Priming Of Monocytes Research Articles

Ly6Chi Monocytes Are Metabolically Reprogrammed in the Blood during Inflammatory Stimulation and Require Intact OxPhos for Chemotaxis and Monocyte to Macrophage Differentiation.

Acute inflammation is a rapid and dynamic process involving the recruitment and activation of multiple cell types in a coordinated and precise manner. Here, we investigate the origin and transcriptional reprogramming of monocytes using a model of acute inflammation, zymosan-induced peritonitis. Monocyte trafficking and adoptive transfer experiments confirmed that monocytes undergo rapid phenotypic change as they exit the blood and give rise to monocyte-derived macrophages that persist during the resolution of inflammation. Single-cell transcriptomics revealed significant heterogeneity within the surface marker-defined CD11b+Ly6G-Ly6Chi monocyte populations within the blood and at the site of inflammation. We show that two major transcriptional reprogramming events occur during the initial six hours of Ly6Chi monocyte mobilisation, one in the blood priming monocytes for migration and a second at the site of inflammation. Pathway analysis revealed an important role for oxidative phosphorylation (OxPhos) during both these reprogramming events. Experimentally, we demonstrate that OxPhos via the intact mitochondrial electron transport chain is essential for murine and human monocyte chemotaxis. Moreover, OxPhos is needed for monocyte-to-macrophage differentiation and macrophage M(IL-4) polarisation. These new findings from transcriptional profiling open up the possibility that shifting monocyte metabolic capacity towards OxPhos could facilitate enhanced macrophage M2-like polarisation to aid inflammation resolution and tissue repair.

Interrupting an IFN-γ-dependent feedback loop in the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne

ObjectivesTo study the molecular pathogenesis of PAPA (pyogenic arthritis, pyoderma gangrenosum and acne) syndrome, a debilitating hereditary autoinflammatory disease caused by dominant mutation in PSTPIP1.MethodsGene knock-out and knock-in mice were...

Interferons and epigenetic mechanisms in training, priming and tolerance of monocytes and hematopoietic progenitors.

Training and priming of innate immune cells involve preconditioning by PAMPs, DAMPs, and/or cytokines that elicits stronger induction of inflammatory genes upon secondary challenge. Previous models distinguish training and priming based upon whether immune activation returns to baseline prior to secondary challenge. Tolerance is a protective mechanism whereby potent stimuli induce refractoriness to secondary challenge. Training and priming are important for innate memory responses that protect against infection, efficacy of vaccines, and maintaining innate immune cells in a state of readiness; tolerance prevents toxicity from excessive immune activation. Dysregulation of these processes can contribute to pathogenesis of autoimmune/inflammatory conditions, post-COVID-19 hyperinflammatory states, or sepsis-associated immunoparalysis. Training, priming, and tolerance regulate similar "signature" inflammatory genes such as TNF, IL6, and IL1B and utilize overlapping epigenetic mechanisms. We review how interferons (IFNs), best known for activating JAK-STAT signaling and interferon-stimulated genes, also play a key role in regulating training, priming, and tolerance via chromatin-mediated mechanisms. We present new data on how monocyte-to-macrophage differentiation modulates IFN-γ-mediated priming, affects regulation of AP-1 and CEBP activity, and attenuates superinduction of inflammatory genes. We present a "training-priming continuum" model that integrates IFN-mediated priming into current concepts about training and tolerance and proposes a central role for STAT1 and IRF1.

Early monocyte response following local ablation in hepatocellular carcinoma.

Local ablative therapies are established treatment modalities in the treatment of early- and intermediate-stage hepatocellular carcinoma (HCC). Systemic effects of local ablation on circulating immune cells may contribute to patients' response. Depending on their activation, myeloid cells are able to trigger HCC progression as well as to support anti-tumor immunity. Certain priming of monocytes may already occur while still in the circulation. By using flow cytometry, we analyzed peripheral blood monocyte cell populations from a prospective clinical trial cohort of 21 HCC patients following interstitial brachytherapy (IBT) or radiofrequency ablation (RFA) and investigated alterations in the composition of monocyte subpopulations and monocytic myeloid-derived suppressor cells (mMDSCs) as well as receptors involved in orchestrating monocyte function. We discovered that mMDSC levels increased following both IBT and RFA in virtually all patients. Furthermore, we identified varying alterations in the level of monocyte subpopulations following radiation compared to RFA. (A) Liquid biopsy liquid biopsy of circulating monocytes in the future may provide information on the inflammatory response towards local ablation as part of an orchestrated immune response.

