Immunologic profile of experimental autoimmune encephalomyelitis

4 Český abstrakt Experimentální autoimunitní encefalomyelitida (EAE) je uznávána jako myší model lidského autoimunitního onemocnění roztroušené sklerózy. Myší EAE je aktivně indukována imunizací vhodným myelinovým antigenem. Po imunizaci dochází k akumulaci CD4+ pomocných T lymfocytů (angl. T helper cell) Th1 a Th17 v centrální nervové tkáni, které produkcí svých cytokinů zprostředkovávají zánětlivou reakci vedoucí k destrukci myelinu. Hlavním cílem této práce bylo navození EAE s klinicky pozorovatelnými příznaky a sledování změn v počtech a změně fenotypu buněk, především NK buněk a T lymfocytů. NK buňky na svém povrchu nesou širokou škálu inhibičních a aktivačních receptorů. V obou skupinách má své zastoupení skupina receptorů C-lektinové rodiny NKR-P1. Ligand aktivační formy NKR-P1C není dosud znám, z tohoto důvodu bylo sledováno jeho zapojení v EAE. Dalším cílem bylo použití léčiva s ohledem na zlepšení průběhu choroby. Pro účel diplomové práce byly nejprve použity dva inbrední kmeny myší lišící se expresí NKR-P1 receptoru na NK buňkách. SJL/J myši (exprimují inhibiční NKR­P1B) a C57BL/6 (exprimují aktivační NKR­P1C). U SJL myší byla pozorována střídavě recidivující encefalomyelitida a u C57BL/6 chronická forma EAE. U obou kmenů myší byly detekovány změny počtu NK buněk i v ...5 Anglický abstrakt Experimental autoimmune encephalomyelitis (EAE) is widely accepted as a murine model of human multiple sclerosis autoimmune disease. Murine EAE is usually actively induced by immunization with a suitable myelin antigen. Following immunization, CD4+ T helper lymphocytes Th1 and Th17 accumulate in the nervous tissue and via the production of cytokines, they mediate an inflammatory reaction and the subsequent destruction of myelin. The main goal of this study was the induction of EAE with clinically observable symptoms and the observation of changes in the counts and phenotypes of cells, mainly NK and T cells. NK cells express a wide range of inhibitory and activation receptors from the C-lectin-like receptor superfamily. The specific ligand of the activating NKR-P1C isoform is still unknown and thus this receptor's involvement in EAE was also observed. Another goal was the use of medication with regard to the disease progress improvement. For the purposes of this study, two inbred murine strains with distinct NKR-P1 surface expression were used - the SJL/J strain (expressing inhibitory NKR-P1B) and C57BL/6 (expression activating NKR-P1C). SJL mice elicited a relapse-remitting of EAE, while C57BL/6 had chronic EAE. Both mouse strains exerted changes in the counts of NK...Department of Cell BiologyKatedra buněčné biologieFaculty of SciencePřírodovědecká fakult

Immunologic profile of experimental autoimmune encephalomyelitis

5 Anglický abstrakt Experimental autoimmune encephalomyelitis (EAE) is widely accepted as a murine model of human multiple sclerosis autoimmune disease. Murine EAE is usually actively induced by immunization with a suitable myelin antigen. Following immunization, CD4+ T helper lymphocytes Th1 and Th17 accumulate in the nervous tissue and via the production of cytokines, they mediate an inflammatory reaction and the subsequent destruction of myelin. The main goal of this study was the induction of EAE with clinically observable symptoms and the observation of changes in the counts and phenotypes of cells, mainly NK and T cells. NK cells express a wide range of inhibitory and activation receptors from the C-lectin-like receptor superfamily. The specific ligand of the activating NKR-P1C isoform is still unknown and thus this receptor's involvement in EAE was also observed. Another goal was the use of medication with regard to the disease progress improvement. For the purposes of this study, two inbred murine strains with distinct NKR-P1 surface expression were used - the SJL/J strain (expressing inhibitory NKR-P1B) and C57BL/6 (expression activating NKR-P1C). SJL mice elicited a relapse-remitting of EAE, while C57BL/6 had chronic EAE. Both mouse strains exerted changes in the counts of NK..

Chlorophyll-Mediated Changes in the Redox Status of Pancreatic Cancer Cells Are Associated with Its Anticancer Effects

Nutritional factors which exhibit antioxidant properties, such as those contained in green plants, may be protective against cancer. Chlorophyll and other tetrapyrrolic compounds which are structurally related to heme and bilirubin (a bile pigment with antioxidant activity) are among those molecules which are purportedly responsible for these effects. Therefore, the aim of our study was to assess both the antiproliferative and antioxidative effects of chlorophylls (chlorophyll a/b, chlorophyllin, and pheophytin a) in experimental pancreatic cancer. Chlorophylls have been shown to produce antiproliferative effects in pancreatic cancer cell lines (PaTu-8902, MiaPaCa-2, and BxPC-3) in a dose-dependent manner (10–125 μmol/L). Chlorophylls also have been observed to inhibit heme oxygenase (HMOX) mRNA expression and HMOX enzymatic activity, substantially affecting the redox environment of pancreatic cancer cells, including the production of mitochondrial/whole-cell reactive oxygen species, and alter the ratio of reduced-to-oxidized glutathione. Importantly, chlorophyll-mediated suppression of pancreatic cancer cell viability has been replicated in in vivo experiments, where the administration of chlorophyll a resulted in the significant reduction of pancreatic tumor size in xenotransplanted nude mice. In conclusion, this data suggests that chlorophyll-mediated changes on the redox status of pancreatic cancer cells might be responsible for their antiproliferative and anticancer effects and thus contribute to the decreased incidence of cancer among individuals who consume green vegetables

Intestinal Microbiota Promotes Psoriasis-Like Skin Inflammation by Enhancing Th17 Response.

