Single-cell Western Blot Research Articles

ACKR1+ Endothelial Cells Mediate Leukocyte Infiltration and Synergize with SFRP2/ASPN+ Fibroblasts to Promote Skin Fibrosis in Systemic Sclerosis.

Skin fibrosis is the most typical pathological manifestation of systemic sclerosis (SSc) and localized scleroderma (LS) with unclear etiology and few effective treatments. Though excessive collagen secretion by fibroblasts is the primary cause of skin fibrosis, many lines of evidence suggested that vascular damage was the initiating event and various cell types along with fibroblasts worked together to contribute to the pathogenesis of skin fibrosis. We sought to explore the relationships between vascular endothelial cell lesions and immune cell infiltration, along with the cell-cell interactions among various cell types within the fibrotic skin ecosystem. Single-cell RNA-seq (10x Genomics) was performed on skin biopsies of 3 healthy donors and 7 SSc patients in Chinese. The additional 3 localized scleroderma patients' data from NCBI database (GSE160536) were integrated by Harmony. CellChat package (v1.5.0) was applied to analyze cell communication network. Transwell assay and subcutaneous bleomycin (BLM) injection in mice were used to explore the role of ACKR1 on immune cell infiltration. Milo single-cell western blot was applied to show the activation of fibroblast subclusters. A total of 62,295 cells were obtained and subpopulations of stromal and immune cells were identified. Interaction network analysis revealed that multiple chemokines secreted by macrophages, pericytes, and pro-inflammatory fibroblasts could bind with Duffy antigen/receptor for chemokines (ACKR1), which is highly expressed on ACKR1+ endothelial cells of lesion skin. Transwell assay revealed that over-expressed ACKR1 in HUVEC facilitated leukocyte infiltration under the treatment of IL8. The BLM mice showed enhanced ACKR1 expression, massive immune cell infiltration, and fibrosis in skin, which could be attenuated by ACKR1 inhibition. Furthermore, infiltrated macrophages with TGFB1 or PDGFB high production could activate SFRP2/ASPN+ fibroblasts to contribute to excessive accumulation of extracellular matrix (ECM), and the SOX4-ASPN axis plays an important role in the TGF-β signaling cascade and the etiology of skin fibrosis. Our results reveal that highly expressed ACKR1 in endothelial cells of fibrotic skin tissue promotes immune cell infiltration, and SFRP2/ASPN+ fibroblasts synergize to exacerbate skin fibrosis.

DropBlot: single-cell western blotting of chemically fixed cancer cells

Archived patient-derived tissue specimens play a central role in understanding disease and developing therapies. To address specificity and sensitivity shortcomings of existing single-cell resolution proteoform analysis tools, we introduce a hybrid microfluidic platform (DropBlot) designed for proteoform analyses in chemically fixed single cells. DropBlot serially integrates droplet-based encapsulation and lysis of single fixed cells, with on-chip microwell-based antigen retrieval, with single-cell western blotting of target antigens. A water-in-oil droplet formulation withstands the harsh chemical (SDS, 6 M urea) and thermal conditions (98 °C, 1-2 hr) required for effective antigen retrieval, and supports analysis of retrieved protein targets by single-cell electrophoresis. We demonstrate protein-target retrieval from unfixed, paraformaldehyde-fixed (PFA), and methanol-fixed cells. Key protein targets (HER2, GAPDH, EpCAM, Vimentin) retrieved from PFA-fixed cells were resolved and immunoreactive. Relevant to biorepositories, DropBlot profiled targets retrieved from human-derived breast tumor specimens archived for six years, offering a workflow for single-cell protein-biomarker analysis of sparing biospecimens.

Photosensitive Hydrogel with Temperature-Controlled Reversible Nano-Apertures for Single-Cell Protein Analysis.

