scholarly journals Flow cytofluorimetric detection and immunophenotyping of platelet-monocyte complexes in peripheral blood

2021 ◽  
Vol 23 (2) ◽  
pp. 401-410
Author(s):  
O. V. Pavlov ◽  
S. V. Chepanov ◽  
A. V. Selutin ◽  
M. S. Zainulina ◽  
D. R. Eremeeva ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Pregnancy Complications ◽  
Ex Vivo ◽  
Flow Cytometric Analysis ◽  
First Trimester ◽  
Immunocytochemical Staining ◽  
Adhesive Properties ◽  
Pathophysiological Mechanisms ◽  
Pathological Conditions

Activated platelets aggregate with monocytes by binding membrane bound molecules. Platelet-monocyte interaction is considered to underlie pathophysiological mechanisms bridging thrombosis and inflammation. Detection and analysis of platelet-monocyte complexes (PMC) provide means for revealing their physiological and pathogenetic roles and are instrumental in the diagnostics of various pathological conditions including obstetric complications. The aim of the study was to develop the method of quantitative determination of peripheral blood PMC, that preserve phenotypic features of platelets and monocytes, and to reveal their changes by ex vivo analysis. The suggested procedure includes immediate fixation of blood sample, immunocytochemical staining with fluorochrome-conjugated specific antibodies against markers of activation and differentiation followed by lysis of erythrocytes, and flow cytometric analysis. Fourteen samples of peripheral blood from patients with history of pregnancy complication were obtained in first trimester of ongoing pregnancy and analyzed. It was demonstrated that quantitative and qualitative in vivo characteristics of PMC remained unchanged in fixed samples, whereas the number of PMC and expression levels of the markers of platelet and monocyte activation dramatically increased in the unfixed blood. The set of monoclonal antibodies and gating strategies, used in this study, ensure phenotyping and evaluation of percentage/absolute count of PMC in the total monocyte population (CD45+CD14+) and in the subpopulations of classical (CD14+CD16-), intermediate (CD14+CD16+), and non-classical (CD14lowCD16+) monocytes. This approach provides insight into the participation of different monocyte subsets in the formation of PMC and their roles in physiological and pathophysiological processes. In some samples, elevated PMC proportion was observed, accompanied by significant increase in the expression of platelet activation marker CD62P and decrease in the expression of its monocytic ligand CD162. These changes suggested altered activation of PMC and their participation in the pathophysiological mechanisms of some pregnancy complications. Immunophenotyping of PMC affords an opportunity to characterize their proinflammatory, procoagulant and adhesive properties; these results can be used for research and diagnostics. In particular, the method is suitable for detection and phenotyping of PMC in pregnancy complications and other pathological conditions associated with the disorders of hemostasis and thrombosis.

Abstract 1144: Successful Surface-Aminated Nanofiber Expansion of Human Umbilical Cord-Derived CD133+ Cells Leads to Augmentation of Angiogenic Functionality In Vitro and In Vivo

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Hiranmoy Das ◽  
Matthew Joseph ◽  
Nasreen Abdulhameed ◽  
Hai-Quan Mao ◽  
Vincent J Pompili
Keyword(s):  
Umbilical Cord ◽  
Hind Limb ◽  
Ex Vivo ◽  
Flow Cytometric Analysis ◽  
Surface Expression ◽  
Capillary Density ◽  
Therapeutic Angiogenesis ◽  
Human Umbilical Cord

