Functions of Platelet Microparticles in Inflammatory Arthritis

Abstract Abstract SCI-35 Besides their pivotal role in thrombosis and wound repair, platelets can participate in inflammatory responses via a broad arsenal that includes CD40L and CD40, cyclooxygenase-1 mediated prostaglandins, leukotrienes, IL-1, RANTES, serotonin, platelet factor 4, matrix metalloproteinases -2 and -9, P-selectin, adenosine diphosphate, and reactive oxygen species. Rheumatoid arthritis (RA) is amongst the most common autoimmune chronic inflammation that affects the joints. Interestingly, we found copious amounts of submicron particles that harbor platelet integrins in the synovial fluid of patients suffering from RA, pointing to evidence of platelet activation in this inflammatory autoimmune disease. Importantly, we identified the collagen receptor glycoprotein VI as a key trigger for platelet microparticle generation in arthritis pathophysiology. In addition to the transcellular collaboration between platelets and fibroblast-like synoviocytes in generation of pro-inflammatory prostacyclin (1), we found that platelets contribute to synovitis by production of IL-1-rich platelet microparticles (2). These recent advances in understanding of the platelet activities in inflammation notwithstanding, the mechanisms by which platelet microparticles invade the diseased joint in RA remain obscure. Using synovial biopsies from patients suffering from RA in addition to in vivo imaging strategies in a murine model of arthritis, our current work aspires to dissect the means of transportation of platelet microparticles during RA. Given their pro-inflammatory properties, to understand the process by which microparticles invade the synovial joint is of great clinical interest. Disclosures: No relevant conflicts of interest to declare.

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Silencing of junctional adhesion molecule-like protein attenuates atherogenesis and enhances plaque stability in ApoE−/− mice

Clinical Science ◽

10.1042/cs20180561 ◽

2019 ◽

Vol 133 (11) ◽

pp. 1215-1228 ◽

Cited By ~ 1

Author(s):

Yu Sun ◽

Juan Guan ◽

Yunfeng Hou ◽

Fei Xue ◽

Wei Huang ◽

...

Keyword(s):

Adhesion Molecule ◽

Wound Repair ◽

Inflammatory Responses ◽

Atherosclerotic Lesion ◽

Atherosclerotic Plaques ◽

Plaque Stability ◽

Fibrous Cap ◽

Enhanced Expression

Abstract Background: Although junctional adhesion molecule-like protein (JAML) has recently been implicated in leukocyte recruitment during inflammation and wound repair, its role in atherosclerosis remains to be elucidated. Methods and results: First, we showed that JAML was strongly expressed in atherosclerotic plaques of cardiovascular patients. Similar results were obtained with atherosclerotic plaques of ApoE−/− mice. Co-immunofluorescence staining showed that JAML was mainly expressed in macrophages. Enhanced expression of JAML in cultured macrophages was observed following exposure of the cells to oxLDL. The functional role of JAML in atherosclerosis and macrophages function was assessed by interference of JAML with shRNA in vivo and siRNA in vitro. Silencing of JAML in mice significantly attenuated atherosclerotic lesion formation, reduced necrotic core area, increased plaque fibrous cap thickness, decreased macrophages content and inflammation. In addition, histological staining showed that JAML deficiency promoted plaques to stable phenotype. In vitro, JAML siRNA treatment lowered the expression of inflammatory cytokines in macrophages treated with oxLDL. The mechanism by which JAML mediated the inflammatory responses may be related to the ERK/NF-κB activation. Conclusions: Our results demonstrated that therapeutic drugs which antagonize the function of JAML may be a potentially effective approach to attenuate atherogenesis and enhance plaque stability.

