Old and new generation lipid mediators in acute inflammation and resolution

Originally regarded as just membrane constituents and energy storing molecules, lipids are now recognised as potent signalling molecules that regulate a multitude of cellular responses via receptor-mediated pathways, including cell growth and death, and inflammation/infection. Derived from polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), each lipid displays unique properties, thus making their role in inflammation distinct from that of other lipids derived from the same PUFA. The diversity of their actions arises because such metabolites are synthesised via discrete enzymatic pathways and because they elicit their response via different receptors. This review will collate the bioactive lipid research to date and summarise the findings in terms of the major pathways involved in their biosynthesis and their role in inflammation and its resolution. It will include lipids derived from AA (prostanoids, leukotrienes, 5-oxo-6,8,11,14-eicosatetraenoic acid, lipoxins and epoxyeicosatrienoic acids), EPA (E-series resolvins), and DHA (D-series resolvins, protectins and maresins).     

肠道菌群与肠易激综合征分型及患者血清细胞间黏附分子 1表达的关系

目的分析肠道菌群与IBS分型及患者血清细胞间黏附分子 1(ICAM 1)表达的关系,为后续研究提供参考。方法选择2018年12月至2020年1月我院收治的152例符合标准的肠易激综合征(IBS)患者作为观察组,并依照罗马Ⅲ标准将观察组患者分为腹泻型组(62例)、便秘型组(56例)、混合型组(34例);选择同期我院健康体检者30例为对照组。检测受试者粪便中双歧杆菌、乳杆菌、肠杆菌和肠球菌数量,同时检测受试者血清ICAM 1水平。采用Pearson相关性分析患者肠道菌群与血清ICAM 1关系,并采用Logistics回归模型分析肠道菌群与IBS各亚型的关系。结果观察组患者血清ICAM 1、肠道双歧杆菌、乳杆菌水平显著低于对照组,肠道肠杆菌和肠球菌水平显著高于对照组,差异均有统计学意义(均P<0.05)。不同亚型IBS患者血清ICAM 1、肠道双歧杆菌、乳杆菌、肠杆菌和肠球菌水平存在明显差异,其中混合组患者血清ICAM 1、肠杆菌、肠球菌数量最高,双歧杆菌、乳杆菌数量最低;腹泻型组肠道肠杆菌、肠球菌数量最低,双歧杆菌、乳杆菌数量最高(均P<0.05)。肠道中双歧杆菌、乳杆菌、肠杆菌、肠球菌数量是IBS分型的独立影响因素(均P<0.05)。患者肠道双歧杆菌、乳杆菌数量与ICAM 1水平呈显著负相关,而肠道肠杆菌和肠球菌数量与ICAM 1水平呈显著正相关(均P<0.05)。结论肠道菌群可影响IBS患者分型,且肠道中部分菌群与ICAM 1水平存在显著相关关系。     

Quantitative detection of cell surface protein expression by time-resolved fluorimetry.

A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging.     

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.     

A model of the complex between the PfEMP1 malaria protein and the human ICAM-1 receptor

Malaria is caused by protozoan parasites of the genus Plasmodium. Four species of Plasmodium can infect humans: P. falciparum, P. malariae, P. vivax, and P. ovale. P. falciparum is the only able to cytoadhere to the surface of postcapillary endothelial cells. A key role in cytoadherence is played by the interaction between the PfEMP1 P. falciparum protein and the human intracellular adhesion molecule (ICAM-1) although very little is known about the molecular details of this complex. Here we propose a model for this interaction on the basis of a homology model of the functional domain of PfEMP1 and of the ICAM-1 three dimensional structures. Our model is consistent with the results of many experimental observations, provides a rational explanation for the different binding abilities of different strains of P. falciparum and explains the reduced binding affinity of the A4 strain of P. falciparum for the ICAM-1(Kilifi) polymorphism. On the basis of our model, we can also explain why the murine ICAM-1, although sharing 70% sequence similarity with its human homologue, does not bind PfEMP1, and why the binding of fibrinogen and PfEMP1 to ICAM-1 is mutually exclusive. The model of the complex proposed here can serve as a useful tool for the design and interpretation of biochemical and immunological experimental results.     

Altered expression of glycoproteins on the cell surface of Jurkat cells during etoposide-induced apoptosis: Shedding and intracellular translocation of glycoproteins

Background

The glycoproteins on the cell surface are altered during apoptosis and play an important role in phagocytic clearance of apoptotic cells.

Methods

We classified Jurkat cells treated with etoposide as viable and early apoptotic cells, late apoptotic cells or secondary necrotic cells based on propidium iodide staining and scattered grams and estimated the expression levels of glycoproteins on the cell surface.

