Monocyte Function Research Articles

Preterm ETs Are Significantly Reduced Compared with Adults and Partially Reduced Compared with Term Infants.

The release of DNA by cells during extracellular trap (ET) formation is a defense function of neutrophils and monocytes. Neutrophil ET (NET) formation in term infants is reduced compared to adults. Objective: The aim was to quantify NET and monocyte ET (MET) release and the respective key enzymes myeloperoxidase (MPO) and neutrophil elastase (NE) in preterm infants. In this prospective explorative study, ET induction was stimulated by N-formylmethionine-leucyl-phenylalanine (fMLP), phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), and lipoteichoic acid (LTA) in the cord blood of preterm infants (n = 55, 23–36 weeks) compared to term infants and adults. METs were quantified by microscopy, and NETs by microscopy and flow cytometry. We also determined the MPO levels within NETs and the intracellular concentrations of NE and MPO in neutrophils. The percentage of neutrophils releasing ET was significantly reduced for preterm infants compared to adults for all stimulants, and with a 68% further reduction for PMA compared to term infants (p = 0.0141). The NET area was not reduced except for when fMLP was administered. The amount of MPO in NET-producing cells was reduced in preterm infants compared to term infants. For preterm infants, but not term infants, the percentage of monocytes releasing ETs was significantly reduced compared to healthy adults for LTA and LPS stimulation. Conclusion: In preterm infants, ETs are measurable parts of the innate immune system, but are released in a reduced percentage of cells compared to adults.

Two week interruption of statin therapy results in an exaggerated inflammatory phenotype in young myocardial infarction patients without standard modifiable risk factors

Abstract Background In patients with myocardial infarction (MI) it is recommended to start high intensity statin therapy, as early as possible, irrespective of lipid levels at presentation. In ST-elevated myocardial infarction (STEMI) patients that do not have standard modifiable risk factors (SMuRFs) there is lower use of statins at discharge compared to patients with at least 1 risk factor, and this is associated with a worse prognosis. Beside lipid lowering capacities, statins are known for their anti-inflammatory effects. Atherosclerosis is characterized as an inflammatory disease in which monocytes play a pivotal role. Circulating monocytes of atherosclerosis patients with SMuRFs have a pro-inflammatory phenotype. In the absence of other modifiable factors in young SMurfless patients, targeting inflammation seems crucial. The effects of (short term) cessation of statin treatment in young SMuRFless MI patients are not known. Purpose The aim of this study was to assess the short term effect of cessation of statin treatment on the inflammatory profile in young SMuRFless MI patients. Methods We included 19 SMuRFless patients (low-density Lipoprotein cholesterol (LDLc) <4.5 mmol/L), no hypertension, no diabetes mellitus, body mass index <30 kg/m2) who suffered a myocardial infarction between 1 and 4 years ago at an age <50 years, and were actively receiving statin therapy. Blood was drawn prior to- and after 14 days of statin cessation. At both time points we assessed circulating high sensitive-C-reactive protein (hs-CRP) levels and leukocyte counts. Using flow cytometry we determined monocyte (subset) phenotype. Monocyte function was studied by measuring cytokine production of peripheral blood mononuclear cells (PBMCs) after isolation and 24-hours incubation with Toll-like receptor (TLR) agonists. Results Short-term interruption of statin treatment resulted in a significant increase in circulating hs-CRP (0.6 vs 0.7 mg/L, p=0.03). Flow cytometry analysis revealed that short term cessation of statin treatment caused a shift in monocyte subset distribution (Figure 1). The CD14++CD16+ “intermediate” monocyte subset became significantly upregulated (p=0.026). Statin interruption also resulted in altered PBMC function after stimulation. No significant differences were seen in PBMCs stimulated with lipopolysaccharide (LPS). Pam3CysK4 (P3C) stimulated cells produces more IL-6 (p=0.004), IL-10 (p=0.010) and IL1Ra (p=0.047) after cessation of statin treatment (Figure 2). Conclusions In young SMuRFless MI patients, short-term interruption of statin treatment results in a heightened inflammatory profile, as demonstrated by elevated circulating CRP, a switch in monocyte phenotype and exaggerated monocyte cytokine production capacity. This difference was already noticeable 14 days after cessation of statin. This emphasizes the importance of statins therapy, irrespective of lipid levels or other SMuRFs. Funding Acknowledgement Type of funding sources: None.

