Publications by authors named "João P Pereira"

27 Publications

  • Page 1 of 1

GIMAP5 maintains liver endothelial cell homeostasis and prevents portal hypertension.

J Exp Med 2021 Jul 6;218(7). Epub 2021 May 6.

Department of Internal Medicine (Digestive Diseases), Yale School of Medicine, New Haven, CT.

Portal hypertension is a major contributor to decompensation and death from liver disease, a global health problem. Here, we demonstrate homozygous damaging mutations in GIMAP5, a small organellar GTPase, in four families with unexplained portal hypertension. We show that GIMAP5 is expressed in hepatic endothelial cells and that its loss in both humans and mice results in capillarization of liver sinusoidal endothelial cells (LSECs); this effect is also seen when GIMAP5 is selectively deleted in endothelial cells. Single-cell RNA-sequencing analysis in a GIMAP5-deficient mouse model reveals replacement of LSECs with capillarized endothelial cells, a reduction of macrovascular hepatic endothelial cells, and places GIMAP5 upstream of GATA4, a transcription factor required for LSEC specification. Thus, GIMAP5 is a critical regulator of liver endothelial cell homeostasis and, when absent, produces portal hypertension. These findings provide new insight into the pathogenesis of portal hypertension, a major contributor to morbidity and mortality from liver disease.
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http://dx.doi.org/10.1084/jem.20201745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105721PMC
July 2021

Hematopoietic Stem Cell Niches and Signals Controlling Immune Cell Development and Maintenance of Immunological Memory.

Front Immunol 2020 26;11:600127. Epub 2020 Nov 26.

Department of Immunobiology and Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, United States.

Studies over the last couple of decades have shown that hematopoietic stem cells (HSCs) are critically dependent on cytokines such as Stem Cell Factor and other signals provided by bone marrow niches comprising of mesenchymal stem and progenitor cells (MSPCs) and endothelial cells (ECs). Because of their critical roles in HSC maintenance the niches formed by MSPCs and ECs are commonly referred to as HSC niches. For the most part, the signals required for HSC maintenance act in a short-range manner, which imposes the necessity for directional and positional cues in order for HSCs to localize and be retained properly in stem cell niches. The chemokine CXCL12 and its Gαi protein coupled receptor CXCR4, besides promoting HSC quiescence directly, also play instrumental roles in enabling HSCs to access bone marrow stem cell niches. Recent studies have revealed, however, that HSC niches also provide a constellation of hematopoietic cytokines that are critical for the production of most, if not all, blood cell types. Some hematopoietic cytokines, namely IL-7 and IL-15 produced by HSC niches, are not only required for lymphopoiesis but are also essential for memory T cell maintenance. Consequently, hematopoietic progenitors and differentiated immune cells, such as memory T cell subsets, also depend on the CXCL12/CXCR4 axis for migration into bone marrow and interactions with MSPCs and ECs. Similarly, subsets of antibody-secreting plasma cells also reside in close association with CXCL12-producing MSPCs in the bone marrow and require the CXCR4/CXCL12 axis for survival and long-term maintenance. Collectively, these studies demonstrate a broad range of key physiological roles, spanning blood cell production and maintenance of immunological memory, that are orchestrated by stem cell niches through a common and simple mechanism: CXCL12/CXCR4-mediated cell recruitment followed by receipt of a maintenance and/or instructive signal. A fundamental flaw of this type of cellular organization is revealed by myeloid and lymphoid leukemias, which target stem cell niches and induce profound transcriptomic changes that result in reduced hematopoietic activity and altered mesenchymal cell differentiation.
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http://dx.doi.org/10.3389/fimmu.2020.600127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726109PMC
November 2020

Differential DNA methylation in familial hypercholesterolemia.

EBioMedicine 2020 Nov 21;61:103079. Epub 2020 Oct 21.

Department of Vascular Medicine, Amsterdam UMC, Location AMC, Meibergdreef 9, Amsterdam 1105AZ, The Netherlands. Electronic address:

Background: Familial hypercholesterolemia (FH) is a monogenic disorder characterized by elevated low-density lipoprotein cholesterol (LDL-C). A FH causing genetic variant in LDLR, APOB, or PCSK9 is not identified in 12-60% of clinical FH patients (FH mutation-negative patients). We aimed to assess whether altered DNA methylation might be associated with FH in this latter group.

Methods: In this study we included 78 FH mutation-negative patients and 58 FH mutation-positive patients with a pathogenic LDLR variant. All patients were male, not using lipid lowering therapies and had LDL-C levels >6 mmol/L and triglyceride levels <3.5 mmol/L. DNA methylation was measured with the Infinium Methylation EPIC 850 K beadchip assay. Multiple linear regression analyses were used to explore DNA methylation differences between the two groups in genes related to lipid metabolism. A gradient boosting machine learning model was applied to investigate accumulated genome-wide differences between the two groups.

