Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 112
Filter
1.
Proc Natl Acad Sci U S A ; 120(39): e2309328120, 2023 09 26.
Article in English | MEDLINE | ID: mdl-37729200

ABSTRACT

We used electrophysiology and Ca2+ channel tethering to evaluate the performance of jGCaMP8 genetically encoded Ca2+ indicators (GECIs). Orai1 Ca2+ channel-jGCaMP8 fusions were transfected into HEK 293A cells and jGCaMP8 fluorescence responses recorded by simultaneous total internal reflection fluorescence microscopy and whole-cell patch clamp electrophysiology. Noninactivating currents from the Orai1 Y80E mutant provided a steady flux of Ca2+ controlled on a millisecond time scale by step changes in membrane potential. Test pulses to -100 mV produced Orai1 Y80E-jGCaMP8f fluorescence traces that unexpectedly declined by ~50% over 100 ms before reaching a stable plateau. Testing of Orai1-jGCaMP8f using unroofed cells further demonstrated that rapid and partial fluorescence inactivation is a property of the indicator itself, rather than channel function. Photoinactivation spontaneously recovered over 5 min in the dark, and recovery was accelerated in the absence of Ca2+. Mutational analysis of residues near the tripeptide fluorophore of jGCaMP8f pointed to a mechanism: Q69M/C70V greatly increased (~90%) photoinactivation, reminiscent of fluorescent protein fluorophore cis-trans photoswitching. Indeed, 405-nm illumination of jGCaMP8f or 8m/8s/6f led to immediate photorecovery, and simultaneous illumination with 405 and 488-nm light blocked photoinactivation. Subsequent mutagenesis produced a variant, V203Y, that lacks photoinactivation but largely preserves the desirable properties of jGCaMP8f. Our results point to caution in interpreting rapidly changing Ca2+ signals using jGCaMP8 and earlier series GECIs, suggest strategies to avoid photoswitching, and serve as a starting point to produce more photostable, and thus more accurate, GECI derivatives.


Subject(s)
Fluorescent Dyes , Lighting , Heart Rate , Ionophores , Membrane Potentials
3.
Sci Signal ; 15(738): eabq5594, 2022 06 14.
Article in English | MEDLINE | ID: mdl-35700266

ABSTRACT

Calcium (Ca2+) signaling has long been known to be crucial for T cell activation. Erdogmus et al. tested the function of voltage-gated Ca2+ channel (CaV) proteins and discovered a nonchannel function mediated by an accessory subunit but found no evidence for CaV channel activity in T cells.


Subject(s)
Calcium , T-Lymphocytes , Calcium/metabolism , Ion Channels/metabolism , Signal Transduction , T-Lymphocytes/metabolism
4.
Cell Rep ; 38(13): 110600, 2022 03 29.
Article in English | MEDLINE | ID: mdl-35354026

ABSTRACT

Several mental illnesses, characterized by aberrant stress reactivity, often arise after early-life adversity (ELA). However, it is unclear how ELA affects stress-related brain circuit maturation, provoking these enduring vulnerabilities. We find that ELA increases functional excitatory synapses onto stress-sensitive hypothalamic corticotropin-releasing hormone (CRH)-expressing neurons, resulting from disrupted developmental synapse pruning by adjacent microglia. Microglial process dynamics and synaptic element engulfment were attenuated in ELA mice, associated with deficient signaling of the microglial phagocytic receptor MerTK. Accordingly, selective chronic chemogenetic activation of ELA microglia increased microglial process dynamics and reduced excitatory synapse density to control levels. Notably, selective early-life activation of ELA microglia normalized adult acute and chronic stress responses, including stress-induced hormone secretion and behavioral threat responses, as well as chronic adrenal hypertrophy of ELA mice. Thus, microglial actions during development are powerful contributors to mechanisms by which ELA sculpts the connectivity of stress-regulating neurons, promoting vulnerability to stress and stress-related mental illnesses.