Monocyte and endothelial dysfunction induced by hyperglycemia is reversed by Empagliflozin mainly through glucose-transport independent mechanisms

Abstract Type II Diabetes mellitus (T2DM) leads via hyperglycemia (HG)-associated inflammation and oxidative stress to vascular cell dysfunction. The EMPA-REG trial revealed a reduction of cardiovascular mortality in T2DM patients by Empagliflozin, a sodium-glucose co-transporter-2 (SGLT-2) inhibitor. SGLT-2 is expressed on monocytes and endothelial cells (EC), therefore the aim of this study was to investigate the potential role of Empagliflozin for glucose transport regulation and improvement of HG-induced dysfunction of monocytes and EC. HUVEC and HCAEC were used as EC model cells. Primary human monocytes were isolated from T2DM patients and healthy controls. The cells were exposed to HG conditions in vitro in presence of 40 or 100 ng/ml Empagliflozin. Using RT-qPCR and FACS, expression levels of SGLT-2 were analysed. Glucose uptake assays were performed with a fluorescent derivative of glucose, 2-NBDG. To measure reactive oxygen species (ROS) accumulation, we used the H2DFFDA method via FACS and fluorescence microscopy. Monocyte and EC chemotaxis was analysed using modified Boyden chamber assays. We could prove an expression of SGLT-2 in primary human monocytes and EC. The transcripts of SGLT-2 in T2DM monocytes did not show a difference. SGLT2-levels in monocytes and EC were not altered by HG. In presence of GLUT inhibitor in vitro glucose uptake assays revealed the ability of SGLT-2 inhibition to mildly supress the glucose uptake, whereas uptake by monocytes and EC remained comparable. Nevertheless, in presence of Empagliflozin HG-induced ROS accumulation in monocytes (1.3-fold, p=0.021) and ECs (1.5-fold, p=0.003) was reduced. HG monocytes and EC readily exhibited an impaired chemotaxis behaviour. Treating the cells in HG with Empagliflozin reversed the PlGF-1 resistance phenotype of HG monocytes (1.4-fold, p=0.002). Comparably, Empagliflozin was able to rescued the blunted VEGF-A responses of HG ECs (1.35-fold, p=0.028), which could be ascribed to restoration of VEGFR-2 receptor levels on the EC surface. Most of the aberrant phenotypes exhibited by HG monocytes and EC were entirely recapitulated by induction of oxidative stress. We could mimic these effects of Empagliflozin using the general antioxidant N-acetyl-L-cysteine (NAC). Our study reveals a beneficial role of Empagliflozin for reversing HG-induced vascular cell dysfunction. The expressed SGLT-2 on monocytes and EC does not serve as primary glucose transporter. So it is highly to speculate that HG-mediated enhanced glucotoxicity in those cells is not prevented by Empagliflozin through inhibition of glucose uptake itself. Reduction of oxidative stress by Empagliflozin was revealed as central mechanism for an improved function of HG monocytes and EC in vitro. Finally, we conclude that antioxidant properties of Empagliflozin reverse vascular cell dysfunction independently of glucose transport. This is likely to support the beneficial effect of Empagliflozin in cardiovascular disease. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Medical Faculty, University of Münster, Germany

Primary human monocytes are stimulated in a pro-atherosclerotic manner in hyperhomocysteine conditions by attenuated PTEN phosphatase function

