Implementation of flow cytometry testing on rare matrix samples: Special considerations and best practices when the sample is unique or difficult to obtain.

The publication of Clinical and Laboratory Standards Institute's guideline H62 has provided the flow cytometry community with much-needed guidance on development and validation of flow cytometric assays (CLSI, 2021). It has also paved the way for additional exploration of certain topics requiring additional guidance. Flow cytometric analysis of rare matrices, or unique and/or less frequently encountered specimen types, is one such topic and is the focus of this manuscript. This document is the result of a collaboration subject matter experts from a diverse range of backgrounds and seeks to provide best practice consensus guidance regarding these types of specimens. Herein, we define rare matrix samples in the setting of flow cytometric analysis, address validation implications and challenges with these samples, and describe important considerations of using these samples in both clinical and research settings.

Cellular senescence is a double-edged sword in regulating aged immune responses to influenza.

Clearance of senescent cells has demonstrated therapeutic potential in the context of chronic age-related diseases. Little is known, however, how clearing senescent cells affects the ability to respond to an acute infection and form quality immunological memory. We aimed to probe the effects of clearing senescent cells in aged mice on the immune response to influenza (flu) infection. We utilized a p16 trimodality reporter mouse model (p16-3MR) to allow for identification and selective clearance of p16-expressing cells upon administration of ganciclovir (GCV). While p16-expressing cells may exacerbate dysfunctional responses to a primary infection, our data suggest they may play a role in fostering memory cell generation. We demonstrate that although clearance of p16-expressing cells enhanced viral clearance, this also severely limited antibody production in the lungs of flu-infected aged mice. 30 days later, there were fewer flu-specific CD8 memory T cells and lower levels of flu-specific antibodies in the lungs of GCV-treated mice. Furthermore, GCV-treated mice were unable to mount an optimal memory response and demonstrated increased viral load following heterosubtypic challenge. These results suggest that targeting senescent cells may potentiate primary responses while limiting the ability to form durable and protective immune memory with age.

IL-10 Differentially Promotes Mast Cell Responsiveness to IL-33, Resulting in Enhancement of Type 2 Inflammation and Suppression of Neutrophilia.

Mast cells (MCs) play critical roles in the establishment of allergic diseases. We recently demonstrated an unexpected, proinflammatory role for IL-10 in regulating MC responses. IL-10 enhanced MC activation and promoted IgE-dependent responses during food allergy. However, whether these effects extend to IgE-independent stimuli is not clear. In this article, we demonstrate that IL-10 plays a critical role in driving IL-33-mediated MC responses. IL-10 stimulation enhanced MC expansion and degranulation, ST2 expression, IL-13 production, and phospho-relA upregulation in IL-33-treated cells while suppressing TNF-α. These effects were partly dependent on endogenous IL-10 and further amplified in MCs coactivated with both IL-33 and IgE/Ag. IL-10's divergent effects also extended in vivo. In a MC-dependent model of IL-33-induced neutrophilia, IL-10 treatment enhanced MC responsiveness, leading to suppression of neutrophils and decreased TNF-α. In contrast, during IL-33-induced type 2 inflammation, IL-10 priming exacerbated MC activity, resulting in MC recruitment to various tissues, enhanced ST2 expression, induction of hypothermia, recruitment of eosinophils, and increased MCPT-1 and IL-13 levels. Our data elucidate an important role for IL-10 as an augmenter of IL-33-mediated MC responses, with implications during both allergic diseases and other MC-dependent disorders. IL-10 induction is routinely used as a prognostic marker of disease improvement. Our data suggest instead that IL-10 can enhance ST2 responsiveness in IL-33-activated MCs, with the potential to both aggravate or suppress disease severity depending on the inflammatory context.

Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

Astrocyte activation is associated with neuropathology and the production of tissue inhibitor of metalloproteinase-1 (TIMP1). TIMP1 is a pleiotropic extracellular protein that functions both as a protease inhibitor and as a growth factor. Astrocytes that lack expression of Timp1 do not support rat oligodendrocyte progenitor cell (rOPC) differentiation, and adult global Timp1 knockout (Timp1KO) mice do not efficiently remyelinate following a demyelinating injury. Here, we performed an unbiased proteomic analysis and identified a fibronectin-derived peptide called Anastellin (Ana) that was unique to the Timp1KO astrocyte secretome. Ana was found to block rOPC differentiation in vitro and enhanced the inhibitory influence of fibronectin on rOPC differentiation. Ana is known to act upon the sphingosine-1-phosphate receptor 1, and we determined that Ana also blocked the pro-myelinating effect of FTY720 (or fingolimod) on rOPC differentiation in vitro. Administration of FTY720 to wild-type C57BL/6 mice during MOG35-55-experimental autoimmune encephalomyelitis ameliorated clinical disability while FTY720 administered to mice lacking expression of Timp1 (Timp1KO) had no effect. Analysis of Timp1 and fibronectin (FN1) transcripts from primary human astrocytes from healthy and multiple sclerosis (MS) donors revealed lower TIMP1 expression was coincident with elevated FN1 in MS astrocytes. Last, analyses of proteomic databases of MS samples identified Ana peptides to be more abundant in the cerebrospinal fluid (CSF) of human MS patients with high disease activity. A role for Ana in MS as a consequence of a lack of astrocytic TIMP-1 production could influence both the efficacy of fingolimod responses and innate remyelination potential in the MS brain.

Activation of endothelial TRPM2 exacerbates blood-brain barrier degradation in ischemic stroke.

AIMS: Damage of the blood-brain barrier (BBB) is a hallmark of brain injury during the early stages of ischemic stroke. The subsequent endothelial hyperpermeability drives the initial pathological changes and aggravates neuronal death. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable nonselective cation channel activated by oxidative stress. However, whether TRPM2 is involved in BBB degradation during ischemic stroke remains unknown. We aimed to investigate the role of TRPM2 in BBB degradation during ischemic stroke and the underlying molecular mechanisms. METHODS AND RESULTS: Specific deletion of Trpm2 in endothelial cells using Cdh5 Cre produces a potent protective effect against brain injury in mice subjected to middle cerebral artery occlusion (MCAO), which is characterized by reduced infarction size, mitigated plasma extravasation, suppressed immune cell invasion, and inhibited oxidative stress. In vitro experiments using cultured cerebral endothelial cells (CECs) demonstrated that either Trpm2 deletion or inhibition of TRPM2 activation attenuates oxidative stress, Ca2+ overload, and endothelial hyperpermeability induced by oxygen-glucose deprivation (OGD) and CD36 ligand thrombospondin-1 (TSP1). In transfected HEK293T cells, OGD and TSP1 activate TRPM2 in a CD36-dependent manner. Noticeably, in cultured CECs, deleting Trpm2 or inhibiting TRPM2 activation also suppresses the activation of CD36 and cellular dysfunction induced by OGD or TSP1. CONCLUSIONS: In conclusion, our data reveal a novel molecular mechanism in which TRPM2 and CD36 promote the activation of each other, which exacerbates endothelial dysfunction during ischemic stroke. Our study suggests that TRPM2 in endothelial cells is a promising target for developing more effective and safer therapies for ischemic stroke.

The Implant-Induced Foreign Body Response Is Limited by CD13-Dependent Regulation of Ubiquitination of Fusogenic Proteins.