Subject(s)
Corticotropin-Releasing Hormone , Neural Stem Cells , Animals , Mice , Microglia/physiology , Neurons/physiology , Synapses/physiology
5.
Alzheimers Dement ; 18(10): 1765-1778, 2022 10.
Article in English | MEDLINE | ID: mdl-35142046

ABSTRACT

The P522R variant of PLCG2, expressed by microglia, is associated with reduced risk of Alzheimer's disease (AD). Yet, the impact of this protective mutation on microglial responses to AD pathology remains unknown. Chimeric AD and wild-type mice were generated by transplanting PLCG2-P522R or isogenic wild-type human induced pluripotent stem cell microglia. At 7 months of age, single-cell and bulk RNA sequencing, and histological analyses were performed. The PLCG2-P522R variant induced a significant increase in microglial human leukocyte antigen (HLA) expression and the induction of antigen presentation, chemokine signaling, and T cell proliferation pathways. Examination of immune-intact AD mice further demonstrated that the PLCG2-P522R variant promotes the recruitment of CD8+ T cells to the brain. These data provide the first evidence that the PLCG2-P522R variant increases the capacity of microglia to recruit T cells and present antigens, promoting a microglial transcriptional state that has recently been shown to be reduced in AD patient brains.


Subject(s)
Alzheimer Disease , Induced Pluripotent Stem Cells , Animals , Humans , Mice , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Antigen Presentation , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , Chemokines/metabolism , Disease Models, Animal , Induced Pluripotent Stem Cells/metabolism , Mice, Transgenic , Microglia/metabolism
6.
Elife ; 112022 02 22.
Article in English | MEDLINE | ID: mdl-35191835

ABSTRACT

The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD). Because abnormalities in Ca2+ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca2+ signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca2+ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y12 and P2Y13 receptors, results in greater release of Ca2+ from the endoplasmic reticulum stores, which triggers sustained Ca2+ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca2+ probe, Salsa6f, we found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca2+ using a P2Y12 receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca2+ response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility.


Subject(s)
Alzheimer Disease , Induced Pluripotent Stem Cells , Adenosine Diphosphate/metabolism , Alzheimer Disease/metabolism , Calcium/metabolism , Calcium Signaling , Humans , Induced Pluripotent Stem Cells/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Microglia/metabolism , Receptors, Immunologic/metabolism , Receptors, Purinergic/metabolism
7.
Bio Protoc ; 11(19): e4170, 2021 Oct 05.
Article in English | MEDLINE | ID: mdl-34722817

ABSTRACT

Elevations in cytosolic calcium (Ca2+) drive a wide array of immune cell functions, including cytokine production, gene expression, and cell motility. Live-cell imaging of cells loaded with ratiometric chemical Ca2+ indicators remains the gold standard for visualization and quantification of intracellular Ca2+ signals; ratiometric imaging can be accomplished with dyes such as Fura-2, the combination of Fluo-4 and Fura-Red, or, alternatively, by expressing genetically-encoded Ca2+ indicators (GECI) such as GCaMPs. Here, we describe a detailed protocol for Ca2+ imaging of T cells in vitro using genetically encoded or chemical indicators that can also be applied to a wide variety of cell types. The protocol addresses the challenge of facilitating T cell attachment on various substrates prepared on glass-bottom dishes to enable T cell imaging on an inverted microscope. The protocol also emphasizes cell preparation steps that ensure optimal cell viability - an essential requirement for recording dynamic changes in cytosolic Ca2+ levels - and that ensure reproducibility between multiple samples. Finally, we describe a simple algorithm to analyze single-cell Ca2+ signals over time using Fiji (ImageJ) software.

8.
Sci Adv ; 7(28)2021 07.
Article in English | MEDLINE | ID: mdl-34233878

ABSTRACT

T lymphocytes encounter complex mechanical cues during an immune response. The mechanosensitive ion channel, Piezo1, drives inflammatory responses to bacterial infections, wound healing, and cancer; however, its role in helper T cell function remains unclear. In an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we found that mice with genetic deletion of Piezo1 in T cells showed diminished disease severity. Unexpectedly, Piezo1 was not essential for lymph node homing, interstitial motility, Ca2+ signaling, T cell proliferation, or differentiation into proinflammatory T helper 1 (TH1) and TH17 subsets. However, Piezo1 deletion in T cells resulted in enhanced transforming growth factor-ß (TGFß) signaling and an expanded pool of regulatory T (Treg) cells. Moreover, mice with deletion of Piezo1 specifically in Treg cells showed significant attenuation of EAE. Our results indicate that Piezo1 selectively restrains Treg cells, without influencing activation events or effector T cell functions.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Multiple Sclerosis , Animals , Cell Differentiation , Encephalomyelitis, Autoimmune, Experimental/pathology , Ion Channels/genetics , Lymphocyte Activation , Mice , Mice, Inbred C57BL , T-Lymphocytes, Regulatory , Th1 Cells
9.
Nat Commun ; 12(1): 3256, 2021 05 31.
Article in English | MEDLINE | ID: mdl-34059671

