Myeloid-biased HSC require Semaphorin 4A from the bone marrow niche for self-renewal under stress and life-long persistence

Tissue stem cells are hierarchically organized. Those that are most primitive serve as key drivers of regenerative response but the signals that selectively preserve their functional integrity are largely unknown. Here, we identify a secreted factor, Semaphorin 4A (Sema4A), as a specific regulator of myeloid-biased hematopoietic stem cells (myHSC), which are positioned at the top of the HSC hierarchy. Lack of Sema4A leads to exaggerated myHSC (but not downstream balanced HSC) proliferation after acute inflammatory stress, indicating that Sema4A enforces myHSC quiescence. Strikingly, aged Sema4A knock-out myHSC expand but almost completely lose reconstitution capacity. The effect of Sema4A is non cell-autonomous, since upon transplantation into Sema4A-deficient environment, wild-type myHSC excessively proliferate but fail to engraft long-term. Sema4A constrains inflammatory signaling in myHSC and acts via a surface receptor Plexin-D1. Our data support a model whereby the most primitive tissue stem cells critically rely on a dedicated signal from the niche for self-renewal and life-long persistence.

Genetic barcoding systematically comparing genes in del(5q) MDS reveals a central role for CSNK1A1 in clonal expansion

How genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSC) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, a systematic comparison was carried out on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1-/+ multipotent progenitors showed a pro-proliferative gene signature and HSCs a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1-/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. A crucial role for Csnk1a1 haploinsufficiency in the selective advantage of the 5q- HSC is therefore proposed. It is implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress.

AGEs-Induced and Endoplasmic Reticulum Stress/Inflammation-Mediated Regulation of GLUT4 Expression and Atherogenesis in Diabetes Mellitus

In recent decades, complex and exquisite pathways involved in the endoplasmic reticulum (ER) and inflammatory stress responses have been demonstrated to participate in the development and progression of numerous diseases, among them diabetes mellitus (DM). In those pathways, several players participate in both, reflecting a complicated interplay between ER and inflammatory stress. In DM, ER and inflammatory stress are involved in both the pathogenesis of the loss of glycemic control and the development of degenerative complications. Furthermore, hyperglycemia increases the generation of advanced glycation end products (AGEs), which in turn refeed ER and inflammatory stress, contributing to worsening glycemic homeostasis and to accelerating the development of DM complications. In this review, we present the current knowledge regarding AGEs-induced and ER/inflammation-mediated regulation of the expression of GLUT4 (solute carrier family 2, facilitated glucose transporter member 4), as a marker of glycemic homeostasis and of cardiovascular disease (CVD) development/progression, as a leading cause of morbidity and mortality in DM.

Gênero e hidratação como mediadores relevantes para restauração de processos inflamatórios em atletas jovens

