Homeostatic Conditions Research Articles

Cellular Proteomic Profiling Using Proximity Labeling by TurboID-NES in Microglial and Neuronal Cell Lines

Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, invivo proteomic labeling with proximity-dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without the need for cellular isolation. To complement invivo proximity labeling approaches, invitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole-cell proteome and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID labeled endolysosome, translation, vesicle, and signaling proteins in BV2 microglia and synaptic, neuron projection, and microtubule proteins in N2A neurons. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2Acells and did not affect lipopolysaccharide-mediated cytokine production or resting cellular respiration in BV2 cells. MS analysis of the microglial biotin-labeled proteins captured the impact of lipopolysaccharide treatment (>500 differentially abundant proteins) including increased canonical proinflammatory proteins (Il1a, Irg1, and Oasl1) and decreased anti-inflammatory proteins (Arg1 and Mgl2).

Continuous ex vivo glucose sensing in human physiological fluids using an enzymatic sensor in a vein replica

Managing blood glucose can affect important clinical outcomes during the intraoperative phase of surgery. However, currently available instruments for glucose monitoring during surgery are few and not optimized for the specific application. Here we report an attempt to exploit an enzymatic sensor in a vein replica that could continuously monitor glucose level in an authentic human bloodstream. First, detailed investigations of the superficial venous systems of volunteers were carried out using ocular and palpating examinations, as well as advanced ultrasound measurements. Second, a tubular glucose-sensitive biosensor mimicking a venous system was designed and tested. Almost ideal linear dependence of current output on glucose concentration in phosphate buffer saline was obtained in the range 2.2–22.0 mM, whereas the dependence in human plasma was less linear. Finally, the developed biosensor was investigated in whole blood under homeostatic conditions. A specific correlation was found between the current output and glucose concentration at the initial stage of the biodevice operation. However, with time, blood coagulation during measurements negatively affected the performance of the biodevice. When the experimental results were remodeled to predict the response without the influence of blood coagulation, the sensor output closely followed the blood glucose level.

Abstract LB344: Complement-mediated signaling during cross-presentation of tumor antigen augments T cell responses

Abstract Production of complement C3a and C5a locally at the immune synapse by activated dendritic cells (DCs) enhances T cell responses in multiple experimental models. Complement signaling through cognate C3a receptor (C3aR) and C5a receptor (C5aR) modulate effector T cell stability, proliferation and differentiation. We have previously shown that complement C3 plays a mechanistic role in driving synergistic anti-tumor effects of blocking indoleamine 2,3-dioxygenase (IDO) during radiation and chemotherapy in a murine brain tumor model that expresses the gp10025-33 peptide antigen. We hypothesize that, when anti-tumor responses are allowed to happen, complement C3 production by local DCs acts as a costimulation factor, operating through C3aR and C5aR on the T cell surface to allow optimal T cell activation and proliferation. To study the role of complement C3 as a costimulation factor during T cell responses against a nominal tumor antigen (gp10025-33 peptide, expressed by B16F10 melanoma), we used T cells from syngeneic pmel-1 mice (TCR transgenic with CD8 T cells that recognize the cross-presented gp10025-33 peptide) co-cultured with gp10025-33 peptide and dendritic cells from either wild-type (WT) or C3-defiecient (C3-KO) mice. In addition, we used in vitro culture conditions where gp10025-33 tumor peptide was in stringent limited supply to model homeostatic conditions of the tumor microenvironment. We found that, when stimulated by DCs from C3-KO mice, pmel-1 CD8 T cells had decreased activation marker expression, proliferation, and cytokine effector function relative to stimulation by DCs from WT mice. Next, we bred pmel-1 mice onto a syngeneic background deficient in receptors for both C3a and C5a complement factors (C3aR-KO/C5aR-KO double knock-out mice). We used CD8 T cells from the resulting mouse strain (pmel-1/C3aR-KO/C5aR-KO) co-cultured with WT DCs to study the role of complement signaling in T cells during responses to tumor antigen. Using the same stringent antigen conditions, CD8 T cells from the pmel-1/C3aR-KO/C5aR-KO mice had decreased activation marker expression and proliferation, and differences in cytokine function compared to CD8 T cells from the pmel-1 mice. These findings suggest that lack of local complement production by the DCs in the tumor microenvironment may inhibit anti-tumor immune responses, and that complement signaling through C3aR and C5aR on T cells may play an important role during such anti-tumor responses. Citation Format: Caryn L. Bird, Gabriela A. Pacholczyk, Theodore S. Johnson. Complement-mediated signaling during cross-presentation of tumor antigen augments T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB344.

