Ly6Chigh Monocytes Research Articles

Pravastatin attenuates atherosclerosis after myocardial infarction by inhibiting inflammatory Ly6Chigh monocytosis in apolipoprotein E knockout mice.

ObjectiveTo evaluate the protective effect of pravastatin on atherosclerotic development and inflammatory monocyte subset in atherosclerotic apolipoprotein E (ApoE)−/− mice after myocardial infarction (MI).MethodsMale ApoE−/− mice (8 weeks old) were fed a high-fat diet for 14 weeks throughout the experiment. A MI model was produced using 18-week-old ApoE−/− mice. They were randomly divided into three groups: sham group, MI group, and MI+Pra group (40 mg/kg/day pravastatin). After 4 weeks (at the end of the study period), the mice were sacrificed and cardiac function was evaluated by echocardiography. Aortic lesion areas were evaluated using oil red O staining. Plaque macrophage in aortic sinus was analyzed by immunofluorescence staining. Flow cytometry was used to explore the proportions of monocyte subsets in the blood, spleen, and bone marrow．ResultsPravastatin improved cardiac function and reduced lesion areas. It also attenuated the supply of monocytes in spleen, especially the inflammatory Ly6Chigh monocyte subset. Pravastatin also subsequently reduced macrophage accumulation in atherosclerotic lesions.ConclusionsMI accelerated chronic atherosclerosis progress. Pravastatin suppressed atherosclerotic development and inhibited inflammatory monocytosis after MI in ApoE−/− mice.

Gut Microbial Dysbiosis after Traumatic Brain Injury Induces Alterations in the Immune Response and Fear Memory

The influence of the gut microbiota on traumatic brain injury (TBI) is presently unknown. This knowledge gap is of paramount clinical significance as TBI patients are highly susceptible to alterations in the gut microbiota by antibiotic exposure. Antibiotic-induced gut microbial dysbiosis established prior to TBI significantly worsened neuronal loss and reduced microglia activation in the injured hippocampus with concomitant changes in fear memory response. Importantly, microbial dysbiosis after TBI reduced hippocampal infiltration of Ly6Chigh monocytes followed by a decreased presence of CD4+ and CD8+ T cells that persisted through 1 month post-injury, and increase of microglial pro-inflammatory markers. By 3 months, these mice had a larger percentage of microglia with a persistent amoeboid morphology, increased neuronal loss, and a maladaptive fear memory response. These data demonstrate the important role the gut microbiota plays in neuroinflammation and recovery after TBI and implicate gut microbial modulation as a potential therapeutic intervention.

2B4 controls gut immune homeostasis by promoting IL-1β expression in Ly6Chigh monocytes that trigger IL-22/IL-18-mediated epithelial regeneration

Abstract Impaired signaling, downstream of 2B4, is associated with colitis in human patients with XLP ; however, the precise mechanism by which 2B4 maintains gut integrity remains unknown. In dextran sodium sulfate (DSS)-fed reciprocal chimera mice, massive tissue destruction was observed due to lack of 2B4 in hematopoietic compartments, in particular Ly6ChighCD11b+ monocyte subsets within the lamina propria leukocytes. Not only did the number of Ly6ChighCD11b+ monocytes get significantly reduced, their intrinsic capacity to produce IL-1β during DSS stimulation was also drastically impaired in 2B4−/− mice. These defects led to an impaired IL-22 secretion in ILCs and IL-18 production in gut epithelial cells in the 2B4−/− mice. Furthermore, severe inflammatory phenotypes seen in 2B4−/− mice could only be rescued by adoptive transfer of Ly6ChighCD11b+ monocytes, but not with BMDC or NK cells. Therefore, 2B4 functions as an important receptor in inflammatory monocytes in response to commensal microbiota during acute colonic inflammation.

Myriocin and d-PDMP ameliorate atherosclerosis in ApoE-/- mice via reducing lipid uptake and vascular inflammation.

Sphingolipids have been implicated in the etiology of atherosclerosis. The commonly used sphingolipid inhibitors, myriocin (a ceramide inhibitor) and d-PDMP (d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glycosphingolipid inhibitor), have shown therapeutic potential but their efficacy and their underlying mechanisms remain unclear. Here, apolipoprotein E-deficient (apoE-/-) mice were fed a high-fat diet (HFD) and treated with a control, myriocin, d-PDMP, or atorvastatin for 12 weeks. We analyzed the effects of these drugs on the size and detailed composition of atherosclerotic plaques. Molecular biological approaches were used to explore how the inhibitors affect lipid metabolism and foam-cell formation. Treatment with myriocin or d-PDMP led to smaller and less vulnerable atherosclerotic lesions and was almost as effective as atorvastatin. Sphingolipid inhibitors down-regulated the expression of monocyte chemotactic protein 1 (MCP-1) and its receptor chemoattractant cytokine receptor 2 (CCR2), which play a key role in monocyte recruitment. They also decreased pro-inflammatory Ly-6chigh monocytes and influenced the uptake of modified LDL by down-regulating the expression of cluster of differentiation 36 (CD36) and lectin-like oxidized LDL (ox-LDL) receptor-1 (LOX-1). The inhibitors exhibited the advantage of maintaining normal glucose homeostasis compared with atorvastatin. These findings reveal for the first time that the modulation of sphingolipid synthesis can effectively alleviate atherosclerosis progression by preventing lipid uptake and reducing inflammatory responses in the arterial walls.