Gut commensal Limosilactobacillus reuteri induces atypical memory-like phenotype in human dendritic cells in vitro

ABSTRACT Memory-like responses in innate immune cells confer nonspecific protection against secondary exposures. A number of microbial agents have been found to induce enhanced or diminished recall responses in innate cells, however, studies investigating the ability of probiotic bacteria to trigger such effects are lacking. Here, we show that priming of human monocytes with a secretome from the gut probiotic bacterium Limosilactobacillus (L.) reuteri induces a mixed secondary response phenotype in monocyte-derived dendritic cells (mo-DCs), with a strong IL-6 and IL-1β response but low TNFα, IL-23 and IL-27 secretion. Instead, blood DC priming with L. reuteri-secretome resembles a tolerant state upon secondary exposure. A similar pattern was found in conventional and gut-like (retinoic acid exposed) DCs, although retinoic acid hampered TNFα and IL-6 production and enrichment of histone modifications in L. reuteri-secretome primed mo-DC cultures. Further, we show that the memory-like phenotype of mo-DCs, induced by priming stimuli, is important for subsequent T helper (Th) cell differentiation pathways and might determine the inflammatory nature of Th cells. We also show enhanced recall responses characterized by robust inflammatory cytokines and lactate production in the gut-like mo-DCs derived from β-glucan primed monocytes. Such responses were accompanied with enriched histone modifications at the promoter of genes associated with a trained phenotype in myeloid cells. Altogether, we demonstrate that a gut commensal-derived secretome prompts recall responses in human DCs which differ from that induced by classical training agents such as β-glucan. Our results could be beneficial for future therapeutic interventions where T cell responses are needed to be modulated.

Single-Cell Analysis of the In Vivo Dynamics of Host Circulating Immune Cells Highlights the Importance of Myeloid Cells in Avian Flaviviral Infection.

Ducks are an economically important waterfowl but a natural reservoir for some zoonotic pathogens, such as influenza virus and flaviviruses. Our understanding of the duck immune system and its interaction with viruses remains incomplete. In this study, we constructed the transcriptomic landscape of duck circulating immune cells, the first line of defense in the arthropod-borne transmission of arboviruses, using high-throughput single-cell transcriptome sequencing, which defined 14 populations of peripheral blood leukocytes (PBLks) based on distinct molecular signatures and revealed differences in the clustering of PBLks between ducks and humans. Taking advantage of in vivo sex differences in the susceptibility of duck PBLks to avian tembusu virus (TMUV) infection, a mosquito-borne flavivirus newly emerged from ducks with a broad host range from mosquitos to mammals, a comprehensive comparison of the in vivo dynamics of duck PBLks upon TMUV infection between sexes was performed at the single-cell level. Using this in vivo model, we discovered that TMUV infection reprogrammed duck PBLks differently between sexes, driving the expansion of granulocytes and priming granulocytes and monocytes for antiviral immune activation in males but decreasing the antiviral immune activity of granulocytes and monocytes by restricting their dynamic transitions from steady states to antiviral states with a decrease in the abundance of circulating monocytes in females. This study provides insights into the initial immune responses of ducks to arthropod-borne flaviviral infection and provides a framework for studying duck antiviral immunity.

Fingolimod Phosphate (FTY720-P) Activates Protein Phosphatase 2A in Human Monocytes and Inhibits Monosodium Urate Crystal-Induced Interleukin-1β Production.

Gout is a chronic inflammatory arthritis caused by monosodium urate monohydrate (MSU) crystal deposits in joints of lower limbs. Phagocytic uptake of MSU crystals by joint-resident macrophages and recruited circulating monocytes results in IL-1β expression and production. Current acute gout treatments have serious toxicities and suffer suboptimal clinical outcomes. Protein phosphatase 2A (PP2A) plays an important role in regulating signaling pathways relevant to inflammation. We hypothesized that innate immune danger signals, e.g., lipopolysaccharide (LPS) and soluble uric acid (sUA), prime human monocytes toward MSU crystal phagocytosis and that increased IL-1β production mediated by a reduction in PP2A activity and restoring PP2A activity exerts an anti-inflammatory effect in this setting. Priming monocytes with LPS + sUA increased cytosolic pro-IL-1β and mature IL-1β and enhanced MSU crystal phagocytosis and its downstream IL-1β expression (P < 0.001). A combination of LPS + sUA priming and MSU crystals reduced PP2A activity in monocytes by 60% (P = 0.013). PP2A catalytic subunit gene knockdown reduced PP2A activity and exacerbated MSU crystal-induced IL-1β expression and secretion (P < 0.0001). Fingolimod (FTY720) and its active metabolite, fingolimod phosphate (FTY720-P), were evaluated for their ability to activate PP2A in human monocytes over 24 hours. FTY720 and FTY720-P activated PP2A to a similar extent, and maximal enzyme activity occurred at 24 hours for FTY720 and at 6 hours for FTY720-P. FTY720-P (2.5 μM) reduced pro-IL-1β production and IL-1β secretion in primed and MSU crystal-stimulated monocytes (P < 0.0001) without changing the magnitude of crystal phagocytosis. We conclude that PP2A is a promising new target in acute gout. SIGNIFICANCE STATEMENT: The activity of protein phosphatase 2A (PP2A) is implicated in the enhanced expression and production of IL-1β by human monocytes in response to priming with soluble uric acid and lipopolysaccharide and phagocytosis of monosodium urate monohydrate (MSU) crystals. Fingolimod phosphate activates PP2A in human monocytes and reduces cytosolic pro-IL-1β content and its conversion to biologically active IL-1β in human monocytes exposed to MSU crystals.