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Histones, Like Platelet Factor 4 (PF4), Affect Generation of Activated Protein C: Implications for the Pathogenesis of Severe Sepsis

Blood ◽

10.1182/blood.v118.21.530.530 ◽

2011 ◽

Vol 118 (21) ◽

pp. 530-530

Author(s):

M. Anna Kowalska ◽

Guohua Zhao ◽

George David ◽

Mortimer Poncz

Keyword(s):

Severe Sepsis ◽

Protein C ◽

Conflicts Of Interest ◽

Platelet Factor 4 ◽

Endothelial Protein C Receptor ◽

Maximal Increase ◽

Platelet Factor ◽

Positively Charged ◽

Formation Of Complexes

Abstract Abstract 530 Platelet factor 4 (PF4) increases aPC generation by the thrombin (IIa)/thrombomodulin (TM) complex and may impact outcome in sepsis. PF4's effect on aPC generation follows a biphasic curve when tested in solution, on human TM expressing HEK293, and on primary endothelial cells (ECs) with a peak concentration at around 25 μg/ml. Formation of complexes at a specific molar ratio between positively-charged tetramers of PF4 and negatively-charged chondroitin sulfate (CS) on the TM glycosaminoglycan (GAG) is crucial for the increase in aPC generation. Other positively-charged molecules like protamine sulfate (PRT) affect aPC generation in a similar manner, and heparin, which is known to bind PF4 and PRT more avidly than CS, lowers effective PF4 or PRT concentrations. Here we examined whether histones, that are also small positively-charged molecules, affect aPC generation. Histones released from cells in sepsis are cytotoxic toward ECs and lethal when injected into mice, and aPC reverses this lethality. May histones affect aPC generation by the same mechanism as other positively-charged molecules, and how does the presence of PF4 or heparin influence this effect? We have addressed these questions both in solution and with TM-expressing cells, in the absence or presence of endothelial protein C receptor. We found that individual, or mixed histones affect aPC formation following a similar biphasic curve seen with PF4 with a peak effect at around 10 μg/ml but to lesser extent (2-fold maximal increase compared to 6-fold for PF4). Histones and PF4 are additive at low concentrations; however, more importantly, histones only decreased aPC generation when tested in the presence of optimal or higher PF4 concentration (>25 μg/ml). Just as with PF4, added heparin decreased effective histone concentration and shifted the curve for aPC generation to the right, both in the absence or presence of PF4. We hypothesize that normally PF4 released from platelets augments aPC generation and low concentration of histones have similar effect. But when histones are released in sepsis in high concentrations, their interaction with CS on TM blocks formation of complexes between PF4 and TM's CS that are optimal for maximal increase of aPC generation. Further we tested the effect of histones on aPC generation in vivo. Injection of histones in mice increased IIa-induced (2U/kg) aPC generation in plasma. This increase was concentration dependent (at 1 to 20 mg/kg increasing aPC generation up to 10-fold), but injection of higher amount of histones (40 mg/kg) became lethal. Mice that were overexpressing human PF4 had an increased lethality when histones at 40 mg/kg were co-injected with thrombin (2U/kg) over the littermate mice deficient in murine PF4 (60% vs. 0% mortality, respectively, n=5 for each group) suggesting that in vivo histones may also act additively with PF4 on aPC generation. We propose that in severe septic patients, especially those with high levels of released PF4, concurrently available histones suppress aPC generation. By binding to the excess of PF4 and/or histones, heparin may be beneficial in severe sepsis by allowing improved aPC generation. Disclosures: No relevant conflicts of interest to declare.

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A Mouse Model for XLP-2 Disease Uncovers a Critical Function for IL-1beta and TNF in Driving Hyper-Inflammation

Blood ◽

10.1182/blood.v124.21.1403.1403 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1403-1403

Author(s):

Philipp J. Jost ◽

Monica Yabal ◽

Heiko Adler ◽

Nathalie Knies ◽

Christina Groß ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Lymphocytes ◽

