Targeting of the Enhanced Green Fluorescent Protein Reporter to Adrenergic Cells in Mice

Adrenaline and noradrenaline are important neurotransmitter hormones that mediate physiological stress responses in adult mammals, and are essential for cardiovascular function during a critical period of embryonic/fetal development. In this study, we describe a novel mouse model system for identifying and characterizing adrenergic cells. Specifically, we generated a reporter mouse strain in which a nuclear-localized enhanced green fluorescent protein gene (nEGFP) was inserted into exon 1 of the gene encoding Phenylethanolamine n-methyltransferase (Pnmt), the enzyme responsible for production of adrenaline from noradrenaline. Our analysis demonstrates that this knock-in mutation effectively marks adrenergic cells in embryonic and adult mice. We see expression of nEGFP in Pnmt-expressing cells of the adrenal medulla in adult animals. We also note that nEGFP expression recapitulates the restricted expression of Pnmt in the embryonic heart. Finally, we show that nEGFP and Pnmt expressions are each induced in parallel during the in vitro differentiation of pluripotent mouse embryonic stem cells into beating cardiomyocytes. Thus, this new mouse genetic model should be useful for the identification and functional characterization of adrenergic cells in vitro and in vivo.     

Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes.     

Differential Capacity of Human Skin Dendritic Cells to Polarize CD4+T Cells into IL-17, IL-21 and IL-22 Producing Cells

Accumulating evidence suggests a contribution of T cell-derived IL-17, IL-21 and IL-22 cytokines in skin immune homeostasis as well as inflammatory disorders. Here, we analyzed whether the cytokine-producing T lymphocytes could be induced by the different subsets of human skin dendritic cells (DCs), i.e., epidermal Langerhans cells (LCs), dermal CD1c⁺CD14⁻ and CD14⁺ DCs (DDCs). DCs were purified following a 2-day migration from separated epidermal and dermal sheets and co-cultured with allogeneic T cells before cytokine secretion was explored. Results showed that no skin DCs could induce substantial IL-17 production by naïve CD4⁺ or CD8⁺T lymphocytes whereas all of them could induce IL-17 production by memory T cells. In contrast, LCs and CD1c⁺CD14⁻DDCs were able to differentiate naïve CD4⁺T lymphocytes into IL-22 and IL-21-secreting cells, LCs being the most efficient in this process. Intracellular cytokine staining showed that the majority of IL-21 or IL-22 secreting CD4⁺T lymphocytes did not co-synthesized IFN-γ, IL-4 or IL-17. IL-21 and IL-22 production were dependent on the B7/CD28 co-stimulatory pathway and ICOS-L expression on skin LCs significantly reduced IL-21 level. Finally, we found that TGF-β strongly down-regulates both IL-21 and IL-22 secretion by allogeneic CD4⁺ T cells. These results add new knowledge on the functional specialization of human skin DCs and might suggest new targets in the treatment of inflammatory skin disorders.     

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.     

B Cells Contribute to Heterogeneity of IL-17 Producing Cells in Rheumatoid Arthritis and Healthy Controls

Secretion of the proinflammatory cytokine Interleukin-17A (IL-17A) is the hallmark of a unique lineage of CD4 T cells designated Th17 cells, which may play a crucial role in the pathogenesis of rheumatoid arthritis (RA) and many autoimmune diseases. Recently, IL-17-producing cells other than T cells have been described, including diverse innate immune cells. Here, we show that the cellular sources of IL-17A in RA include a significant number of non-T cells. Multicolour fluorescence analysis of IL-17-expressing peripheral blood mononuclear cells (PBMC) revealed larger proportions of IL-17⁺CD3^- non-T cells in RA patients than in healthy controls (constitutive, 13.6% vs. 8.4%, and after stimulation with PMA/ionomycin 17.4% vs. 7.9% p < 0.001 in both cases). The source of IL-17 included CD3^-CD56⁺ NK cells, CD3^-CD14⁺ myeloid cells as well as the expected CD3⁺CD4⁺ Th17 cells and surprisingly a substantial number of CD3^-CD19⁺ B cells. The presence of IL-17A-expressing B cells was confirmed by specific PCR of peripheral MACS-sorted CD19⁺ B cells, as well as by the analysis of different EBV-transformed B cell lines. Here we report for the first time that in addition to Th17 cells and different innate immune cells B cells also contribute to the IL-17A found in RA patients and healthy controls.     

