Exosomes from adipose-derived mesenchymal stem cells alleviate sepsis-induced lung injury in mice by inhibiting the secretion of IL-27 in macrophages

Acute lung injury (ALI) represents a frequent sepsis-induced inflammatory disorder. Mesenchymal stromal cells (MSCs) elicit anti-inflammatory effects in sepsis. This study investigated the mechanism of exosomes from adipose-derived MSCs (ADMSCs) in sepsis-induced ALI. The IL-27r−/− (WSX-1 knockout) or wild-type mouse model of sepsis was established by cecal ligation and puncture (CLP). The model mice and lipopolysaccharide (LPS)-induced macrophages were treated with ADMSC-exosomes. The content of Dil-labeled exosomes in pulmonary macrophages, macrophages CD68+ F4/80+ in whole lung tissues, and IL-27 content in macrophages were detected. The mRNA expression and protein level of IL27 subunits P28 and EBI3 in lung tissue and the levels of IL-6, TNF-α, and IL-1β were measured. The pulmonary edema, tissue injury, and pulmonary vascular leakage were measured. In vitro, macrophages internalized ADMSC-exosomes, and ADMSC-exosomes inhibited IL-27 secretion in LPS-induced macrophages. In vivo, IL-27 knockout attenuated CLP-induced ALI. ADMSC-exosomes suppressed macrophage aggregation in lung tissues and inhibited IL-27 secretion. ADMSC-exosomes decreased the contents of IL-6, TNF-α, and IL-1β, reduced pulmonary edema and pulmonary vascular leakage, and improved the survival rate of mice. Injection of recombinant IL-27 reversed the protective effect of ADMSC-exosomes on sepsis mice. Collectively, ADMSC-exosomes inhibited IL-27 secretion in macrophages and alleviated sepsis-induced ALI in mice.

Temporal Profiling of the Cortical Synaptic Mitochondrial Proteome Identifies Ageing Associated Regulators of Stability

Synapses are particularly susceptible to the effects of advancing age, and mitochondria have long been implicated as organelles contributing to this compartmental vulnerability. Despite this, the mitochondrial molecular cascades promoting age-dependent synaptic demise remain to be elucidated. Here, we sought to examine how the synaptic mitochondrial proteome (including strongly mitochondrial associated proteins) was dynamically and temporally regulated throughout ageing to determine whether alterations in the expression of individual candidates can influence synaptic stability/morphology. Proteomic profiling of wild-type mouse cortical synaptic and non-synaptic mitochondria across the lifespan revealed significant age-dependent heterogeneity between mitochondrial subpopulations, with aged organelles exhibiting unique protein expression profiles. Recapitulation of aged synaptic mitochondrial protein expression at the Drosophila neuromuscular junction has the propensity to perturb the synaptic architecture, demonstrating that temporal regulation of the mitochondrial proteome may directly modulate the stability of the synapse in vivo.

Nrf2 plays a critical role in the metabolic response during and after spaceflight

Space travel induces stresses that contribute to health problems, as well as inducing the expression of Nrf2 (NF-E2-related factor-2) target genes that mediate adaptive responses to oxidative and other stress responses. The volume of epididymal white adipose tissue (eWAT) in mice increases during spaceflight, a change that is attenuated by Nrf2 knockout. We conducted metabolome analyses of plasma from wild-type and Nrf2 knockout mice collected at pre-flight, in-flight and post-flight time points, as well as tissues collected post-flight to clarify the metabolic responses during and after spaceflight and the contribution of Nrf2 to these responses. Plasma glycerophospholipid and sphingolipid levels were elevated during spaceflight, whereas triacylglycerol levels were lower after spaceflight. In wild-type mouse eWAT, triacylglycerol levels were increased, but phosphatidylcholine levels were decreased, and these changes were attenuated in Nrf2 knockout mice. Transcriptome analyses revealed marked changes in the expression of lipid-related genes in the liver and eWAT after spaceflight and the effects of Nrf2 knockout on these changes. Based on these results, we concluded that space stress provokes significant responses in lipid metabolism during and after spaceflight; Nrf2 plays critical roles in these responses.

