Functional In Vivo Imaging of Macrophages.

Resident tissue macrophages (RTMs) are specialized phagocytes that are widely distributed throughout the body and are responsible for maintaining homeostasis. Recent advances in experimental techniques have enabled us to gain a greater insight into the actual in vivo biology of RTMs by observing their spatiotemporal dynamics directly in their native environment. Here, we detail a method for live tracking macrophages in a prototypical stromal tissue with high spatial and temporal resolution and great experimental versatility. Our approach builds on a custom intravital imaging platform and straightforward surgical preparation to gain access to an intact stromal compartment in order to analyze the morphological and behavioral dynamics of RTMs at single-cell resolution before and after experimental intervention. Furthermore, our versatile approach can also be utilized for live visualization of intracellular signaling and even for tracking cell organelles at subcellular resolution, and can be combined with downstream analyses such as multiplex confocal imaging, providing a unique insight into macrophage biology in vivo.

Tongyang Huoxue decoction (TYHX) ameliorating hypoxia/reoxygenation-induced disequilibrium of calcium homeostasis via regulating ß-tubulin in rabbit sinoatrial node cells.

ETHNOPHARMACOLOGICAL RELEVANCE: ß-tubulin is a skeletal protein of sinoatrial node cells (SANCs) that maintains the physiological structure of SANCs and inhibits calcium overload. Tongyang Huoxue decoction (TYHX) is widely used to treat sick sinus syndrome (SSS) owing to its effects on calcium channels regulation and SANCs protection. AIM OF THE STUDY: This study focuses on the mechanism of TYHX in improving the hypoxia/reoxygenation (H/R)-induced disequilibrium of calcium homeostasis in SANCs via regulating ß-tubulin. MATERIALS AND METHODS: Real-Time PCR (RT-PCR) and Western Blot were adopted to detect the mRNA and protein expression levels of calcium channel regulatory molecules. Laser confocal method was employed to examine ß-tubulin structure and fluorescence expression levels in SANCs, as well as calcium wave and calcium release levels. RESULTS: It was found that the fluorescence expression level decreased and the ß-tubulin structure of SANCs was damaged after H/R treatment. The mRNA and protein expression levels of SERCA2a/CaV1.3/NCX and ß-tubulin decreased, while the mRNA and protein expression of RyR2 increased. The results of calcium wave and calcium transient experiments showed that the fluorescence expression level of Ca2+ increased and calcium overload occurred in SANCs. After treatment with TYHX, the mRNA and protein expression levels of SERCA2a/CaV1.3/NCX and ß-tubulin increased, while the mRNA and protein expression levels of RyR2 decreased and the cell structure was restored. Interestingly, the regulation of TYHX on calcium homeostasis was further enhanced after Ad-ß-tubulin treatment and counteracted after siRNA-ß-tubulin treatment. These results suggest that TYHX could maintain calcium homeostasis via regulating ß-tubulin, thus protecting against H/R-induced SANCs injury, which may be a new target for SSS treatment.

Dietary sources of branched-chain fatty acids and their biosynthesis, distribution, and nutritional properties.

Branched-chain fatty acids (BCFAs) consist of a wide variety of fatty acids with alkyl branching of methyl group. The most common BCFAs are the types with one methyl group (mmBCFA) on the penultimate carbon (iBCFA) or the antepenultimate carbon (aiBCFA). Long-chain mmBCFAs are widely existing in animal fats, milks and are mostly derived from bacteria in the diet or animal digestive system. Recent studies show that BCFAs benefit human intestinal health and immune homeostasis, but the connection between their content, distribution in the human and their nutritional functions are not well established. In this paper, we reviewed BCFAs from various dietary sources focused on their molecular species. The BCFAs biosynthesis in bacteria, Caenorhabditis elegans, mammals and their distribution in human tissues are summarized. This paper also discusses the nutritional properties of BCFAs including influences on intestinal health, immunoregulatory effects, anti-carcinoma, and anti-obesity activities, by highlighting the most recent research progress.

Macrophage Development and Function.