TGF-BETA STIMULATES PERIOSTEAL OSTEOGENIC ACTIVITY

Osteoprogenitors on the inner layer of periosteum are the major cellular contributors to appositional bone growth and bone repair by callus formation. Previous work showed that periosteal-derived cells have little or no osteogenic activity under standard in vitro osteogenic culture conditions. This study was conducted to determine what growth factor(s) can activate periosteal osteogenic capacity.This study was conducted with IACUC approval. Periosteum from five equine donors was digested in collagenase for 3-4 hours at 37C. Isolated periosteal cells were maintained in DMEM/10% FBS medium and exposed to PDGF, Prostaglandin E2, BMP-2 and TGF-b3 at a range of concentrations for 72 hours. Changes in osteogenic gene expression (Runx2, OSX and ALP) were measured by qPCR. Periosteal cells were pre-treated with TGF-b3 or maintained in control medium were transferred into basal or osteogenic medium. Osteogenic status was assessed by Alizarin Red staining for mineralized matrix, ALP enzymatic activity and induction of osteogenic genes.PDGF, PgE2 and BMP-2 had little impact on expression of osteogenic markers by periosteal cells. In contrast, TGF-b3 stimulated significant increases in Osterix (over 100-fold) ALP expression (over 70-fold). Pre-treating periosteal cells with TGF-b3 for 72 hours stimulated rapid cell aggregation and aggregate mineralization once cells were transferred to osteogenic medium, while cells not exposed to TGF-b3 exhibited minimal evidence of osteogenic activity.This study indicate that TGF-b signaling is vital for periosteal osteogenic activity. Transient ‘priming’ of periosteal cells through TGF-b exposure was sufficient to activate subsequent osteogenesis without requiring ongoing growth factor stimulation. TGF beta ligands are secreted by many cell types, including periosteal progenitors and osteocytes, providing opportunities for both autocrine and paracrine pathways to regulate periosteal bone formation under homeostatic and reparative conditions.

Intestinal α-Defensins Play a Minor Role in Modulating the Small Intestinal Microbiota Composition as Compared to Diet.

The intestinal microbiota is at the interface between the host and its environment and thus under constant exposure to host-derived and external modulators. While diet is considered to be an important external factor modulating microbiota composition, intestinal defensins, one of the major classes of antimicrobial peptides, have been described as key host effectors that shape the gut microbial community. However, since dietary compounds can affect defensin expression, thereby indirectly modulating the intestinal microbiota, their individual contribution to shaping gut microbiota composition remains to be defined. To disentangle the complex interaction among diet, defensins, and small-intestinal microbiota, we fed wild-type (WT) mice and mice lacking functionally active α-defensins (Mmp7-/- mice) either a control diet or a Western-style diet (WSD) that is rich in saturated fat and simple carbohydrates but low in dietary fiber. 16S rDNA sequencing and robust statistical analyses identified that bacterial composition was strongly affected by diet while defensins had only a minor impact. These findings were independent of sample location, with consistent results between the lumen and mucosa of the jejunum and ileum, in both mouse genotypes. However, distinct microbial taxa were also modulated by α-defensins, which was supported by differential antimicrobial activity of ileal protein extracts. As the combination of WSD and defensin deficiency exacerbated glucose metabolism, we conclude that defensins only have a fine-tuning role in shaping the small-intestinal bacterial composition and might instead be important in protecting the host against the development of diet-induced metabolic dysfunction. IMPORTANCE Alterations in the gut microbial community composition are associated with many diseases, and therefore identifying factors that shape the microbial community under homeostatic and diseased conditions may contribute to the development of strategies to correct a dysbiotic microbiota. Here, we demonstrate that a Western-style diet, as an extrinsic parameter, had a stronger impact on shaping the small intestinal bacterial composition than intestinal defensins, as an intrinsic parameter. While defensins have been previously shown to modulate bacterial composition in young mice, our study supplements these findings by showing that defensins may be less important in adult mice that harbor a mature microbial community. Nevertheless, we observed that defensins did affect the abundance of distinct bacterial taxa in adult mice and protected the host from aggravated diet-induced glucose impairments. Consequently, our study uncovers a new angle on the role of intestinal defensins in the development of metabolic diseases in adult mice.