Findings: Candidate gene analysis revealed one significantly hypomethylated CpG site in CPT1A (cg00574958) in FH mutation-negative patients, while no differences in methylation in other lipid genes were observed. The machine learning model did distinguish the two groups with a mean Area Under the Curve (AUC)±SD of 0.80±0.17 and provided two CpG sites (cg26426080 and cg11478607) in genes with a possible link to lipid metabolism (PRDM16 and GSTT1).

Interpretation: FH mutation-negative patients are characterized by accumulated genome wide DNA methylation differences, but not by major DNA methylation alterations in known lipid genes compared to FH mutation-positive patients.

Funding: ZonMW grant (VIDI no. 016.156.445).
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http://dx.doi.org/10.1016/j.ebiom.2020.103079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581877PMC
November 2020

Application of the Quartz Crystal Microbalance Method for Measuring Mercury in the Air of Working Environments Involved to Artisanal and Small-scale Gold Mining (ASGM).

Anal Sci 2020 Dec 21;36(12):1515-1519. Epub 2020 Aug 21.

International Mercury Laboratory (IML).

Artisanal small gold mining (ASGM) is responsible for approximately 40% of the total Hg emissions into the atmosphere worldwide. In developing countries, many people are engaged in ASGM activities. We developed a small, simple Hg measuring device, which detects Hg in the air based on the change of the oscillation frequency of an Au electrode on a quartz crystal microbalance (QCM). This device is called QCM-Hg. We tested the viability of the QCM-Hg in various work settings including a gold mining area and gold shops. In working environments with an airborne Hg concentration of several μg m, the changing rate of the oscillation frequency for either 2 or 3 min corresponded with the Hg concentrations measured using the conventional method of gold amalgamation and cold vapor atomic absorption spectrometer (CVAAS). The results revealed that the QCM-Hg is a useful device for real-time Hg monitoring in actual working environments related to ASGM activities and Hg treatment facilities.
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http://dx.doi.org/10.2116/analsci.20P178DOI Listing
December 2020

Improved cardiovascular risk prediction using targeted plasma proteomics in primary prevention.

Eur Heart J 2020 11;41(41):3998-4007

Department of Vascular Medicine, Amsterdam University Medical Centers, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

Aims: In the era of personalized medicine, it is of utmost importance to be able to identify subjects at the highest cardiovascular (CV) risk. To date, single biomarkers have failed to markedly improve the estimation of CV risk. Using novel technology, simultaneous assessment of large numbers of biomarkers may hold promise to improve prediction. In the present study, we compared a protein-based risk model with a model using traditional risk factors in predicting CV events in the primary prevention setting of the European Prospective Investigation (EPIC)-Norfolk study, followed by validation in the Progressione della Lesione Intimale Carotidea (PLIC) cohort.

Methods And Results: Using the proximity extension assay, 368 proteins were measured in a nested case-control sample of 822 individuals from the EPIC-Norfolk prospective cohort study and 702 individuals from the PLIC cohort. Using tree-based ensemble and boosting methods, we constructed a protein-based prediction model, an optimized clinical risk model, and a model combining both. In the derivation cohort (EPIC-Norfolk), we defined a panel of 50 proteins, which outperformed the clinical risk model in the prediction of myocardial infarction [area under the curve (AUC) 0.754 vs. 0.730; P < 0.001] during a median follow-up of 20 years. The clinically more relevant prediction of events occurring within 3 years showed an AUC of 0.732 using the clinical risk model and an AUC of 0.803 for the protein model (P < 0.001). The predictive value of the protein panel was confirmed to be superior to the clinical risk model in the validation cohort (AUC 0.705 vs. 0.609; P < 0.001).

Conclusion: In a primary prevention setting, a proteome-based model outperforms a model comprising clinical risk factors in predicting the risk of CV events. Validation in a large prospective primary prevention cohort is required to address the value for future clinical implementation in CV prevention.
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http://dx.doi.org/10.1093/eurheartj/ehaa648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672529PMC
November 2020

Should I Stay or Should I Flow: HSCs Are on the Move!

Cell Stem Cell 2020 08;27(2):189-190

Department of Immunobiology and Yale Stem Cell Center, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06519, USA. Electronic address:

Stem cell biologists have been yearning to visualize hematopoietic stem cells (HSCs) in live animals since Kiel et al. (2005) first visualized them in bone cavities. With two recent papers from Christodoulou et al. (2020) and Upadhaya et al. (2020), we can all now see how HSCs behave in their niches!
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http://dx.doi.org/10.1016/j.stem.2020.07.014DOI Listing
August 2020

Effector T17 Cells Give Rise to Long-Lived T Cells that Are Essential for an Immediate Response against Bacterial Infection.

