Pro-inflammatory Monocyte Subsets Research Articles

Bone marrow macrophages are involved in the ineffective hematopoiesis of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective hematopoiesis. Accumulating evidence has shown that macrophages (MΦs) are important components in the regulation of tumor progression and hematopoietic stem cells (HSCs). However, the roles of bone marrow (BM) MΦsin regulating normal and malignant hematopoiesis in different clinical stages of MDS are largely unknown. Age-paired patients with lower-risk MDS (N = 15), higher-risk MDS (N = 15), de novo acute myeloid leukemia (AML) (N = 15), and healthy donors (HDs) (N = 15) were enrolled. Flow cytometry analysis showed increased pro-inflammatory monocyte subsets and a decreased classically activated (M1) MΦs/alternatively activated (M2) MΦs ratio in the BM of patients with higher-risk MDS compared to lower-risk MDS. BM MФs from patients with higher-risk MDS and AML showed impaired phagocytosis activity but increased migration compared with lower-risk MDS group. AML BM MΦs showed markedly higher S100A8/A9 levels than lower-risk MDS BM MΦs. More importantly, coculture experiments suggested that the HSC supporting abilities of BM MΦs from patients with higher-risk MDS decreased, whereas the malignant cell supporting abilities increased compared with lower-risk MDS. Gene Ontology enrichment comparing BM MΦs from lower-risk MDS and higher-risk MDS for genes was involved in hematopoiesis- and immunity-related pathways. Our results suggest that BM MΦs are involved in ineffective hematopoiesis in patients with MDS, which indicates that repairing aberrant BM MΦs may represent a promising therapeutic approach for patients with MDS.

Deciphering the molecular and cellular atlas of immune cells in septic patients with different bacterial infections

BackgroundSepsis is a life-threatening organ dysfunction caused by abnormal immune responses to various, predominantly bacterial, infections. Different bacterial infections lead to substantial variation in disease manifestation and therapeutic strategies. However, the underlying cellular heterogeneity and mechanisms involved remain poorly understood.MethodsMultiple bulk transcriptome datasets from septic patients with 12 types of bacterial infections were integrated to identify signature genes for each infection. Signature genes were mapped onto an integrated large single-cell RNA (scRNA) dataset from septic patients, to identify subsets of cells associated with different sepsis types, and multiple omics datasets were combined to reveal the underlying molecular mechanisms. In addition, an scRNA dataset and spatial transcriptome data were used to identify signaling pathways in sepsis-related cells. Finally, molecular screening, optimization, and de novo design were conducted to identify potential targeted drugs and compounds.ResultsWe elucidated the cellular heterogeneity among septic patients with different bacterial infections. In Escherichia coli (E. coli) sepsis, 19 signature genes involved in epigenetic regulation and metabolism were identified, of which DRAM1 was demonstrated to promote autophagy and glycolysis in response to E. coli infection. DRAM1 upregulation was confirmed in an independent sepsis cohort. Further, we showed that DRAM1 could maintain survival of a pro-inflammatory monocyte subset, C10_ULK1, which induces systemic inflammation by interacting with other cell subsets via resistin and integrin signaling pathways in blood and kidney tissue, respectively. Finally, retapamulin was identified and optimized as a potential drug for treatment of E. coli sepsis targeting the signature gene, DRAM1, and inhibiting E. coli protein synthesis. Several other targeted drugs were also identified in other types of sepsis, including nystatin targeting C1QA in Neisseria sepsis and dalfopristin targeting CTSD in Streptococcus viridans sepsis.ConclusionOur study provides a comprehensive overview of the cellular heterogeneity and underlying mechanisms in septic patients with various bacterial infections, providing insights to inform development of stratified targeted therapies for sepsis.

Assessing the Sustainability of an Integrated Rural Sanitation and Hygiene Approach: A Repeated Cross-Sectional Evaluation in 10 Countries.