ABSTRACT

Macrophages perform diverse functions within tissues during immune responses to pathogens and injury, but molecular mechanisms by which physical properties of the tissue regulate macrophage behavior are less well understood. Here, we examine the role of the mechanically activated cation channel Piezo1 in macrophage polarization and sensing of microenvironmental stiffness. We show that macrophages lacking Piezo1 exhibit reduced inflammation and enhanced wound healing responses. Additionally, macrophages expressing the transgenic Ca2+ reporter, Salsa6f, reveal that Ca2+ influx is dependent on Piezo1, modulated by soluble signals, and enhanced on stiff substrates. Furthermore, stiffness-dependent changes in macrophage function, both in vitro and in response to subcutaneous implantation of biomaterials in vivo, require Piezo1. Finally, we show that positive feedback between Piezo1 and actin drives macrophage activation. Together, our studies reveal that Piezo1 is a mechanosensor of stiffness in macrophages, and that its activity modulates polarization responses.


Subject(s)
Biocompatible Materials/adverse effects , Foreign-Body Reaction/immunology , Ion Channels/metabolism , Macrophages/immunology , Wound Healing/immunology , Actins/metabolism , Animals , Cells, Cultured , Cellular Microenvironment/immunology , Disease Models, Animal , Feedback, Physiological , Female , Humans , Ion Channels/genetics , Macrophage Activation , Macrophages/metabolism , Male , Mechanotransduction, Cellular/immunology , Mice , Primary Cell Culture , Subcutaneous Tissue/surgery
10.
Nat Commun ; 11(1): 5370, 2020 10 23.
Article in English | MEDLINE | ID: mdl-33097708

ABSTRACT

The discovery of TREM2 as a myeloid-specific Alzheimer's disease (AD) risk gene has accelerated research into the role of microglia in AD. While TREM2 mouse models have provided critical insight, the normal and disease-associated functions of TREM2 in human microglia remain unclear. To examine this question, we profile microglia differentiated from isogenic, CRISPR-modified TREM2-knockout induced pluripotent stem cell (iPSC) lines. By combining transcriptomic and functional analyses with a chimeric AD mouse model, we find that TREM2 deletion reduces microglial survival, impairs phagocytosis of key substrates including APOE, and inhibits SDF-1α/CXCR4-mediated chemotaxis, culminating in an impaired response to beta-amyloid plaques in vivo. Single-cell sequencing of xenotransplanted human microglia further highlights a loss of disease-associated microglial (DAM) responses in human TREM2 knockout microglia that we validate by flow cytometry and immunohistochemistry. Taken together, these studies reveal both conserved and novel aspects of human TREM2 biology that likely play critical roles in the development and progression of AD.


Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Gene Expression Regulation , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Microglia/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Amyloid beta-Peptides/metabolism , Animals , Brain/metabolism , Cell Death , Cell Line , Chemokine CXCL12/metabolism , Chemotaxis , Disease Models, Animal , Female , Gene Knockout Techniques , Genetic Predisposition to Disease/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Transgenic , Phagocytosis , Plaque, Amyloid/metabolism , Receptors, CXCR4/metabolism , Transcriptome
11.
Article in English | MEDLINE | ID: mdl-32984268

ABSTRACT

The changes in intracellular calcium concentration ([Ca2+]) following laser-induced cell injury in nearby cells were studied in primary mouse astrocytes selectively expressing the Ca2+ sensitive GFAP-Cre Salsa6f fluorescent tandem protein, in an Ast1 astrocyte cell line, and in primary mouse astrocytes loaded with Fluo4. Astrocytes in these three systems exhibit distinct changes in [Ca2+] following induced death of nearby cells. Changes in [Ca2+] appear to result from release of Ca2+ from intracellular organelles, as opposed to influx from the external medium. Salsa6f expressing astrocytes displayed dynamic Ca2+ changes throughout the phagocytic response, including lamellae protrusion, cytosolic signaling during vesicle formation, vesicle maturation, and vesicle tract formation. Our results demonstrate local changes in [Ca2+] are involved in the process of phagocytosis in astrocytes responding to cell corpses and/or debris.

