CRISPR/Cas9 mediated dual fluorescent reporter mice to study spatial bone genomics of osteoblasts and osteoclasts in the local in vivo environment

AbstractInterleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.

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Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis

Science Advances ◽

10.1126/sciadv.abd6167 ◽

2021 ◽

Vol 7 (8) ◽

pp. eabd6167

Author(s):

Capucine L. Grandjean ◽

Zacarias Garcia ◽

Fabrice Lemaître ◽

Béatrice Bréart ◽

Philippe Bousso

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

Critical Path ◽

B Cell Lymphoma ◽

Antibody Dependent Cellular Cytotoxicity ◽

Fluorescent Reporter ◽

Cd20 Antibody ◽

Anti Cd20 ◽

Partial Depletion

Anti-CD20 antibody (mAb) represents an effective strategy for the treatment of B cell malignancies, possibly involving complement activity, antibody-dependent cellular cytotoxicity and phagocytosis (ADP). While ADP by Kupffer cells deplete circulating tumors, mechanisms targeting non-circulating tumors remain unclear. Using intravital imaging in a model of B cell lymphoma, we establish here the dominance and limitations of ADP in the bone marrow (BM). We found that tumor cells were stably residing in the BM with little evidence for recirculation. To elucidate the mechanism of depletion, we designed a dual fluorescent reporter to visualize phagocytosis and apoptosis. ADP by BM-associated macrophages was the primary mode of tumor elimination but was no longer active after one hour, resulting in partial depletion. Moreover, macrophages were present at low density in tumor-rich regions, targeting only neighboring tumors. Overcoming spatiotemporal bottlenecks in tumor-targeting Ab therapy thus represents a critical path towards the design of optimized therapies.

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Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages

Cell Death and Disease ◽

10.1038/s41419-021-03872-9 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Andreas Lindhorst ◽

Nora Raulien ◽

Peter Wieghofer ◽

Jens Eilers ◽

Fabio M. V. Rossi ◽

...

Keyword(s):

Inflammatory Response ◽

Metabolic Activation ◽

Degradation Process ◽

Critical Threshold ◽

Low Grade ◽

Blood Monocytes ◽

Threshold Size ◽

Laser Injury ◽

Reporter Mice

AbstractA chronic low-grade inflammation within adipose tissue (AT) seems to be the link between obesity and some of its associated diseases. One hallmark of this AT inflammation is the accumulation of AT macrophages (ATMs) around dead or dying adipocytes, forming so-called crown-like structures (CLS). To investigate the dynamics of CLS and their direct impact on the activation state of ATMs, we established a laser injury model to deplete individual adipocytes in living AT from double reporter mice (GFP-labeled ATMs and tdTomato-labeled adipocytes). Hence, we were able to detect early ATM-adipocyte interactions by live imaging and to determine a precise timeline for CLS formation after adipocyte death. Further, our data indicate metabolic activation and increased lipid metabolism in ATMs upon forming CLS. Most importantly, adipocyte death, even in lean animals under homeostatic conditions, leads to a locally confined inflammation, which is in sharp contrast to other tissues. We identified cell size as cause for the described pro-inflammatory response, as the size of adipocytes is above a critical threshold size for efferocytosis, a process for anti-inflammatory removal of dead cells during tissue homeostasis. Finally, experiments on parabiotic mice verified that adipocyte death leads to a pro-inflammatory response of resident ATMs in vivo, without significant recruitment of blood monocytes. Our data indicate that adipocyte death triggers a unique degradation process and locally induces a metabolically activated ATM phenotype that is globally observed with obesity.

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Enhancement and Segmentation Workflow for the Developing Zebrafish Vasculature

Journal of Imaging ◽

10.3390/jimaging5010014 ◽

2019 ◽

Vol 5 (1) ◽

pp. 14 ◽

Cited By ~ 1

Author(s):

Elisabeth Kugler ◽

Karen Plant ◽

Timothy Chico ◽

Paul Armitage

Keyword(s):

Open Source Software ◽

Light Sheet ◽

Visual Assessment ◽

Cardiovascular Development ◽

Fluorescent Reporter ◽

Enhancement Method ◽

Vertebrate Model ◽

Vascular Enhancement ◽

Light Sheet Fluorescence Microscopy

Zebrafish have become an established in vivo vertebrate model to study cardiovascular development and disease. However, most published studies of the zebrafish vascular architecture rely on subjective visual assessment, rather than objective quantification. In this paper, we used state-of-the-art light sheet fluorescence microscopy to visualize the vasculature in transgenic fluorescent reporter zebrafish. Analysis of image quality, vascular enhancement methods, and segmentation approaches were performed in the framework of the open-source software Fiji to allow dissemination and reproducibility. Here, we build on a previously developed image processing pipeline; evaluate its applicability to a wider range of data; apply and evaluate an alternative vascular enhancement method; and, finally, suggest a work-flow for successful segmentation of the embryonic zebrafish vasculature.

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A Fluorescent Reporter Mouse for Inflammasome Assembly Demonstrates an Important Role for Cell-Bound and Free ASC Specks during In Vivo Infection

Cell Reports ◽

10.1016/j.celrep.2016.06.011 ◽

2016 ◽

Vol 16 (2) ◽

pp. 571-582 ◽

Cited By ~ 35

Author(s):

Te-Chen Tzeng ◽

Stefan Schattgen ◽

Brian Monks ◽

Donghai Wang ◽

Anna Cerny ◽

...

