Intestinalni i sistemski imunski efekti oralnog unosa kadmijuma kod pacova

Abstract

Kadmijum (Cd) je teški metal koji se nalazi u svim delovima životne sredine, nema poznatu biološku funkciju i ima štetno dejstvo na žive sisteme. Najčešći put izloženosti Cd je oralni, preko kontaminirane vode i hrane, kada je primarna meta toksičnosti ovog metala gastrointestinalni trakt. Poznato je da Cd oštećuje tkivo creva i remeti funkciju epitelne barijere koja je neophodna za održavanje imunske homeostaze, međutim mehanizmi imunotoksičnosti u ovoj regiji nisu dovoljno ispitani. Dodatno, poznato je da toksični efekti Cd mogu da zavise i od genetske osnove / soja eksperimentalnih životinja, ali sojne razlike u intestinalnoj toksičnosti oralnog unosa Cd do sada nisu ispitane. Ova disertacija je imala za cilj karakterizaciju efekta subhronične oralne primene Cd na imunski sistem creva pacova. Pacovi su bili 30 dana oralno (u vodi za piće) izloženi Cd u obliku kadmijum hlorida (CdCl2) u koncentraciji 5 ppm (5 mg Cd/l) i 50 ppm (50 mg Cd/l) Cd, što odgovara dozama prisutnim u životnoj sredini. U okviru lokalnog imunomodulatornog efekta Cd ispitivani su osnovni parametri imunskog odgovora u duodenumu (kao mestu najveće apsorpcije Cd) i mezenteričnim limfnim čvorovima (MLČ) koji dreniraju intestinum. U duodenumu su ispitani pokazatelji tkivnog oštećenja, oksidativnog stresa i zapaljenskih promena, a u MLČ su ispitane osnovne fenotpiske karakteristike i parametri aktivnosti ćelija ovog limfnog tkiva (celularnost, proliferacija, citokinski odgovor, urođeno-imunska aktivnost ćelija). Pored lokalnog, ispitan je i sistemski odgovor na oralni unos Cd uključujući humoralne i ćelijske parametre zapaljenske reakcije u krvi (promene hematoloških parametara, prisustvo medijatora inflamacije i oksidativnog stresa), kao i oksidativni stres i osnovne karakteristike urođenog i adaptivnog imunskog odgovora u slezini, limfnom organu u kome se uspostavlja imunski odgovor na antigene iz krvi. U cilju ispitivanja uticaja genetske osnove na intestinalnu i sistemsku imunotoksičnost oralne izloženosti Cd, lokalni i sistemski efekti su analizirani kod dva soja pacova, Dark Agouti (DA) i Albino Oxford (AO), koji uspostavljaju kvalitativno i / ili kvantitativno različit imunski odgovor na iste stimuluse. Studija je pokazala da oralni tretman Cd dovodi do dozno zavisne akumulacije metala u crevu, MLČ i slezini, slično kod DA i AO pacova, što pokazuje da genetska osnova ne utiče na nivoe deponovanog metala. I pored sličnih koncentracija Cd u crevu oba soja, izraženiji infiltrat mononuklearnih leukocita u tkivu creva, veći stepen nekroze enterocita [meren povećanjem markera nekroze HMGB1 (engl., High Mobility Group Box 1) molekula] i povećana produkcija proinflamatornih citokina (faktora nekroze tumora/TNF, interferona-gama/IFN-γ, interleukina-17/IL-17) pokazani su u tkivu creva DA pacova u odnosu na pacove AO soja. Promena aktivnosti osnovnih enzima antioksidativne odbrane (superoksid dismutaze/SOD i katalaze/CAT), u crevu DA pacova i SOD kod AO pacova, koja predstavlja pokušaj domaćina da ukloni reaktivne kiseonične vrste (engl., Reactive Oxygen Species/ROS) i ograniči oštećenje tkiva, kao i glutation-s-transferaze/GST (enzima odgovornog za vezivanje elektrofilnih supstanci kao što je Cd za redukovani glutation, GSH) bila je takođe izraženija kod pacova DA soja. Ovakav tip promena je verovatno u osnovi povećanja nivoa lipidnih peroksida (mereno promenama malondialdehida/MDA) kod AO pacova. Oralni tretman Cd dovodi do smanjene zastupljenosti laktobacila u crevu DA pacova što može doprineti razvoju proinflamatornog citokinskog odgovora na Cd, dok relativno očuvanje komensalne flore kod AO pacova (uz značajan porast zastupljenosti L. johnsonii i L. murinus) može biti od značaja za očuvanje integriteta crevne barijere i izostanak lokalnog inflamatornog citokinskog odgovora kod ovog soja pacova. Oralni tretman Cd je uticao na aktivnost MLČ kod oba soja pacova (porast mase i celularnosti MLČ, produkcija ROS). Ipak, Cd je indukovao ekspresiju gena za metalotioneine/MT kao i proinflamatorni imunski odgovor (proliferativna aktivnost ćelija MLČ, oksidativne aktivnosti, Th1/Tip1 i Th17/tip17 odgovor) i inhibirao antiinflamatorni odgovor (ekspresija gena i produkcija interleukina-10/IL-10) samo u MLČ DA pacova. Veći stepen oštećenja i nekroze epitela creva koji je zapažen kod tretiranih DA (u odnosu na AO pacove) je najverovatniji faktor koji je doveo do stimulacije proinflamatornog odgovora u MLČ kod DA pacova. Promene u MLČ DA pacova ukazuju da Cd remeti tolerogenu imunsku sredinu u crevu indukcijom kako urođenog