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Abstract
Anemia of β-thalassemias is caused by ineffective erythropoiesis and reduced red cell survival. Several lines of evidence indicate that iron/heme restriction is a potential therapeutic strategy for the disease. Glycine is a key initial substrate for heme and globin synthesis. We provide evidence that bitopertin, a glycine transport inhibitor administered orally, improves anemia, reduces hemolysis, diminishes ineffective erythropoiesis, and increases red cell survival in a mouse model of β-thalassemia (Hbbth3/+ mice). Bitopertin ameliorates erythroid oxidant damage as indicated by a reduction in membrane-associated free α–globin chain aggregates, in reactive oxygen species cellular content, in membrane-bound hemichromes and in HRI activation and elF2α phosphorylation. The improvement of β-thalassemic ineffective erythropoiesis is associated with diminished mTOR activation, Rab5, Lamp1, and p62 accumulation, indicating an improved autophagy. Bitopertin also upregulates liver hepcidin and diminishes liver iron overload. The hematologic improvements achieved by bitopertin are blunted by the concomitant administration of the iron chelator deferiprone, suggesting that an excessive restriction of iron availability might negate the beneficial effects of bitopertin. These data provide important and clinically relevant insights into glycine restriction and reduced heme synthesis strategies for the treatment of β-thalassemia.
Authors
Alessandro Matte, Enrica Federti, Michael Winter, Annette Koerner, Anja Harmeier, Norman Mazer, Tomas Tomka, Maria Luisa Di Paolo, Luigia De Falco, Immacolata Andolfo, Elisabetta Beneduce, Achille Iolascon, Alejandra Macias-Garcia, Jane-Jane Chen, Anne Janin, Christophe Leboeuf, Francesco Turrini, Carlo Brugnara, Lucia De Franceschi
Abstract
Platelet inositol hexakisphosphate kinase 1 (IP6K1) has been shown to control systemic inflammation. Herein, we examined if platelets and IP6K1 regulate pancreatic tissue injury via formation of NETs in experimental models of acute pancreatitis (AP) in mice. By use of electron microscopy abundant NET formation was observed in the inflamed pancreas. These NETs contained numerous microparticles (MP) expressing CD41 or Mac-1. Platelet depletion reduced deposition of NET-MP complexes in the inflamed pancreas. Circulating platelet-neutrophil aggregates (PNA) were increased and inhibition of P-selectin not only disrupted PNA formation but also reduced NETs formation in the inflamed pancreas. NETs depleted of MPs had lower capacity to provoke amylase secretion and STAT-3 phosphorylation in acinar cells. Taurocholate-induced NETs formation, inflammation and tissue damage in the pancreas were decreased in IP6K1-deficient mice. Thrombin stimulation of mixtures of wild-type platelets and neutrophils resulted in NETs formation but not when IP6K1-deficient platelets were incubated with wild-type neutrophils. Polyphosphate rescue restored thrombin-induced NET formation in mixtures of IP6K1-deficient platelets and wild-type neutrophils. Platelet IP6K1 regulates NET-MP complex formation in the pancreas of mice during induction of AP. Targeting platelet IP6K1 might useful to decrease NET-dependent pancreatic tissue inflammation and tissue injury in patients with AP.
Authors
Raed Madhi, Milladur Rahman, Dler Taha, Johan Linders, Mohammed Merza, Yongzhi Wang, Matthias Mörgelin, Henrik Thorlacius
Abstract
The red blood cell (RBC) storage lesion is a multi-parametric response that occurs during storage at 4°C, but its impact on transfused patients remains unclear. In studies of the RBC storage lesion, the temperature transition from cold storage to normal body temperature that occurs during transfusion has received limited attention. We hypothesized that multiple deleterious events might occur in this period of increasing temperature. We show dramatic alterations in several properties of therapeutic blood units stored at 4°C after warming them to normal body temperature (37°C), as well as febrile temperature (40°C). In particular, the intracellular content and redox state of nicotinamide adenine dinucleotide phosphate [NADP(H)] were directly affected by post-storage incubation at 37°C, as well as by pro-oxidant storage conditions. Modulation of the NADPH-producing pentose phosphate pathway, but not the prevention of hemoglobin autoxidation by conversion of oxyhemoglobin to carboxyhemoglobin, provided protection against storage-induced alterations in RBCs, demonstrating the central role of NADPH in mitigating increased susceptibility of stored RBCs to oxidative stress. We propose that assessing RBCs oxidative status after restoration of body temperature provides a sensitive tool to detect storage-related alterations, and has the potential to improve the quality of stored RBCs for transfusion.
