Defining the role of CD47 and SIRPα in murine B cell homeostasis

B cell development is a highly organized process, which commences in the fetal liver during embryogenesis and in the bone marrow (BM) after birth. Surface IgM+ immature B cells emigrate from the BM via the blood stream to the spleen and finally differentiate into conventional mature follicular B (FoB) cells and marginal zone (MZ) B cells. Conversely, some sIgM+ immature B cells can also mature into IgD+ FoB cells in the BM. The ubiquitously expressed cell surface glycoprotein CD47 and its receptor signal regulatory protein α (SIRPα) are members of the immunoglobulin superfamily. Both individually and upon their interaction, CD47 and SIRPα have been found to play important role in the homeostasis of T lymphocytes or CD8­ conventional dendritic cells (cDCs) in secondary lymphoid organs. However, their role in regulating B cell homeostasis has remained unknown. The present study describes important roles of CD47 and SIRPα in B cell homeostasis. Lack of SIRPα signaling in adult SIRPα mutant (MT - cytoplasmic domain deletion) mice resulted in an impaired B cell maturation in the BM and spleen, which was also reflected in the blood. In the BM and spleen of SIRPα MT mice, reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells were observed, while earlier BM B cell progenitors or splenic transitional B cells remained unaltered. In SIRPα MT mice, maturing B cells in BM and spleen were found to express higher levels of the pro-apoptotic protein BIM and contained an increased level of apoptotic cells. In contrast to that for FoB cells, the splenic MZ B cell population was increased with age in SIRPα MT mice without showing an increased level of activation markers. Immunohistochemical analysis revealed an increased follicular localization of MZ B cells in the spleens of SIRPα MT mice. In addition, MZ macrophages and marginal metallophilic macrophages were not restricted to their normal position in SIRPα MT spleens. Interestingly, CD47-deficient (CD47-/-) mice mimicked the FoB cell phenotype observed in SIRPα MT mice and had a reduced number of  FoB cells in the BM, blood and the spleen at 5­6 months of age, but not in younger mice. Similar to SIRPα MT mice, CD47-/- mice also displayed an increased number of splenic MZ B cells. Sera form both mouse strains did not show any signs of an increased production of autoantibodies or antinuclear antigens. BM reconstitution experiments identified a requirement for non-hematopoietic SIRPα signaling for normal B cell maturation in the BM and to maintain normal numbers and retention of MZ B cells in the splenic MZ. On the contrary, hematopoietic SIRPα signaling appeared to be important for FoB cell maturation in the spleen. Interestingly, hematopoietic SIRPα was required for normal MZ retention of MZ macrophages while normal distribution of metallophilic macrophages required non­hematopoietic SIRPα signaling.  Collectively, these findings revealed an important role of CD47 and of SIRPα signaling in B cell homeostasis in different lymphoid organs