While many studies have implemented programs to increase sanitation coverage throughout the world, there are limited rigorous studies on the sustainability of these sanitation programs. Between 2014 and 2018, the rural Sustainable Sanitation and Hygiene for All (SSH4A) approach was implemented by SNV in sub-Saharan Africa and Asia. Repeated cross-sectional household surveys were administered annually throughout program implementation and 1 to 2 years following completion of program activities. We characterize to what extent sanitation coverage was sustained 1 to 2 years after implementation of this SSH4A intervention. Surveys were conducted in 12 program areas in 10 countries, with 22,666 households receiving a post-implementation survey. Six of 12 program areas (Bhutan, Ghana, Kenya, both Nepal sites, Tanzania) had similar coverage levels of basic sanitation 1-2 years post-implementation, whereas there were varying levels of slippage in the other program areas (both Ethiopia sites, Indonesia, Mozambique, Uganda, Zambia), ranging from a drop of 63 percentage points in coverage in Ethiopia to a drop of only 4 percentage points in Indonesia. In countries that experienced losses in the coverage of household sanitation, sanitation sharing among neighbors generally did not increase, whereas open defecation did increase. In each of the areas where slippage occurred, the sanitation coverage levels at the final time point were all still higher than the initial time point before SNV started working in these areas. We found several factors to be associated with the sustainability of sanitation coverage, including household socioeconomic status, having household members with disabilities, baseline sanitation coverage levels of the program areas, and rate of change of coverage during program activities. Data revealed sustained gains in sanitation coverage in some program areas, yet slippage in other areas. This work may serve to benchmark the sustainability of sanitation interventions in sub-Saharan Africa and Asia.

LDL associates with pro-inflammatory monocyte subset differentiation and increases in chemokine receptor profile expression in African Americans

BackgroundIn the United States, African Americans (AAs) have greater risk for Class III obesity and cardiovascular disease (CVD). Previous reports suggest that AAs have a different immune cell profile when compared to Caucasians. MethodsThe immune cell profile of AAs was characterized by flow cytometry using two experimental setups: ex vivo (N = 40) and in vitro (N = 10). For ex vivo experiments, PBMC were treated with participant serum to understand how lipid contents may contribute to monocyte phenotypic differences. For in vitro experiments, monocytes were low-density lipoprotein (LDL)- or vehicle-treated for four hours and subsequently analyzed by flow cytometry and RT-qPCR. ResultsWhen PBMCs were treated with participant sera, subsequent multivariable regression analysis revealed that serum triglycerides and LDL levels were associated with monocyte subset differences. In vitro LDL treatment of monocytes induced a phenotypic switch in monocytes away from classical monocytes accompanied by subset-specific chemokine receptor CCR2 and CCR5 expression changes. These observed changes are partially translation-dependent as determined by co-incubation with cycloheximide. ConclusionsLDL treatment of monocytes induces a change in monocyte subsets and increases CCR2/CCR5 expression in a subset-specific manner. Understanding the molecular mechanisms could prove to have CVD-related therapeutic benefits, especially in high-risk populations with hyperlipidemia and increased risk for CVD.

Gene expression study in monocytes: evidence of inflammatory dysregulation in early-onset obsessive-compulsive disorder

Obsessive-compulsive disorder (OCD) has a complex etiology that seems to include immune dysfunction and alterations in circulating monocytes. To investigate the immune basis and the functional dysregulation of monocytes in this disease, we analyzed gene expression in the peripheral monocytes of pediatric patients with OCD (N = 102) compared to controls (N = 47). We examined gene expression in primary cultures of peripheral monocytes from participants, under basal conditions and under exposure to lipopolysaccharide (LPS) to stimulate immune response. Whole-genome expression was assessed in 8 patients and 8 controls. Differentially expressed genes were identified followed by protein-protein interaction network construction and functional annotation analysis to identify the genes and biological processes that are altered in the monocytes of OCD patients. We also explored the expression levels of selected genes in monocytes from the other participants using qPCR. Several changes in gene expression were observed in the monocytes of OCD patients, with several immune processes involved under basal conditions (antigen processing and presentation, regulation of immune system and leukocyte cell adhesion) and after LPS stimulation (immune and inflammatory response, cytokine production and leukocyte activation). Despite the qPCR analysis provided no significant differences between patients and controls, high correlations were observed between the expression levels of some of the genes and inflammatory markers (i.e., T helper 17 and regulatory T cell levels, total monocyte and proinflammatory monocyte subset levels, and the cytokine production by resting and stimulated monocytes) of the study participants. Our findings provide more evidence of the involvement of monocyte dysregulation in early-onset OCD, indicating a proinflammatory predisposition and an enhanced immune response to environmental triggers.

