The mandatory role of complement in the endotoxin-induced synthesis of tissue thromboplastin in blood monocytes

Osteoclasts are bone-resorptive cells that differentiate from hematopoietic precursors upon receptor activator of nuclear factor κB ligand (RANKL) activation. Previous studies demonstrated that IL-6 indirectly stimulates osteoclastogenesis through the production of RANKL by osteoblasts. However, few data described the direct effect of IL-6 on osteoclasts. To investigate this effect, we used several models: murine RAW264.7 cells, mouse bone marrow, and human blood monocytes. In the three models used, the addition of IL-6 inhibited RANKL-induced osteoclastogenesis. Furthermore, IL-6 decreased the expression of osteoclast markers and up-modulated macrophage markers. To elucidate this inhibition, signal transducer and activator of transcription (STAT) 3, the main signaling molecule activated by IL-6, was analyzed. Addition of two STAT3 inhibitors completely abolished RANKL-induced osteoclastogenesis, revealing a key role of STAT3. We demonstrated that a basal level of phosphorylated-STAT3 on Serine727 associated with an absence of phosphorylation on Tyrosine705 is essential for osteoclastogenesis. Furthermore, a decrease of Serine727 phosphorylation led to an inhibition of osteoclast differentiation, whereas an increase of Tyrosine705 phosphorylation upon IL-6 stimulation led to the formation of macrophages instead of osteoclasts. In conclusion, we showed for the first time that IL-6 inhibits RANKL-induced osteoclastogenesis by diverting cells into the macrophage lineage, and demonstrated the functional role of activated-STAT3 and its form of phosphorylation in the control of osteoclastogenesis.

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AP-1 (Activated Protein-1) Transcription Factor JunD Regulates Ischemia/Reperfusion Brain Damage via IL-1β (Interleukin-1β)

Stroke ◽

10.1161/strokeaha.118.023739 ◽

2019 ◽

Vol 50 (2) ◽

pp. 469-477 ◽

Cited By ~ 10

Author(s):

Candela Diaz-Cañestro ◽

Martin F. Reiner ◽

Nicole R. Bonetti ◽

Luca Liberale ◽

Mario Merlini ◽

...

Keyword(s):

Ischemic Stroke ◽

Brain Injury ◽

Acute Ischemic Stroke ◽

Peripheral Blood ◽

Ischemia Reperfusion ◽

Interleukin 1Β ◽

Blood Monocytes ◽

Peripheral Blood Monocytes

Background and Purpose— Inflammation is a major pathogenic component of ischemia/reperfusion brain injury, and as such, interventions aimed at inhibiting inflammatory mediators promise to be effective strategies in stroke therapy. JunD—a member of the AP-1 (activated protein-1) family of transcription factors—was recently shown to regulate inflammation by targeting IL (interleukin)-1β synthesis and macrophage activation. The purpose of the present study was to assess the role of JunD in ischemia/reperfusion-induced brain injury. Methods— WT (wild type) mice randomly treated with either JunD or scramble (control) siRNA were subjected to 45 minutes of transient middle cerebral artery occlusion followed by 24 hours of reperfusion. Stroke size, neurological deficit, plasma/brain cytokines, and oxidative stress determined by 4-hydroxynonenal immunofluorescence staining were evaluated 24 hours after reperfusion. Additionally, the role of IL-1β was investigated by treating JunD siRNA mice with an anti–IL-1β monoclonal antibody on reperfusion. Finally, JunD expression was assessed in peripheral blood monocytes isolated from patients with acute ischemic stroke. Results— In vivo JunD knockdown resulted in increased stroke size, reduced neurological function, and increased systemic inflammation, as confirmed by higher neutrophil count and lymphopenia. Brain tissue IL-1β levels were augmented in JunD siRNA mice as compared with scramble siRNA, whereas no difference was detected in IL-6, TNF-α (tumor necrosis factor-α), and 4-hydroxynonenal levels. The deleterious effects of silencing of JunD were rescued by treating mice with an anti–IL-1β antibody. In addition, JunD expression was decreased in peripheral blood monocytes of patients with acute ischemic stroke at 6 and 24 hours after onset of stroke symptoms compared with sex- and age-matched healthy controls. Conclusions— JunD blunts ischemia/reperfusion-induced brain injury via suppression of IL-1β.

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Phosphatase Shp2 exacerbates intestinal inflammation by disrupting macrophage responsiveness to interleukin-10

Journal of Experimental Medicine ◽

10.1084/jem.20181198 ◽

2019 ◽

Vol 216 (2) ◽

pp. 337-349 ◽

Cited By ~ 10

Author(s):

Peng Xiao ◽

Huilun Zhang ◽

Yu Zhang ◽

Mingzhu Zheng ◽

Rongbei Liu ◽

...