HCV Specific IL-21 Producing T Cells but Not IL-17A Producing T Cells Are Associated with HCV Viral Control in HIV/HCV Coinfection

BackgroundDecreased hepatitis C virus (HCV) clearance, faster cirrhosis progression and higher HCV RNA levels are associated with Human Immunodeficiency virus (HIV) coinfection. The CD4⁺ T helper cytokines interleukin (IL)-21 and IL-17A are associated with virus control and inflammation, respectively, both important in HCV and HIV disease progression. Here, we examined how antigen-specific production of these cytokines during HCV mono and HIV/HCV coinfection was associated with HCV virus control.MethodsWe measured HCV-specific IL-21 and IL-17A production by transwell cytokine secretion assay in PBMCs from monoinfected and coinfected individuals. Viral control was determined by plasma HCV RNA levels.ResultsIn acutely infected individuals, those able to establish transient/complete HCV viral control tended to have stronger HCV-specific IL-21-production than non-controllers. HCV-specific IL-21 production also correlated with HCV viral decline in acute infection. Significantly stronger HCV-specific IL-21 production was detected in HAART-treated coinfected individuals. HCV-specific IL-17A production was not associated with lower plasma HCV RNA levels in acute or chronic HCV infection and responses were stronger in HIV coinfection. HCV-specific IL-21/ IL-17A responses did not correlate with microbial translocation or fibrosis. Exogenous IL-21 treatment of HCV-specific CD8⁺ T cells from monoinfected individuals enhanced their function although CD8⁺ T cells from coinfected individuals were somewhat refractory to the effects of IL-21.ConclusionsThese data show that HCV-specific IL-21 and IL-17A-producing T cells are induced in HIV/HCV coinfection. In early HIV/HCV coinfection, IL-21 may contribute to viral control, and may represent a novel tool to enhance acute HCV clearance in HIV/HCV coinfected individuals.     

Loss of Function of Intestinal IL-17 and IL-22 Producing Cells Contributes to Inflammation and Viral Persistence in SIV-Infected Rhesus Macaques

In HIV/SIV-infected humans and rhesus macaques (RMs), a severe depletion of intestinal CD4⁺ T-cells producing interleukin IL-17 and IL-22 associates with loss of mucosal integrity and chronic immune activation. However, little is known about the function of IL-17 and IL-22 producing cells during lentiviral infections. Here, we longitudinally determined the levels and functions of IL-17, IL-22 and IL-17/IL-22 producing CD4⁺ T-cells in blood, lymph node and colorectum of SIV-infected RMs, as well as how they recover during effective ART and are affected by ART interruption. Intestinal IL-17 and IL-22 producing CD4⁺ T-cells are polyfunctional in SIV-uninfected RMs, with the large majority of cells producing four or five cytokines. SIV infection induced a severe dysfunction of colorectal IL-17, IL-22 and IL-17/IL-22 producing CD4⁺ T-cells, the extent of which associated with the levels of immune activation (HLA-DR⁺CD38⁺), proliferation (Ki-67⁺) and CD4⁺ T-cell counts before and during ART. Additionally, Th17 cell function during ART negatively correlated with residual plasma viremia and levels of sCD163, a soluble marker of inflammation and disease progression. Furthermore, IL-17 and IL-22 producing cell frequency and function at various pre, on, and off-ART experimental points associated with and predicted total SIV-DNA content in the colorectum and blood. While ART restored Th22 cell function to levels similar to pre-infection, it did not fully restore Th17 cell function, and all cell types were rapidly and severely affected—both quantitatively and qualitatively—after ART interruption. In conclusion, intestinal IL-17 producing cell function is severely impaired by SIV infection, not fully normalized despite effective ART, and strongly associates with inflammation as well as SIV persistence off and on ART. As such, strategies able to preserve and/or regenerate the functions of these CD4⁺ T-cells central for mucosal immunity are critically needed in future HIV cure research.     

