英语国际会议省公共课一等奖全国赛课获奖课件.pptx

1Macrophages from patients with atopic dermatitis show a reduced CXCL10 expression in response to staphylococcal a-toxin.第1页CONTENTSCONTENTSIntroductionResultsMaterials and Methods2Discussion第2页3Introductionkeywordspresent studypurposebackground第3页Keywordsatopic dermatitis;(特应性皮炎)chemokines;（趋化因子）human macrophages;（人巨噬细胞）IP-10/CXCL10;MDC/CCL22;psoriasis;（牛皮癣；银屑病）Staphylococcus aureus;（金黄色葡萄球菌）-toxin.（-毒素）4第4页Background:Patients with atopic dermatitis(AD)and psoriasis are frequently colonized with Staphylococcus aureus(S.aureus).one-third of them producing-toxin,which is cor-related with the severity of eczema in AD.Distinct expression of chemokine(C-C motif)ligand(配体CCL)and chemokine(C-X-C motif)ligand(配体CXCL)chemokines has been documented in both diseases.the effects of a-toxin on human macrophages in patients with AD and psoriasis?5第5页Present study6Recent studies demonstrated that infiltration of inflammatory cells into tissues is regulated by chemokines.Two main subfamilies,recently renamed chemokine(C-C motif)ligand(CCL)and chemokine(C-X-C motif)ligand(CXCL)chemo-kines,have been distinguished.Several studies have suggested a crucial involvement of CCL chemokines in inflammation.Chemokines and their receptors are implicated in the development of symptoms of AD and psoriasis.CXCL10 plays a role in pathogenesis of psoriasis.第6页Purpose7The effects of-toxin on human macrophages still remain unclear.We show here for the first time that -toxin induces the Th1-related chemokine CXCL10 in human macrophages.we studied the effects of-toxin on Th1-and Th2-related chemokines in macrophages from patients with AD and psoriasis where the intrinsic abnormal and different chemokines production profile is well defined.第7页Materials and Methods8Materials and MethodsPatientsCell isolation and cultureStatistical analysisCytokine assessment by ELISAWestern blot第8页Methods9macrophage-derived chemokine(MDC)/CCL22 Cell surface markers expression and chemotaxis IFN-c-induced protein of 10-kDa(IP-10)/CXCL10qRTPCR or ELISA flow cytometry Boyden chamber technique第9页10Patients healthy donors and patients with AD or psoriasis Cell isolation and culturePeripheral blood mononuclear cells(PBMCs)were isolated by Lymphoprep density-gradient centrifugation from healthy donors and from patients with AD and psoriasisChemotaxis assay The chemotactic activity of supernatants from a-toxin stimulated macrophages on lymphocytes(CD4+T cells)was determined using a Boyden chamber technique 第10页11Western blot macrophages were prestimulated with a-toxin(100 ng/ml),IFN-c(10 ng/ml),TNF-a(10 ng/ml)or LPS.Cell extracts were subjected to Western blot analysis Cytokine assessment by ELISACell-free culture supernatants were harvested and analysed for CXCL10 or CCL22,using a commercially available enzyme-linked immunosorbent assay(ELISA)system.第11页ResultsResults12One Induction of IP-10/CXCL10 by staphylococcal a-toxin in human macrophagesThreeLow effect of a-toxin on CXCL10 induction(Th1-related chemokine)in macrophages from patients with ADTwo a-Toxin-stimulated macrophages could induce the migration of human CD4+T cells via CXCR3Four Effect of a-toxin on MDC production(Th2-related chemokine)in macrophages from patients with chronic inflammatory skin diseases第12页Induction of IP-10/CXCL10 by staphylococcal Induction of IP-10/CXCL10 by staphylococcal a-toxin in human macrophagesa-toxin in human macrophages13Microarray analysis revealed that CXCL10 was the most strongly up-regulated gene in macrophages.An increased expression was not observed for other chemokines.The mean ratio for all these chemokines was between 0.2 and 2 when comparing not stimulated with a-toxin-stimulated macrophages.第13页14 Figure 1 Induction of chemokine(C-X-C motif)ligand(CXCL)10 following a-toxin stimulation in human macrophages at the mRNA level.Figure 2 Induction of chemokine(C-X-C motif)ligand(CXCL)10 following a-toxin stimulation in human macrophages at the protein level.第14页15 Figure 3 Punch biopsies(3 mm)from healthy individuals were left either unstimulated(A)or stimulated with a-toxin(100 ng/ml)(B)or IFN-c(100 ng/ml)(C)for 24 h at 37C.5-lm paraffin sections were stained for CXCL10 along with appropriate isotype as well as CD68.第15页 a-Toxin-stimulated macrophages could a-Toxin-stimulated macrophages could induce the migration of human CD4+T cells induce the migration of human CD4+T cells via CXCR3via CXCR316Both inhibitors(anti-IP-10 antibody&anti-CXCR3 antibody)abrogated the migration induced by a-toxin-treated macrophage supernatants.a-Toxin in cell culture medium without macrophages did not directly induce the migration of human CD4+lymphocytes.第16页17 Figure 4 Chemotactic activity of a-toxin-stimulated macrophages and inhibition of chemokine(C-X-C motif)ligand(CXCL)10-induced chemotaxis using a neutralizing anti-CXCL10 or anti-CXCR3 Ab.第17页Low effect of a-toxin on CXCL10 induction Low effect of a-toxin on CXCL10 induction(Th1-related chemokine)in macrophages(Th1-related chemokine)in macrophages from patients with ADfrom patients with AD18 a-toxin induced significantly lower levels of CXCL10 expression or secretion in patients with AD compared with healthy controls as well as psoriasis at all time points and doses tested.第18页19 Figure 5 Macrophages from patients with atopic dermatitis(AD)and psoriasis(PSO)as well as from healthy controls(HC)were left either unstimulated(medium control)or stimulated with a-toxin as indicated or with IFN-c(10 ng/ml)or TNF-a(20 ng/ml),respectively,for the indicated periods of time.第19页 Effect of a-toxin on MDC production(Th2-Effect of a-toxin on MDC production(Th2-related chemokine)in macrophages from related chemokine)in macrophages from patients with chronic infammatory skin patients with chronic infammatory skin diseasesdiseases20Macrophages from patients with AD as well as patients with psoriasis showed an intrinsically higher CCL22 production compared to healthy controls by trend.On the whole,a-toxin did not regulate CCL22 secretion.第20页21Thank you第21页