Gata2-regulated Gfi1b expression controls endothelial programming during endothelial-to-hematopoietic transition.

The first hematopoietic stem cells (HSCs) are formed through endothelial-to-hematopoietic transition (EHT) during embryonic development. The transcription factor GATA2 is a crucial regulator of EHT and HSC function throughout life. Because patients with GATA2 haploinsufficiency have inborn mutations, prenatal defects are likely to influence disease development. In mice, Gata2 haploinsufficiency (Gata2+/-) reduces the number and functionality of embryonic hematopoietic stem and progenitor cells (HSPCs) generated through EHT. However, the embryonic HSPC pool is heterogeneous and the mechanisms underlying this defect in Gata2+/- embryos remain unclear. Here, we investigated whether Gata2 haploinsufficiency selectively affects a cellular subset undergoing EHT. We showed that Gata2+/- HSPCs initiate, but cannot fully activate, hematopoietic programming during EHT. In addition, due to the reduced activity of the endothelial repressor Gfi1b, Gata2+/- HSPCs cannot repress endothelial identity to complete maturation. Finally, we showed that hematopoietic-specific induction of gfi1b could restore HSC production in gata2b-null (gata2b-/-) zebrafish embryos. This study illustrates the pivotal role of Gata2 in the regulation of the transcriptional network governing HSPC identity throughout the EHT.

Immune composition and its association with hematologic recovery after chemotherapeutic injury in acute myeloid leukemia.

Chemotherapy-induced bone marrow (BM) injury is a significant cause of morbidity and mortality in acute myeloid leukemia (AML). Time to hematologic recovery after standard ("7 + 3") myeloablative chemotherapy can vary considerably among patients, but the factors that drive or predict BM recovery remain incompletely understood. Here, we assessed the composition of innate and adaptive immune subsets in the regenerating BM (day 17) after induction chemotherapy and related it to hematologic recovery in AML. T cells, and in particular the CD4 central memory (CD4CM) T-cell subset, were significantly enriched in the BM after chemotherapy, suggesting the relative chemoresistance of cells providing long-term memory for systemic pathogens. In contrast, B cells and other hematopoietic subsets were depleted. Higher frequencies of the CD4CM T-cell subset were associated with delayed hematopoietic recovery, whereas a high frequency of natural killer (NK) cells was related to faster recovery of neutrophil counts. The NK/CD4CM ratio in the BM after chemotherapy was significantly associated with the time to subsequent neutrophil recovery (Spearman's ρ = -0.723, p < 0.001, false discovery rate <0.01). The data provide novel insights into adaptive immune cell recovery after injury and identify the NK/CD4CM index as a putative predictor of hematopoietic recovery in AML.

Identification of osteolineage cell-derived extracellular vesicle cargo implicated in hematopoietic support.

Osteolineage cell-derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non-stimulatory osteolineage EVs by next-generation sequencing and mass spectrometry analyses revealed distinct microRNA and protein signatures identifying EV-derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood-derived CD34+ HSPCs with stimulatory EVs-altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study gives novel insights into the complex biological role of EVs in osteolineage cell-HSPC crosstalk and promotes the utility of EVs and their cargo as therapeutic agents in regenerative medicine.

CD1d deficiency inhibits the development of abdominal aortic aneurysms in LDL receptor deficient mice.

An abdominal aortic aneurysm (AAA) is a dilatation of the abdominal aorta leading to serious complications and mostly to death. AAA development is associated with an accumulation of inflammatory cells in the aorta including NKT cells. An important factor in promoting the recruitment of these inflammatory cells into tissues and thereby contributing to the development of AAA is angiotensin II (Ang II). We demonstrate that a deficiency in CD1d dependent NKT cells under hyperlipidemic conditions (LDLr-/-CD1d-/- mice) results in a strong decline in the severity of angiotensin II induced aneurysm formation when compared with LDLr-/- mice. In addition, we show that Ang II amplifies the activation of NKT cells both in vivo and in vitro. We also provide evidence that type I NKT cells contribute to AAA development by inducing the expression of matrix degrading enzymes in vSMCs and macrophages, and by cytokine dependently decreasing vSMC viability. Altogether, these data prove that CD1d-dependent NKT cells contribute to AAA development in the Ang II-mediated aneurysm model by enhancing aortic degradation, establishing that therapeutic applications which target NKT cells can be a successful way to prevent AAA development.

Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells.

Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Using next-generation sequencing we show that human osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of human umbilical cord blood-derived CD34(+) HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34(+) cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and successfully engraft in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders.

Magnetic Resonance Detection of CD34+ Cells from Umbilical Cord Blood Using a 19F Label.

Impaired homing and delayed recovery upon hematopoietic stem cell transplantation (HSCT) with hematopoietic stem cells (HSC) derived from umbilical cord blood (UCB) is a major problem. Tracking transplanted cells in vivo will be helpful to detect impaired homing at an early stage and allows early interventions to improve engraftment and outcome after transplantation. In this study, we show sufficient intracellular labeling of UCB-derived CD34+ cells, with 19F-containing PLGA nanoparticles which were detectable with both flow cytometry and magnetic resonance spectroscopy (MRS). In addition, labeled CD34+ cells maintain their capacity to proliferate and differentiate, which is pivotal for successful engraftment after transplantation in vivo. These results set the stage for in vivo tracking experiments, through which the homing efficiency of transplanted cells can be studied.

Wnt3a protein reduces growth factor-driven expansion of human hematopoietic stem and progenitor cells in serum-free cultures.

Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34+ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with Lin-CD34+CD38lowCD45RA-CD90+ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC.

Interruption of the OX40-OX40 ligand pathway in LDL receptor-deficient mice causes regression of atherosclerosis.

Patients suffering from cardiovascular disease have well-established atherosclerotic lesions, rendering lesion regression of therapeutic interest. The OX40 (TNFRSF4)-OX40 ligand (OX40L; TNFSF4) pathway is important for the proliferation and survival of T cells, stimulates B cells, and is associated with cardiovascular disease. We hypothesized that interference with the OX40-OX40L pathway, in combination with decreases in cholesterol, may induce regression of atherosclerosis. LDLr(-/-) mice were fed a Western-type diet for 10 wk, after which they received chow diet and were treated with anti-OX40L or PBS for 10 wk. A significant regression of lesions was observed in the aorta and aortic arch of anti-OX40L-treated mice compared with control mice. Interference of the OX40-OX40L pathway reduced Th2 responses, as shown by decreases in GATA-3 and IL-4 levels. Also, IgE levels were decreased, as demonstrated by reduced mast cell presence and activation. Notably, IL-5 production by T and B1 cells was increased, thus enhancing atheroprotective oxidized low-density lipoprotein-specific IgM production. The increase in IL-5 production and IgM was mediated by IL-33 production by APCs upon OX40L blockade. We conclude that interruption of the OX40-OX40L signaling pathway, combined with decreases in dietary cholesterol, induces the regression of atherosclerosis through induction of IL-5-producing T cells and oxidized low-density lipoprotein-specific IgM and reductions in Th2 and mast cells.

T-cell immunoglobulin and mucin domain 3 acts as a negative regulator of atherosclerosis.

OBJECTIVE: Atherosclerosis is a chronic autoimmune-like disease in which lipids and fibrous elements accumulate in the arterial blood vessels. T cells are present within atherosclerotic plaques, and their activation is partially dependent on costimulatory signals, which can either provide positive or negative signals that promote T-cell activation or limit T-cell responses, respectively. T-cell immunoglobulin and mucin domain 3 (Tim-3) is a coinhibitory type 1 transmembrane protein that affects the function of several immune cells involved in atherosclerosis, such as monocytes, macrophages, effector T cells, and regulatory T cells. In the present study, we determined the role of Tim-3 in the development of atherosclerosis. APPROACH AND RESULTS: Western-type diet-fed low-density lipoprotein receptor-deficient (LDLr(-/-)) mice were treated with an anti-Tim-3 antibody for 3 and 8 weeks. Anti-Tim-3 administration increased fatty streak formation with 66% and increased atherosclerotic plaque formation after 8 weeks with 35% in the aortic root and with 50% in the aortic arch. Furthermore, blockade of Tim-3 signaling increased percentages of circulating monocytes with 33% and lesional macrophages with 20%. In addition, anti-Tim-3 administration increased CD4(+) T cells with 17%, enhanced their activation status, and reduced percentages of regulatory T cells with 18% and regulatory B cells with 37%. CONCLUSIONS: It is known that Tim-3 acts as a negative regulator of both innate and adaptive immune responses, and in the present study, we show that anti-Tim-3 treatment augments lesion development, accompanied by an increase in the number of monocytes/macrophages and CD4(+) T cells and by decreased regulatory T cells and regulatory B cells.