Single cell western blot (scWB) is one of the most important methods for cellular heterogeneity profiling. However, current scWB based on conventional photoactive polyacrylamide hydrogel material suffers from the tradeoff between in-gel probing and separation resolution. Here, a highly sensitive temperature-controlled single-cell western blotting (tc-scWB) method is introduced, which is based on a thermo/photo-dualistic-sensitive polyacrylamide hydrogel, namely acrylic acid-functionalized graphene oxide (AFGO) assisted, N-isopropylacrylamide modified polyacrylamide (ANP) hydrogel. The ANP hydrogel is contracted at high-temperature to constrain protein band diffusion during microchip electrophoretic separation, while the gel aperture is expanded under low-temperature for better antibody penetration into the hydrogel. The tc-scWB method enables the separation and profiling of small-molecule-weight proteins with highly crosslinked gel (12% T) in SDS-PAGE. The tc-scWB is demonstrated on three metabolic and ER stress-specific proteins (CHOP, MDH2 and FH) in four pancreatic cell subtypes, revealing the expression of key enzymes in the Krebs cycle is upregulated with enhanced ER stress. It is found that ER stress can regulate crucial enzyme (MDH2 and FH) activities of metabolic cascade in cancer cells, boosting aerobic respiration to attenuate the Warburg effect and promote cell apoptosis. The tc-scWB is a general toolbox for the analysis of low-abundance small-molecular functional proteins at the single-cell level.

Immunoblot Analysis from Single Cells Using Milo™ Single-Cell Western Platform.

In this new era of precision medicine, characterization of single-cell subpopulations to better understand disease etiology is paramount. It is thus an opportune time to explore techniques that allow molecular analysis of single cells and to better understand the basis of pathogenesis of diseases like cancer. Single-cell western blotting is one such method that allows analysis of single cells at the protein level. In contrast to traditional western blotting, which relies heavily on bulk analysis of lysates generated from tissues and is often indicative of the population average, this technique allows analysis of lysates from single-cell subpopulations thereby providing a glimpse into cell heterogeneity. The method entails the use of a chip containing 30μm thick photoactivated polyacrylamide gel spotted with nearly 6400 microwells. Single cells loaded on the chip are captured in the microwells by passive gravity and are then lysed and electrophoresed using the MILO™ single-cell western platform. This method forgoes the use of transfer of proteins on a PVDF and a nitrocellulose membrane, as performed in traditional western blotting, and all other steps including probing of primary and fluorescent secondary antibodies against the protein of interest are performed directly on the chip. The proteins of interest can then be visualized by scanning a chip with the use of a microarray scanner. The entire procedure can be performed in as less as 4-6h, and thus this method provides several advantages over traditional western blotting.

Multiplex Protein Profiling by Low-Signal-Loss Single-Cell Western Blotting with Fluorescent-Quenching Aptamers.

Single-cell western blotting (scWB) is a prevalent technique for high-resolution protein analysis on low-abundance cell samples. However, the extensive signal loss during repeated antibody stripping precludes multiplex protein detection. Herein, we introduce Fluorescent-quenching Aptamer-based Single-cell Western Blotting (FAS-WB) for multiplex protein detection at single-cell resolution. The minimal size of aptamer probes allows rapid in-gel penetration, diffusion, and elution. Meanwhile, the fluorophore-tagged aptamers, coordinated with complementary quenching strands, avoid the massive signal loss conventionally caused by antibody stripping during repeated staining. Such a strategy also facilitates multiplex protein analysis with a limited number of fluorescent tags. We demonstrated FAS-WB for co-imaging four biomarker proteins (EpCAM, PTK7, HER2, CA125) at single-cell resolution with lower signal loss and enhanced signal-to-noise ratio compared to conventional antibody-based scWB. Being more time-saving (less than 25 min per cycle) and economical (1/1000 cost of conventional antibody probes), FAS-WB offers a highly efficient platform for profiling multiplex proteins at single-cell resolution.

Xinmaikang-mediated mitophagy attenuates atherosclerosis via the PINK1/Parkin signaling pathway