Effect of Si-Miao-Yong-An decoction on the differentiation of monocytes, macrophages, and regulatory T cells in ApoE−/− mice

Ethnopharmacological relevanceSi-Miao-Yong-An decoction (SMYAD) is a renowned traditional Chinese medicinal formula. SMYAD was originally recorded in the “Shi Shi Mi Lu”, which was edited by medical scientist Chen Shi'duo during the Qing Dynasty. SMYAD has been traditionally used to treat thromboangiitis obliterans. At present, it is mainly used in clinical applications and research of cardiovascular diseases. Aim of the studyTo explore the effects of SMYAD on the pathological changes of atherosclerosis (AS) and the differentiation of monocytes, macrophages, and regulatory T (Treg) cells in apolipoprotein E knockout (ApoE−/−) mice. Materials and methodsEight C57BL/6J mice, which were fed with normal diet for 16 weeks, were used as control group. Forty ApoE−/− mice were randomly divided into model group, atorvastatin group, SMYAD low-dose (SMYAD-LD) group, SMYAD medium-dose (SMYAD-MD) group, and SMYAD high-dose (SMYAD-HD) group. ApoE−/− mice were fed with western diet (WD) for 8 weeks, and the drugs were continuously administered for 8 weeks. The levels of serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured by the esterase method. Morphological changes of the aortic sinus in mice were observed by hematoxylin-eosin (HE) staining, the lipid infiltration of the aorta and aortic sinus were observed by oil red O staining, and the spleen index was calculated. The proportion of Ly6Chigh and Ly6Clow monocyte subsets, macrophages, and their M1 phenotype, as well as Treg cells in spleen were measured by flow cytometry. The expressions of cluster of differentiation 36 (CD36), scavenger receptor A1 (SRA1), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), F4/80, and fork head frame protein 3 (FOXP3) in aortic sinus were assessed by immunohistochemical staining. The serum levels of oxidized low density lipoprotein (ox-LDL), interleukin-1β (IL-1β), IL-18, transforming growth factor-β (TGF-β), and IL-10 were measured by enzyme-linked immunosorbent assays (ELISA). ResultsCompared with the model group, the level of serum TC and LDL-C decreased in the SMYAD group, the pathological changes of aortic sinus decreased, and lipid infiltration of aorta and aortic sinus also decreased. These decreases were accompanied by a significant downregulation of CD36, SRA1, and LOX-1. Furthermore, the proportions of Ly6Chigh pro-inflammatory monocyte subsets, macrophages, and their M1 phenotypes in spleen decreased significantly, while the proportion of Treg cells increased. In addition, while the expression of F4/80 decreased, the expression of FOXP3 increased in the aorta sinus. The levels of serum pro-inflammatory factors IL-1β and IL-18 decreased. ConclusionsSMYAD can improve the pathological changes associated with AS and can inhibit lipid deposition in ApoE−/− mice induced by WD diet. The likely mechanism is the inhibition of the differentiation and recruitment of monocytes and macrophages, the promotion of the differentiation and recruitment of Treg cells, as well as the reduction of the secretion of pro-inflammatory factors.

Meta-inflammation in monocytes of patients with Acute Coronary Syndrome

Abstract Background Several studies suggest that an alteration of monocyte metabolism might be implicated in inflammatory diseases. Enhanced glycolysis might be a hallmark of pro-inflammatory monocyte subsets. Improved glycolysis enables the immune cells to generate sufficient ATP and biosynthetic intermediates to carry out its particular effector functions. For macrophages this includes phagocytosis and inflammatory cytokine production. Pyruvate Kinase isozyme M2 (PKM-2) catalyzes the final step of glycolysis producing pyruvate and ATP. Latest studies have shown that a member of Jumonji family (JMJD8) acts as a positive regulator in TNF-induced NF-kB signaling leading to pro-inflammatory pathways in macrophages and is involved in angiogenesis and cellular metabolism through interacting with PKM-2 in endothelial cells. Purpose The aims of the study are to assess the expression of the glycolytic key enzyme PKM-2 in CD14+ monocytes obtained from patients with non-ST-elevation myocardial infarction (NSTEMI) or with stable angina (SA). Furthermore, the expression of JMJD8 was evaluated. Methods 30 patients with NSTEMI and 30 patients with SA were enrolled. Peripheral blood mononuclear cells were obtained from whole blood samples. For cytoplasmatic protein identification, cells were fixed and permeabilized and then incubated with fluorochrome-conjugated mAbs anti-CD14, anti-PKM-2 and anti-JMJD8. For analysis we used Two-tailed Mann-Whitney non parametric Comparison test. Results CD14+ monocytes from NSTEMI patients showed reduced expression of the key glycolytic enzyme PKM-2 as compared to CD14+ monocytes from SA patients (p=0.02) (Figure 1). JMJD8 expression in NSTEMI patients is increased compared with SA patients (p=0.02) (Figure 2). Conclusion This study introduces a role for immune-metabolism in the immunity dysregulation described in ACS patients and provides novel insights into the mechanisms responsible for coronary instability. Taking their potential interaction into account, our data suggest that in acute setting glycolysis key enzyme PKM2 expression is downregulated. Besides, JMJD8 protein levels increase in NSTEMI patients acting as potential limiting factor of PKM2 function. Moreover, our data propose the potential roles of immune-metabolism to detect novel therapeutic targets, associated with an accurate patient stratification based on immune-metabolic profiles, for prevention and treatment of atherosclerosis, in the perspective of a personalized medicine approach. Funding Acknowledgement Type of funding source: Private hospital(s). Main funding source(s): Fondazione Policlinico A. Gemelli