Implanted medical devices, from artificial heart valves and arthroscopic joints to implantable sensors, often induce a foreign body response (FBR), a form of chronic inflammation resulting from the inflammatory reaction to a persistent foreign stimulus. The FBR is characterized by a subset of multinucleated giant cells (MGCs) formed by macrophage fusion, the foreign body giant cells (FBGCs), accompanied by inflammatory cytokines, matrix deposition, and eventually deleterious fibrotic implant encapsulation. Despite efforts to improve biocompatibility, implant-induced FBR persists, compromising the utility of devices and making efforts to control the FBR imperative for long-term function. Controlling macrophage fusion in FBGC formation presents a logical target to prevent implant failure, but the actual contribution of FBGCs to FBR-induced damage is controversial. CD13 is a molecular scaffold, and in vitro induction of CD13KO bone marrow progenitors generates many more MGCs than the wild type, suggesting that CD13 regulates macrophage fusion. In the mesh implant model of FBR, CD13KO mice produced significantly more peri-implant FBGCs with enhanced TGF-ß expression and increased collagen deposition versus the wild type. Prior to fusion, increased protrusion and microprotrusion formation accompanies hyperfusion in the absence of CD13. Expression of fusogenic proteins driving cell-cell fusion was aberrantly sustained at high levels in CD13KO MGCs, which we show is due to a novel CD13 function, to our knowledge, regulating ubiquitin/proteasomal protein degradation. We propose CD13 as a physiologic brake limiting aberrant macrophage fusion and the FBR, and it may be a novel therapeutic target to improve the success of implanted medical devices. Furthermore, our data directly implicate FBGCs in the detrimental fibrosis that characterizes the FBR.

Host extracellular vesicles confer cytosolic access to systemic LPS licensing non-canonical inflammasome sensing and pyroptosis.

Intracellular surveillance for systemic microbial components during homeostasis and infections governs host physiology and immunity. However, a long-standing question is how circulating microbial ligands become accessible to intracellular receptors. Here we show a role for host-derived extracellular vesicles (EVs) in this process; human and murine plasma-derived and cell culture-derived EVs have an intrinsic capacity to bind bacterial lipopolysaccharide (LPS). Remarkably, circulating host EVs capture blood-borne LPS in vivo, and the LPS-laden EVs confer cytosolic access for LPS, triggering non-canonical inflammasome activation of gasdermin D and pyroptosis. Mechanistically, the interaction between the lipid bilayer of EVs and the lipid A of LPS underlies EV capture of LPS, and the intracellular transfer of LPS by EVs is mediated by CD14. Overall, this study demonstrates that EVs capture and escort systemic LPS to the cytosol licensing inflammasome responses, uncovering EVs as a previously unrecognized link between systemic microbial ligands and intracellular surveillance.

CD8+ T cell depletion prevents neuropathology in a mouse model of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) or Krabbe's disease is a fatal genetic demyelinating disease of the central nervous system caused by loss-of-function mutations in the galactosylceramidase (galc) gene. While the metabolic basis for disease is known, the understanding of how this results in neuropathology is not well understood. Herein, we report that the rapid and protracted elevation of CD8+ cytotoxic T lymphocytes occurs coincident with clinical disease in a mouse model of GLD. Administration of a function-blocking antibody against CD8α effectively prevented disease onset, reduced morbidity and mortality, and prevented CNS demyelination in mice. These data indicate that subsequent to the genetic cause of disease, neuropathology is driven by pathogenic CD8+ T cells, thus offering novel therapeutic potential for treatment of GLD.

Cellular Senescence is a Double-Edged Sword in Regulating Aged Immune Responses to Influenza.

Clearance of senescent cells has demonstrated therapeutic potential in the context of chronic age-related diseases. Little is known, however, how clearing senescent cells affects the ability to respond to an acute infection and form quality immunological memory. We aimed to probe the effects of clearing senescent cells in aged mice on the immune response to influenza (flu) infection. We utilized a p16 trimodality reporter mouse model (p16-3MR) to allow for identification and selective deletion of p16-expressing senescent cells upon administration of ganciclovir (GCV). While p16-expressing senescent cells may exacerbate dysfunctional responses to a primary infection, our data suggest they may play a role in fostering memory cell generation. We demonstrate that although deletion of p16-expressing cells enhanced viral clearance, this also severely limited antibody production in the lungs of flu-infected aged mice. 30 days later, there were fewer flu-specific CD8 memory T cells and lower levels of flu-specific antibodies in the lungs of GCV treated mice. GCV treated mice were unable to mount an optimal memory response and demonstrated increased viral load following a heterosubtypic challenge. These results suggest that targeting senescent cells may potentiate primary responses while limiting the ability to form durable and protective immune memory with age.