BackgroundThe Chinese herbal compound Xinmaikang (XMK) is effective in treating atherosclerosis (AS), although the associated mechanisms of action remain unclear. We hypothesize that XMK increases mitophagy via the PINK1/Parkin signaling pathway and decreases reactive oxygen species (ROS), thus treating AS. PurposeTo explore the above-mentioned mechanisms of action of XMK in AS. Materials and methodsUltra-performance liquid chromatography assay was performed to clarify the composition of XMK. A 16-week high-fat diet was fed to APOE−/− mice to form an AS model. Next, mice were given XMK(0.95 g/kg/d, 1.99 g/kg/d, 3.98 g/kg/d, i.g.) or Atorvastatin(3 mg/kg/d, i.g.) or Rapamycin(4 mg/kg/d, i.p.) or XMK with Mdivi-1(40 mg/kg/d, i.p.) or an equivalent amount of normal saline for 4 weeks. Then mice were examined for AS plaque area, lesion area, collagen fiber, pro-inflammatory cytokines, lipid level, ROS level and mitophagy level. We assessed AS using Oil Red O, hematoxylin and eosin, and Sirius red staining, as well as ROS measurements. Mitophagy was evaluated by transmission electron microscopy, real-time quantitative polymerase chain reaction (RT-qPCR), Western blot, single-cell Western blot, and immunofluorescence staining. In vitro, by oxidizing low-density lipoprotein, formation of RAW264.7 macrophage-derived foam cells induced. we induced foam cell formation in RAW264.7 macrophages. Then cells were incubated with XMK-medicated serum with or without Mdivi-1. We examined foam cell formation, ROS level, mitophagy level in cells. Finally, we knocked down the PINK1, and examined foam cell formation and PINK1/Parkin level in RAW264.7 macrophages. ResultsUPLC analysis revealed 102 main ingredients in XMK. In vivo, XMK at medium-dose or high-dose significantly reduced AS plaques, lipids, pro-inflammatory cytokines, and ROS and increased mitophagy. In further study, Single-cell western blot showed that mitophagy level in macrophages sorted from AS mice was lower than the control mice. While XMK improved mitophagy level. In vitro, XMK reduced foam cell formation and ROS and increased mitophagy. When PINK1 was knocked down, XMK's effects on foam cell formation and PINK1/Parkin pathway activation were reduced. ConclusionThe study shows that XMK is effective against AS by mediating macrophage mitophagy via the PINK1/Parkin signaling pathway. For the treatment of AS and drug discovery, it provides an experimental basis and target.

Single-Cell Western Blotting to Determine Cell Heterogeneity

Single-Cell Western Blotting to Determine Cell Heterogeneity

Single-Cell Western Blotting to Determine Cell Heterogeneity

Single-Cell Western Blotting to Determine Cell Heterogeneity

Presence of immunogenic alternatively spliced insulin gene product in human pancreatic delta cells

Aims/hypothesisTranscriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets.MethodsAlternative splicing of insulin pre-mRNA was determined by PCR analysis performed on human islet RNA and single-cell RNA-seq analysis. Antisera were generated to detect insulin variants in human pancreatic tissue using immunohistochemistry, electron microscopy and single-cell western blot to confirm the expression of insulin variants. Cytotoxic T lymphocyte (CTL) activation was determined by MIP-1β release.ResultsWe identified an alternatively spliced INS product. This variant encodes the complete insulin signal peptide and B chain and an alternative C-terminus that largely overlaps with a previously identified defective ribosomal product of INS. Immunohistochemical analysis revealed that the translation product of this INS-derived splice transcript was detectable in somatostatin-producing delta cells but not in beta cells; this was confirmed by light and electron microscopy. Expression of this alternatively spliced INS product activated preproinsulin-specific CTLs in vitro. The exclusive presence of this alternatively spliced INS product in delta cells may be explained by its clearance from beta cells by insulin-degrading enzyme capturing its insulin B chain fragment and a lack of insulin-degrading enzyme expression in delta cells.Conclusions/interpretationOur data demonstrate that delta cells can express an INS product derived from alternative splicing, containing both the diabetogenic insulin signal peptide and B chain, in their secretory granules. We propose that this alternative INS product may play a role in islet autoimmunity and pathology, as well as endocrine or paracrine function or islet development and endocrine destiny, and transdifferentiation between endocrine cells. INS promoter activity is not confined to beta cells and should be used with care when assigning beta cell identity and selectivity.Data availabilityThe full EM dataset is available via www.nanotomy.org (for review: http://www.nanotomy.org/OA/Tienhoven2021SUB/6126-368/). Single-cell RNA-seq data was made available by Segerstolpe et al [13] and can be found at https://sandberglab.se/pancreas. The RNA and protein sequence of INS-splice was uploaded to GenBank (BankIt2546444 INS-splice OM489474).Graphical abstract

2058-P: Single-Cell Transcriptome Atlas of the Aging Mouse Pancreatic Islet Reveals Resilience to Diet-Induced Obesity