Authors
Aline Roch, Nicholas J. Magon, Jessica Maire, Cacang Suarna, Anita Ayer, Sophie Waldvogel, Beat. A. Imhof, Mark J. Koury, Roland Stocker, Marc Schapira
Abstract
Islet transplantation can restore lost glycemic control in type 1 diabetes subjects, but is restricted in its clinical application by limiting supplies of islets and the need for heavy immune suppression to prevent rejection. TNFAIP3, encoding the ubiquitin editing enzyme A20, regulates the activation of immune cells by raising NF-κB signalling thresholds. Here we show that increasing A20 expression in allogeneic islet grafts resulted in permanent survival for approximately 45% of recipients, and > 80% survival when combined with subtherapeutic rapamycin. Allograft survival was dependent upon regulatory T cells, was antigen-specific and grafts showed reduced expression of inflammatory factors. Transplantation of islets with A20 containing a loss-of-function variant (I325N) resulted in increased RIPK1 ubiquitination and NF-κB signalling, graft hyper-inflammation and acute allograft rejection. Overexpression of A20 in human islets potently reduced expression of inflammatory mediators with no impact on glucose stimulated insulin secretion. Therapeutic administration of A20 raises inflammatory signalling thresholds to favour immune tolerance and promotes islet allogeneic survival. Clinically this would allow for reduced immunosuppression and support the use of alternate islet sources.
Authors
Nathan W. Zammit, Stacey N. Walters, Karen L. Seeberger, Philip J. O’Connell, Gregory S. Korbutt, Shane T. Grey
Abstract
Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of idiopathic pulmonary fibrosis (IPF). The aim of this study was to test the therapeutic effects of MSC-extracellular vesicles/exosomes (MEx) in a bleomycin-induced pulmonary fibrosis model and investigate putative mechanisms of action. Exosomes were isolated from media conditioned by human bone marrow MSCs. Adult mice (C57BL/6 strain) were challenged with endotracheal instillation of bleomycin and treated with MEx concurrently or for reversal models, at day 7 or 21. Experimental groups were assessed at day 7 and/or at day 14 or 28. Bleomycin-challenged mice presented with severe septal thickening and prominent fibrosis, and this was effectively prevented or reversed by a single dose of MEx. Furthermore, MEx therapy modulated whole lung macrophage phenotype and shifted the proportion of lung ‘proinflammatory’ classical monocytes, non-classical monocytes and alveolar macrophages to favor the monocyte/macrophage profiles of untreated-control mice. A parallel immunomodulatory effect was demonstrated in the bone marrow. Notably, transplantation of MEx-preconditioned bone marrow-derived monocytes alleviated core features of pulmonary fibrosis and lung inflammation. Proteomic analysis further revealed a signature enriched in non-inflammatory monocyte genes following MEx therapy supporting the immuno-regulatory, anti-inflammatory effect of MEx.We conclude that a bolus dose of MEx prevents and reverts core features of bleomycin-induced pulmonary fibrosis, and that the beneficial actions of MEx may be mediated via systemic modulation of monocyte phenotypes.
