Response Of Myeloid Cells Research Articles

Immature myeloid/myeloid-suppressor cells and myeloid cell necroptosis mediate lethal pulmonary tularemia

Abstract Inhalation of Francisella tularensis (Ft) causes acute and fatal pneumonia. The lung cytokine milieu favors exponential Ft replication, but mechanisms underlying acute pathogenesis and death remain unknown. Evaluation of the sequential and systemic host immune response in pulmonary tularemia reveals that an overt innate cellular response to Ft, not bacterial burden per se, drives tissue pathology and host mortality. Lethal infection with Ft elicits medullary and extra-medullary myelopoiesis supporting recruitment of large numbers of immature myeloid cells and MDSC to the lungs. These cells fail to mature, but subsequently die leading to necrotic lung damage, loss of pulmonary function, and host death. Ensuing tissue damage and host mortality is Necrostatin sensitive and depletion of dying immature Ly6G+ cells is beneficial, implicating a contribution of necroptotic cell death in tularemia pathogenesis. Accelerated recruitment of immature myeloid cells and/or myeloid cell death may account for the rapid mortality seen with a virulent strain of Ft SchuS4. In contrast, during sub-lethal infection with Ft LVS the pulmonary cellular response is characterized by a predominance of mature neutrophils and monocytes required for protection, suggesting a required threshold for lethal bacterial infection. Further, eliciting a mature phagocyte response provides transient, but dramatic, innate protection against Ft SchuS4. This study reveals that the nature of the innate myeloid cell response may be the primary determinant of host mortality versus survival following Francisella infection

Gli-1/PI3K/AKT/NF-kB pathway mediates resistance to radiation and is a target for reversion of responses in refractory acute myeloid leukemia cells.

Total body irradiation combined with chemotherapy is currently the most effective procedure as a preparative myeloablative regimen. However, resistance to radiotherapy and chemotherapy in refractory acute myeloid leukemia is associated with short-time recurrence after allogeneic hematopoietic stem cell transplantation. To address this issue, we used three cell lines, HL60, HL60/ADR (adriamycin-resistant cells), and HL60/RX (a radiation-resistant cell line established from HL60 cells), as cellular models to investigate the mechanism of the Hedgehog (Hh) signaling pathway resulting in radioresistance, and the efficacy of LDE225 (an inhibitor of the Hh pathway) to enhance radiation sensitivity. Our results indicated that HL60/RX and HL60/ADR cells showed an increased in radioresistance and elevated activity of Hh pathway proteins compared with HL60 cells (P<0.001). In addition, LDE225 significantly reduced clonogenic survival with a sensitivity enhancement ratio (SER) of 1.283 for HL60/ADR and 1.245 for HL60/RX cells. The combination of LDE225 with irradiation significantly increased radiation-induced apoptosis and expression of γ-H2AX and BAK compared with single-treatment groups in both HL60/RX and HL60/ADR cells (P<0.001). In vivo, the combination of LDE225 with irradiation exerted a significant antitumor effect compared with the control and single agents in HL60/RX- and HL60/ADR-xenografted mouse models (P<0.001). Furthermore, our data obtained from western blot and IHC analyses showed that the activation of pAKT and NF-kB was reduced by LDE225 treatment in both HL60/ADR and HL60/RX cells. This demonstrates that the Gli-1/PI3K/AKT/NF-kB pathway plays a key role in resistance to radiation, and that inhibition of the Hh pathway sensitizes cells to radiation by overcoming radioresistance.

Fibronectin-modified surfaces for evaluating the influence of cell adhesion on sensitivity of leukemic cells to siRNA nanoparticles.

This study aimed to create fibronectin (FN)-grafted polymeric surfaces to investigate the influence of leukemic cell adhesion on siRNA treatment. FN was grafted on plasma-treated PTFE surfaces using chemical crosslinkers. Adhesion and growth of chronic myeloid leukemia K562 cells on modified surfaces were investigated. The silencing effect of siRNA/lipid-polymers nanoparticles on cells grown on FN-grafted surfaces was evaluated. Crosslinker-mediated immobilization showed significant FN grafting on surfaces, which provided K562 cell adhesion and growth advantage. siRNA nanoparticle silencing was similarly effective on FN-adhered and suspension-growing K562 cells. This study provided initial data to develop a cell-adhesive system to investigate therapeutic effects on leukemic cells. The response of chronic myeloid leukemia cells to siRNA nanoparticles was independent on cell attachment.