CD74: a prospective marker for reactive microglia?

CD74: a prospective marker for reactive microglia?

Mast cells in type 2 skin inflammation: Maintenance and function.

Mast cells (MCs) are immune cells residing in tissues and playing indispensable roles in maintaining homeostasis and inflammatory states. Skin lesions associated with atopic dermatitis (AD) and type 2 skin inflammation display an increment in MCs, which have both pro- and anti-inflammatory effects. The direct and indirect activations of skin MCs by environmental factors such as Staphylococcus aureus can instigate type 2 skin inflammation in AD with poorly understood mechanisms. Furthermore, both IgE-dependent and -independent degranulation of MCs contribute to pruritus in AD. Conversely, MCs suppress type 2 skin inflammation by promoting Treg expansion through IL-2 secretion in the spleen. Moreover, skin MCs can upregulate gene expression involved in skin barrier function, thus mitigating AD-like inflammation. These functional variances of MCs in AD could stem from differences in experimental systems, their localization, and origins. In this review, we will focus on how MCs are maintained in the skin under homeostatic and inflammatory conditions, and how they are involved in the pathogenesis of type 2 skin inflammation.

Mitigation of Cardiovascular Disease and Toxicity through NRF2 Signalling.

Cardiovascular toxicity and diseases are phenomena that have a vastly detrimental impact on morbidity and mortality. The pathophysiology driving the development of these conditions is multifactorial but commonly includes the perturbance of reactive oxygen species (ROS) signalling, iron homeostasis and mitochondrial bioenergetics. The transcription factor nuclear factor erythroid 2 (NFE2)-related factor 2 (NRF2), a master regulator of cytoprotective responses, drives the expression of genes that provide resistance to oxidative, electrophilic and xenobiotic stresses. Recent research has suggested that stimulation of the NRF2 signalling pathway can alleviate cardiotoxicity and hallmarks of cardiovascular disease progression. However, dysregulation of NRF2 dynamic responses can be severely impacted by ageing processes and off-target toxicity from clinical medicines including anthracycline chemotherapeutics, rendering cells of the cardiovascular system susceptible to toxicity and subsequent tissue dysfunction. This review addresses the current understanding of NRF2 mechanisms under homeostatic and cardiovascular pathophysiological conditions within the context of wider implications for this diverse transcription factor.

A tryptophan metabolite prevents depletion of circulating endothelial progenitor cells in systemic low-grade inflammation.

Chronic systemic inflammation reduces the bioavailability of circulating endothelial progenitor cells (EPCs). Indoleamine 2,3-dioxygenase 1 (IDO1), a key enzyme of immune tolerance catalyzing the initial step of tryptophan degradation along the so-called l-kynurenine (l-kyn) pathway, that is induced by inflammatory stimuli and exerts anti-inflammatory effects. A specific relationship between IDO1 activity and circulating EPC numbers has not yet been investigated. In this study, circulating EPCs were examined in mice treated with low doses of lipopolysaccharide (LPS) to mimic low-grade inflammation. Moreover, the association between IDO1 activity and circulating EPCs was studied in a cohort of 277 patients with variable systemic low-grade inflammation. Repeated low doses of LPS caused a decrease in circulating EPCs and l-kyn supplementation, mimicking IDO1 activation, significantly increased EPC numbers under homeostatic conditions preventing EPC decline in low-grade endotoxemia. Accordingly, in patients with variable systemic low-grade inflammation, there was a significant interaction between IDO1 activity and high-sensitivity C-reactive protein (hs-CRP) in predicting circulating EPCs, with high hs-CRP associated with significantly lower EPCs at low IDO1 activity but not at high IDO1 activity. Overall, these findings demonstrate that systemic low-grade inflammation reduces circulating EPCs. However, high IDO1 activity and l-kyn supplementation limit circulating EPC loss in low-grade inflammation.

