Recruitment Of Classical Monocytes Research Articles

B cells mediate lung ischemia/reperfusion injury by recruiting classical monocytes via synergistic B cell receptor/TLR4 signaling.

Ischemia/reperfusion injury-mediated (IRI-mediated) primary graft dysfunction (PGD) adversely affects both short- and long-term outcomes after lung transplantation, a procedure that remains the only treatment option for patients suffering from end-stage respiratory failure. While B cells are known to regulate adaptive immune responses, their role in lung IRI is not well understood. Here, we demonstrated by intravital imaging that B cells are rapidly recruited to injured lungs, where they extravasate into the parenchyma. Using hilar clamping and transplant models, we observed that lung-infiltrating B cells produce the monocyte chemokine CCL7 in a TLR4-TRIF-dependent fashion, a critical step contributing to classical monocyte (CM) recruitment and subsequent neutrophil extravasation, resulting in worse lung function. We found that synergistic BCR-TLR4 activation on B cells is required for the recruitment of CMs to the injured lung. Finally, we corroborated our findings in reperfused human lungs, in which we observed a correlation between B cell infiltration and CM recruitment after transplantation. This study describes a role for B cells as critical orchestrators of lung IRI. As B cells can be depleted with currently available agents, our study provides a rationale for clinical trials investigating B cell-targeting therapies.

THE ANNEXIN-A1 MIMETIC RTP-026 PROMOTES ACUTE CARDIOPROTECTION THROUGH MODULATION OF IMMUNE CELL ACTIVATION

The cardio-protective and immuno-regulatory properties of RTP-026, a synthetic peptide that spans the Annexin-A1 (AnxA1) N-terminal region, were tested in rat acute myocardial infarction. In vitro, selective activation of formyl-peptide receptor type 2 (FPR2) by RTP-026 occurred with apparent EC50 in the 10-30nM range. With human primary cells, RTP-026 counteracted extension of neutrophil life-span and augmented phagocytosis of fluorescent E.coli by blood myeloid cells. An in vivo model of rat acute infarction was used to quantify tissue injury and phenotype immune cells in myocardium and blood. The rat left anterior descending coronary artery was occluded and then reopened for 2-hour or 24-hour reperfusion. For the 2-hour reperfusion protocol, RTP-026 (25-500µg/kg; given i.v. at the start of reperfusion) significantly reduced infarct size by ∼50 %, with maximal efficacy at 50µg/kg. Analyses of cardiac immune cells showed that RTP-026 reduced neutrophil and classical monocyte recruitment to the damaged heart. In the blood, RTP-026 (50µg/kg) attenuated activation of neutrophils and monocytes monitored through CD62L and CD54 expression. Modulation of vascular inflammation by RTP-026 was demonstrated by reduction in plasma levels of mediators like TNF-α, IL-1β, KC, PGE2 and PGF2α⊡ For the 24-hour reperfusion protocol, RTP-026 (30µg/kg given i.v. at 0, 3 and 6h reperfusion) reduced necrotic myocardium by ∼40 %. RTP-026 modulate immune cell responses and decreases infarct size of the heart in preclinical settings. Tempering over-exuberant immune cell activation by RTP-026 is a suitable approach to translate the biology of AnxA1 for therapeutic purposes.

Interleukin 4 Controls the Pro-Tumoral Role of Macrophages in Mammary Cancer Pulmonary Metastasis in Mice.

