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TP53 is a master regulator of many signaling and apoptotic pathways involved in: aging, cell cycle progression, gene regulation, growth, apoptosis, cellular senescence, DNA repair, drug resistance, malignant transformation, metastasis, and metabolism. Most pancreatic cancers are classified as pancreatic ductal adenocarcinomas (PDAC). The tumor suppressor gene

B cell–activating factor (BAFF), part of a tumor necrosis factor family of cytokines, was recently identified as a regulator of atherosclerosis; however, its role in aortic aneurysm has not been determined. Here, the study examined the effect of selective BAFF antagonism using an anti-BAFF antibody (blocks binding of BAFF to receptors BAFF receptor 3, transmembrane activator and CAML interactor, and B-cell maturation antigen) and mBaffR-mFc (blocks binding of BAFF to BAFF receptor 3) on a murine model of abdominal aortic aneurysm (AAA). In a prevention strategy, the antagonists were injected before the induction of AAA, and in an intervention strategy, the antagonists were injected after the induction of AAA. Both strategies attenuated the formation of AAA. In the intervention group, BAFF antagonism depleted most of the mature B-cell subsets in spleen and circulation, leading to enhanced resolution of inflammation in AAA as indicated by decreased infiltration of B cells and proinflammatory macrophages and a reduced number of apoptotic cells. In AAA tissues, B cells and macrophages were found in close contact. In vitro, B cells, irrespective of treatment with BAFF, impaired the efferocytosis activity of macrophages, suggesting a direct innate role of B cells on macrophage function. Altogether, BAFF antagonism affects survival of the mature B cells, promotes resolution of inflammation in the aorta, and attenuates the growth of AAA in mice.

Adults with type 2 diabetes have a two-to-three-fold increased risk of cardiovascular diseases. B cell activating factor (BAFF), a tumor necrosis superfamily member, is implicated both in diabetes and cardiovascular diseases. BAFF deficiency in mice retains glucose homeostasis despite high-fat diet-induced obesity. We examined if neutralization of BAFF with an anti-BAFF antibody improves glucose utilization in high-fat diet-induced obese mice. Male C57BL/6J mice fed with a high-fat diet (60% calories from fat) for 12 weeks were segregated into two groups based on their glucose intolerance (GI) determined by a glucose tolerance test (GTT). Each group received a total of 4 injections of anti-BAFF or an isotype control antibody, and GTT was performed every two weeks. BAFF neutralization was confirmed by depletion of B2 B cells in the blood two weeks after the first anti-BAFF antibody injection. Although GI increased after continuous feeding of a high-fat diet, no differences in GI was observed between the groups till 18 weeks on diet. At 20 weeks, GI was significantly higher in the anti-BAFF treated group compared to the control group. Extensive cellular phenotyping using flow cytometry revealed that BAFF neutralization depleted mature B2 B cells in the spleen and blood but did not affect the CD4+ T cells and CD8+ T cells. No differences were found in fat accumulation in the liver, whole body fat content, visceral adipose tissue (VAT) weight, or in the number of VAT-infiltrated M1 and M2 macrophages, eosinophils, natural killer T cells, CD4+ T cells, and CD8+ T cells. Unexpectedly, in the VAT of anti-BAFF antibody-treated mice, B2 cells were retained, whereas, the number of CD4+ T cells and dead adipocytes was higher. Real-time RT-PCR analysis of stromal vascular fraction from VAT revealed increased expression of both the M1 and M2 macrophage markers IL-1β, IL-6, IL-10, Arg1, and Mrc1 in the anti-BAFF treated mice. Altogether, these results suggest BAFF neutralization promotes inflammation in the VAT of obese mice and exacerbates systemic glucose intolerance. Furthermore, our results highlight the need for more studies on the use of anti-BAFF biologics for the treatment of systemic lupus erythematosus patients who are diabetic.

Objective Resolvins have been shown to attenuate inflammation, whereas NETosis, the process of neutrophils releasing neutrophil extracellular traps (NETs), produces increased inflammation. It is hypothesized that treatment of animals with resolvin D1 (RvD1) would reduce abdominal aortic aneurysm (AAA) formation by inhibiting NETosis. Methods Wild-type 8- to 12-week-old C57BL/6 male mice (n = 47) and apolipoprotein E-deficient (ApoE−/−) mice (n = 20) were used in two models to demonstrate the effects of RvD1 on AAA growth. In the topical elastase AAA model, wild-type mice were divided into three groups: a deactivated elastase control group, in which sham surgery was performed using deactivated elastase and mice were intravenously injected with phosphate-buffered saline (PBS) once a day until harvest; an elastase group, in which active elastase was used to induce AAA and mice were injected with PBS daily until harvest; and an RvD1-treated group, in which AAA was induced and mice were injected with RvD1 daily until harvest. In the angiotensin II (Ang II)-induced AAA model, ApoE−/− mice were fed a high-fat diet and implanted with osmotic infusion pumps containing Ang II (1000 ng/kg/min). The Ang II model was divided into two groups: an Ang II control group, in which Ang II was delivered and mice were injected with PBS daily until harvest; and an RvD1-treated group, in which Ang II was delivered and mice were injected with RvD1 daily until harvest. On postoperative day 3, day 14, or day 28, aortic and blood samples were collected for Western blot, histology, cytokine array, enzyme-linked immunosorbent assay, and gelatin zymography after aortic diameter measurement. Results The day 14 RvD1-treated group demonstrated 42% reduced AAA diameter compared with the elastase group (P Conclusions RvD1-mediated inhibition of NETosis may represent a future medical treatment for the attenuation of AAA growth.