Abstract Purpose Monocytes are essential for atherosclerosis and hereby crucial for the detrimental consequences of cardiovascular diseases like coronary heart disease. Interestingly, homocysteine (HCY) was identified as an idependent risk factor for atherosclerosis development. However, the specific implication of HCY on monocytes has not yet been clarified and was therefore investigated in the present study. Methods Primary human monocytes, isolated from peripheral blood through the immuno-magnetic method, were treated with a clinically relevant dose of 400 μM HCY for 24 hours. Monocyte migration was investigated with transwell migration assays. Adhesion to inflamed endothelium (HUVECs) was studied under static and physiological flow conditions. Expression of relevant molecules was quantified with Western Blot, RT-qPCR and FACS. 5-azacytidine (AZA) was used to inhibit DNA methyltransferase 1 (DNMT1) and SF1670 to inhibit PTEN. Results First, we studied monocyte migration phenotype. Under hyperhomocysteine conditons monocytes revealed elevated chemokinesis and enhance chemotaxis towards MCP-1. In addition, monocytes show a pro-adhesive phenotype under HCY treatment which was indicated by heightened adhesion to inflamed endothelial cells, both in static and physiological flow conditions. These data could be sustained by observation of HCY-induced augmented expression of adhesion molecule CD11a on primary human monocytes. Interestingly, we observed decreased PTEN phosphatase function and activity in HCY-treated monocytes, which was reflected downstream in increased activation of AKT at serine 473. Based on these data, it was also possible to induce the pro-migratory phenotype in monocytes by pharmacological inhibition of PTEN alone. Since HCY can modify the methylation status of PTEN and hereby regulates its expression, we inhibited DNMT1 with AZA which could prevent downregulation of PTEN expression by HCY treatment. In addition, through inhibition of DNMT1 HCY-induced aberrant migration and adhesion was rescued. In line with that, HCY-induced pro-migratory and pro-adhesive phenotype was also rescued by co-treatment with the cofactors (30 μM Vitamin B12 and 3 μM folic acid) responsible for homocysteine to methionine catabolism due to normalized PTEN expression. Conclusions The present work deciphers a previously unknown mechanism how an increased concentration of HCY induces a pro-atherosclerotic activation of monocytes. Accumulation of HCY leads to a methylation-dependent inactivation of PTEN phosphatase. This subsequently causes increased phosphorylation of AKT at serine 473, which results in an augmented migration behaviour of monocytes. At the same time, we were able to reduce HCY-induced aberrant monocyte activation by inhibiting DNTM1 or by interfering with HCY metabolism by adding vitamin B12 or folic acid. In summary, these findings provide a new approach to reset pro-atherosclerotic monocytes in hyperhomocysteinemic conditions. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): IZKF SEED Project 14/20

Inflammatory and diabetic conditions trigger SHP2 tyrosine phosphatase expression and subsequent aberrant activation of primary human monocytes

Abstract Purpose Atherosclerosis is an inflammatory process that is particularly accelerated in diabetics, leading to increased incidence of cardiovascular diseases such as CAD and PAD in diabetic patients. Monocytes are the main component of atherosclerosis development. SHP-2 tyrosine phosphatase has been identified as an important regulator of monocyte function. The present study therefore aims to investigate the regulation of SHP-2 in inflammatory and diabetic conditions. Methods Primary human monocytes were isolated from the peripheral blood of type 2 Diabetes mellitus (T2DM) patients and healthy individuals. Monocytes were incubated with pro-inflammatory cytokine TNFa. For diabetic conditions, monocytes were incubated with methylglyoxal (MG), a highly reactive side product of glycolysis, or Receptor for advanced glycation end product (RAGE) ligand AGE-bovine serum (AGE-BSA). Monocyte migration was studied with Transwell migration assays. Expression of important molecules was investigated with Western Blot, RT-qPCR or FACS. Pharmacological inhibitors for SHP2, RAGE or NFκB were used. Results First, we could detect a significant correlation between SHP-2 mRNA and TNFa levels in T2DM monocytes in comparison to monocytes from healthy individuals. In line with that, incubation of monocytes with TNFa lead to an enhanced expression of SHP-2. Co-incubation with NFκB-inhibitor blocked TNFa-induced SHP-2 upregulation. Interestingly, incubation of monocytes with methylglyoxal caused increased release of TNFa and also augmented expression of SHP-2, indicating a pro-inflammatory effect of diabetic conditions. Moreover, AGE-BSA treatment induced enhanced SHP-2 expression, reflecting an inflammatory-independent pathway which regulates SHP-2 additionally. This could be supported by the observation that pharmacological inhibition of RAGE attenuated both AGE-BSA and MG-induced SHP-2 activation. On a functional level, increased expression of SHP-2 in each treatment resulted in a pro-migratory phenotype that could be completely reversed by inhibition of RAGE, respectively. Fittingly, monocytes from T2DM patients showed increased migration, which normalized to an ordinary level after application of a SHP-2 inhibitor. Conclusions The present results reveal a new mechanism for accelerated atherosclerosis development in diabetic patients. MG and advanced glycated end products, as crucial components of the diabetic milieu, lead to increased expression of SHP-2 via the RAGE-NFkB signalling axis. Interestingly, this diabetic environment causes an increased inflammatory response through the release of TNFa cytokine, which itself leads to enhanced SHP-2 expression through activation of the NFkB transcription factor. Finally, by pharmacological inhibition of each component in this outlined SHP-2 regulatory pathway, we were able to prevent the pro-migratory activation of monocytes, offering a new approach to the treatment of diabetes-induced atherosclerosis. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): IZKF SEED Project 14/20

UVA1 radiation attenuates pro-inflammatory functions in human monocytes.

