Locally administered lentivirus-mediated siRNA inhibits wear debris-induced inflammation in murine air pouch model

Lentivirus(LV)-mediated small interfering RNA (siRNA) targeting tumor necrosis factor-α (TNF-α) was locally administered into the air pouch of mice to inhibit inflammation induced by titanium alloy particles. The lentiviral vector expressed green fluorescent protein (GFP) as a reporter gene. Down-regulation of TNF-α in pouch area was confirmed by real-time PCR and ELISA, resulting in significantly decreased local inflammatory responses (P < 0.01). This approach was proven safe by localized GFP fluorescence and invariant TNF-α expression in peripheral blood, liver, spleen, kidney, lung and brain of mouse. In conclusion, locally administered siRNA provides an effective and safe method for inhibiting particle-induced inflammation.     

Fusion genetic analysis of jasmonate-signalling mutants in Arabidopsis

Jasmonates induce plant-defence responses and act to regulate defence-related genes including positive feedback of the lipoxygenase 2 (LOX2) gene involved in jasmonate synthesis. To identify jasmonate-signalling mutants, we used a fusion genetic strategy in which the firefly luciferase (FLUC) and Escherichia coli beta-glucuronidase (GUS) reporters were expressed under control of the jasmonate-responsive LOX2 promoter. Spatial and temporal patterns of reporter expression were determined initially, and revealed that JA-responsive expression from the LOX2 promoter required de novo protein synthesis. Reporter activity was also induced by the protein kinase inhibitor staurosporine and antagonized by the protein phosphatase inhibitor okadaic acid. FLUC bio-imaging, RNA gel-blot analysis and progeny analyses identified three recessive mutants that underexpress the FLUC reporter, designated jue1, 2 and 3, as well as two recessive mutants, designated joe1 and 2, that overexpress the reporter. Genetic analysis indicated that reporter overexpression in the joe mutants requires COI. joe1 responded to MeJA with increased anthocyanin accumulation, while joe2 responded with decreased root growth inhibition. In addition, reporter induction and endogenous LOX2 expression by staurosporine was absent in joe2.     

Peritoneal M2 macrophage transplantation as a potential cell therapy for enhancing renal repair in acute kidney injury

Acute kidney injury (AKI) is a clinical condition that is associated with high morbidity and mortality. Inflammation is reported to play a key role in AKI. Although the M2 macrophages exhibit antimicrobial and anti-inflammatory activities, their therapeutic potential has not been evaluated for AKI. This study aimed to investigate the protective effect of peritoneal M2 macrophage transplantation on AKI in mice. The macrophages were isolated from peritoneal dialysates of mice. The macrophages were induced to undergo M2 polarization using interleukin (IL)-4/IL-13. AKI was induced in mice by restoring the blood supply after bilateral renal artery occlusion for 30 minutes. The macrophages were injected into the renal cortex of mice. The changes in renal function, inflammation and tubular proliferation were measured. The M2 macrophages were co-cultured with the mouse primary proximal tubular epithelial cells (PTECs) under hypoxia/reoxygenation conditions in vitro. The PTEC apoptosis and proliferation were analysed. The peritoneal M2 macrophages effectively alleviated the renal injury and inflammatory response in mice with ischaemia-reperfusion injury (IRI) and promoted the PTEC proliferation in vivo and in vitro. These results indicated that the peritoneal M2 macrophages ameliorated AKI by decreasing inflammatory response and promoting PTEC proliferation. Hence, the peritoneal M2 macrophage transplantation can serve as a potential cell therapy for renal diseases.     

Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression

Unstable variants of green fluorescent protein (GFP) tagged with C-terminal extensions, which are targets for a tail specific protease, have been described in Escherichia coli and Pseudomonas putida [Appl. Envir. Microbiol. 64 (1998) 2240]. We investigated whether similar modifications to flow cytometer optimised GFP (GFPmut2) could be used to generate unstable variants of GFP for gene expression studies in mycobacteria. We constructed GFP variants in a mycobacterial shuttle vector under the control of the regulatory region of the inducible Mycobacterium smegmatis acetamidase gene. GFP expression was induced by the addition of acetamide and the stability of the GFP variants in M. smegmatis, following the removal of the inducer to switch off their expression, was determined using spectrofluorometry and flow cytometry. We demonstrate that, compared to the GFPmut2 (half-lives>7 days), the modified GFP variants exhibit much lower half-lives (between 70 and 165 min) in M. smegmatis. To investigate their utility in the measurement of mycobacterial gene expression, we cloned the promoter region of a putative amino acid efflux pump gene, lysE (Rv1986), from Mycobacterium tuberculosis together with the divergently transcribed, putative lysR-type regulator gene (Rv1985c) upstream of one of the unstable GFP variants. We found that the expression kinetics of the lysRE-gfp fusion were identical throughout the M. smegmatis growth curve to those measured using a conventional lysRE-xylE reporter fusion, peaking upon entry into stationary phase. In addition, it was established that the tagged GFP variants were also unstable in Mycobacterium bovis BCG. Thus, we have demonstrated that unstable GFP variants are suitable reporter genes for monitoring transient gene expression in fast- and slow-growing mycobacteria.     