Quantifying Stochastic Noise in Cultured Circadian Reporter Cells

Stochastic noise at the cellular level has been shown to play a fundamental role in circadian oscillations, influencing how groups of cells entrain to external cues and likely serving as the mechanism by which cell-autonomous rhythms are generated. Despite this importance, few studies have investigated how clock perturbations affect stochastic noise—even as increasing numbers of high-throughput screens categorize how gene knockdowns or small molecules can change clock period and amplitude. This absence is likely due to the difficulty associated with measuring cell-autonomous stochastic noise directly, which currently requires the careful collection and processing of single-cell data. In this study, we show that the damping rate of population-level bioluminescence recordings can serve as an accurate measure of overall stochastic noise, and one that can be applied to future and existing high-throughput circadian screens. Using cell-autonomous fibroblast data, we first show directly that higher noise at the single-cell results in faster damping at the population level. Next, we show that the damping rate of cultured cells can be changed in a dose-dependent fashion by small molecule modulators, and confirm that such a change can be explained by single-cell noise using a mathematical model. We further demonstrate the insights that can be gained by applying our method to a genome-wide siRNA screen, revealing that stochastic noise is altered independently from period, amplitude, and phase. Finally, we hypothesize that the unperturbed clock is highly optimized for robust rhythms, as very few gene perturbations are capable of simultaneously increasing amplitude and lowering stochastic noise. Ultimately, this study demonstrates the importance of considering the effect of circadian perturbations on stochastic noise, particularly with regard to the development of small-molecule circadian therapeutics.     

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.     

人乳腺癌MCF-7细胞在放射线处理的SCID小鼠中转移的实验研究

目的建立人乳腺癌MCF-7细胞SCID(Severe combined immunodeficiency,SCID)小鼠转移动物模型。方法采用人乳腺癌细胞株MCF-7细胞悬液,分别接种于5只经放射线处理的SCID小鼠腋背部皮下。记录肿瘤生长情况,处死荷瘤鼠并做病理切片,观察各脏器转移情况。结果接种SCID小鼠后6～10d成瘤,成瘤率为5/5只,潜伏期平均(7.4±1.3)d。接种后5只鼠分别于第60～68天拉颈处死,检测荷瘤,平均直径为(26.6±2.2)mm,平均重量为5.28g。病理学检查,转移脏器有3个部位,出现肺转移的为4/5只、骨转移的为3/5只和淋巴结转移的为1/5只。结论建立了人乳腺癌SCID小鼠转移动物模型,该模型可为肿瘤转移研究提供重要的实验工具。     

Identification of TRPM5 Ion Channels in Type-II Taste Cells of Mice

TRPM5 are ion channels belonging to the TRP family, which demonstrate a nonselective permeability for monovalent cations and are activated by an increase in the intracellular calcium level. TRPM5 are present in taste receptor cells of type II responsible for reception of bitter, sweet, and umami taste sensations. Knockout of the trpm5 gene in mice results in a nearly complete loss of sensitivity to taste stimuli of the above-mentioned modalities (taste blindness). The physiological activity of TRPM5 in taste receptive cells has practically not been studied. Using a patch-clamp technique, we carried out a comparative analysis of the properties of recombinant TRPM5 and Ca²⁺-activated membrane channels in type-II taste cells in mice. Dialysis of the studied cells with a high-Ca²⁺ solution and application of a calcium ionophore, ionomycin, caused activation of outward-rectification ion channels permeable for Na⁺, Cs⁺, and K⁺ in CHO-strain cells with exogenous TRPM5. These channels were blocked by 100 μM triphenylphosphine oxide (TPPO). Calcium-activated channels in type-II taste cells also possessed analogous properties. Application of the calcium ionophore ionomycin or a stepwise increase in the intracellular Ca²⁺ level using photolysis (uncaging) caused activation of channels nonselective with respect to Na⁺ and Cs⁺ and impermeable for N-methyl-D-glucamine (NMDG⁺). These channels had the current–voltage characteristics of outward rectification and a high thermosensitivity (Q₁₀ = 6.7 ± 0.5); they could be blocked by TPPO. It should be emphasized that TRPM5 were specific with respect to type-II cells. An increase in the intracellular calcium level induced the appearance of Cl– current in type-I cells and did not influence the basic current in type-III cells.     

小鼠脂肪来源干细胞体外培养、诱导分化及鉴定

目的:观察C57小鼠ASCs (Adipose-derived stem cells,ASCs)体外培养的生物学特性,探讨其成脂成骨诱导分化能力.方法:无菌条件下切取C57小鼠腹股沟处脂肪组织,0.25％Ⅰ型胶原酶消化,分离培养ASCs,37℃,5％CO2饱和湿度孵箱培养,细胞融合达80％时消化传代.观察其细胞形态;MTT比色法测细胞生长曲线;流式细胞仪检测细胞表面标志物;取第2代细胞行成骨及成脂诱导培养,3周后分别茜素红染色和油红O染色鉴定.结果:从C57小鼠脂肪组织中分离获取的ASCs呈长梭形,成纤维细胞样.细胞生长曲线呈“S”型,证明ASCs具有很强的增殖能力;流式细胞术分析结果:CD29、CD44、CD90阳性表达,CD31、CD34、CD45阴性表达;成骨诱导后茜素红染色阳性,成脂诱导后油红O染色阳性.结论:本试验分离培养的细胞为ASCs,具有确切分化能力.     