Objective Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of β-adrenergic receptor (βAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis. Methods In an immortalized brown pre-adipocytes cell line, Panx1 currents were measured using patch-clamp electrophysiology. Flow cytometry was used for assessment of dye uptake and luminescence assays for adenosine triphosphate (ATP) release, and cellular temperature measurement was performed using a ratiometric fluorescence thermometer. We used RNA interference and expression plasmids to manipulate expression of wild-type and mutant Panx1. We used previously described adipocyte-specific Panx1 knockout mice (Panx1Adip-/-) and generated brown adipocyte-specific Panx1 knockout mice (Panx1BAT-/-) to study pharmacological or cold-induced thermogenesis. Glucose uptake into brown adipose tissue was quantified by positron emission tomography (PET) analysis of 18F-fluorodeoxyglucose (18F-FDG) content. BAT temperature was measured using an implantable telemetric temperature probe. Results In brown adipocytes, Panx1 channel activity was induced either by apoptosis-dependent caspase activation or by β3AR stimulation via a novel mechanism that involves Gβγ subunit binding to Panx1. Inactivation of Panx1 channels in cultured brown adipocytes resulted in inhibition of β3AR-induced lipolysis, UCP-1 expression, and cellular thermogenesis. In mice, adiponectin-Cre-dependent genetic deletion of Panx1 in all adipose tissue depots resulted in defective β3AR agonist- or cold-induced thermogenesis in BAT and suppressed beigeing of white adipose tissue. UCP1-Cre-dependent Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis without affecting beigeing of white adipose tissue and aggravated diet-induced obesity and insulin resistance. Conclusions These data demonstrate that Gβγ-dependent Panx1 channel activation is involved in β3AR-induced thermogenic regulation in brown adipocytes. Identification of Panx1 channels in BAT as novel thermo-regulatory elements downstream of β3AR activation may have therapeutic implications., Highlights • Panx1 channel activation plays a critical role in thermogenic regulation in brown adipocytes. • Panx1 channel activity in brown adipocytes was induced by β3 adrenergic receptor stimulation. • Inactivation of Panx1 channels in cultured brown adipocytes resulted in defective cellular thermogenesis. • In mice, genetic deletion of Panx1 in all adipose tissue depots resulted in defective β3AR agonist- or cold-induced thermogenesis. • Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis and aggravated diet-induced obesity and insulin resistance in mice.

Glycogen synthase kinase-3 (GSK-3) is a regulator of signaling pathways. KRas is frequently mutated in pancreatic cancers. The growth of certain pancreatic cancers is KRas-dependent and can be suppressed by GSK-3 inhibitors, documenting a link between KRas and GSK-3. To further elucidate the roles of GSK-3β in drug-resistance, we transfected KRas-dependent MIA-PaCa-2 pancreatic cells with wild-type (WT) and kinase-dead (KD) forms of GSK-3β. Transfection of MIA-PaCa-2 cells with WT-GSK-3β increased their resistance to various chemotherapeutic drugs and certain small molecule inhibitors. Transfection of cells with KD-GSK-3β often increased therapeutic sensitivity. An exception was observed with cells transfected with WT-GSK-3β and sensitivity to the BCL2/BCLXL ABT737 inhibitor. WT-GSK-3β reduced glycolytic capacity of the cells but did not affect the basal glycolysis and mitochondrial respiration. KD-GSK-3β decreased both basal glycolysis and glycolytic capacity and reduced mitochondrial respiration in MIA-PaCa-2 cells. As a comparison, the effects of GSK-3 on MCF-7 breast cancer cells, which have mutant PIK3CA, were examined. KD-GSK-3β increased the resistance of MCF-7 cells to chemotherapeutic drugs and certain signal transduction inhibitors. Thus, altering the levels of GSK-3β can have dramatic effects on sensitivity to drugs and signal transduction inhibitors which may be influenced by the background of the tumor.

Objective— Neutrophils promote experimental abdominal aortic aneurysm (AAA) formation via a mechanism that is independent from MMPs (matrix metalloproteinases). Recently, we reported a dominant role of IL (interleukin)-1β in the formation of murine experimental AAAs. Here, the hypothesis that IL-1β–induced neutrophil extracellular trap formation (NETosis) promotes AAA was tested. Approach and Results— NETs were identified through colocalized staining of neutrophil, Cit-H3 (citrullinated histone H3), and DNA, using immunohistochemistry. NETs were detected in human AAAs and were colocalized with IL-1β. In vitro, IL-1RA attenuated IL-1β–induced NETosis in human neutrophils. Mechanistically, IL-1β treatment of isolated neutrophils induced nuclear localization of ceramide synthase 6 and synthesis of C16-ceramide, which was inhibited by IL-1RA or fumonisin B1, an inhibitor of ceramide synthesis. Furthermore, IL-1RA or fumonisin B1 attenuated IL1-β–induced NETosis. In an experimental model of murine AAA, NETs were detected at a very early stage–day 3 of aneurysm induction. IL-1β–knockout mice demonstrated significantly lower infiltration of neutrophils to aorta and were protected from AAA. Adoptive transfer of wild-type neutrophils promoted AAA formation in IL-1β–knockout mice. Moreover, treatment of wild-type mice with Cl-amidine, an inhibitor NETosis, significantly attenuated AAA formation, whereas, treatment with deoxyribonuclease, a DNA digesting enzyme, had no effect on AAA formation. Conclusions— Altogether, the results suggest a dominant role of IL-1β–induced NETosis in AAA formation.

Adipose tissue macrophages (ATMs) adapt their metabolic phenotype either to maintain lean tissue homeostasis or drive inflammation and insulin resistance in obesity. However, the factors in the adipose tissue microenvironment that control ATM phenotypic polarization and bioenergetics remain unknown. We have recently shown that oxidized phospholipids (OxPL) uniquely regulate gene expression and cellular metabolism in Mox macrophages, but the presence of the Mox phenotype in adipose tissue has not been reported. Here we show, using extracellular flux analysis, that ATMs isolated from lean mice are metabolically inhibited. We identify a unique population of CX3CR1neg/F4/80low ATMs that resemble the Mox (Txnrd1+HO1+) phenotype to be the predominant ATM phenotype in lean adipose tissue. In contrast, ATMs isolated from obese mice had characteristics typical of the M1/M2 (CD11c+CD206+) phenotype with highly activated bioenergetics. Quantifying individual OxPL species in the stromal vascular fraction of murine adipose tissue, using targeted liquid chromatography-mass spectrometry, revealed that high fat diet-induced adipose tissue expansion led to a disproportional increase in full-length over truncated OxPL species. In vitro studies showed that macrophages respond to truncated OxPL species by suppressing bioenergetics and up-regulating antioxidant programs, mimicking the Mox phenotype of ATMs isolated from lean mice. Conversely, full-length OxPL species induce proinflammatory gene expression and an activated bioenergetic profile that mimics ATMs isolated from obese mice. Together, these data identify a redox-regulatory Mox macrophage phenotype to be predominant in lean adipose tissue and demonstrate that individual OxPL species that accumulate in adipose tissue instruct ATMs to adapt their phenotype and bioenergetic profile to either maintain redox homeostasis or to promote inflammation.