Detection of promoter activity by flow cytometric analysis of GFP reporter expression

Low efficiency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially difficult to efficiently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quantified by flow cytometry. In this system, green fluorescent protein (GFP) reporter constructs are co-transfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection efficiency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color fluorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low efficiency.     

稳定表达CD19-FLUC-GFP的CT26细胞系的构建及鉴定

实体瘤缺乏明确的嵌合抗原受体T细胞(chimeric antigen receptor T-cell, CAR-T)治疗靶点。因此,通过慢病毒将已经明确的靶点分子CD19带入实体瘤细胞系,研究CD19 CAR-T细胞对其的杀伤,能够为CAR-T细胞针对实体瘤的治疗提供潜在的支撑。本研究利用三质粒慢病毒系统构建了稳定表达CD19、萤火虫荧光素酶(firefly luciferase, FLUC)和绿色荧光蛋白(green fluorescent protein, GFP)的结肠癌CT26细胞系CT26-CD19-FLUC-GFP。该细胞系与CT26细胞系的生长活性一致。通过流式细胞术检测不同代次CT26-CD19-FLUC-GFP细胞,证实了CT26-CD19-FLUC-GFP细胞连续传代至第5、10、22代后CD19及GFP的稳定表达。进一步证实,连续传代至第22代的CT26-CD19-FLUC-GFP细胞中的CD19 mRNA及FLUC表达水平显著高于对照组CT26细胞。与T细胞相比,CD19 CAR-T细胞能够显著杀伤CT26-CD19-FLUC-GFP细胞及MC38-CD19细胞。CT26-CD19-FLUC-GFP细胞腹腔植入小鼠体内1周后,通过活体成像仪可以检测到腹腔区域的FLUC表达。上述结果表明,成功构建了稳定表达CD19-FLUC-GFP的CT26细胞系,且该细胞系能够被CD19 CAR-T细胞特异性杀伤。     

Arachidonate 15-lipoxygenase type B knockdown leads to reduced lipid accumulation and inflammation in atherosclerosis

Inflammation in the vascular wall is important for development of atherosclerosis. We have shown previously that arachidonate 15-lipoxygenase type B (ALOX15B) is more highly expressed in human atherosclerotic lesions than in healthy arteries. This enzyme oxidizes fatty acids to substances that promote local inflammation and is expressed in lipid-loaded macrophages (foam cells) present in the atherosclerotic lesions. Here, we investigated the role of ALOX15B in foam cell formation in human primary macrophages and found that silencing of human ALOX15B decreased cellular lipid accumulation as well as proinflammatory cytokine secretion from macrophages. To investigate the role of ALOX15B in promoting the development of atherosclerosis in vivo, we used lentiviral shRNA silencing and bone marrow transplantation to knockdown mouse Alox15b gene expression in LDL-receptor-deficient (Ldlr(-/-)) mice. Knockdown of mouse Alox15b in vivo decreased plaque lipid content and markers of inflammation. In summary, we have shown that ALOX15B influences progression of atherosclerosis, indicating that this enzyme has an active proatherogenic role.     

In vivo cell tracking of mouse embryonic myoblasts and fast fibers during development

Fast and slow TnI are co‐expressed in E11.5 embryos, and fast TnI is present from the very beginning of myogenesis. A novel green fluorescent protein (GFP) reporter mouse lines (FastTnI/GFP lines) that carry the primary and secondary enhancer elements of the mouse fast troponin I (fast TnI), in which reporter expression correlates precisely with distribution of the endogenous fTnI protein was generated. Using the FastTnI/GFP mouse model, we characterized the early myogenic events in mice, analyzing the migration of GFP+ myoblasts, and the formation of primary and secondary myotubes in transgenic embryos. Interestingly, we found that the two contractile fast and slow isoforms of TnI are expressed during the migration of myoblasts from the somites to the limbs and body wall, suggesting that both participate in these events. Since no sarcomeres are present in myoblasts, we speculate that the function of fast TnI in early myogenesis is, like Myosin and Tropomyosin, to participate in cell movement during the initial myogenic stages. genesis 52:793–808, 2014. © 2014 Wiley Periodicals, Inc.     