12.
Proc Natl Acad Sci U S A ; 117(33): 20088-20099, 2020 08 18.
Article in English | MEDLINE | ID: mdl-32732436

ABSTRACT

T lymphocyte motility and interaction dynamics with other immune cells are vital determinants of immune responses. Regulatory T (Treg) cells prevent autoimmune disorders by suppressing excessive lymphocyte activity, but how interstitial motility patterns of Treg cells limit neuroinflammation is not well understood. We used two-photon microscopy to elucidate the spatial organization, motility characteristics, and interactions of endogenous Treg and Th17 cells together with antigen-presenting cells (APCs) within the spinal cord leptomeninges in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Th17 cells arrive before the onset of clinical symptoms, distribute uniformly during the peak, and decline in numbers during later stages of EAE. In contrast, Treg cells arrive after Th17 cells and persist during the chronic phase. Th17 cells meander widely, interact with APCs, and exhibit cytosolic Ca2+ transients and elevated basal Ca2+ levels before the arrival of Treg cells. In contrast, Treg cells adopt a confined, repetitive-scanning motility while contacting APCs. These locally confined but highly motile Treg cells limit Th17 cells from accessing APCs and suppress Th17 cell Ca2+ signaling by a mechanism that is upstream of store-operated Ca2+ entry. Finally, Treg cell depletion increases APC numbers in the spinal cord and exaggerates ongoing neuroinflammation. Our results point to fundamental differences in motility characteristics between Th17 and Treg cells in the inflamed spinal cord and reveal three potential cellular mechanisms by which Treg cells regulate Th17 cell effector functions: reduction of APC density, limiting access of Th17 cells to APCs, and suppression of Th17 Ca2+ signaling.


Subject(s)
Calcium Signaling/physiology , Spinal Cord/metabolism , Th17 Cells/metabolism , Animals , Autoantigens , Female , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Green Fluorescent Proteins , Male , Mice , Mice, Inbred C57BL , Myelin Sheath , T-Lymphocytes, Regulatory
13.
J Gen Physiol ; 152(9)2020 09 07.
Article in English | MEDLINE | ID: mdl-32589186

ABSTRACT

Upon Ca2+ store depletion, Orai1 channels cluster and open at endoplasmic reticulum-plasma membrane (ER-PM) junctions in signaling complexes called puncta. Little is known about whether and how Orai1 channel activity may vary between individual puncta. Previously, we developed and validated optical recording of Orai channel activity, using genetically encoded Ca2+ indicators fused to Orai1 or Orai3 N or C termini. We have now combined total internal reflection fluorescence microscopy with whole-cell recording to map functional properties of channels at individual puncta. After Ca2+ store depletion in HEK cells cotransfected with mCherry-STIM1 and Orai1-GCaMP6f, Orai1-GCaMP6f fluorescence increased progressively with increasingly negative test potentials and robust responses could be recorded from individual puncta. Cell-wide fluorescence half-rise and -fall times during steps to -100 mV test potential indicated probe response times of <50 ms. The in situ Orai1-GCaMP6f affinity for Ca2+ was 620 nM, assessed by monitoring fluorescence using buffered Ca2+ solutions in "unroofed" cells. Channel activity and temporal activation profile were tracked in individual puncta using image maps and automated puncta identification and recording. Simultaneous measurement of mCherry-STIM1 fluorescence uncovered an unexpected gradient in STIM1/Orai1 ratio that extends across the cell surface. Orai1-GCaMP6f channel activity was found to vary across the cell, with inactive channels occurring in the corners of cells where the STIM1/Orai1 ratio was lowest; low-activity channels typically at edges displayed a slow activation phase lasting hundreds of milliseconds. Puncta with high STIM1/Orai1 ratios exhibited a range of channel activity that appeared unrelated to the stoichiometric requirements for gating. These findings demonstrate functional heterogeneity of Orai1 channel activity between individual puncta and establish a new experimental platform that facilitates systematic comparisons between puncta composition and activity.


