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Purified anti-mouse CD16/32 antibody

RRID:AB_312801

Antibody ID

AB_312801

Target Antigen

CD16/32 mouse

Proper Citation

(BioLegend Cat# 101302, RRID:AB_312801)

Clonality

monoclonal antibody

Comments

Applications: FC, IP

Clone ID

Clone 93

Host Organism

rat

Deconstructing Adipogenesis Induced by β3-Adrenergic Receptor Activation with Single-Cell Expression Profiling.

  • Burl RB
  • Cell Metab.
  • 2018 Jun 9

Literature context:


Abstract:

Recruitment of brown/beige adipocytes (BAs) in white adipose tissue (WAT) involves proliferation and differentiation of adipocyte stem cells (ASCs) in concert with close interactions with resident immune cells. To deconvolve stromal cell heterogeneity in a comprehensive and unbiased fashion, we performed single-cell RNA sequencing (scRNA-seq) of >33,000 stromal/vascular cells from epididymal WAT (eWAT) and inguinal WAT (iWAT) under control conditions and during β3-adrenergic receptor (ADRB3) activation. scRNA-seq identified distinct ASC subpopulations in eWAT and iWAT that appeared to be differentially poised to enter the adipogenic pathway. ADRB3 activation triggered the dramatic appearance of proliferating ASCs in eWAT, whose differentiation into BAs could be inferred from a single time point. scRNA-seq identified various immune cell types in eWAT, including a proliferating macrophage subpopulation that occupies adipogenic niches. These results demonstrate the power of scRNA-seq to deconstruct adipogenic niches and suggest novel functional interactions among resident stromal cell subpopulations.

Funding information:
  • NCI NIH HHS - U24 CA126480(United States)

High-Dimensional Single-Cell Mapping of Central Nervous System Immune Cells Reveals Distinct Myeloid Subsets in Health, Aging, and Disease.

  • Mrdjen D
  • Immunity
  • 2018 Feb 20

Literature context:


Abstract:

Individual reports suggest that the central nervous system (CNS) contains multiple immune cell types with diverse roles in tissue homeostasis, immune defense, and neurological diseases. It has been challenging to map leukocytes across the entire brain, and in particular in pathology, where phenotypic changes and influx of blood-derived cells prevent a clear distinction between reactive leukocyte populations. Here, we applied high-dimensional single-cell mass and fluorescence cytometry, in parallel with genetic fate mapping systems, to identify, locate, and characterize multiple distinct immune populations within the mammalian CNS. Using this approach, we revealed that microglia, several subsets of border-associated macrophages and dendritic cells coexist in the CNS at steady state and exhibit disease-specific transformations in the immune microenvironment during aging and in models of Alzheimer's disease and multiple sclerosis. Together, these data and the described framework provide a resource for the study of disease mechanisms, potential biomarkers, and therapeutic targets in CNS disease.

Funding information:
  • NHLBI NIH HHS - HL086621(United States)

Pseudomonas expression of an oxygen sensing prolyl hydroxylase homologue regulates neutrophil host responses in vitro and in vivo.

  • Dickinson RS
  • Wellcome Open Res
  • 2018 Feb 2

Literature context:


Abstract:

Background: Pseudomonas species are adapted to evade innate immune responses and can persist at sites of relative tissue hypoxia, including the mucus-plugged airways of patients with cystic fibrosis and bronchiectasis.  The ability of these bacteria to directly sense and respond to changes in local oxygen availability is in part consequent upon expression of the 2-oxoglutarate oxygenase, Pseudomonas prolyl hydroxylase (PPHD), which acts on elongation factor Tu (EF-Tu), and is homologous with the human hypoxia inducible factor (HIF) prolyl hydroxylases. We report that PPHD expression regulates the neutrophil response to acute pseudomonal infection. Methods:In vitro co-culture experiments were performed with human neutrophils and PPHD-deficient and wild-type bacteria and supernatants, with viable neutrophil counts determined by flow cytometry. In vivo consequences of infection with PPHD deficient P. aeruginosa were determined in an acute pneumonia mouse model following intra-tracheal challenge. Results: Supernatants of PPHD-deficient bacterial cultures contained higher concentrations of the phenazine exotoxin pyocyanin and induced greater acceleration of neutrophil apoptosis than wild-type PAO1 supernatants in vitro.  In vivo infection with PPHD mutants compared to wild-type PAO1 controls resulted in increased levels of neutrophil apoptosis and impaired control of infection, with higher numbers of P. aeruginosa recovered from the lungs of mice infected with the PPHD-deficient strain.  This resulted in an overall increase in mortality in mice infected with the PPHD-deficient strain. Conclusions: Our data show that Pseudomonas expression of its prolyl hydroxylase influences the outcome of host-pathogen interactions in vitro and in vivo, demonstrating the importance of considering how both host and pathogen adaptations to hypoxia together define outcomes of infection. Given that inhibitors for the HIF prolyl hydroxylases are in late stage trials for the treatment of anaemia and that the active sites of PPHD and human HIF prolyl hydroxylases are closely related, the results are of current clinical interest.