Distinct Dasatinib-Induced Mechanisms of Apoptotic Response and Exosome Release in Imatinib-Resistant Human Chronic Myeloid Leukemia Cells.

Although dasatinib is effective in most imatinib mesylate (IMT)-resistant chronic myeloid leukemia (CML) patients, the underlying mechanism of its effectiveness in eliminating imatinib-resistant cells is only partially understood. This study investigated the effects of dasatinib on signaling mechanisms driving-resistance in imatinib-resistant CML cell line K562 (K562RIMT). Compared with K562 control cells, exsomal release, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling and autophagic activity were increased significantly in K562RIMT cells and mTOR-independent beclin-1/Vps34 signaling was shown to be involved in exosomal release in these cells. We found that Notch1 activation-mediated reduction of phosphatase and tensin homolog (PTEN) was responsible for the increased Akt/mTOR activities in K562RIMT cells and treatment with Notch1 γ-secretase inhibitor prevented activation of Akt/mTOR. In addition, suppression of mTOR activity by rapamycin decreased the level of activity of p70S6K, induced upregulation of p53 and caspase 3, and led to increase of apoptosis in K562RIMT cells. Inhibition of autophagy by spautin-1 or beclin-1 knockdown decreased exosomal release, but did not affect apoptosis in K562RIMT cells. In summary, in K562RIMT cells dasatinib promoted apoptosis through downregulation of Akt/mTOR activities, while preventing exosomal release and inhibiting autophagy by downregulating expression of beclin-1 and Vps34. Our findings reveal distinct dasatinib-induced mechanisms of apoptotic response and exosomal release in imatinib-resistant CML cells.

An Immature Myeloid/Myeloid-Suppressor Cell Response Associated with Necrotizing Inflammation Mediates Lethal Pulmonary Tularemia.

Inhalation of Francisella tularensis (Ft) causes acute and fatal pneumonia. The lung cytokine milieu favors exponential Ft replication, but the mechanisms underlying acute pathogenesis and death remain unknown. Evaluation of the sequential and systemic host immune response in pulmonary tularemia reveals that in contrast to overwhelming bacterial burden or cytokine production, an overt innate cellular response to Ft drives tissue pathology and host mortality. Lethal infection with Ft elicits medullary and extra-medullary myelopoiesis supporting recruitment of large numbers of immature myeloid cells and MDSC to the lungs. These cells fail to mature and die, leading to subsequent necrotic lung damage, loss of pulmonary function, and host death that is partially dependent upon immature Ly6G+ cells. Acceleration of this process may account for the rapid lethality seen with Ft SchuS4. In contrast, during sub-lethal infection with Ft LVS the pulmonary cellular response is characterized by a predominance of mature neutrophils and monocytes required for protection, suggesting a required threshold for lethal bacterial infection. Further, eliciting a mature phagocyte response provides transient, but dramatic, innate protection against Ft SchuS4. This study reveals that the nature of the myeloid cell response may be the primary determinant of host mortality versus survival following Francisella infection.

Role of Granulocyte-Macrophage Colony-Stimulating Factor Signaling in Regulating Neutrophil Antifungal Activity and the Oxidative Burst During Respiratory Fungal Challenge.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that plays a critical role in regulating myeloid cell host defense. In this study, we demonstrated that GM-CSF signaling plays an essential role in antifungal defense against Aspergillus fumigatus. Mice that lack the GM-CSF receptor β chain (GM-CSFRβ) developed invasive hyphal growth and exhibited impaired survival after pulmonary challenge with A. fumigatus conidia. GM-CSFRβ signaling regulated the recruitment of inflammatory monocytes to infected lungs, but not the recruitment of effector neutrophils. Cell-intrinsic GM-CSFRβ signaling mediated neutrophil and inflammatory monocyte antifungal activity, because lung GM-CSFRβ(-/-) leukocytes exhibited impaired conidial killing compared with GM-CSFRβ(+/+) counterparts in mixed bone marrow chimeric mice. GM-CSFRβ(-/-) neutrophils exhibited reduced (hydrogenated) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in vivo. Conversely, administration of recombinant GM-CSF enhanced neutrophil NADPH oxidase function, conidiacidal activity, and lung fungal clearance in A. fumigatus-challenged mice. Thus, our study illustrates the functional role of GM-CSFRβ signaling on lung myeloid cell responses against inhaled A. fumigatus conidia and demonstrates a benefit for systemic GM-CSF administration.

