Research
Abstract
Dystrophic muscle is characterised by chronic injury, and a steady recruitment of inflammatory Ly6Chi monocytes. Recent studies have identified the spleen as the dominant reservoir of these cells during chronic inflammation. Here we investigated the hitherto unexplored contribution of splenic Ly6Chi monocytes to dystrophic muscle pathology. Using the mdx mouse model of muscular dystrophy, we show that Ly6Chi monocytes accumulate in great numbers in the spleen over the course of the disease. The chemokine receptor CCR2 was upregulated on Ly6Chi monocytes in mdx spleen before disease onset, thereby enabling their recruitment to dystrophic muscle. Splenectomy performed before disease onset significantly reduced the number of Ly6Chi monocytes infiltrating dystrophic limb muscle. Moreover, in the absence of splenic Ly6Chi monocytes there was a significant reduction in dystrophic muscle inflammation and necrosis, along with improved regeneration during early disease. However, during late disease, lack of splenic Ly6Chi monocytes adversely affected muscle fiber repair, due to a delay in the phenotypic shift of pro-inflammatory F4/80+/Ly6Chi/CD206lo to anti-inflammatory F4/80+/Ly6Clo/CD206+ macrophages. Overall, we show that the spleen is an indispensable source of Ly6Chi monocytes in muscular dystrophy, and that splenic monocytes are critical players in both muscle fiber injury and repair.
Authors
Giuseppe Rizzo, Rosanna Di Maggio, Anna Benedetti, Jacopo Morroni, Marina Bouche, Biliana Lozanoska-Ochser
Abstract
Anti-PD1 therapy has become an immunotherapeutic backbone for treating many cancer types. While many studies have aimed to characterize the immune response to anti-PD1 therapy in the tumor and in the peripheral blood, relatively less is known about the changes in the tumor draining lymph nodes (TDLNs). TDLNs are primary sites of tumor antigen exposure that are critical to both regulation and cross-priming of the antitumor immune response. We employed multi-panel mass cytometry to obtain a high-parameter proteomic (39 total unique markers) immune profile of the TDLN in a well-studied PD1-responsive immunocompetent mouse model. Based on combined hierarchal gating and unsupervised clustering analyses, we found that anti-PD1 therapy enhances remodeling of both B and T cell compartments toward memory phenotypes. Functionally, expression of checkpoint markers was increased in conjunction with production of IFNγ, TNFα, or IL2 in key cell types, including B and T cell subtypes and rarer subsets such as Tregs and NKT cells. A deeper profiling of the immunologic changes that occur in the TDLN milieu during effective anti-PD1 therapy may lead to the discovery of novel biomarkers for monitoring response and provide key insights toward developing combination immunotherapeutic strategies.
Authors
Won Jin Ho, Mark Yarchoan, Soren Charmsaz, Rebecca M. Munday, Ludmila Danilova, Marcelo B. Sztein, Elana J. Fertig, Elizabeth M. Jaffee
Abstract
Chronic sympathoexcitation is implicated in ventricular arrhythmogenesis (VAs) following myocardial infarction (MI), but the critical neural pathways involved are not well understood. Cardiac adrenergic function is partly regulated by sympathetic afferent reflexes, transduced by spinal afferent fibers expressing the TRPV1 channel. The role of chronic TRPV1 afferent signaling in VAs is not known. We hypothesized that persistent TRPV1 afferent neurotransmission promotes VAs after MI. Using epicardial Resiniferatoxin (RTX) to deplete cardiac TRPV1-expressing fibers, we dissected the role of this neural circuit in VAs after chronic MI in a porcine model. We examined the underlying mechanisms using molecular approaches, immunohistochemistry, in vitro and in vivo cardiac electrophysiology, and simultaneous cardio-neural mapping. Epicardial RTX depleted cardiac TRPV1 afferent fibers and abolished functional responses to TRPV1 agonists. Ventricular tachycardia/fibrillation (VT/VF) was readily inducible in MI subjects by programmed electrical stimulation or cesium chloride administration, however, TRPV1 afferent depletion prevented VT/VF induced by either method. Mechanistically, TRPV1 afferent depletion neither altered cardiomyocyte action potentials and calcium transients; nor the expression of ion channels and calcium handling proteins. However, it attenuated fibrosis and mitigated electrical instability in the scar-border zone. In vivo recordings of cardiovascular-related stellate ganglion neurons (SGNs) revealed that MI enhances SGN function and disrupts integrated neural processing. Depleting TRPV1 afferents normalized these processes. Taken together, these data indicate that after MI, TRPV1 afferent-induced adrenergic dysfunction promotes fibrosis, adverse cardiac remodeling, and worsens border zone electrical heterogeneity, resulting in electrically unstable ventricular myocardium. We propose targeting TRPV1-expressing afferent to reduce VT/VF following MI.