Simple SummaryMetastasis is the main cause of death from breast cancer. In mouse models of breast cancer lung metastasis, macrophages enhance metastasis by promoting tumor cell seeding and persistent growth. Here, we show that interleukin-4 (IL4) is required for this process as IL4 receptor (IL4rα)-null mice develop fewer and smaller lung metastases. This deficiency is partially rescued by adoptive transfer of wild-type monocytes. IL4 signaling in macrophages upregulates the expression of the chemokine receptor CXCR2, necessary for IL4-mediated tumor cell extravasation in vitro. In addition, expression of several other genes already causally associated with lung metastasis including Ccl2, Csf1, Ccr1, Hgf and Flt1 are upregulated in macrophages. High-resolution intravital imaging at the time of metastatic seeding showed reduced physical interaction between tumor cells and IL4rα-deficient macrophages, showing the dependence on IL4. We conclude that IL4 signaling in monocytes and macrophages is important during seeding and growth of breast metastasis in the lung.Metastasis is the systemic manifestation of cancer and the main cause of death from breast cancer. In mouse models of lung metastases, recruitment of classical monocytes from blood to the lung and their differentiation to metastasis-associated macrophages (MAMs) facilitate cancer cell extravasation, survival and growth. Ablation of MAMs or their monocytic progenitors inhibits metastasis. We hypothesized that factors controlling macrophage polarization modulate tumor cell extravasation in the lung. We evaluated whether signaling by Th1 or Th2 cytokines in macrophages affected transendothelial migration of tumor cells in vitro. Interferon gamma and LPS inhibited macrophage-dependent tumor cell extravasation while the Th2 cytokine interleukin-4 (IL4) enhanced this process. We demonstrated that IL4 receptor (IL4rα)-null mice developed fewer and smaller lung metastasis in E0771-LG mammary cancer models of this disease. Adoptive transfer of wild-type monocytes to IL4rα-deficient mice partially rescued this phenotype. IL4 signaling in macrophages controlled the expression of the chemokine receptor CXCR2, necessary for IL4-mediated tumor cell extravasation in vitro. Furthermore, IL4 signaling in macrophages regulated the transcript abundance of several other genes already causally associated with mammary cancer lung metastasis including Ccl2, Csf1, Ccr1, Hgf and Flt1. The central role of IL4 signaling in MAMs was confirmed by high-resolution intravital imaging of the lung in mice at the time of metastatic seeding, which showed reduced physical interaction between tumor cells and IL4rα-deficient macrophages. This interaction with wild-type MAMs enhanced tumor cell survival and seeding, which was lost in the IL4rα mice. These data indicate that IL4 signaling in monocytes and macrophages is key during seeding and growth of breast metastasis in the lung, as it regulates pro-tumoral paracrine signaling between cancer cells and macrophages.

Resolvin D1 reduces cancer growth stimulating a protective neutrophil-dependent recruitment of anti-tumor monocytes

BackgroundInnovative therapies to target tumor-associated neutrophils (PMN) are of clinical interest, since these cells are centrally involved in cancer inflammation and tumor progression. Resolvin D1 (RvD1) is a lipid autacoid that promotes resolution of inflammation by regulating the activity of distinct immune and non-immune cells. Here, using human papilloma virus (HPV) tumorigenesis as a model, we investigated whether RvD1 modulates PMN to reduce tumor progression.MethodsGrowth-curve assays with multiple cell lines and in vivo grafting of two distinct HPV-positive cells in syngeneic mice were used to determine if RvD1 reduced cancer growth. To investigate if and how RvD1 modulates PMN activities, RNA sequencing and multiplex cytokine ELISA of human PMN in co-culture with HPV-positive cells, coupled with pharmacological depletion of PMN in vivo, were performed. The mouse intratumoral immune cell composition was evaluated through FACS analysis. Growth-curve assays and in vivo pharmacological depletion were used to evaluate anti-tumor activities of human and mouse monocytes, respectively. Bioinformatic analysis of The Cancer Genome Atlas (TCGA) database was exploited to validate experimental findings in patients.ResultsRvD1 decreased in vitro and in vivo proliferation of human and mouse HPV-positive cancer cells through stimulation of PMN anti-tumor activities. In addition, RvD1 stimulated a PMN-dependent recruitment of classical monocytes as key determinant to reduce tumor growth in vivo. In human in vitro systems, exposure of PMN to RvD1 increased the production of the monocyte chemoattractant protein-1 (MCP-1), and enhanced transmigration of classical monocytes, with potent anti-tumor actions, toward HPV-positive cancer cells. Consistently, mining of immune cells infiltration levels in cervical cancer patients from the TCGA database evidenced an enhanced immune reaction and better clinical outcomes in patients with higher intratumoral monocytes as compared to patients with higher PMN infiltration.ConclusionsRvD1 reduces cancer growth by activating PMN anti-cancer activities and encouraging a protective PMN-dependent recruitment of anti-tumor monocytes. These findings demonstrate efficacy of RvD1 as an innovative therapeutic able to stimulate PMN reprogramming to an anti-cancer phenotype that restrains tumor growth.

Crosstalk between nonclassical monocytes and alveolar macrophages mediates transplant ischemia-reperfusion injury through classical monocyte recruitment.