Background: Umbilical cord blood (UCB) and marrow-derived CD133+ cells have been shown to mediate encouraging effects on therapeutic angiogenesis in both animal models and early clinical trials. Low numbers of CD133+ cells derived from a single donor have been a limitation of use of these cells in cardiovascular therapy. We hypothesized that an ex vivo aminated nanofiber system combined with cytokine supplementation would provide optimized topographical and biochemical signals to allow the expansion and potential functional augmentation of CD133+ cells without promoting terminal differentiation. Methods and Results: Human UCB derived CD133+ progenitor cells were isolated by MACS sorting and ex vivo expanded on aminated nanofiber plates with cytokine rich media. Cells harvested 10 days after expansion demonstrated a 225X increase in total number. Flow cytometric analysis demonstrated CD133–24%, CD34–93%, CXCR4–97%, LFA-97% surface expression. The expanded cells can uptake AcLDL efficiently and demonstrate a 2.3X increase in transwell migration to SDF-1 as compared to fresh UCB CD133+ cells. In vitro analysis revealed that expanded cells have potential to differentiate into endothelial or smooth muscle phenotype as demonstrated with CD31, vWF, VCAM-1 and F-pholloidin, α-actin, and SM myosin heavy chain immunocytochemistry when re-cultured for 14d in EGM2 or SMBM respectively. RT-QPCR analysis of 1% O 2 exposed (hypoxic) cells demonstrated a 2X increase in VEGF and 3X increase in IL-8 gene expression compared to normoxic control. In vivo functionality in a NOD/SCID mouse hind limb ischemic model demonstrated that mice treated with 5 x 10 6 expanded cells (n=7) augmented blood flow ratio (ischemic/control limb) as compared to mice treated with CD133+ cells (n=7) and control (n=7) at 28d. (control 0.32±.02 vs. UCB133+ 0.37±.02 vs. expanded cells 0.50±.04 p<0.01) Capillary density in ischemic hind-limb was increased at 28d (control 62.5±5.4 vs. expanded cell 97.6±2.5 p< 0.01) Conclusions: These studies demonstrate successful high level expansion of UCB derived CD133+ cells into functionally potent stem cells which have the capacity to differentiate into vascular cells and promote in vivo neovascularization.

Expression of CD10 by Human T Cells That Undergo Apoptosis Both In Vitro and In Vivo

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3067-3076 ◽  
Author(s):  
Giovanna Cutrona ◽  
Nicolò Leanza ◽  
Massimo Ulivi ◽  
Giovanni Melioli ◽  
Vito L. Burgio ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Lymphoid Cells ◽  
Normal Peripheral Blood ◽  
Apoptotic Process ◽  
Cd10 Expression ◽  
T Cell Lines

Abstract This study shows that human postthymic T cells express CD10 when undergoing apoptosis, irrespective of the signal responsible for initiating the apoptotic process. Cells from continuous T-cell lines did not normally express CD10, but became CD10+ when induced into apoptosis by human immunodeficiency virus (HIV) infection and exposure to CD95 monoclonal antibody, etoposide, or staurosporin. Inhibitors of caspases blocked apoptosis and CD10 expression. Both CD4+ and CD8+ T cells purified from normal peripheral blood expressed CD10 on apoptotic induction. CD10 was newly synthesized by the apoptosing cells because its expression was inhibited by exposure to cycloheximide and CD10 mRNA became detectable by reverse transcription-polymerase chain reaction in T cells cultured under conditions favoring apoptosis. To show CD10 on T cells apoptosing in vivo, lymph node and peripheral blood T cells from HIV+ subjects were used. These suspensions were composed of a substantial, although variable, proportion of apoptosing T cells that consistently expressed CD10. In contrast, CD10+ as well as spontaneously apoptosing T cells were virtually absent in peripheral blood from normal individuals. Collectively, these observations indicate that CD10 may represent a reliable marker for identifying and isolating apoptosing T cells in vitro and ex vivo and possibly suggest novel functions for surface CD10 in the apoptotic process of lymphoid cells.

Ex Vivo Expansion of Genetically Marked Rhesus Peripheral Blood Progenitor Cells Results in Diminished Long-Term Repopulating Ability

Blood ◽  
1998 ◽  
Vol 92 (4) ◽  
pp. 1131-1141 ◽  
Author(s):  
J.F. Tisdale ◽  
Y. Hanazono ◽  
S.E. Sellers ◽  
B.A. Agricola ◽  
M.E. Metzger ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Ex Vivo ◽  
Retroviral Vectors ◽  
Ex Vivo Expansion ◽  
Us Government ◽  
Government Work ◽  
Stromal Support ◽  
Short And Long Term