Astrocytic TIMP-1 regulates production of Anastellin, a novel inhibitor of oligodendrocyte differentiation and FTY720 responses.

Astrocyte activation is associated with neuropathology and the production of tissue inhibitor of metalloproteinase-1 (TIMP1). TIMP1 is a pleiotropic extracellular protein that functions both as a protease inhibitor and as a growth factor. We have previously demonstrated that murine astrocytes that lack expression of Timp1 do not support rat oligodendrocyte progenitor cell (rOPC) differentiation, and adult global Timp1 knockout ( Timp1 KO ) mice do not efficiently remyelinate following a demyelinating injury. To better understand the basis of this, we performed unbiased proteomic analyses and identified a fibronectin-derived peptide called anastellin that is unique to the murine Timp1 KO astrocyte secretome. Anastellin was found to block rOPC differentiation in vitro and enhanced the inhibitory influence of fibronectin on rOPC differentiation. Anastellin is known to act upon the sphingosine-1-phosphate receptor 1 (S1PR1), and we determined that anastellin also blocked the pro-myelinating effect of FTY720 (or fingolimod) on rOPC differentiation in vitro . Further, administration of FTY720 to wild-type C57BL/6 mice during MOG 35-55 -EAE ameliorated clinical disability while FTY720 administered to mice lacking expression of Timp1 in astrocytes ( Timp1 cKO ) had no effect. Analysis of human TIMP1 and fibronectin ( FN1 ) transcripts from healthy and multiple sclerosis (MS) patient brain samples revealed an inverse relationship where lower TIMP1 expression was coincident with elevated FN1 in MS astrocytes. Lastly, we analyzed proteomic databases of MS samples and identified anastellin peptides to be more abundant in the cerebrospinal fluid (CSF) of human MS patients with high versus low disease activity. The prospective role for anastellin generation in association with myelin lesions as a consequence of a lack of astrocytic TIMP-1 production could influence both the efficacy of fingolimod responses and the innate remyelination potential of the the MS brain. Significance Statement: Astrocytic production of TIMP-1 prevents the protein catabolism of fibronectin. In the absence of TIMP-1, fibronectin is further digested leading to a higher abundance of anastellin peptides that can bind to sphingosine-1-phosphate receptor 1. The binding of anastellin with the sphingosine-1-phosphate receptor 1 impairs the differentiation of oligodendrocytes progenitor cells into myelinating oligodendrocytes in vitro , and negates the astrocyte-mediated therapeutic effects of FTY720 in the EAE model of chronic CNS inflammation. These data indicate that TIMP-1 production by astrocytes is important in coordinating astrocytic functions during inflammation. In the absence of astrocyte produced TIMP-1, elevated expression of anastellin may represent a prospective biomarker for FTY720 therapeutic responsiveness.

Targeted inCITE-Seq Analysis Identifies the Loss of Nuclear TDP-43 in Endothelium as a Mediator of Blood Brain Barrier Signaling Pathway Dysfunction in Neurodegeneration.