Macrophages were first described over a hundred years ago. Throughout the years, they were shown to be essential players in their tissue-specific environment, performing various functions during homeostatic and disease conditions. Recent reports shed more light on their ontogeny as long-lived, self-maintained cells with embryonic origin in most tissues. They populate the different tissues early during development, where they help to establish and maintain homeostasis. In this chapter, the history of macrophages is discussed. Furthermore, macrophage ontogeny and core functions in the different tissues are described.

Isolation and Flow Cytometry Analysis of Macrophages from the Kidney.

Renal macrophages help maintain homeostasis, participate in tissue injury and repair, and play a vital role in immune surveillance [1-3]. Kidney macrophages can be broken down into two subsets, infiltrating macrophages, which can be further broken down into Ly6Chi and Ly6Clo cells, and kidney resident macrophages. While recent studies have shed light on the differing origins and niches of these cells, a more thorough understanding of kidney macrophage populations and how they may respond to various conditions is needed. This protocol describes how to efficiently isolate murine kidney macrophage populations for flow cytometry analysis.

Isolation and Flow Cytometry Analysis of Macrophages from White Adipose Tissue.

Macrophages are one of the prominent leukocyte populations in white adipose tissue (WAT) and play an important role during WAT homeostasis and remodeling. Macrophage function in WAT is determined by ontogeny and the local tissue environment. Here, we present a protocol to analyze different macrophage populations from murine WAT using flow cytometry.

From Cells to Environment: Exploring the Interplay between Factors Shaping Bone Health and Disease.

The skeletal system is an extraordinary structure that serves multiple purposes within the body, including providing support, facilitating movement, and safeguarding vital organs. Moreover, it acts as a reservoir for essential minerals crucial for overall bodily function. The intricate interplay of bone cells plays a critical role in maintaining bone homeostasis, ensuring a delicate balance. However, various factors, both intrinsic and extrinsic, can disrupt this vital physiological process. These factors encompass genetics, aging, dietary and lifestyle choices, the gut microbiome, environmental toxins, and more. They can interfere with bone health through several mechanisms, such as hormonal imbalances, disruptions in bone turnover, direct toxicity to osteoblasts, increased osteoclast activity, immune system aging, impaired inflammatory responses, and disturbances in the gut-bone axis. As a consequence, these disturbances can give rise to a range of bone disorders. The regulation of bone's physiological functions involves an intricate network of continuous processes known as bone remodeling, which is influenced by various intrinsic and extrinsic factors within the organism. However, our understanding of the precise cellular and molecular mechanisms governing the complex interactions between environmental factors and the host elements that affect bone health is still in its nascent stages. In light of this, this comprehensive review aims to explore emerging evidence surrounding bone homeostasis, potential risk factors influencing it, and prospective therapeutic interventions for future management of bone-related disorders.

Beyond Ion Homeostasis: Hypomagnesemia, Transient Receptor Potential Melastatin Channel 7, Mitochondrial Function, and Inflammation.

As the second most abundant intracellular divalent cation, magnesium (Mg2+) is essential for cell functions, such as ATP production, protein/DNA synthesis, protein activity, and mitochondrial function. Mg2+ plays a critical role in heart rhythm, muscle contraction, and blood pressure. A significant decline in Mg2+ intake has been reported in developed countries because of the increased consumption of processed food and filtered/deionized water, which can lead to hypomagnesemia (HypoMg). HypoMg is commonly observed in cardiovascular diseases, such as heart failure, hypertension, arrhythmias, and diabetic cardiomyopathy, and HypoMg is a predictor for cardiovascular and all-cause mortality. On the other hand, Mg2+ supplementation has shown significant therapeutic effects in cardiovascular diseases. Some of the effects of HypoMg have been ascribed to changes in Mg2+ participation in enzyme activity, ATP stabilization, enzyme kinetics, and alterations in Ca2+, Na+, and other cations. In this manuscript, we discuss new insights into the pathogenic mechanisms of HypoMg that surpass previously described effects. HypoMg causes mitochondrial dysfunction, oxidative stress, and inflammation. Many of these effects can be attributed to the HypoMg-induced upregulation of a Mg2+ transporter transient receptor potential melastatin 7 channel (TRMP7) that is also a kinase. An increase in kinase signaling mediated by HypoMg-induced TRPM7 transcriptional upregulation, independently of any change in Mg2+ transport function, likely seems responsible for many of the effects of HypoMg. Therefore, Mg2+ supplementation and TRPM7 kinase inhibition may work to treat the sequelae of HypoMg by preventing increased TRPM7 kinase activity rather than just altering ion homeostasis. Since many diseases are characterized by oxidative stress or inflammation, Mg2+ supplementation and TRPM7 kinase inhibition may have wider implications for other diseases by acting to reduce oxidative stress and inflammation.