tako i stečenog imunskog odgovora. Iako je ispitivanjem sistemskog efekta oralno unetog Cd ukazano, generalno, na slab uticaj metala na osnovne hematološke i biohemijske parametare u perifernoj krvi, zapažen je efekat na antioksidativne aktivnosti eritrocita kod oba soja pacova. Povećani nivoi HMGB1 molekula u krvi samo DA pacova sugerišu veći stepen oštećenja organa i inflamatorni karakter imunskog odgovora na Cd kod ovog soja pacova. Iako se ista količina Cd deponuje u MLČ i slezini, odgovor u ova dva imunska organa se razlikuje čime je pokazano da je za praćenje efekata Cd od velike važnosti i mikrosredina ispitivanog tkiva. Za razliku od MLČ, u slezini oralni tretman Cd nije uticao na masu i celularnost ali je doveo do smanjene vijabilnosti i smanjene proliferativne aktivnosti ćelija slezine, dok je slično kao i u MLČ, zabeležena povećana oksidativna aktivnost ćelija slezine (aktivnost mijeloperoksidaze/MPO, produkcija azotoksida/ NO) i povećana nestimulisana produkcija proinflamatornih citokina (interleukina-1 beta/IL-1ß, IFN-γ, IL-17) i to samo kod DA pacova. Ovaj proinflamatorni citokinski odgovor ćelija slezine kod DA pacova može se dovesti u vezu sa povećanim nivoom HMGB1 molekula u slezini. Izraženiji proinflamatorni odgovor na oralni tretman Cd u crevu kao i u MLČ i slezini DA u odnosu na AO pacove (kod kojih su nađene slične koncentracije deponovanog Cd) pokazuju da su DA pacovi podložniji oralnom subhroničnom tretmanu ovim metalom. Rezultati ove studije doprinose razumevanju mehanizama koji leže u osnovi toksičnih efekata Cd na imunski odgovor u crevu, pokazujući po prvi put uticaj ovog metala na homeostazu urođenih i adaptivnih komponenti imunskog odgovora u MLČ. Ona je istovremeno pokazala i značaj tkiva u ispoljavanju imunotoksičnosti Cd. Takođe, ova studija je po prvi put ukazala na uticaj genetske osnove na efekte oralno unešenog Cd, sugerišući na značaj pažljivog odabira soja eksperimentalnih životinja koji će se koristiti za analizu imunotoksičnih efekata ovog metala.

Cadmium (Cd) is a heavy metal which is found in every part of the environment, it does not have any known biological function and has an adverse effect upon the living systems. The most common way of Cd exposure is orally, through contaminated water and food, where the prime target of this metal toxicity is the gastrointestinal tract. It is known that Cd causes damage to intestinal tissue and disrupts the epithelial barrier function which is necessary for maintaining immune homeostasis, however, the immunotoxicity mechanisms in this region have not been examined sufficiently. Additionally, it is known that Cd toxic effects may depend also on genetic background / strain of experimental animals, but the strain-dependent differences in intestinal toxicity of oral Cd intake have not been examined to date. This dissertation was aimed at characterization of the effect of subchronic oral Cd administration on rat’s intestinal immune system. Rats were, for 30 days, orally (in drinking water) exposed to Cd in the form of cadmium chloride (CdCl2) at concentration of 5 ppm (5 mg Cd/l) and 50 ppm and (50 mg Cd/l) of Cd, which corresponds to the doses present in the environment. Within the local immunomodulatory Cd effect, basic parameters of immune response in duodenum (region of greatest Cd absorption) and in mesenteric lymph nodes (MLN) which drain intestine, were investigated. The indicators of tissue damage, oxidative stress and inflammatory changes were tested in duodenum, whereas in the MLN basic phenotype characteristics and parameters of this lymph tissue cells' activities (cellularity, proliferation, cytokine responses, and innate-immune cell activity) were tested. Besides the local, the systemic response to oral Cd intake was tested as well, including humoral and cellular parameters of the inflammatory reaction in blood (changes in hematological parameters, presence of inflammatory mediators and oxidative stress), as well as oxidative stress and basic characteristics of the innate and adaptive immune response in the spleen, a lymph organ in which immune response to blood borne antigens are generated. Aiming to test the contribution of genetic background to intestinal and systemic immunotoxicity of oral Cd exposure, local and systemic effects were analyzed in two rat strains, Dark Agouti (DA) and Albino Oxford (AO), which establish qualitatively and/or quantitative different immune response to the same stimuli. The study has shown that oral Cd treatment leads to dose-dependent accumulation of this metal in intestine, MLN and spleen, similarly in DA and AO rats, which demonstrates