Despite the importance of the endothelium in the regulation of the blood brain barrier (BBB) in aging and neurodegenerative disease, difficulties in extracting endothelial cell (EC) nuclei have limited analysis of these cells. In addition, nearly all Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Degeneration (FTD), and a large portion of Alzheimer's Disease (AD) exhibit neuronal TDP-43 aggregation, leading to loss of nuclear function, but whether TDP-43 is similarly altered in human BBB ECs is unknown. Here we utilize a novel technique for the enrichment of endothelial and microglial nuclei from human cortical brain tissues, combined with inCITE-seq, to analyze nuclear proteins and RNA transcripts in a large cohort of healthy and diseased donors. Our findings reveal a unique transcriptional signature in nearly half of the capillary endothelial cells across neurodegenerative states, characterized by reduced levels of nuclear ß-Catenin and canonical downstream genes, and an increase in TNF/NF-kB target genes. We demonstrate that this does not correlate with increased nuclear p65/NF-kB, but rather a specific loss of nuclear TDP-43 in these disease associated ECs. Comparative analysis in animal models with targeted disruption of TDP-43 shows that this is sufficient to drive these transcriptional alterations. This work reveals that TDP-43 is a critical governor of the transcriptional output from nuclear p65/NF-kB, which has paradoxical roles in barrier maintenance and also barrier compromising inflammatory responses, and suggests that disease specific loss in ECs contributes to BBB defects observed in the progression of AD, ALS and FTD.

Loss of Endothelial TDP-43 Leads to Blood Brain Barrier Defects in Mouse Models of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

Loss of nuclear TDP-43 occurs in a wide range of neurodegenerative diseases, and specific mutations in the TARDBP gene that encodes the protein are linked to familial Frontal Temporal Lobar Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS). Although the focus has been on neuronal cell dysfunction caused by TDP-43 variants, TARDBP mRNA transcripts are expressed at similar levels in brain endothelial cells (ECs). Since increased permeability across the blood brain barrier (BBB) precedes cognitive decline, we postulated that altered functions of TDP-43 in ECs contributes to BBB dysfunction in neurodegenerative disease. To test this hypothesis, we examined EC function and BBB properties in mice with either knock-in mutations found in ALS/FTLD patients (TARDBPG348C and GRNR493X) or EC-specific deletion of TDP-43 throughout the endothelium (Cdh5(PAC)CreERT2; Tardbpff) or restricted to brain endothelium (Slco1c1(BAC)CreERT2; Tardbpff). We found that TARDBPG348C mice exhibited increased permeability to 3kDa Texas Red dextran and NHS-biotin, relative to their littermate controls, which could be recapitulated in cultured brain ECs from these mice. Nuclear levels of TDP-43 were reduced in vitro and in vivo in ECs from TARDBPG348C mice. This coincided with a reduction in junctional proteins VE-cadherin, claudin-5 and ZO-1 in isolated ECs, supporting a cell autonomous effect on barrier function through a loss of nuclear TDP-43. We further examined two models of Tardbp deletion in ECs, and found that the loss of TDP-43 throughout the endothelium led to systemic endothelial activation and permeability. Deletion specifically within the brain endothelium acutely increased BBB permeability, and eventually led to hallmarks of FTD, including fibrin deposition, microglial and astrocyte activation, and behavioral defects. Together, these data show that TDP-43 dysfunction specifically within brain ECs would contribute to the BBB defects observed early in the progression of ALS/FTLD.

Complementary anti-cancer pathways triggered by inhibition of sideroflexin 4 in ovarian cancer.

DNA damaging agents are a mainstay of standard chemotherapy for ovarian cancer. Unfortunately, resistance to such DNA damaging agents frequently develops, often due to increased activity of DNA repair pathways. Sideroflexin 4 (SFXN4) is a little-studied inner mitochondrial membrane protein. Here we demonstrate that SFXN4 plays a role in synthesis of iron sulfur clusters (Fe-S) in ovarian cancer cells and ovarian cancer tumor-initiating cells, and that knockdown of SFXN4 inhibits Fe-S biogenesis in ovarian cancer cells. We demonstrate that this has two important consequences that may be useful in anti-cancer therapy. First, inhibition of Fe-S biogenesis triggers the accumulation of excess iron, leading to oxidative stress. Second, because enzymes critical to multiple DNA repair pathways require Fe-S clusters for their function, DNA repair enzymes and DNA repair itself are inhibited by reduction of SFXN4. Through this dual mechanism, SFXN4 inhibition heightens ovarian cancer cell sensitivity to DNA-damaging drugs and DNA repair inhibitors used in ovarian cancer therapy, such as cisplatin and PARP inhibitors. Sensitization is achieved even in drug resistant ovarian cancer cells. Further, knockout of SFXN4 decreases DNA repair and profoundly inhibits tumor growth in a mouse model of ovarian cancer metastasis. Collectively, these results suggest that SFXN4 may represent a new target in ovarian cancer therapy.