Primary graft dysfunction (PGD) is the predominant cause of early graft loss following lung transplantation. We recently demonstrated that donor pulmonary intravascular nonclassical monocytes (NCM) initiate neutrophil recruitment. Simultaneously, host-origin classical monocytes (CM) permeabilize the vascular endothelium to allow neutrophil extravasation necessary for PGD. Here, we show that a CCL2-CCR2 axis is necessary for CM recruitment. Surprisingly, although intravital imaging and multichannel flow cytometry revealed that depletion of donor NCM abrogated CM recruitment, single cell RNA sequencing identified donor alveolar macrophages (AM) as predominant CCL2 secretors. Unbiased transcriptomic analysis of murine tissues combined with murine KOs and chimeras indicated that IL-1β production by donor NCM was responsible for the early activation of AM and CCL2 release. IL-1β production by NCM was NLRP3 inflammasome dependent and inhibited by treatment with a clinically approved sulphonylurea. Production of CCL2 in the donor AM occurred through IL-1R–dependent activation of the PKC and NF-κB pathway. Accordingly, we show that IL-1β–dependent paracrine interaction between donor NCM and AM leads to recruitment of recipient CM necessary for PGD. Since depletion of donor NCM, IL-1β, or IL-1R antagonism and inflammasome inhibition abrogated recruitment of CM and PGD and are feasible using FDA-approved compounds, our findings may have potential for clinical translation.

Abstract IA001: Deconvoluting the tumor microenvironment to enhance immunotherapy

Abstract Cancer evolution to metastasis (the metastatic cascade) involves not only the acquisition of mutations or epigenetic modifications in oncogenes and tumor suppressor gene but also an interplay with the surrounding tumor microenvironment (TME). The TME promotes every step of the metastatic cascade. In this TME immune cells of both the acquired and innate system are highly represented and generally these act in a pro-tumoral fashion or are inert. Much work has been performed on primary tumors and studies of metastasis have lagged behind despite the fact that ~90% of deaths from solid tumors are caused by this process. We have addressed this issue using mouse models of breast cancer lung and bone metastasis. Our studies have particularly focussed upon metastasis associated macrophages (MAMs) that are distinct from the tumor associated macrophages in primary tumors. We have shown that MAMs and their progenitors promote metastatic seeding and persistent growing in both organs. In the lung particularly we have described complex signalling pathways including the regulation of recruitment of classical monocytes to the metastatic site and stages of MAM differentiation form these monocytes. Our data show not only direct pro-tumoral effects of these cells on tumor cells such as conferring survival signals but also indirect ones via suppression of tumor cell cytotoxic T and NK cell activity (Kitamura et al., 2017; Kitamura et al., 2019; Ma et al., 2020). In parallel we have profiled TAMs in primary human breast and endometrial cancers and shown that they are different from circulating monocytes but surprisingly, are also different from each other. Further bioinformatic analysis and multiplex immunohistochemistry using breast cancer data sets shows that TAM signatures can be derived that predict poor disease specific overall survival and disease sub-classes. These TAMs have many different subtypes within a tumor and thus may play independent roles including anti-tumoral roles particularly in the face of immunotherapy. Exploiting these possibilities for therapy will be discussed (Lopez-Yrigoyen et al., 2020). Kitamura, T., Doughty-Shenton, D., Cassetta, L., Fragkogianni, S., Brownlie, D., Kato, Y., Carragher, N., and Pollard, J.W. (2017). Monocytes Differentiate to Immune Suppressive Precursors of Metastasis-Associated Macrophages in Mouse Models of Metastatic Breast Cancer. Frontiers in immunology 8, 2004. Kitamura, T., Kato, Y., Brownlie, D., Soong, D.Y.H., Sugano, G., Kippen, N., Li, J., Doughty-Shenton, D., Carragher, N., and Pollard, J.W. (2019). Mammary Tumor Cells with High Metastatic Potential Are Hypersensitive to Macrophage-Derived HGF. Cancer immunology research 7, 2052-2064. Lopez-Yrigoyen, M., Cassetta, L., and Pollard, J.W. (2020). Macrophage targeting in cancer. Ann. N. Y. Acad. Sci. Ma, R.Y., Zhang, H., Li, X.F., Zhang, C.B., Selli, C., Tagliavini, G., Lam, A.D., Prost, S., Sims, A.H., Hu, H.Y., et al. (2020). Monocyte-derived macrophages promote breast cancer bone metastasis outgrowth. J. Exp. Med. 217. Citation Format: Jeffrey W. Pollard. Deconvoluting the tumor microenvironment to enhance immunotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA001.