that genetic background has no effect on the metal deposition levels. Despite similar Cd concentrations in the intestine of both rat strains, more pronounced mononuclear leukocytes infiltrates in intestinal tissue, higher degree of enterocytes necrosis [measured by increased necrosis marker HMGB1 (High mobility group box 1) molecule] and the increased production of proinflammatory cytokines (tumor necrosis factor/TNF, interferone-gamma/IFN-γ, interleukin-17/IL-17) were shown in intestinal tissue of DA rats compared to rats of AO strain. The change in activity of basic enzymes of anti-oxidative defense (superoxide dismutase/SOD and catalase/CAT), in intestine of DA rats and SOD in AO rats, which depicts host tissue attempt to counterattack reactive oxygen species (ROS) and limit tissue damage, as well as glutathione-s-transferase/GST (the enzyme responsible for electrophilic substances binding, such as Cd, to the reduced form of glutathione, GSH), was also more expressed in rats of DA strain. Such type of changes is probably in the background of the increased level of lipid peroxides (measured by the changes of malondialdehyde/MDA) in AO rats. Oral Cd treatment results in reduced prevalence of lactobacilli in DA rats' intestine which may contribute to the development of proinflammatory cytokine response to Cd, whereas relative preservation of commensal flora in AO rats (along with significant increase in prevalence of L. johnsonii and L. Murinus) might be of significance for preservation of gut barrier integrity and absence of the local inflammatory cytokine response in this strain of rats. Oral Cd treatment affected the MLN activity in both strains of rats (increase of MLN mass and cellularity, ROS production). Nevertheless, Cd induced metalotioneinas/MT gene expression as well as proinflammatory immune response (MLN cells' proliferative activity, oxidative activities, Th1/Type1 and Th17/Type17 response) and inhibited anti-inflammatory response (interleukine-10/IL-10 gene expression and production) only in MLN of DA rats. Higher degree of intestinal epithelium damage and necrosis observed in DA treated rats (compared to AO rats) is the most likely factor which stimulated proinflammatory response in MLN of DA rats. The changes in MLN of DA rats indicate that Cd disrupts the tolerogenic immune environment in the gut by induction of both innate and the acquired immune responses. Although the investigation of the systemic effect of orally administered Cd indicated, in general, slight effect of the metal on the basic hematological and biochemical parameters in the peripheral blood, the effect on anti-oxidative activities of erythrocytes in both strains of rats was noticed. The increased levels of HMGB1 molecule in blood of DA rats only, suggests higher degree of organs damage and inflammatory character of immune response to Cd in this strain of rats. Although the same quantity of Cd is deposited in MLN and spleen, the response in these two immune organs differs, showing that the micro environment of the examined tissue is also of great importance in observing Cd effects. Unlike MLN, the oral Cd administration did not affect spleen mass and cellularity but it resulted in reduced viability and reduced proliferative activity of spleen cells, whereas, similar as in MLN, the increased oxidative activity of spleen cells (activity of myeloperoxidase/MPO, nitrogen-oxide/NO production) was noted and the increase of non-stimulated production of proinflammatory cytokines (interleukine-1 beta/ IL-1ß, IFN-γ, IL-17) was noted, only in DA rats. This proinflammatory cytokine response of spleen cells in DA rats may be associated with increased level of HMBG1 molecule in the spleen. More pronounced proinflammatory response to oral Cd administration in gut as well as in MLN and spleen of DA rats in comparison to AO rats (in which similar concentrations of deposited Cd were found) show that DA rats are more susceptible to oral subchronic administration of this metal. The results of this study contribute to the understanding of the mechanisms which underlie the toxic Cd effects on immune response in intestine, for the first time showing the effect of this metal on homeostasis of innate and adaptive components of immune response in MLN. At the same time, this study has also shown the significance of tissue in expressing Cd toxicity. Also, for the first time, this study has indicated the effect of genetic background on orally administered Cd effects, suggesting the significance of careful selection of experimental animals to be used in the analysis of immunotoxic effects of this metal.