Authors
Koji Yoshie, Pradeep S. Rajendran, Louis Massoud, Janki Mistry, M. Amer Swid, Xiaohui Wu, Tamer Sallam, Rui Zhang, Joshua I. Goldhaber, Siamak Salavatian, Olujimi A. Ajijola
Abstract
Vision loss in age-related macular degeneration (AMD) stems from disruption of photoreceptor cells in the macula, the central retinal area required for high-acuity vision. Mice and rats have no macula, but surgical insertion of a subretinal implant can induce localized photoreceptor degeneration due to chronic separation from retinal pigment epithelium, simulating a key aspect of AMD. We find that the implant-induced loss of photoreceptors in rat retina leads to local changes in the physiology of downstream retinal ganglion cells (RGCs), similar to changes in RGCs of rodent models of retinitis pigmentosa (RP), an inherited disease causing retina-wide photoreceptor degeneration. The local implant-induced changes in RGCs include enhanced intrinsic excitability leading to accelerated spontaneous firing, increased membrane permeability to fluorescent dyes, and enhanced photosensitization by azobenzene photoswitches. The local physiological changes are correlated with an increase in Retinoic Acid Receptor (RAR)-induced gene transcription, the key process underlying retinal remodeling in mouse models of RP. Hence the loss of photoreceptors, whether by local physical perturbation or by inherited mutation, leads to a stereotypical set of pathophysiological consequences in RGCs. These findings implicate RAR as a possible common therapeutic target for reversing the signal-corrupting effects of retinal remodeling in both RP and AMD.
Authors
Bristol Denlinger, Zachary Helft, Michael Telias, Henri Lorach, Daniel Palanker, Richard H. Kramer
Abstract
Duchenne muscular dystrophy (DMD) is a devastating genetic muscle disease resulting in progressive muscle degeneration and wasting. Glucocorticoids, specifically prednisone/prednisolone and deflazacort, are commonly used by DMD patients. Emerging DMD therapeutics include those targeting the muscle wasting factor, myostatin (Mstn). The aim of this study was to investigate how chronic glucocorticoid treatment impacts the efficacy of Mstn inhibition in the D2.mdx mouse model of DMD. We report that chronic treatment of dystrophic mice with prednisolone (Pred) causes significant muscle wasting, entailing both activation of the ubiquitin-proteasome degradation pathway and inhibition of muscle protein synthesis. Combining Pred with Mstn inhibition, using a modified Mstn propeptide (dnMstn), completely abrogates the muscle hypertrophic effects of Mstn inhibition independent of Mstn expression or SMAD3 activation. Transcriptomic analysis identified that combining Pred with dnMstn treatment affects gene expression profiles associated with inflammation, metabolism, and fibrosis. Additionally, we demonstrate that Pred-induced muscle atrophy is not prevented by Mstn ablation. Therefore, glucocorticoids interfere with potential muscle mass benefits associated with targeting Mstn, and the ramifications of glucocorticoid use should be a consideration during clinical trial design for DMD therapeutics. These results have significant implications for past and future Mstn inhibition trials in DMD.