In adipose tissues of obese mice, macrophages accumulate in crown-like structures (CLS) formed around dying adipocytes. The CLS macrophages are thought to clear the dead adipocytes by exophagy, which involves exocytosis of lysosomes and digestion of apoptotic adipocytes. After digesting the plasma membrane and other cytosolic contents of the adipocyte, CLS macrophages come in contact with the large lipid droplet, however, it is unknown how macrophages response to such acellular adipocytes. We hypothesized that the acelular adipocytes in CLSs promote inflammation and metabolically activate the macrophages to promote clearance of the lipid. To mimic the in vivo scenario, we exposed cultured murine bone marrow-derived macrophages to lipid droplets isolated from the adipocytes of high-fat diet-induced obese C57BL/6 mice. In response to adipocyte lipid, macrophages accumulated lipid droplets, which were similar in size and number to lipid droplets of CLS macrophages in vivo . Acellular lipid exposure significantly increased TNF-α gene expression, which was suppressed by the CD36 inhibitor sulfo-N-succinimidyl oleate, supporting CD36-mediated inflammatory response. Moreover, lipid exposure significantly lowered metabolic activity of macrophages and impaired clearance of apoptotic bodies from 3T3-L1 adipocytes. Using specific inhibitors, unexpectedly we found that lipid droplet accumulation in macrophages was independent of CD36 activity or processes involved in exophagy such as exocytosis of lysosomes, extracellular lipase activity, lipolysis and phagocytosis. Interestingly, lipid droplet accumulation was dependent on acetyl-CoA carboxylase (ACC) as determined by use of ACC inhibitors soraphen A and TOFA, and siRNA knock-down of ACC in macrophages. Furthermore, confocal microscopy of whole mount adipose tissue revealed expression of ACC in CLS macrophages. Altogether, using a novel in vitro model we demonstrate that acelular adipocytes suppress metabolic activity, but induce inflammation and de novo lipogenesis-mediated lipid droplet formation in macrophages.

Marginal zone and follicular B cells together constitute the B2 cell population, which is known to promote cardiovascular diseases by secretion of pathogenic antibodies. However, it is not completely understood how B2 cells are activated. Here, we tested the hypothesis that B cell activating factor (BAFF) activates B2 cells and promote abdominal aortic aneurysm (AAA) formation. Since BAFF can either exist as a 3mer or multimerize to a highly active 60mer, we further examined if the 60mer is critical for B2 cell-mediated pathogenicity. Anti-BAFF antibody (Ab) Sandy-2 was injected to C57BL/6 male mice at 1 mg/kg once in every 14 days. AAA was induced by topical elastase model after 14 days of Sandy-2 injection. Native PAGE and ELISA methods were used to determine binding of Abs to recombinant BAFF 3mer and 60mer. For in vitro experiments, B cells were isolated from murine spleens. Activation of B cells was examined by Western blotting and RNA sequencing and by surface expression of CD23 and MHC II by flow cytometry. Metabolic reprogramming of B cells by BAFF was determined by extracellular flux analysis using a Seahorse XF24 Flux Analyzer. Sandy-2 bound to both 3mer and 60mer, resulting in suppressed AAA formation (n=8, pIn vitro , the 60mer, but not the 3mer, significantly activated both NF-kB1 and -kB2 signaling, and induced expression of B2 cell activation markers and anti-apoptotic genes in B cells. Inhibitors of NF-kB signaling decreased B cell activation in response to 60mer. The 60mer treatment significantly increased mitochondrial respiration and glycolysis in B cells, supporting an activated status. An antibody against multimerization site of BAFF (anti-multiBAFF) significantly suppressed B cell activation relative to a control Ab, in a neutrophil:B cell co-culture model. The effect of the anti-multiBAFF Ab on AAA formation is currently being tested in our laboratory. Altogether, our results suggest a critical role for BAFF 60mer in skewing B cells to an activated B2 cell phenotype, supporting a pathogenic role of B2 cells in AAA.

Objective Defective glucose uptake in adipocytes leads to impaired metabolic homeostasis and insulin resistance, hallmarks of type 2 diabetes. Extracellular ATP-derived nucleotides and nucleosides are important regulators of adipocyte function, but the pathway for controlled ATP release from adipocytes is unknown. Here, we investigated whether Pannexin 1 (Panx1) channels control ATP release from adipocytes and contribute to metabolic homeostasis. Methods We assessed Panx1 functionality in cultured 3T3-L1 adipocytes and in adipocytes isolated from murine white adipose tissue by measuring ATP release in response to known activators of Panx1 channels. Glucose uptake in cultured 3T3-L1 adipocytes was measured in the presence of Panx1 pharmacologic inhibitors and in adipocytes isolated from white adipose tissue from wildtype (WT) or adipocyte-specific Panx1 knockout (AdipPanx1 KO) mice generated in our laboratory. We performed in vivo glucose uptake studies in chow fed WT and AdipPanx1 KO mice and assessed insulin resistance in WT and AdipPanx1 KO mice fed a high fat diet for 12 weeks. Panx1 channel function was assessed in response to insulin by performing electrophysiologic recordings in a heterologous expression system. Finally, we measured Panx1 mRNA in human visceral adipose tissue samples by qRT-PCR and compared expression levels with glucose levels and HOMA-IR measurements in patients. Results Our data show that adipocytes express functional Pannexin 1 (Panx1) channels that can be activated to release ATP. Pharmacologic inhibition or selective genetic deletion of Panx1 from adipocytes decreased insulin-induced glucose uptake in vitro and in vivo and exacerbated diet-induced insulin resistance in mice. Further, we identify insulin as a novel activator of Panx1 channels. In obese humans Panx1 expression in adipose tissue is increased and correlates with the degree of insulin resistance. Conclusions We show that Panx1 channel activity regulates insulin-stimulated glucose uptake in adipocytes and thus contributes to control of metabolic homeostasis., Graphical abstract, Highlights • Adipocytes express Pannexin 1 channels that can be activated to release ATP. • Inhibition of Pannexin 1 decreased insulin-induced glucose uptake in adipocytes. • Adipocyte Pannexin 1 knockout mice are more insulin resistant on high fat diet. • We identify insulin as a novel activator of Pannexin 1 channels. • Pannexin 1 expression in human adipose tissue correlates with insulin resistance.