Altered Dynamics of Monocyte Subpopulations and Pro-Inflammatory Signaling Pathways in Polycythemia Vera Revealed By Mass Cytometry

Introduction: Polycythemia vera (PV) is a clonal hematologic malignancy characterized by overproduction of mature erythrocytes that exhibits a propensity for transformation to myelofibrosis (MF) and/or secondary acute myeloid leukemia (sAML). Like the majority of myeloproliferative neoplasms (MPN), the defining genetic feature of PV is the JAK2 V617F mutation, occurring in >95% of patients, resulting in overactive JAK-STAT signaling which drives myeloproliferation and can contribute to inflammation. We have demonstrated that aberrant NFkB pathway signaling plays an integral role in maintaining monocyte-derived inflammatory cytokine production in MF (Fisher et al, Leukemia 2017 & 2019). We have recently shown that inflammation is a hallmark of PV as well, but the signaling pathways and cell populations responsible have not been elucidated (Fowles et al, Leukemia 2019). Mass cytometry (CyTOF) enables the simultaneous measurement of 40+ markers with single cell resolution, thereby allowing comprehensive analysis of intracellular signaling networks in defined hematopoietic cell populations. We utilized mass cytometry to investigate cell populations and inflammatory signaling pathways in a cohort of PV patients including those who transformed to MF or secondary acute myeloid leukemia (sAML). Methods: Plasma cytokine levels were measured with the U-PLEX Human Cytokine 10plex assay (MSD) from 20 normal donors, 22 PV, 92 MF and 6 sAML patients, as well as serial samples from PV patients transformed to MF/sAML. Cryopreserved cells bone marrow (BM) and/or peripheral blood (PB) from 15 PV patients and 12 normal donors from were incubated with or without TPO or TNF and then stained with surface and intracellular antibodies prior to being subjected to mass cytometric analysis. Cell population frequency and intracellular signaling were identified and calculated through viSNE analysis and manual gating in Cytobank. Results: Plasma levels of inflammatory cytokines IL-6, IL-8, MIP-1a, and TNF increased with disease severity from PV to MF and sAML. For serial samples, an increased number of elevated cytokines was observed in post-transformation samples, suggesting a role for inflammation in MPN disease progression. Mass cytometry analysis revealed expansion of CD14+CD16+ intermediate monocytes and CD16+CD14-CD33+ non-classical monocytes in PV PB compared to both normal BM and PB. A reduction in CD14+CD16- classical monocytes was observed in PV compared to normal in both BM and PB, suggesting a shift in pro-inflammatory subsets in PV. Heightened NFKB activation in response to TNF stimulation was observed in lineage negative CD34+ cells in the majority of PV PB and BM samples tested compared to normal. Similarly, abnormally elevated pSTAT3/5 levels were frequently observed in response to TPO stimulation. In monocyte populations, basal elevations in several signaling markers including pNFkB, pSTAT1, and pMAPKAP2 were identified. Notably, in serial samples from patients transformed to MF or sAML, evidence of progressive NFkB hyperactivation associated with disease evolution was observed. Conclusions: Inflammatory cytokines are known to be elevated in MPN in part through constitutive JAK-STAT signaling, and recently we identified NFkB signaling as an important contributor in MF. To our knowledge NFkB has not previously been investigated in PV. This study suggests that NFkB signaling is similarly important to PV and might play a role in disease transformation. Our plasma cytokine data identified a correlation with higher cytokine levels associated with disease severity from PV to MF and sAML. This is consistent with prior studies associating IL-6 and IL-8 with leukemic transformation of MF. The observed expansion of CD14+CD16+ monocytes in PV is notable because studies have associated this population with increased endothelial cell adhesion and atherosclerosis. These associations have also been reported in JAK2 V617F-mutant red blood cells and neutrophils, raising the question of how the JAK2 mutation affects the function of pro-inflammatory monocyte subsets. Integration of this study with molecular and gene expression data to further investigate the role of inflammation in MPN progression are ongoing. Disclosures Oh: Novartis: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees.

