Immunomodulatory activity of recombinant banana lectin isoform in large intestine of BALB/c mice under physiological and pathological conditions

Lektin banane (BanLec) pripada podgrupi lektina koji vezuju glukozu ili manoza- u okviru familije lektina sličnih žakalinu. U plodu banane, koji predstavlja prirodni izvor BanLec-a, javlja se u više izoformi, a može se proizvesti i rekombinantnom DNK tehnologijom. Rekombinantna izoforma BanLec-a (rBanLec) je po svojim strukturnim karakteristikama i specifičnosti vrlo slična prirodnim izoformama. Kao i većina biljnih lektina, (r)BanLec ne podleže brzo denaturaciji / razgradnji u uslovima digestivnog trakta, a pokazano je da se vezuje za epitel tankog creva i postepeno prolazi u subepitelni prostor. Danas je poznato da BanLec, nezavisno od izoforme, ima imunomodulatorni potencijal. Dosadašnja istraživanja su se dominantno bavila uticajem (r)BanLec-a na funkcionalne karakteristike limfocita i pokazano je da stimulacije određene ćelijske populacije prirodnim i rekombinantnom izoformom kvalitativno imaju isti ishod. Uticaj (r)BanLec-a na funkcionalne karakteristike drugih ćelija imunskog sistema nije detaljno analiziran. Cilj ove doktorske teze je da se kroz ispitivanja (1) modulatornog dejstva rBanLeca na funkcionalne karakteristike antigen-prezentujućih ćelija (APĆ) BALB/c miša, (2) imunomodulatornog dejstva rBanLec-a u debelom crevu BALB/c miša u fiziološkim uslovima, i (3) efekata profilaktičke i terapijske primene rBanLec-a u modelu TNBS-om indukovanog kolitisa kod BALB/c miša, dobije uvid u imunomodulatorni efekat rBanLec-a na imunski sistem u mukozi debelog creva miševa BALB/c soja pod specifičnim uslovima. Koristeći peritonealne makrofage i dendritske ćelije slezine (DĆ) kao in vitro model sisteme, pokazano je da rBanLec dozno-zavisno moduliše funkcionalne karakteristike APĆ BALB/c miševa. Analiza uticaja rBanLec stimulacije na karakteristike rezidentnih (RM) i tioglikolatom-indukovanh (TGM) peritonealnh makrofaga, pokazala je da ishod stimulacije određene ćelijske populacije nije jednoznačan već zavisi od njihovog funkcionalnog stanja. rBanLec na pozitivan dozno-zavisan način pospešuje proinflamatorni kapacitet RM (povećanje produkcije NO i IL-12 i smanjenje aktivnosti arginaze i sekrecije IL-4), a na isti način smanjuje proinflamatorni kapacitet TGM (smanjenje produkcije NO i IL-12 i povećanje aktivnosti arginaze i sekrecije IL-4). Uticaj rBanLec-a na produkciju TGF-β kod peritonealnih makrofaga takođe zavisi od funkcionalnog stanja makrofaga (kod RM, rBanLec negativno dozno-zavisan, a kod TGM pozitivno dozno-zavisno utiče na produkciju TGF-β). I kod RM i kod TGM, rBanLec na pozitivan dozno-zavisan način podstiče aktivnost mijeloperoksidaze (MPO), produkciju TNF-α i IL-10. Za stimulaciju produkcije proinflamatornih medijatora, nezavisno od funkcionalnog stanja makrofaga, značajno je vezivanje rBanLec-a za TLR2 i TLR4 / CD14. rBanLec utiče na sekreciju efektorskih citokina IFN-γ i IL-4 (negativno dozno-zavisno) i regulatornog citokina IL-10 (pozitivno dozno-zavisno) od strane DĆ slezine. rBanLec TLR2-posredovana stimulacija DĆ slezine nije ključna za promene u ekspresiji Ifn-γ i Il-4...Banana lectin (BanLec) is primarily isolated from the fruit of banana (Musa paradisiac). It is glucose / mannose-specific lectin that belongs to the jackalin superfamily of lectins. There are several naturally occurring BanLec isoforms. Nowadays BanLec can also be produced by recombinant technology (rBanLec). rBanLec possesses structural and functional characteristics that highly resemble those reported for its natural counterparts. As most plant lectins, (r)BanLec is resistant to denaturation/proteolytic cleavage in the gastrointestinal tract. It has been reported that rBanLec attaches specifically to the mucosal surface of small intestine and passes into subepithelial compartment. (r)BanLec has been recognized as a potential immunomodulator. It has been shown that (r)BanLec modulates functional characteristics of lymphocytes but the effects of (r)BanLec stimulation in other immune cells are not yet elucidated. The aim of this study was to investigate immunomodulatory / immunostimulatory activity of rBanLec in the colon of BALB/c mice. This aim was accomplished through evaluation of the impact of rBanLec stimulation on 1) functional characteristics of antigen presenting cells (APC) isolated from BALB/c mice 2) local immune system in the large intestine of healthy BALB/c mice, and 3) the course of TNBS-induced experimental colitis in BALB/c mice. Peritoneal macrophages and spleen dendritic cells (DC) isolated from BALB/c mice were used in vitro for the evaluation of rBanLec influence on functional characteristics of APC. Generally, it is shown that rBanLec in a dose-dependent manner modulated the functional characteristics of APCs. By using resident (RMs) and thioglycollate-elicited (TGMs) peritoneal macrophages, it has been shown that effects of rBanLec stimulation depend on its concentration but also on the functional status of macrophages. Obtained results have clearly shown that rBanLec, in a positive dosedependent manner, promotes pro-inflammatory phenotype with BALB/c RMs (enhancement of NO and IL-12, reduction of arginase activity and IL-4 secretion) while, in the same manner, it tends to skew BALB/c TGMs towards anti-inflammatory profile (reduction of NO and IL-12 production, enhancement of arginase activity and IL-4 secretion). The dose-dependent changes in production of TGF-β by peritoneal macrophages also depended on their functional status: rBanLec stimulated the production of TGF-β by RMs in a negative dose-dependent manner, while in TGMs production of TGF-β positively correlated to the rBanLec concentration. The activity of myeloperoxidase (MPO) and productions of TNF-α and IL-10 were enhanced upon rBanLec stimulation in a positive dose-dependent manner with both RMs and TGMs. Further, it was shown that interactions of rBanLec with TLR2 and TLR4 / CD14 are important for initiations of the production of pro-inflammatory mediators by peritoneal macrophages irrespective to their functional status. rBanLec also stimulated in a specific dose-dependent manner the secretion of effector cytokines IFN-γ and IL-4 (negative dose dependent manner) and regulatory cytokine IL-10 (positive dose-dependent manner) by spleen DCs..