SMAD4 and TGFß are architects of inverse genetic programs during fate determination of antiviral CTLs.

Transforming growth factor ß (TGFß) is an important differentiation factor for cytotoxic T lymphocytes (CTLs) and alters the expression levels of several of homing receptors during infection. SMAD4 is part of the canonical signaling network used by members of the transforming growth factor family. For this study, genetically modified mice were used to determine how SMAD4 and TGFß receptor II (TGFßRII) participate in transcriptional programming of pathogen-specific CTLs. We show that these molecules are essential components of opposing signaling mechanisms, and cooperatively regulate a collection of genes that determine whether specialized populations of pathogen-specific CTLs circulate around the body, or settle in peripheral tissues. TGFß uses a canonical SMAD-dependent signaling pathway to downregulate Eomesodermin (EOMES), KLRG1, and CD62L, while CD103 is induced. Conversely, in vivo and in vitro data show that EOMES, KLRG1, CX3CR1, and CD62L are positively regulated via SMAD4, while CD103 and Hobit are downregulated. Intravascular staining also shows that signaling via SMAD4 promotes formation of long-lived terminally differentiated CTLs that localize in the vasculature. Our data show that inflammatory molecules play a key role in lineage determination of pathogen-specific CTLs, and use SMAD-dependent signaling to alter the expression levels of multiple homing receptors and transcription factors with known functions during memory formation.

Splice factor polypyrimidine tract-binding protein 1 (Ptbp1) primes endothelial inflammation in atherogenic disturbed flow conditions.

NF-κB-mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through adhesion molecules Icam1 and Vcam1. The endothelium is primed for cytokine activation of NF-κB by exposure to low and disturbed blood flow (LDF)but the molecular underpinnings are not fully understood. In an experimental in vivo model of LDF, platelets were required for the increased expression of several RNA-binding splice factors, including polypyrimidine tract binding protein (Ptbp1). This was coordinated with changes in RNA splicing in the NF-κB pathway in primed cells, leading us to examine splice factors as mediators of priming. Using Icam1 and Vcam1 induction by tumor necrosis factor (TNF)-α stimulation as a readout, we performed a CRISPR Cas9 knockout screen and identified a requirement for Ptbp1 in priming. Deletion of Ptbp1 had no effect on cell growth or response to apoptotic stimuli, but reversed LDF splicing patterns and inhibited NF-κB nuclear translocation and transcriptional activation of downstream targets, including Icam1 and Vcam1. In human coronary arteries, elevated PTBP1 correlates with expression of TNF pathway genes and plaque. In vivo, endothelial-specific deletion of Ptbp1 reduced Icam1 expression and myeloid cell infiltration at regions of LDF in atherosclerotic mice, limiting atherosclerosis. This may be mediated, in part, by allowing inclusion of a conserved alternative exon in Ripk1 leading to a reduction in Ripk1 protein. Our data show that Ptbp1, which is induced in a subset of the endothelium by platelet recruitment at regions of LDF, is required for priming of the endothelium for subsequent NF-κB activation, myeloid cell recruitment and atherosclerosis.

TRPM2 deficiency in mice protects against atherosclerosis by inhibiting TRPM2-CD36 inflammatory axis in macrophages.