UVA1 therapy is effective in the treatment of inflammatory and autoimmune skin diseases. The mode of action of UVA1 therapy is not completely understood and especially data on cells of the innate immune system like monocytes, which are critically involved in many inflammatory processes, are sparse. We wanted to answer the question whether UVA1 irradiation alters functional properties of human monocytes. We treated human peripheral blood monocytes in vitro with 2J/cm2 UVA1 light, incubated the cells for 48 h and examined both functional properties and alterations in the gene and protein expression profile. While UVA1 did not alter cell viability or susceptibility to apoptosis inducing agents, it decreased the capacity of monocytes for phagocytosis and to eliminate infectious agents like Leishmania major. Moreover, we measured a significantly reduced production of interleukin (IL)-1β mRNA in lipopolysaccharide activated monocytes after UVA1 treatment. Importantly, UVA1-treated monocytes not only produce less IL-1β, but also upregulate expression of the anti-inflammatory IL-1β decoy receptor. Our data provide evidence that UVA1 radiation not only interferes with fundamental monocyte properties like phagocytosis, pathogen killing and activation, but could also specifically attenuate pro-inflammatory IL-1 effects. This might constitute a hitherto unknown anti-inflammatory mechanism of UVA1 in human monocytes.

Platelets, Macrophages, and Thromboinflammation in Chagas Disease.

Chagas disease (CD) is a major health problem in the Americas and an emerging health problem in Europe and other nonendemic countries. Several studies have documented persistence of the protozoan parasite Trypanosoma cruzi, and oxidative and inflammatory stress are major pathogenic factor. Mural and cardiac thrombi, cardiac arrhythmias, and cardiomyopathy are major clinical features of CD. During T. cruzi infection, parasite-released factors induce endothelial dysfunction along with platelet (PLT) and immune-cell activation. PLTs have a fundamental role in maintaining hemostasis and preventing bleeding after vascular injury. Excessive activation of PLTs and coagulation cascade can result in thrombosis and thromboembolic events, which are recognized to occur in seropositive individuals in early stages of CD when clinically symptomatic heart disease is not apparent. Several host and parasite factors have been identified to signal hypercoagulability and increase the risk of ischemic stroke in early phases of CD. Further, PLT interaction with immune cells and their role in host defense against pathogens and inflammatory processes have only recently been recognized and evolving. In the context of parasitic diseases, PLTs function in directly responding to T. cruzi infection, and PLT interactions with immune cells in shaping the proinflammatory or immunoregulatory function of monocytes, macrophages, and neutrophils remains elusive. How T. cruzi infection alters systemic microenvironment conditions to influence PLT and immune-cell interactions is not understood. In this review, we discuss the current literature, and extrapolate the mechanistic situations to explain how PLT and innate immune cell (especially monocytes and macrophages) interactions might be sustaining hypercoagulability and thromboinflammation in chronic CD.

Brazilian red propolis exerts a cytotoxic action against prostate cancer cells and upregulates human monocyte functions.

Different propolis samples can be obtained in Brazil, such as green, brown and red. Studies related to Brazilian red propolis (BRP) have increased in the last few years, so the aim of this study was to investigate its effects on the prostate cell lines LNCaP and PC-3 and on human monocytes. BRP chemical composition was analyzed by HPLC-DAD, the viability of monocyte and cancer cell by MTT assay. Cytokine production (TNF-α, IL-1β, IL-6, IL-10) by monocytes was quantitated by ELISA, the expression of cell markers (TLR-2, TLR-4, HLA-DR, CD80) and reactive oxygen species by flow cytometry. The candidacidal activity and the effects of supernatant of treated monocytes on tumor cells were assessed. BRP affected LNCaP viability after 48 and 72 h, while PC-3 cells were more resistant over time. BRP upregulated CD80 and HLA-DR expression, and stimulated TNF-α, IL-6 and IL-10 production. BRP enhanced the fungicidal activity of monocytes, displayed an antioxidant action and the supernatant of BRP-treated monocytes diminished LNCaP viability. In the search for new immunomodulatory and antitumoral agents, BRP exerted a selective cytotoxic activity on prostate cancer cells and an immunomodulatory action, suggesting its potential for clinical trials with oncological patients and for the discovery of new immunomodulatory and antitumor drugs.

Circulating monocytes associated with anti-PD-1 resistance in human biliary cancer induce Tcell paralysis.