Subject(s)
Endoplasmic Reticulum , ORAI1 Protein/metabolism , Stromal Interaction Molecule 1/metabolism , Calcium/metabolism , Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , HEK293 Cells , Humans , Neoplasm Proteins
14.
Neurobiol Dis ; 140: 104868, 2020 07.
Article in English | MEDLINE | ID: mdl-32276110

ABSTRACT

Multiple sclerosis (MS) is a chronic, inflammatory autoimmune disease that affects the central nervous system (CNS) for which there is no cure. In MS, encephalitogenic T cells infiltrate the CNS causing demyelination and neuroinflammation; however, little is known about the role of regulatory T cells (Tregs) in CNS tissue repair. Transplantation of neural stem and progenitor cells (NSCs and NPCs) is a promising therapeutic strategy to promote repair through cell replacement, although recent findings suggest transplanted NSCs also instruct endogenous repair mechanisms. We have recently described that dampened neuroinflammation and increased remyelination is correlated with emergence of Tregs following human NPC transplantation in a murine viral model of immune-mediated demyelination. In the current study we utilized the prototypic murine autoimmune model of demyelination experimental autoimmune encephalomyelitis (EAE) to test the efficacy of hNSC transplantation. Eight-week-old, male EAE mice receiving an intraspinal transplant of hNSCs during the chronic phase of disease displayed remyelination, dampened neuroinflammation, and an increase in CNS CD4+CD25+FoxP3+ regulatory T cells (Tregs). Importantly, ablation of Tregs abrogated histopathological improvement. Tregs are essential for maintenance of T cell homeostasis and prevention of autoimmunity, and an emerging role for Tregs in maintenance of tissue homeostasis through interactions with stem and progenitor cells has recently been suggested. The data presented here provide direct evidence for collaboration between CNS Tregs and hNSCs promoting remyelination.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Multiple Sclerosis/therapy , Neural Stem Cells/transplantation , Remyelination , T-Lymphocytes, Regulatory , Animals , Humans , Male , Mice , Myelin Sheath , Stem Cell Transplantation
15.
Cell Mol Gastroenterol Hepatol ; 9(4): 557-567, 2020.
Article in English | MEDLINE | ID: mdl-31786364

ABSTRACT

BACKGROUND & AIMS: Biotin is a water-soluble vitamin that is indispensable for human health. Biotin deficiency can cause failure-to-thrive, immunodeficiency, alopecia, dermatitis, and conjunctivitis. We previously reported that biotin deficiency also can lead to severe colitis in mice, which is completely reversed with supplementation. Our aim in this study was to determine if high-dose biotin supplementation can provide a therapeutic benefit in a preclinical model for inflammatory bowel disease (IBD) and to identify the molecular mechanism by which this occurs. METHODS: Mice were challenged with dextran sodium sulfate to induce colitis and were treated with 1 mmol/L biotin to induce or maintain remission. Clinical response was monitored by the Disease Activity Index and fecal calprotectin levels. The colon tissue was investigated for histology, length, as well as expression of inflammatory cytokines (interleukin 6, tumor necrosis factor-α, interleukin 1ß), intestinal permeability, tight junctions (zonula occludens-1 and claudin-2), and the transcription factor nuclear factor-κB (NF-κB). RESULTS: Biotin therapy led to delayed onset and severity of colitis as well as accelerated healing. There was improvement in the Disease Activity Index, fecal calprotectin levels, colon length, and histology. In addition, biotin-treated mice had reduced expression of inflammatory cytokines, reduced intestinal permeability, and reduced activation of NF-κB. CONCLUSIONS: Oral supplementation with biotin provides benefit for maintenance and induction of remission in the dextran sodium sulfate preclinical model for IBD. Biotin does this by reducing the activation of NF-κB, which prevents the production of inflammatory cytokines and helps maintain the integrity of the intestinal barrier. Clinically, the NF-κB pathway is important in the development of IBD and this finding suggests that biotin may have therapeutic potential for patients with IBD.


Subject(s)
Biotin , Colitis , Animals , Biotin/pharmacology , Biotin/therapeutic use , Colitis/chemically induced , Colitis/drug therapy , Dextran Sulfate , Dietary Supplements , Humans , Kruppel-Like Transcription Factors , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Regeneration , Signal Transduction , Stem Cells/metabolism
16.
J Leukoc Biol ; 105(5): 1027-1039, 2019 05.
Article in English | MEDLINE | ID: mdl-30860634