The composition and roles of gastric stem cells in epithelial homeostasis, regeneration, and tumorigenesis.

The epithelial lining of the stomach undergoes rapid turnover to preserve its structural and functional integrity, a process driven by long-lived stem cells residing in the antral and corpus glands. Several subpopulations of gastric stem cells have been identified and their phenotypic and functional diversities linked to spatiotemporal specification of stem cells niches. Here, we review the biological features of gastric stem cells at various locations of the stomach under homeostatic conditions, as demonstrated by reporter mice, lineage tracing, and single cell sequencing. We also review the role of gastric stem cells in epithelial regeneration in response to injury. Moreover, we discuss emerging evidence demonstrating that accumulation of oncogenic drivers or alteration of stemness signaling pathways in gastric stem cells promotes gastric cancer. Given a fundamental role of the microenvironment, this review highlights the role reprogramming of niche components and signaling pathways under pathological conditions in dictating stem cell fate. Several outstanding issues are raised, such as the relevance of stem cell heterogeneity and plasticity, and epigenetic regulatory mechanisms, to Helicobacter pylori infection-initiated metaplasia-carcinogenesis cascades. With the development of spatiotemporal genomics, transcriptomics, and proteomics, as well as multiplexed screening and tracing approaches, we anticipate that more precise definition and characterization of gastric stem cells, and the crosstalk with their niche will be delineated in the near future. Rational exploitation and proper translation of these findings may bring forward novel modalities for epithelial rejuvenation and cancer therapeutics.

Eosinophilic esophagitis-associated epithelial remodeling may limit esophageal carcinogenesis.

Under homeostatic conditions, esophageal epithelium displays a proliferation/differentiation gradient that is generated as proliferative basal cells give rise to suprabasal cells then terminally differentiated superficial cells. This proliferation/differentiation gradient is often perturbed in esophageal pathologies. Basal cell hyperplasia may occur in patients with gastroesophageal reflux disease (GERD), a condition in which acid from the stomach enters the esophagus, or eosinophilic esophagitis (EoE), an emerging form of food allergy. While GERD is a primary risk factor for esophageal cancer, epidemiological data suggests that EoE patients do not develop esophageal cancer. In order to investigate the impact of EoE and esophageal cancer specifically on the cellular landscape of esophageal epithelium, we perform single cell RNA-sequencing in murine models of EoE and esophageal cancer, specifically esophageal squamous cell carcinoma (ESCC). We further evaluate modules of co-expressed genes in EoE- and ESCC-enriched epithelial cell clusters. Finally, we pair EoE and ESCC murine models to examine the functional relationship between these pathologies. In mice with either EoE or ESCC, we find expansion of cell populations as compared to normal esophageal epithelium. In mice with EoE, we detect distinct expansion of 4 suprabasal populations coupled with depletion of 2 basal populations. By contrast, mice with ESCC display unique expansion of 2 basal populations and 1 suprabasal population, as well as depletion of 2 suprabasal populations. Senescence, glucocorticoid receptor signaling, and granulocyte-macrophage colony-stimulating factor pathways are associated with EoE-enriched clusters while pathways associated with cell proliferation and metabolism are identified in ESCC-enriched clusters. Finally, our in vivo data demonstrate that exposure to EoE inflammation limits tumor burden of esophageal carcinogenesis. Our findings provide the first functional investigation of the relationship between EoE and esophageal cancer and suggest that esophageal epithelial remodeling events occurring in response to EoE inflammation may limit esophageal carcinogenesis. This investigation may have future implications for leveraging allergic inflammation-associated alterations in epithelial biology to prevent and/or treat esophageal cancer.

Molecular tracking of insulin resistance and inflammation development on visceral adipose tissue.