Objective— The impact of leukotriene production by the 5-lipoxygenase (5-LO) pathway in the pathophysiology of abdominal aortic aneurysms (AAAs) has been debated. Moreover, a clear mechanism through which 5-LO influences AAA remains unclear. Approach and Results— Aneurysm formation was attenuated in 5-LO –/– mice, and in lethally irradiated wild-type mice reconstituted with 5-LO –/– bone marrow in an elastase perfusion model. Pharmacological inhibition of 5-LO–attenuated aneurysm formation in both aortic elastase perfused wild-type and angiotensin II–treated LDLr –/– (low-density lipoprotein receptor) mice, with resultant preservation of elastin and fewer 5-LO and MMP9 (matrix metalloproteinase)-producing cells. Separately, analysis of wild-type mice 7 days after elastase perfusion showed that 5-LO inhibition was associated with reduced polymorphonuclear leukocyte infiltration to the aortic wall. Importantly, 5-LO inhibition initiated 3 days after elastase perfusion in wild-type mice arrested progression of small AAA. Human AAA and control aorta corroborated these elastin and 5-LO expression patterns. Conclusions— Inhibition of 5-LO by pharmacological or genetic approaches attenuates aneurysm formation and prevents fragmentation of the medial layer in 2 unique AAA models. Administration of 5-LO inhibitor in small AAA slows progression of AAA. Targeted interruption of the 5-LO pathway is a potential treatment strategy in AAA.

B cell activating factor (BAFF) regulates differentiation and survival of B cells by binding to the surface receptors BAFF receptor (BR3), transmembrane activator and CAML interactor (TACI) and B cell maturation antigen (BCMA). During differentiation, intracellular metabolic reprogramming is crucial, such as, naïve B cells are metabolically quiescent, whereas, antibody producing plasma cells are metabolically active. We have reported that depletion of B cells protects mice from abdominal aortic aneurysm (AA), however it is not clear how B cells promote AA growth. BAFF exists as a 3mer (binds only to BR3) or as a 60mer (binds to BR3, TACI and BCMA). Therefore, we hypothesize that BAFF multimerization regulates the immune and metabolic phenotype of B cells by binding to BAFF receptors and modulate AA growth. Immunohistology was performed on AA tissues collected from patients undergoing open AA repair. Experimental AA was induced by elastase perfusion of abdominal aorta or angiotensin II infusion (1000 ng/kg/min) method in 8 weeks old male C57BL/6 or apolipoprotein E knockout mice, respectively. Western blotting, flow cytometry and Seahorse extracellular flux assays were used to determine immune and metabolic changes in B cells in response to recombinant BAFF 3mer and 60mer. BR3+ B cells were detected in the milieu of BAFF in human AAs. Mouse AAs demonstrated significant infiltration (>50/section) of CD138+ plasma B cells, but few (4-10/section) CD20+ B cells. In vitro, BAFF 3mer induced canonical NF-kB, whereas, 60mer induced both canonical and non-canonical NF-kB signaling. Moreover, the 3mer significantly decreased mitochondrial density, oxygen consumption rate, and surface expression of IgD and IgM indicating a metabolically quiescent state of B cells. However, these parameters were significantly increased by the 60mer similar to plasma cells. Anti-BR3 IgG1, but not a control IgG1 antibody decreased BAFF 60mer-induced oxygen consumption rate by 50%. In a pilot study (n=10/group), anti-BR3 IgG1, but not the control IgG1 aggravated angiotensin II-induced AA growth. Altogether, our results suggest that BAFF 3mer and 60mer oppositely regulate immune and metabolic phenotype of B cells and inhibition of BAFF-BR3 signaling is detrimental for AA growth.

Objectives: Abdominal aortic aneurysm (AAA) rupture (RAAA) is associated with high mortality. Intraluminal thrombus and associated inflammation have been implicated in RAAA. We sought to characterize neutrophil extracellular trap (NET) formation, or NETosis, associated with a rat RAAA model that we previously developed. Methods: To stimulate AAA formation, infrarenal abdominal aortas of male SD rats were infused with porcine pancreatic elastase (PPE): active (AAA, n=21) and heat-inactivated (control, n=14). Aortas were harvested 3, 6, and 14d post PPE exposure. To stimulate RAAA, a separate group of animals was administered β-aminopropionitrile (BAPN, n=12) daily. Those animals that did not rupture (NRAAA-14d, n=6) were harvested 14d post PPE exposure. Paraffin embedded sections were analyzed using immunofluorescence. To evaluate evidence of primarily neutrophil activity and NET generation, myeloperoxidase (MPO) and peptidylarginine deiminase 4 (PAD4) were quantified utilizing Cy3 and Alexa-488 secondary antibodies, respectively. To determine the presence of NETs, citrullinated histone H3 (H3Cit, Cy3) was visualized. DAPI was utilized to detect nuclei. Aortic sections were imaged with fluorescent microscopy, and quantification of percent area stained (PAS) was performed by threshold analysis. Results: Greater MPO PAS was observed for AAA-6d compared with control-6d (p=0.03). Greater PAD4 PAS was observed for AAA-3d (p=0.02) and -6d (p=0.05) compared with control-3d and -6d, respectively. Greater H3Cit PAS was observed for AAA-6d (p Conclusions: Increased PAD4 expression by AAAs compared with controls at 3 and 6d post PPE exposure, results in increased H3Cit expression at 6 and 14d post PPE exposure. Comparable H3Cit expression by AAA-6d and RAAA demonstrates the acute nature of this rupture model; however, greater H3Cit expression by RAAAs suggests a role for NETosis in RAAA. Further evaluation of NETosis as it pertains to human RAAA is necessary. Therapeutic inhibition of NETosis may slow AAA progression and prevent RAAA.

