Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells.

Humoral immunity depends on intrinsic B cell developmental programs guided by systemic signals that convey physiologic needs. Aberrant cues or their improper interpretation can lead to immune insufficiency or a failure of tolerance and autoimmunity. The means by which such systemic signals are conveyed remain poorly understood. Hence, further insight is essential to understanding and treating autoimmune diseases and to the development of improved vaccines. ST6Gal-1 is a sialyltransferase that constructs the α2,6-sialyl linkage on cell surface and extracellular glycans. The requirement for functional ST6Gal-1 in the development of humoral immunity is well documented. Canonically, ST6Gal-1 resides within the intracellular ER-Golgi secretory apparatus and participates in cell-autonomous glycosylation. However, a significant pool of extracellular ST6Gal-1 exists in circulation. Here, we segregate the contributions of B cell intrinsic and extrinsic ST6Gal-1 to B cell development. We observed that B cell-intrinsic ST6Gal-1 is required for marginal zone B cell development, while B cell non-autonomous ST6Gal-1 modulates B cell development and survival at the early transitional stages of the marrow and spleen. Exposure to extracellular ST6Gal-1 ex vivo enhanced the formation of IgM-high B cells from immature precursors, and increased CD23 and IgM expression. Extrinsic sialylation by extracellular ST6Gal-1 augmented BAFF-mediated activation of the non-canonical NF-kB, p38 MAPK, and PI3K/AKT pathways, and accelerated tyrosine phosphorylation after B cell receptor stimulation. in vivo, systemic ST6Gal-1 did not influence homing of B cells to the spleen but was critical for their long-term survival and systemic IgG levels. Circulatory ST6Gal-1 levels respond to inflammation, infection, and malignancy in mammals, including humans. In turn, we have shown previously that systemic ST6Gal-1 regulates inflammatory cell production by modifying bone marrow myeloid progenitors. Our data here point to an additional role of systemic ST6Gal-1 in guiding B cell development, which supports the concept that circulating ST6Gal-1 is a conveyor of systemic cues to guide the development of multiple branches of immune cells.

Treatment of ongoing autoimmune encephalomyelitis with activated B-cell progenitors maturing into regulatory B cells.

The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here, we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes, where they trap T cells by releasing the CCR7 ligand, CCL19, and to inflamed central nervous system, where they locally limit immunopathogenesis through interleukin-10 production, thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment, where proB cells develop, is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition, these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases.

Interleukin-10 From Marginal Zone Precursor B-Cell Subset Is Required for Costimulatory Blockade-Induced Transplantation Tolerance.

BACKGROUND: Blocking CD40-CD40L costimulatory signals induces transplantation tolerance. Although B-cell depletion prevents alloantibody formation, nonhumoral functions of B cells in tolerance have not been well characterized. We investigated whether specific subsets of B cell or B cell-derived interleukin (IL)-10 contribute to tolerance. METHODS: Wild type C57BL/6, or B cell-specific interleukin (IL)-10 (CD19-Cre::IL-10) mice, received vascularized BALB/c cardiac allografts. BALB/c donor-specific splenocyte transfusion and anti-CD40L monoclonal antibody were used as tolerogen. B cells were depleted with antimouse CD20 monoclonal antibody. Various B-cell subsets were purified and characterized by flow cytometry, reverse transcription polymerase chain reaction, and adoptive transfer. RESULTS: B-cell depletion prevented costimulatory blockade-induced allogeneic tolerance. Costimulatory blockade increased IL-10 in marginal zone precursor (MZP) B cells, but not other subsets. In particular, costimulatory blockade did not change other previously defined regulatory B-cell subsets (Breg), including CD5CD1d Breg or expression of TIM1 or TIM4 on these Breg or other Breg cell subsets. Costimulatory blockade also induced IL-21R expression in MZP B cells, and IL-21R MZP B cells expressed even more IL-10. B-cell depletion or IL-10 deficiency in B cells prevented tolerance in a cardiac allograft model, resulting in rapid acute cardiac allograft rejection. Adoptive transfer of wild type MZP B cells but not other subsets to B cell-specific IL-10 deficient mice prevented graft rejection. CONCLUSIONS: CD40 costimulatory blockade induces MZP B cell IL-10 which is necessary for tolerance. These observations have implications for understanding tolerance induction and how B cell depletion may prevent tolerance.

Platelet-rich plasma in the pathologic processes of cartilage: review of basic science evidence.

PURPOSE: The purpose of this study was to systematically review the basic science evidence for the use of platelet-rich plasma (PRP) in the treatment of pathologic processes of cartilage, both as an adjunct to cartilage repair and as a conservative management strategy for osteoarthritis, with the intent of determining the effect of PRP and whether a proof of concept for its use has been established to facilitate further investigation at a clinical level. METHODS: Using the terms "platelet-rich plasma OR PRP OR autologous conditioned plasma OR ACP AND cartilage OR chondrocytes OR chondrogenesis OR osteoarthritis OR arthritis" we searched EMBASE and PubMed/Medline in April 2012. Two authors performed the search, 3 authors independently assessed the studies for inclusion, and 2 authors extracted the data. Extracted data included cytologic analysis of PRP, study design, and results. RESULTS: Twenty-one studies (12 in vitro, 8 in vivo, one in vitro and in vivo) met the inclusion criteria. The effects of PRP in these studies included increasing chondrocyte and mesenchymal stem cell proliferation, proteoglycan deposition, and type II collagen deposition. PRP was also found to increase the cell viability of chondrocytes and the migration and chondrogenic differentiation of mesenchymal stem cells (MSCs) and to inhibit the effect of catabolic cytokines. In vivo, PRP was used as an adjunct to concomitant surgical management, including microfracture surgery and implant, scaffold, and graft insertion. Not all studies concluded that PRP has a positive effect on cartilage repair. CONCLUSIONS: The current basic science evidence suggests that PRP has several potential effects on cartilage repair and osteoarthritis, and a proof of concept has been established. Well-designed randomized controlled trials (RCTs) are needed to extrapolate this evidence to the clinical setting.

miR-221 redirects precursor B cells to the BM and regulates their residence.

