by
Meredith E. Fay;
David Myers;
Amit Kumar;
Cory T. Turbyfield;
Rebecca Byler;
Kaci Crawford;
Robert Mannino;
Alvin Laohapant;
Erika A. Tyburski;
Yumiko Sakurai;
Michael J. Rosenbluth;
Neil A. Switz;
Todd A. Sulchek;
Michael D. Graham;
Wilbur Lam
Leukocytes normally marginate toward the vascular wall in large vessels and within the microvasculature. Reversal of this process, leukocyte demargination, leads to substantial increases in the clinical white blood cell and granulocyte count and is a welldocumented effect of glucocorticoid and catecholamine hormones, although the underlying mechanisms remain unclear. Here we show that alterations in granulocyte mechanical properties are the driving force behind glucocorticoid- and catecholamine-induced demargination. First, we found that the proportions of granulocytes from healthy human subjects that traversed and demarginated from microfluidic models of capillary beds and veins, respectively, increased after the subjects ingested glucocorticoids.
Also, we show that glucocorticoid and catecholamine exposure reorganizes cellular cortical actin, significantly reducing granulocyte stiffness, as measured with atomic force microscopy. Furthermore, using simple kinetic theory computational modeling, we found that this reduction in stiffness alone is sufficient to cause granulocyte demargination. Taken together, our findings reveal a biomechanical answer to an old hematologic question regarding how glucocorticoids and catecholamines cause leukocyte demargination. In addition, in a broader sense, we have discovered a temporally and energetically efficient mechanism in which the innate immune system can simply alter leukocyte stiffness to fine tune margination/demargination and therefore leukocyte trafficking in general. These observations have broad clinically relevant implications for the inflammatory process overall as well as hematopoietic stem cell mobilization and homing.
The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α-And γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.
by
Lílian Cataldi Rodrigues;
Adriana Secatto;
Carlos A. Sorgi;
Naiara N. Dejani;
Alexandra I. Medeiros;
Morgana Kelly Borges Prado;
Simone Gusmão Ramos;
Richard D. Cummings;
Sean Stowell;
Lúcia Helena Faccioli;
Marcelo Dias-Baruffi
Histoplasma capsulatum is a dimorphic fungus that develops a yeast-like morphology in host's tissue, responsible for the pulmonary disease histoplasmosis. The recent increase in the incidence of histoplasmosis in immunocompromised patients highlights the need of understanding immunological controls of fungal infections. Here, we describe our discovery of the role of endogenous galectin-1 (Gal-1) in the immune pathophysiology of experimental histoplasmosis. All infected wild-type (WT) mice survived while only 1/3 of Lgals1-/- mice genetically deficient in Gal-1 survived 30 days after infection. Although infected Lgals1-/- mice had increased proinflammatory cytokines, nitric oxide (NO), and elevations in neutrophil pulmonary infiltration, they presented higher fungal load in lungs and spleen. Infected lung and infected macrophages from Lgals1-/- mice exhibited elevated levels of prostaglandin E2 (PGE2, a prostanoid regulator of macrophage activation) and prostaglandin E synthase 2 (Ptgs2) mRNA. Gal-1 did not bind to cell surface of yeast phase of H. capsulatum, in vitro, suggesting that Gal-1 contributed to phagocytes response to infection rather than directly killing the yeast. The data provides the first demonstration of endogenous Gal-1 in the protective immune response against H. capsulatum associated with NO and PGE2 as an important lipid mediator in the pathogenesis of histoplasmosis.
Homeostatic regulation is essential for the maintenance of synaptic strength within the physiological range. The current study is the first to demonstrate that both induction and reversal of homeostatic upregulation of synaptic vesicle release can occur within seconds of blocking or unblocking acetylcholine receptors at the mouse neuromuscular junction. Our data suggest that the homeostatic upregula-tion of release is due to Ca2+-dependent increase in the size of the readily releasable pool (RRP). Blocking vesicle refilling prevented upregulation of quantal content (QC), while leaving baseline release relatively unaffected. This suggested that the upregulation of QC was due to mobilization of a distinct pool of vesicles that were rapidly recycled and thus were dependent on continued vesicle refilling. We term this pool the “homeostatic reserve pool.” A detailed analysis of the time course of vesicle release triggered by a presynaptic action potential suggests that the homeostatic reserve pool of vesicles is normally released more slowly than other vesicles, but the rate of their release becomes similar to that of the major pool during homeostatic upregulation of QC. Remarkably, instead of finding a generalized increase in the recruitment of vesicles into RRP, we identified a distinct homeostatic reserve pool of vesicles that appear to only participate in synchronized release following homeostatic upregulation of QC. Once this small pool of vesicles is depleted by the block of vesicle refilling, homeostatic upregulation of QC is no longer observed. This is the first identification of the population of vesicles responsible for the blockade-induced upregulation of release previously described.