Aberrant activation of bone marrow Ly6Chighmonocytes in diabetic mice contributes to impaired glucose tolerance.

Accumulating evidence indicates that diabetes and obesity are associated with chronic low-grade inflammation and multiple organ failure. Tissue-infiltrated inflammatory M1 macrophages are aberrantly activated in these conditions and contribute to hyperglycemia and insulin resistance. However, it is unclear at which stage these cells become aberrantly activated: as precursor monocytes in the bone marrow or as differentiated macrophages in tissues. We examined the abundance, activation state, and function of bone marrow-derived Ly6Chigh monocytes in mice with diabetes and/or obesity. Ly6Chigh monocytes were FACS-purified from six groups of male mice consisting of type 2 diabetes model db/db mice, streptozotocin (STZ) induced insulin depletion mice, high fat diet (HFD) induced obesity mice and each control mice. Ly6Chigh monocytes were then analyzed for the expression of inflammation markers by qRT-PCR. In addition, bone marrow-derived Ly6Chigh monocytes from db/+ and db/db mice were fluorescently labeled and injected into groups of db/db recipient mice. Cell trafficking to tissues and levels of markers were examined in the recipient mice. The expression of many inflammation-related genes was significantly increased in Ly6Chigh monocytes from db/db mice, compared with the control. Bone marrow-derived Ly6Chigh monocytes isolated from db/db mice, but not from db/+ mice, displayed prominent infiltration into peripheral tissues at 1 week after transfer into db/db mice. The recipients of db/db Ly6Chigh monocytes also exhibited significantly increased serum glucose levels and worsening tolerance compared with mice receiving db/+ Ly6Chigh monocytes. These novel observations suggest that activated Ly6Chigh monocytes may contribute to the glucose intolerance observed in diabetes.

Extracellular vesicles from human cardiovascular progenitors trigger a reparative immune response in infarcted hearts.

The cardioprotective effects of human induced pluripotent stem cell-derived cardiovascular progenitor cells (CPC) are largely mediated by the paracrine release of extracellular vesicles (EV). We aimed to assess the immunological behaviour of EV-CPC, which is a prerequisite for their clinical translation. Flow cytometry demonstrated that EV-CPC expressed very low levels of immune relevant molecules including HLA Class I, CD80, CD274 (PD-L1), and CD275 (ICOS-L); and moderate levels of ligands of the natural killer (NK) cell activating receptor, NKG2D. In mixed lymphocyte reactions, EV-CPC neither induced nor modulated adaptive allogeneic T cell immune responses. They also failed to induce NK cell degranulation, even at high concentrations. These in vitro effects were confirmed in vivo as repeated injections of EV-CPC did not stimulate production of immunoglobulins or affect the interferon (IFN)-γ responses from primed splenocytes. In a mouse model of chronic heart failure, intra-myocardial injections of EV-CPC, 3 weeks after myocardial infarction, decreased both the number of cardiac pro-inflammatory Ly6Chigh monocytes and circulating levels of pro-inflammatory cytokines (IL-1α, TNF-α, and IFN-γ). In a model of acute infarction, direct cardiac injection of EV-CPC 2 days after infarction reduced pro-inflammatory macrophages, Ly6Chigh monocytes, and neutrophils in heart tissue as compared to controls. EV-CPC also reduced levels of pro-inflammatory cytokines IL-1α, IL-2, and IL-6, and increased levels of the anti-inflammatory cytokine IL-10. These effects on human macrophages and monocytes were reproduced in vitro; EV-CPC reduced the number of pro-inflammatory monocytes and M1 macrophages, while increasing the number of anti-inflammatory M2 macrophages. EV-CPC do not trigger an immune response either in in vitro human allogeneic models or in immunocompetent animal models. The capacity for orienting the response of monocyte/macrophages towards resolution of inflammation strengthens the clinical attractiveness of EV-CPC as an acellular therapy for cardiac repair.

MSCs Contribute to the Conversion of Ly6Chigh Monocytes into Ly6Clow Subsets under AMI.