Lentivirus‐mediated bifunctional cell labeling for in vivo melanoma study

Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long‐term culture and colony formation of several LV‐labeled mouse melanoma cells showed that promoters derived from mammalian house‐keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase–green fluorescence protein fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP‐labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP‐positive cells can be isolated from the tumors by fluorescence‐activated cell sorter. Pol2‐Luc/GFP labeling, while lower in activity, was more sustainable than FerH‐Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol‐2‐Luc/GFP labeling allows long‐term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models.     

M2 macrophages induced by prostaglandin E2 and IL-6 from cervical carcinoma are switched to activated M1 macrophages by CD4+ Th1 cells

Monocytes attracted by tumor-induced chronic inflammation differentiate to APCs, the type of which depends on cues in the local tumor milieu. In this work, we studied the influence of human cervical cancer cells on monocyte differentiation and showed that the majority of cancer cells either hampered monocyte to dendritic cell differentiation or skewed their differentiation toward M2-like macrophages. Blocking studies revealed that M2 differentiation was caused by tumor-produced PGE(2) and IL-6. TGF-β, IL-10, VEGF, and macrophage colony-stimulating factor did not play a role. Notably, these CD14(+)CD163(+) M2 macrophages were also detected in situ. Activation of cancer cell-induced M2-like macrophages by several TLR-agonists revealed that compared with dendritic cells, these M2 macrophages displayed a tolerogenic phenotype reflected by a lower expression of costimulatory molecules, an altered balance in IL-12p70 and IL-10 production, and a poor capacity to stimulate T cell proliferation and IFN-γ production. Notably, upon cognate interaction with Th1 cells, these tumor-induced M2 macrophages could be switched to activated M1-like macrophages that expressed high levels of costimulatory molecules, produced high amounts of IL-12 and low amounts of IL-10, and acquired the lymphoid homing marker CCR7. The effects of the interaction between M2 macrophages and Th1 cells could partially be mimicked by activation of these APCs via CD40 in the presence of IFN-γ. Our data on the presence, induction, and plasticity of tumor-induced tolerogenic APCs in cervical cancer suggest that tumor-infiltrated Th1 cells can stimulate a tumor-rejecting environment by switching M2 macrophages to classical proinflammatory M1 macrophages.     

Novel highly sensitive IL-10-beta-lactamase reporter mouse reveals cells of the innate immune system as a substantial source of IL-10 in vivo

In this study, we report on a novel, highly sensitive IL-10 reporter mouse based on the reporter enzyme β-lactamase and the fluorescence resonance energy transfer substrate coumarin-cephalosporin-fluorescein (4). In contrast to an IL-10 reporter mouse model that we generated by using enhanced GFP as reporter and allowed tracking IL-10 expression only in T cells, the IL-10-β-lactamase reporter (ITIB) mouse enables us to easily analyze and quantify IL-10 production at the single-cell level in all myeloid and lymphoid cell types. Furthermore, the ITIB mouse allows studying of the kinetics of IL-10 expression on a single-cell basis and provides a valuable tool for in vivo screening of cell type-specific IL-10-modulating drugs. Remarkably, the ITIB mouse revealed that, although a significant portion of each myeloid and lymphoid cell type produces IL-10, macrophages represent the major IL-10 producer population in several organs of naive mice. Moreover, using the examples of bacterial infection and transplantable skin melanoma models, we demonstrate the exceptional applicability of the ITIB mouse for the identification of IL-10-producing cells during immune responses in vivo. In this study, we identified tumor-infiltrating F4/80(+) macrophages as the major source for IL-10 in B16-F10 melanoma in vivo. During systemic infection with Yersinia enterocolitica, although the proportion of IL-10(+) cells increased in each myeloid and lymphoid cell type population, infiltrating CD11b(+)Ly6G(+) neutrophils represent a majority among IL-10-producing cells at the site of infection. We conclude that cells of the innate immune system that are involved in immune homeostasis or immune responses are substantial sources of IL-10.     