Abstract The possibility of primitive hematopoietic cell ex vivo expansion is of interest for both gene therapy and transplantation applications. The engraftment of autologous rhesus peripheral blood (PB) progenitors expanded 10 to 14 days were tracked in vivo using genetic marking. Stem cell factor (SCF)/granulocyte colony-stimulating factor (G-CSF)–mobilized and CD34-enriched PB cells were divided into two equal aliquots and transduced with one of two retroviral vectors carrying the neomycin-resistance gene (neo) for 4 days in the presence of interleukin-3 (IL-3), IL-6, and SCF in the first 5 animals, IL-3/IL-6/SCF/Flt-3 ligand (FLT) in 2 subsequent animals, or IL-3/IL-6/SCF/FLT plus an autologous stromal monolayer (STR) in the final 2. At the end of transduction period, one aliquot (nonexpanded) from each animal was frozen, whereas the other was expanded under the same conditions but without vector for a total of 14 days before freezing. After total body irradiation, both the nonexpanded and expanded transduced cells were reinfused. Despite 5- to 13-fold higher cell and colony-forming unit (CFU) doses from the expanded fraction of marked cells, there was greater short- and long-term marking from the nonexpanded cells in all animals. In animals receiving cells transduced and expanded in the presence of IL-3/IL-6/SCF/FLT, engraftment by the marked expanded cells was further diminished. This discrepancy was even more pronounced in the animals who received cells transduced and expanded in the presence of FLT and autologous stroma, with no marking detectable from the expanded cells. Despite lack of evidence for expansion of engrafting cells, we found that the addition of FLT and especially STR during the initial brief transduction period increased engraftment with marked cells into a clinically relevant range. Levels of marked progeny cells originating from the nonexpanded aliqouts were significantly higher than that seen in previous 4 animals receiving cells transduced in the presence of IL-3/IL-6/SCF, with levels of 10% to 20% confirmed by Southern blotting from the nonexpanded IL-3/IL-6/SCF/FLT/STR graft compared with 0.01% in the original IL-3/IL-6/SCF cohort. These results suggest that, although expansion of PB progenitors is feasible ex vivo, their contribution towards both short- and long-term engraftment is markedly impaired. However, a brief transduction in the presence of specific cytokines and stromal support allows engraftment with an encouraging number of retrovirally modified cells. This is a US government work. There are no restrictions on its use.

A New Animal Model for Anemia Induced by Environmental Low-Temperature: Physiology of Erythrocyte Production and Circulation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4770-4770
Author(s):  
Shun Maekawa ◽  
Hitomi Iemura ◽  
Yuko Kuramochi ◽  
Nami Kosaka-Nogawa ◽  
Hironori Nishikawa ◽  
...  
Keyword(s):  
Low Temperature ◽  
Peripheral Blood ◽  
Erythropoietin Receptor ◽  
Immunocytochemical Staining ◽  
Chain Reactions ◽  
Cell Counts ◽  
New Perspective ◽  
Low Temperature Exposure ◽  
Peripheral Erythrocyte

Abstract To survive, organisms must adapt to changes in the ambient environment. Here, we describe a new model of anemia based on exposure of African clawed frog, Xenopus laevis to low-temperature. Frogs exposed at low-temperature (5ºC) for five days had decreased numbers of peripheral blood erythrocytes, leukocytes, and thrombocytes as well as low hemoglobin levels. By contrast, spleen erythrocytes increased in number. Cell counts returned to normal in frogs re-warmed at ambient temperature (22ºC) for two days. To confirm these observations in vivo, we labeled peripheral blood cells with fluorescent reagent CFSE. During five days at 5ºC, labeled erythrocytes in peripheral blood decreased in number while those in spleen increased. When the temperature was raised to 22ºC, however, their numbers increased in peripheral blood. The findings suggested that exposure to low-temperature resulted in splenic pooling of peripheral erythrocytes. Accordingly, we looked at recovery from anemia induced by phenylhydrazine (PHZ) in this model. PHZ-treated frogs maintained at 22ºC decreased numbers of peripheral erythrocytes that were minimal on day 8, and increased gradually thereafter. In the liver, we found erythrocyte progenitors expressing erythropoietin receptor and GATA1-A detected by reverse transcription polymerase chain reactions and immunocytochemical staining but no mature forms. In PHZ-treated frogs exposed to 5ºC, peripheral erythrocyte counts remained minimal from day 8, and reversibly recovered when temperature returned to 22ºC. Erythrocyte progenitors were present in liver on day 8 but absent on day 12. Conversely, mature erythrocytes were absent in liver on day 8 but present on day 12. Finally, to learn whether the progenitors proliferate and differentiate without migrating from liver to peripheral blood, we treated frogs with thymidine analog bromodeoxyuridine (BrdU). In frogs kept at 22 ºC, BrdU-labeled erythrocytes were abundant in both liver and peripheral blood. However, frogs cooled at 5ºC had labeled cells in liver but few in peripheral blood. The findings suggest low-temperature exposure cause this anemia by impairing migration of mature/immature erythrocytes from the liver. In summary, this amphibian model offers a new perspective for investigating physiological effects of environmental temperature on vertebrate erythropoiesis.