Remote Postischemic Conditioning Promotes Stroke Recovery by Shifting Circulating Monocytes to CCR2+ Proinflammatory Subset.

Brain injury from stroke is typically considered an event exclusive to the CNS, but injury progression and repair processes are profoundly influenced by peripheral immunity. Stroke stimulates an acute inflammatory response that results in a massive infiltration of peripheral immune cells into the ischemic area. While these cells contribute to the development of brain injury, their recruitment has been considered as a key step for tissue repair. The paradoxical role of inflammatory monocytes in stroke raises the possibility that the manipulation of peripheral immune cells before infiltration into the brain could influence stroke outcome. One such manipulation is remote ischemic limb conditioning (RLC), which triggers an endogenous tolerance mechanism. We observed that mice subjected to poststroke RLC shifted circulating monocytes to a CCR2+ proinflammatory monocyte subset and had reduced acute brain injury, swelling, and improved motor/gait function in chronic stroke. The RLC benefits were observed regardless of injury severity, with a greater shift to a CCR2+ subset in severe stroke. Adoptive transfer of CCR2-deficient monocytes abolished RLC-mediated protection. The study demonstrates the importance of RLC-induced shift of monocytes to a CCR2+ proinflammatory subset in attenuating acute injury and promoting functional recovery in chronic stroke. The defined immune-mediated mechanism underlying RLC benefits allows for an evidence-based framework for the development of immune-based therapeutic strategies for stroke patients.SIGNIFICANCE STATEMENT Stroke is the leading cause of physical disability worldwide but has few treatment options for patients. Because remote ischemic limb conditioning (RLC) elicits endogenous tolerance in neither an organ- nor a tissue-specific manner, the immune system has been considered a mediator for an RLC-related benefit. Application of RLC after stroke increased a proinflammatory CCR2+ monocyte subset in the blood and the brain. RLC reduced acute stroke injury and promoted motor/gait function during the recovery phase. The RLC benefits were absent in mice that received CCR2-deficient monocytes. This preclinical study shows the importance of CCR2+ proinflammatory monocytes in RLC benefits in stroke and provides a therapeutic RLC platform as a novel immune strategy to improve outcomes in stroke patients.

Elevated monocyte-specific type I interferon signalling correlates positively with cardiac healing in myocardial infarct patients but interferon alpha application deteriorates myocardial healing in rats

Monocytes are involved in adverse left ventricular (LV) remodelling following myocardial infarction (MI). To provide therapeutic opportunities we aimed to identify gene transcripts in monocytes that relate to post-MI healing and evaluated intervention with the observed gene activity in a rat MI model. In 51 MI patients treated by primary percutaneous coronary intervention (PCI), the change in LV end-diastolic volume index (EDVi) from baseline to 4-month follow-up was assessed using cardiovascular magnetic resonance imaging (CMR). Circulating monocytes were collected at day 5 (Arterioscler Thromb Vasc Biol 35:1066–1070, 2015; Cell Stem Cell 16:477–487, 2015; Curr Med Chem 13:1877–1893, 2006) after primary PCI for transcriptome analysis. Transcriptional profiling and pathway analysis revealed that patients with a decreased LV EDVi showed an induction of type I interferon (IFN) signalling (type I IFN pathway: P value < 0.001; false discovery rate < 0.001). We subsequently administered 15,000 Units of IFN-α subcutaneously in a rat MI model for three consecutive days following MI. Cardiac function was measured using echocardiography and infarct size/cardiac inflammation using (immuno)-histochemical analysis. We found that IFN-α application deteriorated ventricular dilatation and increased infarct size at day 28 post-MI. Moreover, IFN-α changed the peripheral monocyte subset distribution towards the pro-inflammatory monocyte subset whereas in the myocardium, the presence of the alternative macrophage subset was increased at day 3 post-MI. Our findings suggest that induction of type I IFN signalling in human monocytes coincides with adverse LV remodelling. In rats, however, IFN-α administration deteriorated post-MI healing. These findings underscore important but also contradictory roles for the type I IFN response during cardiac healing following MI.