Objetivos: Poucos estudos tem dado a devida importância ao estado de hidratação e as alterações hematológicas em menores de 18 anos. O presente estudo investigou estas alterações em menores de 18 anos na prova de 3000 metros para ambos os gêneros. Método: Foram incluídos os seis melhores participantes das Olimpíadas Nacionais do Ensino Médio (menores de 18 anos) no Brasil. Trata-se de um estudo observacional e transversal. Os atletas correram 3.000 metros em uma pista oval de 400 metros. Foi realizado leucograma total e específico, eritrograma e contagem de plaquetas em quatro diferentes tempos (jejum, pré, pós e recuperação). Os cálculos de comparação entre tempos, apresentados, foram adaptados do modelo matemático de variação percentual. Resultados: Quando comparadas as médias em relação a cada tempo, pode-se notar que os eritrócitos, hemoglobina, hematócrito, leucócitos totais e basófilos aumentaram significativamente após a corrida para ambos os gêneros. Além disso, as plaquetas também aumentaram para ambos os gêneros sem significância. O coeficiente de correlação de Pearson mostrou importante interação leucócitos-plaquetas. Os homens recuperaram seu estado de hidratação em quase metade do tempo em relação as mulheres. Em relação ao sistema imune e resposta inflamatória, as atletas do gênero feminino tiveram um aumento relativo de mais que o dobro para leucócitos e não recuperaram estes valores, além de apresentarem um aumento de eosinófilos neste mesmo tempo, enquanto os atletas do gênero masculino apresentaram diminuição. As mulheres apresentaram um aumento de quase o dobro para linfócitos, enquanto os homens tiveram este comportamento para os fagócitos. Conclusões: O exercício induz alteração do estado de hidratação e sistema imune, tendo diferenças em relação ao gênero. Quando bem conduzido, gera efeitos antiinflamatórios a longo prazo. Quando não há controle sobre o volume, intensidade e recuperação pode causar imunossupressão e promover a suceptibilidade para infecções. Foi possível observar que os atletas do gênero masculino tem um menor estresse inflamatório e recuperam o seu estado de hidratação e a resposta inflamatória mais rápido que as meninas após uma corrida de 3000 metros. Objectives: Few studies have given due importance to the hydration status and hematological alterations in children under 18 years of age. The present study investigated these alterations in children under 18 years old in the 3000 meters test for both genders. Method: The six best participants of Brazil's National High School Olympiads (under 18 years old) were included. This is an observational and cross-sectional study. Athletes ran 3,000 meters on a 400 meters oval track. Total and specific white blood cell count, erythrogram, and platelet count were performed four times (fasting, pre, post, and recovery). The time comparison calculations presented were adapted from the percentage variation mathematical model. Results: When comparing the means for each time, it can be noted that erythrocytes, hemoglobin, hematocrit, total leukocytes, and basophils increased significantly after the race for both genders. Furthermore, platelets also increased for both genders without significance. Pearson's correlation coefficient showed a significant leukocyte-platelet interaction. Men regained their hydration status in almost half the time compared to women. Regarding the immune system and inflammatory response, female athletes had a relative increase of more than double for leukocytes and did not recover these values, in addition to showing an increase in eosinophils simultaneously, while male athletes showed a decrease. Women showed an increase of almost double for lymphocytes, while men showed this behavior for phagocytes. Conclusions: Exercise induces changes in hydration status and immune system, with differences concerning gender. When done well, it generates long-term anti-inflammatory effects. When there is no control over volume, intensity, and recovery, it can cause immunosuppression and increase infection susceptibility. It was observed that male athletes have lower inflammatory stress and recover their hydration status and inflammatory response faster than girls after a 3000 meter run.

Ketone bodies: from enemy to friend and guardian angel

During starvation, fasting, or a diet containing little digestible carbohydrates, the circulating insulin levels are decreased. This promotes lipolysis, and the breakdown of fat becomes the major source of energy. The hepatic energy metabolism is regulated so that under these circumstances, ketone bodies are generated from β-oxidation of fatty acids and secreted as ancillary fuel, in addition to gluconeogenesis. Increased plasma levels of ketone bodies thus indicate a dietary shortage of carbohydrates. Ketone bodies not only serve as fuel but also promote resistance to oxidative and inflammatory stress, and there is a decrease in anabolic insulin-dependent energy expenditure. It has been suggested that the beneficial non-metabolic actions of ketone bodies on organ functions are mediated by them acting as a ligand to specific cellular targets. We propose here a major role of a different pathway initiated by the induction of oxidative stress in the mitochondria during increased ketolysis. Oxidative stress induced by ketone body metabolism is beneficial in the long term because it initiates an adaptive (hormetic) response characterized by the activation of the master regulators of cell-protective mechanism, nuclear factor erythroid 2-related factor 2 (Nrf2), sirtuins, and AMP-activated kinase. This results in resolving oxidative stress, by the upregulation of anti-oxidative and anti-inflammatory activities, improved mitochondrial function and growth, DNA repair, and autophagy. In the heart, the adaptive response to enhanced ketolysis improves resistance to damage after ischemic insults or to cardiotoxic actions of doxorubicin. Sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors may also exert their cardioprotective action via increasing ketone body levels and ketolysis. We conclude that the increased synthesis and use of ketone bodies as ancillary fuel during periods of deficient food supply and low insulin levels causes oxidative stress in the mitochondria and that the latter initiates a protective (hormetic) response which allows cells to cope with increased oxidative stress and lower energy availability. Keywords Ketogenic diet, Ketone bodies, Beta hydroxybutyrate, Insulin, Obesity, Type 2 diabetes, Inflammation, Oxidative stress, Cardiovascular disease, SGLT2, Hormesis