Background Ly6Chigh monocytes are inflammatory cells that accumulate in an infarcted myocardium, and Ly6Clow monocytes are believed to be reparative and curb myocardial remodeling. NR4A1 is a novel target for modulating the inflammatory phenotype of monocytes during atherogenesis. Objectives We aimed to investigate whether MSCs can contribute to the heterogeneity of Ly6Chigh monocytes differentiated into Ly6Clow monocytes and whether this regulation is related to nuclear receptor NR4A1. Methods Ly6Chigh/low monocytes were first cocultured with MSCs. C57BL/6CX3CR1-/- mice and C57BL/6 wild-type mice were then used to construct AMI models, and survival functions in the two groups were further compared. Ly6Chigh/low monocytes in circulation and in MI tissue of C57BL/6CX3CR1-/- AMI mice with or without MSC transplantation were determined by flow cytometry at day 1 and day 3. NR4A1 expression was further determined by Western blot. Apoptosis of cardiac myocytes in the infarct border zone at day 3 and day 7 was identified by TUNEL kits. Angiogenesis in the AMI heart at day 7 and day 21 was determined through immunohistochemistry by CD31. Results We first demonstrated that the percentage of Ly6Clow monocytes increased greatly after 3 days of coculture with MSCs (12.8% ± 3.77% vs. 3.69% ± 0.74%, p < 0.001). The expression of NR4A1 in Ly6Chigh/low monocytes was also significantly elevated at that time (1.81 ± 0.46 vs. 0.43 ± 0.09, p < 0.001). Following AMI, the percentage of circulating Ly6Clow monocytes in C57BL/6CX3CR1-/- mice was significantly lower than that in C57BL/6 wild-type mice (4.36% ± 1.27% vs. 12.17% ± 3.81%, p < 0.001). The survival rate of C57BL/6CX3CR1-/- mice (25%) was significantly lower than that of C57BL/6 wild-type mice (56.3%) after AMI (χ2 = 4.343, p = 0.037). After MSCs were transplanted, we observed a significant increase in Ly6Clow monocytes both in circulation (16.7% ± 3.67% vs. 3.22% ± 0.44%, p < 0.001) and in the MI heart (3.31% ± 0.69% vs. 0.42% ± 0.21%, p < 0.001) of C57BL/6CX3CR1-/- mice. Western blot analysis further showed that the expression level of NR4A1 in the MI hearts of C57BL/6CX3CR1-/- mice increased significantly under MSC transplantation (0.39 ± 0.10 vs. 0.11 ± 0.04, p < 0.001). We also found significantly decreased TUNEL+ cardiac myocytes (15.45% ± 4.42% vs. 22.78% ± 6.40%, p < 0.001) in mice with high expression levels of NR4A1 compared to mice with low expression levels. Meanwhile, we further identified increased capillary density in the infarct zones of mice with high expression levels of NR4A1 (0.193 ± 0.036 vs. 0.075 ± 0.019, p < 0.001) compared to mice with low expression levels 21 days after AMI. Conclusions MSCs can control the heterogeneity of Ly6Chigh monocyte differentiation into Ly6Clow monocytes and further reduce inflammation after AMI. The underlying mechanism might be that MSCs contribute to the increased expression of NR4A1 in Ly6Chigh/low monocytes.

Monocytes mediate homing of circulating microvesicles to the pulmonary vasculature during low-grade systemic inflammation

ABSTRACT Microvesicles (MVs), a plasma membrane-derived subclass of extracellular vesicles, are produced and released into the circulation during systemic inflammation, yet little is known of cell/tissue-specific uptake of MVs under these conditions. We hypothesized that monocytes contribute to uptake of circulating MVs and that their increased margination to the pulmonary circulation and functional priming during systemic inflammation produces substantive changes to the systemic MV homing profile. Cellular uptake of i.v.-injected, fluorescently labelled MVs (J774.1 macrophage-derived) in vivo was quantified by flow cytometry in vascular cell populations of the lungs, liver and spleen of C57BL6 mice. Under normal conditions, both Ly6Chigh and Ly6Clow monocytes contributed to MV uptake but liver Kupffer cells were the dominant target cell population. Following induction of sub-clinical endotoxemia with low-dose i.v. LPS, MV uptake by lung-marginated Ly6Chigh monocytes increased markedly, both at the individual cell level (~2.5-fold) and through substantive expansion of their numbers (~8-fold), whereas uptake by splenic macrophages was unchanged and uptake by Kupffer cells actually decreased (~50%). Further analysis of MV uptake within the pulmonary vasculature using a combined model approach of in vivo macrophage depletion, ex vivo isolated perfused lungs and in vitro lung perfusate cell-based assays, indicated that Ly6Chigh monocytes possess a high MV uptake capacity (equivalent to Kupffer cells), that is enhanced directly by endotoxemia and ablated in the presence of phosphatidylserine (PS)-enriched liposomes and β3 integrin receptor blocking peptide. Accordingly, i.v.-injected PS-enriched liposomes underwent a redistribution of cellular uptake during endotoxemia similar to MVs, with enhanced uptake by Ly6Chigh monocytes and reduced uptake by Kupffer cells. These findings indicate that monocytes, particularly lung-marginated Ly6Chigh subset monocytes, become a dominant target cell population for MVs during systemic inflammation, with significant implications for the function and targeting of endogenous and therapeutically administered MVs, lending novel insights into the pathophysiology of pulmonary vascular inflammation.