Radiation Impacts Early Atherosclerosis by Suppressing Intimal LDL Accumulation

Rationale: Bone marrow transplantation (BMT) is used frequently to study the role of hematopoietic cells in atherosclerosis, but aortic arch lesions are smaller in mice after BMT. Objective: To identify the earliest stage of atherosclerosis inhibited by BMT and elucidate potential mechanisms. Methods and Results: Ldlr-/- mice underwent total body γ-irradiation, bone marrow reconstitution and 6-week recovery. Atherosclerosis was studied in the ascending aortic arch and compared to mice without BMT. In BMT mice neutral lipid and myeloid cell topography were lower in lesions after feeding a cholesterol-rich diet (CRD) for 3, 6 and 12 weeks. Lesion coalescence and height were suppressed dramatically in mice post-BMT, whereas lateral growth was inhibited minimally. Targeted radiation to the upper thorax alone reproduced the BMT phenotype. Classical monocyte recruitment, intimal myeloid cell proliferation and apoptosis did not account for the post-BMT phenotype. Neutral lipid accumulation was reduced in 5-day lesions, thus we developed quantitative assays for LDL accumulation and paracellular leakage using DiI-labeled human LDL and rhodamine B-labeled 70kD dextran. LDL accumulation was dramatically higher in the intima of Ldlr-/- relative to Ldlr+/+ mice, and was inhibited by injection of HDL mimics, suggesting a regulated process. LDL, but not dextran, accumulation was lower in mice post-BMT both at baseline and in 5-day lesions. Since the transcript abundance of molecules implicated in LDL transcytosis was not significantly different in the post-BMT intima, transcriptomics from whole aortic arch intima, and at single cell resolution, was performed to give insights into pathways modulated by BMT. Conclusions: Radiation exposure inhibits LDL entry into the aortic intima at baseline and the earliest stages of atherosclerosis. Single cell transcriptomic analysis suggests that LDL uptake by endothelial cells is diverted to lysosomal degradation and reverse cholesterol transport pathways. This reduces intimal accumulation of lipid and impacts lesion initiation and growth.

Live Imaging of Monocyte Subsets in Immune Complex-Mediated Glomerulonephritis Reveals Distinct Phenotypes and Effector Functions.

Immune complexes within glomerular capillary walls cause crescentic GN (CrGN). Monocytes and macrophages are important in mediating CrGN, but little work has been done to phenotype the subpopulations involved and determine their respective contributions to glomerular inflammation. Live glomerular imaging using confocal microscopy monitored intravascular monocyte subset behavior during nephrotoxic nephritis (NTN) in a novel WKY-hCD68-GFP monocyte/macrophage reporter rat strain. Flow cytometry and qPCR further analyzed ex vivo the glomerular leukocyte infiltrate during NTN. Non-classical monocytes surveyed the glomerular endothelium via lymphocyte function-associated antigen 1 (LFA-1) in the steady state. During NTN, non-classical monocytes were recruited first, but subsequent recruitment and retention of classical monocytes was associated with glomerular damage. Monocytes recruited to the glomerular vasculature did not undergo transendothelial migration. This finding suggests that inflammation in immune complex-mediated CrGN is predominantly intravascular, driven by dynamic interactions between intravascular blood monocytes and the endothelium. Glomerular endothelium and non-classical monocytes overexpressed a distinct chemokine axis, which may orchestrate inflammatory myeloid cell recruitment and expression of damage mediators. Reduced classical monocyte recruitment in Lewis rats during NTN confirmed a role for CD16 in mediating glomerular damage. Monocyte subsets with distinct phenotypes and effector functions may be important in driving inflammation in experimental CrGN resulting from immune complexes formed within the glomerular capillary wall. LFA-1-dependent endothelial surveillance by non-classical monocytes may detect immune complexes through CD16, orchestrating the inflammatory response through intravascular retention of classical monocytes, which results in glomerular damage and proteinuria.