Funding information:
  • NEI NIH HHS - K22 EY016875-01(United States)

Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.

  • Gaya M
  • Cell
  • 2018 Jan 25

Literature context:


Abstract:

B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity.

Funding information:
  • NICHD NIH HHS - R01 HD049808(United States)
  • Wellcome Trust - UM1 AI100663()

Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor Engagement.

  • Freeman SA
  • Cell
  • 2018 Jan 11

Literature context:


Abstract:

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion, however, can be obstructed by transmembrane proteins ("pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44, an abundant transmembrane protein capable of indirect association with F-actin, hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments, curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages, where receptor mobility was minimal. Conversely, receptors were most mobile at the leading edge, where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan, anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier, enabling receptors to engage their targets.

Funding information:
  • Howard Hughes Medical Institute - (United States)
  • NIGMS NIH HHS - P50 GM085273()
  • NIGMS NIH HHS - R01 GM059907()
  • NIGMS NIH HHS - R35 GM119619()

Abnormal Microglia and Enhanced Inflammation-Related Gene Transcription in Mice with Conditional Deletion of Ctcf in Camk2a-Cre-Expressing Neurons.

  • McGill BE
  • J. Neurosci.
  • 2018 Jan 3

Literature context:


Abstract:

CCCTC-binding factor (CTCF) is an 11 zinc finger DNA-binding domain protein that regulates gene expression by modifying 3D chromatin structure. Human mutations in CTCF cause intellectual disability and autistic features. Knocking out Ctcf in mouse embryonic neurons is lethal by neonatal age, but the effects of CTCF deficiency in postnatal neurons are less well studied. We knocked out Ctcf postnatally in glutamatergic forebrain neurons under the control of Camk2a-Cre. CtcfloxP/loxP;Camk2a-Cre+ (Ctcf CKO) mice of both sexes were viable and exhibited profound deficits in spatial learning/memory, impaired motor coordination, and decreased sociability by 4 months of age. Ctcf CKO mice also had reduced dendritic spine density in the hippocampus and cerebral cortex. Microarray analysis of mRNA from Ctcf CKO mouse hippocampus identified increased transcription of inflammation-related genes linked to microglia. Separate microarray analysis of mRNA isolated specifically from Ctcf CKO mouse hippocampal neurons by ribosomal affinity purification identified upregulation of chemokine signaling genes, suggesting crosstalk between neurons and microglia in Ctcf CKO hippocampus. Finally, we found that microglia in Ctcf CKO mouse hippocampus had abnormal morphology by Sholl analysis and increased immunostaining for CD68, a marker of microglial activation. Our findings confirm that Ctcf KO in postnatal neurons causes a neurobehavioral phenotype in mice and provide novel evidence that CTCF depletion leads to overexpression of inflammation-related genes and microglial dysfunction.SIGNIFICANCE STATEMENT CCCTC-binding factor (CTCF) is a DNA-binding protein that organizes nuclear chromatin topology. Mutations in CTCF cause intellectual disability and autistic features in humans. CTCF deficiency in embryonic neurons is lethal in mice, but mice with postnatal CTCF depletion are less well studied. We find that mice lacking Ctcf in Camk2a-expressing neurons (Ctcf CKO mice) have spatial learning/memory deficits, impaired fine motor skills, subtly altered social interactions, and decreased dendritic spine density. We demonstrate that Ctcf CKO mice overexpress inflammation-related genes in the brain and have microglia with abnormal morphology that label positive for CD68, a marker of microglial activation. Our findings suggest that inflammation and dysfunctional neuron-microglia interactions are factors in the pathology of CTCF deficiency.