Authors
Nahal Mansouri, Gareth R. Willis, Angeles Fernandez-Gonzalez, Monica Reis, Sina Nassiri, Alex Mitsialis, Stella Kourembanas
Abstract
The acute respiratory distress syndrome (ARDS) is an inflammatory lung disorder that frequently complicates critical illness, and most commonly occurs in the setting of sepsis. Although a number of clinical and environmental risk factors for ARDS have been described, not all patients with risk factors develop the syndrome, raising the possibility of genetic underpinnings for ARDS susceptibility. We have previously reported that circulating cell-free hemoglobin (CFH) is elevated during sepsis, and higher levels are associated with worse outcomes. CFH is rapidly scavenged by the plasma protein haptoglobin (Hp). A common HP genetic variant HP2 is unique to humans and represents 60% of the HP allele frequency in populations of European ancestry. The HP2 gene product has reduced ability to inhibit CFH-mediated inflammation and oxidative stress compared to the alternative HP1. We hypothesized that the HP2 variant increases ARDS susceptibility during sepsis when plasma CFH levels are elevated. In a murine model of sepsis with elevated CFH levels, transgenic mice homozygous for Hp2 had increased lung inflammation, pulmonary vascular permeability, lung apoptosis, and mortality compared to mice homozygous for the alternative allele Hp1. We then tested the clinical relevance of our findings in a prospective observational cohort study of 496 septic critically ill adults, and found that the HP2 variant was significantly associated with increased ARDS susceptibility (odds ratio 1.41 per HP2 allele, 95% confidence interval 1.06 – 1.88, P = 0.018) after controlling for clinical risk factors and plasma CFH. This relationship between the HP2 genetic variant and ARDS risk was only seen in patients with elevated plasma CFH levels. These observations identify the HP2 variant as a novel genetic ARDS risk factor during sepsis, and may have important implications in the study and treatment of ARDS.
Authors
V. Eric Kerchberger, Julie A. Bastarache, Ciara M. Shaver, Hiromasa Nagata, J. Brennan McNeil, Stuart R. Landstreet, Nathan D. Putz, Wen-Kuang Yu, Jordan Jesse, Nancy E. Wickersham, Tatiana N. Sidorova, David R. Janz, Chirag R. Parikh, Edward D. Siew, Lorraine B. Ware
Abstract
Background: Hydroxymethyl-glutaryl-coenzyme A reductase inhibitors (‘statins’) are prescribed to millions of people. Statins are anti-inflammatory independent of their cholesterol-reducing effects. To date, most reports on the immune effects of statins have assayed a narrow array of variables and have focused on cell lines, rodent models, or patient cohorts. We sought to define the effect of rosuvastatin on the ‘immunome’ of healthy, normocholesterolemic subjects. Methods: Prospective study of rosuvastatin (20 mg/day x 28 days) in 18 statin-naïve adults with low density lipoprotein-cholesterol <130 mg/dL. A panel of >180 immune/biochemical/endocrinologic variables was measured at baseline, and days 14, 28, and 42 (14 days after drug withdrawal). Drug effect was evaluated using linear mixed effects models. Potential interactions between drug and baseline high-sensitivity C-reactive protein (hsCRP) were evaluated. Results: A wide array of immune measures changed (nominal p<0.05) during rosuvastatin treatment, although the changes were modest in magnitude and few met a false discovery rate of 0.05. Among changes noted were a concordant increase in pro-inflammatory cytokines (IFNγ, IL-1β, IL-5, IL-6, TNFα) and peripheral blood neutrophil frequency, and a decline in activated T regulatory cell frequency. Several drug effects were significantly modified by baseline hsCRP, and some did not resolve after drug withdrawal. Among other unexpected rosuvastatin effects were changes in erythrocyte indices, glucose-regulatory hormones, CD8+ T cells, and haptoglobin. Conclusion: Rosuvastatin induces modest changes in immunologic and metabolic measures in normocholesterolemic subjects, with several effects dependent upon baseline CRP. Future, larger studies are warranted to validate these changes and their physiological significance.
Authors
Peer W. F. Karmaus, Min Shi, Shira Perl, Angélique Biancotto, Julián Candia, Foo Cheung, Yuri Kotliarov, Neal Young, Michael B. Fessler
Abstract
Accumulation of lysosomal storage material and late-stage neurodegeneration are hallmarks of lysosomal storage disorders (LSDs) affecting the brain. Yet, for most LSDs, including CLN3 disease, the most common form of childhood dementia, it is unclear what mechanisms drive neurologic symptoms. Do deficits arise from loss of function of the mutated protein or toxicity from storage accumulation? Here, using in vitro voltage sensitive dye imaging and in vivo electrophysiology, we find progressive hippocampal dysfunction occurs prior to notable lysosomal storage and neuronal loss in two CLN3 disease mouse models. Pharmacologic reversal of lysosomal storage deposition in young mice does not rescue this circuit dysfunction. Additionally, we find that CLN3 disease mice lose an electrophysiologic marker of new memory encoding – hippocampal sharp wave ripples. This discovery, which is also seen in Alzheimer’s disease, suggests the possibility of a shared electrophysiologic signature of dementia. Overall, our data describes new insights into previously unknown network-level changes occurring in LSDs affecting the central nervous system, and highlight the need for new therapeutic interventions targeting early circuit defects.