Authors
David W. Hammers, Cora C. Hart, Andreas Patsalos, Michael K. Matheny, Lillian A. Wright, Laszlo Nagy, H. Lee Sweeney
LXRs regulate features of age-related macular degeneration and may be a potential therapeutic target
Abstract
Effective treatments and animal models for the most prevalent neurodegenerative form of blindness in the elderly, called age-related macular degeneration (AMD), are lacking. Genome-wide association studies have identified lipid metabolism and inflammation as AMD-associated pathogenic pathways. Given liver x receptors, encoded by NR1H3 and NR1H2, are master regulators of these pathways, herein we investigated the role of LXR in human and mouse eyes as a function of age and disease, and tested the therapeutic potential of targeting LXR. We identified immunopositive LXR fragments in human extracellular early dry AMD lesions and a decrease in LXR expression within the retinal pigment epithelium (RPE) as a function of age. Aged mice, lacking LXR presented with isoform dependent ocular pathologies. Specifically, loss of the Nr1h3 isoform results in pathobiologies aligned with AMD, supported by compromised visual function, accumulation of native and oxidized lipids in the outer retina, and upregulation of ocular inflammatory cytokines, while absence of Nr1h2 is associated with ocular lipoidal degeneration. Therapeutically, LXR activation, ameliorated lipid accumulation and oxidant-induced injury in RPE cells in vitro, and decreased ocular inflammatory markers and lipid deposition in a mouse model, in vivo, providing translational support for pursuing LXR-active pharmaceuticals as potential therapies for dry AMD.
Authors
Mayur Choudhary, Ebraheim N. Ismail, Pei-Li Yao, Faryan Tayyari, Roxana A. Radu, Steven Nusinowitz, Michael E. Boulton, Rajendra S. Apte, Jeffrey W. Ruberti, James T. Handa, Peter Tontonoz, Goldis Malek
Abstract
High-density lipoproteins (HDL) contain hundreds of lipid species and proteins and exert many potentially vasoprotective and anti-diabetogenic activities on cells. To resolve structure-function-disease relationships of HDL we characterized HDL of 51 healthy subjects and 98 patients with diabetes (T2DM), coronary heart disease (CHD), or both for protein and lipid composition as well as functionality in five cell types. The integration of 40 clinical characteristics, 34 NMR features, 182 proteins, 227 lipid species, and 12 functional read-outs by high-dimensional statistical modelling revealed first that CHD and T2DM are associated with different changes of HDL in size distribution, protein and lipid composition as well as function. Second, different cellular functions of HDL are weakly correlated with each other and determined by different structural components. Cholesterol efflux capacity was no proxy of other functions. Third, three novel determinants of HDL function were identified and validated by the use of artificially reconstituted HDL, namely the sphingadienine-based sphingomyelin SM 42:3 and glycosylphosphatidylinositol-phospholipase D1 for the ability of HDL to inhibit starvation induced apoptosis of human aortic endothelial cells and apolipoprotein F for the ability of HDL to promote maximal respiration of brown adipocytes.
Authors
Mathias Cardner, Mustafa Yalcinkaya, Sandra Goetze, Edlira Luca, Miroslav Balaz, Monika Hunjadi, Johannes Hartung, Andrej Shemet, Nicolle Kraenkel, Silvija Radosavljevic, Michaela Keel, Alaa Othman, Gergely Karsai, Thorsten Hornemann, Manfred Claassen, Gerhard Liebisch, Erick Carreira, Andreas Ritsch, Ulf Landmesser, Jan Krützfeldt, Christian Wolfrum, Bernd Wollscheid, Niko Beerenwinkel, Lucia Rohrer, Arnold von Eckardstein
Abstract
Pancreatic ductal adenocarcinoma (PDAC) has dismal five-year survival (<9%). We hypothesize that exposure of tumors to conventional therapies may preferentially modulate immune biomarkers in the tumor microenvironment in PDAC. PDAC patients who underwent upfront surgical resection or who received neoadjuvant FOLFIRINOX with or without neoadjuvant radiotherapy followed by surgical resection were selected for study. Total expression of immunologically relevant transcripts and spatially resolved expression of immunologically relevant proteins was quantitated using multiplexed methods (Nanostring nCounter and GeoMX platforms). This analysis identified numerous differentially expressed transcripts associated with the type of neoadjuvant therapy received. Moreover, we identified significant alterations in the expression and/or spatial distribution of immunologically relevant proteins in different regions (tumor cell rich, immune cell rich, stromal cell rich) of the TME. These data provide insight into the immunological effects of clinically relevant neoadjuvant therapy for resectable/borderline-resectable PDAC, by describing significant differences in the expression of key immunologic biomarkers within the PDAC microenvironment that were associated with the type of treatment patients received prior to surgical resection. This represents a comprehensive analysis of numerous biomarkers conducted on the PDAC microenvironment. This work may guide strategic new combination therapies for pancreatic cancer.
