Neuroinflammation underlies the development of social stress induced cognitive deficit in sickle cell disease.

Cognitive deficit is a debilitating complication of SCD with multifactorial pathobiology. Here we show that neuroinflammation and dysregulation in lipidomics and transcriptomics profiles are major underlying mechanisms of social stress-induced cognitive deficit in SCD. Townes sickle cell (SS) mice and controls (AA) were exposed to social stress using the repeat social defeat (RSD) paradigm concurrently with or without treatment with minocycline. Mice were tested for cognitive deficit using novel object recognition (NOR) and fear conditioning (FC) tests. SS mice exposed to RSD without treatment had worse performance on cognitive tests compared to SS mice exposed to RSD with treatment or to AA controls, irrespective of their RSD or treatment disposition. Additionally, compared to SS mice exposed to RSD with treatment, SS mice exposed to RSD without treatment had significantly more cellular evidence of neuroinflammation coupled with a significant shift in the differentiation of neural progenitor cells towards astrogliogenesis. Additionally, brain tissue from SS mice exposed to RSD was significantly enriched for genes associated with blood-brain barrier dysfunction, neuron excitotoxicity, inflammation, and significant dysregulation in sphingolipids important to neuronal cell processes. We demonstrate in this study that neuroinflammation and lipid dysregulation are potential underlying mechanisms of social stress-related cognitive deficit in SS mice.

Characterizing human mesenchymal stromal cells' immune-modulatory potency using targeted lipidomic profiling of sphingolipids.

Cell therapies are expected to increase over the next decade owing to increasing demand for clinical applications. Mesenchymal stromal cells (MSCs) have been explored to treat a number of diseases, with some successes in early clinical trials. Despite early successes, poor MSC characterization results in lessened therapeutic capacity once in vivo. Here, we characterized MSCs derived from bone marrow (BM), adipose tissue and umbilical cord tissue for sphingolipids (SLs), a class of bioactive lipids, using liquid chromatography/tandem mass spectrometry. We found that ceramide levels differed based on the donor's sex in BM-MSCs. We detected fatty acyl chain variants in MSCs from all three sources. Linear discriminant analysis revealed that MSCs separated based on tissue source. Principal component analysis showed that interferon-γ-primed and unstimulated MSCs separated according to their SL signature. Lastly, we detected higher ceramide levels in low indoleamine 2,3-dioxygenase MSCs, indicating that sphingomyelinase or ceramidase enzymatic activity may be involved in their immune potency.

Reassessing the clinical spectrum associated with hereditary leiomyomatosis and renal cell carcinoma syndrome in French FH mutation carriers.

We addressed uncertainties regarding hereditary leiomyomatosis and renal cell carcinoma (HLRCC) by exploring all French cases, representing the largest series to date. Fumarate hydratase (FH) germline testing was performed with Sanger sequencing and qPCR/MLPA. Enzyme activity was measured when necessary. We carried out whenever possible a pathology review of RCC and S-(2-succino)-cysteine (2SC)/fumarate hydratase immunohistochemistry. We estimated survival using non-parametric Kaplan-Meier. There were 182 cases from 114 families. Thirty-seven RCC were diagnosed in 34 carriers (19%) at a median age of 40. Among the 23 RCC with pathology review, 13 were papillary type 2. There were 4 papillary RCC of unspecified type, 3 unclassified, 2 tubulocystic, and 1 collecting duct (CD) RCC, all 2SC+ and most (8/10) FH-. Of the remaining 14, papillary type 2, papillary unspecified, CD, and clear cell histologies were reported. The vast majority of RCC (82%) were metastatic at diagnosis or rapidly became metastatic. Median survival for metastatic disease was 18 months (95%CI: 11-29). 133 cases (73%) had a history of cutaneous leiomyomas, 3 developed skin leiomyosarcoma. Uterine leiomyomas were frequent in women (77%), but no sarcomas were observed. Only 2 cases had pheochromocytomas/paraganglioma. CONCLUSION: Our findings have direct implications regarding the identification and management of HLRCC patients.

Regulation of embryonic/fetal globin genes by nuclear hormone receptors: a novel perspective on hemoglobin switching.