Fingolimod (FTY720) Activates Protein Phosphatase‐2A in Human Monocytes and Inhibits Uric Acid Crystal‐Induced Interleukin‐1 Beta Production

IntroductionGout is a chronic inflammatory disease due to monosodium urate (MSU) crystal deposits in articular joints. Phagocytosis of MSU crystals by monocytes/macrophages results in downstream expression and production of interleukin‐1 beta (IL‐1β). The two‐step process of gout inflammation involves 1) priming of monocytes with danger signals e.g. lipopolysaccharide (LPS) or soluble uric acid (UA) and 2) MSU phagocytosis and activation of the NLRP3 inflammasome in monocytes. Protein‐phosphatase‐2A (PP2A) is a serine/threonine phosphatase, and a reduction in intracellular macrophage PP2A activity was shown to aggravate LPS‐induced proinflammatory cytokine production. The prodrug Fingolimod (FTY720) and its phosphorylated active metabolite (p‐FTY720) are efficacious in multiple sclerosis and their mechanism of action involves activation of intracellular PP2A. The objective of this study is to investigate the role of PP2A in regulating monocyte priming and activation by MSU crystals and whether intracellular PP2A activation exerts an anti‐inflammatory effect in MSU‐stimulated monocytes.MethodsHuman THP‐1 monocytes (ATCC; 500,000 cells per well) were treated with UA (50 mg/dL) and LPS (10ng/mL) for 24 hours. Subsequently, cells were treated with MSU crystals (100ug/mL) and MSU phagocytosis was determined at 4 hours using flow cytometry and the percentage of cells displaying side‐scattering above a threshold value, indicative of crystal phagocytosis, were determined. Intracellular PP2A activity was determined at 6 hours following MSU stimulation using the PP2A Immunoprecipitation Phosphatase Assay Kit (Fisher Scientific). IL‐1β expression and protein levels at 6 hours were determined using quantitative polymerase chain reaction and ELISA, respectively. THP‐1 monocytes were treated with FTY720 or p‐FTY720 (2.5mM for both treatments; Cayman Chemicals) and PP2A activity was determined at 1, 3, 6 and 24 hours. THP‐1 monocytes were treated with UA+LPS followed by MSU ± p‐FTY720 (2.5mM for 6 hours) and IL‐1β expression and protein levels were determined. Statistical analyses were performed using analysis of variance (ANOVA) with Tukey’s post‐hoc test.ResultsUA+LPS pre‐treatment increased MSU phagocytosis by THP‐1 monocytes (fig. 1A and 1B; p&lt;0.001) and IL‐1β expression (fig. 1C; p&lt;0.001) and production (fig. 1D; p&lt;0.001). This effect was associated with a reduction in intracellular PP2A activity (fig. 1E; p&lt;0.001). FTY720 and p‐FTY720 increased intracellular PP2A activity in monocytes (fig. 2A; p&lt;0.001) with a higher peak effect achieved at 1 hour for p‐FTY720 (p&lt;0.01). p‐FTY720 treatment reduced IL‐1β expression (fig. 2B; p&lt;0.001) and mature IL‐1β production (fig. 2C; p&lt;0.001) in UA+LPS pre‐treated human monocytes following MSU stimulation.ConclusionUA and LPS enhanced MSU phagocytosis, expression and production of IL‐1β, potentially via a reduction in intracellular PP2A activity. FTY720 and p‐FTY720 activated PP2A in monocytes with an onset of effect of 1 hour and correspondingly reduced IL‐1β expression and production. PP2A is a novel therapeutic target for gout treatment.Impact of soluble uric acid (UA) and lipopolysaccharide (LPS) pre‐treatments on THP‐1 monocyte phagocytic activity towards monosodium urate (MSU) crystals, interleukin‐1 beta (IL‐1β) expression and production and association with intracellular protein phosphatase‐2A (PP2A) activity. A) Representative flow cytometry scatter plots showing enhanced MSU crystal phagocytosis (as shown by more cells with increased side‐scatter in P2 region of interest) by THP‐1 monocytes following UA+LPS pre‐treatment. B) UA and UA+LPS pre‐treatment increased the percentage of THP‐1 monocytes positive for MSU phagocytosis. C) UA+LPS pre‐treatment increased IL‐1β expression by THP‐1 monocytes following MSU challenge. D) UA+LPS pre‐treatment increased IL‐1β production by THP‐1 monocytes following MSU challenge. E) Intracellular PP2A activity was reduced in UA or UA+LPS pre‐treated THP‐1 monocytes. *p&lt;0.001; **p&lt;0.01; n.s.: non significant.Figure 1Impact of FTY720 (Fingolimod; prodrug) and phosphorylated FTY720 (p‐FTY‐720; active metabolite) treatments on protein phosphatase‐2A (PP2A) activity in human THP‐1 monocytes and impact of p‐FTY720 treatment on interleukin‐1 beta (IL‐1β) expression and production by monosodium urate (MSU) crystal stimulated THP‐1 monocytes ± soluble uric acid (UA) and lipopolysaccharide (LPS) pre‐treatment. Treatments were conducted using 2.5μM concentrations. A) FTY720 and p‐FTY720 increased intracellular PP2A activity in THP‐1 monocytes. B) p‐ FTY720 reduced IL‐1β expression in THP‐1 monocytes. C) p‐FTY720 reduced IL‐1β production in THP‐1 monocytes. *p&lt;0.001; **p&lt;0.01; n.s.: non significant.Figure 2