Proinflammatory GM-CSF-producing B cells in multiple sclerosis and B cell depletion therapy.

B cells are not limited to producing protective antibodies; they also perform additional functions relevant to both health and disease. However, the relative contribution of functionally distinct B cell subsets in human disease, the signals that regulate the balance between such subsets, and which of these subsets underlie the benefits of B cell depletion therapy (BCDT) are only partially elucidated. We describe a proinflammatory, granulocyte macrophage-colony stimulating factor (GM-CSF)-expressing human memory B cell subset that is increased in frequency and more readily induced in multiple sclerosis (MS) patients compared to healthy controls. In vitro, GM-CSF-expressing B cells efficiently activated myeloid cells in a GM-CSF-dependent manner, and in vivo, BCDT resulted in a GM-CSF-dependent decrease in proinflammatory myeloid responses of MS patients. A signal transducer and activator of transcription 5 (STAT5)- and STAT6-dependent mechanism was required for B cell GM-CSF production and reciprocally regulated the generation of regulatory IL-10-expressing B cells. STAT5/6 signaling was enhanced in B cells of untreated MS patients compared with healthy controls, and B cells reemerging in patients after BCDT normalized their STAT5/6 signaling as well as their GM-CSF/IL-10 cytokine secretion ratios. The diminished proinflammatory myeloid cell responses observed after BCDT persisted even as new B cells reconstituted. These data implicate a proinflammatory B cell/myeloid cell axis in disease and underscore the rationale for selective targeting of distinct B cell populations in MS and other human autoimmune diseases.

ID: 73: Type I interferon-associated signaling in T cells: The novel functions of STING

The cytoplasmic protein Stimulator of Interferon Genes (STING) plays an essential role in sensing cytoplasmic DNA of intracellular pathogens and initiating IFN type I responses in myeloid cells. It can also directly bind cyclic dinucleotides produced by intracellular bacteria or by the DNA-binding cyclic GMP-AMP (cGAMP) synthase. To date, the signaling mechanisms involving STING activation has been studied in macrophages and dendritic cells. Taking into consideration the fact that STING is highly expressed in the thymus and spleen, we decided to investigate its functional role in T cells. We revealed that STING can be activated in T cells by DMXAA (5,6-dimethylxanthenone-4-acetic acid), which binds to the same site in murine STING as cGAMP and triggers phosphorylation of TBK1 and IRF3 resulting in IFNβ production and increased expression of interferon stimulated genes. In contrast to T lymphocytes obtained from C57B6 WT mice, STING−/− T cells displayed no phosphorylation of TBK1/IRF3, and produced no detectable IFNβ upon stimulation. DMXAA treatment also resulted in IFNgamma secretion by WT cells, while DMXAA-treated knockouts produced only a small amount of IFNgamma comparable to unstimulated WT T cells. Both CD4+ and CD8+ subsets of WT T cells responded to DMXAA with phosphorylation of TBK1/IRF3 and IFNβ production, but only CD4+ cells produced IFNgamma. By performing RNA sequencing, numerous differences in gene expression (including interferon stimulated genes) between WT and STING−/− T cells before and after DMXAA stimulation were identified. The work was supported by the Russian Science Foundation (grant no. 115-15-00100 ).

Cytotoxicity of HBD3 for dendritic cells, normal human epidermal keratinocytes, hTERT keratinocytes, and primary oral gingival epithelial keratinocytes in cell culture conditions.