Viral Infections ◽

Inflammatory Responses ◽

Conflicts Of Interest ◽

Human Peripheral Blood ◽

Critical Function ◽

Deficient Mice

Abstract The hyper-inflammatory syndrome X-linked lymphoproliferative syndrome type 2 (XLP-2) is defined by mutations in BIRC4 (XIAP). XLP-2 is often diagnosed in paediatric patients and is characterized by hyper-inflammation triggered by common viral infections. Symptoms include splenomegaly, HLH, fevers, and chronic haemorrhagic colitis among others. Recent work has also shown that mutations in BIRC4 predispose to the development of early-onset IBD, which is not necessarily associated with symptoms of systemic hyper-inflammation. Symptoms of XLP-2 are mostly attributed to the aberrant activation of macrophages and dendritic cells (DC) and the subsequent accumulation of activated T-lymphocytes. We have characterized the inflammatory response of mice deficient for BIRC4 (XIAP) to viral infections with the murine herpes virus 68 (MHV-68) as the closest murine model for human EBV-driven mononucleosis. Xiap-/- mice were capable of clearing the virus normally during early infection (day 6, 16), but failed to do so during the course of the infection measured as elevated viral genomic loads during late (day 43) and very late (day 84) latency. Xiap-/- mice responded to intranasal application of the virus with systemic hyper-inflammation exemplified by elevated IL-1beta levels, splenomegaly and increased activation of peripheral T lymphocytes such as CD4+ effector T cells, regulatory T cells (Treg), and IFNg+ T cells. In previous work, we have shown that TNF is critically required to drive the hyper-inflammatory phenotype of macrophages and dendritic cells of XIAP-deficient mice. Indeed, genetic deletion of TNF in vivo or, alternatively, anti-TNF treatment in vivo using Eternacept (Enbrel) ameliorated the symptoms of XIAP-deficient mice in response to viral infection. Elevated IL-1beta levels were also observed in human peripheral blood-derived monocytes from XLP-2 patients (7 patients from 5 different families) when compared to healthy controls. In conclusion, this data supports the notion that anti-TNF treatment might be able to ameliorate the hyper-inflammatory responses in XLP-2 patients, when used early during an infection. Disclosures No relevant conflicts of interest to declare.

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Platelet Mediated Monocyte/Macrophage Immune Training

Blood ◽

10.1182/blood-2021-151243 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3127-3127

Author(s):

Chen Li ◽

Preeti Maurya ◽

Benjamin Nieves-Lopez ◽

Sara Ture ◽

Craig N. Morrell

Keyword(s):

Bacterial Infections ◽

Inflammatory Responses ◽

Conflicts Of Interest ◽

Immune Memory ◽

Adult Mice ◽

Monocytes And Macrophages ◽

Artery Disease ◽

Critical Components ◽

Inflammation Resolution

Abstract Platelets are essential mediators of vascular and immune homeostasis as well as mediators of thrombosis. Platelet functions in hemostasis and thrombosis have received great attention in both basic and clinical research, however, emerging research indicates platelets are also critical components of the immune system. Platelets have important roles in many inflammation-associated diseases including sepsis, atherosclerosis, and peripheral artery disease. In recent years, much interest has focused on the acute outcomes of interactions between platelets and monocytes/macrophages in immune responses to bacterial infections, as several studies have demonstrated that platelets contribute to bacteria clearance and inflammation resolution by regulating monocyte/macrophage responses and their immune differentiation. However, macrophages are long-lived cells and past stimulation and immune interactions can change their responses to future stimuli - a concept termed 'innate immune memory'. Whether platelet interactions with monocytes/macrophages alters their inflammatory responses to secondary stimuli in a long-lasting manner remains unclear. We have now found that platelet interactions with monocytes and macrophages as a first stimulus, changed their responses to secondary stimuli, such as lipopolysaccharide (LPS) and unmethylated cytosine-phosphate-guanine (CpG). We incubated monocytes or macrophages with control buffer or platelets overnight and then removed the platelets prior to the addition of LPS or CpG as secondary stimulation. LPS and CpG induced IL-6 and TNFα were reduced by platelet pre-incubation compared to platelet naïve macrophages. Despite reduced cytokine secretion, platelet pre-incubation increased monocyte and macrophage proliferation in response to secondary stimuli. Furthermore, the platelet mediated macrophage secondary stimuli phenotype was preserved for several days after platelet exposure indicating a genetic reprogramming mediated mechanism. Circulating monocytes from platelet deficient TPOR -/- mice and adult mice made platelet deficient also had higher Il6 expression and secrete more IL-6 in response to stimulation compared to monocytes from WT mice indicating in vivo platelet mediated monocyte immune-training. Furthermore, the genetic reprogramming may be dependent on a HIF1α dependent process, as platelet pre-incubation increased macrophage Hif1α expression, a known mediator of trained immunity. Monocytes and macrophages, are 'immune plastic' and adapt to their environment in a functional manner. These novel data indicate that platelets 'reprogram' monocytes/macrophages and shape their responses to future stimulation. Disclosures No relevant conflicts of interest to declare.