Our group has previously shown that oxidized phospholipids (OxPL) induce a unique macrophage (MΦ) phenotype known as “Mox,” distinct from pro- and anti-inflammatory M1 and M2 phenotypes. We have also shown that Mox make up 30% of the MΦs found in atherosclerotic lesions. OxPL induce TLR2-dependent inflammatory gene expression in MΦs. TLR2 activation by peptidoglycan was shown to induce accumulation of ceramides, which can alter bioenergetics by inhibiting the mitochondria. Recently, it has been shown that spleen tyrosine kinase (Syk) is phosphorylated upon TLR2 agonist Pam3CSK stimulation in MΦs. The effect of OxPL on bioenergetics has never before been studied. Here we test the hypothesis that OxPL change MΦ bioenergetics and inflammatory capacity via a TLR2-Syk-Ceramide pathway. Using flow cytometry, we found that in mice fed a high-fat diet, more than 20% of all adipose tissue MΦs can be described as Mox. Concomitantly, using immunohistochemistry and liquid-chromatography mass spectrometry, we measured increased levels of OxPL in the stromal vascular fraction of obese murine adipose tissue, as compared to lean controls. We treated bone marrow-derived MΦs (BMDMs) from WT, TLR2-KO, and Syk-KO mice with OxPL and measured bioenergetics using a Seahorse Flux Analyzer. Our results show that OxPL decrease oxygen consumption rate (OCR), a measure of oxidative phosphorylation, and decrease extracellular acidification rate (ECAR), a measure of glycolytic capacity. These MΦs can be described as quiescent, but they still engage in low-level cytokine production. Moreover, OxPL result in the accumulation of ceramides, as quantified by mass spectrometry. Finally, using Syk-KO MΦs and Syk inhibitors, we show that OxPL-induced inflammatory gene expression and ceramide accumulation are dependent on Syk. In summary, our results demonstrate that OxPL accumulate in obese adipose tissue and induce a change in the inflammatory and metabolic profiles of MΦs, involving a TLR2-ceramide-Syk dependent pathway. These results suggest that OxPL are triggers of adipose tissue inflammation and subsequent development of insulin resistance. Furthermore, we identify Syk as a therapeutic target for inhibiting diet-induced adipose tissue inflammation and insulin resistance.

Objective— B-cell depletion therapy is widely used for treatment of cancers and autoimmune diseases. B cells are abundant in abdominal aortic aneurysms (AAA); however, it is unknown whether B-cell depletion therapy affects AAA growth. Using experimental models of murine AAA, we aim to examine the effect of B-cell depletion on AAA formation. Approach and Results— Wild-type or apolipoprotein E–knockout mice were treated with mouse monoclonal anti-CD20 or control antibodies and subjected to an elastase perfusion or angiotensin II infusion model to induce AAA, respectively. Anti-CD20 antibody treatment significantly depleted B1 and B2 cells, and strikingly suppressed AAA growth in both models. B-cell depletion resulted in lower circulating IgM levels, but did not affect the levels of IgG or cytokine/chemokine levels. Although the total number of leukocyte remained unchanged in elastase-perfused aortas after anti-CD20 antibody treatment, the number of B-cell subtypes was significantly lower. Interestingly, plasmacytoid dendritic cells expressing the immunomodulatory enzyme indole 2,3-dioxygenase were detected in the aortas of B-cell–depleted mice. In accordance with an increase in indole 2,3-dioxygenase+ plasmacytoid dendritic cells, the number of regulatory T cells was higher, whereas the expression of proinflammatory genes was lower in aortas of B-cell–depleted mice. In a coculture model, the presence of B cells significantly lowered the number of indole 2,3-dioxygenase+ plasmacytoid dendritic cells without affecting total plasmacytoid dendritic cell number. Conclusions— The present results demonstrate that B-cell depletion protects mice from experimental AAA formation and promotes emergence of an immunosuppressive environment in aorta.

Activation of the transcription factor NF-E2-related factor 2 (Nrf2) by oxidative stress induces the expression of a variety of antioxidant and anti-inflammatory genes. Yet, genetic ablation of Nrf2 was shown to protect mice from high-fat diet (HFD)-induced obesity and insulin resistance. The mechanisms that underlie this seemingly paradoxical finding remain largely unexplored. Here we examined whether Nrf2 deficiency in myeloid cells contributes to protection against HFD-induced metabolic changes by decreasing adipose tissue inflammation. In vitro, induction of IL-1β by inflammatory stimuli was significantly reduced in Nrf2-deficient macrophages. Whereas inflammatory gene expression in the stromal vascular fraction was reduced in both global and chimeric Nrf2 KO mice, only global Nrf2-deficient, and not bone marrow-transplanted Nrf2 chimeric, mice were protected against HFD-induced adipose tissue inflammation. Whereas global Nrf2 deficiency resulted in significantly decreased expression of inflammatory genes and PPARγ2, there was no difference when Nrf2 was absent only from myeloid cells. In vitro coculture with adipocytes demonstrated that macrophage Nrf2 regulated inflammatory gene expression in macrophages; however, it was not required to induce inflammatory gene expression in adipocytes. Finally, in contrast to global Nrf2 knockout, Nrf2 deficiency in myeloid cells did not protect against HFD-induced insulin resistance. Together, our data demonstrate a dominant role for nonmyeloid Nrf2 in controlling HFD-induced adipose tissue inflammation and the development of insulin resistance.

Large-volume protein crystals are a prerequisite for neutron diffraction studies and their production represents a bottleneck in obtaining neutron structures. Many protein crystals that permit the collection of high-resolution X-ray diffraction data are inappropriate for neutron diffraction owing to a plate-type morphology that limits the crystal volume. Human fibroblast growth factor 1 crystallizes in a plate morphology that yields atomic resolution X-ray diffraction data but has insufficient volume for neutron diffraction. The thin physical dimension has been identified as corresponding to the b cell edge and the X-ray structure identified a solvent-mediated crystal contact adjacent to position Glu81 that was hypothesized to limit efficient crystal growth in this dimension. In this report, a series of mutations at this crystal contact designed to both reduce side-chain entropy and replace the solvent-mediated interface with direct side-chain contacts are reported. The results suggest that improved crystal growth is achieved upon the introduction of direct crystal contacts, while little improvement is observed with side-chain entropy-reducing mutations alone.