Cell 2019 08;178(5):1176-1188.e15

Department of Immunobiology, School of Medicine, Yale University, New Haven, CT, USA; Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA. Electronic address:

Adaptive immunity provides life-long protection by generating central and effector memory T cells and the most recently described tissue resident memory T (T) cells. However, the cellular origin of CD4 T cells and their contribution to host defense remain elusive. Using IL-17A tracking-fate mouse models, we found that a significant fraction of lung CD4 T cells derive from IL-17A-producing effector (T17) cells following immunization with heat-killed Klebsiella pneumonia (Kp). These exT17 T cells are maintained in the lung by IL-7, produced by lymphatic endothelial cells. During a memory response, neither antibodies, γδ T cells, nor circulatory T cells are sufficient for the rapid host defense required to eliminate Kp. Conversely, using parabiosis and depletion studies, we demonstrated that exT17 T cells play an important role in bacterial clearance. Thus, we delineate the origin and function of airway CD4 T cells during bacterial infection, offering novel strategies for targeted vaccine design.
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http://dx.doi.org/10.1016/j.cell.2019.07.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057720PMC
August 2019

Cell circuits and niches controlling B cell development.

Immunol Rev 2019 05;289(1):142-157

Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut.

Studies over the last decade uncovered overlapping niches for hematopoietic stem cells (HSCs), multipotent progenitor cells, common lymphoid progenitors, and early B cell progenitors. HSC and lymphoid niches are predominantly composed by mesenchymal progenitor cells (MPCs) and by a small subset of endothelial cells. Niche cells create specialized microenvironments through the concomitant production of short-range acting cell-fate determining cytokines such as interleukin (IL)-7 and stem cell factor and the potent chemoattractant C-X-C motif chemokine ligand 12. This type of cellular organization allows for the cross-talk between hematopoietic stem and progenitor cells with niche cells, such that niche cell activity can be regulated by the quality and quantity of hematopoietic progenitors being produced. For example, preleukemic B cell progenitors and preB acute lymphoblastic leukemias interact directly with MPCs, and downregulate IL-7 expression and the production of non-leukemic lymphoid cells. In this review, we discuss a novel model of B cell development that is centered on cellular circuits formed between B cell progenitors and lymphopoietic niches.
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http://dx.doi.org/10.1111/imr.12749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6464388PMC
May 2019

Cell circuits between B cell progenitors and IL-7 mesenchymal progenitor cells control B cell development.

J Exp Med 2018 10 29;215(10):2586-2599. Epub 2018 Aug 29.

Department of Immunobiology, Yale University School of Medicine, Yale University, New Haven, CT

B cell progenitors require paracrine signals such as interleukin-7 (IL-7) provided by bone marrow stromal cells for proliferation and survival. Yet, how B cells regulate access to these signals in vivo remains unclear. Here we show that proB and IL-7 cells form a cell circuit wired by IL-7R signaling, which controls CXCR4 and focal adhesion kinase (FAK) expression and restricts proB cell movement due to increased adhesion to IL-7CXCL12 cells. PreBCR signaling breaks this circuit by switching the preB cell behavior into a fast-moving and lower-adhesion state via increased CXCR4 and reduced FAK/α4β1 expression. This behavioral change reduces preB cell exposure to IL-7, thereby attenuating IL-7R signaling in vivo. Remarkably, IL-7 production is downregulated by signals provided by preB cells with unrepaired double-stranded DNA breaks and by preB acute lymphoblastic leukemic cells. Combined, these studies revealed that distinct cell circuits control the quality and homeostasis of B cell progenitors.
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http://dx.doi.org/10.1084/jem.20180778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170173PMC
October 2018

Active mTORC2 Signaling in Naive T Cells Suppresses Bone Marrow Homing by Inhibiting CXCR4 Expression.

J Immunol 2018 08 22;201(3):908-915. Epub 2018 Jun 22.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06519;

Recirculation of naive T cells between secondary lymphoid organs to receive survival cues and scan for signs of infection or other pathologic conditions is important for immune homeostasis and effective immune responses. Although the mechanisms that specifically guide the entry of naive T cells into secondary lymphoid organs are well studied, the mechanisms that keep them from fluxing into inappropriate or undesirable compartments, such as healthy tissues or bone marrow, are less well understood. In this study, we report an unexpected finding that under steady state, bone marrow homing of naive T cells is actively suppressed by mTORC2 signaling. We found that in mice, T cell-specific deletion of an essential mTORC2 component Sin1 results in increased accumulation of naive T cells in the bone marrow. Mechanistically, we show that loss of mTORC2 signaling in naive T cells results in enhanced FOXO1 activity, which leads to increased CXCR4 expression and chemotactic response to CXCL12, a key chemokine that promotes bone marrow homing and retention of T cells. Together, the results of our study reveal a novel role of mTORC2 in T cell homeostasis via active suppression of naive T cell bone marrow homing by the mTORC2-FOXO1-CXCR4 axis.
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http://dx.doi.org/10.4049/jimmunol.1800529DOI Listing
August 2018

Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation.