Aging is a key risk factor for type 2 diabetes (T2D). We examined aging mouse pancreatic islets by single-cell RNA-seq sampled across young (1 month), middle (13 months) to old (24 months) age groups. By applying single-cell Western blotting, we further verified that aged mice had increased islet beta-cell percentage, which was accompanied by an increased in vivo insulin secretion. Young and old groups were fed with a six-month high-fat diet. Age-associated enhancements in insulin secretion prevented old mice from diet-induced glucose intolerance. Together, this single-cell atlas allows a comprehensive exploration of all transcriptional states in the aging islets and provides insight into the pathogenesis of T2D. Importantly, our findings indicate that age-dependent hyperinsulinemia protects against diet-induced obesity in mice and exhibit the resilience of the islet in response to stressors. Disclosure J. Nie: None. N. Musi: None. Funding American Federation for Aging Research; San Antonio Claude D. Pepper Older Americans Independence Center (P30AG044271)

Advances of Single-Cell Protein Analysis.

Proteins play a significant role in the key activities of cells. Single-cell protein analysis provides crucial insights in studying cellular heterogeneities. However, the low abundance and enormous complexity of the proteome posit challenges in analyzing protein expressions at the single-cell level. This review summarizes recent advances of various approaches to single-cell protein analysis. We begin by discussing conventional characterization approaches, including fluorescence flow cytometry, mass cytometry, enzyme-linked immunospot assay, and capillary electrophoresis. We then detail the landmark advances of microfluidic approaches for analyzing single-cell protein expressions, including microfluidic fluorescent flow cytometry, droplet-based microfluidics, microwell-based assay (microengraving), microchamber-based assay (barcoding microchips), and single-cell Western blotting, among which the advantages and limitations are compared. Looking forward, we discuss future research opportunities and challenges for multiplexity, analyte, throughput, and sensitivity of the microfluidic approaches, which we believe will prompt the research of single-cell proteins such as the molecular mechanism of cell biology, as well as the clinical applications for tumor treatment and drug development.

IFN-γ-induced nitric oxide leads to inhibition of Th17 differentiation via induction of iNOS and nitration of ROR-γ T

Abstract Elucidating the molecular mechanisms involved in pleotropic cytokine signaling can lead to novel treatment approaches for autoimmune diseases that target proinflammatory functions while maintaining immune-regulatory functions. To that end, we sought to further investigate the immune-regulatory roles of the pleotropic cytokine IFN-γ. Previous studies demonstrated that IFN-γ prevents the differentiation of pathogenic Th17 cells. In addition, nitric oxide generated via inducible nitric oxide synthase (iNOS) prevents differentiation of Th17 cells by nitration of ROR-γT. However, it remains unresolved whether IFN-γ prevents Th17 differentiation directly via inducing nitric oxide (NO) and nitration of ROR-γT. In line with previous observations, we found an increase in the frequency of autoreactive Th17 cells and a decrease in iNOS expression and NO in the absence of IFN-γ in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Additionally, the increase in Th17 cells in the absence of IFN-γ was reversible both in vitro and in vivo with treatment with a NO donor. This indicates a role for IFN-γ in inhibiting the differentiation of Th17 cells via iNOS-derived NO. We are currently elucidating the underlying mechanisms of NO mediated suppression and the molecular link between ROR-γt-nitration and decreased Th17 differentiation by flow cytometry, single-cell western blotting of key transcription factors, and in vivo adoptive transfer studies. Our studies may lead to a better understanding of the role of IFN-g and NO/iNOS axis in Th17 cell differentiation and autoimmune diseases.

Separation-encoded microparticles for single-cell western blotting.

Direct measurement of proteins from single cells has been realized at the microscale using microfluidic channels, capillaries, and semi-enclosed microwell arrays. Although powerful, these formats are constrained, with the enclosed geometries proving cumbersome for multistage assays, including electrophoresis followed by immunoprobing. We introduce a hybrid microfluidic format that toggles between a planar microwell array and a suspension of microparticles. The planar array is stippled in a thin sheet of polyacrylamide gel, for efficient single-cell isolation and protein electrophoresis of hundreds-to-thousands of cells. Upon mechanical release, array elements become a suspension of separation-encoded microparticles for more efficient immunoprobing due to enhanced mass transfer. Dehydrating microparticles offer improved analytical sensitivity owing to in-gel concentration of fluorescence signal for high-throughput single-cell targeted proteomics.