Ex Vivo Expansion of Human Mobilized Peripheral Blood Stem Cells Using Epigenetic Modifiers

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1920-1920
Author(s):  
Santosh Saraf ◽  
Hiroto Araki ◽  
Benjamin Petro ◽  
Kazumi G Yoshinaga ◽  
Simona Taioli ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Transcript Levels ◽  
Epigenetic Modifiers ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood

Abstract Abstract 1920 Currently, a significant percentage of hematopoietic stem cell (HSC) transplantations are being performed using growth factor mobilized peripheral blood (MPB) grafts. Unfortunately, about 5 to 40% of patients are unable to benefit from HSC transplantation due to failure to mobilize and harvest an adequate graft (> 2 × 106 CD34+ cells/kg). Epigenetic modifications are thought to be important in determining the fate of HSC including self renewal and differentiation. We have previously demonstrated that sequential addition of chromatin modifying agents (CMA), 5-aza-2'-deoxyctidine (5azaD) and trichostatin A (TSA), is capable of expanding transplantable HSC 7-fold from human cord blood (CB), likely by preventing the silencing of genes which promote HSC self renewal divisions (Araki et al. Blood 2007). Using the same protocol we have also previously shown that 5azaD/TSA can expand CD34+CD90+ cells containing in vivo repopulating capacity from human bone marrow (BM) 2.5-fold (Milhem et al. Blood 2004). The objectives of our current studies were to assess whether CMA can also expand HSCs present in MPB. In order to test this hypothesis, CD34+ cells were isolated from MPB products from three healthy donors and were expanded ex vivo using 5azaD/TSA for 9 days as described previously (Araki et al. Blood 2007). Following culture, expansion of primitive CD34+CD90+ cells, colony forming unit mixed lineages (CFU-mix), and long term (5 weeks) cobblestone area forming cells (CAFC) were assessed. A 3.74 ± 0.77 fold expansion of CD34+CD90+ cells was observed in 5azaD/TSA expanded MPB cells while only a 0.93 ± 0.23 fold expansion was observed in control cultures (p = 0.025). The 5azaD/TSA expanded MPB cells had a 10.1-fold increase in the number of CFU-mix in comparison to no expansion in the control cultures (p = 0.0055). A 2.26-fold expansion of CAFC numbers was observed in 5azaD/TSA expanded MPB cells in comparison to 0.19-fold expansion in control cultures. Taken together, our data indicate that 5azaD/TSA can expand MPB CD34+CD90+ cells 3.74-fold which also possess the functional capacity to generate primitive CFU-mix and long term CAFCs. This expansion of primitive MPB CD34+CD90+ cells appears to be at an intermediate level (3.74 fold) in comparison to BM and CB which had 2.5-fold and 10.5-fold expansion, respectively. We have previously demonstrated that CD34+CD90+ expanded CB cells are exclusively responsible for reconstituting blood cells following transplantation (Araki et al. Exp Hematol 2006). Currently, the frequency of in vivo repopulating units for CMA expanded MPB is being determined in contrast to expanded BM and CB cells. However, it remains to be investigated what determines the limit for ex vivo expansion of HSC by epigenetic modifiers based on their ontogeny. Towards this goal we analyzed transcription levels of several genes implicated for HSC self renewal/expansion including HoxB4, GATA 2, and Ezh2, which were compared between MPB cells prior to and following expansion in 5azaD/TSA or control cultures. Significantly higher transcript levels were detected for HoxB4 (p = 0.003), GATA 2 (p = 0.0002), and Ezh2 (p = 0.0001) by real time quantitative RT PCR in the 5azaD/TSA expanded MPB graft in comparison to control cultures. Interestingly the transcript levels of HoxB4 and GATA 2 but not Ezh2 were significantly lower in expanded cells in contrast to unmanipulated primary MPB cells. This is in sharp contrast to our earlier results from CB in which 5azaD/TSA expanded cells displayed much higher transcript levels of HoxB4 and GATA 2 compared to primary unmanipulated CB cells. Previously we have demonstrated that environmental conditions can influence the degree of expansion of transplantable HSC from CB (Araki et al. Exp Hematol 2009). Using the same protocol we expanded MPB cells in the presence or absence of CMA using either optimal (SCF, TPO, FLT3L) or suboptimal cytokine cocktails (SCF, TPO, FLT3L with IL-3 and IL-6). Interestingly, unlike CB cells no significant difference in expansion between the two cytokine groups with or without CMA was observed (4.5 versus 4.3-fold expansion of CD34+CD90+ cells, respectively). Corresponding to this, transcript levels of HoxB4 and Ezh2 did not vary between MPB cells expanded with 5azaD/TSA in the two different cytokine environments. Our studies have the potential to be used to expand HSC from poor mobilizers in order to optimize MPB grafts for transplantation. Disclosures: No relevant conflicts of interest to declare.