Extracellular vesicles derived from Krüppel-Like Factor 2-overexpressing endothelial cells attenuate myocardial ischemia-reperfusion injury by preventing Ly6Chigh monocyte recruitment.

Background: The ischemia/reperfusion (I/R) process in patients with ST-segment elevation myocardial infarction (STEMI) triggers an immune response, resulting in myocyte death. Krüppel-Like Factor 2 (KLF2), which is highly expressed in endothelial cells (ECs) under laminar flow, exerts anti-inflammatory effects. In this study, we explored the role of small extracellular vesicles (EVs) from KLF2-overexpressing ECs (KLF2-EVs) in the immunomodulation and its implications in myocardial I/R injury.Methods and Results: The small EVs were isolated from KLF2-overexpressing ECs' supernatant using gradient centrifugation. Mice were subjected to 45 min of ischemia followed by reperfusion, and KLF2-EVs were administrated through intravenous injection. KLF2-EVs ameliorated I/R injury and alleviated inflammation level in the serum and heart. We employed the macrophage depletion model and splenectomy and showed that Ly6Chigh monocyte recruitment from bone marrow was the main target of KLF2-EVs. miRNA-sequencing of KLF2-EVs and bioinformatics analysis implicated miRNA-24-3p (miR-24-3p) as a potent candidate mediator of monocyte recruitment and CCR2 as a downstream target. miR-24-3p mimic inhibited the migration of Ly6Chigh monocytes, and miR-24-3p antagomir reversed the effect of KLF2-EVs in myocardial I/R.Conclusion: Our data demonstrated that KLF2-EVs attenuated myocardial I/R injury in mice via shuttling miR-24-3p that restrained the Ly6Chigh monocyte recruitment. Thus, KLF2-EVs could be a potential therapeutic agent for myocardial I/R injury.

Acute peripheral inflammation and post-traumatic sleep differ between sexes after experimental diffuse brain injury.

Identifying differential responses between sexes following traumatic brain injury (TBI) can elucidate the mechanisms behind disease pathology. Peripheral and central inflammation in the pathophysiology of TBI can increase sleep in male rodents, but this remains untested in females. We hypothesized that diffuse TBI would increase inflammation and sleep in males more so than in females. Diffuse TBI was induced in C57BL/6J mice and serial blood samples were collected (baseline, 1, 5, 7days post-injury [DPI]) to quantify peripheral immune cell populations and sleep regulatory cytokines. Brains and spleens were harvested at 7DPI to quantify central and peripheral immune cells, respectively. Mixed-effects regression models were used for data analysis. Female TBI mice had 77%-124% higher IL-6 levels than male TBI mice at 1 and 5DPI, whereas IL-1β and TNF-α levels were similar between sexes at all timepoints. Despite baseline sex differences in blood-measured Ly6Chigh monocytes (females had 40% more than males), TBI reduced monocytes by 67% in TBI mice at 1DPI. Male TBI mice had 31%-33% more blood-measured and 31% more spleen-measured Ly6G+ neutrophils than female TBI mice at 1 and 5DPI, and 7DPI, respectively. Compared with sham, TBI increased sleep in both sexes during the first light and dark cycles. Male TBI mice slept 11%-17% more than female TBI mice, depending on the cycle. Thus, sex and TBI interactions may alter the peripheral inflammation profile and sleep patterns, which might explain discrepancies in disease progression based on sex.

Ly-6Chigh inflammatory-monocyte recruitment is regulated by p38 MAPK/MCP-1 activation and promotes ventilator-induced lung injury

Lymphocyte antigen 6Chigh (Ly-6Chigh) inflammatory monocytes, as novel mononuclear cells in the innate immune system, participate in infectious diseases. In this study, we investigated the potential role of these monocytes in ventilator-induced lung injury (VILI) and the possible mechanism involved in their migration to lung tissue. Our results showed that mechanical ventilation with high tidal volume (HTV) increased the accumulation of Ly-6Chigh inflammatory monocytes in lung tissues and that blocking C‑C chemokine receptor 2 (CCR2) could significantly reduce Ly-6Chigh inflammatory-monocyte migration and attenuate the degree of inflammation of lung tissues. In addition, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) activity could decrease the secretion of monocyte chemoattractant protein 1 (MCP-1), which in turn decreased the migration of Ly-6Chigh inflammatory monocytes into lung tissue. We also demonstrated that high ventilation caused Ly-6Chigh inflammatory monocytes in the bone marrow to migrate into and aggregate in the lungs, creating inflammation, and that the mechanism was quite different from that of infectious diseases. Ly-6Chigh inflammatory monocytes might play a pro-inflammatory role in VILI, and blocking their infiltration into lung tissue might become a new target for the treatment of this injury.