Visceral adipose tissue (VAT) is one of the most important sources of proinflammatory molecules in obese people and it conditions the appearance of insulin resistance and diabetes. Thus, understanding the synergies between adipocytes and VAT-resident immune cells is essential for the treatment of insulin resistance and diabetes. We collected information available on databases and specialized literature to construct regulatory networks of VAT resident cells, such as adipocytes, CD4+ T lymphocytes and macrophages. These networks were used to build stochastic models based on Markov chains to visualize phenotypic changes on VAT resident cells under several physiological contexts, including obesity and diabetes mellitus. Stochastic models showed that in lean people, insulin produces inflammation in adipocytes as a homeostatic mechanism to downregulate glucose intake. However, when the VAT tolerance to inflammation is exceeded, adipocytes lose insulin sensitivity according to severity of the inflammatory condition. Molecularly, insulin resistance is initiated by inflammatory pathways and sustained by intracellular ceramide signaling. Furthermore, our data show that insulin resistance potentiates the effector response of immune cells, which suggests its role in the mechanism of nutrient redirection. Finally, our models show that insulin resistance cannot be inhibited by anti-inflammatory therapies alone. Insulin resistance controls adipocyte glucose intake under homeostatic conditions. However, metabolic alterations such as obesity, enhances insulin resistance in adipocytes, redirecting nutrients to immune cells, permanently sustaining local inflammation in the VAT.

Epithelial Responses in Radiation-Induced Lung Injury (RILI) Allow Chronic Inflammation and Fibrogenesis.

Radiation models, such as whole thorax lung irradiation (WTLI) or partial-body irradiation (PBI) with bone-marrow sparing, have shown that affected lung tissue displays a continual progression of injury, often for months after the initial insult. Undoubtably, a variety of resident and infiltrating cell types either contribute to or fail to resolve this type of progressive injury, which in lung tissue, often develops into lethal and irreversible radiation-induced pulmonary fibrosis (RIPF), indicating a failure of the lung to return to a homeostatic state. Resident pulmonary epithelium, which are present at the time of irradiation and persist long after the initial insult, play a key role in the maintenance of homeostatic conditions in the lung and have often been described as contributing to the progression of radiation-induced lung injury (RILI). In this study, we took an unbiased approach through RNA sequencing to determine the in vivo response of the lung epithelium in the progression of RIPF. In our methodology, we isolated CD326+ epithelium from the lungs of 12.5 Gy WTLI C57BL/6J female mice (aged 8-10 weeks and sacrificed at regular intervals) and compared irradiated and non-irradiated CD326+ cells and whole lung tissue. We subsequently verified our findings by qPCR and immunohistochemistry. Transcripts associated with epithelial regulation of immune responses and fibroblast activation were significantly reduced in irradiated animals at 4 weeks postirradiation. Additionally, alveolar type-2 epithelial cells (AEC2) appeared to be significantly reduced in number at 4 weeks and thereafter based on the diminished expression of pro-surfactant protein C (pro-SPC). This change is associated with a reduction of Cd200 and cyclooxygenase 2 (COX2), which are expressed within the CD326 populations of cells and function to suppress macrophage and fibroblast activation under steady-state conditions, respectively. These data indicate that either preventing epithelial cell loss that occurs after irradiation or replacing important mediators of immune and fibroblast activity produced by the epithelium are potentially important strategies for preventing or treating this unique injury.

Epithelial Responses in Radiation-Induced Lung Injury (RILI) Allow Chronic Inflammation and Fibrogenesis.

Radiation models, such as whole thorax lung irradiation (WTLI) or partial-body irradiation (PBI) with bone-marrow sparing, have shown that affected lung tissue displays a continual progression of injury, often for months after the initial insult. Undoubtably, a variety of resident and infiltrating cell types either contribute to or fail to resolve this type of progressive injury, which in lung tissue, often develops into lethal and irreversible radiation-induced pulmonary fibrosis (RIPF), indicating a failure of the lung to return to a homeostatic state. Resident pulmonary epithelium, which are present at the time of irradiation and persist long after the initial insult, play a key role in the maintenance of homeostatic conditions in the lung and have often been described as contributing to the progression of radiation-induced lung injury (RILI). In this study, we took an unbiased approach through RNA sequencing to determine the in vivo response of the lung epithelium in the progression of RIPF. In our methodology, we isolated CD326+ epithelium from the lungs of 12.5 Gy WTLI C57BL/6J female mice (aged 8-10 weeks and sacrificed at regular intervals) and compared irradiated and non-irradiated CD326+ cells and whole lung tissue. We subsequently verified our findings by qPCR and immunohistochemistry. Transcripts associated with epithelial regulation of immune responses and fibroblast activation were significantly reduced in irradiated animals at 4 weeks postirradiation. Additionally, alveolar type-2 epithelial cells (AEC2) appeared to be significantly reduced in number at 4 weeks and thereafter based on the diminished expression of pro-surfactant protein C (pro-SPC). This change is associated with a reduction of Cd200 and cyclooxygenase 2 (COX2), which are expressed within the CD326 populations of cells and function to suppress macrophage and fibroblast activation under steady-state conditions, respectively. These data indicate that either preventing epithelial cell loss that occurs after irradiation or replacing important mediators of immune and fibroblast activity produced by the epithelium are potentially important strategies for preventing or treating this unique injury.