Epigenetic Profiling Of Leukemia Stem Cells In a Model Of TET2/FLT3-Mutant AML

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 476-476
Author(s):  
Alan H. Shih ◽  
Yanwen Jiang ◽  
Kaitlyn Shank ◽  
Suveg Pandey ◽  
Agnes Viale ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Targeted Therapies ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
In Vivo Models ◽  
Flow Cytometric

Specific combinations of Acute Myeloid Leukemia (AML) somatic mutations are associated with distinct clinical and biologic features. However, in vivo models do not exist for the majority of common, poor-prognosis genotypes. Concurrent mutations in FLT3 and TET2 are associated with adverse outcome. We hypothesized that activating mutations in FLT3 would cooperate with inactivating mutations in TET2to induce AML in vivo, and that we could investigate AML pathogenesis and therapeutic response using a genetic model of this poor-risk AML genotype. To understand how these genes cooperate to induce AML, we generated Vav+Tet2fl/flFlt3-ITD mice, which resulted in fully penetrant, lethal disease in all recipient mice. Flow cytometric analysis revealed expansion of mac1+ cells in the peripheral blood, with progressive expansion of a c-Kit+, blast population which was apparent in the blood and bone marrow at 28 days, leading to lethal AML in all Vav+Tet2fl/flFlt3-ITD mice with a median survival of 12 months. Consistent with genetic data demonstrating most AML patients have monoallelic TET2 mutations, Vav+Tet2fl/+Flt3-ITD mice also develop AML, suggesting haploinsufficiency for Tet2 is sufficient to cooperate with the Flt3-ITD mutation to induce AML. All mice developed leukocytosis (median 85K/uL), splenomegaly (median 554mg) and hepatomegaly (median 2900mg) with evidence of extramedullary disease cell infiltration by leukemic blasts. Flow cytometric analysis of stem/progenitor populations revealed expansion of the granulocyte-macrophage progenitor (GMP) population and the lin- sca+ kit+ (LSK) stem cell population. Detailed analysis of the LSK population revealed a decrease in the LT-HSC population (LSK CD150+ CD48-) that was replaced by a monomorphic CD48+ CD150- multipotent progenitor population. Given previous studies have shown that LSK and GMP cells can contain leukemia stem cells (LSC) in other models of AML, we performed secondary transplant studies with LSK and GMP populations. LSK (CD48+ CD150-) cells, but not GMP cells, were able to induce disease in secondary and tertiary recipients in vivo. In order to assess the sensitivity of Tet2/Flt3-mutant AML and specifically the LSCs, to targeted therapies, we treated primary and transplanted mice with chronic administration of AC220, a FLT3 inhibitor in late-stage clinical trials. AC220 treatment inhibited FLT3 signaling in vivo, and reduced peripheral blood counts/splenomegaly. However, FLT3 inhibition did not reduce the proportion of AML cells in the bone marrow and peripheral blood. AC220 therapy in vivo reduced the proportion of GMP cells, but not LSK cells, demonstrating LSCs in this model are resistant to FLT3-targeted anti-leukemic therapy. We hypothesized that Tet2/Flt3-mutant LSCs possess a distinct epigenetic/transcriptional signature that contributes to leukemic cell self-renewal and therapeutic resistance. We performed RNA-seq using the Lifetech Proton sequencer to profile the expression landscape of Vav+Tet2fl/flFlt3-ITD mutant LSKs compared to normal stem cells. We were able to obtain an average of 62 million reads per sample. We identified over 400 genes differentially expressed in LSCs relative to normal hematopoietic stem cells (FC>2.5, padj <0.05). Of note, we found that genes involved in normal myelo-erythroid differentiation, including GATA1, GATA2, and EPOR, were transcriptionally silenced in LSCs relative to normal stem cells, consistent with their the impaired differentiation and increased self-renewal observed in LSCs. Enhanced representation bisulfite sequencing revealed a subset of these genes were marked by increased promoter methylation. The number of hyper differentially methylated regions (HyperDMRs, 10% methylation difference, FDR<0.2) was significantly greater in Vav+Tet2fl/flFlt3-ITD cells (787 HyperDMRs) compared to Vav+Tet2fl/fl cells (76 DMRs) suggesting FLT3 activation and TET2 loss cooperate to alter the epigenetic landscape in hematopoietic cells. Our data demonstrate that TET and FLT3 mutations can cooperate to induce AML in vivo, with a defined LSC population that is resistant to targeted therapies and characterized by site-specific changes in DNA methylation and gene expression. Current studies are aimed to assess the functional role of specific gene targets in LSC survival, and at defining therapeutic liabilities that can be translated to the clinical context. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Apoptotic cell death, detectedex vivoin peripheral blood lymphocytes of HIV-1 infected persons