Circulating Mcp-1 Associated With Prolonged Cycling In Hot Temperature

PURPOSE: The purpose of this investigation was to examine the recruitment of classical monocytes during prolonged aerobic exercise in high temperature and humidity conditions. METHODS: Seven recreationally active men (23.4 ± 3.0 yrs; 180.9 ± 5.8 cm; 85.1 ± 11.3 kg; 3.7 ± 0.27 L∙min-1) completed five trials: a graded exercise test, and four cycling trials in 37°C/23% Relative Humidity (RH)(HTLH), 37°C/33%RH (HTMH), 24°C/38%RH (MTMH), and 24°C/51%RH (MTHH) in a counterbalanced fashion. During the exercise protocol, participants rested supine for 15 minutes before completing 60-min of cycling at 60% VO2max, a 15-min rest, and cycling until exhaustion at 90% VO2max (TTE), before 60 minutes of recovery. Blood samples were obtained prior to exercise (PRE), after 60 minutes of cycling (60), after the TTE (90) and following one hour of recovery (REC). Blood was assessed for plasma concentrations of Monocyte Chemoattractant Protein 1 (MCP-1) via ELISA, and CCR2 expression on classical monocytes (CD14++CD16-) via flow cytometry. Briefly, CCR2 expression was determined as fold change over fluorescence minus one (FMO). Data were analyzed using within-subjects repeated measures ANOVA. RESULTS: A main effect for time was observed (F = 8.9; p = 0.003; η2p = 0.560) for MCP-1 in circulation. MCP-1 increased from PRE (183.8 ± 59.7 pg.mL-1) to 60 (215.8 ± 74.1 pg.mL-1; p = 0.009), 90 (231.6 ± 83.8 pg.mL-1; p = 0.006), and REC (216.0 ± 88.3 pg.mL-1; p = 0.019). Concentrations also increased from 60 to 90 (p = 0.018). A main effect of time was also observed (F = 11.6; p = 0.009; η2p = 0.659) for CCR2 expression on classical monocytes. No differences in CCR2 expression were observed between PRE (147.4 ± 50.6), and 60 (146.0 ± 70.8; p = 0.869), however decreases from PRE were observed at 90 (131.1 ± 39.7; p = 0.023), and REC (102.2 ±37.1; p < 0.001). CONCLUSION: These data indicate that high temperature and/or humidity conditions do not impact recruitment of classical monocytes. Furthermore, prolonged cycling appears to increase circulating MCP-1 and decrease CCR2 expression on classical monocytes. Collectively, this may indicate a limited effect of aerobic exercise on the overall recruitment of classical monocytes, although further research is warranted. This investigation was partially funded by the Kent State University Research Council.

Mechanical strain determines the site-specific localization of inflammation and tissue damage in arthritis

Many pro-inflammatory pathways leading to arthritis have global effects on the immune system rather than only acting locally in joints. The reason behind the regional and patchy distribution of arthritis represents a longstanding paradox. Here we show that biomechanical loading acts as a decisive factor in the transition from systemic autoimmunity to joint inflammation. Distribution of inflammation and erosive disease is confined to mechano-sensitive regions with a unique microanatomy. Curiously, this pathway relies on stromal cells but not adaptive immunity. Mechano-stimulation of mesenchymal cells induces CXCL1 and CCL2 for the recruitment of classical monocytes, which can differentiate into bone-resorbing osteoclasts. Genetic ablation of CCL2 or pharmacologic targeting of its receptor CCR2 abates mechanically-induced exacerbation of arthritis, indicating that stress-induced chemokine release by mesenchymal cells and chemo-attraction of monocytes determines preferential homing of arthritis to certain hot spots. Thus, mechanical strain controls the site-specific localisation of inflammation and tissue damage in arthritis.

Spleen-derived classical monocytes mediate lung ischemia-reperfusion injury through IL-1β.

Ischemia-reperfusion injury, a form of sterile inflammation, is the leading risk factor for both short-term mortality following pulmonary transplantation and chronic lung allograft dysfunction. While it is well recognized that neutrophils are critical mediators of acute lung injury, processes that guide their entry into pulmonary tissue are not well understood. Here, we found that CCR2+ classical monocytes are necessary and sufficient for mediating extravasation of neutrophils into pulmonary tissue during ischemia-reperfusion injury following hilar clamping or lung transplantation. The classical monocytes were mobilized from the host spleen, and splenectomy attenuated the recruitment of classical monocytes as well as the entry of neutrophils into injured lung tissue, which was associated with improved graft function. Neutrophil extravasation was mediated by MyD88-dependent IL-1β production by graft-infiltrating classical monocytes, which downregulated the expression of the tight junction-associated protein ZO-2 in pulmonary vascular endothelial cells. Thus, we have uncovered a crucial role for classical monocytes, mobilized from the spleen, in mediating neutrophil extravasation, with potential implications for targeting of recipient classical monocytes to ameliorate pulmonary ischemia-reperfusion injury in the clinic.

The Effect of Post-Resistance Exercise Amino Acids on Plasma MCP-1 and CCR2 Expression.

The recruitment and infiltration of classical monocytes into damaged muscle is critical for optimal tissue remodeling. This study examined the effects of an amino acid supplement on classical monocyte recruitment following an acute bout of lower body resistance exercise. Ten resistance-trained men (24.7 ± 3.4 years; 90.1 ± 11.3 kg; 176.0 ± 4.9 cm) ingested supplement (SUPP) or placebo (PL) immediately post-exercise in a randomized, cross-over design. Blood samples were obtained at baseline (BL), immediately (IP), 30-min (30P), 1-h (1H), 2-h (2H), and 5-h (5H) post-exercise to assess plasma concentrations of monocyte chemoattractant protein 1 (MCP-1), myoglobin, cortisol and insulin concentrations; and expressions of C-C chemokine receptor-2 (CCR2), and macrophage-1 antigen (CD11b) on classical monocytes. Magnitude-based inferences were used to provide inferences on the true effects of SUPP compared to PL. Changes in myoglobin, cortisol, and insulin concentrations were similar between treatments. Compared to PL, plasma MCP-1 was “very likely greater” (98.1% likelihood effect) in SUPP at 2H. CCR2 expression was “likely greater” at IP (84.9% likelihood effect), “likely greater” at 1H (87.7% likelihood effect), “very likely greater” at 2H (97.0% likelihood effect), and “likely greater” at 5H (90.1% likelihood effect) in SUPP, compared to PL. Ingestion of SUPP did not influence CD11b expression. Ingestion of an amino acid supplement immediately post-exercise appears to help maintain plasma MCP-1 concentrations and augment CCR2 expression in resistance trained men.