Immunity 2018 01;48(1):120-132.e8

Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, 141 86 Stockholm, Sweden; Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA. Electronic address:

Group 3 innate lymphoid cells (ILC3s) sense environmental signals and are critical for tissue integrity in the intestine. Yet, which signals are sensed and what receptors control ILC3 function remain poorly understood. Here, we show that ILC3s with a lymphoid-tissue-inducer (LTi) phenotype expressed G-protein-coupled receptor 183 (GPR183) and migrated to its oxysterol ligand 7α,25-hydroxycholesterol (7α,25-OHC). In mice lacking Gpr183 or 7α,25-OHC, ILC3s failed to localize to cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Gpr183 deficiency in ILC3s caused a defect in CP and ILF formation in the colon, but not in the small intestine. Localized oxysterol production by fibroblastic stromal cells provided an essential signal for colonic lymphoid tissue development, and inflammation-induced increased oxysterol production caused colitis through GPR183-mediated cell recruitment. Our findings show that GPR183 promotes lymphoid organ development and indicate that oxysterol-GPR183-dependent positioning within tissues controls ILC3 activity and intestinal homeostasis.
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http://dx.doi.org/10.1016/j.immuni.2017.11.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772175PMC
January 2018

A Chemoattractant-Guided Walk Through Lymphopoiesis: From Hematopoietic Stem Cells to Mature B Lymphocytes.

Adv Immunol 2017 18;134:47-88. Epub 2017 Mar 18.

Yale University School of Medicine, New Haven, CT, United States. Electronic address:

B lymphocytes develop from hematopoietic stem cells (HSCs) in specialized bone marrow niches composed of rare mesenchymal lineage stem/progenitor cells (MSPCs) and sinusoidal endothelial cells. These niches are defined by function and location: MSPCs are mostly perisinusoidal cells that together with a small subset of sinusoidal endothelial cells express stem cell factor, interleukin-7 (IL-7), IL-15, and the highest amounts of CXCL12 in bone marrow. Though rare, MSPCs are morphologically heterogeneous, highly reticular, and form a vast cellular network in the bone marrow parenchyma capable of interacting with large numbers of hematopoietic cells. HSCs, downstream multipotent progenitor cells, and common lymphoid progenitor cells utilize CXCR4 to fine-tune access to critical short-range growth factors provided by MSPCs for their long-term maintenance and/or multilineage differentiation. In later stages, developing B lymphocytes use CXCR4 to navigate the bone marrow parenchyma, and predominantly cannabinoid receptor-2 for positioning within bone marrow sinusoids, prior to being released into peripheral blood circulation. In the final stages of differentiation, transitional B cells migrate to the spleen where they preferentially undergo further rounds of differentiation until selection into the mature B cell pool occurs. This bottleneck purges up to 97% of all developing B cells in a peripheral selection process that is heavily controlled not only by the intensity of BCR signaling and access to BAFF but also by the proper functioning of the B cell motility machinery.
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http://dx.doi.org/10.1016/bs.ai.2017.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5784265PMC
April 2017

Inflammatory Cell Migration in Rheumatoid Arthritis: A Comprehensive Review.

Clin Rev Allergy Immunol 2016 Aug;51(1):59-78

Department of Immunobiology, Yale University School of Medicine, New Haven, CT, 06519, USA.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that primarily affects the joints. Self-reactive B and T lymphocytes cooperate to promote antibody responses against self proteins and are major drivers of disease. T lymphocytes also promote RA independently of B lymphocytes mainly through the production of key inflammatory cytokines, such as IL-17, that promote pathology. While the innate signals that initiate self-reactive adaptive immune responses are poorly understood, the disease is predominantly caused by inflammatory cellular infiltration and accumulation in articular tissues, and by bone erosions driven by bone-resorbing osteoclasts. Osteoclasts are giant multinucleated cells formed by the fusion of multiple myeloid cells that require short-range signals, such as the cytokines MCSF and RANKL, for undergoing differentiation. The recruitment and positioning of osteoclast precursors to sites of osteoclast differentiation by chemoattractants is an important point of control for osteoclastogenesis and bone resorption. Recently, the GPCR EBI2 and its oxysterol ligand 7a, 25 dihydroxycholesterol, were identified as important regulators of osteoclast precursor positioning in proximity to bone surfaces and of osteoclast differentiation under homeostasis. In chronic inflammatory diseases like RA, osteoclast differentiation is also driven by inflammatory cytokines such as TNFa and IL-1, and can occur independently of RANKL. Finally, there is growing evidence that the chemotactic signals guiding osteoclast precursors to inflamed articular sites contribute to disease and are of great interest. Furthering our understanding of the complex osteoimmune cell interactions should provide new avenues of therapeutic intervention for RA.
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http://dx.doi.org/10.1007/s12016-015-8520-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785098PMC
August 2016

Deletion of Rac in Mature Osteoclasts Causes Osteopetrosis, an Age-Dependent Change in Osteoclast Number, and a Reduced Number of Osteoblasts In Vivo.

