retroviral-overexpression-cxcr4-on-murine-b-1a-cells-adoptive

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

University of Virginia

Proinflammatory cytokines are thought to play a significant role in the pathogenesis of type 2 diabetes (T2D) and are elevated in the circulation even before the onset of the disease. However, the full complement of cytokines involved in the development of T2D is not known. In this study, 32 serum cytokines were measured from diabetes-prone BKS.Cg-m+/+Lepr(db)/J (db/db) mice and heterozygous age-matched control mice at 5 weeks (non-diabetic/non-obese), 6-7 weeks (transitional-to-diabetes), or 11 weeks (hyperglycemic/obese) and then correlated with body weight, blood glucose, and fat content. Among these 32 cytokines, C-X-C motif ligand 1 (CXCL1) showed the greatest increase (+78%) in serum levels between db/db mice that were hyperglycemic (blood glucose: 519±23 mg/dl, n=6) and those that were non-hyperglycemic (193±13 mg/dl, n=8). Similarly, increased CXCL1 (+68%) and CXCL5 (+40%) were associated with increased obesity in db/db mice; note that these effects could not be entirely separated from age. We then examined whether islets could be a source of these chemokines. Exposure to cytokines mimicking low-grade systemic inflammation (10 pg/ml IL1β+20 pg/ml IL6) for 48 h upregulated islet CXCL1 expression by 53±3-fold and CXCL5 expression by 83±10-fold (n=4, P<0.001). Finally, overnight treatment with the combination of CXCL1 and CXCL5 at serum levels was sufficient to produce a significant decrease in the peak calcium response to glucose stimulation, suggesting reduced islet function. Our findings demonstrated that CXCL1 and CXCL5 i) are increased in the circulation with the onset of T2D, ii) are produced by islets under stress, and iii) synergistically affect islet function, suggesting that these chemokines participate in the pathogenesis of T2D.

Increased serum CXCL1 and CXCL5 are linked to obesity, hyperglycemia, and impaired islet function.

Cardiovascular diseases are the main cause of death worldwide and pose an immense economical burden. In most cases, the underlying problem is vascular occlusion by atherosclerotic plaques. Importantly, different cell types of the vascular wall and the immune system play crucial roles in atherosclerosis at different stages of the disease. Furthermore, atherosclerosis and conditions recognized as risk factors are characterized by a reduced availability of the vasoprotective molecule nitric oxide and an increase in reactive oxygen species, so-called oxidative stress. Transcription factors function as intracellular signal integrators and relays and thus, play a central role in cellular responses to changing conditions. Recent Advances: Work on specific transcriptional regulators has uncovered many of their functions and the upstream pathways modulating their activity in response to reactive oxygen and nitrogen species. Here, we have reviewed for a few selected examples how this can contribute not only to protection against atherosclerosis development but also to disease progression and the occurrence of clinical manifestations, such as plaque rupture.

Regulation of Transcription Factors by Reactive Oxygen Species and Nitric Oxide in Vascular Physiology and Pathology.

Adipose tissue surrounding major arteries (Perivascular adipose tissue or PVAT) has long been thought to exist to provide vessel support and insulation. Emerging evidence suggests that PVAT regulates artery physiology and pathology, such as, promoting atherosclerosis development through local production of inflammatory cytokines. Yet the immune subtypes in PVAT that regulate inflammation are poorly characterized. B cells have emerged as important immune cells in the regulation of visceral adipose tissue inflammation and atherosclerosis. B cell-mediated effects on atherosclerosis are subset-dependent with B-1 cells attenuating and B-2 cells aggravating atherosclerosis. While mechanisms whereby B-2 cells aggravate atherosclerosis are less clear, production of immunoglobulin type M (IgM) antibodies is thought to be a major mechanism whereby B-1 cells limit atherosclerosis development. B-1 cell-derived IgM to oxidation specific epitopes (OSE) on low density lipoproteins (LDL) blocks oxidized LDL-induced inflammatory cytokine production and foam cell formation. However, whether PVAT contains B-1 cells and whether atheroprotective IgM is produced in PVAT is unknown. Results of the present study provide clear evidence that the majority of B cells in and around the aorta are derived from PVAT. Interestingly, a large proportion of these B cells belong to the B-1 subset with the B-1/B-2 ratio being 10-fold higher in PVAT relative to spleen and bone marrow. Moreover, PVAT contains significantly greater numbers of IgM secreting cells than the aorta. ApoE mice with B cell-specific knockout of the gene encoding the helix-loop-helix factor Id3, known to have attenuated diet-induced atherosclerosis, have increased numbers of B-1b cells and increased IgM secreting cells in PVAT relative to littermate controls. Immunostaining of PVAT on human coronary arteries identified fat associated lymphoid clusters (FALCs) harboring high numbers of B cells, and flow cytometry demonstrated the presence of T cells and B cells including B-1 cells. Taken together, these results provide evidence that murine and human PVAT harbor B-1 cells and suggest that local IgM production may serve to provide atheroprotection.

Perivascular Adipose Tissue Harbors Atheroprotective IgM-Producing B Cells.

Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease.

c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells.

Cell- and Sex-Specific Role of FcγR (Fcγ Receptor) IIb in Experimental Atherosclerosis.