Transcriptional control of ILC identity.

Innate lymphoid cells (ILCs) are heterogeneous innate immune cells which participate in host defense, mucosal repair and immunopathology by producing effector cytokines similarly to their adaptive immune cell counterparts. The development of ILC1, 2, and 3 subsets is controlled by core transcription factors: T-bet, GATA3, and RORγt, respectively. ILCs can undergo plasticity and transdifferentiate to other ILC subsets in response to invading pathogens and changes in local tissue environment. Accumulating evidence suggests that the plasticity and the maintenance of ILC identity is controlled by a balance between these and additional transcription factors such as STATs, Batf, Ikaros, Runx3, c-Maf, Bcl11b, and Zbtb46, activated in response to lineage-guiding cytokines. However, how interplay between these transcription factors leads to ILC plasticity and the maintenance of ILC identity remains hypothetical. In this review, we discuss recent advances in understanding transcriptional regulation of ILCs in homeostatic and inflammatory conditions.

Lineage Tracing of RGS5-CreER-Labeled Cells in Long Bones During Homeostasis and Injury.

Regulator of G protein signaling 5 (RGS5) is a GTPase activator for heterotrimeric G-protein α-subunits, shown to be a marker of pericytes. Bone marrow stromal cell population (BMSCs) is heterogeneous. Populations of mesenchymal progenitors, cells supportive of hematopoiesis, and stromal cells regulating bone remodeling have been recently identified. Periosteal and bone marrow mesenchymal stem cells (MSCs) are participating in fracture healing, but it is difficult to distinguish the source of cells within the callus. Considering that perivascular cells exert osteoprogenitor potential, we generated an RGS5 transgenic mouse model (Rgs5-CreER) which when crossed with Ai9 reporter animals (Rgs5/Tomato), is suitable for lineage tracing during growth and post-injury. Flow cytometry analysis and histology confirmed the presence of Rgs5/Tomato+ cells within CD31+ endothelial, CD45+ hematopoietic, and CD31-CD45- mesenchymal/perivascular cells. A tamoxifen chase showed expansion of Rgs5/Tomato+ cells expressing osterix within the trabeculae positioned between mineralized matrix and vasculature. Long-term chase showed proportion of Rgs5/Tomato+ cells contributes to mature osteoblasts expressing osteocalcin. Following femoral fracture, Rgs5/Tomato+ cells are observed around newly formed bone within the BM cavity and expressed osterix and osteocalcin, while contribution within periosteum was low and limited to fibroblastic callus with very few positive chondrocytes. In addition, BM injury model confirmed that RGS5-Cre labels population of BMSCs expands during injury and participates in osteogenesis. Under homeostatic conditions, lineage-traced RGS5 cells within the trabecular area demonstrate osteoprogenitor capacity that in an injury model contributes to new bone formation primarily within the BM niche.

Water exchange rates measure active transport and homeostasis in neural tissue.