The genetic correction of induced pluripotent stem cells (iPSCs) induced from somatic cells of patients with sensorineural hearing loss (caused by hereditary factors) is a promising method for its treatment. The correction of gene mutations in iPSCs could restore the normal function of cells and provide a rich source of cells for transplantation. In the present study, iPSCs were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T; P-iPSCs), the asymptomatic father of the patient (MYO7A c.1184G>A mutation; CF-iPSCs), and a normal donor (MYO7AWT/WT; C-iPSCs). One of MYO7A mutation sites (c.4118C>T) in the P-iPSCs was corrected using CRISPR/Cas9. The corrected iPSCs (CP-iPSCs) retained cell pluripotency and normal karyotypes. Hair cell-like cells induced from CP-iPSCs showed restored organization of stereocilia-like protrusions; moreover, the electrophysiological function of these cells was similar to that of cells induced from C-iPSCs and CF-iPSCs. These results might facilitate the development of iPSC-based gene therapy for genetic disorders.
Intracellular accumulation of wild type tau is a hallmark of sporadic Alzheimer's disease (AD). However, the molecular mechanisms underlying tau toxicity is not fully understood. Here, we detected mitophagy deficits evidenced by the increased levels of mitophagy markers, including COX IV, TOMM20, and the ratio of mtDNA to genomic DNA indexed as mt-Atp6/Rpl13, in the AD brains and in the human wild type full-length tau (htau) transgenic mice. More interestingly, the mitophagy deficit was only shown in the AD patients who had an increased total tau level. Further studies demonstrated that overexpression of htau induced mitophagy deficits in HEK293 cells, the primary hippocampal neurons and in the brains of C57 mice. Upon overexpression of htau, the mitochondrial membrane potential was increased and the levels of PTENinduced kinase 1 (PINK1) and Parkin decreased in the mitochondrial fraction, while upregulation of Parkin attenuated the htau-induced mitophagy deficits. Finally, we detected a dose-dependent allocation of tau proteins into the mitochondrial outer membrane fraction along with its cytoplasmic accumulation. These data suggest that intracellular accumulation of htau induces mitophagy deficits by direct inserting into the mitochondrial membrane and thus increasing the membrane potential, which impairs the mitochondrial residence of PINK1/Parkin. Our findings reveal a novel mechanism underlying the htau-induced neuronal toxicities in AD and other tauopathies.
Background: Abnormal branched myofibers within skeletal muscles are commonly found in diverse animal models of muscular dystrophy as well as in patients. Branched myofibers from dystrophic mice are more susceptible to break than unbranched myofibers suggesting that muscles containing a high percentage of these myofibers are more prone to injury. Previous studies showed ubiquitous over-expression of mouse olfactory receptor 23 (mOR23), a G protein-coupled receptor, in wild type mice decreased myofiber branching. Whether mOR23 over-expression specifically in skeletal muscle cells is sufficient to mitigate myofiber branching in dystrophic muscle is unknown.
Methods: We created a novel transgenic mouse over-expressing mOR23 specifically in muscle cells and then bred with dystrophic (mdx) mice. Myofiber branching was analyzed in these two transgenic mice and membrane integrity was assessed by Evans blue dye fluorescence.
Results: mOR23 over-expression in muscle led to a decrease of myofiber branching after muscle regeneration in non-dystrophic mouse muscles and reduced the severity of myofiber branching in mdx mouse muscles. Muscles from mdx mouse over-expressing mOR23 significantly exhibited less damage to eccentric contractions than control mdx muscles.