Defining the role of CD47 and SIRPα in murine B cell homeostasis

B cell development is a highly organized process, which commences in the fetal liver during embryogenesis and in the bone marrow (BM) after birth. Surface IgM+ immature B cells emigrate from the BM via the blood stream to the spleen and finally differentiate into conventional mature follicular B (FoB) cells and marginal zone (MZ) B cells. Conversely, some sIgM+ immature B cells can also mature into IgD+ FoB cells in the BM. The ubiquitously expressed cell surface glycoprotein CD47 and its receptor signal regulatory protein α (SIRPα) are members of the immunoglobulin superfamily. Both individually and upon their interaction, CD47 and SIRPα have been found to play important role in the homeostasis of T lymphocytes or CD8­ conventional dendritic cells (cDCs) in secondary lymphoid organs. However, their role in regulating B cell homeostasis has remained unknown. The present study describes important roles of CD47 and SIRPα in B cell homeostasis. Lack of SIRPα signaling in adult SIRPα mutant (MT - cytoplasmic domain deletion) mice resulted in an impaired B cell maturation in the BM and spleen, which was also reflected in the blood. In the BM and spleen of SIRPα MT mice, reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells were observed, while earlier BM B cell progenitors or splenic transitional B cells remained unaltered. In SIRPα MT mice, maturing B cells in BM and spleen were found to express higher levels of the pro-apoptotic protein BIM and contained an increased level of apoptotic cells. In contrast to that for FoB cells, the splenic MZ B cell population was increased with age in SIRPα MT mice without showing an increased level of activation markers. Immunohistochemical analysis revealed an increased follicular localization of MZ B cells in the spleens of SIRPα MT mice. In addition, MZ macrophages and marginal metallophilic macrophages were not restricted to their normal position in SIRPα MT spleens. Interestingly, CD47-deficient (CD47-/-) mice mimicked the FoB cell phenotype observed in SIRPα MT mice and had a reduced number of  FoB cells in the BM, blood and the spleen at 5­6 months of age, but not in younger mice. Similar to SIRPα MT mice, CD47-/- mice also displayed an increased number of splenic MZ B cells. Sera form both mouse strains did not show any signs of an increased production of autoantibodies or antinuclear antigens. BM reconstitution experiments identified a requirement for non-hematopoietic SIRPα signaling for normal B cell maturation in the BM and to maintain normal numbers and retention of MZ B cells in the splenic MZ. On the contrary, hematopoietic SIRPα signaling appeared to be important for FoB cell maturation in the spleen. Interestingly, hematopoietic SIRPα was required for normal MZ retention of MZ macrophages while normal distribution of metallophilic macrophages required non­hematopoietic SIRPα signaling.  Collectively, these findings revealed an important role of CD47 and of SIRPα signaling in B cell homeostasis in different lymphoid organs

Activated, Pro-Inflammatory Th1, Th17, and Memory CD4+ T Cells and B Cells Are Involved in Delayed-Type Hypersensitivity Arthritis (DTHA) Inflammation and Paw Swelling in Mice

Delayed-type hypersensitivity arthritis (DTHA) is a recently established experimental model of rheumatoid arthritis (RA) in mice with pharmacological values. Despite an indispensable role of CD4+ T cells in inducing DTHA, a potential role for CD4+ T cell subsets is lacking. Here we have quantified CD4+ subsets during DTHA development and found that levels of activated, pro-inflammatory Th1, Th17, and memory CD4+ T cells in draining lymph nodes were increased with differential dynamic patterns after DTHA induction. Moreover, according to B-cell depletion experiments, it has been suggested that this cell type is not involved in DTHA. We show that DTHA is associated with increased levels of B cells in draining lymph nodes accompanied by increased levels of circulating IgG. Finally, using the anti-rheumatoid agents, methotrexate (MTX) and the anti-inflammatory drug dexamethasone (DEX), we show that MTX and DEX differentially suppressed DTHA-induced paw swelling and inflammation. The effects of MTX and DEX coincided with differential regulation of levels of Th1, Th17, and memory T cells as well as B cells. Our results implicate Th1, Th17, and memory T cells, together with activated B cells, to be involved and required for DTHA-induced paw swelling and inflammation

Cellular metabolism dictates T cell effector function in health and disease

In a healthy person, metabolically quiescent T lymphocytes (T cells) circulate between lymph nodes and peripheral tissues in search of antigens. Upon infection, some T cells will encounter cognate antigens followed by proliferation and clonal expansion in a context‐dependent manner, to become effector T cells. These events are accompanied by changes in cellular metabolism, known as metabolic reprogramming. The magnitude and variation of metabolic reprogramming are, in addition to antigens, dependent on factors such as nutrients and oxygen to ensure host survival during various diseases. Herein, we describe how metabolic programmes define T cell subset identity and effector functions. In addition, we will discuss how metabolic programs can be modulated and affect T cell activity in health and disease using cancer and autoimmunity as examples