For its size, the brain is the most metabolically active organ in the body. Most of its energy demand is used to maintain stable homeostatic physiological conditions. Altered homeostasis and active states are hallmarks of many diseases and disorders. Yet there is currently no direct and reliable method to assess homeostasis and absolute basal activity of cells in the tissue noninvasively without exogenous tracers or contrast agents. We propose a novel low-field, high-gradient diffusion exchange nuclear magnetic resonance (NMR) method capable of directly measuring cellular metabolic activity via the rate constant for water exchange across cell membranes. Exchange rates are s under normal conditions in viable ex vivo neonatal mouse spinal cords. High repeatability across samples suggest that values are absolute and intrinsic to the tissue. Using temperature and drug (ouabain) perturbations, we find that the majority of water exchange is metabolically active and coupled to active transport by the sodium-potassium pump. We show that this water exchange rate is sensitive primarily to tissue homeostasis and provides distinct functional information. In contrast, the apparent diffusion coefficient (ADC) measured with submillisecond diffusion times is sensitive primarily to tissue microstructure but not activity. Water exchange appears independently regulated from microstructural and oxygenation changes reported by ADC and relaxation measurements in an oxygen-glucose deprivation model of stroke; exchange rates remain stable for 30-40 min before dropping to levels similar to the effect of ouabain and never completely recovering when oxygen and glucose are restored.

How to implement constrained mixture growth and remodeling algorithms for soft biological tissues

Biological soft tissues are constantly adapting to their mechanical environment and remodel to restore certain mechanobiological homeostatic conditions. These effects can be modeled using the constrained mixture theory, that assumes degradation of material over time and the gradual replacement of extant material by newly deposited material. While this theory presents an elegant way to grasp phenomena of growth and remodeling in soft biological tissues, implementation difficulties may arise. Therefore, we give a detailed overview of the mathematical description of the constrained mixture theory and its homogenized equivalent, and present practical suggestions to numerically implement the theories. These implementations are thoroughly tested with multiple example growth and remodeling models. Results show a good correspondence between both theories, with the homogenized theory favored in terms of time efficiency. Results of a step time convergence study show the importance of choosing a small enough time step, especially when using the classical theory.

Microfluidic Wound-Healing Assay for ECM and Microenvironment Properties on Microglia BV2 Cells Migration.

Microglia cells, as the resident immune cells of the central nervous system (CNS), are highly motile and migratory in development and pathophysiological conditions. During their migration, microglia cells interact with their surroundings based on the various physical and chemical properties in the brain. Herein, a microfluidic wound-healing chip is developed to investigate microglial BV2 cell migration on the substrates coated with extracellular matrixes (ECMs) and substrates usually used for bio-applications on cell migration. In order to generate the cell-free space (wound), gravity was utilized as a driving force to flow the trypsin with the device. It was shown that, despite the scratch assay, the cell-free area was created without removing the extracellular matrix coating (fibronectin) using the microfluidic assay. It was found that the substrates coated with Poly-L-Lysine (PLL) and gelatin stimulated microglial BV2 migration, while collagen and fibronectin coatings had an inhibitory effect compared to the control conditions (uncoated glass substrate). In addition, the results showed that the polystyrene substrate induced higher cell migration than the PDMS and glass substrates. The microfluidic migration assay provides an in vitro microenvironment closer to in vivo conditions for further understanding the microglia migration mechanism in the brain, where the environment properties change under homeostatic and pathological conditions.

Regeneration after blood loss and acute inflammation proceeds without contribution of primitive HSCs

AbstractHematopoietic stem cells (HSCs) are the ultimate source of blood and immune cells, and transplantation reveals their unique potential to regenerate all blood lineages lifelong. HSCs are considered a quiescent reserve population under homeostatic conditions, which can be rapidly activated by perturbations to fuel blood regeneration. In accordance with this concept, inflammation and loss of blood cells were reported to stimulate the proliferation of HSCs, which is associated with a decline in their transplantation potential. To investigate the contribution of primitive HSCs to the hematopoietic stress response in the native environment, we use fate mapping and proliferation tracking mouse models. Although primitive HSCs were robustly activated by severe myeloablation, they did not contribute to the regeneration of mature blood cells in response to prototypic hematopoietic emergencies, such as acute inflammation or blood loss. Even chronic inflammatory stimulation, which triggered vigorous HSC proliferation, only resulted in a weak contribution of HSCs to mature blood cell production. Thus, our data demonstrate that primitive HSCs do not participate in the hematopoietic recovery from common perturbations and call for the reevaluation of the concept of HSC-driven stress responses.