Pluripotent hematopoietic stem cells and multipotent myeloid/lymphoid progenitors express miR-221 and miR-222. When Pax5 expression commits these progenitors to monopotent pre-B lymphocytes the two microRNAs (miRNAs) are downregulated. Upon transplantation, stem cells and progenitors can reside in the BM, while pre-B cells, after their commitment, no longer do so. Retrovirally transduced, doxycycline-induced overexpression of either miR-221 or miR-222 in pre-B-I cells does not revert their monopotency to multipotency. However, upon transplantation miR-221, but not miR-222, transduced pre-B-I cells regain the capacity to reside in the BM. Upon subsequent termination of miR-221-expression by removal of doxycycline, the transplanted cells leave the BM again. Microarray analyses identified 25 downregulated miR-221-target genes, which could function to localize phases of B-lymphocyte development in BM before and after commitment.

Innate pro-B-cell progenitors protect against type 1 diabetes by regulating autoimmune effector T cells.

Diverse hematopoietic progenitors, including myeloid populations arising in inflammatory and tumoral conditions and multipotent cells, mobilized by hematopoietic growth factors or emerging during parasitic infections, display tolerogenic properties. Innate immune stimuli confer regulatory functions to various mature B-cell subsets but immature B-cell progenitors endowed with suppressive properties per se or after differentiating into more mature regulatory B cells remain to be characterized. Herein we provide evidence for innate pro-B cells (CpG-proBs) that emerged within the bone marrow both in vitro and in vivo upon Toll-like receptor-9 activation and whose adoptive transfer protected nonobese diabetic mice against type 1 diabetes (T1D). These cells responded to IFN-γ released by activated effector T cells (Teffs), by up-regulating their Fas ligand (FasL) expression, which enabled them to kill Teffs through apoptosis. In turn, IFN-γ derived from CpG-proBs enhanced IFN-γ while dramatically reducing IL-21 production by Teffs. In keeping with the crucial pathogenic role played by IL-21 in T1D, adoptively transferred IFN-γ-deficient CpG-proBs did not prevent T1D development. Additionally, CpG-proBs matured in vivo into diverse pancreatic and splenic suppressive FasL(high) B-cell subsets. CpG-proBs may become instrumental in cell therapy of autoimmune diseases either on their own or as graft complement in autologous stem cell transplantation.

Restoring specific lactobacilli levels decreases inflammation and muscle atrophy markers in an acute leukemia mouse model.

The gut microbiota has recently been proposed as a novel component in the regulation of host homeostasis and immunity. We have assessed for the first time the role of the gut microbiota in a mouse model of leukemia (transplantation of BaF3 cells containing ectopic expression of Bcr-Abl), characterized at the final stage by a loss of fat mass, muscle atrophy, anorexia and inflammation. The gut microbial 16S rDNA analysis, using PCR-Denaturating Gradient Gel Electrophoresis and quantitative PCR, reveals a dysbiosis and a selective modulation of Lactobacillus spp. (decrease of L. reuteri and L. johnsonii/gasseri in favor of L. murinus/animalis) in the BaF3 mice compared to the controls. The restoration of Lactobacillus species by oral supplementation with L. reuteri 100-23 and L. gasseri 311476 reduced the expression of atrophy markers (Atrogin-1, MuRF1, LC3, Cathepsin L) in the gastrocnemius and in the tibialis, a phenomenon correlated with a decrease of inflammatory cytokines (interleukin-6, monocyte chemoattractant protein-1, interleukin-4, granulocyte colony-stimulating factor, quantified by multiplex immuno-assay). These positive effects are strain- and/or species-specific since L. acidophilus NCFM supplementation does not impact on muscle atrophy markers and systemic inflammation. Altogether, these results suggest that the gut microbiota could constitute a novel therapeutic target in the management of leukemia-associated inflammation and related disorders in the muscle.

Pim1 and Myc reversibly transform murine precursor B lymphocytes but not mature B lymphocytes.

The proto-oncogenes Myc and Pim1, which are deregulated in many types of cancers, are known to cooperate in B lymphoma development. Here we show that overexpression of retrovirally transduced, doxycycline-inducible Myc alone in IL-7-deprived, growth-arrested pre-B cells enhanced cell cycle entry without impairing apoptosis. Overexpression of Pim1 decreased apoptosis, but had no effect on cell cycle entry. Co-expression of Pim1 and Myc inhibited apoptosis and led to IL-7-independent proliferation of the transduced pre-B cells in vitro, while blocking their differentiation to IgM(+) immature cells. Transplantation of Pim1/Myc overexpressing pre-BI cells into B-cell-deficient mice expanded the pre-B-cell compartments up to 100-fold within 4-8 weeks. Transformation remained dependent on the expression of both oncogenes, as removal of doxycycline in vitro and in vivo terminated proliferation and induced differentiation to IgM(+) B cells. In contrast, Pim1/Myc-transduced mature B cells that developed from the oncogene-transduced pre-BI cells in the absence of oncogene overexpression in vivo were not capable of long-term proliferation after induction of Pim and Myc overexpression, neither in vivo nor in vitro, neither with nor without stimulation by polyclonal activators.