J Bone Miner Res 2016 Apr 18;31(4):864-73. Epub 2015 Nov 18.

Department of Internal Medicine, Endocrinology, Yale School of Medicine, New Haven, CT, USA.

Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1(flox/flox) mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2(-/-) mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4- to 6-week-old DKO mice at all sites when measured by dual-energy X-ray absorptiometry (DXA) and serum cross-linked C-telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4- to 6-week-old DKO animals. In 14- to 16-week-old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once-daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism.
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http://dx.doi.org/10.1002/jbmr.2733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4826801PMC
April 2016

Oxysterols and EBI2 promote osteoclast precursor migration to bone surfaces and regulate bone mass homeostasis.

J Exp Med 2015 Oct 5;212(11):1931-46. Epub 2015 Oct 5.

Department of Immunobiology and Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, CT 06510

Bone surfaces attract hematopoietic and nonhematopoietic cells, such as osteoclasts (OCs) and osteoblasts (OBs), and are targeted by bone metastatic cancers. However, the mechanisms guiding cells toward bone surfaces are essentially unknown. Here, we show that the Gαi protein-coupled receptor (GPCR) EBI2 is expressed in mouse monocyte/OC precursors (OCPs) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-OHC) is secreted abundantly by OBs. Using in vitro time-lapse microscopy and intravital two-photon microscopy, we show that EBI2 enhances the development of large OCs by promoting OCP motility, thus facilitating cell-cell interactions and fusion in vitro and in vivo. EBI2 is also necessary and sufficient for guiding OCPs toward bone surfaces. Interestingly, OCPs also secrete 7α,25-OHC, which promotes autocrine EBI2 signaling and reduces OCP migration toward bone surfaces in vivo. Defective EBI2 signaling led to increased bone mass in male mice and protected female mice from age- and estrogen deficiency-induced osteoporosis. This study identifies a novel pathway involved in OCP homing to the bone surface that may have significant therapeutic potential.
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http://dx.doi.org/10.1084/jem.20150088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4612084PMC
October 2015

Immature B Cell Egress from Bone Marrow Is SOCS3 Independent.

PLoS One 2015 14;10(8):e0136061. Epub 2015 Aug 14.

Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, United States of America.

Suppressor of cytokine signaling (SOCS)-3 has been suggested to regulate CXCR4 signaling in a variety of human cell lines. In mice, conditional SOCS3 inactivation in hematopoietic cells including B-lineage lymphocytes has been reported to exacerbate CXCR4-signaling and focal adhesion kinase phosphorylation, which resulted in altered immature B cell distribution in bone marrow (BM) due to sustained α4β1 integrin-mediated adhesion to the extracellular matrix. However, a recent study examining conditional SOCS3 deletion specifically in B-lineage cells failed to detect significant roles in B-lineage cell retention in BM. In this study we carefully examined the role played by SOCS3 in CXCR4 signaling in developing B cell subsets. We show that in mice conditionally deficient in SOCS3 exclusively in B cells (Socs3fl/fl Mb1cre/+) there was no detectable difference in B cell development in BM and in periphery. We show that SOCS3 deficient and sufficient immature B cell subsets are similarly distributed between BM parenchyma and sinusoids, and are equally competent at exiting BM into peripheral blood. Furthermore, we found no significant differences in CXCR4 desensitization upon ligand exposure in developing B lymphocyte subsets. Consequently, SOCS3-deficient and sufficient B-lineage cell migration towards CXCL12 in vitro was undistinguishable, and B-lineage cell amoeboid motility within BM parenchyma was also unaffected by SOCS3-deficiency. Thus we conclude that SOCS3 has no detectable influence on biological processes known to be controlled by CXCR4 signaling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136061PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537204PMC
May 2016

CXCR4 and a cell-extrinsic mechanism control immature B lymphocyte egress from bone marrow.

J Exp Med 2014 Dec 17;211(13):2567-81. Epub 2014 Nov 17.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520

Leukocyte residence in lymphoid organs is controlled by a balance between retention and egress-promoting chemoattractants sensed by pertussis toxin (PTX)-sensitive Gαi protein-coupled receptors (GPCRs). Here, we use two-photon intravital microscopy to show that immature B cell retention within bone marrow (BM) was strictly dependent on amoeboid motility mediated by CXCR4 and CXCL12 and by α4β1 integrin-mediated adhesion to VCAM-1. However, B lineage cell egress from BM is independent of PTX-sensitive GPCR signaling. B lineage cells expressing PTX rapidly exited BM even though their motility within BM parenchyma was significantly reduced. Our experiments reveal that when immature B cells are near BM sinusoids their motility is reduced, their morphology is predominantly rounded, and cells reverse transmigrate across sinusoidal endothelium in a largely nonamoeboid manner. Immature B cell egress from BM was dependent on a twofold CXCR4 down-regulation that was antagonized by antigen-induced BCR signaling. This passive mode of cell egress from BM also contributes significantly to the export of other hematopoietic cells, including granulocytes, monocytes, and NK cells, and is reminiscent of erythrocyte egress.
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http://dx.doi.org/10.1084/jem.20140457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267240PMC
December 2014

Dynamin 2-dependent endocytosis is required for sustained S1PR1 signaling.