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Therapeutic Effects of Hypoxic and Pro-Inflammatory Priming of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Inflammatory Arthritis

International Journal of Molecular Sciences ◽

10.3390/ijms23010126 ◽

2021 ◽

Vol 23 (1) ◽

pp. 126

Author(s):

Alasdair G. Kay ◽

Kane Treadwell ◽

Paul Roach ◽

Rebecca Morgan ◽

Rhys Lodge ◽

...

Keyword(s):

T Cell ◽

Extracellular Vesicles ◽

Inflammatory Responses ◽

Therapeutic Potential ◽

Histopathological Analysis ◽

Therapeutic Effects ◽

Synovial Joint ◽

Paracrine Signalling

Mesenchymal stem cells (MSCs) immunomodulate inflammatory responses through paracrine signalling, including via secretion of extracellular vesicles (EVs) in the cell secretome. We evaluated the therapeutic potential of MSCs-derived small EVs in an antigen-induced model of arthritis (AIA). EVs isolated from MSCs cultured normoxically (21% O2, 5% CO2), hypoxically (2% O2, 5% CO2) or with a pro-inflammatory cytokine cocktail were applied into the AIA model. Disease pathology was assessed post-arthritis induction through swelling and histopathological analysis of synovial joint structure. Activated CD4+ T cells from healthy mice were cultured with EVs or MSCs to assess deactivation capabilities prior to application of standard EVs in vivo to assess T cell polarisation within the immune response to AIA. All EVs treatments reduced knee-joint swelling whilst only normoxic and pro-inflammatory primed EVs improved histopathological outcomes. In vitro culture with EVs did not achieve T cell deactivation. Polarisation towards CD4+ helper cells expressing IL17a (Th17) was reduced when normoxic and hypoxic EV treatments were applied in vitro. Normoxic EVs applied into the AIA model reduced Th17 polarisation and improved Regulatory T cell (Treg):Th17 homeostatic balance. Normoxic EVs present the optimal strategy for broad therapeutic benefit. EVs present an effective novel technology with the potential for cell-free therapeutic translation.

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Platelets Are the Major Source of Circulating TGF-β1 in Mice.

Blood ◽

10.1182/blood.v114.22.4021.4021 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4021-4021

Author(s):

Jasimuddin Ahamed ◽

Joseph S. Palumbo ◽

Jay L. Degen ◽

Barry S. Coller

Keyword(s):