Calcium Homeostasis and Psychiatric Disorders: A Mendelian Randomization Study.

Observational studies have investigated the impact of calcium homeostasis on psychiatric disorders; however, the causality of associations is yet to be established. Bidirectional Mendelian randomization (MR) analysis of calcium homeostasis hormones was conducted on nine psychiatric disorders. Calcium, serum 25-hydroxyvitamin D levels (25OHD), parathyroid hormone, and fibroblast growth factor 23 are the major calcium homeostasis hormones. The causality was evaluated by the inverse variance weighted method (IVW) and the MR Steiger test, while Cochran's Q test, the MR-Egger intercept test, funnel plot, and the leave-one-out method were used for sensitivity analyses. Bonferroni correction was used to determine the causative association features (p < 6.94 × 10-4). Schizophrenia (SCZ) was significantly associated with decreased 25OHD concentrations with an estimated effect of -0.0164 (Prandom-effect IVW = 2.39 × 10-7). In the Multivariable MR (MVMR) analysis adjusting for potentially confounding traits including body mass index, obesity, mineral supplements (calcium, fish oil, and vitamin D) and outdoor time (winter and summer), the relationship between SCZ and 25OHD remained. The genetically predicted autism spectrum disorder and bipolar disorder were also nominally associated with decreased 25OHD. This study provided evidence for a causal effect of psychiatric disorders on calcium homeostasis. The clinical monitoring of 25OHD levels in patients with psychiatric disorders is beneficial.

The metabolic and functional roles of sensory nerves in adipose tissues.

Homeostatic regulation of adipose tissue is critical for the maintenance of energy balance and whole-body metabolism. The peripheral nervous system provides bidirectional neural communication between the brain and adipose tissue, thereby providing homeostatic control. Most research on adipose innervation and nerve functions has been limited to the sympathetic nerves and their neurotransmitter norepinephrine. In recent years, more work has focused on adipose sensory nerves, but the contributions of subsets of sensory nerves to metabolism and the specific roles contributed by sensory neuropeptides are still understudied. Advances in imaging of adipose innervation and newer tissue denervation techniques have confirmed that sensory nerves contribute to the regulation of adipose functions, including lipolysis and browning. Here, we summarize the historical and latest findings on the regulation, function and plasticity of adipose tissue sensory nerves that contribute to metabolically important processes such as lipolysis, vascular control and sympathetic axis cross-talk.

Sound stimulation using the individual's heart rate to improve the stability and homeostasis of the autonomic nervous system.

OBJECTIVES: In this study, we explain the role of enhancing the stability and homeostasis of the autonomic nervous system (ANS) by proposing the average heart rate sound resonance (aHRSR), a sound stimulation to prevent imbalance of ANS due to dynamic movement. The effect of aHRSR on ANS was analyzed through the time and frequency domain of heart rate variability (HRV) using the photoplethysmogram data (PPG) of 22 participants (DUIRB-202109-12). METHOD: When the subjects performed dynamic movements that could cause changes in the ANS, HRV indicators using PPG data for 5 min before and after the movements were analyzed according to the presence or absence of aHRSR. The standard deviation of the NN intervals (SDNN), the square root of the mean squared differences of the NN intervals (RMSSD), low-frequency band (LF), and high-frequency band (HF), which represent sympathetic and parasympathetic nerve activity, were used as indicators, where SNDD and LF represent total ANS and sympathetic activity, while RMSSD and HF represent parasympathetic activity. RESULTS: As the effects of aHRSR on dynamic movement, the recovery time of RR interval was advanced by about 15 s, SDNN increased from ([44.16 ± 13.11] to [47.85 ± 15.16]) ms, and RMSSD increased from ([23.73 ± 9.95] to [31.89 ± 12.48]) ms (p < 0.05), increasing the stability of the ANS and reducing instability. The effect of homeostasis of the ANS according to aHRSR is also shown in reducing the change rate of LF from (-13.83 to -8.83) %, and the rate of change of HF from (10.59 to 3.27) %. CONCLUSIONS: These results suggest that aHRSR can affect the cardiovascular system by assisting physiological movements that occur during dynamic movement.

