High molecular weight components containing N-linked oligosaccharides of Ascaris suum extract inhibit the dendritic cells activation through DC-SIGN and MR

Helminths, as well as their secretory/excretory products, induce a tolerogenic immune microenvironment. High molecular weight components (PI) from Ascaris suum extract down-modulate the immune response against ovalbumin (OVA). The PI exerts direct effect on dendritic cells (DCs) independent of TLR 2, 4 and MyD88 molecule and, thus, decreases the T lymphocytes response. Here, we studied the glycoconjugates in PI and the role of C-type lectin receptors (CLRs), DC-SIGN and MR, in the modulation of DCs activity. Our data showed the presence of glycoconjugates with high mannose- and complex-type N-linked oligosaccharide chains and phosphorylcholine residues on PI. In addition, these N-linked glycoconjugates inhibited the DCs maturation induced by LPS. The binding and internalization of PI-Alexa were decreased on DCs previously incubated with mannan, anti-DC-SIGN and/or anti-MR antibodies. In agreement with this, the incubation of DCs with mannan, anti-DC-SIGN and/or anti-MR antibodies abolished the down-modulatory effect of PI on these cells. It was also observed that the blockage of CLRs, DC-SIGN and MR on DCs reverted the inhibitory effect of PI in in vitro T cells proliferation. Therefore, our data show the involvement of DC-SIGN and MR in the recognition and consequent modulatory effect of N-glycosylated components of PI on DCs.Fil: Favoretto, Bruna C.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Casabuono, Adriana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Portes Junior, José A.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Jacysyn, Jacqueline F.. Universidade de Sao Paulo; BrasilFil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Faquim Mauro, Eliana L.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; Brasi

Crotoxin from <i>Crotalus durissus terrificus</i> Is Able to Down-Modulate the Acute Intestinal Inflammation in Mice

<div><p>Inflammatory bowel diseases (IBD) is the result of dysregulation of mucosal innate and adaptive immune responses. Factors such as genetic, microbial and environmental are involved in the development of these disorders. Accordingly, animal models that mimic human diseases are tools for the understanding the immunological processes of the IBD as well as to evaluate new therapeutic strategies. Crotoxin (CTX) is the main component of <i>Crotalus durissus terrificus</i> snake venom and has an immunomodulatory effect. Thus, we aimed to evaluate the modulatory effect of CTX in a murine model of colitis induced by 2,4,6- trinitrobenzene sulfonic acid (TNBS). The CTX was administered intraperitoneally 18 hours after the TNBS intrarectal instillation in BALB/c mice. The CTX administration resulted in decreased weight loss, disease activity index (DAI), macroscopic tissue damage, histopathological score and myeloperoxidase (MPO) activity analyzed after 4 days of acute TNBS colitis. Furthermore, the levels of TNF-α, IL-1β and IL-6 were lower in colon tissue homogenates of TNBS-mice that received the CTX when compared with untreated TNBS mice. The analysis of distinct cell populations obtained from the intestinal lamina propria showed that CTX reduced the number of group 3 innate lymphoid cells (ILC3) and Th17 population; CTX decreased IL-17 secretion but did not alter the frequency of CD4⁺Tbet⁺ T cells induced by TNBS instillation in mice. In contrast, increased CD4⁺FoxP3⁺ cell population as well as secretion of TGF-β, prostaglandin E₂ (PGE₂) and lipoxin A₄ (LXA₄) was observed in TNBS-colitis mice treated with CTX compared with untreated TNBS-colitis mice. In conclusion, the CTX is able to modulate the intestinal acute inflammatory response induced by TNBS, resulting in the improvement of clinical status of the mice. This effect of CTX is complex and involves the suppression of the pro-inflammatory environment elicited by intrarectal instillation of TNBS due to the induction of a local anti-inflammatory profile in mice.</p></div

CD4⁺Tbet⁺ cell population and IFN-γ secretion of mice with acute colitis induced by TNBS treated or not with CTX.