Keyword(s):

Intestinal Inflammation ◽

Interleukin 10 ◽

Blood Monocytes ◽

Stat3 Signaling ◽

Tnf Α ◽

Mouse Macrophages ◽

Inflammatory Bowel ◽

Anti Inflammatory ◽

Further Development

Inflammatory cytokines produced by activated macrophages largely contribute to the pathological signs of inflammatory bowel disease (IBD). Interleukin-10 (IL-10) is the predominant anti-inflammatory cytokine in the intestine, and its therapeutic efficacy for IBD has been clinically tested. Nevertheless, how the function of IL-10 is regulated in the intestinal microenvironment remains unknown, which largely hinders the further development of IL-10–based therapeutic strategies. Here, we found that the expression of phosphatase Shp2 was increased in colonic macrophages and blood monocytes from IBD patients compared with those from healthy controls. Shp2 deficiency in macrophages protects mice from colitis and colitis-driven colon cancer. Mechanistically, Shp2 disrupts IL-10–STAT3 signaling and its dependent anti-inflammatory response in human and mouse macrophages. Furthermore, a Shp2-inducing role of TNF-α is unveiled in our study. Collectively, our work identifies Shp2 as a detrimental factor for intestinal immune homeostasis and hopefully will be helpful in the future exploitation of IL-10 immunotherapy for IBD.

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Akt Activation Regulates Macrophage Survival and Differentiation: Role of M-CSF and Endogenous ROS.

Blood ◽

10.1182/blood.v106.11.2208.2208 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2208-2208

Author(s):

Yijie Wang ◽

Mandy M. Zeigler ◽

Greg K. Lam ◽

Melissa G. Hunter ◽

Tim D. Eubank ◽

...

Keyword(s):

Bone Marrow ◽

Cell Survival ◽

Kinase Inhibitors ◽

Critical Role ◽

Annexin V ◽

Mononuclear Phagocytes ◽

Ros Generation ◽

Blood Monocytes ◽

Human Alveolar Macrophage

Abstract Previous reports from our laboratory showed M-CSF promotes PI 3-kinase activation resulting in the production of reactive oxygen species (ROS) and PI 3-kinase inhibitors, and the antioxidants diphenyleneiodonium (DPI) and n-acetyl cysteine (NAC) suppressed M-CSF-stimulated Erk activation. In this study, we hypothesized that M-CSF-induced generation of ROS affected Akt1 activation and sought to define the role of Akt1 in monocyte/macrophage survival and differentiation. We found that the production of ROS following M-CSF-treatment was inhibited by the antioxidant DPI. The addition of either DPI or NAC to the monocytes in the presence of M-CSF resulted in decrease cell survival as measured by Annexin V/PI and DNA fragmentation. In the cells treated with the antioxidants, there was a reduction in pAKT protein levels compared to M-CSF alone treatment suggesting that ROS contributed to Akt activity and cell survival. Macrophages from p47phox −/− mice, lacking a key component of the NADPH oxidase complex required for ROS generation were examined for M-CSF-induced survival and Akt1 activation. Bone marrow macrophages from p47phox −/ − mice and wild type (WT) littermates were isolated and differentiated in RPMI-1640 medium in the presence of 20 ng/ml of M-CSF for 5 days. We observed a reduction in Akt1 phosphorylation, cellular survival and increase in apoptosis measured by Annexin V/PI staining in p47phox −/ − macrophages compared to WT controls. Since macrophages from the p47phox−/− mice had reduced Akt1 activity and cell survival to M-CSF stimulation, we next wanted to independently evaluate the role of Akt in macrophage survival and differentiation. We therefore examined macrophages from mice that had targeted expression of activated Akt1 (Myr-Akt1) in mononuclear phagocytes. Bone marrow from Myr-Akt1 mice and (WT) littermates was isolated and cultured in the presence of 20 ng/ml of M-CSF for 5 days. We found that Myr-Akt1 cells had enhanced survival and reduced apoptosis versus WT cells. Interestingly, Myr-Akt1 mice had normal circulating numbers of monocytes, but had splenomegaly and increased numbers of mature macrophages in their spleens by CD68 staining. Since the expression of Myr-Akt1 in BMM enhanced cell survival, we were interested in the effect in human monocytes. Peripheral blood monocytes (PBM) were transiently transfected with Myr-Akt1 constructs. We observed a decrease in Annexin V/PI staining indicating promotion of cell survival in PBM expressing Myr-Akt compared to untransfected PBM. Since Akt1 appeared to be important for macrophage survival/differentiation, we examined its contribution in human alveolar macrophage homeostasis and found that freshly isolated macrophages had constitutive Akt1 activity and had increased levels of Akt1 protein. This data support a critical role for Akt1 in macrophage differentiation in mice. Together, our findings may provide insight in the pathogenesis of monocyte/macrophage homeostasis.