Monocyte NOTCH2 expression predicts IFN-β immunogenicity in multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-β is an established treatment for MS; however, up to 30% of IFN-β-treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14+ monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-β. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-β administration. Reduced monocyte NOTCH2 expression in nADA+ MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA+ patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-β administration.

Frequency of Monocyte Subtypes and TLR4 Expression Correlate with Clinical Outcomes After Mechanical Circulatory Device Implantation

After mechanical circulatory support device (MCSD) implantation, a subset of patients will progress to multiorgan dysfunction. Noninvasive monitoring of immune cells holds promise to understand the mechanism behind adverse clinical outcomes. This is of special importance in the growing group of older patients with advanced heart failure. We hypothesized that pro-inflammatory monocyte subsets and decreased anti-bacterial activity would be associated with adverse clinical outcomes after MCSD.

Prior Exposure to Zika Virus Significantly Enhances Peak Dengue-2 Viremia in Rhesus Macaques

Structural and functional homologies between the Zika and Dengue viruses’ envelope proteins raise the possibility that cross-reactive antibodies induced following Zika virus infection might enhance subsequent Dengue infection. Using the rhesus macaque model we show that prior infection with Zika virus leads to a significant enhancement of Dengue-2 viremia that is accompanied by neutropenia, lympocytosis, hyperglycemia, and higher reticulocyte counts, along with the activation of pro-inflammatory monocyte subsets and release of inflammatory mediators. Zika virus infection induced detectable Dengue cross-reactive serum IgG responses that significantly amplified after Dengue-2 virus infection. Serum from Zika virus immune animals collected prior to Dengue-2 infection showed significant capacity for in vitro antibody dependent enhancement of Dengue-1, 2, 3 and 4 serotypes suggesting that pre-existing immunity to Zika virus could potentially enhance infection by heterologous Dengue serotypes. Our results provide first in vivo evidence that prior exposure to Zika virus infection can enhance Dengue infection, which has implications for understanding pathogenesis and the development of vaccines.

CD154-CD40 T-cell co-stimulation pathway is a key mechanism in kidney ischemia-reperfusion injury

Ischemia-reperfusion occurs in a great many clinical settings and contributes to organ failure or dysfunction. CD154-CD40 signaling in leukocyte–endothelial cell interactions or T-cell activation facilitates tissue inflammation and injury. Here we tested a siRNA anti-CD40 in rodent warm and cold ischemia models to check the therapeutic efficacy and anti-inflammatory outcome of in vivo gene silencing. In the warm ischemia model different doses were used, resulting in clear renal function improvement and a structural renoprotective effect. Renal ischemia activated the CD40 gene and protein expression, which was inhibited by intravenous siRNA administration. CD40 gene silencing improved renal inflammatory status, as seen by the reduction of CD68 and CD3 T-cell infiltrates, attenuated pro-inflammatory, and enhanced anti-inflammatory mediators. Furthermore, siRNA administration decreased a spleen pro-inflammatory monocyte subset and reduced TNFα secretion by splenic T cells. In the cold ischemia model with syngeneic and allogeneic renal transplantation, the most effective dose induced similar functional and structural renoprotective effects. Our data show the efficacy of our siRNA in modulating both the local and the systemic inflammatory milieu after an ischemic insult. Thus, CD40 silencing could emerge as a novel therapeutic strategy in solid organ transplantation.

Abstract TMP68: A Pro-inflammatory Monocyte Subset (ccr2+/ly6c hi ) Is Dominantly Recruited Into Acute Ischemic Brain.

Post-ischemic inflammation has been associated with ischemic brain injury. Infiltrated peripheral immune cells including monocytes/macrophages (MMs) contribute to the stroke-induced inflammation. MM mobilization from the periphery to the brain (MM trafficking) involves spleen, as an immediate storage of monocytes and the circulatory system as a conduit for their transport; however, there has been no systematic analysis of MM trafficking in stroke. There are two monocyte subsets, a pro-inflammatory (CCR2+/Ly6C hi ) and an anti-inflammatory (CCR2-/Ly6C lo ). These are sequentially recruited to the injury site in a controlled manner to elicit inflammation and repair/healing respectively. Current study determines the extent of trafficking of the monocyte subsets upon stroke and the role of the subsets on stroke-induced brain injury. Eleven week-old C57BL mice were subjected to transient middle cerebral artery occlusion and then leukocytes were isolated from blood, spleen, and brain prior to, 1d-, and 3d-post ischemia. The cells were incubated with antibodies (lineage marker or CD45 and CD11b to detect MMs, and Ly6C) and then analyzed using flow cytometry/FACS. We observed decreased spleen size following ischemia (&gt;40% reduction in spleen weight at 1d- and 3d-post). Both Ly6C hi and Ly6C lo MM number in the spleen and blood were significantly decreased in 1d-post ischemic mice, and these levels were sustained until 3d-post ischemia. On the other hand, brain MM numbers were increased at 1d-post and further increased at 3d-post ischemia (Fig. 1). Importantly, the Ly6C hi subset was dominantly increased in the ischemic brain compared to Ly6C lo subset (Fig. 2). The findings of MM trafficking and predominant increase in Ly6C hi MMs in the ischemic brain indicate that the pro-inflammatory monocyte subset might have a critical role in acute ischemic brain injury. Selective targeting of pro-inflammatory subset is suggested to reduce acute stroke-induced brain injury.