Human Cytomegalovirus Latency-Associated Proteins Elicit Immune-Suppressive IL-10 Producing CD4+ T Cells

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4⁺ T cell mediated. These UL138-specific CD4⁺ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CD4⁺ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4⁺ T cell responses included CD4⁺ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4⁺ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4⁺ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4⁺ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.     

Identification of an IL-7-dependent pre-T committed population in the spleen

Several extrathymic T cell progenitors have been described but their various contributions to the T cell lineage puzzle are unclear. In this study, we provide evidence for a splenic Lin(-)Thy1.2(+) T cell-committed population, rare in B6 mice, abundant in TCRalpha(-/-), CD3epsilon(-/-), and nude mice, and absent in IL-7- and Rag-2-deficient mice. Neither B nor myeloid cells are generated in vivo and in vitro. The incidence of these pre-T cells is under the control of thymus and/or mature T cells, as revealed by graft experiments. Indeed, IL-7 consumption by mature T cells inhibits the growth of these pre-T cells. Moreover, the nude spleen contains an additional Lin(-)Thy1.2(+)CD25(+) subset which is detected in B6 mice only after thymectomy. We establish that the full pre-T cell potential and proliferation capacity are only present in the c-kit(low) fraction of progenitors. We also show that most CCR9(+) progenitors are retained in the spleen of nude mice, but present in the blood of B6 mice. Thus, our data describe a new T cell lineage restricted subset that accumulates in the spleen before migration to the thymus.     

Expression Pattern of the LacZ Reporter in Secretogranin III Gene-trapped Mice

Secretogranin III (SgIII) is a granin protein involved in secretory granule formation in peptide-hormone-producing endocrine cells. In this study, we analyzed the expression of the LacZ reporter in the SgIII knockout mice produced by gene trapping (SgIII-gtKO) for the purpose of comprehensively clarifying the expression patterns of SgIII at the cell and tissue levels. In the endocrine tissues of SgIII-gtKO mice, LacZ expression was observed in the pituitary gland, adrenal medulla, and pancreatic islets, where SgIII expression has been canonically revealed. LacZ expression was extensively observed in brain regions, especially in the cerebral cortex, hippocampus, hypothalamic nuclei, cerebellum, and spinal cord. In peripheral nervous tissues, LacZ expression was observed in the retina, optic nerve, and trigeminal ganglion. LacZ expression was particularly prominent in astrocytes, in addition to neurons and ependymal cells. In the cerebellum, at least four cell types expressed SgIII under basal conditions. The expression of SgIII in the glioma cell lines C6 and RGC-6 was enhanced by excitatory glutamate treatment. It also became clear that the expression level of SgIII varied among neuron and astrocyte subtypes. These results suggest that SgIII is involved in glial cell function, in addition to neuroendocrine functions, in the nervous system:     

Role of Mast Cells in Inflammatory Bowel Disease and Inflammation-Associated Colorectal Neoplasia in IL-10-Deficient Mice

Background

Inflammatory bowel disease (IBD) is hypothesized to result from stimulation of immune responses against resident intestinal bacteria within a genetically susceptible host. Mast cells may play a critical role in IBD pathogenesis, since they are typically located just beneath the intestinal mucosal barrier and can be activated by bacterial antigens.

Methodology/Principal Findings

This study investigated effects of mast cells on inflammation and associated neoplasia in IBD-susceptible interleukin (IL)-10-deficient mice with and without mast cells. IL-10-deficient mast cells produced more pro-inflammatory cytokines in vitro both constitutively and when triggered, compared with wild type mast cells. However despite this enhanced in vitro response, mast cell-sufficient Il10 ^−/− mice actually had decreased cecal expression of tumor necrosis factor (TNF) and interferon (IFN)-γ mRNA, suggesting that mast cells regulate inflammation in vivo. Mast cell deficiency predisposed Il10 ^−/− mice to the development of spontaneous colitis and resulted in increased intestinal permeability in vivo that preceded the development of colon inflammation. However, mast cell deficiency did not affect the severity of IBD triggered by non-steroidal anti-inflammatory agents (NSAID) exposure or helicobacter infection that also affect intestinal permeability.

Conclusions/Significance

Mast cells thus appear to have a primarily protective role within the colonic microenvironment by enhancing the efficacy of the mucosal barrier. In addition, although mast cells were previously implicated in progression of sporadic colon cancers, mast cells did not affect the incidence or severity of colonic neoplasia in this inflammation-associated model.