Transgenic Mice ◽

Wound Repair ◽

Conflicts Of Interest ◽

Left Ventricular ◽

Conditional Knockout ◽

Platelet Factor ◽

Blood Drawing ◽

Wide Range ◽

Granule Proteins ◽

Tgf Β1

Abstract 4021 Poster Board III-957 TGF-β1 is a multifunctional cytokine with profound effects on several biological processes, including malignancy, immunity, wound repair, and tissue fibrosis. In addition to local generation of TGF-β1, it also circulates in plasma where it has the potential to produce systemic effects. The source(s) of plasma TGF-β1 have not, however, been defined, and a very wide range of normal values have been reported (∼1.0 to 50.0 ng/mL). Since platelets contain 40-100 times more TGF-β1 than other cells, release of platelet TGF-β1 during blood drawing and/or sample preparation can influence the results. Citrated plasma prepared from blood samples obtained from C57Bl/6 mice by the retrobulbar capillary technique showed evidence of variable release of the platelet-specific α granule proteins platelet factor 4 (PF4) and thrombospondin-1 (TSP-1), and the degree of release correlated with the total TGF-β1 levels measured by an ELISA (Table 1). Adding PGE1 to the anticoagulant reduced but did not completely eliminate the release of platelet granule proteins. In contrast, plasma prepared from free-flowing blood obtained using ultrasound guidance to percutaneously enter the left ventricle inflow tract with a 27 gauge needle showed minimal or no evidence of release of platelet granule proteins and had a total TGF-β1 level of 2.1 ± 1.2 ng/mL. To more firmly assess the role of platelets in circulating TGF-β1, we measured the platelet, plasma, and serum TGF-β1 levels in mice with a megakaryocyte-specific conditional knockout of TGF-β1 (achieved by crossing mice carrying a floxed TGF-β1 allele with transgenic mice expressing a Cre-recombinase under the control of a PF4 promoter) and littermate control mice (WT; WT/ PF4Cre+; TGF-β1flox/flox/PF4Cre-). Compared to the controls, TGF-β1flox/flox/PF4Cre+ mice exhibited an ∼80 to 90% reduction of TGF-β1 in both platelets and serum as well as an ∼45% reduction in plasma TGF-β1 (Table 2). As a complementary test of the contribution of platelets to plasma TGF-β1, we generated profoundly thrombocytopenic mice by i.v. injection of the hamster anti-mouse αIIbβ3 mAb 1B5 (1.0 mg/kg). 24 hours later the platelet count decreased by >98% and plasma TGF-β1 levels in these thrombocytopenic animals were reduced by more than 70% (0.6 ± 0.2 ng/mL) compared to pretreatment values (3.2 ± 0.4 ng/mL; p=0.002) or values after giving saline or control polyclonal hamster IgG (3.2 ± 2.0 ng/mL; p=0.002). We conclude that platelets are the major source of plasma TGF-β1 in mice. In addition, based on the rapid decrease of plasma TGF-β1 levels after the induction of thrombocytopenia, we infer that plasma TGF-β1 has a relatively short survival time. Our data have potential implications for the systemic contribution of platelet-derived TGF-β1 in regulating various normal and disease states, including wound healing and organ fibrosis. Table 1 Effect of Blood Drawing Technique on Plasma Levels of TGF-β1, PF4, and TSP-1 Protein Retrobulbar Retrobulbar + PGE1 Left Ventricular TGF-β1 (ng/mL) n=25, 10, 15 6.0 ± 3.8 2.2 ± 0.8* 2.1 ± 1.2* PF4 Antigen (AU), n=22, 7, 12 41.0 ± 30 8.0 ± 5.0* 5.6 ± 3.0* TSP-1 Antigen (AU), n=22, 7, 12 71 ± 34 28 ± 14* 21. ± 5.0* AU: arbitrary unit; *P<0.005 vs. retrobulbar Table 2 TGF-β1 Levels in Control and Transgenic Mice Mice TGF-β1 Platelet (ng/109 platelets) Serum (ng/mL) Plasma (ng/mL) Controls [(WT; WT/ PF4Cre+; TGF-β1flox/flox/PF4Cre-) (n=9, 21, 10)] 120 ± 43 91.0 ± 23 4.5 ± 1.5 PF4Cre+/ TGF-β1flox/flox (n=4, 9, 10) 26 ± 8** 12.2 ± 1.5** 2.4 ± 0.6* * P<0.005 vs. WT; ** P<0.001 vs. WT Disclosures: No relevant conflicts of interest to declare.