Mesenchymal stem cell–derived conditioned medium attenuate angiotensin II‐induced aortic aneurysm growth by modulating macrophage polarization

Mesenchymal stem cells (MSCs) exhibit therapeutic benefits on aortic aneurysm (AA); however, the molecular mechanisms are not fully understood. The current study aimed to investigate the therapeutic effects and potential mechanisms of murine bone marrow MSC (BM‐MSCs)–derived conditioned medium (MSCs‐CM) on angiotensin II (AngII)‐induced AA in apolipoprotein E‐deficient (apoE−/−) mice. Murine BM‐MSCs, MSCs‐CM or control medium were intravenously administrated into AngII‐induced AA in apoE−/− mice. Mice were sacrificed at 2 weeks after injection. BM‐MSCs and MSCs‐CM significantly attenuated matrix metalloproteinase (MMP)‐2 and MMP‐9 expression, aortic elastin degradation and AA growth at the site of AA. These treatments with BM‐MSCs and MSCs‐CM also decreased Ly6chigh monocytes in peripheral blood on day 7 and M1 macrophage infiltration in AA tissues on day 14, whereas they increased M2 macrophages. In addition, BM‐MSCs and MSCs‐CM reduced MCP‐1, IL‐1Ra and IL‐6 expression and increased IL‐10 expression in AA tissues. In vitro, peritoneal macrophages were co‐cultured with BM‐MSCs or fibroblasts as control in a transwell system. The mRNA and protein expression of M2 macrophage markers were evaluated. IL‐6 and IL‐1β were reduced, while IL‐10 was increased in the BM‐MSC systems. The mRNA and protein expression of M2 markers were up‐regulated in the BM‐MSC systems. Furthermore, high concentration of IGF1, VEGF and TGF‐β1 was detected in MSCs‐CM. Our results suggest that MSCs‐CM could prevent AA growth potentially through regulating macrophage polarization. These results may provide a new insight into the mechanisms of BM‐MSCs in the therapy of AA.

P6598MicroRNA 21 and Hypoxia Inducible Factor 1 synchronize the impact of B lymphocytes on cardiac function after acute myocardial infarction

Abstract Background Myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Mature B lymphocytes have been shown to exacerbate tissue injury and deterioration of cardiac function after MI. However, the cellular and molecular mechanisms governing B cell deleterious effects in the ischemic milieu remain to be defined. Purpose In this study, we speculate that endogenous activation of the miR21/HIFα-related pathways mediates the effect of B lymphocytes on post-ischemic cardiac remodeling. Methods Acute MI was induced by permanent ligation of the left anterior descending artery in mice. Cardiac function and remodeling was determined by echocardiography and immunohistochemistry. Inflammatory cell number and phenotype were defined by FACS analysis. To evaluate the role of HIFα isoforms in B cells, we generated mice with B cell lineage specific (Cd79aCre/+) conditional deletion of HIF1α (HIF1αflox/flox), HIF2α (HIF2αflox/flox), or both isoforms (HIF1α-HIF2αflox/flox). Results Acute MI increased miR21 levels in B cells. miR21 deficient mice showed reduced B cell numbers in the spleen, blood and subsequently in the injured cardiac tissue. Transplantation of bone marrow derived cells isolated from miR21-deficient mice (miR21−/−) improved cardiac function and remodeling when compared to administration of wild-type (WT) bone marrow cells. Similarly, in Rag1−/− immunodeficient mice with acute MI, re-supplementation with miR21−/− B lymphocytes restored cardiac repair and function when compared to injection of WT B cells. miR21 abrogated PTEN contents and subsequently enhanced HIF1α levels in cultured B cells. B cell deletion of HIF1α, but not that of HIF2α, reduced B cell accumulation and improved cardiac function after MI. Mice, which were equally deficient in HIF1α and HIF2α, also exhibited abrogation of adverse ventricular remodeling and showed recovery of cardiac function after MI. Toll like receptor agonist, CpG, fostered the release of the monocyte chemo-attractant protein, Ccl7, in cultured WT B cells but not in miR21- or HIF1α- deficient B cells. Ccl7 circulating levels were also reduced in miR21−/− and Cd79aCre/+/HIF1α flox/flox animals after acute MI. Ccl7 down-regulation hampered Ly6Chigh monocyte infiltration in the ischemic myocardium, leading to decreased infarct size and interstitial fibrosis, supporting cardiac repair. Conclusion This work reveals a novel function for miR21/HIF1α related pathways in B lymphocyte dependent effect on cardiac function and remodeling in the setting of acute MI.