Human β-defensin 3 (HBD3) is a prominent host defense peptide. In our recent work, we observed that HBD3 modulates pro-inflammatory agonist-induced chemokine and cytokine responses in human myeloid dendritic cells (DCs), often at 20.0 μM concentrations. Since HBD3 can be cytotoxic in some circumstances, it is necessary to assess its cytotoxicity for DCs, normal human epidermal keratinocytes (NHEKs), human telomerase reverse transcriptase (hTERT) keratinocytes, and primary oral gingival epithelial (GE) keratinocytes in different cell culture conditions. Cells, in serum free media with resazurin and in complete media with 10% fetal bovine serum and resazurin, were incubated with 5, 10, 20, and 40 μM HBD3. Cytotoxicity was determined by measuring metabolic conversion of resazurin to resorufin. The lethal dose 50 (LD50, mean μM±Std Err) values were determined from the median fluorescent intensities of test concentrations compared to live and killed cell controls. The LD50 value range of HBD3 was 18.2-35.9 μM in serum-free media for DCs, NHEKs, hTERT keratinocytes, and GE keratinocytes, and >40.0 μM in complete media. Thus, HBD3 was cytotoxic at higher concentrations, which must be considered in future studies of HBD3-modulated chemokine and cytokine responses in vitro.

Altered myeloid and lymphoid immune cell responses following repeated social defeat stress

Dysregulation of the innate immune response has frequently been described in stress-associated psychiatric disorders such as major depressive disorder (MDD). However the kinetics and cellular origin of cytokine and chemokine expression are not well understood. Aim of this study was to elucidate stress-induced changes in the immune response in mice. After 2, 6, or 10 daily social defeats, mice were tested for social interaction, expression levels of immune-related genes were assessed in brain and spleen tissues, and splenocytes and mononuclear cells isolated from the brain were analyzed by flow cytometry. Mice that developed social avoidance showed a decreased expression of genes encoding C-C chemokines accompanied by a reduction in the percentages of activated T lymphocytes and reduced IFN-γ expression levels in the spleens of defeated mice. Also, diminished percentages of CD4+FoxP3+ regulatory T cells (Treg) and lower TGF-beta mRNA levels accumulated in the spleens of these animals. In contrast, enhanced percentages of CD45hiCD11b+ cells representing peripheral myeloid cells immigrated into the brain of defeated animals. In summary, stress induced social avoidance was accompanied by an altered expression of CC chemokine genes and reduced T cell activation in the spleen, associated with an increased brain invasion of myeloid cells. Further studies will address the underlying molecular mechanisms, the cell types involved, and the immune cell phenotype in patients with MDD. This study was supported by Innovative Medizinische Forschung (IMF AM211307)

CCL17-deficiency alters myeloid cell-responses and prevents cognitive decline in APP/PS1-mice

The CC-chemokine CCL17 regulates leukocyte trafficking during inflammation, however, its role in Alzheimerʼs disease (AD) pathogenesis remains undefined. Here we show that CCL17 is enhanced in AD patients when compared to healthy controls. In a mouse model for AD we found that CCL17 controls amyloid β (Aβ) deposition, neuroinflammation, and cognitive decline. CCL17 deficient APP/PS1 mice (APP/PS1-CCL17E/E) showed reduced Aβ brain levels, and were protected against neuronal loss and cognitive deficits. Enhanced microgliosis and brain recruitment of Ly6C+CCR2+ macrophages expressing mannose receptors associated with elevated brain IL-10 levels pointed to beneficial immune responses in these mice. In the absence of CCL17 we observed accelerated uptake of Aβ, enhanced IL-10 release by activated microglia and upregulated Aβ-degrading enzyme neprilysin (NEP) in APP/PS1 brains. These newly identified roles for CCL17 in regulating microglia function and memory loss suggest that targeting this chemokine may harbor therapeutic potential for the treatment of AD.

Stromal-Derived Factors Modulate Ex Vivo Drug Responses of Primary Acute Myeloid Leukemia Cells

Stromal-Derived Factors Modulate Ex Vivo Drug Responses of Primary Acute Myeloid Leukemia Cells

Adenosine Modulates NR4A Orphan Nuclear Receptors To Attenuate Hyperinflammatory Responses in Monocytic Cells.