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IFNs Upregulate Sca-1 and Block Proliferation in Murine Hematopoietic Stem and Progenitor Cells,

Blood ◽

10.1182/blood.v118.21.3394.3394 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3394-3394

Author(s):

Kaitlyn Shank ◽

Yusup Shin ◽

Carson Wills ◽

Nicole Cunningham ◽

Alevtina Domashenko ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Caspase 3 ◽

Inflammatory Responses ◽

Conflicts Of Interest ◽

Brdu Incorporation ◽

Apparent Paradox ◽

Hematopoietic Stem

Abstract Abstract 3394 Hematopoietic stem cells (HSC) replenish the cellular components of the blood throughout life by a homeostatic process in which the majority of HSCs remain quiescent while a small percentage enter the cell cycle to either self-review or differentiate. During inflammatory responses to infections, Interferons (IFNa, IFNg) perturb HSC homeostasis, presumably in response to the demand for increased numbers of inflammatory cells. Previous studies have highlighted an apparent paradox, i.e. IFNs suppress the proliferation of normally cycling murine hematopoietic progenitor cells (HPCs), yet increase the fraction of normally quiescent Sca+ HSCs that proliferate. To investigate the mechanisms underlying this paradox, we dissected the dynamics of cell surface phenotypes, cell cycle kinetics, pro- and anti-apoptotic pathways within the HSC and HPC compartments in response to pIpC and IFNs both in vivo and in vitro. Forty-eight hours after pIpC injection, bone marrow (BM) cellularity declined by 60%, the proportion of Sca- kit+ HPCs fell from 0.45% to 0.05%, while the proportion of BM cells with the Sca+ kit+ HSC phenotype increased from 0.17 to 0.26%. To determine whether the increase in Sca+kit+ cells was due to proliferation of HSCs or upregulation of Sca-1 on HPCs, we cultured purified CD150+ Sca-Kit+ HPCs and CD150+Sca+kit+ HSCs in vitro with IFNa, IFNg, or PBS. Sca expression was induced on previously Sca- HPCs, and the level of Sca expression on HSCs was also increased. This induction was detectable as early as 6 hours after treatment and accompanied by an increase in Sca mRNA. BrdU incorporation into both HPC and HSC populations decreased from pre-treatment baselines, further indicating that the increase in cells with the HSC phenotype was not due to HSC proliferation, but rather the appearance of cycling HPCs within the HSC staining gate following IFN-induced upregulation of Sca. Staining with FITC-DEVD-FMK identified active cleaved capase-3 in pIpC- or IFN-treated cells, suggesting that the reduced cellularity following IFN reflected a cellular stress that killed Lin+ precursors cells and some HPCs, but spared HSCs. In contrast to lin+kit- precursors, all kit + HPCs and HSCs expressed bcl-2, suggesting that expression of anti-apoptotic proteins may prevent IFN-induced stress from resulting in HSC/HPC apoptosis despite the initial triggering of caspase-3 cleavage. In summary, acute treatment with IFNs has anti-proliferative effects on all hematopoietic cells, including precursors, HPCs and HSCs, with the apparent increase in HSC proliferation the result of HPCs masquerading as Sca+HSCs after exposure to IFN. Unlike precursors, HSCs and some HPCs survive treatment to IFNs despite activation of cleaved caspase-3, possibly due to their expression of bcl-2, and likely related anti-apoptotic regulators. The previously observed increase in HSC proliferation days and weeks following IFN treatment is most likely due to the homeostatic response of HSCs to the depopulation of the precursor and HPCs caused by acute IFN exposure. Disclosures: No relevant conflicts of interest to declare.

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Selective Disruption of PU.1 in Mature Dendritic Cells Affects Their Tissue Distribution and T Cell Homeostasis

Blood ◽

10.1182/blood.v118.21.518.518 ◽

2011 ◽

Vol 118 (21) ◽

pp. 518-518

Author(s):

Tadafumi Iino ◽

Hiromi Iwasaki ◽

Kentaro Kohno ◽

Shin-ichi Mizuno ◽

Yojiro Arinobu ◽

...

