Group 2 Innate Lymphoid Cells (ILC2) play an important role in immune responses at barrier surfaces, notably in the lung during airway allergic inflammation or asthma. Several studies have described methods to isolate ILC2s from wild-type naive mice, most of them using cell sorting to obtain a pure population. Here, we describe in detail, a simple, efficient method for isolation and culture of lung mouse ILC2s. Lungs from Rag2-/- mice pretreated with IL-33 are collected and processed into single cell suspensions. Lymphoid cells are then recovered by density gradient separation. Lin-CD45+ cells are selected by depletion of lineage positive cells followed by positive selection of CD45+ cells. Culture of the isolated cells for several days results in a highly purified ILC2 population expressing typical cell surface markers (CD90.2, Sca1, CD25, CD127, and IL-33R). These cells can be expanded in culture for up to 10 days and used for diverse ex vivo assays or in vivo adoptive transfer experiments.
Copyright © 2018 The Authors; exclusive licensee Bio-protocol LLC.

Angiogenesis is a critical feature of plaque development in atherosclerosis and might play a key role in both the initiation and later rupture of plaques. The precursory molecular or cellular pro-angiogenic events that initiate plaque growth and that ultimately contribute to plaque instability, however, cannot be detected directly with any current diagnostic modality. This study was designed to investigate the feasibility of ultrasound molecular imaging of endothelial αvβ3 expression in vitro and in vivo using αvβ3-targeted ultrasound contrast agents (UCAs). In the in vitro study, αvβ3 expression was confirmed by immunofluorescence in a murine endothelial cell line and detected using the targeted UCA and ultrasound imaging at 18-MHz transmit frequency. In the in vivo study, expression of endothelial αvβ3 integrin in murine carotid artery vessels and microvessels of the salivary gland was quantified using targeted UCA and high-frequency ultrasound in seven animals. Our results indicated that endothelial αvβ3 expression was significantly higher in the carotid arterial wall containing atherosclerotic lesions than in arterial segments without any lesions. We also found that the salivary gland can be used as an internal positive control for successful binding of targeted UCA to αvβ3 integrin. In conclusion, αvβ3-targeted UCA allows non-invasive assessment of the expression levels of αvβ3 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.
Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

The actual occurrence of spontaneous plaque rupture in mice has been a matter of debate. We report on an in vivo observation of the actual event of possible plaque disruption in a living ApoE(-/-) mouse.
During live contrast-enhanced ultrasonography of a 50-week-old ApoE(-/-) male mouse, symptoms suggesting plaque disruption in the brachiocephalic artery were observed. Histological analysis confirmed the presence of advanced atherosclerotic lesions with dissections and intraplaque hemorrhage in the affected brachiocephalic trunk, pointing towards plaque rupture as the cause of the observed event. However, we did not detect a luminal thrombus or cap rupture, which is a key criterion for plaque rupture in human atherosclerosis.
This study reports the real-time occurrence of a possible plaque rupture in a living ApoE(-/-) mouse.

Lymphocytes play a significant role in the immunological processes of the bovine mammary gland and were found to be the dominant cell population in the milk of healthy udder quarters. The objective of this study was to investigate the quantitative relationship between CD2(+) T and CD21(+) B lymphocytes using flow cytometry. In a first study, quarter foremilk samples from apparently healthy udder quarters [somatic cell counts (SCC) ≤100,000 cells/mL; n=65] were analyzed and compared with diseased quarters (SCC >100,000 cells/mL; n=15). Percentages of CD2(+) T cells were significantly higher in milk samples with SCC ≤100,000 cells/mL than in those with SCC >100,000 cells/mL, whereas percentages of CD21(+) B cells developed in the opposite direction. As a result of this opposing trend, a new variable, the CD2/CD21 index-representing the percentages of CD2(+) cells per CD21(+) cells-was defined. Although diseased quarters with SCC >100,000 cells/mL and the detection of major pathogens revealed generally CD2/CD21 indices <10, values >10 were observed in apparently healthy quarters. Hence, a CD2/CD21 index cutoff value of 10 may be suitable to aid differentiation between unsuspicious and microbiologically suspicious or diseased udder quarters. To test whether CD2/CD21 indices <10 were primarily related to pathogens, quarters with SCC ≤100,000 cells/mL and >100,000 cells/mL with different bacteriological status (culture negative, or minor or major pathogens) were selectively examined in a second biphasic study. In the first trial, 63 udder quarters were analyzed and 55 of these quarters were able to be sampled again in the second trial carried out 14 d later. In both trials, results of the first study were confirmed. Indeed, CD2/CD21 indices <10 were also found in quarters showing SCC ≤100,000 cells/mL and containing minor or major pathogens at the time of the current or previous bacteriological analysis. The results of our examinations indicated a clear relationship between the CD2/CD21 index and the bacteriological status of the mammary gland. In combination with SCC, it offers a new marker for quick differentiation of unsuspicious and microbiologically suspicious or diseased udder quarters.
Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Deficiency of the signaling adapter protein DAP12 or its associated receptor TREM2 is associated with abnormal OC development in humans. Here we examine the role of TREM2 in mouse OC development and function, including migration and resorption in vitro. These results provide new evidence that TREM2 regulates OC function independent of its effects on multinucleated OC differentiation.
TREM2 (triggering receptor expressed in myeloid cells-2) associates with the signaling adapter DAP12 in osteoclasts (OCs). Genetic mutation or deletion of either the TYROBP (DAP12) or TREM2 gene is associated with the human disorder of brain and bone, Nasu-Hakola disease. We and others recently showed the critical requirement for immunoreceptor tyrosine-based activation motif (ITAM) signals through DAP12 and the Fc Receptor gamma chain (FcRgamma) during OC development. Here, we further define the role of TREM2 in OC differentiation and describe a role for TREM2 in OC migration and bone resorption.
We generated monoclonal anti-mouse TREM2 antibodies (mAb), analyzed pre-osteoclasts and mature OCs for TREM2 surface expression, and determined the effect of antibody ligation on in vitro OC differentiation, resorption, and migration. TREM2 RNA interference (RNAi) was used to disrupt expression of TREM2 in pre-osteoclasts.
Using flow cytometry, our studies reveal that TREM2 is weakly expressed on C57BL/6 bone marrow macrophages (BMMs) and is upregulated during culture with RANKL and macrophage-colony stimulating factor (M-CSF). The expression of TREM2 is unaltered in DAP12-deficient OCs. Using C57BL/6 BMMs or RAW264.7 precursors, anti-TREM2 mAb treatment with RANKL and M-CSF enhances the formation of multinuclear TRACP+ OCs compared with control mAb treatment. In contrast, these agents have no effect on DAP12-deficient precursors. Monoclonal Ab blockade of TREM2 on OCs generated from C57BL/6 BMMs results in decreased resorption of artificial calcium-phosphate substrate and dentine. Reduction of TREM2 expression in RAW264.7 cells by RNAi results in loss of OC formation in response to RANKL and M-CSF. Anti-TREM2 cross-linking enhances migration of C57BL/6 OCs and RAW246.7 OCs in response to M-CSF.
Our studies indicate that the TREM2 receptor regulates OC multinucleation as well as resorption and migration of mature OCs. Thus, TREM2-DAP12 signals regulate both OC formation and function.