Adenosine receptor-mediated regulation of monocyte/macrophage inflammatory responses is critical in the maintenance of tissue homeostasis. In this study, we reveal that adenosine potently modulates the expression of NR4A1, 2, and 3 orphan nuclear receptors in myeloid cells, and this modulation is primarily through the adenosine A2a receptor subtype. We demonstrate that A2a receptor activation of NR4A1-3 receptor synthesis is further enhanced in TLR4-stimulated monocytes. After TLR4 stimulation, NR4A receptor-depleted monocyte/macrophage cells display significantly altered expression of cell-surface markers and produce increased inflammatory cytokine and chemokine secretion rendering the cells an enhanced proinflammatory phenotype. Exposure of TLR4 or TNF-α-stimulated monocytes to adenosine analogs directs changes in the expression of MIP-3α and IL-23p19, with NR4A2 depletion leading to significantly enhanced expression of these factors. Furthermore, we establish that nuclear levels of NF-κB/p65 are increased in TLR/adenosine-stimulated NR4A2-depleted cells. We show that, after TLR/adenosine receptor stimulation, NR4A2 depletion promotes significant binding of NF-κB/p65 to a κB consensus binding motif within the MIP-3α proximal promoter leading to increased protein secretion, confirming a pivotal role for NF-κB activity in controlling cellular responses and gene expression outcomes in response to these mediators. Thus, these data demonstrate that during an inflammatory response, adenosine modulation of NR4A receptor activity acts to limit NF-κB-mediated effects and that loss of NR4A2 expression leads to enhanced NF-κB activity and hyperinflammatory responses in myeloid cells.

Abstract CT223: CCX872: Pharmacodynamic study of a potent and selective CCR2 antagonist in human volunteers and plans for phase Ib trial in patients with pancreatic cancer

Abstract Recent clinical results have highlighted the importance of myeloid-derived suppressor cells in enhancing the ability of the host immune system to limit the growth of tumors. Chemokine receptors control the directed trafficking and persistence of peripheral blood mononuclear cells, including effector and suppressor cells, to the tumor microenvironment. Myeloid derived suppressor cells in particular express high levels of the chemokine receptor CCR2, and pre-clinical data suggests that CCR2 plays an important role in the recruitment of these suppressor cells to tumors. Here we report pharmacodynamic details of a potent and selective small molecule antagonist of CCR2, CCX872, and our plans for a phase Ib trial in patients with pancreatic cancer. CCX872 has been successfully evaluated in a phase I single and multiple ascending dose study (3 to 300 mg) in 40 healthy volunteers. All dose levels were well tolerated and safe. CCX872-B showed a dose-linear PK profile. Ex vivo CCR2 occupancy and internalization assays revealed that blood monocytes from CCX872, but not placebo-treated subjects, were impaired in their ability to bind to or internalize exogenously added CCL2, indicating that CCX872 blocked CCR2 in the treated subjects. The 300 mg daily dose of CCX872 exhibited 104 ± 3% blockade at 2 hours and 93 ± 7% blockade at 24 hours. We calculated that 150 mg twice a day would provide over 90% CCR2 inhibition at all times. Analysis of data from the Tumor Cancer Genome Atlas revealed high levels of expression of CCL2 in pancreatic tumors, and co-expression of CCR2 with monocyte/macrophage markers such as CD68 and CSF1R. In pre-clinical studies, we found that a CCR2 antagonist reduced the growth of the pancreatic tumor cell line, PancO2, in rodent xenograft models. Based in part on these data we have initiated a phase Ib single arm, open label, multicenter study of CCX872 in patients with un-resectable pancreatic cancer. In Part A, subjects will receive a single dose of CCX872 for pharmacokinetic and pharmacodynamics analyses. In Part B, up to 50 subjects will be treated for at least 12 weeks with CCX872 in addition to FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin). Responders and those with stable disease after 12 weeks will continue treatment until disease progression, unacceptable toxicity or death. The primary endpoints are safety/tolerability and efficacy (progression-free rate at 6 months based on RECIST criteria). Other endpoints include overall survival, and effect on the tumor microenvironment and immunological biomarkers in the blood. This trial will test the hypothesis that CCR2 plays an important role in enabling the myeloid suppressor cell response to pancreatic cancer, and will determine if addition of a CCR2 antagonist can enhance the standard of care for these patients Citation Format: Anne-Marie Duliege, Stefan Sleijfer, Ashley Bischof, Joanne Tan, Penglie Zhang, Lisa Seitz, Daniel Dairaghi, Pirow Bekker, Israel Charo, Thomas Schall. CCX872: Pharmacodynamic study of a potent and selective CCR2 antagonist in human volunteers and plans for phase Ib trial in patients with pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT223. doi:10.1158/1538-7445.AM2015-CT223