Testosterone is theorized to play a major role in the pathophysiology of abdominal aortic aneurysms (AAAs) because this disease occurs primarily in men. The role of the androgen receptor (AR) in the formation of AAAs has not been well elucidated, and therefore, it is hypothesized that androgen blockade will attenuate experimental aortic aneurysm formation.Aortas of 8- to 12-week-old male C57Bl/6 wild-type (WT) mice or male AR knockout (AR(-/-)) mice were perfused with purified porcine pancreatic elastase (0.35 U/mL) to induce AAA formation. Two groups of WT male mice were treated with the AR blockers flutamide (50 mg/kg) or ketoconazole (150 mg/kg) twice daily by intraperitoneal injection. Aortas were harvested on day 14 after video micrometry was used to measure AAA diameter. Cytokine arrays and histologic analysis were performed on aortic tissue. Groups were compared using an analysis of variance and a Tukey post hoc test.Flutamide and ketoconazole treatment (mean ± standard error of the mean) attenuated AAA formation in WT mice (84.2% ± 22.8% [P = .009] and 91.5% ± 18.2% [P = .037]) compared with WT elastase (121% ± 5.23%). In addition, AR(-/-) mice showed attenuation of AAA growth (64.4% ± 22.7%; P .0001) compared with WT elastase. Cytokine arrays of aortic tissue revealed decreased levels of proinflammatory cytokines interleukin (IL)-α, IL-6, and IL-17 in flutamide-treated and AR(-/-) groups compared with controls.Pharmacologic and genetic AR blockade cause attenuation of AAA formation. Therapies for AR blockade used in prostate cancer may provide medical treatment to halt progression of AAAs in humans.

We thank Dr Usui and colleagues for their insightful comments on the upstream mechanism of interleukin-1β (IL-1β) production in aortic aneurysms. As mentioned, we have demonstrated that IL-1β is key in aortic aneurysm formation and progression.1 IL-1β binds to its receptor to incite a host of inflammatory cytokines. Inhibition of IL-1β by genetic deletion or pharmacological inhibition reduces experimental aortic aneurysm size and halts small aneurysm growth. Because IL-1β triggers multiple inflammatory cytokines and pathways, targeting downstream mediators may have limited effect. Therefore, upstream triggers of IL-1β release must be identified for further potential treatment …

Background— Thoracic aortic aneurysms (TAAs) are common, but experimental TAA models are limited and the role of interleukin-1β (IL-1β) is undetermined. Methods and Results— IL-1β protein was measured in human TAAs and control aortas, and IL-1β protein was increased ≈20-fold in human TAAs. To develop an experimental model of TAAs, 8- to10-week-old male C57Bl/6 mice (wild type [WT]) underwent thoracotomy with application of periadventitial elastase (WT TAA) or saline (WT control; n=30 per group). Elastase treatment to thoracic aortas resulted in progressive dilation until day 14 with maximal dilation of 99.6±24.7% compared with 14.4±8.2% for WT saline control ( P P P P =0.01). Conclusions— Periadventitial application of elastase to murine thoracic aortas reproducibly produced aneurysms with molecular and histological features consistent with TAA disease. Genetic and pharmacological inhibition of IL-1β decreased TAA formation and progression, indicating that IL-1β may be a potential target for TAA treatment.

Objective: We have previously shown that circulating neutrophils are required for experimental aortic aneurysm (AA) formation via a non-MMP2/9-mediated mechanism. In other experiments we documented that IL-1β signaling is required for AA formation. Therefore, we hypothesized that increased levels of IL-1β in AA triggers inflammatory ceramide synthesis and thus extracellular trap (NET) formation (NETosis) by neutrophils, thereby augmenting inflammation. Methods: Immunohistochemical techniques were used to identify neutrophils and NETs. Ceramides from human AA and control aortas from transplant donors were quantified using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Neutrophils isolated from human blood were treated with IL-1β to determine if IL-1β induces ceramide synthesis and NETosis in vitro. Experimental AAs were induced by elastase perfusion in C57BL/6 wild-type (WT) and IL-1β knock-out (KO) mice and cellular content of AA was analyzed using flow cytometry. Groups were compared using two-tailed unpaired t-test. Results: Immunohistochemical staining showed that neutrophils were scattered or grouped near lymphoid-like structures in the adventitia of human AA. Three-dimensional confocal imaging showed NETs in the adventitia of human AA. Importantly, the levels of long chain ceramides C16 and C24:1 were higher (2.6 and 1.8-fold, respectively), whereas, the levels of short chain ceramides C4, C6 and C8 were lower (4.3, 3.9 and 3.5-fold, respectively) in human AA compared to controls. In isolated neutrophils, IL-1β treatment induced synthesis of long chain ceramides and discharge of NETs. Neutrophils and NETs were also detected in aortic media at day 7 after induction of AA in WT mice. On day 7 following elastase perfusion, despite the increase in number of circulating immune cells, AA size was smaller (91.08±11.7% vs 53.8±5.4%, p=0.03), and, infiltration of neutrophils (12179±2697 vs 3046±628, p=0.01) and B cells was lower in the aorta of IL-1β KO mice compared to WT mice. Conclusions: These data document for the first time the presence of NETs in human AAs. Furthermore, the data suggests that neutrophils augment inflammation in AA by increasing pro-inflammatory long chain ceramide synthesis and NETosis in response to IL-1β.