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The Role of Beta 2 Integrins in Macrophage Migration During Resolution of Inflammation.

Blood ◽

10.1182/blood.v114.22.3600.3600 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3600-3600 ◽

Cited By ~ 1

Author(s):

Yevgeniya Kushchayeva ◽

Darya Mishchuk ◽

Tatiana Ugarova

Keyword(s):

Lymph Nodes ◽

Conflicts Of Interest ◽

Fold Increase ◽

Initial Population ◽

Blood Monocytes ◽

Resident Cell ◽

Low Levels ◽

Inflammatory Macrophages ◽

Β2 Integrins

Abstract Abstract 3600 Poster Board III-537 The mobilization of blood monocytes and their differentiation into macrophages during the immune-inflammatory response helps to prepare the tissue for resolution. During the resolution phase of inflammation macrophages do not die locally: some cells emigrate by draining lymphatics whereas some remain at the site of inflammation. The major myelo-monocytic integrin αMβ2 (Mac-1, CD11b/CD18), together with two related integrins αDβ2 (CD11d/CD18) and αXβ2 (CD11c/CD18), mediate critical adhesive reactions of monocyte/macrophages. However, the roles of these adhesion receptors in control of macrophage retention at sites of inflammation and their emigration to lymph nodes are unclear. Using a mouse model of sterile peritonitis induced by thioglycollate injection, we examined the dynamics of macrophage β2 integrins during the resolution phase of inflammation. Macrophages were defined by FACS analyses as a population of cells expressing αMβ2high, αDβ2+ and CD115+. The initial population of resident β2, positive for βDβ2 and negative for αXβ2. The thioglycollate-challenged mice showed a ∼4-fold increase in macrophages on day 3 followed by a progressive decrease to normal resident cell numbers by day 13. Expression of αMβ2 on macrophages on day 3 decreased by 2.5-fold as a result of dilution of the initial population of αMβ2high resident macrophages by infiltrating blood monocytes expressing αMβ2low. However, after day 3, the density of αMβ2 on macrophages gradually increased and by day 13 returned to the high levels characteristic of resident macrophages. By contrast, expression of αDβ2 and αXβ2 on inflammatory macrophages increased by 2-fold by day 6-9 compared to that on resident macrophages and then returned to the resident levels by day 3. Thus, although the number of macrophages decreased from day 3 to day 9 by several fold, the population of macrophages which remained in the peritoneum was enriched in cells expressing the high levels of αMβ2 and αDα2. Tracking migration of fluorescently labeled peritoneal cells demonstrated that a population of macrophages which leaves the inflamed peritoneum and enters lymph nodes consists of cells expressing low levels of αMβ2 and αDβ2. These data suggested that upregulation of β2 integrins, especially αMβ2, may be responsible for the retention of macrophages in the peritoneum. Indeed, the rate of macrophage emigration from the peritoneum in the αMβ2-deficient mice was significantly higher than that in wild-type mice. The results indicate that macrophage emigration from the inflamed site is controlled by the level of integrin αMβ2 and αDβ2 with low expressors being migratory and high expressors remaining in the peritoneum. The data also highlight the importance of integrins αDβ2 and αXβ2 as specific markers of inflammatory macrophages. Disclosures: No relevant conflicts of interest to declare.

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Investigate the role of GPR15/BOB in the SLE patients

American Journal of BioMedicine ◽

10.18081/2333-5106/018-19-32 ◽

2018 ◽

Vol 6 (1) ◽

pp. 19-32

Author(s):

Caroline G. Jackson ◽

Donald R. Kwan

Keyword(s):

Peripheral Blood ◽

Messenger Rna ◽

Viral Envelope ◽

Orphan Receptor ◽

Blood Monocytes ◽

Rt Pcr ◽

Peripheral Blood Monocytes ◽

Healthy Donors ◽

Hiv 1

GPR15 functions as a cellular co-receptor for some isolates of HIV-1, HIV-2, and SIV through interactions with several viral envelope proteins. The objective of this study was to investigate the expression of orphan receptor GPR15/BOB in the serum of SLE patients and non-SLE healthy people. GPR15/BOB expression was analysed by flow cytometry while, GPR15/BOB messenger RNA was examined in peripheral blood monocytes by RT-PCR. GPR15/BOB mRNA was detected in all periphral blood of SLE patients examined. Further, a significant increase in GPR15/BOB expression as measured by mean fluorescence intensity was observed on SLE PB neutrophils compared to these cell populations from healthy donors. We concluded that GPR15/BOB is expressed in monocytes and neutrophils in peripheral blood, and expression is up-regulated in SLE patients compared to controls. GPR15/BOB may play a role in SLE pathogenesis.

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