miR-122过表达转基因小鼠质粒构建及其功能验证

目的:比较两种miR-122转基因小鼠过表达载体构建方法,为建立miR-122过表达转基因小鼠奠定基础。方法:PCR扩增长约291bp的pre-miR-122的序列,分别定向克隆到pBROAD3-GFP载体GFP基因上游内含子或下游3'UTR区域,两种质粒分别转染293T细胞,Q-PCR检测miR-122和GFP的表达水平,并观察GFP绿色荧光。miR-122 sensor reporter是将3个miR-122成熟序列的反义序列串联克隆至psiCHECK2载体luciferase 3'UTR中,然后分别与2种miR-122过表达质粒载体共转染293T细胞,最后检测荧光素酶活性来鉴定miR-122调控功能。结果:2种构建方法的miR-122表达水平都明显增高,而只有插入到GFP基因3'UTR的质粒表达GFP功能正常。结论:构建microRNA过表达载体时,microRNA位于报告基因3'UTR区域不会影响microRNA和报告基因的功能;构建的两种miR-122过表达质粒载体都可应用到转基因小鼠研究中,而将miR-122插入到GFP下游的方法则更利于miR-122的表达。     

ADAMDEC1 promotes skin inflammation in rosacea via modulating the polarization of M1 macrophages

Rosacea is a chronic inflammatory cutaneous disease which mainly affects central face, leading to cosmetic disfigurement and compromised social psychology in billions of rosacea patients. Though the exact etiology of rosacea remains elusive, accumulating evidence has highlighted the dysfunction of innate immunity and inflammation in rosacea pathogenesis. Disintegrin Metalloprotease ADAM-like Decysin-1 (ADAMDEC1) is an orphan ADAM-like metalloprotease which is believed to be closely related to inflammation. Here for the first time, we reported that Adamdec1 expression was significantly increased in the skin lesions of rosacea patients and LL37–induced rosacea-like mouse models. Immunofluorescence analysis revealed co-localization of ADAMDEC1 and macrophages in patient and mouse biopsies. In cellular experiment, the expression of ADAMDEC1 was prominently elevated in M1 but not M2 macrophages. Knocking down of ADAMDEC1 significantly blunted M1 polarization in macrophages induced from human monocytes and THP-1 cell lines. Furthermore, silencing of Adamdec1 in LL-37-induced mouse model also suppressed the expression of M1 signature genes such as IL-6, iNOS and TNF-α, resulting in attenuated rosacea-like phenotype and inflammation. Taken together, our results demonstrate that ADAMDEC1 plays a pro-inflammatory role in rosacea via modulating the M1 polarization of macrophages.     

肥胖与脂肪组织巨噬细胞的研究进展

肥胖被认为是一种慢性促炎症疾病。近年来巨噬细胞在肥胖的发生过程中起的重要作用越来越被研究者们所重视。研究发现脂肪组织巨噬细胞(ATMs)的极化和招募在肥胖的发生过程中扮演着重要角色:在肥胖的脂肪组织中,巨噬细胞M1/M2的比例出现失衡即M1促炎巨噬细胞比例上调M2抑炎巨噬细胞比例下调导致脂肪组织慢性炎症;脂肪组织的局部炎症发生时周边组织巨噬细胞招募至脂肪组织也能够促进肥胖的发展进程。本文就肥胖的发生与脂肪组织巨噬细胞的极化和招募的关系作一综述。     

An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile

Cardiac tissue macrophages (cTMs) are a previously uncharacterised cell type that we have identified and characterise here as an abundant GFP(+) population within the adult Cx(3)cr1(GFP/+) knock-in mouse heart. They comprise the predominant myeloid cell population in the myocardium, and are found throughout myocardial interstitial spaces interacting directly with capillary endothelial cells and cardiomyocytes. Flow cytometry-based immunophenotyping shows that cTMs exhibit canonical macrophage markers. Gene expression analysis shows that cTMs (CD45(+)CD11b(+)GFP(+)) are distinct from mononuclear CD45(+)CD11b(+)GFP(+) cells sorted from the spleen and brain of adult Cx(3)cr1(GFP/+) mice. Gene expression profiling reveals that cTMs closely resemble alternatively-activated anti-inflammatory M2 macrophages, expressing a number of M2 markers, including Mrc1, CD163, and Lyve-1. While cTMs perform normal tissue macrophage homeostatic functions, they also exhibit a distinct phenotype, involving secretion of salutary factors (including IGF-1) and immune modulation. In summary, the characterisation of cTMs at the cellular and molecular level defines a potentially important role for these cells in cardiac homeostasis.