Authors
Rebecca C. Ahrens-Nicklas, Luis Tecedor, Arron Hall, Elena Lysenko, Akiva S. Cohen, Beverly L. Davidson, Eric D. Marsh
Abstract
Background: In this study, we aimed to identify the lipidomic predictors of early type-2 diabetic kidney disease (DKD) progression which are currently undefined DKD progression. Methods: This longitudinal study included 92 American Indians with type-2 diabetes. Serum lipids (406 from 18 classes) were quantified using mass spectrometry from baseline samples when iothalamate glomerular filtration rate (GFR) was ≥90 mL/min. Affymetrix GeneChip Array was used to measure renal transcript expression. DKD Progression was defined as ≥40% decline in GFR during follow up. Results: Participants had a mean age of 45±9 years and median urine albumin-creatinine ratio of 43 (interquartile range 11 to 144). The 32 progressors had significantly higher relative abundance of polyunsaturated triacylglycerols (TAG)s and a lower abundance of C16-20 acylcarnitines (AC)s (p<0.001). In a Cox regression model the main effect terms of unsaturated free fatty acids and phosphatidylethanolamines and the interaction terms of C16-20 ACs and short, low-double-bond TAGs by categories of albuminuria independently predicted progression of DKD. Renal expression of acetyl-CoA carboxylase encoding gene (ACACA) correlated with serum diacylglycerols in the glomerular compartment (r=0.36, p=0.006), and with low-double-bond TAGs in the tubulointerstitial compartment (r=0.52, p<0.001). Conclusion: Collectively, the findings reveal a previously unrecognized link between lipid markers of impaired mitochondrial β-oxidation and enhanced lipogenesis, with DKD progression, in individuals with preserved GFR. Renal acetyl-CoA carboxylase activation accompanies these lipidomic changes and suggests that it may be the underlying mechanism linking lipid abnormalities to DKD progression. Funding: R24DK082841, K08DK106523, R03DK121941, P30DK089503, P30DK081943, P30DK020572
Authors
Farsad Afshinnia, Viji Nair, Jiahe Lin, Thekkelnaycke M. Rajendiran, Tanu Soni, Jaeman Byun, Kumar Sharma, Patrice E. Fort, Thomas W. Gardner, Helen C. Looker, Robert G. Nelson, Frank C. Brosius, Eva L. Feldman, George Michailidis, Matthias Kretzler, Subramaniam Pennathur
Abstract
Kabuki syndrome 1 (KS1) is a Mendelian disorder of the epigenetic machinery caused by mutations in the gene encoding KMT2D, which methylates lysine 4 on histone H3 (H3K4). KS1 is characterized by intellectual disability, postnatal growth retardation, and distinct craniofacial dysmorphisms. A mouse model (Kmt2d+/bGeo) exhibits features of the human disorder and has provided insight into other phenotypes; however, the mechanistic basis of skeletal abnormalities and growth retardation remains elusive. Using high-resolution micro-computed tomography we show that Kmt2d+/bGeo mice have shortened long bones and ventral bowing of skulls. In vivo expansion of growth plates within skulls and long bones suggests disrupted endochondral ossification as a common disease mechanism. Stable chondrocyte cell lines harboring inactivating mutations in Kmt2d exhibit precocious differentiation, further supporting this mechanism. A known inducer of chondrogenesis, SOX9, and its targets show markedly increased expression in Kmt2d-/- chondrocytes. By transcriptome profiling, we identify Shox2 as a putative KMT2D target. We propose that decreased KMT2D-mediated H3K4me3 at Shox2 releases Sox9 inhibition and thereby leads to enhanced chondrogenesis, providing a novel and plausible explanation for precocious chondrocyte differentiation. Our findings provide insight into the pathogenesis of growth retardation in KS1 and suggest novel therapeutic approaches for this and related disorders.
Authors
Jill A. Fahrner, Wan-Ying Lin, Ryan C. Riddle, Leandros Boukas, Valerie B. DeLeon, Sheetal Chopra, Susan E. Lad, Teresa Romeo Luperchio, Kasper D. Hansen, Hans T. Bjornsson
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