Suppressive myeloid cells can contribute to immunotherapy resistance, but their role in response to checkpoint inhibition (CPI) in anti-PD-1 refractory cancers, such as biliary tract cancer (BTC), remains elusive. We use multiplexed single-cell transcriptomic and epitope sequencing to profile greater than 200,000 peripheral blood mononuclear cells from advanced BTC patients (n= 9) and matched healthy donors (n= 8). Following anti-PD-1 treatment, CD14+ monocytes expressing high levels of immunosuppressive cytokines and chemotactic molecules (CD14CTX) increase in the circulation of patients with BTC tumors that are CPI resistant. CD14CTX can directly suppress CD4+ Tcells and induce SOCS3 expression in CD4+ Tcells, rendering them functionally unresponsive. The CD14CTX gene signature associates with worse survival in patients with BTC as well as in other anti-PD-1 refractory cancers. These results demonstrate that monocytes arising after anti-PD-1 treatment can induce Tcell paralysis as a distinct mode of tumor-mediated immunosuppression leading to CPI resistance.

HSP90α induces immunosuppressive myeloid cells in melanoma via TLR4 signaling

BackgroundTumor cells modulate host immunity by secreting extracellular vesicles (EV) and soluble factors. Their interactions with myeloid cells lead to the generation of myeloid-derived suppressor cells (MDSC), which inhibit the...

How Can We Improve Vaccination Response in Old People? Part I: Targeting Immunosenescence of Innate Immunity Cells.

Vaccination, being able to prevent millions of cases of infectious diseases around the world every year, is the most effective medical intervention ever introduced. However, immunosenescence makes vaccines less effective in providing protection to older people. Although most studies explain that this is mainly due to the immunosenescence of T and B cells, the immunosenescence of innate immunity can also be a significant contributing factor. Alterations in function, number, subset, and distribution of blood neutrophils, monocytes, and natural killer and dendritic cells are detected in aging, thus potentially reducing the efficacy of vaccines in older individuals. In this paper, we focus on the immunosenescence of the innate blood immune cells. We discuss possible strategies to counteract the immunosenescence of innate immunity in order to improve the response to vaccination. In particular, we focus on advances in understanding the role and the development of new adjuvants, such as TLR agonists, considered a promising strategy to increase vaccination efficiency in older individuals.

Production of MHCII-expressing classical monocytes increases during aging in mice and humans.

Aging is associated with increased monocyte production and altered monocyte function. Classical monocytes are heterogenous and a shift in their subset composition may underlie some of their apparent functional changes during aging. We have previously shown that mouse granulocyte‐monocyte progenitors (GMPs) produce “neutrophil‐like” monocytes (NeuMo), whereas monocyte‐dendritic cell progenitors (MDPs) produce monocyte‐derived dendritic cell (moDC)‐producing monocytes (DCMo). Here, we demonstrate that classical monocytes from the bone marrow of old male and female mice have higher expression of DCMo signature genes (H2‐Aa, H2‐Ab1, H2‐Eb1, Cd74), and that more classical monocytes express MHCII and CD74 protein. Moreover, we show that bone marrow MDPs and classical monocytes from old mice yield more moDC. We also demonstrate higher expression of Aw112010 in old monocytes and that Aw112010 lncRNA activity regulates MHCII induction in macrophages, which suggests that elevated Aw112010 levels may underlie increased MHCII expression during monocyte aging. Finally, we show that classical monocyte expression of MHCII is also elevated during healthy aging in humans. Thus, aging‐associated changes in monocyte production may underlie altered monocyte function and have implications for aging‐associated disorders.

β-hydroxybutyrate impairs monocyte function via the ROS-NLR family pyrin domain-containing three inflammasome (NLRP3) pathway in ketotic cows.