Gastric epithelial stem cells in development, homeostasis and regeneration.

The stem/progenitor cell pool is indispensable for the development, homeostasis and regeneration of the gastric epithelium, owing to its defining ability to self-renew whilst supplying the various functional epithelial lineages needed to digest food efficiently. A detailed understanding of the intricacies and complexities surrounding the behaviours and roles of these stem cells offers insights, not only into the physiology of gastric epithelial development and maintenance, but also into the pathological consequences following aberrations in stem cell regulation. Here, we provide an insightful synthesis of the existing knowledge on gastric epithelial stem cell biology, including the in vitro and in vivo experimental techniques that have advanced such studies. We highlight the contributions of stem/progenitor cells towards patterning the developing stomach, specification of the differentiated cell lineages and maintenance of the mature epithelium during homeostasis and following injury. Finally, we discuss gaps in our understanding and identify key research areas for future work.

Effect of agents affecting bone homeostasis on short- and long-term implant failure.

OBJECTIVES: To review the current evidence on the relationship between agents that affect bone homeostasis and dental implant failures. MATERIALS AND METHODS: Electronic searches for bisphosphonates, denosumab, methotrexate, corticosteroids, romosozumab, sunitinib, and bevacizumab were performed using PubMed, MEDLINE (OVID), EMBASE (OVID), Cochrane Central Register of Controlled Trials (Cochrane Library), Cochrane Oral Health Group Trials Register (Cochrane Library) and Web of Science (Thomson Reuters). Manual searches were also conducted to complement the digital searches for recent issues. RESULTS: Previous publications suggested that bisphosphonates do not compromise the survival of dental implants. However, one study documented an increased risk of implant failure in patients who had received high-dose of intravenous bisphosphonate therapy after implant rehabilitation. There has been an issue of MRONJ around implants in patients who have successfully received implant therapy before and after antiresorptive therapy, leading to late implant failure. Despite evidence on the detrimental effects of denosumab, methotrexate and corticosteroids on bone metabolism, their role in implant survival is not conclusive. CONCLUSIONS: At present, there is insufficient evidence to establish a potential connection between agents that affects bone homeostasis and implant failure. However, some studies have reported negative results for implant therapy. In addition, implant-related sequestration in patients who received anti-resorptive therapy, despite of successful osseointegration, is also noticeable. Although limited studies are available at present, clinicians should still carefully consider the potential hazards and take appropriate precautions to minimize the risks associated with the medications and implant therapy.

Bifidobacterium longum 68S mediated gut-skin axis homeostasis improved skin barrier damage in aging mice.

BACKGROUND: Bifidobacterium as probiotics, play important roles in skin status, while the potential mechanisms interaction remains unknown. The study further explored the potential mechanism of B. longum 68S in ameliorating skin barrier damage from the perspective of the gut-skin axis in aging mice. METHODS: B. longum 68S supplied natural aging mouse model and fecal microbiota transplantation (FMT) experiment proves the key role of intestinal microbiota in B. longum 68S up-regulating the production of ceramide synthesis key enzyme (SPT1) and ceramide level and improving skin barrier damage. Moreover, B. longum 68S supplied SPT1 gene deletion mouse model to investigate the mechanism of B. longum 68S on improving skin barrier damage. RESULTS: Transcriptome analysis and 16S rRNA high-throughput pyrosequencing demonstrated that aging mice exhibited skin barrier dysfunction and intestinal dysbiosis. Meanwhile, aging mice exhibited an up-regulation in the trans epidermal water loss (TEWL) and a down-regulation in the level of SPT1, ceramide and skin barrier-related proteins (Loricrin, Keratin 10 and Desmoglein 1). Similarity, the FMT from aging mice to normal mice and SPT1 gene deletion mice could rebuild skin barrier damage and B. longum 68S supplementation exerted a positive effect on it. Further, B. longum 68S-mediated SPT1-derived ceramide production prevented impaired ceramide synthesis-induced endoplasmic reticulum stress and apoptotic response, ultimately improving skin barrier damage in vitro. CONCLUSION: Emerging anti-aging therapies are necessary given the poor safety profiles of current pharmaceutical drugs. B. longum 68S may be better alternatives, considering the association between the gut microbiota and healthy aging. The findings suggested that B. longum 68S-mediated gut-skin axis homeostasis, thereby exhibiting an anti-aging effect and facilitate a better understanding of the mechanisms governing the various beneficial effects of B. longum 68S.