Authors
Matthew R. Farren, Layal Sayegh, Michael Brandon Ware, Hsiao-Rong Chen, Jingjing Gong, Yan Liang, Alyssa Krasinskas, Shishir K. Maithel, Mohammad Zaidi, Juan M. Sarmiento, David Kooby, Pretesh Patel, Bassel El-Rayes, Walid Shaib, Gregory B. Lesinski
Abstract
Small molecule inhibitors of Dual Specificity, Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A), including harmine and others, are able to drive human beta cell regeneration. While DYRK1A is certainly a target of this class, whether it is the only, or the most important target, is uncertain. Here, we employ a combined pharmacologic and genetic approach to refine the potential mitogenic targets of the DYRK1A inhibitor family in human islets. A combination of human beta cell RNAseq, DYRK1A inhibitor kinome screens, pharmacologic inhibitors, and targeted silencing of candidate genes confirms that DYRK1A is a central target. Surprisingly, however, DYRK1B also proves to be an important target: silencing DYRK1A results in an increase in DYRK1B; simultaneous silencing of both DYRK1A and DYRK1B yields greater beta cell proliferation than silencing either individually. Importantly, other potential kinases, such as the CLK and the GSK3 families, are excluded as important harmine targets. Finally, we describe adenoviruses that are able to silence up to seven targets simultaneously. Collectively, we report that inhibition of both DYRK1A and DYRK1B is required for induction of maximal rates of human beta cell proliferation, and provide clarity for future efforts at structure-based drug design for human beta cell regenerative drugs.
Authors
Courtney Ackeifi, Ethan Swartz, Kunal Kumar, Hongtao Liu, Suebsuwong Chalada, Esra Karakose, Donald K. Scott, Adolfo Garcia-Ocaña, Roberto Sanchez, Robert J. DeVita, Andrew F. Stewart, Peng Wang
Abstract
Subpial demyelination is a specific hallmark of multiple sclerosis (MS) and a correlate of disease progression. Although the mechanism(s) that mediate pathogenesis in the subpial compartment remain unclear, it has been speculated that inflammation in the overlying meninges may be associated with subpial injury. Here we show that adoptive transfer of proteolipid protein-primed Th17 cells into SJL/J recipient mice induces subpial demyelination associated with microglial/macrophage activation, disruption of the glial limitans and evidence of an oxidative stress response. This pathology was topologically associated with foci of immune cells in the meninges and occurred in the absence of measurable anti-MOG IgM or IgG antibodies. To test the role of brain-infiltrating leukocytes on subpial injury, we modulated sphingosine 1-phosphate (S1P) receptor1,5 activity with BAF312 (siponimod) treatment. Administration of BAF312, even after adoptively transferred T cells had entered the brain, significantly ameliorated clinical EAE and diminished subpial pathology, concomitant with a selective reduction in the capacity of transferred T cells to make Th17 cytokines. We conclude that sustained subpial cortical injury is associated with the capacity for brain-resident T cells to produce Th17 cytokines, and this pathological process occurs in an S1P receptor1,5-dependent manner.
Authors
Lesley A. Ward, Dennis S.W. Lee, Anshu Sharma, Angela Wang, Ikbel Naouar, Xianjie I. Ma, Natalia Pikor, Barbara Nuesslein-Hildesheim, Valeria Ramaglia, Jennifer L. Gommerman
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