Recruitment of classical monocytes can be inhibited by disturbing heteromers of neutrophil HNP1 and platelet CCL5.

In acute and chronic inflammation, neutrophils and platelets, both of which promote monocyte recruitment, are often activated simultaneously. We investigated how secretory products of neutrophils and platelets synergize to enhance the recruitment of monocytes. We found that neutrophil-borne human neutrophil peptide 1 (HNP1, α-defensin) and platelet-derived CCL5 form heteromers. These heteromers stimulate monocyte adhesion through CCR5 ligation. We further determined structural features of HNP1-CCL5 heteromers and designed a stable peptide that could disturb proinflammatory HNP1-CCL5 interactions. This peptide attenuated monocyte and macrophage recruitment in a mouse model of myocardial infarction. These results establish the in vivo relevance of heteromers formed between proteins released from neutrophils and platelets and show the potential of targeting heteromer formation to resolve acute or chronic inflammation.

Human T Cell Leukemia Virus Type 1 Infection of the Three Monocyte Subsets Contributes to Viral Burden in Humans.

Because the viral DNA burden correlates with disease development, we investigated the contribution of monocyte subsets (classical, intermediate, and nonclassical monocytes) to the total viral burden in 22 human T cell leukemia virus type 1 (HTLV-1)-infected individuals by assessing their infectivity status, frequency, as well as chemotactic and phagocytic functions. All three monocyte subsets sorted from HTLV-1-infected individuals were positive for viral DNA, and the frequency of classical monocytes was lower in the blood of HTLV-1-infected individuals than in that of uninfected individuals, while the expression levels of the chemokine receptors CCR5, CXCR3, and CX3CR1 in classical monocytes were higher in HTLV-1-infected individuals than uninfected individuals; the percentage of intermediate monocytes and their levels of chemokine receptor expression did not differ between HTLV-1-infected and uninfected individuals. However, the capacity of intermediate monocytes to migrate to CCL5, the ligand for CCR5, was higher, and a higher proportion of nonclassical monocytes expressed CCR1, CXCR3, and CX3CR1. The level of viral DNA in the monocyte subsets correlated with the capacity to migrate to CCL2, CCL5, and CX3CL1 for classical monocytes, with lower levels of phagocytosis for intermediate monocytes, and with the level of viral DNA in CD8(+) and CD4(+) T cells for nonclassical monocytes. These data suggest a model whereby HTLV-1 infection augments the number of classical monocytes that migrate to tissues and become infected and the number of infected nonclassical monocytes that transmit virus to CD4(+) and CD8(+) T cells. These results, together with prior findings in a macaque model of HTLV-1 infection, support the notion that infection of monocytes by HTLV-1 is likely a requisite for viral persistence in humans. Monocytes have been implicated in immune regulation and disease progression in patients with HTLV-1-associated inflammatory diseases. We detected HTLV-1 DNA in all three monocyte subsets and found that infection impacts surface receptor expression, migratory function, and subset frequency. The frequency of nonclassical patrolling monocytes is increased in HTLV-1-infected individuals, and they have increased expression of CCR1, CXCR3, and CX3CR1. The viral DNA level in nonclassical monocytes correlated with the viral DNA level in CD4(+) and CD8(+) T cells. Altogether, these data suggest an increased recruitment of classical monocytes to inflammation sites that may result in virus acquisition and, in turn, facilitate virus dissemination and viral persistence. Our findings thus provide new insight into the importance of monocyte infection in viral spread and suggest targeting of monocytes for therapeutic intervention.

Circulation Research “In This Issue” Anthology

<i>Circulation Research</i> “In This Issue” Anthology

Joint UK societies' 2014 consensus statement on renal denervation for resistant hypertension.

