Uticaj Sistemske Administracije Lipopolisaharida na Strukturne, Celularne i Molekularne Karakteristike Slezine miša

Abstract

Uvod: Lipopolisaharid (LPS) je glavna komponenta spoljašnje membrane Gram negativnih bakterija. Prepoznavanje LPS-a inicira intracelularne signalne puteve koji dovode do transkripcije gena i oslobaĊanja proinflamatornih medijatora. Dobro je poznato da kljuĉnu ulogu u imunskom odgovoru na LPS ima receptor TNFR1 (tumor necrosis factor receptor-1), kao i da TNFR1-/- (tumor necrosis factor receptor-1knockout) miševi pokazuju rezistenciju na endotoksiĉni šok ĉak i nakon tretmana letalnim dozama LPS-a. Dosadašnja istraţivanja su pokazala da LPS izaziva migraciju nekoliko ćelijskih tipova unutar slezine. MeĊutim, nema podataka o uticaju LPS-a na populaciju B i T limfocita koji se nalaze u crvenoj pulpi slezine miša. Nedavna studija istakla je znaĉaj hemokina CCL20 u brzoj akumulaciji B limfocita u slezini miša nakon sistemske administracije Nod1 agoniste, koji izaziva sistemsku inflamaciju in vivo.

Cilj:Ispitivanje uticaja sistemske administracije LPS-a na broj B i T limfocita u crvenoj pulpi slezine miša, kao i na strukturnu organizaciju bele pulpe slezine, 24 h nakon tretmana. Osim toga, cilj ove studije bio je da ispita uticaj sistemske administracije LPS-a na ekspresiju gena za hemokine i proinflamatorne citokine u slezini miša 1 h, 2 h i 24 h nakon tretmana. Dodatni cilj bio je da se ispitaju potencijalne promene u ekspresiji hemokina CCL20 u slezini miša 2 h nakon sistemske administracije LPS-a, kao i da se odredi lokalizacija ćelija koje bi u ovim uslovima proizvodile CCL20.

Materijal i metode:Eksperimenti su izvoĊeni na wild-type miševima soja C57BL/6 i TNFR1-/- miševima istog soja. Za ispitivanje uticaja sistemske administracije LPS-a na broj B i T limfocita u crvenoj pulpi slezine, kao i na strukturnu organizaciju bele pulpe slezine, formirane su ĉetiri grupe ţivotinja: netretirani wild-type miševi (n=5), tretirani wild-type miševi (n=5), netretirani TNFR1-/- miševi (n=5) i tretirani TNFR1-/- miševi (n=5). Tretman ţivotinja podrazumevao je ubrizgavanje u repnu venu LPS-a E. coli (soj 055:B5) rastvorenog u fosfatnom puferu, u dozi 2,5 μg/g telesne mase. Miševi su ţrtvovani 24 h nakon tretmana. Na kriostatskim iseĉcima slezine primenjene su imunohistohemijske metode (bojenje na B220, TCRβ i F4/80) i morfometrijska analiza. Za ispitivanje uticaja sistemske administracije LPS-a na ekspresiju gena za hemokine i proinflamatorne citokine u slezini, formirane su ĉetiri grupe ţivotinja: netretirani miševi (n=9), tretirani miševi ţrtvovani 1 h nakon intravenske injekcije LPS-a (n=6), tretirani miševi ţrtvovani 2 h nakon intravenske injekcije LPS-a (n=9) i tretirani miševi ţrtvovani 24 h nakon intravenske injekcije LPS-a (n=6). Kvantitativnom real-time PCR (qRT-PCR) analizom, u tkivu slezine ispitivana je relativna ekspresija gena 18 citokina/hemokina, 1 h, 2 h i 24 h nakon tretmana LPS-om. Za ispitivanje uticaja sistemske administracije LPS-a na ekspresiju gena za hemokin CCL20 u razliĉitim odeljcima tkiva slezine, kao i na lokalizaciju CCL20-produkujućih ćelija u slezini miša, korišćene su slezine 3 netretirana miša i 3 tretirana miša ţrtvovana 2 h nakon intravenske administracije LPS-a. Uzorci tkiva slezine isecani su laserskom mikrodisekcijom na tkivne odeljke u kojima je vršena qRT-PCR analiza. Lokalizacija CCL20-produkujućih ćelija u slezini ispitivana je primenom imunofluorescentnih metoda.