Results

The cell surface expression levels of intercellular adhesion molecules (ICAM)-2 and -3 on the apoptotic cells were markedly lower, while those of calnexin, calreticulin, and lysosome-associated membrane proteins (LAMP)-1 and -2 were significantly higher compared to non-apoptotic cells. These decreases in ICAM-2 and -3 on the apoptotic cell surface were reduced in the presence of metalloproteinase inhibitors and caspase inhibitors, respectively. Confocal microscopic analysis revealed that calnexin and calreticulin were assembled around fragmented nuclei of blebbed apoptotic cells.

Conclusions

These results suggest that alteration of glycoproteins on the cell surface during apoptosis is associated with shedding and intracellular translocation of glycoproteins.     

Monocytes induce proximal tubular epithelial-mesenchymal transition through NF-kappa B dependent upregulation of ICAM-1

Inflammatory cell infiltration plays a key role in the pathogenesis of tubulointerstitial damage in chronic renal diseases. In addition to secreting the profibrotic cytokines, monocytes themselves have been demonstrated to be directly associated with renal fibrogenesis. However, how infiltrating monocytes interact with resident cells and the underlying mechanisms remain elusive. In this study we investigated the effects of monocytes on phenotypic changes of human proximal tubular HK-2 cells. The typical epithelial cell morphology of HK-2 cells disappeared after co-culture with monocytes, accompanied by decreased E-cadherin expression, and increased α-SMA and fibronectin expression, suggesting that HK-2 cells undergo epithelial-mesenchymal transition (EMT). Further analysis revealed that the effects were dependent on direct contact of the two types of cells as conditioned medium had no effects. Interestingly, administration of CD18 antibody directly inhibited this process. Furthermore, by microarray and RT-PCR we found that NF-kB signaling may play a role in this process and blockade of this signaling pathway in HK-2 cells could inhibit ICAM-1 expression and EMT phenotypes. Taken together, these findings suggest that monocytes infiltration could directly induce EMT of HK-2 cells via upregulation ICAM-1 through NF-kB signaling pathway.     

Model of the alphaLbeta2 integrin I-domain/ICAM-1 DI interface suggests that subtle changes in loop orientation determine ligand specificity

The interaction of the alphaLbeta2 integrin with its cellular ligand the intercellular adhesion molecule-1 (ICAM-1) is critical for the tight binding interaction between most leukocytes and the vascular endothelium before transendothelial migration to the sites of inflammation. In this article we have modeled the alphaL subunit I-domain in its active form, which was computationally docked with the D1 domain of the ICAM-1 to probe potential protein-protein interactions. The experimentally observed key interaction between the carboxylate of Glu 34 in the ICAM-1 D1 domain and the metal ion-dependent adhesion site (MIDAS) in the open alphaL I-domain was consistently reproduced by our calculations. The calculations reveal the nature of the alphaLbeta2/ICAM-1 interaction and suggest an explanation for the increased ligand-binding affinity in the "open" versus the "closed" conformation of the alphaL I-domain. A mechanism for substrate selectivity among alphaL, alphaM, and alpha2 I-domains is suggested whereby the orientation of the loops within the I-domain is critical in mediating the interaction of the Glu 34 carboxylate of ICAM-1 D1 with the MIDAS.     

ICAM在着床期小鼠PBLC及EC/DC中表达的研究(英文)

细胞粘附分子（ＣＡＭ）可介导细胞间及细胞与间质之间的相互作用并传导信息,参与机体胚胎发育、免疫调节、炎症反应、组织修复及肿瘤转移等生理和病理过程。细胞间粘附分子１（ＩＣＡＭ１）是主要的ＣＡＭ分子之一,可表达于活化的细胞,内皮细胞等。人膜是母体与胚胎滋养层直接接触的特殊组织、已发现蜕膜细胞在着床过程中参与了局部免疫耐受的形成,但对着床期ＩＣＡＭ１在蜕膜细胞表达的动态研究鲜见报道。本研究采用免疫荧光、多参数流式细胞术,分别从整体和局部角度、着床过程中外周血淋巴细胞（ＰＢＬＣ）及子宫内膜／蜕膜（ＥＣ／ＤＣ）细胞ＩＣＡＭ１的不同表达特点进行了动态观察和对比性分析。结果发现,ＩＣＡＭ１在着床期ＰＢＬＣ及ＥＣ／ＤＣ中的表达均存在明显的动态变化（Ｔａｂ．１;Ｆｉｇｓ．１＆２）。ＩＣＡＭ１在ＰＢＬＣ中的表达于妊娠第一天（Ｄ１）即开始降低,Ｄ２降至最低;与此不同,ＩＣＡＭ１在ＥＣ／ＤＣ中的表达于Ｄ２开始降低,Ｄ４降至最低,Ｄ５开始恢复,但尚未恢复到对照水平。结果表明,ＩＣＡＭ１在蜕膜局部的表达调节方式不同于外周血;ＩＣＡＭ１表达阳性的外周血,淋巴细胞和蜕膜细胞均代表着活化的功能性细胞,这些细胞表面ＩＣＡＭ