Markers of inflammation and CD8 T‐cell activation, but not monocyte activation, are associated with subclinical carotid artery disease in HIV‐infected individuals

The aim of the study was to explore the relationships between lymphocyte and monocyte activation, inflammation, and subclinical vascular disease among HIV-1-infected patients on antiretroviral therapy (ART). Baseline mean common carotid artery (CCA) intima-media thickness (IMT) and carotid plaque (IMT > 1.5 cm) were evaluated in the first 60 subjects enrolled in the Stopping Atherosclerosis and Treating Unhealthy Bone with Rosuvastatin in HIV (SATURN-HIV) trial. All subjects were adults on stable ART with evidence of heightened T-cell activation (CD8(+)CD38(+)HLA-DR(+) ≥ 19%) or increased inflammation (high-sensitivity C-reactive protein ≥ 2 mg/L). All had fasting low-density lipoprotein (LDL) cholesterol ≤ 130 mg/dL. Seventy-eight per cent of patients were men and 65% were African-American. Median (interquartile range) age and CD4 count were 47 (43, 52) years and 648 (511, 857) cells/μL, respectively. All had HIV-1 RNA < 400 HIV-1 RNA copies/mL. Mean CCA-IMT was correlated with log-transformed CD8(+)CD38(+)HLA-DR(+) percentage (r = 0.326; P = 0.043), and concentrations of interleukin-6 (r = 0.283; P = 0.028), soluble vascular cell adhesion molecule (sVCAM; r = 0.434; P = 0.004), tumour necrosis factor-α receptor-I (TNFR-I; r = 0.591; P < 0.0001) and fibrinogen (r = 0.257; P = 0.047). After adjustment for traditional cardiovascular disease (CVD) risk factors, the association with TNFR-I (P = 0.007) and fibrinogen (P = 0.033) remained significant. Subjects with plaque (n = 22; 37%) were older [mean (standard deviation) 51 (7.7) vs. 43 (9.4) years, respectively; P = 0.002], and had a higher CD8(+)CD38(+)HLA-DR(+) percentage [median (interquartile range) 31% (24, 41%) vs. 23% (20, 29%), respectively; P = 0.046] and a higher sVCAM concentration [mean (standard deviation) 737 (159) vs. 592 (160) ng/mL, respectively; P = 0.008] compared with those without plaque. Pro-inflammatory monocyte subsets and serum markers of monocyte activation (soluble CD163 and soluble CD14) were not associated with CCA-IMT or plaque. Participants in SATURN-HIV have a high level of inflammation and immune activation that is associated with subclinical vascular disease despite low serum LDL cholesterol.

Effects of Two Weeks of High-intensity Interval Training (HIIT) on Monocyte TLR2 and TLR4 Expression in High BMI Sedentary Men

Monocyte TLR expression has been shown to be reduced after a combination of aerobic and resistance exercise, but more studies considering the influences of different exercise intensities, type and duration on TLR expression are needed. Although there is an agreement about the importance of physical exercise, the minimal amount needed to improve health status is uncertain. Therefore, the aim of this study was to analyse the influence of 2 weeks of high-intensity intermittent exercise training on CD14+ monocyte TLR4 expression in a sedentary, high BMI population. As a secondary purpose, this study covers the influence of exercise on classical and pro-inflammatory monocytes and the TLR4 expression before and after a training period in these monocyte subsets. Six high-intensity interval training (HIIT) sessions over a 2 week period (three sessions per week) were completed by 11 sedentary participants (24 ± 5 years old). Blood samples were taken at the beginning and end of the training period for analysis of haematocrit, haemoglobin, total white blood cell (leukocyte), monocyte counts, monocyte CD14+ TLR4 expression and monocyte subsets. Two weeks of high-intensity intermittent exercise training increased VO2peak and total CD14+ monocyte TLR4 expression in a sedentary, high BMI population. There was no influence of training on the proportions of classical and pro-inflammatory monocyte subsets, but TLR4 expression in the majority of these monocyte subsets (apart from CD14++CD16+) was higher after the six training sessions.