P5988Non-classical Ly6C-low monocytes contribute to aortic valve stenosis development in mice

Abstract Introduction Aortic valve stenosis (AS) is one of the most common valvular diseases in the western world. Once patients with AS develop symptoms mortality increases up to 50% over two years. For many years AS was believed to be a passive degenerative process. Recent findings suggest a more complex pathomechanism with a strict molecular and cellular regulation involving local inflammation and invasion of immune cells. Specifically, pro-inflammatory macrophages have been shown to play an important role in disease progression. On the other hand, monocyte counts in the peripheral blood of patients with severe AS are significantly decreased and there is an inverse correlation between valve orifice area and circulating monocyte count. In order to elucidate the exact function of pro-inflammatory classical (Ly6C-high) and non-classical (Ly6C-low) monocytes in disease initiation processes, we conducted a series of experiments in a model of murine AS. Methods In this study 10–12 weeks old male C57BL6/J or CX3CR1-deficient mice were investigated. For AS induction, a coronary springwire was introduced into the left ventricle, pushed and rotated over the aortic valve under echocardiographic guidance. In sham operated mice the wire was only inserted into the left carotid artery without manipulation of the aortic valve. Development of AS was confirmed via weekly ultrasound examinations. Immune cells in the aortic valve were quantified using flow cytometry and immunofluorescence microscopy. Results Ultrasound analysis after two weeks confirmed the development of AS in the injured wildtype mice. Transaortic peak velocity levels were significantly increased compared to sham operated mice. Flow cytometry of explanted aortic valves showed a strong cellular immunoreaction in the stenotic valves. The amount of anti-inflammatory Ly6C-low monocytes was significantly increased compared to sham-mice, whereas the number of pro-inflammatory Ly6C-high monocytes remained stable. Interestingly CD-4 positive T-cells were augmented in stenotic aortic valves. Immunofluorescence confirmed Ly6C-low monocytes infiltration in the valve, in close proximity to the endothelial cells on the ventricular and the aortic side. The expression of recruitment and adhesion markers CD11b and CX3CR1 were significantly increased on the surface of the Ly6C-low monocytes of AS mice. To investigate the role of Ly6C-low monocytes in AS development, CX3CR1 deficient mice, which are not able to recruit non-classical monocytes via CX3CR1, were subjected to aortic valve wire injury. After two weeks, peak velocity levels in CX3CR1−/− mice remained nearly unchanged and were significantly decreased compared to wildtype mice. Conclusion Our data suggest that non-classical Ly6Clow monocytes play a crucial role in AS development in mice. Therefore, they may be targets for future therapeutic interventions. Acknowledgement/Funding S.T.N. was funded by Else-Kröner-Fresenius-Foundation of the Medical Faculty of the University of Bonn

Role and mechanism of Ly6Chigh monocyte in ventilator-induced lung injury in mice

To investigate the role and mechanism of Ly6Chigh monocyte in mice with ventilator-induced lung injury (VILI). Forty-eight healthy male SPF C57BL/6 mice were divided into spontaneous breathing group (n = 8), normal tidal volume (VT) group (VT was 8 mL/kg, n = 8), and high VT group (VT was 20 mL/kg, n = 32). The mice in the high VT group were subdivided into 1, 2, 3 and 4 hours subgroups, with 8 mice in each subgroup. All mice underwent direct tracheal intubation, those in the spontaneous breathing group maintained spontaneous breathing, and those in the normal VT group and high VT group were mechanically ventilated with different VT. After ventilation for 4 hours, bronchoalveolar lavage fluid (BALF) was collected to determine total protein, and the levels of inflammatory factors including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were determined by enzyme-linked immune sorbent assay (ELISA). The lung tissues were harvested to determine the wet/dry (W/D) ratio, and lung tissue injury was assessed in terms of lung histopathologic examination after hematoxylin-eosin (HE) staining under the light microscope. The protein expressions of monocyte chemotactic protein-1 (MCP-1) and CC-chemokine receptor 2 (CCR2) in lung tissues were determined by Western Blot. Flow cytometry was used to detect the proportion of Ly6Chigh monocyte in lung tissue. The histopathology of lung tissue structures was normal in the spontaneous breathing group and the normal VT group. Inflammatory reaction began to appear at 2 hours of high VT ventilation, and inflammatory reaction was gradually aggravated with the time extension. Compared with the spontaneous breathing group, the total protein, TNF-α, and IL-1β levels in BALF, the lung W/D ratio and MCP-1 expression were increased from 2 hours of high VT ventilation [total protein in BALF (g/L): 1.05±0.13 vs. 0.58±0.11, TNF-α in BALF (ng/L): 116.86±16.14 vs. 38.27±8.00, IL-1β in BALF (ng/L): 178.98±10.41 vs. 117.56±23.40, lung W/D ratio: 5.76±0.27 vs. 4.98±0.39, MCP-1/GAPDH: 0.87±0.19 vs. 0.29±0.12, all P < 0.05], and CCR2 expression and the proportion of Ly6Chigh monocyte was significantly increased from 3 hours of high VT ventilation [CCR2/GAPDH: 0.84±0.19 vs. 0.24±0.11, Ly6Chigh monocyte proportion: (9.01±2.47)% vs. (1.06±0.35)%, both P < 0.05], and they all showed an increased tendency with the time extension. There was no significant difference in the parameters mentioned above among the spontaneous breathing group, normal VT group and high VT ventilation 1-hour group. Ly6Chigh monocytes are involved in VILI, which aggravate VILI by activating the MCP-1/CCR2 axis.