Resistant hypertension continues to pose a major challenge to clinicians worldwide and has serious implications for patients who are at increased risk of cardiovascular morbidity and mortality with this diagnosis. Pharmacological therapy for resistant hypertension follows guidelines-based regimens although there is surprisingly scant evidence for beneficial outcomes using additional drug treatment after three antihypertensives have failed to achieve target blood pressure. Recently there has been considerable interest in the use of endoluminal renal denervation as an interventional technique to achieve renal nerve ablation and lower blood pressure. Although initial clinical trials of renal denervation in patients with resistant hypertension demonstrated encouraging office blood pressure reduction, a large randomised control trial (Symplicity HTN-3) with a sham-control limb, failed to meet its primary efficacy end point. The trial however was subject to a number of flaws which must be taken into consideration in interpreting the final results. Moreover a substantial body of evidence from non-randomised smaller trials does suggest that renal denervation may have an important role in the management of hypertension and other disease states characterised by overactivation of the sympathetic nervous system. The Joint UK Societies does not recommend the use of renal denervation for treatment of resistant hypertension in routine clinical practice but remains committed to supporting research activity in this field. A number of research strategies are identified and much that can be improved upon to ensure better design and conduct of future randomised studies.

95: Sex-differences in classical monocyte recruitment during inflammation

Background Certain human cardiovascular diseases are subject to gender bias with more prevalence in men. Sexual dimorphisms in murine leukocyte responses during inflammation have previously been described, with males recruiting more neutrophils [1] . This study aimed to determine sex-differences in storage, trafficking and responses of classical monocytes (CMs), in an acute inflammatory model. Materials and methods Peritonitis was induced by intraperitoneal administration of zymosan (1 mg) to CX3 CR 1 + / gfp mice. Leukocytes were isolated from blood, peritoneal cavity, bone marrow (BM) and spleen. CMs were identified as CX3CR1+ Gr1+ by flow cytometry. Results Equal basal circulating, peritoneal and BM CM numbers were observed between the sexes, however males exhibited a greater splenic storage pool. Expression of key chemokine receptors was similar on splenic CMs from both sexes, as was the basal splenic chemokine environment. In response to zymosan, male mice demonstrated 2-fold greater classical monocytosis at 3 h post-administration than females, which translated into greater recruitment to the peritoneum. Reduction in BM cells at 3 h indicated mobilization; this was equal in both sexes. Equivalent spleen CM numbers at 3 h suggest greater outward flux in males than females. Female resident peritoneal macrophages were more numerous however expression of zymosan-sensing TLR2–TLR6 was equal to males and no sex-differences were seen in the basal or inflamed peritoneal cytokine environments. Conclusions These data indicate that male mice have greater splenic stores of CMs and furthermore recruit more CMs to the tissue during peritonitis. Sex-differences in classical monocyte trafficking during inflammation may translate to monocyte/macrophage-dependant pathologies more prevalent in males, like atherosclerosis, and may provide a more personalised method of targeting such diseases.

Footprints of Neutrophil Extracellular Traps as Predictors of Cardiovascular Risk

Cardiovascular events, such as myocardial infarction and stroke, are leading causes of mortality and morbidity in Western societies. Biomarkers could help to predict the risk of a patient and to advance the treatment of people at risk by allowing fine-tuning of the intensity of therapy.1 Although more recent studies have used proteomic, metabolomic, and genetic approaches to identify biomarkers, Borissoff et al2 here investigate the importance of double-stranded DNA, nucleosomes, thrombin-antithrombin complex complexes, and myeloperoxidase (MPO)–DNA complexes in predicting the severity of atherosclerosis and the risk of future cardiovascular events. Their data indicate that these markers were elevated in patients with severe coronary atherosclerosis or calcified coronary arteries. Furthermore, plasma nucleosome levels were found to be associated with an increased risk of coronary stenosis, whereas MPO–DNA complexes predicted the occurrence of major adverse cardiac events. Double-stranded DNA, nucleosomes, and MPO–DNA complexes are all markers of neutrophil extracellular traps (NETs), a mesh …