Salt increases monocyte CCR2 expression and inflammatory responses in humans.

Inflammation may play a role in the link between high salt intake and its deleterious consequences. However, it is unknown whether salt can induce proinflammatory priming of monocytes and macrophages in humans. We investigated the effects of salt on monocytes and macrophages in vitro and in vivo by performing a randomized crossover trial in which 11 healthy human subjects adhered to a 2-week low-salt and high-salt diet. We demonstrate that salt increases monocyte expression of CCR2, a chemokine receptor that mediates monocyte infiltration in inflammatory diseases. In line with this, we show a salt-induced increase of plasma MCP-1, transendothelial migration of monocytes, and skin macrophage density after high-salt diet. Macrophages demonstrate signs of an increased proinflammatory phenotype after salt exposure, as represented by boosted LPS-induced cytokine secretion of IL-6, TNF, and IL-10 in vitro, and by increased HLA-DR expression and decreased CD206 expression on skin macrophages after high-salt diet. Taken together, our data open up the possibility for inflammatory monocyte and macrophage responses as potential contributors to the deleterious effects of high salt intake.

Extracellular vesicles from regenerative human cardiac cells act as potent immune modulators by priming monocytes

BackgroundNano-sized vesicles, so called extracellular vesicles (EVs), from regenerative cardiac cells represent a promising new therapeutic approach to treat cardiovascular diseases. However, it is not yet sufficiently understood how cardiac-derived EVs facilitate their protective effects. Therefore, we investigated the immune modulating capabilities of EVs from human cardiac-derived adherent proliferating (CardAP) cells, which are a unique cell type with proven cardioprotective features.ResultsDifferential centrifugation was used to isolate EVs from conditioned medium of unstimulated or cytokine-stimulated (IFNγ, TNFα, IL-1β) CardAP cells. The derived EVs exhibited typical EV-enriched proteins, such as tetraspanins, and diameters mostly of exosomes (< 100 nm). The cytokine stimulation caused CardAP cells to release smaller EVs with a lower integrin ß1 surface expression, while the concentration between both CardAP-EV variants was unaffected. An exposure of either CardAP-EV variant to unstimulated human peripheral blood mononuclear cells (PBMCs) did not induce any T cell proliferation, which indicates a general low immunogenicity. In order to evaluate immune modulating properties, PBMC cultures were stimulated with either Phytohemagglutin or anti-CD3. The treatment of those PBMC cultures with either CardAP-EV variant led to a significant reduction of T cell proliferation, pro-inflammatory cytokine release (IFNγ, TNFα) and increased levels of active TGFβ. Further investigations identified CD14+ cells as major recipient cell subset of CardAP–EVs. This interaction caused a significant lower surface expression of HLA-DR, CD86, and increased expression levels of CD206 and PD-L1. Additionally, EV-primed CD14+ cells released significantly more IL-1RA. Notably, CardAP-EVs failed to modulate anti-CD3 triggered T cell proliferation and pro-inflammatory cytokine release in monocultures of purified CD3+ T cells. Subsequently, the immunosuppressive feature of CardAP-EVs was restored when anti-CD3 stimulated purified CD3+ T cells were co-cultured with EV-primed CD14+ cells. Beside attenuated T cell proliferation, those cultures also exhibited a significant increased proportion of regulatory T cells.ConclusionsCardAP-EVs have useful characteristics that could contribute to enhanced regeneration in damaged cardiac tissue by limiting unwanted inflammatory processes. It was shown that the priming of CD14+ immune cells by CardAP-EVs towards a regulatory type is an essential step to attenuate significantly T cell proliferation and pro-inflammatory cytokine release in vitro.