Cows with ketosis display severe metabolic stress and immune dysfunction which renders them more susceptible to infections. Monocytes, one of the major subtypes of white blood cells, play an important role in innate immune defense against infections. Thus, the aim of this study was to investigate alterations in immune function, reactive oxygen species (ROS) production and activity of the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway in monocytes (CD14+) of cows with clinical ketosis (CK). Twelve healthy multiparous Holstein cows [blood β-hydroxybutyrate (BHB) concentration < 1.2 mM] and 12 cows with CK (BHB > 3.0 mM) at 3 to 14 days in milk were used for blood sample collection. To determine effects of BHB on phagocytosis, ROS and protein abundance of the NLRP3 inflammasome pathway in vitro, monocytes isolated from healthy cows were treated with 3.0 mM BHB for 0, 6, 12 or 24 h. Dry matter intake (22.7 vs. 19.0 kg) was lower in cows with CK. Serum concentrations of fatty acids (0.30 vs. 0.88 mM) and BHB (0.52 vs. 3.78 mM) were greater in cows with CK, whereas concentration of glucose was lower (4.09 vs. 2.23 mM). The adhesion, migration and phagocytosis of monocytes were lower in cows with CK, but apoptosis and ROS content were greater. Protein abundance of NLRP3, cysteinyl aspartate specific proteinase 1 (caspase 1) and interleukin-1B p17 (IL1B p17) were greater in monocytes of cows with CK, while abundance of NADPH oxidase isoform 2 (NOX2) was lower. Compared with 0 h BHB, ROS content and apoptosis were greater in the monocytes challenged for 6, 12 or 24 h BHB. Compared with 0 h BHB, protein abundance of NLRP3, caspase 1, IL1B p17 and concentration of IL1B in medium were greater in the monocytes challenged for 6, 12 or 24 h BHB. However, compared with 0 h BHB, protein abundance of NOX2 and phagocytosis of monocytes were lower in the monocytes challenged for 6, 12 or 24 h BHB. Overall, the data suggested that exogenous BHB activated the ROS-NLRP3 pathway, which might be partly responsible for immune dysfunction of dairy cows with CK.

The relationship between TLR4/NF-κB/IL-1β signaling, cognitive impairment, and white-matter integrity in patients with stable chronic schizophrenia.

ObjectivePrevious studies have implicated intricate interactions between innate immunity and the brain in schizophrenia. Monocytic Toll-like receptor (TLR) 4 signaling, a crucial “sensor” of innate immunity, was reported to be over-activated in link with cognitive impairment in schizophrenia. As TLR4 is predominantly expressed on gliocytes prior to expression in neurons, we hypothesized that higher TLR4 levels may contribute to cognitive deterioration by affecting white matter microstructure.MethodsForty-four patients with stable chronic schizophrenia (SCS) and 59 healthy controls (HCs) were recruited in this study. The monocytic function was detected with lipopolysaccharide (LPS) stimulation to simulate bacterial infection. Basal and LPS- stimulated levels of TLR4, nuclear factor-kappa B (NF-κB), and interleukin (IL)-1β were quantified with flow cytometry. Cognitive function was assessed by the MATRICS Consensus Cognitive Battery (MCCB) and psychopathological symptoms were evaluated by the Positive and Negative Syndrome Scale (PANSS). We employed diffusion tensor imaging with a 3-T scanner and evaluated white-matter integrity with fractional anisotropy (FA). Subcortical volume and cortical thickness were also assessed.ResultsThe TLR4/NF-κB/IL-1β signaling pathway was activated in patients with SCS, but responded sluggishly to LPS stimulation when compared with HCs. Furthermore, monocytic TLR4 expressions were inversely correlated with cognitive function and white matter FA, but not with cortical thickness or subcortical gray matter volume in schizophrenia.ConclusionOur findings support altered TLR4 signaling pathway activity in association with deficits in cognition and white matter integrity in schizophrenia.

Intracellular pyruvate levels positively correlate with cytokine production capacity in tolerant monocytes from patients with pneumonia

BackgroundCommunity-acquired pneumonia (CAP) is responsible for a high morbidity and mortality worldwide. Monocytes are essential for pathogen recognition and the initiation of an innate immune response. Immune cells induce intracellular glycolysis upon activation to support several functions. ObjectiveTo obtain insight in the metabolic profile of blood monocytes during CAP, with a focus on glycolysis and branching metabolic pathways, and to determine a possible association between intracellular metabolite levels and monocyte function. MethodsMonocytes were isolated from blood of patients with CAP within 24 h of hospital admission and from control subjects matched for age, sex and chronic comorbidities. Changes in glycolysis, oxidative phosphorylation (OXPHOS), tricarboxylic acid (TCA) cycle and the pentose phosphate pathway were investigated through RNA sequencing and metabolomics measurements. Monocytes were stimulated ex vivo with lipopolysaccharide (LPS) to determine their capacity to produce tumor necrosis factor (TNF), interleukin (IL)-1β and IL-10. Results50 patients with CAP and 25 non-infectious control subjects were studied. When compared with control monocytes, monocytes from patients showed upregulation of many genes involved in glycolysis, including PKM, the gene encoding pyruvate kinase, the rate limiting enzyme for pyruvate production. Gene set enrichment analysis of OXPHOS, the TCA cycle and the pentose phosphate pathway did not reveal differences between monocytes from patients and controls. Patients' monocytes had elevated intracellular levels of pyruvate and the TCA cycle intermediate α-ketoglutarate. Monocytes from patients were less capable of producing cytokines upon LPS stimulation. Intracellular pyruvate (but not α-ketoglutarate) concentrations positively correlated with IL-1β and IL-10 levels released by patients' (but not control) monocytes upon exposure to LPS. ConclusionThese results suggest that elevated intracellular pyruvate levels may partially maintain cytokine production capacity of hyporesponsive monocytes from patients with CAP.