J Exp Med 2014 Apr 17;211(4):685-700. Epub 2014 Mar 17.

Department of Immunobiology, 2 Department of Cell Biology, 3 Program in Cellular Neuroscience, Neurodegeneration, and Repair, and 4 Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520.

Sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) is critical for lymphocyte egress from lymphoid organs. Lymphocytes encounter low S1P concentrations near exit sites before transmigration, yet S1PR1 signaling is rapidly terminated after exposure to S1P. How lymphocytes maintain S1PR1 signaling in a low S1P environment near egress sites is unknown. Here we identify dynamin 2, an essential component of endocytosis, as a novel regulator of T cell egress. Mice with T cell-specific dynamin 2 deficiency had profound lymphopenia and impaired egress from lymphoid organs. Dynamin 2 deficiency caused impaired egress through regulation of S1PR1 signaling, and transgenic S1PR1 overexpression rescued egress in dynamin 2 knockout mice. In low S1P concentrations, dynamin 2 was essential for S1PR1 internalization, which enabled continuous S1PR1 signaling and promoted egress from both thymus and lymph nodes. In contrast, dynamin 2-deficient cells were only capable of a pulse of S1PR1 signaling, which was insufficient for egress. Our results suggest a possible mechanism by which T lymphocytes positioned at exit portals sense low S1P concentrations, promoting their egress into circulatory fluids.
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http://dx.doi.org/10.1084/jem.20131343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3978280PMC
April 2014

The role of aggressions suffered by healthcare workers as predictors of burnout.

J Clin Nurs 2013 Nov 17;22(21-22):3120-9. Epub 2012 Sep 17.

Authors: Santiago Gascon, PhD, Assistant Professor, Department of Psychology, Zaragoza University, Teruel, Spain; Michael P Leiter, PhD, Professor, Centre for Organisational and Development Research, Acadia University, Wolfville, NS, Canada; Joao P Pereira, PhD, Assistant Professor, Instituto Superior de Maia, Castelo da Maia; María J Cunha, PhD, Assistant Professor, Instituto Superior de Maia, Castelo da Maia, Portugal; Agustín Albesa, PhD Student, Psychologist and Lawyer, Department of Psychology, Zaragoza University, Zaragoza; Jesus Montero-Marín, PhD, Assistant Professor, Zaragoza University, Zaragoza; Javier García-Campayo, PhD, Professor, Department of Psychiatry Zaragoza University, Zaragoza, Spain.

Aims And Objectives: To examine the prevalence of aggression against healthcare professionals and to determine the possible impact that violent episodes have on healthcare professionals in terms of loss of enthusiasm and involvement towards work. The objective was to analyse the percentage of occupational assault against professionals' aggression in different types of healthcare services, differentiating between physical and verbal aggression as a possible variable in detecting burnout in doctors and nursing professionals.

Background: Leiter and Maslach have explored a double process model of burnout not only based on exhaustion by overload, but also based on personal and organisational value conflicts (community, rewards or values). Moreover, Whittington has obtained conclusive results about the possible relationship between violence and burnout in mental health nurses.

Design: A retrospective study was performed in three hospitals and 22 primary care centres in Spain (n = 1·826).

Methods: Through different questionnaires, we have explored the relationship between aggression suffered by healthcare workers and burnout.

Results: Eleven percent of respondents had been physically assaulted on at least one occasion, whilst 34·4% had suffered threats and intimidation on at least one occasion and 36·6% had been subjected to insults. Both forms of violence, physical and non-physical aggression, showed significant correlations with symptoms of burnout (emotional exhaustion, depersonalisation and inefficacy).

Conclusions: The survey showed evidence of a double process: (1) by which excess workload helps predict burnout, and (2) by which a mismatch in the congruence of values, or interpersonal conflict, contributes in a meaningful way to each of the dimensions of burnout, adding overhead to the process of exhaustion-cynicism-lack of realisation. Relevance to clinical practice.  Studies indicate that health professionals are some of the most exposed to disorders steaming from psychosocial risks and a high comorbidity: anxiety, depression, etc. There is a clear need for accurate instruments of evaluation to detect not only the burnout but also the areas that cause it. Professional exhaustion caused by aggression or other factors can reflect a deterioration in the healthcare relationship.
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http://dx.doi.org/10.1111/j.1365-2702.2012.04255.xDOI Listing
November 2013

EBI2 guides serial movements of activated B cells and ligand activity is detectable in lymphoid and nonlymphoid tissues.

J Immunol 2011 Sep 15;187(6):3026-32. Epub 2011 Aug 15.

Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94143, USA.

EBV-induced gene 2 (EBI2) was recently shown to direct the delayed movement of activated B cells to interfollicular and outer follicular regions of secondary lymphoid organs and to be required for mounting a normal T-dependent Ab response. In this study, we show that EBI2 promotes an early wave of Ag-activated B cell migration to the outer follicle in mice. Later, when B cells have moved to the T zone in a CCR7-dependent manner, EBI2 helps distribute the cells along the B zone-T zone boundary. Subsequent EBI2-dependent movement to the outer follicle coincides with CCR7 downregulation and is promoted by CD40 engagement. Using a bioassay, we identify a proteinase K-resistant, hydrophobic EBI2 ligand activity in lymphoid and nonlymphoid tissues. Production of EBI2 ligand activity by a cell line is sensitive to statins, suggesting production in a 3-hydroxy-3-methyl-glutaryl-CoA reductase-dependent manner. CD40-activated B cells show sustained EBI2-dependent responsiveness to the bioactivity. These findings establish a role for EBI2 in helping control B cell position at multiple stages during the Ab response and they suggest that EBI2 responds to a broadly distributed lipid ligand.
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http://dx.doi.org/10.4049/jimmunol.1101262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3169736PMC
September 2011

Oxysterols direct immune cell migration via EBI2.

Nature 2011 Jul 27;475(7357):524-7. Epub 2011 Jul 27.

Euroscreen S.A., 6041 Gosselies, Belgium.

Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2-oxysterol signalling pathway has an important role in the adaptive immune response.
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http://dx.doi.org/10.1038/nature10280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297623PMC
July 2011

T-bet-dependent S1P5 expression in NK cells promotes egress from lymph nodes and bone marrow.

J Exp Med 2009 Oct 6;206(11):2469-81. Epub 2009 Oct 6.

Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA.

During a screen for ethylnitrosourea-induced mutations in mice affecting blood natural killer (NK) cells, we identified a strain, designated Duane, in which NK cells were reduced in blood and spleen but increased in lymph nodes (LNs) and bone marrow (BM). The accumulation of NK cells in LNs reflected a decreased ability to exit into lymph. This strain carries a point mutation within Tbx21 (T-bet), which generates a defective protein. Duane NK cells have a 30-fold deficiency in sphingosine-1-phosphate receptor 5 (S1P5) transcript levels, and S1P5-deficient mice exhibit an egress defect similar to Duane. Chromatin immunoprecipitation confirms binding of T-bet to the S1pr5 locus. S1P-deficient mice exhibit a more severe NK cell egress block, and the FTY720-sensitive S1P1 also plays a role in NK cell egress from LNs. S1P5 is not inhibited by CD69, a property that may facilitate trafficking of activated NK cells to effector sites. Finally, the accumulation of NK cells within BM of S1P-deficient mice was associated with reduced numbers in BM sinusoids, suggesting a role for S1P in BM egress. In summary, these findings identify S1P5 as a T-bet-induced gene that is required for NK cell egress from LNs and BM.
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http://dx.doi.org/10.1084/jem.20090525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2768857PMC
October 2009

EBI2 mediates B cell segregation between the outer and centre follicle.

Nature 2009 Aug 13;460(7259):1122-6. Epub 2009 Jul 13.

Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California San Francisco, California 94143, USA.

B cell follicles are specialized microenvironments that support events necessary for humoral immunity. After antigen encounter, activated B cells initially seek T-cell help at the follicle-T-zone boundary and then move to interfollicular and T-zone distal (outer) regions of the follicle. Subsequently, some cells move to the follicle centre, become germinal centre B cells and undergo antibody affinity maturation. Although germinal centres within follicles were described in 1885 (ref. 12), the molecular cues mediating segregation of B cells between the outer and centre follicle have remained undefined. Here we present a role for the orphan G-protein-coupled receptor, Epstein-Barr virus induced molecule-2 (EBI2, also known as GPR183), in this process. EBI2 is expressed in mature B cells and increases in expression early after activation, before being downregulated in germinal centre B cells. EBI2 deficiency in mice led to a reduction in the early antibody response to a T-dependent antigen. EBI2-deficient B cells failed to move to the outer follicle at day 2 of activation, and instead were found in the follicle centre, whereas EBI2 overexpression was sufficient to promote B cell localization to the outer follicle. In mixed bone marrow chimaeras, EBI2-deficient B cells phenocopied germinal centre B cells in preferentially localizing to the follicle centre. When downregulation of EBI2 in wild-type B cells was antagonized, participation in the germinal centre reaction was impaired. These studies identify an important role for EBI2 in promoting B cell localization in the outer follicle, and show that differential expression of this receptor helps position B cells appropriately for mounting T-dependent antibody responses.
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http://dx.doi.org/10.1038/nature08226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2809436PMC
August 2009

Cannabinoid receptor 2 mediates the retention of immature B cells in bone marrow sinusoids.