BLTR1 in Monocytes Emerges as a Therapeutic Target For Vascular Inflammation With a Subsequent Intimal Hyperplasia in a Murine Wire-Injured Femoral Artery

Given the importance of high-mobility group box 1 (HMGB1) and 5-lipoxygenase (5-LO) signaling in vascular inflammation, we investigated the role of leukotriene signaling in monocytes on monocyte-to-macrophage differentiation (MMD) induced by HMGB1, and on vascular inflammation and subsequent intimal hyperplasia in a mouse model of wire-injured femoral artery. In cultured primary bone marrow-derived cells (BMDCs) stimulated with HMGB1, the number of cells with macrophage-like morphology was markedly increased in association with an increased expression of CD11b/Mac-1, which were attenuated in cells pre-treated with Zileuton, a 5-LO inhibitor as well as in 5-LO-deficient BMDCs. Of various leukotriene receptor inhibitors examined, which included leukotriene B4 receptors (BLTRs) and cysteinyl leukotriene receptors (cysLTRs), the BLTR1 inhibitor (U75302) exclusively suppressed MMD induction by HMGB1. The importance of BLTR1 in HMGB1-induced MMD was also observed in BMDCs isolated from BLTR1-deficient mice and BMDCs transfected with BLTR1 siRNA. Although leukotriene B4 (LTB4) had minimal direct effects on MMD in control and 5-LO-deficient BMDCs, MMD attenuation by HMGB1 in 5-LO-deficient BMDCs was significantly reversed by exogenous LTB4, but not in BLTR1-deficient BMDCs, suggesting that LTB4/BLTR1-mediated priming of monocytes is a prerequisite of HMGB1-induced MMD. In vivo, both macrophage infiltration and intimal hyperplasia in our wire-injured femoral artery were markedly attenuated in BLTR1-deficient mice as compared with wild-type controls, but these effects were reversed in BLTR1-deficient mice transplanted with monocytes from control mice. These results suggest that BLTR1 in monocytes is a pivotal player in MMD with subsequent macrophage infiltration into neointima, leading to vascular remodeling after vascular injury.

Uric acid priming in human monocytes is driven by the AKT–PRAS40 autophagy pathway

Metabolic triggers are important inducers of the inflammatory processes in gout. Whereas the high serum urate levels observed in patients with gout predispose them to the formation of monosodium urate (MSU) crystals, soluble urate also primes for inflammatory signals in cells responding to gout-related stimuli, but also in other common metabolic diseases. In this study, we investigated the mechanisms through which uric acid selectively lowers human blood monocyte production of the natural inhibitor IL-1 receptor antagonist (IL-1Ra) and shifts production toward the highly inflammatory IL-1β. Monocytes from healthy volunteers were first primed with uric acid for 24 h and then subjected to stimulation with lipopolysaccharide (LPS) in the presence or absence of MSU. Transcriptomic analysis revealed broad inflammatory pathways associated with uric acid priming, with NF-κB and mammalian target of rapamycin (mTOR) signaling strongly increased. Functional validation did not identify NF-κB or AMP-activated protein kinase phosphorylation, but uric acid priming induced phosphorylation of AKT and proline-rich AKT substrate 40 kDa (PRAS 40), which in turn activated mTOR. Subsequently, Western blot for the autophagic structure LC3-I and LC3-II (microtubule-associated protein 1A/1B-light chain 3) fractions, as well as fluorescence microscopy of LC3-GFP-overexpressing HeLa cells, revealed lower autophagic activity in cells exposed to uric acid compared with control conditions. Interestingly, reactive oxygen species production was diminished by uric acid priming. Thus, the Akt-PRAS40 pathway is activated by uric acid, which inhibits autophagy and recapitulates the uric acid-induced proinflammatory cytokine phenotype.