Atherosclerosis is the major cause of ischemic heart disease and stroke, the leading causes of mortality worldwide. The central pathological features of atherosclerosis include macrophage infiltration and foam cell formation. However, the detailed mechanisms regulating these two processes remain unclear. Here we show that oxidative stress-activated Ca2+-permeable transient receptor potential melastatin 2 (TRPM2) plays a critical role in atherogenesis. Both global and macrophage-specific Trpm2 deletion protect Apoe -/- mice against atherosclerosis. Trpm2 deficiency reduces oxidized low-density lipoprotein (oxLDL) uptake by macrophages, thereby minimizing macrophage infiltration, foam cell formation and inflammatory responses. Activation of the oxLDL receptor CD36 induces TRPM2 activity, and vice versa. In cultured macrophages, TRPM2 is activated by CD36 ligands oxLDL and thrombospondin-1 (TSP1), and deleting Trpm2 or inhibiting TRPM2 activity suppresses the activation of CD36 signaling cascade induced by oxLDL and TSP1. Our findings establish the TRPM2-CD36 axis as a molecular mechanism underlying atherogenesis, and suggest TRPM2 as a potential therapeutic target for atherosclerosis.

A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue.

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

Targeting p21Cip1 highly expressing cells in adipose tissue alleviates insulin resistance in obesity.

Insulin resistance is a pathological state often associated with obesity, representing a major risk factor for type 2 diabetes. Limited mechanism-based strategies exist to alleviate insulin resistance. Here, using single-cell transcriptomics, we identify a small, critically important, but previously unexamined cell population, p21Cip1 highly expressing (p21high) cells, which accumulate in adipose tissue with obesity. By leveraging a p21-Cre mouse model, we demonstrate that intermittent clearance of p21high cells can both prevent and alleviate insulin resistance in obese mice. Exclusive inactivation of the NF-κB pathway within p21high cells, without killing them, attenuates insulin resistance. Moreover, fat transplantation experiments establish that p21high cells within fat are sufficient to cause insulin resistance in vivo. Importantly, a senolytic cocktail, dasatinib plus quercetin, eliminates p21high cells in human fat ex vivo and mitigates insulin resistance following xenotransplantation into immuno-deficient mice. Our findings lay the foundation for pursuing the targeting of p21high cells as a new therapy to alleviate insulin resistance.

Appearance of claudin-5+ leukocyte subtypes in the blood and CNS during progression of EAE.

BACKGROUND: Tight junctions (TJs) are membrane specializations characteristic of barrier-forming membranes, which function to seal the aqueous pathway between endothelial cells or epithelial cells and, thereby, obstruct intercellular solute and cellular movement. However, previous work from our laboratory found that claudin-5 (CLN-5), a TJ protein prominent at the blood-brain barrier (BBB), was also detected, ectopically, on leukocytes (CLN-5+) in the blood and central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a neuroinflammatory, demyelinating disease that is a model for multiple sclerosis. CLN-5 was further shown to be transferred from endothelial cells to circulating leukocytes during disease, prompting consideration this action is coupled to leukocyte transendothelial migration (TEM) into the CNS by fostering transient interactions between corresponding leukocyte and endothelial junctional proteins at the BBB. METHODS: To begin clarifying the significance of CLN-5+ leukocytes, flow cytometry was used to determine their appearance in the blood and CNS during EAE. RESULTS: Flow cytometric analysis revealed CLN-5+ populations among CD4 and CD8 T cells, B cells, monocytes and neutrophils, and these appeared with varying kinetics and to different extents in both blood and CNS. CLN-5 levels on circulating T cells further correlated highly with activation state. And, the percentage of CLN-5+ cells among each of the subtypes analyzed was considerably higher in CNS tissue than in blood, consistent with the interpretation that CLN-5+ leukocytes gain preferred access to the CNS. CONCLUSION: Several leukocyte subtypes variably acquire CLN-5 in blood before they enter the CNS, an event that may represent a novel mechanism to guide leukocytes to sites for paracellular diapedesis across the BBB.