<p><b>(A)</b> Representative dot plots of CD4⁺Tbet⁺ cells in the lamina propria of distinct group of mice. <b>(B)</b> CD4⁺Tbet⁺ cells were expressed as a mean of the absolute number of cells ± SEM. The samples were prepared from a pool of cells from 4–5 animals/group performed in duplicate. The results are from 3 independent experiments. <b>(C)</b> Secretion of IFN-γ in colonic tissue homogenates determined by ELISA. The results represent the mean obtained in the individual samples/group ± SEM. * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001; (n = 4–5 animals/group).</p

Secretion of pro-inflammatory cytokines in colonic tissue homogenates of TNBS-mice treated or not with CTX.

<p>Production of IL-1β (A), IL-6 (B) and TNF-α (C) was measured in homogenates of colonic segments by ELISA. The results represent the mean of the cytokine secretion in individual mice/group ± SEM. * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001 (n = 4–5 animals/group).</p

Effect of CTX on Th17 cells, ILC3 and IL-17 secretion of mice with acute colitis induced by TNBS.

<p>Cell suspensions were prepared from lamina propria of distinct experimental groups after 4 days of TNBS-induced colitis and that received or not the CTX. The TCRß⁺CD4⁺RORγt⁺, TCRß⁺CD4⁺IL-17⁺ and Lin^-CD90⁺IL-17⁺ cells were analyzed by flow cytometry. <b>(A, B and C)</b> Strategy for the analysis of TCRß⁺CD4⁺RORγt⁺ and TCRß⁺CD4⁺IL-17⁺ cells in the lamina propria obtained from each group of mice. The results of TCRß⁺CD4⁺RORγt⁺<b>(D)</b> and TCRß⁺CD4⁺IL-17⁺ cells <b>(E)</b> expressed as the mean of the absolute number ± SEM. The samples were prepared from a pool of cells from 4–5 animals/group performed in duplicate. The results are from 2–3 independent experiments. <b>(F)</b> Strategy for the analysis of the Lin^-CD90⁺IL-17⁺ cells. <b>(G)</b> The results of Lin^-CD90⁺IL-17⁺ cells were expressed as a mean of the absolute number ± SEM. The samples were prepared from a pool of cells from 4–5 animals/group performed in duplicate. The results are from 2 independent experiments. <b>(H)</b> Secretion of IL-17 in colonic tissue homogenates determined by ELISA. The results represent the mean obtained in the individual samples/group ± SEM. * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001 (n = 4–5 animals/group).</p

Analysis of CD4⁺FoxP3⁺ cells and anti-inflammatory mediators in TNBS-mice treated or not with CTX.

<p>Cell suspensions were prepared from lamina propria of distinct experimental groups after 4 days of TNBS-induced colitis for the analysis of CD4⁺FoxP3⁺ cells by flow cytometry. The samples were prepared from a pool of cells from 4–5 animals/group performed in duplicate. The results are from 2 independent experiments. <b>(A)</b> Representative dot plots of CD4⁺FoxP3⁺ cells in the lamina propria obtained from distinct experimental groups. <b>(B)</b> Results of CD4⁺FoxP3⁺ cells expressed as a mean of the absolute number of cells in duplicate of 2 independent experiments ± SEM. Secretion of TGF-β <b>(C)</b> and IL-10 <b>(D)</b> in colonic tissue homogenates determined by ELISA. Production of PGE₂<b>(E)</b> and LXA₄<b>(F)</b> was performed by ELISA in colonic tissue homogenates. The results represent the mean obtained in individual mice/group ± SEM. * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001; (n = 4–5 animals/group).</p

Effect of CTX treatment on the colitis induced by TNBS instillation in BALB/c mice.