B-1 cell-derived natural IgM antibodies against oxidation-specific epitopes on low-density lipoprotein are anti-inflammatory and atheroprotective. Bone marrow (BM) B-1a cells contribute abundantly to IgM production, yet the unique repertoire of IgM antibodies generated by BM B-1a and the factors maintaining the BM B-1a population remain unexplored. CXCR4 (C-X-C motif chemokine receptor 4) has been implicated in human cardiovascular disease and B-cell homeostasis, yet the role of B-1 cell CXCR4 in regulating atheroprotective IgM levels and human cardiovascular disease is unknown.

Diversification and CXCR4-Dependent Establishment of the Bone Marrow B-1a Cell Pool Governs Atheroprotective IgM Production Linked to Human Coronary Atherosclerosis.

Atherosclerosis-the major underlying pathology of cardiovascular disease-is characterized by accumulation and subsequent oxidative modification of lipoproteins within the artery wall, leading to inflammatory cell infiltration and lesion formation that can over time result in arterial stenosis, ischemia, and downstream adverse events. The contribution of innate and adaptive immunity to atherosclerosis development is well established, and B cells have emerged as important modulators of both pro- and anti-inflammatory effects in atherosclerosis. Murine B cells can broadly be divided into 2 subsets: (1) B-2 cells, which are bone marrow derived and include conventional follicular and marginal zone B cells, and (2) B-1 cells, which are largely fetal liver derived and persist in adults through self-renewal. B-cell subsets are developmentally, functionally, and phenotypically distinct with unique subset-specific contributions to atherosclerosis development. Mechanisms whereby B cells regulate vascular inflammation and atherosclerosis will be discussed with a particular emphasis on B-1 cells. B-1 cells have a protective role in atherosclerosis that is mediated in large part by IgM antibody production. Accumulating evidence over the last several years has pointed to a previously underappreciated heterogeneity in B-1 cell populations, which may have important implications for understanding atherosclerosis development and potential targeted therapeutic approaches. This heterogeneity within atheroprotective innate B-cell subsets will be highlighted.

2019 Russell Ross Memorial Lecture in Vascular Biology: B Lymphocyte-Mediated Protective Immunity in Atherosclerosis.

Macrophages are important regulators of obesity-associated inflammation and PPARα and -γ agonism in macrophages has anti-inflammatory effects. In this study, we tested the efficacy with which liposomal delivery could target the PPARα/γ dual agonist tesaglitazar to macrophages while reducing drug action in common sites of drug toxicity: the liver and kidney, and whether tesaglitazar had anti-inflammatory effects in an  model of obesity-associated dysmetabolism. : Male leptin-deficient () mice were administered tesaglitazar or vehicle for one week in a standard oral formulation or encapsulated in liposomes. Following the end of treatment, circulating metabolic parameters were measured and pro-inflammatory adipose tissue macrophage populations were quantified by flow cytometry. Cellular uptake of liposomes in tissues was assessed using immunofluorescence and a broad panel of cell subset markers by flow cytometry. Finally, PPARα/γ gene target expression levels in the liver, kidney, and sorted macrophages were quantified to determine levels of drug targeting to and drug action in these tissues and cells. : Administration of a standard oral formulation of tesaglitazar effectively treated symptoms of obesity-associated dysmetabolism and reduced the number of pro-inflammatory adipose tissue macrophages. Macrophages are the major cell type that took up liposomes with many other immune and stromal cell types taking up liposomes to a lesser extent. Liposome delivery of tesaglitazar did not have effects on inflammatory macrophages nor did it improve metabolic parameters to the extent of a standard oral formulation. Liposomal delivery did, however, attenuate effects on liver weight and liver and kidney expression of PPARα and -γ gene targets compared to oral delivery. : These findings reveal for the first time that tesaglitazar has anti-inflammatory effects on adipose tissue macrophage populations . These data also suggest that while nanoparticle delivery reduced off-target effects, yet the lack of tesaglitazar actions in non-targeted cells such (as hepatocytes and adipocytes) and the uptake of drug-loaded liposomes in many other cell types, albeit to a lesser extent, may have impacted overall therapeutic efficacy. This fulsome analysis of cellular uptake of tesaglitazar-loaded liposomes provides important lessons for future studies of liposome drug delivery.

Article	Year
Increased serum CXCL1 and CXCL5 are linked to obesity, hyperglycemia, and impaired islet function. The Journal of endocrinology\| PubMed ID: 24928936	2014
Regulation of Transcription Factors by Reactive Oxygen Species and Nitric Oxide in Vascular Physiology and Pathology. Antioxidants & redox signaling\| PubMed ID: 27841660	2017
Perivascular Adipose Tissue Harbors Atheroprotective IgM-Producing B Cells. Frontiers in physiology\| PubMed ID: 28970806	2017
c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells. Cell reports\| PubMed ID: 31116977	2019
Cell- and Sex-Specific Role of FcγR (Fcγ Receptor) IIb in Experimental Atherosclerosis. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 31242029	2019
Diversification and CXCR4-Dependent Establishment of the Bone Marrow B-1a Cell Pool Governs Atheroprotective IgM Production Linked to Human Coronary Atherosclerosis. Circulation research\| PubMed ID: 31549940	2019
2019 Russell Ross Memorial Lecture in Vascular Biology: B Lymphocyte-Mediated Protective Immunity in Atherosclerosis. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 31852222	2020
liposomal delivery of PPARα/γ dual agonist tesaglitazar in a model of obesity enriches macrophage targeting and limits liver and kidney drug effects. Theranostics\| PubMed ID: 31903139	2020

Aditi Upadhye

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