In This Issue

HomeCirculation ResearchVol. 112, No. 5In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Originally published1 Mar 2013https://doi.org/10.1161/RES.0b013e31828c5b1dCirculation Research. 2013;112:743Macrophage Targeted PET/MRI (p 755)Download figureDownload PowerPointMajmudar et al devise a macrophage imaging technique that could help identify unstable atherosclerotic plaques.Macrophage abundance in atherosclerotic plaques is strongly associated with the risk of plaque rupture, which can cause thrombus formation and even myocardial infarction or stroke. Therefore, it is important to image and quantify macrophages in human plaques to identify high-risk patients and to inform therapeutic interventions. However, existing imaging techniques lack sufficient sensitivity. Therefore, Majmudar and colleagues set out to develop a viable alternative. They generated radioactive dextran nanoparticles that could be efficiently taken up by macrophages. They injected these nanoparticles into the tail veins of mice and, after allowing time for phagocytosis, they tracked their vascular distribution using a combination of positron emission tomography (PET)—to detect the radioactive signal—and MRI—to determine anatomical location. They found that compared with wild-type mice, mice that were prone to atherosclerosis exhibited considerably higher nanoparticle PET signals from their aortas. The team also found that preventing the recruitment of macrophages to inflammation sites lowered these aortic PET signals. Because nanoparticles used in the study were biodegradable, this technique could perhaps be used to monitor atherosclerotic plaques in humans, say the authors.Cathelicidins and Monocyte Adhesion (p 792)Download figureDownload PowerPointWantha et al show how neutrophils recruit monocytes to sites of inflammation.It is currently believed that during early stages of acute inflammation, soluble granule proteins released from neutrophils are required for subsequent recruitment of monocytes to the inflammation site. However, the underlying mechanisms remain unclear. Wantha and colleagues investigated the monocyte-attracting ability of cathelicidin and cathepsin - two neutrophil granule proteins conserved between mice and humans. They found that deletion of cathelicidin in mice reduced the recruitment of classical monocytes to inflammation sites. Mice lacking cathepsin, on the other hand, showed no such defect. In vitro experiments using cathelicidin treated human vascular endothelial cells confirmed that the protein is indeed able to recruit classical monocytes. By blocking different receptor proteins on the surface of monocytes, the team discovered that one specific receptor was responsible for mediating cathelicidin-dependent recruitment. Interaction between cathelicidin and this receptor, FRP2, induced the activation of integrins that enable monocytes to adhere to sites of inflammation. This interaction could thus be targeted to treat for a variety of inflammatory disorders, say the authors.SDF-1 in Heart Failure (p 816)Download figureDownload PowerPointPenn et al report the outcome of a Phase I gene therapy trial for the treatment of ischemic heart failure.The tissue damage that occurs after a myocardial infarction can lead to progressive heart failure and, if left untreated, to death. Mending damaged cardiac tissue with the help of stem cells is the goal of several current therapeutic investigations. One such approach, adopted by Penn and colleagues, is to recruit the body’s own stem cells to the heart. Stromal cell-derived factor 1 (SDF-1) is a naturally occurring cytokine that attracts stem cells and is upregulated in injured tissues. In the infarcted heart tissue, however, SDF-1 action lasts less than a week, after which stem cell recruitment diminishes. Artificially maintaining SDF-1 expression in damaged myocardium has been shown to improve cardiac function in a number of pre-clinical studies. Penn et al now provide preliminary evidence that the same approach works in patients too. They injected a non-viral DNA vector encoding the SDF-1 gene into the peri-infarcted myocardia of 17 heart failure patients. Four months later, these patients exhibited improvements in quality of life, 6 minute walking distance, and in the overall clinical classification of their condition. And 12 months later, these improvements persisted. Although this was a relatively small Phase I trial, the results indicate that a larger, randomized trial is warranted. Previous Back to top Next FiguresReferencesRelatedDetails March 1, 2013Vol 112, Issue 5 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e31828c5b1d Originally publishedMarch 1, 2013 PDF download Advertisement

Distinct functions of chemokine receptor axes in the atherogenic mobilization and recruitment of classical monocytes

We used a novel approach of cytostatically induced leucocyte depletion and subsequent reconstitution with leucocytes deprived of classical (inflammatory/Gr1hi) or non-classical (resident/Gr1lo) monocytes to dissect their differential role in atheroprogression under high-fat diet (HFD). Apolipoprotein E-deficient (Apoe−/−) mice lacking classical but not non-classical monocytes displayed reduced lesion size and macrophage and apoptotic cell content. Conversely, HFD induced a selective expansion of classical monocytes in blood and bone marrow. Increased CXCL1 levels accompanied by higher expression of its receptor CXCR2 on classical monocytes and inhibition of monocytosis by CXCL1-neutralization indicated a preferential role for the CXCL1/CXCR2 axis in mobilizing classical monocytes during hypercholesterolemia. Studies correlating circulating and lesional classical monocytes in gene-deficient Apoe−/− mice, adoptive transfer of gene-deficient cells and pharmacological modulation during intravital microscopy of the carotid artery revealed a crucial function of CCR1 and CCR5 but not CCR2 or CX3CR1 in classical monocyte recruitment to atherosclerotic vessels. Collectively, these data establish the impact of classical monocytes on atheroprogression, identify a sequential role of CXCL1 in their mobilization and CCR1/CCR5 in their recruitment.