Natural killer (NK) cell-derived extracellular-vesicle shuttled microRNAs control T cell responses.

Natural killer (NK) cells recognize and kill target cells undergoing different types of stress. NK cells are also capable of modulating immune responses. In particular, they regulate T cell functions. Small RNA next-generation sequencing of resting and activated human NK cells and their secreted extracellular vesicles (EVs) led to the identification of a specific repertoire of NK-EV-associated microRNAs and their post-transcriptional modifications signature. Several microRNAs of NK-EVs, namely miR-10b-5p, miR-92a-3p, and miR-155-5p, specifically target molecules involved in Th1 responses. NK-EVs promote the downregulation of GATA3 mRNA in CD4+ T cells and subsequent TBX21 de-repression that leads to Th1 polarization and IFN-γ and IL-2 production. NK-EVs also have an effect on monocyte and moDCs (monocyte-derived dendritic cells) function, driving their activation and increased presentation and costimulatory functions. Nanoparticle-delivered NK-EV microRNAs partially recapitulate NK-EV effects in mice. Our results provide new insights on the immunomodulatory roles of NK-EVs that may help to improve their use as immunotherapeutic tools.

Human monocytes differentiate into tumor-associated macrophages upon SKOV3 cells coculture and/or lysophosphatidic acid stimulation

BackgroundSerous ovarian carcinoma is the most common type of ovarian carcinoma. Tumor-associated macrophages (TAMs) promote ovarian cancer progression. Most macrophages are generated by monocyte differentiation. Lysophosphatidic acid (LPA) levels are high in blood, tissues and ascites of patients with ovarian cancer. This study investigated whether human monocytes can directly differentiate into TAMs in the serous ovarian carcinoma microenvironment.MethodsHuman monocytes were isolated and purified from umbilical cord blood. A serous ovarian carcinoma-like microenvironment was generated by coculturing monocytes and SKOV3 cells in 0.4-μm-pore-size Transwell chambers. Additionally, the effect of LPA was assessed. The two cultured cell types and supernatants were evaluated.ResultsThe morphology and function of monocytes cocultured with SKOV3 cells and/or stimulated with LPA were significantly changed compared with those of non-stimulated monocytes. The CD14 + CD163 + and CD206 + phenotype indicated that stimulated cells were TAMs. The induced cells promoted SKOV3 cell proliferation and invasion, further proving that they were TAMs. The level of the cytokine interleukin-6R in the supernatant was significantly elevated in the treatment groups compared to the control monocyte group. Pathway enrichment analysis of ELISA results showed a strong influence of interleukin-6 family signaling, especially the JAK-STAT signaling pathway, further confirming the importance of IL-6R.ConclusionMonocytes can differentiate into TAMs under coculture with SKOV3 cells and/or LPA stimulation. The induced TAMs promote SKOV3 cell proliferation and invasion. The cytokine receptor IL-6sR and the JAK-STAT signaling pathway play an important role in the differentiation of monocytes into TAMs.

Rectal microbiota are coupled with altered cytokine production capacity following community-acquired pneumonia hospitalization

SummaryHuman studies describing the immunomodulatory role of the intestinal microbiota in systemic infections are lacking. Here, we sought to relate microbiota profiles from 115 patients with community-acquired pneumonia (CAP), both on hospital admission and following discharge, to concurrent circulating monocyte and neutrophil function. Rectal microbiota composition did not explain variation in cytokine responses in acute CAP (median 0%, IQR 0.0%–1.9%), but did one month following hospitalization (median 4.1%, IQR 0.0%–6.6%, p = 0.0035). Gene expression analysis of monocytes showed that undisrupted microbiota profiles following hospitalization were associated with upregulated interferon, interleukin-10, and G-protein-coupled-receptor-ligand-binding pathways. While CAP is characterized by profoundly distorted gut microbiota, the effects of these disruptions on cytokine responses and transcriptional profiles during acute infection were absent or modest. However, rectal microbiota were related to altered cytokine responses one month following CAP hospitalization, which may provide insights into potential mechanisms contributing to the high risk of recurrent infections following hospitalization.