Identification and characterization of a novel glutaminase inhibitor

In humans, there are two forms of glutaminase (GLS), designated GLS1 and GLS2. These enzymes catalyse the conversion of glutamine to glutamate. GLS1 exists as two isozymes: kidney glutaminase (KGA) and glutaminase C (GAC). Several GLS inhibitors have been identified, of which DON (6-diazo-5-oxonorleucine), BPTES (bis-2-(5-phenylacetamido-1, 3, 4-thiadiazol-2-yl) ethyl sulphide), 968 (5-(3-Bromo-4-(dimethylamino)phenyl)-2,2-dimethyl-2,3,5,6-tetrahydrobenzo[a]phenanthridin-4(1H)-one) and CB839 (Telaglenastat) are the most widely used. However, these inhibitors have variable efficacy, specificity and bioavailability in research and clinical settings, implying the need for novel and improved GLS inhibitors. Based on this need, a diverse library of 28,000 compounds from Enamine was screened for inhibition of recombinant, purified GAC. From this library, one inhibitor designated compound 19 (C19) was identified with kinetic features revealing allosteric inhibition of GAC in the µm range. Moreover, C19 inhibits anti-CD3/CD28-induced CD4+ T-cell proliferation and cytokine production with similar or greater potency as compared to BPTES. Taken together, our data suggest that C19 has the potential to modulate GLS1 activity and alter metabolic activity of T cells

Analysis of Neurotrophic Factors in Limb and Extraocular Muscles of Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is currently an incurable fatal motor neuron syndrome characterized by progressive weakness, muscle wasting and death ensuing 3–5 years after diagnosis. Neurotrophic factors (NTFs) are known to be important in both nervous system development and maintenance. However, the attempt to translate the potential of NTFs into the therapeutic options remains limited despite substantial number of approaches, which have been tested clinically. Using quantitative RT-PCR (qRT-PCR) technique, the present study investigated mRNA expression of four different NTFs: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4) and glial cell line-derived neurotrophic factor (GDNF) in limb muscles and extraocular muscles (EOMs) from SOD1G93A transgenic mice at early and terminal stages of ALS. General morphological examination revealed that muscle fibres were well preserved in both limb muscles and EOMs in early stage ALS mice. However, in terminal ALS mice, most muscle fibres were either atrophied or hypertrophied in limb muscles but unaffected in EOMs. qRT-PCR analysis showed that in early stage ALS mice, NT-4 was significantly down-regulated in limb muscles whereas NT-3 and GDNF were markedly up-regulated in EOMs. In terminal ALS mice, only GDNF was significantly up-regulated in limb muscles. We concluded that the early down-regulation of NT-4 in limb muscles is closely associated with muscle dystrophy and dysfunction at late stage, whereas the early up-regulations of GDNF and NT-3 in EOMs are closely associated with the relatively well-preserved muscle morphology at late stage. Collectively, the data suggested that comparing NTFs expression between limb muscles and EOMs from different stages of ALS animal models is a useful method in revealing the patho-physiology and progression of ALS, and eventually rescuing motor neuron in ALS patients

Analysis of neurotrophic factors in limb and extraocular muscles of mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is currently an incurable fatal motor neuron syndrome characterized by progressive weakness, muscle wasting and death ensuing 3-5 years after diagnosis. Neurotrophic factors (NTFs) are known to be important in both nervous system development and maintenance. However, the attempt to translate the potential of NTFs into the therapeutic options remains limited despite substantial number of approaches, which have been tested clinically. Using quantitative RT-PCR (qRT-PCR) technique, the present study investigated mRNA expression of four different NTFs: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4) and glial cell line-derived neurotrophic factor (GDNF) in limb muscles and extraocular muscles (EOMs) from SOD1G93A transgenic mice at early and terminal stages of ALS. General morphological examination revealed that muscle fibres were well preserved in both limb muscles and EOMs in early stage ALS mice. However, in terminal ALS mice, most muscle fibres were either atrophied or hypertrophied in limb muscles but unaffected in EOMs. qRT-PCR analysis showed that in early stage ALS mice, NT-4 was significantly down-regulated in limb muscles whereas NT-3 and GDNF were markedly up-regulated in EOMs. In terminal ALS mice, only GDNF was significantly up-regulated in limb muscles. We concluded that the early down-regulation of NT-4 in limb muscles is closely associated with muscle dystrophy and dysfunction at late stage, whereas the early up-regulations of GDNF and NT-3 in EOMs are closely associated with the relatively well-preserved muscle morphology at late stage. Collectively, the data suggested that comparing NTFs expression between limb muscles and EOMs from different stages of ALS animal models is a useful method in revealing the patho-physiology and progression of ALS, and eventually rescuing motor neuron in ALS patients