Rezultati: Dvadeset ĉetiri ĉasa nakon sistemske administracije LPS-a uoĉeno je znaĉajno smanjenje broja B i T limfocita u crvenoj pulpi slezine wild-type i TNFR1-/- miševa. Dodatno, 24 h nakon tretmana LPS-om, uoĉeno je znaĉajno povećanje procentualne zastupljenosti B ćelijske zone i bele pulpe, kao i smanjenje procentualne zastupljenosti marginalne zone u slezini wild-type i TNFR1-/- miševa. Rano nakon sistemske administracije LPS-a, qRT-PCR-a analiza je pokazala znaĉajno povećanje relativne ekspresije gena 11 hemokina i proinflamatornih citokina (Xcl1, Cxcl9, Cxcl10, Ccl3, Ccl4, Ccl5, Ccl17, Ccl20, Ccl22, Tnf i Lta) u slezini miša. Primenom laserske mikrodisekcije i sledstvenog qRT-PCR-a, detektovano je povećanje nivoa iRNK za CCL20 u beloj pulpi slezine miša 2 h nakon tretmana LPS-om. Imunofluorescentnom analizom slezine miša 2 h nakon tretmana LPS-om, utvrĊeno je da su CCL20- produkujuće ćelije smeštene u PALS-u, dok je izvestan broj manjih CCL20- produkujućih ćelija ravnomerno rasporeĊen u B ćelijskoj zoni.

Alternate abstract:

Introduction: Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram negative bacteria. Recognition of LPS initiates intracellular signaling pathways which lead to gene transcription and release of proinflammatory mediators. It is well known that the key role in the immune response to LPS has the receptor TNFR1(tumor necrosis factor receptor-1) and that TNFR1-/- (tumor necrosis factor receptor-1 knockout) mice show resistance to endotoxic shock even after treatment with lethal doses of LPS. Previous research has shown that LPS causes migration of several cell types within the spleen. However, there is no data on the effect of LPS on the population of B and T lymphocytes found in the red pulp of the mouse spleen. A recent study highlighted the importance of chemokine CCL20 in the rapid accumulation of B lymphocytes in the mouse spleen after systemic administration of Nod1 agonist, which induces systemic inflammation in vivo.

Aim:Examination of the effects of systemic administration of LPS on the number of B and T lymphocytes in the red pulp of the mouse spleen, as well as on the structural organization of the white pulp of the spleen, 24 hours after treatment. In addition, the aim of this study was to examine the effect of systemic administration of LPS on the expression of genes for chemokines and proinflammatory cytokines in the mouse spleen 1 h, 2 h, and 24 h after treatment. An additional goal was to investigate the potential changes in the expression of chemokine CCL20 in the mouse spleen 2 h after systemic administration of LPS, and to determine the localization of cells that might produce CCL20 in these conditions.

Material and methods:The experiments were performed on C57BL/6 wild-type and TNFR1-/- mice. In order to examine the effects of systemic administration of LPS on the number of B and T lymphocytes in the red pulp of the spleen, as well as on the structural organization of the splenic white pulp, four groups of animals were formed:untreated wild-type mice (n = 5), treated wild-type mice (n = 5), untreated TNFR1-/- mice (n = 5) and treated TNFR1-/- mice (n = 5). Treatment of animals implied injection into the tail vein of LPS E. coli (strain 055: B5) dissolved in phosphate buffer saline, at a dose of 2.5 μg/g of body weight. Mice were sacrificed 24 hours after the treatment. On the splenic cryostat sections immunohistochemical methods (staining for B220, TCRβ and F4/80) and morphometric analysis were applied. To investigate the effects of systemic LPS administration on gene expression of chemokines and proinflammatory cytokines in the spleen, four groups of animals were formed: untreated mice (n = 9), treated mice sacrificed 1 h after intravenous injection of LPS (n = 6), treated mice sacrificed 2 h after intravenous injection of LPS (n = 9) and treated mice sacrificed 24 h after intravenous injection of LPS (n = 6). Quantitative real-time PCR (qRT-PCR) analysis was used to examine the relative gene expression of 18 cytokines/chemokines in the splenic tissue, 1 h, 2 h, and 24 h after LPS treatment. To investigate the effects of systemic administration of LPS on the gene expression for chemokine CCL20 in different compartements of splenic tissue, as well as the localization of CCL20- producing cells in the mouse spleen, spleens of 3 untreated mice and 3 treated mice sacrificed 2 h after intravenous injection of LPS were used. Samples of splenic tissue were cut by laser microdisection on tissue compartments in which qRT-PCR analysis was performed. The localization of CCL20-producing cells in the spleen was examined using immunofluorescence methods.