR2 and TLR1 circumvent the TRIF-mediated proteolytic degradation. Furthermore, it is important to stress here that the observed inhibitory effect is gene-specific, if not restricted to OPN, as other proinflammatory mediators, such as TNF-α, are induced in our experimental setting. In addition to TRAF3/TBK, TRIF associates with TRAF6 and receptor-interacting protein 1 (RIP-1) and with the help of TNFR1-associated death domain (TRADD) and TAK1, activates late-phase NF-κB and MAPKs [65]. This pathway, together with other yet-unidentified pathway(s), may thus be responsible for the induction of some inhibitory mediator(s), which in our view, must be OPN-specific. In addition to the release of soluble mediators, the engagement of selected TLR pairs may result in synergy or inhibition, also because of intracellular interference among signaling pathways or induction of molecular effectors. In keeping with this hypothesis, we are currently working at the identification and validation of OPN-binding microRNA that are induced upon simultaneous triggering of TLR2 and TLR3/4.

OPN is mainly studied as a secreted protein, but an iOPN was also identified [19, 66]. The characterization of iOPN is fairly advanced in the mouse system. Recent evidences indicate that iOPN is induced following TLR4 stimulation and negatively regulates TLR4-induced IFN-β production in murine macrophages [67]. Moreover, iOPN is involved in TLR9-dependent expression of IFN-α in plasmacytoid DCs [19] and in the downstream signaling of TLR2/dectin-1 pathways in the antifungal response [68]. Because of the role of iOPN in the regulation of TLR signaling pathways, it would be of great interest to evaluate the iOPN involvement in our experimental system. Unfortunately, the current understanding of iOPN expression and functions in human cells is still limited. Based on histology data from human tissues, cytoplasmic OPN is present in human cells, but it is not clear whether the alternative translation initiation site is the same in human and mice iOPN, and it is difficult to discriminate between iOPN and secreted OPN in human cells [66]. For this reason, we could not explore a possible involvement of iOPN in our experimental system.

In this study, we also provide evidence that the observed OPN modulation by different TLR agonists is biologically relevant and may play a critical role in the polarization of the immune response. In fact, we showed that the higher levels of OPN produced by Mo-DCs following stimulation with S. aureus, compared with E. coli, lead to a more robust response of CD4 + T cells in terms of IL-17 production. IL-17, a cytokine critical for the recruitment of phagocytes operating bacterial clearance, is mainly produced by Th17 cells, which have been shown recently to mediate many inflammatory and autoimmune diseases [69]. Moreover, OPN plays a critical role in the differentiation of Th17 cells in rheumatoid synovium [20]. A high expression of OPN regulating IL-17 production in the pathogenesis of hepatitis and acute coronary syndrome was reported [21, 22]. Nevertheless, the exact role of OPN in pathogen-induced human Th17 cells remains poorly defined. Our data indicate that a selective regulation of OPN by pathogens may influence a Th17 response. In line with our observation that TLR2 signaling can be an important mediator of Th17 cell responses, Kim et al. [70] reported that S. aureus can induce Th1 and Th17 inflammation, mainly in a TLR2-dependent manner, and TLR2 signaling has been shown to be an important molecular mediator of effective Th17 cell responses during Mikobakteriya tuberkulyozi infections [71]. In addition, recent data show that OPN represents an important regulatory factor of the protective Th17 immunity in Trypanosoma kruzi infection [72], in which TLR2 has a predominant, immunoregulatory role [73].

All in all, this study provides novel insights into the regulation of OPN production in Mo-DCs (represented in Fig. 7) and indicates that the differential production of OPN in response to TLR agonists may be relevant in shaping the pathogen-induced inflammatory response through the regulation of IL-17 secretion by CD4 + T cells.

Model for the contribution of the MyD88 and TRIF signaling pathways in the TLR-dependent OPN regulation in Mo-DCs. SF, Soluble factor.


Videoni tomosha qiling: texaschainsaw3d tlr1 h1080p (Iyul 2022).


Izohlar:

  1. Rypan

    It's easier to say than to do.

  2. Carvel

    Admin! Atigi 99 rubl uchun arzon domenni xohlaysizmi? Bu yoqqa keling!

  3. Tretan

    Menimcha, siz to'g'ri emassiz. Menga PM yozing, muhokama qilamiz.

  4. Saramar

    Kechirasiz, lekin menimcha, siz noto'g'risiz. Ishonchim komil. Men buni isbotlashga qodirman. Menga PM orqali yozing.

  5. Devlin

    Albatta, kechirim so'rayman, lekin bu menga umuman to'g'ri kelmaydi. Yana kim yordam bera oladi?

  6. Salvadore

    the ideal variant



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