Zymosan tolerizes primary pig monocytes to lipopolysaccharide stimulation

Abstract Exposure of monocytes to β-1,3 glucans from yeast cell wall can enhance the immune response to heterologous innate agonists, a phenomenon termed innate training. This is in contrast to tolerance, which is well described in relation to lipopolysaccharide (LPS) responses. Yeast extracts are fed to food animals for production and immune benefits, but the mechanism of immune modulation provided by dietary yeast is not clearly defined. With the recent limitations to antibiotic use in food production animals, alternatives are sought to maintain animal health and production. To elucidate the immunological effect of yeast β-glucans on swine monocytes when re-exposed to a heterologous agonist, we primed primary swine monocytes with varying amounts and sources of β-glucan for 24h in vitro and restimulated the cells 5d later to LPS. Monocytes primed with media only or LPS, and exposed to LPS 5d later, served as controls to delineate trained versus tolerant responses. Swine monocytes primed with LPS produced IL-1β and TNF-α, and transitioned to a tolerant state, with decreased production of IL-1β and TNF-α upon re-stimulation with LPS. Interestingly, monocytes primed with the commercial β-glucan, zymosan (S. cerevisiae), at 10mg/mL or higher and restimulated with LPS exhibited a tolerized state. Specifically, zymosan primed cells did not produce IL-1β or TNF-α in response to LPS restimulation. Monocytes primed with laminarin (L. digitata) and restimulated with LPS did not exhibit a trained or tolerant state. These data indicate that dosage and source of β-glucan play an important role in the priming of monocytes to secondary LPS exposure, and warrants further study to elucidate differential responses as they relate to commercial dietary β-glucans.

Respiratory Syncytial Virus Infection Triggers Epithelial HMGB1 Release as a Damage-Associated Molecular Pattern Promoting a Monocytic Inflammatory Response.

RSV is a major cause of serious lower respiratory tract infections in young children and causes severe respiratory morbidity and mortality in the elderly. In addition, to date there is no effective treatment or vaccine available for RSV infection. The mechanisms responsible for RSV-induced acute airway disease and associated long-term consequences remain largely unknown. The oxidative stress response in the airways plays a major role in the pathogenesis of RSV. HMGB1 is a ubiquitous redox-sensitive multifunctional protein that serves as both a DNA regulatory protein and an extracellular cytokine signaling molecule that promotes airway inflammation as a damage-associated molecular pattern. This study investigated the mechanism of action of HMGB1 in RSV infection with the aim of identifying new inflammatory pathways at the molecular level that may be amenable to therapeutic interventions.

Moderate-intensity exercise alters markers of alternative activation in circulating monocytes in females: a putative role for PPARγ.

PurposeMonocytes may be primed towards differentiation into classically activated M1 macrophages or alternatively activated M2 macrophages. M1 macrophages greatly contribute to the inflammation which promotes insulin resistance, whereas M2 macrophages resolve inflammation. We have previously shown that exercise increases M2 marker expression in mixed mononuclear cells, possibly via activation of the nuclear transcription factor PPARγ. However, these effects have not been demonstrated specifically within monocytes. Thus, we aimed to investigate whether moderate-intensity exercise elicited similar effects on monocytic M1/M2 marker expression and PPARγ activity to those reported previously in mononuclear cells, so as to further elucidate the mechanisms by which exercise may alter inflammatory status and, accordingly, prevent insulin resistance.Methods/results19 sedentary females completed an 8 week moderate-intensity exercise programme (walking 45 min, thrice weekly). Monocytes were isolated from blood via immunomagnetic separation; monocyte expression of M2 markers (Dectin-1: 2.6 ± 1.9-fold; IL-10: 3.0 ± 2.8-fold) significantly increased, whilst the expression of the M1 marker MCP-1 significantly decreased (0.83 ± 0.2 cf. basal), over the duration of the programme. Serum PPARγ activity levels and PPARγ target-genes (CD36: 1.9 ± 1.5-fold; LXRα: 5.0 ± 4.7-fold) were significantly increased after the 8 week exercise programme. Associated with these effects were significant improvements in systemic insulin sensitivity (McAuley’s ISI: Δ0.98 M/mU/L cf. basal).ConclusionExercise participation suppressed M1 markers and induced M2 markers in monocytes, potentially via PPARγ-triggered signalling, and these effects may contribute (perhaps via priming of monocytes for differentiation into M2 tissue-macrophages) to improved systemic insulin sensitivity in exercising participants. These findings provide an alternative mechanism by which exercise may exert its anti-inflammatory effects in order to prevent insulin resistance and type 2 diabetes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00421-016-3414-y) contains supplementary material, which is available to authorized users.

Altered T Lymphocyte Proliferation upon Lipopolysaccharide Challenge Ex Vivo.