The CCAAT box is one of the conserved motifs found in globin promoters. It binds the CP1 protein. We noticed that the CCAAT-box region of embryonic/fetal, but not adult, globin promoters also contains one or two direct repeats of a short motif analogous to DR-1 binding sites for non-steroid nuclear hormone receptors. We show that a complex previously named NF-E3 binds to these repeats. In transgenic mice, destruction of the CCAAT motif within the human epsilon-globin promoter leads to substantial reduction in epsilon expression in embryonic erythroid cells, indicating that CP1 activates epsilon expression; in contrast, destruction of the DR-1 elements yields striking epsilon expression in definitive erythropoiesis, indicating that the NF-E3 complex acts as a developmental repressor of the epsilon gene. We also show that NF-E3 is immunologically related to COUP-TF orphan nuclear receptors. One of these, COUP-TF II, is expressed in embryonic/fetal erythroid cell lines, murine yolk sac, intra-embryonic splanchnopleura and fetal liver. In addition, the structure and abundance of NF-E3/COUP-TF complexes vary during fetal liver development. These results elucidate the structure as well as the role of NF-E3 in globin gene expression and provide evidence that nuclear hormone receptors are involved in the control of globin gene switching.

p45 NF-E2 regulates expression of thromboxane synthase in megakaryocytes.

Transcription factor p45 NF-E2 is highly expressed in the erythroid and megakaryocytic lineages. Although p45 recognizes regulatory regions of several erythroid genes, mice deficient for this protein display only mild dyserythropoiesis but have abnormal megakaryocytes and lack circulating platelets. A number of megakaryocytic marker genes have been extensively studied, but none of them is regulated by NF-E2. To find target genes for p45 NF-E2 in megakaryopoiesis, we used an in vivo immunoselection assay: genomic fragments bound to p45 NF-E2 in the chromatin of a megakaryocytic cell line were immunoprecipitated with an anti-p45 antiserum and cloned. One of these fragments belongs to the second intron of the thromboxane synthase gene (TXS). We demonstrate that the TXS gene, which is mainly expressed in megakaryocytes, is indeed directly regulated by p45 NF-E2. First, its promoter contains a functional NF-E2 binding site; second, the intronic NF-E2 binding site is located within a chromatin-dependent enhancer element; third, p45-null murine megakaryocytes do not express detectable TXS mRNA, although TXS expression can be detected in other cells. These data, and the structure of the TXS promoter and enhancer, suggest that TXS belongs to a distinct subgroup of genes involved in platelet formation and function.

Analysis of the thrombopoietin receptor (MPL) promoter implicates GATA and Ets proteins in the coregulation of megakaryocyte-specific genes.

The MPL gene codes for the thrombopoietin receptor, whose ligand specifically controls megakaryocytic differentiation. To understand the molecular basis for the megakaryocyte-specific expression of MPL, we analyzed the promoter of this gene. A 200 bp fragment is sufficient for high-level specific expression. This fragment can bind several trans-acting factors in vitro, including GATA-1 and members of the Ets family. GATA-1 binds with low affinity to a unique GATA motif at -70 in the MPL promoter, and destruction of this site yields only a modest decrease in expression level in HEL cells. Ets proteins also bind with low affinity to two sites. One is located at position -15 and its destruction reduces expression to 50%; the other is located immediately downstream of the GATA motif and plays a crucial role in expression of the promoter in HEL cells, as its inactivation reduces expression to 15%. Furthermore, GATA-1 and two Ets proteins, Ets-1 and Fli-1, can trans-activate the MPL promoter in heterologous cells. The effects of GATA-1 and these two Ets proteins are additive. Together with our previous results on the glycoprotein IIb (GpIIb) promoter, this study indicates a molecular basis for the coregulation of early markers of megakaryocyte differentiation.

Transcriptional regulation in megakaryocytes: the thrombopoietin receptor gene as a model.

The MPL gene codes for the thrombopoietin receptor, whose ligand specifically controls megakaryocytic differentiation. In order to understand the molecular basis for the megakaryocyte-specific expression of MPL, we analyzed the regulatory elements of this gene. Two regions are hypersensitive to DNase I in nuclei of cells that express MPL: the promoter and a portion of intron 6. The latter behaves as a chromatin-dependent enhancer. A 200 bp fragment of the promoter is sufficient for high-level specific expression. This fragment can bind several transacting factors in vitro, including GATA-1 and members of the Ets family. GATA-1 binds with low affinity to a unique GATA motif at -70 in the MPL promoter, and destruction of this site yields only a modest decrease in expression level in human erythroleukemia (HEL) cells. Ets proteins also bind with low affinity to two sites. One is located at position -15 and its destruction reduces expression to 50%; the other is located immediately downstream of the GATA motif and plays a crucial role in expression of the promoter in HEL cells, as its inactivation reduces expression to 15%. This study indicates a molecular basis for the coregulation of markers of megakaryocyte differentiation. Finally, we describe other nuclear factor binding sites that may be involved in the cell-type-specific expression of MPL.