Oncostatin M reduces atherosclerosis development in APOE*3Leiden.CETP mice and is associated with increased survival probability in humans

ObjectivePrevious studies indicate a role for Oncostatin M (OSM) in atherosclerosis and other chronic inflammatory diseases for which inhibitory antibodies are in development. However, to date no intervention studies with OSM have been performed, and its relation to coronary heart disease (CHD) has not been studied.Approach and resultsGene expression analysis on human normal arteries (n = 10) and late stage/advanced carotid atherosclerotic arteries (n = 127) and in situ hybridization on early human plaques (n = 9) showed that OSM, and its receptors, OSM receptor (OSMR) and Leukemia Inhibitory Factor Receptor (LIFR) are expressed in normal arteries and atherosclerotic plaques. Chronic OSM administration in APOE*3Leiden.CETP mice (n = 15/group) increased plasma E-selectin levels and monocyte adhesion to the activated endothelium independently of cholesterol but reduced the amount of inflammatory Ly-6CHigh monocytes and atherosclerotic lesion size and severity. Using aptamer-based proteomics profiling assays high circulating OSM levels were shown to correlate with post incident CHD survival probability in the AGES‐Reykjavik study (n = 5457).ConclusionsChronic OSM administration in APOE*3Leiden.CETP mice reduced atherosclerosis development. In line, higher serum OSM levels were correlated with improved post incident CHD survival probability in patients, suggesting a protective cardiovascular effect.

Differential expression of Plg-RKT and its effects on migration of proinflammatory monocyte and macrophage subsets

AbstractMembrane-bound plasmin is used by immune cells to degrade extracellular matrices, which facilitates migration. The plasminogen receptor Plg-RKT is expressed by immune cells, including monocytes and macrophages. Among monocytes and macrophages, distinct subsets can be distinguished based on cell surface markers and pathophysiological function. We investigated expression of Plg-RKT by monocyte and macrophage subsets and whether potential differential expression might have functional consequences for cell migration. Proinflammatory CD14++CD16+ human monocytes and Ly6Chigh mouse monocytes expressed the highest levels of Plg-RKT and bound significantly more plasminogen compared with the other respective subsets. Proinflammatory human macrophages, generated by polarization with lipopolysaccharide and interferon-γ, showed significantly higher expression of Plg-RKT compared with alternatively activated macrophages, polarized with interleukin-4 and interleukin-13. Directional migration of proinflammatory monocytes was plasmin dependent and was abolished by anti–Plg-RKT monoclonal antibody, ε-amino-caproic acid, aprotinin, and the aminoterminal fragment of urokinase-type plasminogen activator. In an in vivo peritonitis model, significantly less Ly6Chigh monocyte recruitment was observed in Plg-RKT−/− compared with Plg-RKT+/+ mice. Immunohistochemical analysis of human carotid plaques and adipose tissue showed that proinflammatory macrophages also exhibited high levels of Plg-RKT in vivo. Our data demonstrate higher expression of Plg-RKT on proinflammatory monocyte and macrophage subsets that impacts their migratory capacity.

Enhanced monocyte recruitment and delayed alternative macrophage polarization accompanies impaired repair following myocardial infarction in C57BL/6 compared to BALB/c mice.