Phosphatidylcholine restores neuronal plasticity of neural stem cells under inflammatory stress

The balances between NSCs growth and differentiation, and between glial and neuronal differentiation play a key role in brain regeneration after any pathological conditions. It is well known that the nervous tissue shows a poor recovery after injury due to the factors present in the wounded microenvironment, particularly inflammatory factors, that prevent neuronal differentiation. Thus, it is essential to generate a favourable condition for NSCs and conduct them to differentiate towards functional neurons. Here, we show that neuroinflammation has no effect on NSCs proliferation but induces an aberrant neuronal differentiation that gives rise to dystrophic, non-functional neurons. This is perhaps the initial step of brain failure associated to many neurological disorders. Interestingly, we demonstrate that phosphatidylcholine (PtdCho)-enriched media enhances neuronal differentiation even under inflammatory stress by modifying the commitment of post-mitotic cells. The pro-neurogenic effect of PtdCho increases the population of healthy normal neurons. In addition, we provide evidences that this phospholipid ameliorates the damage of neurons and, in consequence, modulates neuronal plasticity. These results contribute to our understanding of NSCs behaviour under inflammatory conditions, opening up new venues to improve neurogenic capacity in the brain.

Heat Shock-Related Protein Responses and Inflammatory Protein Changes Are Associated with Mild Prolonged Hypoglycemia

Mild hypoglycemia is common in clinical practice. Severe hypoglycemia results in heat shock protein and associate co-chaperone changes. Whether mild prolonged hypoglycemia elicits a similar response with inflammatory and oxidative-stress responses compared with a severe hypoglycemic event is unclear; therefore, this pilot exploratory study was undertaken. We performed a case–control induced hypoglycemia clamp study, maintaining blood glucose at 2.8 mmol/L (50 mg/dL) for 1 h in 17 subjects (T2D (n = 10); controls (n = 7)). Blood sampling was performed at baseline, hypoglycemia, and 24 h; slow off-rate modified aptamer (SOMA)-scan plasma protein analysis of HSP-related proteins, inflammatory stress markers, and oxidative stress markers was performed. In total, 16 HSPs were analyzed. At baseline, TLR4:MD-2 complex was elevated (p = 0.01), whilst HSPA8 was lower (p < 0.05) in T2D. At hypoglycemia, UBE2N, STIP1, and UBE2L3 increased (all p < 0.05), whilst TLR4:MD-2 and HSPA8 decreased (p < 0.05) in T2D versus baseline. In follow-up after hypoglycemia, HSPs normalized to baseline by 24 h, except UBE2L3 (p < 0.05), which was decreased in controls versus baseline. Correlation of altered inflammatory markers with HSPs revealed the following: at baseline, TLR4:MD-2 correlated with CXCL10 (p < 0.01) and SIGLEC1 (p < 0.05) in controls; HSPA8 negatively correlated with IL5 (p < 0.05) in T2D. A negative correlation between urinary isoprostane 8-iso PGF2α, a marker of oxidative stress, and HSPA1A was seen at 24 h in T2D only (p < 0.05). In conclusion, the HSP changes seen for mild prolonged hypoglycemia were similar to those previously reported for a severe event. However, mild prolonged hypoglycemia appeared to elicit an increased inflammatory response that was associated with heat shock and related proteins.