Induced cell-autonomous neutropenia systemically perturbs hematopoiesis in Cebpa enhancer-null mice

The transcription factor C/EBPa initiates the neutrophil gene expression program in the bone marrow. Knockouts of the Cebpa gene or its +37kb enhancer in mice show two major findings: (1) neutropenia in bone marrow and blood; (2) decrease in long-term hematopoietic stem cell (LT-HSC) numbers. Whether the latter finding is cell autonomous (intrinsic) to the LT-HSCs or an extrinsic event exerted on the stem cell compartment remained an open question. Flow cytometric analysis of the Cebpa +37kb enhancer knockout model revealed that the reduction in LT-HSC numbers observed was proportional to the degree of neutropenia. Single cell transcriptomics of wild type mouse bone marrow showed that Cebpa is predominantly expressed in early myeloid-biased progenitors, but not in LT-HSCs. These observations suggest that the negative effect on LT-HSCs is an extrinsic event caused by neutropenia. We transplanted whole bone marrows from +37kb enhancer deleted mice and found that 40% of the recipient mice acquired full blown neutropenia with severe dysplasia and a significant reduction in the total LT-HSC population. The other 60% showed initial signs of myeloid differentiation defects and dysplasia when they were sacrificed, suggesting they were in an early stage of the same pathological process. This phenotype was not seen in mice transplanted with wild type bone marrow cells. Altogether, these results indicate that Cebpa-enhancer deletion causes cell autonomous neutropenia, which reprograms and disturbs the quiescence of HSCs, leading to a systemic impairment of the hematopoietic process.

Impediment of Gpibα-Mediated Platelet Clearance By an Anti-Gpibβ Antibody Derivative

Background: Glycoprotein (GP)Ib-IX complex plays a critical role in mediating platelet activation and platelet clearance. Recently, we identified the mechanosensory domain (MSD) in the GPIbα subunit, and demonstrated that unfolding of the MSD and subsequent exposure of the Trigger sequence (residues 473-483) therein activates GPIb-IX and induces rapid platelet clearance. This mechanism could explain acute thrombocytopenia induced by activated VWF, anti-GPIbα antibodies, neuraminidase, and ectodomain shedding of GPIbα. Consistently, platelets in IL4R-IbαTg mice, a transgenic strain in which the entire extracellular domain of human GPIbα except the Trigger sequence was replaced with that of the α-subunit of interleukin-4 receptor, exhibit constitutively more filopodia and are cleared much faster than the wild type. Previously, an anti-GPIbβ antibody RAM.1 was developed. RAM.1 significantly inhibits GPIb-IX-mediated filopodia formation and Ca 2+ signaling in platelets. In addition, it could inhibit GPIb-dependent thrombin generation. These results suggest that targeting GPIbβ could inhibit activation of GPIb-IX induced by MSD unfolding. Objectives: To investigate whether targeting GPIbβ with RAM.1 can impede rapid platelet clearance induced by exposed Trigger sequence and ameliorate related thrombocytopenia. Methods: Spontaneous filopodia in platelets and transfected Chinese hamster ovary (CHO) cells were visualized by fluorescence staining of actin and confocal microscopy. Images were quantified by ImageJ. Platelet signaling events, like P-selectin exposure, β-galactose exposure, and Ca 2+ influx, were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488-labeled anti-mouse GPIX antibody. Results: CHO cells stably expressing the same mutant GPIb-IX complex in IL4R-IbαTg mouse platelets have been successfully obtained. Like IL4R-IbαTg platelets, these IL4R-IbαTg CHO cells exhibited spontaneous filopodia in the absence of any GPIbα ligands. RAM.1 could inhibit spontaneous filopodia formation in these CHO cells and IL4R-IbαTg platelets (Fig. 1, 2). Compared to wild-type mouse platelets, IL4R-IbαTg platelets constitutively exhibited increased P-selectin exposure, increased β-galactose exposure, and elevated intracellular Ca 2+, all of which could be inhibited by treatment of RAM.1 (Fig. 3). Recombinant RAM.1-GCN4 protein (rRAM.1-GCN4), in which the Fc region of RAM.1 heavy chain was replaced with the GCN4 coiled coil dimerizing sequence, has been generated and used as an alternative of the divalent RAM.1-Fab2. It retained the ability of RAM.1 antibody to inhibit GPIb-IX signaling. Injecting rRAM.1-GCN4 into IL4R-IbαTg mice dramatically improved the life span of endogenous IL4R-IbαTg platelets (Fig. 4). Conclusion: These results demonstrate that the exposed Trigger sequence is sufficient to activate GPIb-IX in transfected CHO cells, and that RAM.1 derivatives can impede GPIbα-mediated rapid platelet clearance. Targeting GPIbβ may be a novel approach to treat GPIb-related thrombocytopenia. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