Objective: Recent reports have documented abdominal aortic aneurysm (AA) rupture in patients receiving B cell depletion therapy. This highlights the importance of understanding the role of B cells (B1 and B2 subtypes) in the development of AA. We hypothesized that B2 cells are abundant in AA and aggravate experimental aneurysm formation. Methods: AAs were induced in WT (C57BL/6) mice by elastase perfusion of the abdominal aorta and harvested at 14 days. Aortic B1 and B2 cells were quantified, and, intracellular cytokine synthesis was determined using flow cytometry. Next, AAs were induced in B cell deficient (muMT) mice using either elastase perfusion or topical elastase application to determine the impact of loss of B cells in AA. Finally, adoptive transfer experiments were performed on muMT mice by tail vein injection of 25x10^6 isolated splenic B2 cells or saline. Regulatory T cell (Treg) population was identified as CD4+ T cell expressing Foxp3 (forkhead box P3). Results: B cells appeared in abdominal aortas at day 7 after elastase perfusion in WT mice and persisted at least till day 21. Compared to saline perfused or normal aortas, elastase perfused aortas had greater infiltration of mononuclear cells including B cells (3631±578 in elastase vs 1476±524 in saline, p=0.02) and T cells. B2 cells constituted 93% of total B cells in AA. Furthermore, B cells isolated from elastase and saline perfused aortas displayed high, but similar levels of intracellular IL-6 and IL-10 expression. Interestingly, in both experimental models of AA, muMT mice were prone to AA formation similar to WT mice as evidenced by elastin degradation or infiltration of T cells, neutrophils and macrophages. Contradicting our hypothesis, adoptive transfer of B2 cells suppressed AA formation (102.0±7.3% vs 75.2±5.5%, p=0.01) with concomitant increase in Treg cells in spleen (0.24±0.03% vs 0.92± 0.23%, p=0.02) and aorta (0.37±0.09% vs 1.09±0.38%, p=0.08), and decrease in aortic infiltration of mononuclear cells. Conclusions: Our data suggest that B2 cells constitute the largest population of B cells in experimental AA and participate in protecting aorta from aneurysm formation at least in part by increasing the Treg cell population.

Several mutations at Glu81 located on the crystal contact of human acidic fibroblast growth factor were studied in an effort to improve crystal growth. Mutation to Ser and Thr resulted in crystallization of a rather bulky form of the wild type, whereas mutation to Val prohibited crystallization. These results suggest that crystal growth may be controlled by designing a new interface by protein engineering., An attempt has been made to improve a crystal contact of human acidic fibroblast growth factor (haFGF; 140 amino acids) to control the crystal growth, because haFGF crystallizes only as a thin-plate form, yielding crystals suitable for X-ray but not neutron diffraction. X-ray crystal analysis of haFGF showed that the Glu81 side chain, located at a crystal contact between haFGF molecules, is in close proximity with an identical residue related by crystallographic symmetry, suggesting that charge repulsion may disrupt suitable crystal-packing interactions. To investigate whether the Glu residue affects the crystal-packing interactions, haFGF mutants in which Glu81 was replaced by Ala, Val, Leu, Ser and Thr were constructed. Although crystals of the Ala and Leu mutants were grown as a thin-plate form by the same precipitant (formate) as the wild type, crystals of the Ser and Thr mutants were grown with increased thickness, yielding a larger overall crystal volume. X-ray structural analysis of the Ser mutant determined at 1.35 Å resolution revealed that the hydroxy groups of Ser are linked by hydrogen bonds mediated by the formate used as a precipitant. This approach to engineering crystal contacts may contribute to the development of large protein crystals for neutron crystallography.

Activation of the adenosine 2A receptor (A2AR) reduces inflammation in models of acute injury but contribution in development of chronic abdominal aortic aneurysms (AAAs) is unknown. Elastase perfusion to induce AAA formation in A2AR-knockout (A2ARKO) and C57BL6/J wild-type (WT) mice resulted in nearly 100% larger aneurysms in A2ARKO compared to WT at d 14 (P

B lymphocytes are generally considered to be activators of the immune response; however, recent findings have shown that a subtype of B lymphocytes, regulatory B lymphocytes, play a role in attenuating the immune response. Bronchiolitis obliterans remains the major limitation to modern-day lung transplantation. The role of regulatory B lymphocytes in bronchiolitis obliterans has not been elucidated. We hypothesized that regulatory B lymphocytes play a role in the attenuation of bronchiolitis obliterans.We performed a standard heterotopic tracheal transplant model. Tracheas from Balb/c mice were transplanted into C57BL/6 recipients. Rapamycin treatment and dimethyl sulfoxide control groups were each treated for the first 14 days after the transplant. Tracheas were collected on days 7, 14, and 28 post-transplantation. Luminal obliteration was evaluated by hematoxylin-eosin staining and Picrosirius red staining. Immune cell infiltration and characteristics, and secretion of interleukin-10 and transforming growth factor-β1 were accessed by immunohistochemistry. Cytokines and transforming growth factor-β1 were measured using the Luminex assay (Bio-Rad, Hercules, Calif).The results revealed that intraperitoneal injection of rapamycin for 14 days after tracheal transplantation significantly reduced luminal obliteration on day 28 when compared with the dimethyl sulfoxide control group (97.78% ± 3.63% vs 3.02% ± 2.14%, P.001). Rapamycin treatment markedly induced regulatory B lymphocytes (B220(+)IgM(+)IgG(-)IL-10(+)TGF-β1(+)) cells when compared with dimethyl sulfoxide controls. Rapamycin treatment inhibited interleukin-1β, 6, 13, and 17 on days 7 and 14. Rapamycin also greatly increased interleukin-10 and transforming growth factor-β1 production in B cells and regulatory T lymphocytes infiltration on day 28.Mammalian target of rapamycin inhibition decreases the development of bronchiolitis obliterans via inhibition of proinflammatory cytokines and increasing regulatory B lymphocytes cell infiltration, which subsequently produces anti-inflammatory cytokines and upregulates regulatory T lymphocyte cells.