Myeloid-Biased HSC Require Semaphorin4a from the Bone Marrow Niche for Self-Renewal Under Stress and Life-Long Persistence

A subset of hematopoietic stem cells with inherent myeloid and platelet bias (myHSC) is positioned at the top of hematopoietic hierarchy and considered the most primitive. Notably, lower proliferative output of myHSC correlates with a greater capacity for self-renewal indicating that quiescence is essential for their function. Hence, excessive myHSC expansion following inflammatory challenge is likely to put them at a higher risk of proliferation-induced damage. Given that inflammatory stress is unavoidable throughout live, we hypothesized that myHSC may uniquely depend on quiescence-inducing signals for their protection and long-term persistence. However, the nature of these signals remains largely unexplored. We have previously performed proximity-based analysis of the bone marrow niche to identify novel regulators of HSC quiescence (Silberstein et al, Cell Stem Cell 2016). Briefly, we defined the transcriptional profiles of osteolineage cells which were located in closer proximity to a transplanted HSC (proximal cells), and designated secreted factors with higher expression level in proximal cells as putative regulators of HSC quiescence. For the current study, we selected Semaphorin4a (Sema4a) - a known regulator of neural development, angiogenesis and immune response with no previously documented role in hematopoiesis - and examined its impact on myHSC function during inflammatory stress. We found that Sema4a was expressed in the niche cells (endothelium and osteoprogenitors) in mice and humans. Recombinant Sema4a reduced proliferation of mouse and human hematopoietic stem and progenitor cells ex vivo. Baseline analysis of young Sema4aKO mice revealed mild anemia, thrombocytosis, myeloid bias and a slight reduction in the proportion of HSC in the G0 phase suggesting that Sema4a regulates HSC quiescence and differentiation in vivo. Upon Poly(I:C) injection, Sema4aKO myHSC (Lin -Kit +Sca +CD48 -CD34 -CD150 high) displayed markedly increased cycling and upregulation of alpha-interferon and JAK-STAT signaling while "balanced" HSC (Lin -Kit +Sca +CD48 -CD34 -CD150 low) were unaffected. Similar exaggerated proliferative response in Sema4aKO myHSC was observed upon injection with IL-1β. Next, we investigated the long-term impact of inflammation-induced loss of myHSC quiescence. Aged Sema4aKO mice developed anemia, thrombocytosis, neutrophilia. Most significantly, we observed a two-fold expansion of phenotypic myHSC (but not balanced HSC) which displayed proliferative senescence, increased cellular stress and premature differentiation by scRNA-Seq, and a complete loss of reconstitution upon transplantation. In contrast, young Sema4aKO HSC showed a higher level of post-transplant chimerism consistent with their prior "pre-activated" state. Thus, loss of myHSC quiescence leads to increased sensitivity to inflammatory stressors and enhanced myHSC response but eventual collapse of regenerative function. In order to determine if the microenvironment served as a critical source of Sema4a for myHSC, WT myHSC were transplanted into lethally irradiated WT and Sema4aKO hosts. Strikingly, the majority of Sema4aKO recipients died while all WT recipients survived. Intra-vital imaging at 24 hours revealed a greater number of cells and clusters in Sema4aKO recipients suggesting that excessive early myHSC proliferation led to impaired self-renewal and engraftment failure. Finally, we found that Plexin D1 acts as a functional receptor for Sema4a on myHSC, since Plexin D1-deficient myHSC recapitulated the post-transplant phenotype of young Sema4aKO myHSC described above. Taken together, our data demonstrate that under the conditions of increased myeloid demand, protection from proliferative stress is critical for preserving myHSC function, and highlight a critical but previously unrecognized role for Sema4a-PlxnD1 axis in this process. Our study suggests that therapeutic augmentation of myHSC quiescence may alleviate the negative impact in inflammatory signaling, serve to improve marrow function in inflammatory diseases, and prevent development of myeloid malignancy. Disclosures Radtke: Ensoma Inc.: Consultancy; 47 Inc.: Consultancy. Kiem: Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company; Homology Medicines: Consultancy; VOR Biopharma: Consultancy. Scadden: Fate Therapeutics: Current holder of individual stocks in a privately-held company; Editas Medicines: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Dainippon Sumitomo Pharma: Other: sponsored research; FOG Pharma: Consultancy; Agios Pharmaceuticals: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Garuda Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; VCanBio: Consultancy; Inzen Therapeutics: Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.