1996 ◽  
Vol 5 (5) ◽  
pp. 379-381
Author(s):  
L. F. te Velde ◽  
I. Vermes ◽  
C. Haanen ◽  
C. P. M. Reutelingsperger ◽  
C. H. H. ten Napel
Keyword(s):  
Peripheral Blood ◽  
Ex Vivo ◽  
Apoptotic Cell ◽  
Quantitative Information ◽  
Peripheral Blood Lymphocytes ◽  
Median Percentage ◽  
Blood Lymphocytes ◽  
Heterogenous Group ◽  
Hiv 1

In HIV-1 infection the ongoing depletion of CD4+ T-lymphocytes is believed, to a large extent, to be due to apoptosis. Until now quantitative information aboutin vivoapoptosis of lymphocytes in HIV-patients is scarce because of the very nature of the apoptotic process. Successful detection of apoptosisex vivorequires the recognition of the initial phase of this process, because at a later stage the cells may not remain any longer in the circulation. We measured quantitatively the amount of early apoptotic peripheral blood lymphocytes directlyex vivoin HIV-1 infected patients using a recently described flow cytometric assay. With this method we observed in an unselected heterogenous group of twelve HIV-infected individuals a median percentage of apoptotic lymphocytes to be significantly higher than in ten healthy controls. To the best of our knowledge this is the first report ofex vivoobserved increased apoptosis of peripheral blood lymphocytes in HIV-infected persons.

scholarly journals Enhanced Susceptibility of ADAP-Deficient Mice to Listeria monocytogenes Infection Is Associated With an Altered Phagocyte Phenotype and Function

2021 ◽  
Vol 12 ◽  
Author(s):  
Martha A. L. Böning ◽  
Gerald P. Parzmair ◽  
Andreas Jeron ◽  
Henning P. Düsedau ◽  
Olivia Kershaw ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Ex Vivo ◽  
Transcriptional Profiling ◽  
Fatal Outcome ◽  
Flow Cytometric Analysis ◽  
Adaptor Protein ◽  
Phagocytic Capacity ◽  
Inflammatory Monocytes ◽  
Deficient Mice

The adhesion and degranulation-promoting adaptor protein (ADAP) serves as a multifunctional scaffold and is involved in the formation of immune signaling complexes. To date, only limited data exist regarding the role of ADAP in pathogen-specific immunity during in vivo infection, and its contribution in phagocyte-mediated antibacterial immunity remains elusive. Here, we show that mice lacking ADAP (ADAPko) are highly susceptible to the infection with the intracellular pathogen Listeria monocytogenes (Lm) by showing enhanced immunopathology in infected tissues together with increased morbidity, mortality, and excessive infiltration of neutrophils and monocytes. Despite high phagocyte numbers in the spleen and liver, ADAPko mice only inefficiently controlled pathogen growth, hinting at a functional impairment of infection-primed phagocytes in the ADAP-deficient host. Flow cytometric analysis of hallmark pro-inflammatory mediators and unbiased whole genome transcriptional profiling of neutrophils and inflammatory monocytes uncovered broad molecular alterations in the inflammatory program in both phagocyte subsets following their activation in the ADAP-deficient host. Strikingly, ex vivo phagocytosis assay revealed impaired phagocytic capacity of neutrophils derived from Lm-infected ADAPko mice. Together, our data suggest that an alternative priming of phagocytes in ADAP-deficient mice during Lm infection induces marked alterations in the inflammatory profile of neutrophils and inflammatory monocytes that contribute to enhanced immunopathology while limiting their capacity to eliminate the pathogen and to prevent the fatal outcome of the infection.

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