ERO1α primes the ryanodine receptor to respond to arsenite with concentration dependent Ca2+ release sequentially triggering two different mechanisms of ROS formation.

A 6 h exposure of U937 cells to 2.5 µM arsenite stimulates low Ca2+ release from the inositol 1, 4, 5-triphosphate receptor (IP3R), causing a cascade of causally connected events, i.e., endoplasmic reticulum oxidoreductin-1α (ERO1α) expression, activation of the ryanodine receptor (RyR), mitochondrial Ca2+ accumulation, mitochondrial superoxide formation and further ERO1α expression. At greater arsenite concentrations, the release of the cation from the IP3R and the ensuing ERO1α expression remained unchanged but were nevertheless critical to sequentially promote concentration-dependent increases in Ca2+ release from the RyR, NADPH oxidase activation and a third mechanism of ERO1α expression which, in analogy to the one driven by mitochondrial superoxide, was also mediated by reactive oxygen species (ROS) and devoid of effects on Ca2+ homeostasis. Thus, concentration-independent stimulation of Ca2+ release from the IP3R is of pivotal importance for the effects of arsenite on Ca2+ homeostasis. It stimulates the expression of a fraction of ERO1α that primes the RyR to respond to the metalloid with concentration-dependent Ca2+-release, triggering the formation of superoxide in the mitochondrial respiratory chain and via NADPH oxidase activation. The resulting dose-dependent ROS formation was associated with a progressive increase in ERO1α expression, which however failed to affect Ca2+ homeostasis, thereby suggesting that ROS, unlike IP3R-dependent Ca2+ release, promote ERO1α expression in sites distal from the RyR.

Metabolic heterogeneity of tissue-resident macrophages in homeostasis and during helminth infection.

Tissue-resident macrophage populations constitute a mosaic of phenotypes, yet how their metabolic states link to the range of phenotypes and functions in vivo is still poorly defined. Here, using high-dimensional spectral flow cytometry, we observe distinct metabolic profiles between different organs and functionally link acetyl CoA carboxylase activity to efferocytotic capacity. Additionally, differences in metabolism are evident within populations from a specific site, corresponding to relative stages of macrophage maturity. Immune perturbation with intestinal helminth infection increases alternative activation and metabolic rewiring of monocyte-derived macrophage populations, while resident TIM4+ intestinal macrophages remain immunologically and metabolically hyporesponsive. Similar metabolic signatures in alternatively-activated macrophages are seen from different tissues using additional helminth models, but to different magnitudes, indicating further tissue-specific contributions to metabolic states. Thus, our high-dimensional, flow-based metabolic analyses indicates complex metabolic heterogeneity and dynamics of tissue-resident macrophage populations at homeostasis and during helminth infection.

Langerhans cells shape postnatal oral homeostasis in a mechanical-force-dependent but microbiota and IL17-independent manner.

The postnatal interaction between microbiota and the immune system establishes lifelong homeostasis at mucosal epithelial barriers, however, the barrier-specific physiological activities that drive the equilibrium are hardly known. During weaning, the oral epithelium, which is monitored by Langerhans cells (LC), is challenged by the development of a microbial plaque and the initiation of masticatory forces capable of damaging the epithelium. Here we show that microbial colonization following birth facilitates the differentiation of oral LCs, setting the stage for the weaning period, in which adaptive immunity develops. Despite the presence of the challenging microbial plaque, LCs mainly respond to masticatory mechanical forces, inducing adaptive immunity, to maintain epithelial integrity that is also associated with naturally occurring alveolar bone loss. Mechanistically, masticatory forces induce the migration of LCs to the lymph nodes, and in return, LCs support the development of immunity to maintain epithelial integrity in a microbiota-independent manner. Unlike in adult life, this bone loss is IL-17-independent, suggesting that the establishment of oral mucosal homeostasis after birth and its maintenance in adult life involve distinct mechanisms.