HLA-DRhi and CCR9 Define a Pro-Inflammatory Monocyte Subset in IBD

OBJECTIVES:It has been demonstrated that circulating monocytes relocate to the intestinal mucosa during intestinal inflammation, but the phenotype and inflammatory mechanisms of these monocytes remain poorly understood. Here, we have investigated blood monocytes expressing high levels of HLA-DR and CCR9 in patients with inflammatory bowel disease (IBD).METHODS:Fifty-one patients with mild to severe ulcerative colitis (UC; n=31; UC-DAI 3–12) or Crohn's disease (CD; n=20; Harvey–Bradshaw indices (HBI) 2–16) were included together with 14 controls, during IBD therapy for four consecutive weeks. The frequency of CD14+HLA-DRhi monocytes was monitored weekly in peripheral blood, using flow cytometry. The surface phenotype and cytokine profile of these monocytes were established using flow cytometry and real-time PCR. Clinical parameters were assessed weekly in all patients.RESULTS:The frequency of circulating CD14+HLA-DRhi monocytes was significantly higher in IBD patients with moderate to severe disease compared with healthy controls (P<0.001). During treatment with corticosteroids and granulocyte/monocyte apheresis, the proportion of circulating CD14+HLA-DRhi monocytes was significantly reduced. CD14+HLA-DRhi monocytes produced high levels of inflammatory mediators, such as tumor necrosis factor (TNF)-α, and expressed the gut-homing receptor CCR9. Furthermore, we found that the CCR9 ligand, CCL25/TECK, was expressed at high levels in the colonic mucosa in IBD patients with active disease.CONCLUSIONS:CD14+HLA-DRhi blood monocytes were increased in patients with active IBD. These monocytes exhibit a pro-inflammatory, gut-homing phenotype with regard to their TNF-α production and expression of CCR9. Our results suggest that these monocytes are important in mediating intestinal inflammation, and provide potential therapeutic targets in IBD.

PMO-126 The role of vascular-adhesion-protein 1 (vap-1) in mediating monocyte migration across inflamed hepatic sinusoidal endothelium

IntroductionThere is compelling evidence that accumulating monocyte-derived macrophages are pivotally involved in driving liver fibrogenesis. It remains unclear which molecules mediate transmigration of these cells across hepatic sinusoidal endothelial cells...

Toll-like receptor expression on classic and pro-inflammatory blood monocytes after acute exercise in humans

Monocytes are a heterogeneous group of cells, the relative distribution of which change in peripheral blood following a strenuous bout of aerobic exercise. Monocyte subtypes can be identified in blood based on the cell surface expression of CD14 and CD16: classic (CD14++bright/CD16−negative) and the CD16+dim (CD14++bright/CD16+dim) and CD16++bright (CD14+dim/CD16++bright) pro-inflammatory subtypes. Whole monocyte population changes in TLR2, TLR4 and HLA.DR expression have previously been documented after acute exercise without accounting for relative changes in monocyte subpopulations, therefore, this study examined their expression on classic and pro-inflammatory monocyte subsets following 45min of treadmill running at 75% V˙O2max. Mononuclear cells isolated from the peripheral blood of moderately trained male subjects (n=15) before (PRE), immediately after (POST) and 1h after (1H) exercise were assessed for TLR2, TLR4 and HLA.DR expression on blood monocytes and their subpopulations using three-colour flow cytometry. Compared to PRE, the proportion of CD14+/CD16+ monocytes was 27% greater POST and 49% less at 1H and was associated with changes in the CD16++bright pro-inflammatory subtype (p<0.05). TLR2 expression was 12% lower on CD16+dim monocytes POST (p<0.05), whereas TLR4 and HLA.DR expression on total monocytes was 12% and 22% lower at 1H, respectively, and was attributed to changes in the classic (p<0.05) and not the pro-inflammatory subsets (p>0.05). We conclude that acute exercise causes localised changes in TLR2, TLR4 and HLA.DR expression within specific blood monocyte subpopulations, and could therefore be occurring at the cellular level. Such alterations might have significant implications for modulation of post-exercise immune surveillance.