SummaryActivation of the innate immune response following myocardial infarction (MI) is essential for infarct repair. Preclinical models of MI commonly use C57BL/6 mice, which have a type 1‐dominant immune response, whereas other mouse strains such as BALB/c mice have a type 2‐dominant immune response. We compared C57BL/6 and BALB/c mice to investigate whether predisposition towards a proinflammatory phenotype influences the dynamics of the innate immune response to MI and associated infarct healing and the risk of cardiac rupture. MI was induced by permanent coronary artery ligation in 12–15‐week‐old male wild‐type BALB/c and C57BL/6 mice. Prior to MI, C57BL/6 mice had a lower proportion of CD206+ anti‐inflammatory macrophages in the heart and an expanded blood pool of proinflammatory Ly6Chigh monocytes in comparison to BALB/c mice. The systemic inflammatory response in C57BL/6 mice following MI was more pronounced, with greater peripheral blood Ly6Chigh monocytosis, splenic Ly6Chigh monocyte mobilization and myeloid cell infiltration of pericardial adipose tissue. This led to an increased and prolonged macrophage accumulation, as well as delayed transition towards anti‐inflammatory macrophage polarization in the infarct zone and surrounding tissues of C57BL/6 mice. These findings accompanied a higher rate of mortality due to cardiac rupture in C57BL/6 mice compared with BALB/c mice. We conclude that lower post‐MI survival of C57BL/6 mice over BALB/c mice is mediated in part by a more pronounced and prolonged inflammatory response. Outcomes in BALB/c mice highlight the therapeutic potential of modulating resolution of the innate immune response following MI for the benefit of successful infarct healing.

Cardiac Hyaluronan Synthesis Is Critically Involved in the Cardiac Macrophage Response and Promotes Healing After Ischemia Reperfusion Injury.

Immediate changes in the ECM (extracellular matrix) microenvironment occur after myocardial ischemia and reperfusion (I/R) injury. Aim of this study was to unravel the role of the early hyaluronan (HA)-rich ECM after I/R. Genetic deletion of Has2 and Has1 was used in a murine model of cardiac I/R. Chemical exchange saturation transfer imaging was adapted to image cardiac ECM post-I/R. Of note, the cardiac chemical exchange saturation transfer signal was severely suppressed by Has2 deletion and pharmacological inhibition of HA synthesis 24 hours after I/R. Has2 KO ( Has2 deficient) mice showed impaired hemodynamic function suggesting a protective role for endogenous HA synthesis. In contrast to Has2 deficiency, Has1-deficient mice developed no specific phenotype compared with control post-I/R. Importantly, in Has2 KO mice, cardiac macrophages were diminished after I/R as detected by 19F MRI (magnetic resonance imaging) of perfluorcarbon-labeled immune cells, Mac-2/Galectin-3 immunostaining, and FACS (fluorescence-activated cell sorting) analysis (CD45+CD11b+Ly6G-CD64+F4/80+cells). In contrast to macrophages, cardiac Ly6Chigh and Ly6Clow monocytes were unaffected post-I/R compared with control mice. Mechanistically, inhibition of HA synthesis led to increased macrophage apoptosis in vivo and in vitro. In addition, α-SMA (α-smooth muscle actin)-positive cells were reduced in the infarcted myocardium and in the border zone. In vitro, the myofibroblast response as measured by Acta2 mRNA expression was reduced by inhibition of HA synthesis and of CD44 signaling. Furthermore, Has2 KO fibroblasts were less able to contract collagen gels in vitro. The effects of HA/CD44 on fibroblasts and macrophages post-I/R might also affect intercellular cross talk because cardiac fibroblasts were activated by monocyte/macrophages and, in turn, protected macrophages from apoptosis. Increased HA synthesis contributes to postinfarct healing by supporting macrophage survival and by promoting the myofibroblast response. Additionally, imaging of cardiac HA by chemical exchange saturation transfer post-I/R might have translational value.

Intervention of Inflammatory Monocyte Activity Limits Dermal Fibrosis

Monocytes and monocyte-derived cells are important players in the initiation, progression, and resolution of inflammatory skin reactions. As inflammation is a prerequisite for fibrosis development, we focused on the role of monocytes in cutaneous fibrosis, the clinical hallmark of patients suffering from systemic sclerosis. Investigating the function of monocytes in reactive oxygen species-induced dermal fibrosis, we observed that early monocyte depletion partially reduced disease severity. Low numbers of inflammatory Ly6Chigh monocytes, as well as inhibition of CCR2 and CCL2 in wild type animals by a specific L-RNA aptamer, mitigated disease parameters, indicating a pivotal role for CCR2+ inflammatory monocytes and the CCR2/CCL2 axis in fibrosis development. Of note, mice lacking splenic reservoirs failed to recruit monocytes to the skin and developed less fibrosis. Furthermore, enforced monocyte conversion into noninflammatory, patrolling Ly6Clow monocytes by a nuclear receptor Nur77-agonist also resulted in significantly impaired cutaneous inflammation and dermal fibrosis. Most evident, pronounced monocyte conversion in interferon stimulated gene 12-deficient mice with pronounced nuclear Nur77 signaling completely protected from dermal fibrosis. Our study shows that inflammatory monocytes that are recruited from splenic reservoirs play a key role in the development of skin fibrosis and can be therapeutically challenged by forced conversion via the Nur77/interferon stimulated gene 12 axis.