ContextSepsis is characterized by the development of adaptive immune cell alterations, which intensity and duration are associated with increased risk of health-care associated infections and mortality. However, pathophysiological mechanisms leading to such lymphocyte dysfunctions are not completely understood, although both intrinsic lymphocyte alterations and antigen-presenting cells (APCs) dysfunctions are most likely involved.StudyThe aim of the current study was to evaluate whether lipopolysaccharide (LPS, mimicking initial Gram negative bacterial challenge) could directly impact lymphocyte function after sepsis. Therefore, we explored ex-vivo the effect of LPS priming on human T lymphocyte proliferation induced by different stimuli.ResultsWe showed that LPS priming of PBMCs reduced T cell proliferative response and altered IFNγ secretion after stimulation with OKT3 but not with phytohaemagglutinin or anti-CD2/CD3/CD28-coated beads stimulations. Interestingly only LPS priming of monocytes led to decreased T cell proliferative response as opposed to LPS priming of lymphocytes. Importantly, LPS priming was associated with reduced expression of HLA-DR, CD86 and CD64 on monocytes but not with the modification of CD3, CTLA4, PD-1 and CD28 expressions on lymphocytes. Finally, IFNγ stimulation restored monocytes accessory functions and T cell proliferative response to OKT3.ConclusionWe conclude that LPS priming does not directly impact lymphocyte functions but reduces APC’s capacity to activate T cells. This recapitulates ex vivo indirect mechanisms participating in sepsis-induced lymphocyte alterations and suggests that monocyte-targeting immunoadjuvant therapies in sepsis may also help to improve adaptive immune dysfunctions. Direct mechanisms impacting lymphocytes being also at play during sepsis, the respective parts of direct versus indirect sepsis-induced lymphocyte alterations remain to be evaluated in clinic.

Abstract A99: Imprime PGG, a soluble yeast β-glucan, induces dendritic cell maturation, upregulating co-stimulatory and activation markers to enhance antigen presentation and T cell priming

Abstract Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer immunotherapy in combination with monoclonal antibody therapy. Randomized Phase 2 clinical trials of Imprime in the 1st-line treatment of stage IV non-small cell lung cancer have shown promising efficacy in both objective tumor response and survival. Mechanistic work has revealed that Imprime acts as a pathogen-associated molecular pattern (PAMP) to broadly activate the innate immune system, resulting in priming of monocytes and neutrophils, inhibition of immunosuppressive M2 macrophage polarization, and enhanced NK cell activity. Here, we demonstrate that Imprime also binds to various dendritic cell (DC) subsets and promotes DC maturation as well as subsequent T cell activation and acquisition of effector functions. In humans, in vitro treatment of whole blood with Imprime shows binding to CD16+ inflammatory DCs as well as CD1c+ classical DCs. Following i.v. administration in mice, Imprime binds resident and migratory DC subsets within secondary lymphoid organs. In both human and mouse, Imprime increases DC expression of the co-stimulatory molecules CD80/86 as well as MHC class II. Importantly, Imprime-mediated maturation of DCs enhances T cell responses: in mice, immunization with antigen in the presence of Imprime increases the magnitude of the antigen-specific CD8 T cell response as well as improves their ability to degranulate and produce IFN-γ and IL-2. Additionally, we show that Imprime imparts long-lasting effects on human monocytes that sensitize them to subsequent danger signals. Monocytes that are exposed to Imprime in human whole blood prior to their isolation and week-long differentiation into monocyte-derived DCs with GM-CSF and IL-4 show increased upregulation of CD83, CD86, and MHC class II after being matured with LPS and TNF-α. This increased maturation state translates into improved priming and expansion of allogeneic CD4 and CD8 T cells in a mixed-lymphocyte reaction. Altogether, we demonstrate that Imprime behaves as a PAMP to bridge the innate and adaptive immune systems. In a cancer setting, these data suggest that Imprime treatment in conjugation with agents that relieve immunosuppression (e.g. checkpoint inhibitors) could result in superior anti-tumor immunity. Citation Format: Ross B. Fulton, Anissa SH Chan, Steven M. Leonardo, Adria B. Jonas, Xiaohong Qiu, Nadine C. Ottoson, Takashi O. Kangas, Kyle S. Michel, Michael E. Danielson, Jeremy R. Graff, Nandita Bose, Keith Gorden. Imprime PGG, a soluble yeast β-glucan, induces dendritic cell maturation, upregulating co-stimulatory and activation markers to enhance antigen presentation and T cell priming. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A99.