Conclusions: The decrease of myofiber branching in mdx mouse muscles over-expressing mOR23 reduced the amount of membrane damage induced by mechanical stress. These results suggest that modifying myofiber branching in dystrophic patients, while not preventing degeneration, could be beneficial for mitigating some of the effects of the disease process.
by
Allison E. Norlander;
Mohammed A. Saleh;
Nikhil V. Kamat;
Benjamin Ko;
Juan Gnecco;
Linjue Zhu;
Bethany L. Dale;
Yoichiro Iwakura;
Robert Hoover Jr;
Alicia A. McDonough;
Meena S. Madhur
Angiotensin II-induced hypertension is associated with an increase in T-cell production of interleukin-17A (IL-17A). Recently, we reported that IL-17A-/- mice exhibit blunted hypertension, preserved natriuresis in response to a saline challenge, and decreased renal sodium hydrogen exchanger 3 expression after 2 weeks of angiotensin II infusion compared with wild-type mice. In the current study, we performed renal transporter profiling in mice deficient in IL-17A or the related isoform, IL-17F, after 4 weeks of Ang II infusion, the time when the blood pressure reduction in IL-17A-/- mice is most prominent. Deficiency of IL-17A abolished the activation of distal tubule transporters, specifically the sodium-chloride cotransporter and the epithelial sodium channel and protected mice from glomerular and tubular injury.
In human proximal tubule (HK-2) cells, IL-17A increased sodium hydrogen exchanger 3 expression through a serum and glucocorticoid-regulated kinase 1-dependent pathway. In mouse distal convoluted tubule cells, IL-17A increased sodium-chloride cotransporter activity in a serum and glucocorticoid-regulated kinase 1/Nedd4-2-dependent pathway. In both cell types, acute treatment with IL-17A induced phosphorylation of serum and glucocorticoid-regulated kinase 1 at serine 78, and treatment with a serum and glucocorticoid-regulated kinase 1 inhibitor blocked the effects of IL-17A on sodium hydrogen exchanger 3 and sodium-chloride cotransporter. Interestingly, both HK-2 and mouse distal convoluted tubule 15 cells produce endogenous IL-17A. IL17F had little or no effect on blood pressure or renal sodium transporter abundance. These studies provide a mechanistic link by which IL-17A modulates renal sodium transport and suggest that IL-17A inhibition may improve renal function in hypertension and other autoimmune disorders.
The host antiviral programs that are initiated following viral infection form a dynamic and complex web of responses that we have collectively termed as “the antiviral landscape”. Conventional approaches to studying antiviral responses have primarily used reductionist systems to assess the function of a single or a limited subset of molecules. Systems biology is a holistic approach that considers the entire system as a whole, rather than individual components or molecules. Systems biology based approaches facilitate an unbiased and comprehensive analysis of the antiviral landscape, while allowing for the discovery of emergent properties that are missed by conventional approaches. The antiviral landscape can be viewed as a hierarchy of complexity, beginning at the whole organism level and progressing downward to isolated tissues, populations of cells, and single cells. In this review, we will discuss how systems biology has been applied to better understand the antiviral landscape at each of these layers. At the organismal level, the Collaborative Cross is an invaluable genetic resource for assessing how genetic diversity influences the antiviral response. Whole tissue and isolated bulk cell transcriptomics serves as a critical tool for the comprehensive analysis of antiviral responses at both the tissue and cellular levels of complexity. Finally, new techniques in single cell analysis are emerging tools that will revolutionize our understanding of how individual cells within a bulk infected cell population contribute to the overall antiviral landscape.
In mice that express SOD1 mutations found in human motor neuron disease, degeneration begins in the periphery for reasons that remain unknown. At the neuromuscular junction (NMJ), terminal Schwann cells (TSCs) have an intimate relationship with motor terminals and are believed to help maintain the integrity of the motor terminal. Recent evidence indicates that TSCs in some SOD1 mice exhibit abnormal functional properties, but other aspects of possible TSC involvement remain unknown. In this study, an analysis of TSC morphology and number was performed in relation to NMJ innervation status in mice which express the G93A SOD1 mutation. At P30, all NMJs of the fast medial gastrocnemius (MG) muscle were fully innervated by a single motor axon but 50% of NMJs lacked TSC cell bodies and were instead covered by the processes of Schwann cells with cell bodies located on the preterminal axons. NMJs in P30 slow soleus muscles were also fully innervated by single motor axons and only 5% of NMJs lacked a TSC cell body. At P60, about 25% of MG NMJs were denervated and lacked labeling for TSCs while about 60% of innervated NMJs lacked TSC cell bodies. In contrast, 96% of P60 soleus NMJs were innervated while 9% of innervated NMJs lacked TSC cell bodies. The pattern of TSC abnormalities found at P30 thus correlates with the pattern of denervation found at P60. Evidence from mice that express the G85R SOD1 mutation indicate that TSC abnormalities are not unique for mice that express G93A SOD1 mutations. These results add to an emerging understanding that TSCs may play a role in motor terminal degeneration and denervation in animal models of motor neuron disease.