Insight into the molecular mechanism of heme oxygenase-1 induction by docosahexaenoic acid in U937 cells

Heme oxygenase-1 (HO-1) has anti-inflammatory effects on myeloid cells in response to various stimuli. To date, little is known about whether fatty acids can affect HO-1 induction. Here, we report the induction of HO-1 by docosahexaenoic acid (DHA) and the associated molecular mechanisms in human myelomonocytic lymphoma U937 cells. When U937 cells were treated with DHA, eicosapentaenoic acid, palmitic acid or oleic acid, DHA was the most effective inducer of HO-1. The activation of AKT and glycogen synthase kinase-3β did not significantly change after DHA treatment. However, DHA increased the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), but not of other mitogen-activated protein kinases such as p38 and JNK. The increase in HO-1 expression was significantly inhibited by U0126, an ERK1/2 inhibitor. Nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2) and its binding to the HO-1 promoter significantly increased upon DHA treatment. An increase in intracellular reactive oxygen species was detected by dichlorofluorescein diacetate, but not by hydroethidium or 2-[6-(4-hydroxy)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid after DHA treatment. Pretreatment with NAC dramatically inhibited the ERK1/2 activation, binding of Nrf-2 to antioxidant response elements (AREs) located in the HO-1 promoter and the induction of HO-1 by DHA. In conclusion, DHA increased HO-1 expression in U937 cells via activation of ERK1/2 and increased Nrf-2 binding to ARE in the HO-1 promoter. These findings will help develop better strategies for treating inflammatory disorders with DHA.

Lactate and short chain fatty acids produced by microbial fermentation downregulate proinflammatory responses in intestinal epithelial cells and myeloid cells

The use of short chain fatty acids to modulate gastrointestinal inflammatory conditions such as ulcerative colitis has produced encouraging results either in animal models or also in clinical trials. Identifying the key cellular and molecular targets of this activity will contribute to establish the appropriate combinations/targeting strategies to maximize the efficacy of anti-inflammatory interventions. In the present work, we evaluated in vitro the interaction of lactate, acetate, propionate and butyrate on cells relevant for innate immune response of the gastrointestinal tract. All molecules tested regulate the production of proinflammatory cytokines by TLR-4 and TLR-5 activated intestinal epithelial cells in a dose response manner. Furthermore SCFAs and lactate modulate cytokine secretion of TLR-activated bone marrow derived macrophages and also TLR-dependent CD40 upregulation in bone marrow derived dendritic in a dose-dependent manner. Butyrate and propionate have been effective at concentrations of 1 to 5mM whereas acetate and lactate produced modulatory effects at concentrations higher than 20–50mM in different assays. Our results indicate that in concentrations similar to found in large bowel lumen, all SCFAs tested and lactate can modulate activity of relevant sentinel cell types activated by TLR signals. Modulatory activity was not inhibited by pertussis toxin treatment indicating that the effects are not related to Gi signaling. The use of these molecules in combined or separately as intervention strategy in conditions where epithelial or myeloid cells are main triggers of the inflammatory situation seems appropriate.

The Nlrp3 inflammasome admits defeat

The Nlrp3 inflammasome triggers interleukin-1 secretion by myeloid cells in response to endogenous and exogenous danger signals. Two recent studies identified the sulfonylurea MCC950 and the ketone metabolite β-hydroxybutyrate as specific inhibitors of the Nlrp3 inflammasome, with promising therapeutic potential for the treatment of auto-inflammatory diseases.