Funding information:
  • NICHD NIH HHS - U54 HD087011()
  • NIGMS NIH HHS - GM007240(United States)

Staphylococcus aureus Virulent PSMα Peptides Induce Keratinocyte Alarmin Release to Orchestrate IL-17-Dependent Skin Inflammation.

  • Nakagawa S
  • Cell Host Microbe
  • 2017 Nov 8

Literature context:


Abstract:

Staphylococcus aureus commonly colonizes the epidermis, but the mechanisms by which the host senses virulent, but not commensal, S. aureus to trigger inflammation remain unclear. Using a murine epicutaneous infection model, we found that S. aureus-expressed phenol-soluble modulin (PSM)α, a group of secreted virulence peptides, is required to trigger cutaneous inflammation. PSMα induces the release of keratinocyte IL-1α and IL-36α, and signaling via IL-1R and IL-36R was required for induction of the pro-inflammatory cytokine IL-17. The levels of released IL-1α and IL-36α, as well as IL-17 production by γδ T cells and ILC3 and neutrophil infiltration to the site of infection, were greatly reduced in mice with total or keratinocyte-specific deletion of the IL-1R and IL-36R signaling adaptor Myd88. Further, Il17a-/-f-/- mice showed blunted S. aureus-induced inflammation. Thus, keratinocyte Myd88 signaling in response to S. aureus PSMα drives an IL-17-mediated skin inflammatory response to epicutaneous S. aureus infection.

Funding information:
  • NIDA NIH HHS - R01 DA018928(United States)

O-Acetylation of Peptidoglycan Limits Helper T Cell Priming and Permits Staphylococcus aureus Reinfection.

  • Sanchez M
  • Cell Host Microbe
  • 2017 Oct 11

Literature context:


Abstract:

Humans do not usually develop effective immunity to Staphylococcus aureus reinfection. Using a murine model that mimics human infection, we show that lack of protective immunity to S. aureus systemic reinfection is associated with robust interleukin-10 (IL-10) production and impaired protective Th17 responses. In dendritic cell co-culture assays, priming with S. aureus promotes robust T cell proliferation, but limits Th cells polarization and production of IL-1β and other cytokines important for Th1 and Th17 differentiation. We show that O-acetylation of peptidoglycan, a mechanism utilized by S. aureus to block bacterial cell wall breakdown, limits the induction of pro-inflammatory signals required for optimal Th17 polarization. IL-10 deficiency in mice restores protective immunity to S. aureus infection, and adjuvancy with a staphylococcal peptidoglycan O-acetyltransferase mutant reduces IL-10, increases IL-1β, and promotes development of IL-17-dependent, Th cell-transferable protective immunity. Overall, our study suggests a mechanism whereby S. aureus modulates cytokines critical for induction of protective Th17 immunity.

Reactive Neutrophil Responses Dependent on the Receptor Tyrosine Kinase c-MET Limit Cancer Immunotherapy.

  • Glodde N
  • Immunity
  • 2017 Oct 17

Literature context:


Abstract:

Inhibitors of the receptor tyrosine kinase c-MET are currently used in the clinic to target oncogenic signaling in tumor cells. We found that concomitant c-MET inhibition promoted adoptive T cell transfer and checkpoint immunotherapies in murine cancer models by increasing effector T cell infiltration in tumors. This therapeutic effect was independent of tumor cell-intrinsic c-MET dependence. Mechanistically, c-MET inhibition impaired the reactive mobilization and recruitment of neutrophils into tumors and draining lymph nodes in response to cytotoxic immunotherapies. In the absence of c-MET inhibition, neutrophils recruited to T cell-inflamed microenvironments rapidly acquired immunosuppressive properties, restraining T cell expansion and effector functions. In cancer patients, high serum levels of the c-MET ligand HGF correlated with increasing neutrophil counts and poor responses to checkpoint blockade therapies. Our findings reveal a role for the HGF/c-MET pathway in neutrophil recruitment and function and suggest that c-MET inhibitor co-treatment may improve responses to cancer immunotherapy in settings beyond c-MET-dependent tumors.