Psoriasis is a chronic inflammatory skin disease in which Th17 cells play a crucial role. Since indigenous gut microbiota influences the development and reactivity of immune cells, we analyzed the link among microbiota, T cells and the formation of psoriatic lesions in the imiquimod-induced murine model of psoriasis. To explore the role of microbiota, we induced skin inflammation in germ-free (GF), broad-spectrum antibiotic (ATB)-treated or conventional (CV) BALB/c and C57BL/6 mice. We found that both mice reared in GF conditions for several generations and CV mice treated with ATB were more resistant to imiquimod-induced skin inflammation than CV mice. The ATB treatment dramatically changed the diversity of gut bacteria, which remained stable after subsequent imiquimod application; ATB treatment resulted in a substantial increase in the order Lactobacillales and a significant decrease in Coriobacteriales and Clostridiales. Moreover, as compared to CV mice, imiquimod induced a lower degree of local and systemic Th17 activation in both GF and ATB-treated mice. These findings suggest that gut microbiota control imiquimod-induced skin inflammation by altering the T cell response

Absence of microbiota or ATB treatment reduces the production of pro-inflammatory cytokines by T cells from IMQ-treated mice.

<p>Cells isolated from spleen or axillary lymph nodes of (a) GF and CV mice or (b) ATB-treated and control mice were stimulated for 48hours <i>in vitro</i> by plate-bound anti-CD3 antibody and soluble anti-CD28 antibody. Cell culture supernatants were analyzed for IL-17A and INF-γ by ELISA. These data are representative of three independent experiments (n = 5–8 mice per group) with similar results. Statistical significance was determined by unpaired Student t test; *p < 0.05, **p < 0.01.</p

Absence of microbiota or ATB treatment decreases the percentage of γδ T cells and Th17 cells in spleen or axillary lymph nodes of IMQ-treated mice.

<p>Cells from spleen or axillary lymph nodes were isolated and either analyzed for surface γδTCR or intracellular RORγt or stimulated <i>in vitro</i> for 8 h by PMA and Ionomycin, the last 4h in the presence of Brefeldin A and Monensin, and analyzed for intracellular IL-17A and IFNγ production by flow cytometry. Cells were first gated for live cells and CD3⁺ and subsequently on γδTCR⁺, RORγt⁺ or IL-17⁺ and INFγ⁺ cells as shown on the (a) example of gating strategy. These results from one representative experiment (n = 5–8 mice per group) out of three independent experiments with (b) GF versus CV mice or with (c) ATB-treated versus control mice are quantified in the graphs. Statistical significance was determined by unpaired Student t test; *p < 0.05, **p < 0.01.</p

Antibiotic treatment changes the microbiota composition in both BALB/c and C57BL/6 mice.

<p>(a) Comparison of diversity in microbiota between the conventional (CV) and ATB-treated mice using Shannon diversity index. (b) Principal coordinates analysis (PCoA) plot using the unweighted UniFrac distance metric shows the compositional similarity before and after ATB treatment (Day 0, Day 14, Day 21). Each colored orb represents the microbiota composition in feces of one mouse. Each color represents each group of mice at the day 0, 14, 21. (c) The microbial composition in time is displayed as mountain plot before (Day 0) and after ATB treatment (Day 14), and after the induction of skin inflammation (Day 21) in comparison to CV mice. The figure shows (a) means ± SD or (c) means from pool of 5 mice. Statistical significance was determined by unpaired Student t test; *p < 0.05, **p < 0.01.</p

Antibiotic treatment decreases the skin inflammation in both BALB/c and C57BL/6 mice.

<p>Quantification of ear (a) and skin (b) thickness. Quantification of histopathological score (0–2) after H&E staining of the ear (c) a skin (d). The graphs show 18–19 mice per group (a pool of three independent experiments). Statistical significance was determined by unpaired Student t test; *p < 0.05, **p < 0.01.</p

Disease severity grading using light microscopy.

<p>Disease severity grading using light microscopy.</p

Microbiota enhances sensitivity to IMQ-induced skin inflammation in BALB/c and C57BL/6 mice.

<p>(a) Representative macroscopic pictures of healthy (control) and inflamed (IMQ) ear or dorsal skin of BALB/c mice, illustrating the disease severity. Quantification of skin and ear thickness of BALB/c (b) and C57BL/6 (e) mice. Quantification of histopathological score (0–2) after H&E staining of the ear and skin of BALB/c (c) a C57BL/6 (f) mice. (d) Representative H&E-stained ear and skin sections of BALB/c mice (scale bar, 100 μm). The values represent means ± SD as a pool of three independent experiments (n = 10–20 mice per group). *p < 0.05, ***p < 0.001.</p