The role of STING in T lymphocytes (IRM5P.643)

Abstract The cytoplasmic protein Stimulator of Interferon Genes (STING) plays an essential role in sensing intracellular pathogens and initiating type I IFN responses in myeloid cells. STING detects the presence of cytoplasmic DNA presented by various DNA-binding proteins (e.g. IFI16, DDX41, DAI, RNA Pol III); it also directly binds cyclic dinucleotides produced by intracellular bacteria or by the DNA-binding cyclic GAMP synthase (c-GAS). Once activated, STING phosphorylates TBK1 and IRF3 to trigger type I IFN production. To date STING has only been studied in macrophages and dendritic cells, but early reports showed expression of STING in a variety of tissues including the thymus and spleen, prompting us to ask whether STING might have a functional role in T cells, which account for the majority of cells in the thymus and a larger proportion of cells in the spleen than macrophages and dendritic cells. Here, we show that STING can be activated in T cells by the small molecule DMXAA, resulting in IFNβ production and increased expression of numerous interferon stimulated genes (ISG)—the first evidence to our knowledge of type I IFN production by T cells. Furthermore, RNA-sequencing data revealed unanticipated differences between unstimulated wild type and STING-/- T cells, with STING -/- T cells exhibiting increased expression of genes associated with cytotoxic T lymphocytes, indicating that STING-mediated signaling in T cells may regulate the development of particular T cell subsets.

Abstract 220: Deficiency in Triggering Receptor Expressed in Myeloid Cells-1 Substantially Attenuates Atherosclerotic Lesion Development

TREM-1 is a potent amplifier of pro-inflammatory responses in myeloid cells. While the significance of TREM-1 in acute microbial-induced septic shock has long been acknowledged, our previous studies in experimental models of colitis have also suggested an important role for TREM-1 in driving non-infectious and chronic inflammatory diseases. Here, we aim to define the role of TREM-1 in atherosclerosis, which is recognized as a chronic inflammatory disorder of the arteries with a major involvement of monocytes and macrophages. TREM-1-deficient (Trem1-/-) mice were backcrossed with apolipoprotein E-deficient (Apoe-/-) mice and were fed a high cholesterol/fat diet for 16 weeks. Compared with Trem1+/+ Apoe-/- mice, Trem1-/- Apoe-/- mice were substantially protected from diet-induced atherosclerosis. Interestingly, while the number of individual atherosclerotic plaques did not differ between the two groups of mice, the average lesion size and total surface area of atherosclerotic lesions were significantly reduced in Trem1-/- Apoe-/- mice. Moreover, analysis of early atherosclerotic lesions revealed no difference between the two groups. Hence, TREM-1 appears to be involved in the progression, rather than the initiation of atherosclerotic lesions. In line with the reduced lesion size, FACS analysis of aortic cell preparations revealed substantially lower frequencies of infiltrating CD11b+ myeloid cells in Trem1-/- Apoe-/- mice. To gain insight into the underlying mechanisms of TREM-1-mediated effects in atherogenesis, we are currently analyzing mRNA expression profiles of aortic segments derived from Trem1+/+ Apoe-/- and Trem1-/- Apoe-/- mice. These studies will be complemented by immunohistochemistry stainings and by in vitro studies that are aimed to directly determine the impact of TREM-1 on foam cell generation and cholesterol efflux.

Supraphysiological myeloid cell activation during mycobacterial immune reconstitution inflammatory syndrome (IRC4P.456)

Abstract Anti-retroviral therapy (ART) of HIV-infected individuals usually leads to clinical improvement, but some individuals develop a worsening of symptoms following ART, referred to as Immune Reconstitution Inflammatory Syndrome (IRIS). CD4 T cells are thought to be a primary mediator of this paradoxical disease and recent work in patients has correlated activated monocytes with the development of IRIS. Here we use a mouse model of Mycobacterium avium associated IRIS to examine myeloid cell responses in IRIS. We find that CD4 T cell reconstitution of chronically infected TCRaKO mice leads to large increases in neutrophils and Ly6C+ CCR2+ monocytes. Prior to CD4 T cell transfer, myeloid cells in chronically infected TCRaKO mice expressed less iNOS but higher TNF compared to WT mice. Within ~12 days after CD4 T cell reconstitution, multiple myeloid cell subsets dramatically upregulated iNOS and TNF reaching levels far above what is ever observed in WT mice at any time during infection. Transfer of IFNgKO CD4 T cells into chronically infected TCRaKO mice failed to drive myeloid cell expansion or increased function, and mice displayed greatly reduced signs of illness. These data indicate that progression of a chronic mycobacterial infection in the absence of CD4 T cell help predisposes myeloid cells to over respond to T cell stimulation when CD4 T cells are later introduced, indicating myeloid cell hyper responsiveness may underly the immunopathogenesis of IRIS.