Interference of the CD30-CD30L pathway reduces atherosclerosis development.

OBJECTIVE: Costimulatory molecules tightly control immune responses by providing positive signals that promote T-cell activation or by transducing inhibitory signals that limit T-cell responses. CD30 and CD30L are members of the tumor necrosis factor receptor superfamily and are involved in the activation and proliferation of T and B cells, which have been implicated in the initiation and progression of atherosclerosis. In the present study, we thus aimed to determine the role of the CD30-CD30L pathway in the development of atherosclerosis. METHODS AND RESULTS: Western-type diet-fed low-density lipoprotein receptor-deficient mice were treated with an anti-CD30L antibody for 8 weeks, which resulted in a reduction of atherosclerotic lesion formation in the aortic root by 35%. Reduced numbers of adventitial CD3(+) T cells were found in anti-CD30L-treated mice, whereas no differences were observed in collagen and macrophage content of the atherosclerotic lesions. B-cell and mast cell responses were also not affected on anti-CD30L treatment. Interestingly, splenocyte proliferation was reduced by 53%, whereas T-cell numbers were concomitantly reduced in anti-CD30L-treated mice compared with control mice. These data thus indicate that the CD30-CD30L pathway solely exerts its function via inhibition of T-cell responses. CONCLUSIONS: In the present study, we are the first to show that interruption of the CD30-CD30L pathway reduced initial atherosclerosis development by modulating T-cell function.

Adenosine A2B receptor agonism inhibits neointimal lesion development after arterial injury in apolipoprotein E-deficient mice.

OBJECTIVE: The A(2B) adenosine receptor (A(2B)R) is highly expressed in macrophages and vascular smooth muscle cells and has been established as an important regulator of inflammation and vascular adhesion. Recently, it has been demonstrated that A(2B)R deficiency enhances neointimal lesion formation after vascular injury. Therefore, we hypothesize that A(2B)R agonism protects against injury-induced intimal hyperplasia. METHODS AND RESULTS: Apolipoprotein E-deficient mice were fed a Western-type diet for 1 week, after which the left common carotid artery was denuded. Mice were treated with the A(2B) receptor agonist BAY60-6583 or vehicle control for 18 days. Interestingly, lumen stenosis as defined by the neointima/lumen ratio was inhibited by treatment with the A(2B) receptor agonist, caused by reduced smooth muscle cell proliferation. Collagen content was significantly increased in the BAY60-6583-treated mice, whereas macrophage content remained unchanged. In vitro, vascular smooth muscle cell proliferation decreased dose dependently whereas collagen content of cultured smooth muscle cells was increased by BAY60-6583. CONCLUSIONS: Our data show that activation of the adenosine A(2B) receptor protects against vascular injury, while it also enhances plaque stability as indicated by increased collagen content. These outcomes thus point to A(2B) receptor agonism as a new therapeutic approach in the prevention of restenosis.

Tolerance to ingested deamidated gliadin in mice is maintained by splenic, type 1 regulatory T cells.

BACKGROUND & AIMS: Patients with celiac disease have permanent intolerance to gluten. Because of the high frequency of this disorder (approximately 1 in 100 individuals), we investigated whether oral tolerance to gluten differs from that to other food proteins. METHODS: Using transgenic mice that express human HLA-DQ2 and a gliadin-specific, humanized T-cell receptor, we compared gluten-specific T-cell responses with tolerogenic mucosal T-cell responses to the model food protein ovalbumin. RESULTS: Consistent with previous findings, the ovalbumin-specific response occurred in the mesenteric lymph nodes and induced Foxp3(+) regulatory T cells. In contrast, ingestion of deamidated gliadin induced T-cell proliferation predominantly in the spleen but little in mesenteric lymph nodes. The gliadin-reactive T cells had an effector-like phenotype and secreted large amounts of interferon gamma but also secreted interleukin-10. Despite their effector-like phenotype, gliadin-reactive T cells had regulatory functions, because transfer of the cells suppressed a gliadin-induced, delayed-type hypersensitivity response. CONCLUSIONS: Ingestion of deamidated gliadin induces differentiation of tolerogenic, type 1 regulatory T cells in spleens of HLA-DQ2 transgenic mice. These data indicate that under homeostatic conditions, the T-cell response to deamidated gliadin is tolerance, which is not conditioned by the mucosal immune system but instead requires interleukin-10 induction by antigen presentation in the spleen.