Funding information:
  • NIAID NIH HHS - 2-U54-AI-057153(United States)

Mice deficient of Myc super-enhancer region reveal differential control mechanism between normal and pathological growth.

  • Dave K
  • Elife
  • 2017 Jun 6

Literature context:


Abstract:

The gene desert upstream of the MYC oncogene on chromosome 8q24 contains susceptibility loci for several major forms of human cancer. The region shows high conservation between human and mouse and contains multiple MYC enhancers that are activated in tumor cells. However, the role of this region in normal development has not been addressed. Here we show that a 538 kb deletion of the entire MYC upstream super-enhancer region in mice results in 50% to 80% decrease in Myc expression in multiple tissues. The mice are viable and show no overt phenotype. However, they are resistant to tumorigenesis, and most normal cells isolated from them grow slowly in culture. These results reveal that only cells whose MYC activity is increased by serum or oncogenic driver mutations depend on the 8q24 super-enhancer region, and indicate that targeting the activity of this element is a promising strategy of cancer chemoprevention and therapy.

Long-term intravital imaging of the multicolor-coded tumor microenvironment during combination immunotherapy.

  • Qi S
  • Elife
  • 2016 Nov 18

Literature context:


Abstract:

The combined-immunotherapy of adoptive cell therapy (ACT) and cyclophosphamide (CTX) is one of the most efficient treatments for melanoma patients. However, no synergistic effects of CTX and ACT on the spatio-temporal dynamics of immunocytes in vivo have been described. Here, we visualized key cell events in immunotherapy-elicited immunoreactions in a multicolor-coded tumor microenvironment, and then established an optimal strategy of metronomic combined-immunotherapy to enhance anti-tumor efficacy. Intravital imaging data indicated that regulatory T cells formed an 'immunosuppressive ring' around a solid tumor. The CTX-ACT combined-treatment elicited synergistic immunoreactions in tumor areas, which included relieving the immune suppression, triggering the transient activation of endogenous tumor-infiltrating immunocytes, increasing the accumulation of adoptive cytotoxic T lymphocytes, and accelerating the infiltration of dendritic cells. These insights into the spatio-temporal dynamics of immunocytes are beneficial for optimizing immunotherapy and provide new approaches for elucidating the mechanisms underlying the involvement of immunocytes in cancer immunotherapy.

Funding information:
  • NIH HHS - P51 OD011133(United States)

Expression Analysis of CB2-GFP BAC Transgenic Mice.

  • Schmöle AC
  • PLoS ONE
  • 2015 Sep 26

Literature context:


Abstract:

The endocannabinoid system (ECS) is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2). As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg) to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

Funding information:
  • NCRR NIH HHS - P40-RR17072(United States)
  • NIAAA NIH HHS - R01 AA017413(United States)

Cannabinoid receptor 2 deficiency results in reduced neuroinflammation in an Alzheimer's disease mouse model.

  • Schmöle AC
  • Neurobiol. Aging
  • 2015 Feb 3

Literature context:


Abstract:

Several studies have indicated that the cannabinoid receptor 2 (CB2) plays an important role in neuroinflammation associated with Alzheimer's disease (AD) progression. The present study examined the role of CB2 in microglia activation in vitro as well as characterizing the neuroinflammatory process in a transgenic mouse model of AD (APP/PS1 mice). We demonstrate that microglia harvested from CB2(-/-) mice were less responsive to pro-inflammatory stimuli than CB2(+/+) microglia, based on the cell surface expression of ICAM and CD40 and the release of chemokines and cytokines CCL2, IL-6, and TNFα. Transgenic APP/PS1 mice lacking CB2 showed reduced percentages of microglia and infiltrating macrophages. Furthermore, they showed lowered expression levels of pro-inflammatory chemokines and cytokines in the brain, as well as diminished concentrations of soluble Aβ 40/42. The reduction in neuroinflammation did not affect spatial learning and memory in APP/PS1*CB2(-/-) mice. These data suggest a role for the CB2 in Alzheimer's disease-associated neuroinflammation, independent of influencing Aβ-mediated pathology and cognitive impairment.

Funding information:
  • NIGMS NIH HHS - T32 GM007377(United States)