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Inflammatory Responses ◽

Conflicts Of Interest ◽

Critical Role ◽

T Cell Response ◽

T Cell Homeostasis ◽

Cell Homeostasis ◽

Hematopoietic Stem

Abstract Abstract 518 PU.1, a hematopoietic transcription factor, is indispensable for development of conventional dendritic cells (cDCs) from hematopoietic stem cells. However, the function of PU.1 in mature cDC remains unclear. To test the possible role of PU.1 in mature cDCs, we developed mice lacking PU.1 selectively in mature cDCs (DC-PU.1D/D mice) by crossing a PU.1flox mouse line with a transgenic Itgax (CD11c)-Cre strain. In these mice, cDCs were dramatically reduced in spleen, thymus, lymph node, and skin, down to <40%, <25%, <10% and <5% of DCs in control mice respectively, whereas bone marrow cDCs and common dendritic cells progenitors (CDPs) were not affected. Surprisingly, T cell numbers were significantly decreased in DC-PU.1D/D mice, whereas thymic T cell development was normal, suggesting that maintenance of mature T cell pool might be impaired, presumably by dysfunction of PU.1D/D cDCs. In fact, PU.1D/D cDCs failed to efficiently induce ovalbumin-specific T cell response and to produce inflammatory cytokines in response to Toll like receptor (TLR) stimulation both in vitro and in vivo. The intravenous transfer of spleen PU.1D/D cDCs failed to repopulate the spleen of recipient mice, suggesting their poor survival in vivo. Furthermore, the expression of critical molecules for inflammatory responses was downregulated in PU.1D/D cDCs as compared to normal cDCs. These molecules included Myd88 and NFkB that are downstream molecules of TLR signaling, CD86 that is required for T cell stimulation, and CCR7 that is required for cDC migration. These results clearly show that PU.1 is required for development of the functional cDC pool, and the cDC pool plays a critical role in T cell homeostasis. Disclosures: No relevant conflicts of interest to declare.

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Platelet Factor 4 (PF4)-Mediated Neutrophil Extracellular Trap Compaction Limits Endothelial Injury and Promotes Survival Following Lipopolysaccharide Challenge

Blood ◽

10.1182/blood.v130.suppl_1.997.997 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 997-997

Author(s):

Kandace Gollomp ◽

Ian Johnston ◽

Minna Kim ◽

Li Zhai ◽

Guohua Zhao ◽

...

Keyword(s):