Monocyte Metabolism and Function in Patients Undergoing Cardiac Surgery.

ObjectiveCardiopulmonary bypass (CPB) can lead to systemic inflammation, which is associated with higher morbidity. Therefore, we investigated the metabolism of isolated blood monocytes before and after CPB compared to healthy controls.MethodsIn this prospective, monocentric, observational study, we included 30 patients undergoing CPB and 20 controls. We isolated monocytes from heparinized blood and investigated their metabolism by using Seahorse technology before (t0), 4 h (t4), and 24 h (t24) after the start of the CPB. We also examined programmed cell death 1 ligand (PD-L1), PD-L2, V-domain Ig suppressor of T cell activation (VISTA), and human leukocyte antigen-DR isotype (HLA-DR) using fluorescence-activated cell sorting analysis. Additionally, we investigated plasma cytokine levels in patients without and after ex vivo stimulation.ResultsCPB-induced inflammatory responses are shown by significantly elevated plasma interleukin-6 levels in the CPB group compared to baseline and controls [t0: 0 ng/ml (95%CI 0-0 ng/ml); t4: 0.16 ng/ml (95%CI 0.1-0.197 ng/ml), p < 0.0001; t24: 0.11 ng/ml (95% CI 0.1-0.16 ng/ml), p < 0.0001, and controls: 0 ng/ml (95% CI 0-0 ng/ml)]. The cytokine release in the ex vivo stimulation is reduced for lipopolysaccharide stimulation at t4 [t0: 35.68 ng/ml (95% CI 22.17-46.57 ng/ml) vs. t4: 15.02 (95% CI 10.25-24.78 ng/ml), p < 0.0001]. Intracellular metabolism of monocytes after CPB showed a protracted shift to aerobic glycolysis [t0: 179.2 pmol/min (95% CI 138.0-205.1 pmol/min) vs. t24: 250.1 pmol/min (95% CI 94.8-300.2 pmol/min), p < 0.0001]. Additionally, we observed an altered metabolism in monocytes in patients undergoing cardiac surgery compared to controls even before any surgical procedure [t0: 179.2 pmol/min (95% CI 138.0-205.1) vs. controls 97.4 (95% CI 59.13-144.6 pmol/min), p = 0.0031].ConclusionAfter CPB, patients' monocytes show a shift in metabolism from oxidative phosphorylation to aerobic glycolysis, which is associated with energy-demanding and proinflammatory processes. This is the first study to show changes in monocyte immunometabolism in cardiac surgery. Monocytes of patients undergoing cardiac surgery were leaning toward aerobic glycolysis even before any surgical procedure was conducted. Leaving the question of the pathophysiological mechanisms for future studies to be investigated and paving the way for potential therapy approaches preventing inflammatory effects of CPB.

Immune dysfunction in millitary personnel participating in special tasks and the possibility of medical and psychological rehabilitation

The article considers immune dysfunction in military personnel participating in special operations and the possibilities of their medical and psychological rehabilitation. The state of the immune status of 73 servicemen (average age 37.6 4.1 years) who participated in special operations in areas with a difficult operational situation and underwent a 20-day standard course of medical and psychological rehabilitation in the sanatoriums of the sanatorium complexes North Caucasian, Sochi, and Crimean was monitored. Immune status was assessed before and after participation in special operations and after a course of medical and psychological rehabilitation. Immune dysfunction due to quantitative and functional changes in the immune status was established. Soldiers participating in special operations registered a decrease in the synthesis and differentiation of T-lymphocytes, inhibition of the functionality of cytotoxic lymphocytes, reduced cytolytic potential of natural killer cells, impaired antigen-presenting function of monocytes, and a significant decrease in the number of B lymphocytes with preserved antibody-producing function. The rehabilitation course for military personnel participating in special operations led to the normalization of the analyzed parameters, except for the functional activity of natural killer cells and monocytes. Given the lack of full recovery of homeostatic reserves in the immune system, a more in-depth study of the problem and development of a personalized approach to rehabilitation of the military participants with the assessment of immunological markers are necessary.