CD62L(neg)CD38⁺ expression on circulating CD4⁺ T cells identifies mucosally differentiated cells in protein fed mice and in human celiac disease patients and controls.

OBJECTIVES: The aim of this study was to identify new markers of mucosal T cells to monitor ongoing intestinal immune responses in peripheral blood. METHODS: Expression of cell-surface markers was studied in mice on ovalbumin (OVA)-specific T cells in the gut-draining mesenteric lymph nodes (MLN) after OVA feed. The effect of the local mucosal mediators retinoic acid (RA) and transforming growth factor-ß (TGF-ß) on the induction of a mucosal phenotype was determined in in vitro T-cell differentiation assays with murine and human T cells. Tetramer stainings were performed to study gluten-specific T cells in the circulation of patients with celiac disease, a chronic small-intestinal inflammation. RESULTS: In mice, proliferating T cells in MLN were CD62L(neg)CD38(+) during both tolerance induction and abrogation of intestinal homeostasis. This mucosal CD62L(neg)CD38(+) T-cell phenotype was efficiently induced by RA and TGF-ß in mice, whereas for human CD4(+) T cells RA alone was sufficient. The CD4(+)CD62L(neg)CD38(+) T-cell phenotype could be used to identify T cells with mucosal origin in human peripheral blood, as expression of the gut-homing chemokine receptor CCR9 and ß(7) integrin were highly enriched in this subset whereas expression of cutaneous leukocyte-associated antigen was almost absent. Tetramer staining revealed that gluten-specific T cells appearing in blood of treated celiac disease patients after oral gluten challenge were predominantly CD4(+)CD62L(neg)CD38(+). The total percentage of circulating CD62L(neg)CD38(+) of CD4 T cells was not an indicator of intestinal inflammation as percentages did not differ between pediatric celiac disease patients, inflammatory bowel disease patients and respective controls. However, the phenotypic selection of mucosal T cells allowed cytokine profiling as upon restimulation of CD62L(neg)CD38(+) cells interleukin-10 (IL-10) and interferon-γ (IFN-γ) transcripts were readily detected in circulating mucosal T cells. CONCLUSIONS: By selecting for CD62L(neg)CD38(+) expression that comprises 5-10% of the cells within the total CD4(+) T-cell pool we are able to highly enrich for effector T cells with specificity for mucosal antigens. This is of pivotal importance for functional studies as this purification enhances the sensitivity of cytokine detection and cellular activation.

Tumor necrosis factor alpha drives cadherin 11 expression in rheumatoid inflammation.

OBJECTIVE: Cadherin 11 expressed on fibroblast-like synoviocytes (FLS) plays a key role in normal synovial architecture. The purpose of this study was to examine the expression of cadherin 11 in human synovitis. METHODS: Cadherin 11 expression in synovial biopsy samples from patients with various types of arthritis and in lung biopsy samples from patients with interstitial pneumonitis (IP) was examined by immunostaining. The regulation of cadherin 11 expression in human FLS was assessed by quantitative reverse transcription-polymerase chain reaction analysis and Western blotting. Therapeutic modulation of synovial cadherin 11 was assessed before and after effective antiinflammatory therapy. RESULTS: Abundant staining for cadherin 11 was seen in the intimal lining layer and the synovial sublining in inflamed tissues, with discrete staining in noninflammatory osteoarthritic (OA) tissues. The pattern and degree of immunostaining were similar in tissues from patients with rheumatoid arthritis (RA), nonpsoriatic spondylarthritis (SpA), psoriatic arthritis (PsA), and inflammatory OA. Clear staining for cadherin 11 was also observed in lung tissues from RA-associated IP and idiopathic IP patients, but was very limited in normal lung tissue. Cadherin 11 staining correlated strongly with the degree of inflammatory infiltration of the tissue, as well as with the C-reactive protein level and the erythrocyte sedimentation rate in RA patients. In vitro, cadherin 11 expression by FLS was consistently up-regulated by tumor necrosis factor alpha (TNFalpha) at the protein, but not the messenger RNA, level. Cadherin 11 staining in vivo was strongly down-regulated by prednisone treatment in RA patients and by TNFalpha blockade in SpA patients. CONCLUSION: Cadherin 11 expression is regulated by mediators of inflammation, such as TNFalpha. Since cadherin 11 plays an important role in cartilage destruction in experimental arthritis, down-modulation of cadherin 11 by potent antiinflammatory therapies in humans with arthritis may contribute to halting cartilage damage.