DPP-4 inhibition ameliorates atherosclerosis by priming monocytes into M2 macrophages

ObjectiveGlipitins are widely used for the treatment of type 2 diabetic patients. In addition to their improvement of glycemic control, animal studies have suggested an independent anti-atherosclerotic effect of gliptins. Nevertheless, recent clinical trials regarding long-term effects of gliptin therapy on vascular events have been disappointing. This discrepancy led us to better dissect the functional role of SDF-1/CXCR4 signaling as a potential mechanism underlying gliptin action. The study should give improved understanding of the potential of gliptin therapy in the prevention and treatment of atherosclerosis. Methods and resultsIn an ApoE−/− mouse model on high cholesterol diet, long-term treatment with the DPP-4 inhibitor Sitagliptin significantly reduced atherosclerosic plaque load in the aorta. Flow cytometry analyses showed an enrichment of M2 macrophages in the aortic wall under gliptin therapy. Importantly, the number of recruited CD206+ macrophages was inversely correlated with total plaque area while no correlation was found for the overall macrophage population or M1 macrophages. Blockade of CXCR4/SDF-1 signaling by AMD3100 inhibited aortic M2 accumulation and the therapeutic effect of Sitagliptin. Correspondingly, Sitagliptin shifted the polarization profile of macrophages towards a M2-like phenotype. ConclusionSitagliptin-mediated inhibition of early atherosclerosis is based on M2-polarization during monocyte differentiation via the SDF-1/CXCR4 signaling. In contrast to earlier assumptions gliptin treatment might be especially effective in prevention of atherosclerosis.

Bone-Marrow-Resident NK Cells Prime Monocytes for Regulatory Function during Infection

Tissue-infiltrating Ly6C(hi) monocytes play diverse roles in immunity, ranging from pathogen killing to immune regulation. How and where this diversity of function is imposed remains poorly understood. Here we show that during acute gastrointestinal infection, priming of monocytes for regulatory function preceded systemic inflammation and was initiated prior to bone marrow egress. Notably, natural killer (NK) cell-derived IFN-γ promoted a regulatory program in monocyte progenitors during development. Early bone marrow NK cell activation was controlled by systemic interleukin-12 (IL-12) produced by Batf3-dependent dendritic cells (DCs) in the mucosal-associated lymphoid tissue (MALT). This work challenges the paradigm that monocyte function is dominantly imposed by local signals after tissue recruitment, and instead proposes a sequential model of differentiation in which monocytes are pre-emptively educated during development in the bone marrow to promote their tissue-specific function.

Tu2033 Investigating the microbiome pre and post fecal microbiota therapy from active ulcerative colitis patients in a randomized placebo controlled trial

Purpose: A key challenge post genome-wide association studies (GWAS) is mapping the associated disease/trait susceptibility loci to biological pathways and understanding their level of interconnectedness. Aberrant γ-interferon (INFγ) expression is associated with autoimmune diseases. In vitro and in vivo priming of human monocytes with INFγ leads to monocyte activation and augmentation of responses to various stimuli. Method(s): In order to study the early events of priming, RNA-seq was performed in the human monocyte cell line THP-1 at rest and after 1hr and 6hrs of INFγ stimulation. Results: We identified 893 genes with at least 2 fold differential up-regulation at either 1 or 6hrs compared to unstimulated cells. Strikingly, multiple previously unrecognized INFγ induced genes were found to be significantly enriched in autoimmune susceptibility loci including those related to inflammatory bowel disease (IBD) in general or Crohn’s disease or ulcerative colitis (UC) specifically, celiac disease, and multiple sclerosis (MS). For example, ODF3B (MS) and a specific isoform of SPRED2 (IBD) were found to be INFγ responsive. Further analyses revealed an enrichment of INFγ induced genes in metabolic traits including C4ORF32 (type II diabetes), TMEM229B (phospholipids) and FAM117C (HDL). These genes were independently confirmed to be INFγ inducible by qRT-PCR. Interestingly, TMEM229B and FAM117C have also been associated with Crohn’s disease, suggesting a connection between lipid metabolism and IBD via inflammatory signaling. Of note, many of such INFγ responsive genes show altered expression in normal vs. inflamed colonic samples of Crohn’s and/or UC patients. Integrating public CHIP-seq data, we found a significant enrichment in IRF1, STAT1, IRF8, VDR, and HNF4a binding near the INFγ regulated gene-set. The transcriptional regulator HNF4a, which is best characterized in controlling metabolism, is also encoded in an UC susceptibility locus, further suggesting a novel connection betweenmetabolic processes and INFγ responses. In summary, we have connected a number of autoimmune susceptibility and metabolic trait/disease loci to the INFγ signaling pathway. Ongoing functional characterization of these INFγ responsive genes may further elucidate the underpinnings of autoimmunity.