Keyword(s):

Fluorescent Reporter ◽

Reporter Mouse

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In Vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS)

10.1101/2020.10.08.330894 ◽

2020 ◽

Author(s):

Emily K Don ◽

Alina Maschirow ◽

Rowan A W Radford ◽

Natalie M Scherer ◽

Andres Vidal-Itriago ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Disease Onset ◽

Bimolecular Fluorescence Complementation ◽

Aggregate Formation ◽

Reporter Protein ◽

Fluorescent Reporter ◽

Lateral Sclerosis ◽

Fluorescence Complementation ◽

Bimolecular Fluorescence

AbstractAmyotrophic lateral sclerosis (ALS) is a form of motor neuron disease (MND) that is characterized by the progressive loss of motor neurons within the spinal cord, brainstem and motor cortex. Although ALS clinically manifests as a heterogeneous disease, with varying disease onset and survival, a unifying feature is the presence of ubiquitinated cytoplasmic protein inclusion aggregates containing TDP-43. However, the precise mechanisms linking protein inclusions and aggregation to neuronal loss are currently poorly understood.Bimolecular Fluorescence Complementation (BiFC) takes advantage the association of fluorophore fragments (non-fluorescent on their own) that are attached to an aggregation prone protein of interest. Interaction of the proteins of interest allows for the fluorescent reporter protein to fold into its native state and emit a fluorescent signal. Here, we combined the power of BiFC with the advantages of the zebrafish system to validate, optimize and visualize of the formation of ALS-linked aggregates in real time in a vertebrate model. We further provide in vivo validation of the selectivity of this technique and demonstrate reduced spontaneous self-assembly of the non-fluorescent fragments in vivo by introducing a fluorophore mutation. Additionally, we report preliminary findings on the dynamic aggregation of the ALS-linked hallmark proteins Fus and TDP-43 in their corresponding nuclear and cytoplasmic compartments using BiFC.Overall, our data demonstrates the suitability of this BiFC approach to study and characterize ALS-linked aggregate formation in vivo. Importantly, the same principle can be applied in the context of other neurodegenerative diseases and has therefore critical implications to advance our understanding of pathologies that underlie aberrant protein aggregation.

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A SH3_5 Cell Anchoring Domain for Non-recombinant Surface Display on Lactic Acid Bacteria

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2020.614498 ◽

2021 ◽

Vol 8 ◽

Author(s):

Pei Kun Richie Tay ◽

Pei Yu Lim ◽

Dave Siak-Wei Ow

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Surface Display ◽

Bioactive Molecules ◽

Gastric Digestion ◽

Functional Protein ◽

Fluorescent Reporter ◽

Surface Conditions ◽

Bacterial Surface

Lactic acid bacteria (LAB) are a group of gut commensals increasingly recognized for their potential to deliver bioactive molecules in vivo. The delivery of therapeutic proteins, in particular, can be achieved by anchoring them to the bacterial surface, and various anchoring domains have been described for this application. Here, we investigated a new cell anchoring domain (CAD4a) isolated from a Lactobacillus protein, containing repeats of a SH3_5 motif that binds non-covalently to peptidoglycan in the LAB cell wall. Using a fluorescent reporter, we showed that C-terminal CAD4a bound Lactobacillus fermentum selectively out of a panel of LAB strains, and cell anchoring was uniform across the cell surface. Conditions affecting CAD4a anchoring were studied, including temperature, pH, salt concentration, and bacterial growth phase. Quantitative analysis showed that CAD4a allowed display of 105 molecules of monomeric protein per cell. We demonstrated the surface display of a functional protein with superoxide dismutase (SOD), an antioxidant enzyme potentially useful for treating gut inflammation. SOD displayed on cells could be protected from gastric digestion using a polymer matrix. Taken together, our results show the feasibility of using CAD4a as a novel cell anchor for protein surface display on LAB.

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A new transgene mouse model using an extravesicular EGFP tag to elucidate the in vivo function of extracellular vesicles

10.1101/2021.07.05.451120 ◽

2021 ◽

Author(s):

Mikkel Oernfeldt Noegaard ◽

Lasse Bach Steffensen ◽

Didde Hansen ◽

Ernst-Martin Fuchtbauer ◽

Morten Buch Engelund ◽

...

Keyword(s):

Mouse Model ◽

Extracellular Vesicles ◽

Fluorescent Protein ◽

Urine Samples ◽

Egfp Expression ◽

Reporter Mice ◽

Plasma And Urine Samples ◽

Co Precipitation ◽

Green Fluorescent

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate. We therefore created an EV reporter using CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9-EGFP expression in cells did not affect EV size and concentration, but allowed for co-precipitation of EV markers TSG101 and ALIX from cell-conditioned medium by anti-GFP immunoprecipitation. We created a transgenic mouse where CD9-EGFP was inserted in the inverse orientation and double-floxed, ensuring Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (AMHC-Cre) and kidney epithelium (Pax8-Cre), respectively. The mice showed tissue-specific EGFP expression, and plasma and urine samples were used to immunoprecipitate EVs. CD9-EGFP EVs was detected in plasma samples from CMV-Cre/CD9-EGFP and AMHC-Cre/CD9-EGFP mice, but not in PAX8-Cre/CD9-EGFP mice. On the other hand, CD9-EGFP EVs were detected in urine samples from CMV-Cre/CD9-EGFP and PAX8-Cre/CD9-EGFP mice, but not AMHC-Cre/CD9-EGFP, indicating that plasma EVs are not filtered to the urine. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to study cell-specific EVs in vivo and gain new insight into their physiological and pathophysiological function.

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