Nat Immunol 2009 Apr 1;10(4):403-11. Epub 2009 Mar 1.

Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California San Francisco, USA.

Immature B cells developing in the bone marrow are found in the parenchyma and sinusoids. The mechanisms that control the positioning of B cells in the sinusoids are not understood. Here we show that the integrin alpha(4)beta(1) (VLA-4) and its ligand VCAM-1 were required, whereas the chemokine receptor CXCR4 was dispensable, for sinusoidal retention of B cells. Instead, cannabinoid receptor 2 (CB2), a Galpha(i) protein-coupled receptor upregulated in immature B cells, was required for sinusoidal retention. Using two-photon microscopy, we found immature B cells entering and crawling in sinusoids; these immature B cells were displaced by CB2 antagonism. Moreover, CB2-deficient mice had a lower frequency of immunoglobulin lambda-chain-positive B cells in the peripheral blood and spleen. Our findings identify unique requirements for the retention of B cells in the bone marrow sinusoidal niche and suggest involvement of CB2 in the generation of the B cell repertoire.
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http://dx.doi.org/10.1038/ni.1710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2768754PMC
April 2009

Promotion of lymphocyte egress into blood and lymph by distinct sources of sphingosine-1-phosphate.

Science 2007 Apr 15;316(5822):295-8. Epub 2007 Mar 15.

Cardiovascular Research Institute, University of California, San Francisco, 600 16th Street S472D, San Francisco, CA 94143-2240, USA.

Lymphocytes require sphingosine-1-phosphate (S1P) receptor-1 to exit lymphoid organs, but the source(s) of extracellular S1P and whether S1P directly promotes egress are unknown. By using mice in which the two kinases that generate S1P were conditionally ablated, we find that plasma S1P is mainly hematopoietic in origin, with erythrocytes a major contributor, whereas lymph S1P is from a distinct radiation-resistant source. Lymphocyte egress from thymus and secondary lymphoid organs was markedly reduced in kinase-deficient mice. Restoration of S1P to plasma rescued egress to blood but not lymph, and the rescue required lymphocyte expression of S1P-receptor-1. Thus, separate sources provide S1P to plasma and lymph to help lymphocytes exit the low-S1P environment of lymphoid organs. Disruption of compartmentalized S1P signaling is a plausible mechanism by which S1P-receptor-1 agonists function as immunosuppressives.
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http://dx.doi.org/10.1126/science.1139221DOI Listing
April 2007

Infection with Mycobacterium ulcerans induces persistent inflammatory responses in mice.

Infect Immun 2005 Oct;73(10):6299-310

Life and Health Sciences Research Institute, School of Health Sciences (ICVS), University of Minho, Braga, Portugal.

Buruli ulcer (BU) is a devastating, necrotizing, tropical skin disease caused by infections with Mycobacterium ulcerans. In contrast to other mycobacterioses, BU has been associated with minimal or absent inflammation. However, here we show that in the mouse M. ulcerans induces persistent inflammatory responses with virulence-dependent patterns. Mycolactone-positive, cytotoxic strains are virulent for mice and multiply progressively, inducing both early and persistent acute inflammatory responses. The cytotoxicity of these strains leads to progressive destruction of the inflammatory infiltrates by postapoptotic secondary necrosis, generating necrotic acellular areas with extracellular bacilli released by the lysis of infected phagocytes. The necrotic areas, always surrounded by acute inflammatory infiltrates, expand through the progressive invasion of healthy tissues around the initial necrotic lesions by bacteria and by newly recruited acute inflammatory cells. Our observations show that the lack of inflammatory infiltrates in the extensive areas of necrosis seen in advanced infections results from the destruction of continuously produced inflammatory infiltrates and not from M. ulcerans-induced local or systemic immunosuppression. Whether this is the mechanism behind the predominance of minimal or absent inflammatory responses in BU biopsies remains to be elucidated.
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http://dx.doi.org/10.1128/IAI.73.10.6299-6310.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1230890PMC
October 2005

Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients.

Science 2005 Sep;309(5741):1735-9

Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143-0414, USA.

Lymphocyte egress from the thymus and from peripheral lymphoid organs depends on sphingosine 1-phosphate (S1P) receptor-1 and is thought to occur in response to circulatory S1P. However, the existence of an S1P gradient between lymphoid organs and blood or lymph has not been established. To further define egress requirements, we addressed why treatment with the food colorant 2-acetyl-4-tetrahydroxybutylimidazole (THI) induces lymphopenia. We found that S1P abundance in lymphoid tissues of mice is normally low but increases more than 100-fold after THI treatment and that this treatment inhibits the S1P-degrading enzyme S1P lyase. We conclude that lymphocyte egress is mediated by S1P gradients that are established by S1P lyase activity and that the lyase may represent a novel immunosuppressant drug target.
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http://dx.doi.org/10.1126/science.1113640DOI Listing
September 2005