The Relationships between Target Gene Transduction, Engraftment of HSCs and RBC Physiology in Sickle Cell Disease Gene Therapy

The Relationships between Target Gene Transduction, Engraftment of HSCs and RBC Physiology in Sickle Cell Disease Gene Therapy

Co-Expression of SYK and ZAP70 Subverts Negative B-Cell Selection and Enables Oncogenic Signaling in Multiple B-Cell Malignancies

B-cells are under intense selective pressure to eliminate autoreactive or premalignant clones. B-cell receptor (BCR) signals are required for survival, however, BCR-signaling exceeding maximum thresholds often reflects signaling from an autoreactive BCR or a transforming oncogene and triggers negative selection and cell death. The tyrosine kinase SYK initiates BCR-downstream signaling in B-cells while its close relative ZAP70 is almost exclusively expressed in T-cells. Interestingly, the segregation of SYK to B-cells and ZAP70 to T-cells is less confined in malignant lymphopoiesis suggesting that the balance of these related kinases may alter signaling output in disease and contribute to development of leukemia. As previously shown in B-cell chronic lymphocytic leukemia (B-CLL), we identified aberrant ZAP70 expression as a frequent feature in multiple other B-cell malignancies that depend on survival signals from a functional (pre-) BCR (E2A-PBX1+ pre-B ALL, and mantle cell lymphoma) or harbor oncogenic mimics of the BCR (BCR-ABL1+ B-ALL). Studying SYK and ZAP70 expression by single-cell Western blot, co-expression of the two tyrosine kinases was extremely rare in normal B- and T-cell populations. In contrast, >50% of tumor B-cells in mantle cell lymphoma, pre-B ALL and CLL co-expressed SYK and ZAP70. Despite their structural similarities, genetic deletion and engineered reconstitution of SYK and ZAP70 in human B-cell lymphoma cells revealed striking functional differences. Proximity-dependent biotin identification (BioID) analyses identified that SYK, but not ZAP70, engaged the PI3K pathway via interaction with CD19. Consistent with this, reconstitution with SYK and SYK-ZAP70 but not ZAP70 alone promoted survival and proliferation. Detailed analysis of BCR-mediated cascades in lymphoma cells expressing SYK, ZAP70 or SYK-ZAP70 established that ZAP70 is only weakly efficient at propagating BCR-mediated calcium and downstream pathway activation in B-cells. Strikingly, co-expression of ZAP70 with SYK resulted in re-wired BCR-signaling of intermediate strength: compared to cells expressing only SYK, SYK-ZAP70 co-expressing cells had markedly reduced activation of the BLNK-BTK-PLCγ pathway, further reflected in BCR-induced Ca2+ signaling with delayed onset, lower amplitude but longer duration. In this way, we speculated that SYK and ZAP70 may be present within close proximity at the apex of BCR-initiated interactions, and hence compete for downstream substrates resulting in a re-wiring of classic signaling programs propagated normally by SYK. To explore this, we utilized proximity ligation assays (PLA) to monitor the proximity of SYK and ZAP70 in resting or BCR-stimulated B-cells, and found that SYK and ZAP70 co-exist within close proximity consistent with the view that varying levels of these kinases may alter B-cell signaling output. Functional experiments further showed that phosphomimetic activation of SYK, but not ZAP70, induced hyperactivation of PI3K-signaling and acute BTK-mediated cell death in pre-B ALL cells. In line with altered BCR-signaling strength and quality in SYK and ZAP70 co-expressing cells, over-expression of Zap70 in pre-B ALL cells rescued auto-immune checkpoint activation induced by hyper-activation of BCR-associated signaling. To study functional consequences of SYK-ZAP70 co-expression during normal B-cell development, we generated a novel knock in Zap-70+/Mb1-Cre+mouse model, to induce conditional expression of Zap70 in the B cell compartment from the proB stage. Consistent with compromised central tolerance checkpoints, Syk-Zap70 co-expressing pro/pre-B and immature B-cells had reduced spontaneous apoptosis rates and gave rise to autoantibody production against multiple self-antigens. Importantly, our findings highlight a previously unrecognized role for ZAP70 in oncogenic BCR-signaling and we conclude that the co-expression of ZAP70 mitigates the ability of SYK, downstream of an autoreactive BCR or a transforming oncogene, to trigger negative B-cell selection and cell death (Figure 1). Disclosures Weinstock: Celgene: Research Funding. Meffre:AbbVie: Consultancy, Other: Grant.

In Situ Single-Cell Western Blot on Adherent Cell Culture.

Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single-cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin-functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one-step copolymerization process that creates protein-decorated microwells. After single-cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell-based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab-gel western blot, while revealing cell-to-cell heterogeneity in stress responses.

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages.

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Effects of salt‐loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging

Salt-loading (SL) impairs GABAA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K+ /Cl- co-transporter2 (KCC2) and up-regulating Na+ /K+ /Cl- co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl- ]i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl- ]i in response to focal application of the GABAA agonist muscimol (100μmolL-1 ). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P<0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P<0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and β-actin. Neurones that did not respond during chloride imaging did not show KCC2 and β-actin protein expression. The KCC2 antagonist (VU0240551,10μmolL-1 ) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10μmolL-1 ) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl- influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50μmolL-1 ) and protein kinase inhibitor (K252a) (100nmolL-1 ) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl- ]i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.

Single-cell analysis reveals fibroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds

During wound healing in adult mouse skin, hair follicles and then adipocytes regenerate. Adipocytes regenerate from myofibroblasts, a specialized contractile wound fibroblast. Here we study wound fibroblast diversity using single-cell RNA-sequencing. On analysis, wound fibroblasts group into twelve clusters. Pseudotime and RNA velocity analyses reveal that some clusters likely represent consecutive differentiation states toward a contractile phenotype, while others appear to represent distinct fibroblast lineages. One subset of fibroblasts expresses hematopoietic markers, suggesting their myeloid origin. We validate this finding using single-cell western blot and single-cell RNA-sequencing on genetically labeled myofibroblasts. Using bone marrow transplantation and Cre recombinase-based lineage tracing experiments, we rule out cell fusion events and confirm that hematopoietic lineage cells give rise to a subset of myofibroblasts and rare regenerated adipocytes. In conclusion, our study reveals that wounding induces a high degree of heterogeneity among fibroblasts and recruits highly plastic myeloid cells that contribute to adipocyte regeneration.

20 - PKM2 S-glutathionylation promotes the reprogramming of macrophage metabolism and activation states

Introduction Pyruvate Kinase Isoform M2 (PKM2) regulates glycolysis in monocytes and macrophages and is induced and S-glutathionylated in response to diet-induced metabolic stress. We hypothesized that PKM2 S-glutathionylation promotes PKM2 tetramer dissociation into glycolytically-inactive dimers and that nuclear translocation of these dimers activates HIF1a-dependent gene expression, inducing both glycolytic enzymes as well and pro-inflammatory proteins, reprogramming macrophages to a hyper-inflammatory phenotype. Methods Bone marrow derived macrophages (BMDM) overexpressing glutaredoxin-1-EGFP (Grx1) or EGFP only were exposed for 36 h to metabolic stress (high glucose plus native human LDL). PKM2 expression and phosphorylation state (Tyr105) were assessed in cell lysates by Western blot analysis. PKM2 S-glutathionylation was quantified using the Biotin-switch assay. PKM2 oligomerization state was measured by non-reducing SDS-PAGE and Western blot analysis. To confirm the role of PKM2 S-glutathionylation on metabolic reprogramming of monocytes in vivo, we conducted lentiviral gene transfer experiments in mice using CD68 promoter-driven Grx1 and EGFP expression. Mice were fed a HFD for 10 week to induce monocyte priming. We purified blood monocytes from EGFPMactg and Grx1Mactg mice and determined by single-cell Western blot PKM2 induction and phosphorylation, GLUT1, 14-3-3z and IL-1b expression. Results PKM2 induction, -glutathionylation, and phosphorylation were all enhanced in metabolically stressed BMDMs but prevented in Grx1-overexpressing BMDMs. The dimer/tetramer ratio was increased in response to metabolic stress as was HIF1a-dependent expression of GLUT1 and IL-1b. Compared to monocytes isolated from HFD-fed mice, monocyte from Grx1Mactg mice showed reduced priming, reduced expression of PKM2, GLUT1 and IL-1b and diminished PKM2 phosphorylation. Conclusion Metabolic stress-induced PKM2 S-glutathionylation promotes PKM2 tetramer dissociation, phosphorylation and subsequent nuclear translocation, which, via HIF1a-dependent expression of glycolytic enzymes and inflammatory cytokines, reprograms monocyte-derived macrophages into a hyper-inflammatory phenotype. This mechanism may contribute to chronic inflammatory diseases associated with metabolic disorders.