723 WTX-613, a conditionally activated IFNα INDUKINE™ molecule, induces anti-tumor immune responses resulting in strong tumor growth control in syngeneic mouse tumor models

BackgroundInterferon α (IFNα) was the first cytokine clinically tested as a cancer therapy. IFNα is a member of the type-I IFN family and activates immune responses either directly by engaging IFNα receptors (IFNAR) ubiquitously expressed on immune cells or indirectly by inducing chemokines that attract myeloid and lymphoid cells to the tumor site. High dose IFNα therapy was approved for melanoma, lymphoma, and leukemia but its use is limited by systemic toxicity and modest efficacy.MethodsWTX-613 is a novel systemically delivered IFNα2b pro-drug identified using the Predator™ discovery platform. The inducible WTX-613 INDUKINE™ molecule is designed to deliver wild-type IFNα2b in the tumor microenvironment to reduce systemic toxicity. WTX-613 has two identical half-life extension (HLE) domains tethered to IFNα2b via a tumor protease-sensitive linker. The HLE domain supports less frequent systemic administration but importantly also prevents binding of WTX-613 to IFNAR due to steric hindrance until removal of the HLE domains by tumor proteases.ResultsWTX-613 was selected as a lead molecule due to its improved in vitro profile. Since human IFNα is not functional in the mouse, a surrogate WTX-613 molecule was created consisting of mouse IFNα1 to explore anti-tumor responses in mouse syngeneic tumor models. Intraperitoneal (i.p.) administration of the WTX-613 surrogate resulted in anti-tumor responses in the more immunogenic MC38 colon model which was well tolerated by the mice. Furthermore, less immunogenic tumor models such as B16F10 melanoma and EMT6 breast carcinoma, which are generally less responsive to I/O therapy, also responded with similar anti-tumor activity. Importantly, wild-type mouse IFNα1 was only active in mouse models during the dosing period, and tumors grew back once treatment was stopped. However, the WTX-613 surrogate INDUKINE™ molecule had long lasting anti-tumor activity when dosed at equimolar amounts compared to the native IFNα during the dosing period. The WTX-613 surrogate strongly activated NK and CD8+ cell responses and induced APC and effector cell markers in MC38 tumors. Specifically, the WTX-613 surrogate was better than native IFNα1 in inducing CD8+, NK, and DC cells.ConclusionsPreclinical data obtained so far support the continued development of this innovative and differentiated engineered IFNα therapy and progression into clinical trials.Ethics ApprovalAll mouse in vivo work was performed in accordance with current regulations and standards of the U.S. Department of Agriculture and the NIH at Charles River Laboratories (Morrisville, NC and Worcester, MA).

Cortical Spreading Depression Can Be Triggered by Sensory Stimulation in Primed Wild Type Mouse Brain: a Mechanistic Insight to Migraine Aura Generation