Oxidative tissue damage is a hallmark of many chronic inflammatory diseases. However, the precise mechanisms linking oxidative changes to inflammatory reactions remain unclear. Herein we show that Toll-like receptor 2 (TLR2) translates oxidative tissue damage into inflammatory responses by mediating the effects of oxidized phospholipids. Intraperitoneal injection of oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycerophosphorylcholine (OxPAPC) resulted in upregulation of inflammatory genes in wild-type, but not in TLR2(-/-) mice. In vitro, OxPAPC induced TLR2 (but not TLR4)-dependent inflammatory gene expression and JNK and p38 signaling in macrophages. Induction of TLR2-dependent gene expression required reducible functional groups on sn-2 acyl chains of oxidized phospholipids, as well as serum cofactors. Finally, TLR2(-/-) mice were protected against carbon tetrachloride-induced oxidative tissue damage and inflammation, which was accompanied by accumulation of oxidized phospholipids in livers. Together, our findings demonstrate that TLR2 mediates cellular responses to oxidative tissue damage and they provide new insights into how oxidative stress is linked to acute and chronic inflammation.

Rationale : Plasminogen activator inhibitor-1 (PAI-1) is a biomarker for several vascular disease states; however, its target of action within the vessel wall is undefined. Objective : Determine the ability of PAI-1 to regulate myoendothelial junction (MEJ) formation. Methods and Results : MEJs are found throughout the vasculature linking endothelial cells (ECs) and vascular smooth muscle cells. Using a vascular cell coculture we isolated MEJ fractions and performed two-dimensional differential gel electrophoresis. Mass spectrometry identified PAI-1 as being enriched within MEJ fractions, which we confirmed in vivo. In the vascular cell coculture, recombinant PAI-1 added to the EC monolayer significantly increased MEJs. Conversely, addition of a PAI-1 monoclonal antibody to the EC monolayer reduced the number of MEJs. This was also observed in vivo where mice fed a high fat diet had increased PAI-1 and MEJs and the number of MEJs in coronary arterioles of PAI-1 −/− mice was significantly reduced when compared to C57Bl/6 mice. The presence of MEJs in PAI-1 −/− coronary arterioles was restored when their hearts were transplanted into and exposed to the circulation of C57Bl/6 mice. Application of biotin-conjugated PAI-1 to the EC monolayer in vitro confirmed the ability of luminal PAI-1 to translocate to the MEJ. Functionally, phenylephrine-induced heterocellular calcium communication in the vascular cell coculture was temporally enhanced when recombinant PAI-1 was present, and prolonged when PAI-1 was absent. Conclusion : Our data implicate circulating PAI-1 as a key regulator of MEJ formation and a potential target for pharmacological intervention in diseases with vascular abnormalities (eg, diabetes mellitus).

ESAT-6 and CFP-10 form a 1:1 heterodimeric complex which contributes to the virulence of Mycobacterium tuberculosis H37Rv. Based on the structure of CFP-10-ESAT-6 complex, we have selected four point mutations each of CFP-10 and ESAT-6 and have analyzed complex formation for the 25 possible combinations between wild-type and mutant CFP-10 and ESAT-6 proteins. We observed that the mutations L25R or F58R of CFP-10 and L29D or L65D of ESAT-6 lead to disruption of complex formation. We have evaluated the immunogenic responses of the wild-type and mutant CFP-10 and ESAT-6 proteins, the wild-type CFP-10-ESAT-6 complex, six complex-forming and two non-complex-forming combinations of wild-type/mutant CFP-10 and ESAT-6 proteins. CFP-10 mutants I21R, L25R, and W43R were found to have better immunogenic potential than wt-CFP-10, while none of the ESAT-6 mutants were better than wt-ESAT-6. Very interestingly, we have discovered that the non-complex-forming mixture of CFP-10-I21R and ESAT-6-L29D gives a strong immunogenic response.

The 6-kDa early secretory antigenic target (ESAT-6) and culture filtrate protein-10 (CFP-10), expressed from the region of deletion-1 (RD1) of Mycobacterium tuberculosis H37Rv, are known to play a key role in virulence. In this study, we explored the thermodynamic and biochemical changes associated with the formation of the 1 : 1 heterodimeric complex between ESAT-6 and CFP-10. Using isothermal titration calorimetry (ITC), we precisely determined the association constant and free energy change for formation of the complex to be 2 x 10(7) M(-1) and -9.95 kcal.mol(-1), respectively. Strikingly, the thermal unfolding of the ESAT-6-CFP-10 heterodimeric complex was completely reversible, with a T(m) of 53.4 degrees C and DeltaH of 69 kcal.mol(-1). Mixing of ESAT-6 and CFP-10 at any temperature below the T(m) of the complex led to induction of helical conformation, suggesting molecular recognition between specific segments of unfolded ESAT-6 and CFP-10. Enhanced biochemical stability of the complex was indicated by protection of ESAT-6 and an N-terminal fragment of CFP-10 from proteolysis with trypsin. However, the flexible C-terminal of CFP-10 in the complex, which has been shown to be responsible for binding to macrophages and monocytes, was cleaved by trypsin. In the presence of phospholipid membranes, ESAT-6, but not CFP-10 and the complex, showed an increase in alpha-helical content and enhanced thermal stability. Overall, complex formation resulted in structural changes, enhanced thermodynamic and biochemical stability, and loss of binding to phospholipid membranes. These features of complex formation probably determine the physiological role of ESAT-6, CFP-10 and/or the complex in vivo. The ITC and thermal unfolding approach described in this study can readily be applied to characterization of the 11 other pairs of ESAT-6 family proteins and for screening ESAT-6 and CFP-10 mutants.

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Recent reports of rupture in patients with abdominal aortic aneurysm (AAA) receiving B-cell depletion therapy highlight the importance of understanding the role of B cells (B1 and B2 subsets) in the development of AAA. We hypothesized that B2 cells aggravate experimental aneurysm formation. The IHC staining revealed infiltration of B cells in the aorta of wild-type (C57BL/6) mice at day 7 after elastase perfusion and persisted through day 21. Quantification of immune cell types using flow cytometry at day 14 showed significantly greater infiltration of mononuclear cells, including B cells (B2: 93% of total B cells) and T cells in elastase-perfused aortas compared with saline-perfused or normal aortas. muMT (mature B-cell deficient) mice were prone to AAA formation similar to wild-type mice in two different experimental AAA models. Contradicting our hypothesis, adoptive transfer of B2 cells suppressed AAA formation (102.0% ± 7.3% versus 75.2% ± 5.5%; P