<p><b>(A)</b> Body weight changes (%) of BALB/c mice during four days after the intrarectal instillation of TNBS (2.5 mg/animal) in 45% ethanol solution. The control mice received the 45% of ethanol solution. CTX (0.035 mg/kg) was administered i.p. 18 h after TNBS-induced colitis, and saline solution was administered as control. # <i>(p</i><0.05) ETOH versus TNBS and TNBS+CTX; o <i>(p</i><0.05) ETOH+CTX versus TNBS and TNBS+CTX; α <i>(p</i><0.05) TNBS versus TNBS+CTX (n = 4–6 mice/group). <b>(B)</b> Disease activity index calculated as described in material and methods. The results were expressed as mean ± SEM (n = 4–6 mice/group); <b>(C)</b> Macroscopic appearance of colonic portion (4 cm) obtained from each mice/group at 4 days after TNBS-induced colitis; <b>(D)</b> Histological analysis of perirectal segment from mice of distinct experimental groups stained with H&E (Structures: (e) epithelial damage, (i) inflammatory infiltrate and (s) submucosa edema) obtained after 4 days of TNBS-colitis; <b>(E)</b> Histological score of inflammatory reaction perirectal segment of each experimental group of mice (n = 4–5 mice/group); <b>(F)</b> MPO activity of colonic tissue of each experimental mice-group. Groups: ETOH (control- 45% ETOH); ETOH+CTX (45% ethanol group treated with CTX); TNBS (TNBS instillation in 45% ETOH- inflammatory bowel disease) and TNBS+CTX (TNBS-instillation in 45% ETOH that received the CTX) (n = 4–5 animals/group). * <i>p</i><0.05; *** <i>p</i><0.001.</p

Sialic Acid Residues Are Essential for the Anaphylactic Activity of Murine IgG1 Antibodies

Glycosylation of the Ab molecule is essential for maintaining the functional structure of Fc region and consequently for Ab-mediated effector functions, such as binding to cells or complement system activation. Alterations in the composition of the sugar moiety can dramatically influence Ab activity; however, it is not completely clear how differences in the N-linked oligosaccharide structure impact the biological function of Abs. We have described that murine IgG1 Abs can be separated according to their ability to elicit in vivo anaphylaxis in a fraction of anaphylactic and other of non-anaphylactic molecules. Furthermore, we showed that the N-linked oligosaccharide chain is essential for the structural conformation of the anaphylactic IgG1, the binding to Fc gamma RIII on mast cells, and, consequently, for the ability to mediate anaphylactic reactions. In this study, we evaluated the contribution of individual sugar residues to this biological function. Differences in the glycan composition were observed when we analyzed oligosaccharide chains from anaphylactic or non-anaphylactic IgG1, mainly the presence of more sialic acid and fucose residues in anaphylactic molecules. Interestingly, the enzymatic removal of terminal sialic acid residues in anaphylactic IgG1 resulted in loss of the ability to trigger mast cell degranulation and in vivo anaphylactic reaction, similarly to the deglycosylated IgG1 Ab. In contrast, fucose removal did not affect the anaphylactic function. Therefore, we demonstrated that the ability of murine IgG1 Abs to mediate anaphylaxis is directly dependent on the amount of sialic acid residues associated to the oligosaccharide chain attached to the Fc region of these molecules. The Journal of Immunology, 2008, 181: 8308-8314.FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo Conselho Nacional de PesquisaFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CAPES Comissao de Aperfeicoamento de Pessoal de Nivel SuperiorUniversidad de Buenos Aires, ArgentinaUniversidad de Buenos Aires, ArgentinaConsejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET)Consejo Nacional de Investigaciones Cientificas y Tecnicas de Argentina (CONICET

CPDs and 6-4PPs play different roles in UV-induced cell death in normal and NER-deficient human cells

Ultraviolet (UV) light generates two major DNA lesions: cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts (6-4PPs), but the specific participation of these two lesions in the deleterious effects of UV is a longstanding question. In order to discriminate the precise role of unrepaired CPDs and 6-4PPs in UV-induced responses triggering cell death, human fibroblasts were transduced by recombinant adenoviruses carrying the CPD-photolyase or 6-4PP-photolyase cDNAs. Both photolyases were able to prevent UV-induced apoptosis in cells deficient for nucleotide excision repair (NER) to a similar extent, while in NER-proficient cells UV-induced apoptosis was prevented only by CPD-photolyase, with no effects observed when 6-4PPs were removed by the specific photolyase. These results strongly suggest that both CPDs and 6-4PPs contribute to UV-induced apoptosis in NER-deficient cells, while in NER-proficient cells, CPDs are the only lesions responsible for UV-killing, probably due to the rapid repair of 6-4PPs by NER. As a consequence, the difference in skin photosensitivity, including carcinogenesis, of most of the xeroderma pigmentosum patients and of normal people is probably not only a quantitative aspect, but depends on the type of DNA damage induced by sunlight and its rate of repair. (c) 2007 Elsevier B.V. All rights reserved