Background: Unlike the spontaneously appearing aura in migraineurs, experimentally, cortical spreading depression (CSD), the neurophysiological correlate of aura is induced by non-physiological stimuli. Consequently, neural mechanisms involved in spontaneous CSD generation, which may provide insight how migraine starts in an otherwise healthy brain, remains largely unclear. We hypothesized that CSD can be physiologically induced by sensory stimulation in primed mouse brain. Methods: Cortex was made susceptible to CSD with partial inhibition of Na+/K+-ATPase by epidural application of a low dose of Na+/K+-ATPase blocker ouabain that does not induce repetitive CSDs or by knocking-down α2 subunit of Na+/K+-ATPase, which is crucial for K+ and glutamate re-uptake by astrocytes, with shRNA. Stimulation-induced CSDs and extracellular K+ changes were monitored in vivo electrophysiologically or with a K+-sensitive fluoroprobe (IPG-4). Results: After priming with ouabain, photic stimulation increased the CSD incidence compared with non-stimulated animals (44.0 vs. 4.9%, p<0.001). Whisker stimulation was less effective (14.9 vs. 2.4%, p=0.02). Knocking-down Na+/K+-ATPase (50% decrease in mRNA) lowered the CSD threshold in all mice tested but triggered stimulus-induced CSDs in 14.3% and 16.7% of mice with photic and whisker stimulation, respectively. Confirming Na+/K+-ATPase hypofunction, extracellular K+ significantly rose during stimulation after subthreshold ouabain or shRNA treatment unlike controls. In line with higher CSD susceptibility, K+ rise was more prominent after ouabain. To gain insight to preventive mechanisms reducing the incidence of stimulus-induced CSDs, we applied an A1-receptor (DPCPX) or GABA-A (bicuculine) antagonist over the occipital cortex, because adenosine formed during stimulation or inhibitory interneuron activity can reduce CSD susceptibility. DPCPX induced CSDs or CSD-like small-DC shifts during photic stimulation, whereas bicuculine was not effective. Conclusions: Our findings indicate that normal brain is well protected against CSD generation. For CSD to be ignited under physiological conditions, priming and predisposing factors are required as seen in migraine patients. Intense sensory stimulation has the potential to trigger a CSD when co-existing conditions can bring extracellular K+ and glutamate concentrations over threshold via reduced uptake of K+ and glutamate (e.g. inefficient fueling of α2-Na+/K+-ATPase due to reduced glycogen breakdown) or facilitated glutamate release (e.g. reduced presynaptic adenosinergic inhibition).

Genotype- and Age-Dependent Differences in Ultrasound Vocalizations of SPRED2 Mutant Mice Revealed by Machine Deep Learning

Vocalization is an important part of social communication, not only for humans but also for mice. Here, we show in a mouse model that functional deficiency of Sprouty-related EVH1 domain-containing 2 (SPRED2), a protein ubiquitously expressed in the brain, causes differences in social ultrasound vocalizations (USVs), using an uncomplicated and reliable experimental setting of a short meeting of two individuals. SPRED2 mutant mice show an OCD-like behaviour, accompanied by an increased release of stress hormones from the hypothalamic–pituitary–adrenal axis, both factors probably influencing USV usage. To determine genotype-related differences in USV usage, we analyzed call rate, subtype profile, and acoustic parameters (i.e., duration, bandwidth, and mean peak frequency) in young and old SPRED2-KO mice. We recorded USVs of interacting male and female mice, and analyzed the calls with the deep-learning DeepSqueak software, which was trained to recognize and categorize the emitted USVs. Our findings provide the first classification of SPRED2-KO vs. wild-type mouse USVs using neural networks and reveal significant differences in their development and use of calls. Our results show, first, that simple experimental settings in combination with deep learning are successful at identifying genotype-dependent USV usage and, second, that SPRED2 deficiency negatively affects the vocalization usage and social communication of mice.

Imaging of retina cellular and subcellular structures using ptychographic hard X-ray tomography

ABSTRACT Ptychographic hard X-ray computed tomography (PXCT) is a recent method allowing imaging with quantitative electron-density contrast. Here, we imaged, at cryogenic temperature and without sectioning, cellular and subcellular structures of a chemically fixed and stained wild-type mouse retina, including axons and synapses, with complete isotropic 3D information over tens of microns. Comparison with tomograms of degenerative retina from a mouse model of retinitis pigmentosa illustrates the potential of this method for analyzing disease processes like neurodegeneration at sub-200 nm resolution. As a non-destructive imaging method, PXCT is very suitable for correlative imaging. Within the outer plexiform layer containing the photoreceptor synapses, we identified somatic synapses. We used a small region inside the X-ray-imaged sample for further high-resolution focused ion beam/scanning electron microscope tomography. The subcellular structures of synapses obtained with the X-ray technique matched the electron microscopy data, demonstrating that PXCT is a powerful scanning method for tissue volumes of more than 60 cells and sensitive enough for identification of regions as small as 200 nm, which remain available for further structural and biochemical investigations.