Analysis of Neurotrophic Factors in Limb and Extraocular Muscles of Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is currently an incurable fatal motor neuron syndrome characterized by progressive weakness, muscle wasting and death ensuing 3–5 years after diagnosis. Neurotrophic factors (NTFs) are known to be important in both nervous system development and maintenance. However, the attempt to translate the potential of NTFs into the therapeutic options remains limited despite substantial number of approaches, which have been tested clinically. Using quantitative RT-PCR (qRT-PCR) technique, the present study investigated mRNA expression of four different NTFs: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4) and glial cell line-derived neurotrophic factor (GDNF) in limb muscles and extraocular muscles (EOMs) from SOD1G93A transgenic mice at early and terminal stages of ALS. General morphological examination revealed that muscle fibres were well preserved in both limb muscles and EOMs in early stage ALS mice. However, in terminal ALS mice, most muscle fibres were either atrophied or hypertrophied in limb muscles but unaffected in EOMs. qRT-PCR analysis showed that in early stage ALS mice, NT-4 was significantly down-regulated in limb muscles whereas NT-3 and GDNF were markedly up-regulated in EOMs. In terminal ALS mice, only GDNF was significantly up-regulated in limb muscles. We concluded that the early down-regulation of NT-4 in limb muscles is closely associated with muscle dystrophy and dysfunction at late stage, whereas the early up-regulations of GDNF and NT-3 in EOMs are closely associated with the relatively well-preserved muscle morphology at late stage. Collectively, the data suggested that comparing NTFs expression between limb muscles and EOMs from different stages of ALS animal models is a useful method in revealing the patho-physiology and progression of ALS, and eventually rescuing motor neuron in ALS patients

Inflammatory activation of endothelial cells increases glycolysis and oxygen consumption despite inhibiting cell proliferation

Endothelial cell function and metabolism are closely linked to differential use of energy substrate sources and combustion. While endothelial cell migration is promoted by 2-phosphofructokinase-6/fructose-2,6-bisphosphatase (PFKFB3)-driven glycolysis, proliferation also depends on fatty acid oxidation for dNTP synthesis. We show that inflammatory activation of human umbilical vein endothelial cells (HUVECs) by interleukin-1β (IL-1β), despite inhibiting proliferation, promotes a shift toward more metabolically active phenotype. This was reflected in increased cellular glucose uptake and consumption, which was preceded by an increase in PFKFB3 mRNA and protein expression. However, despite a modest increase in extracellular acidification rates, the increase in glycolysis did not correlate with extracellular lactate accumulation. Accordingly, IL-1β stimulation also increased oxygen consumption rate, but without a concomitant rise in fatty acid oxidation. Together, this suggests that the IL-1β-stimulated energy shift is driven by shunting of glucose-derived pyruvate into mitochondria to maintain elevated oxygen consumption in HUVECs. We also revealed a marked donor-dependent variation in the amplitude of the metabolic response to IL-1β and postulate that the donor-specific response should be taken into account when considering targeting dysregulated endothelial cell metabolism

mRNA levels of NTFs in limb muscles and EOMs from early and late stage ALS mice.

<p>Top panels: comparison of NTF expression in limb muscles (A) and EOMs (B) between early and late stages. Bottom panels: comparison of NTF expression between limb muscles and EOMs of early (C) and late stages (D). The expression levels are given using arbitrary units normalized by β-actin expression. Bars represent mean ± SD from analysis of at least 5 separate samples run in triplicate. *: <i>p</i><0.05.</p