Conflicts Of Interest ◽

Cell Damage ◽

Platelet Factor 4 ◽

Neutrophil Extracellular Trap ◽

In Vivo Studies ◽

Microfluidic Channels ◽

Platelet Factor ◽

Osmotic Pumps

Abstract When stimulated by infection or inflammation, neutrophils expel NETs, decondensed chromatin coated with histones and antimicrobial proteins that ensnares pathogens but also damages host tissue. Platelet factor 4 (PF4, CXCL4) is a CXC chemokine stored in platelet alpha-granules and released in high concentrations during platelet activation. Tetrameric PF4 has a very high affinity for polyanionic molecules, including DNA, and we have found that PF4 binds and physically compacts NETs, causing them to have increased resistance to endonuclease digestion. Our group has also observed that PF4 expression leads to enhanced survival in a murine model of sepsis. Based on these findings, we chose to investigate whether PF4-mediated NET compaction is protective in endotoxemia. To study PF4-NET interactions, we developed a microfluidic assay in which neutrophils were adhered to fibronectin-coated channels and then stimulated to release NETs with phorbol myristate acetate(PMA). NETs were visualized by staining with the fluorescent nucleic acid stain SYTOX. Changes in NET morphology and fluorescence were quantified in the presence of varying PF4 concentrations. DNase I was then infused through these channels and the extent of digestion was measured. These experiments showed that the presence of PF4 led to NET compaction and decreased NET degradation following DNase infusion. We then performed in vitro studies examining NET-endothelial interactions in which isolated neutrophils were stimulated to release NETs, incubated with buffer alone or buffer containing PF4, and flowed through human endothelial umbilical vein cell (HUVEC) lined microfluidic channels that had been stimulated with tumor necrosis factor (TNF) α. EC viability was assessed 24-hours post NET exposure and revealed that the presence of PF4 protected HUVECs from NET-induced damage. To further investigate PF4-NET interactions in endotoxemia, we conducted in vivo studies using PF4-deficient mice (mPF4-/-) and wildtype (WT) controls injected with lipopolysaccharide (LPS). Plasma NET markers [cell free DNA (cfDNA), citrullinated histones (cit-His), and myeloperoxidase (MPO)] were quantified via ELISA and Western blot at various time points following LPS injection. mPF4-/- mice were also implanted with PF4-containing osmotic pumps and the NET markers were also assessed following LPS exposure. These experiments revealed that compared to WT mice, LPS injected mPF4-/- mice had significantly higher plasma levels of NET components, including cfDNA, cit-His and MPO. When mPF4-/- mice were implanted with PF4-releasing osmotic pumps prior to LPS injection, they had plasma NET component levels comparable to those observed in WT mice. Based on the results of our in vitro and in vivo studies, we propose that PF4 infusion compacts NETs, decreasing their susceptibility to DNAse lysis, and preventing the release of toxic NET degradation products (NDPs) such as cfDNA and cit-His. We posit that PF4-mediated sequestration of NDPs prevents endothelial cell damage in the HUVEC-lined microfluidic model. We believe that the results of our studies in mPF4-/- mice demonstrate that PF4 has a similarly protective effect in vivo, decreasing NET lysis and reducing NDP generation. These findings suggest that in sepsis, the stabilization rather than the lysis of NETs may be therapeutic. Further investigation should be performed to determine if treatment with PF4 or other small positively-charged proteins such as protamine sulfate that can sequester NDPs, may be beneficial the treatment of sepsis. Disclosures No relevant conflicts of interest to declare.

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Abstract 3377: Modulation of Vessel Stability by the ELR-negative Chemokine IP-10

Circulation ◽

10.1161/circ.118.suppl_18.s_415 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Richard J Bodnar ◽

Cecelia C Yates ◽

Xiaoping Du ◽

Alan Wells

Keyword(s):

Endothelial Cells ◽

Wound Repair ◽

Critical Role ◽

Tissue Development ◽

Platelet Factor ◽

Signaling Mechanism ◽

Extracellular Signaling ◽

Vessel Stability

Angiogenesis plays a critical role in tissue development and wound repair. Regulation of the vascular tree is critical to organogenesis and neo-organogenesis. The initial vigorous vessel proliferation and arborization that occurs during tissue development and wound repair must be pruned to form mature tissue. The signals that trigger this vascular regression are still ill-defined. Mitotic endothelial cells express the CXC receptor 3 (CXCR3), which binds the ELR-negative chemokines IP-10 (CXCL10), platelet factor 4 (PF4, CXCL4), IP-9/I-TAC (CXCL11), and MIG (CXCL9), with IP-9 and IP-10 being expressed during the later remodeling phases of wound repair when vascular involution occurs. We hypothesized that not only does this signaling system limit angiogenesis, but that CXCR3 signaling also compromises endothelial cell tube integrity contributing to regression. Treatment of newly formed tubes/cords on Matrigel (in vitro) or vessels in a subcutaneous Matrigel plug (in vivo) with IP-10 in either presence or absence of VEGF caused a dissociation of the tubes and subsequent vascular involution. This followed CXCR3 induced cleavage of β3 integrin in endothelial cells. CXCR3 triggering of mu-calpain activity caused cleavage of the cytoplasmic tail of human β3 integrins at the calpain cleavage sites NPLY747 and TSTF754. IP-10 stimulation was also found to activate caspase 3, blockage of which prevented cell death but not tube dissociation. Thus, CXCR3 activation in newly formed vessels results in endothelial dissociation from matrix, followed by anoikis. This is the first direct evidence for an extracellular signaling mechanism through CXCR3 causing the dissociation of newly formed blood vessels. This study was supported by funds from VA Medical Research Program and National Institute of General Medical Sciences (NIH, USA).

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