ABSTRACT

CXCL17 is a homeostatic chemokine in the mucosa known to chemoattract dendritic cells and macrophages but can also be expressed elsewhere under inflammatory conditions. Cxcl17-/- mice have lower numbers of macrophages or dendritic cells in mucosal tissues. CXCL17 is also able to chemoattract suppressor myeloid cells that can recruit regulatory T cells. To explore a possible role of Cxcl17 in T cells, we studied T cell populations from Cxcl17-/- or wild-type (WT) littermate mice. Cxcl17-/- mice have higher numbers of CD4+ and CD8+ T cells in spleen and lymph nodes (LNs). Upon activation, they produce higher levels of several proinflammatory cytokines and chemokines. Furthermore, a Cxcl17-/- mouse developed exacerbated disease in a T cell-dependent model of experimental autoimmune encephalomyelitis (EAE). By 18 days after immunization with myelin oligodendrocyte peptide, only 44% of Cxcl17-/- mice were still alive vs. 90% for WT mice. During EAE, Cxcl17-/- mice exhibited higher numbers of lymphoid and myeloid cells in spleen and LNs, whereas they had less myeloid cell infiltration in the CNS. Cxcl17-/- mice also had higher levels of some inflammatory cytokines in serum, suggesting that they may be involved in the poor survival of these mice. Abnormal T cell function may reflect altered myeloid cell migration, or it could be due to altered T cell development in the thymus. We conclude that CXCL17 is a novel factor regulating T cell homeostasis and function.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Chemokines, CXC/genetics , Encephalomyelitis, Autoimmune, Experimental/genetics , Myeloid Cells/immunology , Animals , Antigens, CD/genetics , Antigens, CD/immunology , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/pathology , Cell Movement/immunology , Central Nervous System/immunology , Central Nervous System/pathology , Chemokines, CXC/deficiency , Chemokines, CXC/immunology , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Gene Expression Regulation , Homeostasis/immunology , Immunoglobulins/genetics , Immunoglobulins/immunology , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymphocyte Activation , Mice , Mice, Knockout , Myelin-Oligodendrocyte Glycoprotein/administration & dosage , Myeloid Cells/pathology , Peptide Fragments/administration & dosage , Primary Cell Culture , Spleen/immunology , Spleen/pathology , Survival Analysis , Thymus Gland/immunology , Thymus Gland/pathology
17.
Elife ; 62017 12 14.
Article in English | MEDLINE | ID: mdl-29239723

ABSTRACT

Ca2+ influx through Orai1 channels is crucial for several T cell functions, but a role in regulating basal cellular motility has not been described. Here, we show that inhibition of Orai1 channel activity increases average cell velocities by reducing the frequency of pauses in human T cells migrating through confined spaces, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca2+ indicator, Salsa6f, which permits real-time monitoring of cytosolic Ca2+ along with cell motility, we show that spontaneous pauses during T cell motility in vitro and in vivo coincide with episodes of cytosolic Ca2+ signaling. Furthermore, lymph node T cells exhibited two types of spontaneous Ca2+ transients: short-duration 'sparkles' and longer duration global signals. Our results demonstrate that spontaneous and self-peptide MHC-dependent activation of Orai1 ensures random walk behavior in T cells to optimize immune surveillance.


Subject(s)
Calcium/metabolism , Cell Movement , ORAI1 Protein/metabolism , Signal Transduction , T-Lymphocytes/physiology , Cells, Cultured , Humans , Locomotion , Optical Imaging , Staining and Labeling
18.
Elife ; 62017 12 14.
Article in English | MEDLINE | ID: mdl-29239725

ABSTRACT

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of 'Salsa6f', a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca2+, and a transgenic Ca2+ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca2+ signals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Ca2+ signals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca2+ signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca2+ transients ('sparkles') as cells migrate.


Subject(s)
Calcium Channels/metabolism , Calcium Signaling , Calcium/metabolism , Optical Imaging/methods , T-Lymphocytes/metabolism , Animals , Genes, Reporter , Mice , Mice, Transgenic , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics
19.
Neuron ; 94(2): 278-293.e9, 2017 Apr 19.
Article in English | MEDLINE | ID: mdl-28426964

ABSTRACT

Microglia play critical roles in brain development, homeostasis, and neurological disorders. Here, we report that human microglial-like cells (iMGLs) can be differentiated from iPSCs to study their function in neurological diseases, like Alzheimer's disease (AD). We find that iMGLs develop in vitro similarly to microglia in vivo, and whole-transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia. Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates. iMGLs were used to examine the effects of Aß fibrils and brain-derived tau oligomers on AD-related gene expression and to interrogate mechanisms involved in synaptic pruning. Furthermore, iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo. Together, these findings demonstrate that iMGLs can be used to study microglial function, providing important new insight into human neurological disease.


Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Brain/metabolism , Induced Pluripotent Stem Cells/cytology , Microglia/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Cells, Cultured , Cytokines/metabolism , Disease Models, Animal , Humans , Mice , Peptide Fragments/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...