Genome-wide promoter responses to CRISPR perturbations of regulators reveal regulatory networks in Escherichia coli.

Elucidating genome-scale regulatory networks requires a comprehensive collection of gene expression profiles, yet measuring gene expression responses for every transcription factor (TF)-gene pair in living prokaryotic cells remains challenging. Here, we develop pooled promoter responses to TF perturbation sequencing (PPTP-seq) via CRISPR interference to address this challenge. Using PPTP-seq, we systematically measure the activity of 1372 Escherichia coli promoters under single knockdown of 183 TF genes, illustrating more than 200,000 possible TF-gene responses in one experiment. We perform PPTP-seq for E. coli growing in three different media. The PPTP-seq data reveal robust steady-state promoter activities under most single TF knockdown conditions. PPTP-seq also enables identifications of, to the best of our knowledge, previously unknown TF autoregulatory responses and complex transcriptional control on one-carbon metabolism. We further find context-dependent promoter regulation by multiple TFs whose relative binding strengths determined promoter activities. Additionally, PPTP-seq reveals different promoter responses in different growth media, suggesting condition-specific gene regulation. Overall, PPTP-seq provides a powerful method to examine genome-wide transcriptional regulatory networks and can be potentially expanded to reveal gene expression responses to other genetic elements.

Efficient intervention for pulmonary fibrosis via mitochondrial transfer promoted by mitochondrial biogenesis.

The use of exogenous mitochondria to replenish damaged mitochondria has been proposed as a strategy for the treatment of pulmonary fibrosis. However, the success of this strategy is partially restricted by the difficulty of supplying sufficient mitochondria to diseased cells. Herein, we report the generation of high-powered mesenchymal stem cells with promoted mitochondrial biogenesis and facilitated mitochondrial transfer to injured lung cells by the sequential treatment of pioglitazone and iron oxide nanoparticles. This highly efficient mitochondrial transfer is shown to not only restore mitochondrial homeostasis but also reactivate inhibited mitophagy, consequently recovering impaired cellular functions. We perform studies in mouse to show that these high-powered mesenchymal stem cells successfully mitigate fibrotic progression in a progressive fibrosis model, which was further verified in a humanized multicellular lung spheroid model. The present findings provide a potential strategy to overcome the current limitations in mitochondrial replenishment therapy, thereby promoting therapeutic applications for fibrotic intervention.

The Emerging Concept of Transportome: State of the Art.

The array of ion channels and transporters expressed in cell membranes, collectively referred to as the transportome, is a complex and multifunctional molecular machinery; in particular, at the plasma membrane level it finely tunes the exchange of biomolecules and ions, acting as a functionally adaptive interface that accounts for dynamic plasticity in the response to environmental fluctuations and stressors. The transportome is responsible for the definition of membrane potential and its variations, participates in the transduction of extracellular signals, and acts as a filter for most of the substances entering and leaving the cell, thus enabling the homeostasis of many cellular parameters. For all these reasons, physiologists have long been interested in the expression and functionality of ion channels and transporters, in both physiological and pathological settings and across the different domains of life. Today, thanks to the high-throughput technologies of the postgenomic era, the omics approach to the study of the transportome is becoming increasingly popular in different areas of biomedical research, allowing for a more comprehensive, integrated, and functional perspective of this complex cellular apparatus. This article represents a first effort for a systematic review of the scientific literature on this topic. Here we provide a brief overview of all those studies, both primary and meta-analyses, that looked at the transportome as a whole, regardless of the biological problem or the models they used. A subsequent section is devoted to the methodological aspect by reviewing the most important public databases annotating ion channels and transporters, along with the tools they provide to retrieve such information. Before conclusions, limitations and future perspectives are also discussed.