Olive polyphenols attenuate TNF-α-stimulated M-CSF and IL-6 synthesis in osteoblasts: Suppression of Akt and p44/p42 MAP kinase signaling pathways.

BACKGROUND: Olive oil polyphenols, which possess cytoprotective activities like anti-oxidant and anti-inflammatory effects, could modulate osteoblast functions. The aim of this study is to elucidate the effects and the underlying mechanisms of hydroxytyrosol and oleuropein on the tumor necrosis factor-α (TNF-α)-induced macrophage colony-stimulating factor (M-CSF) and interleukin-6 (IL-6) synthesis in osteoblasts. METHODS: Osteoblast-like MC3T3-E1 cells were pretreated with hydroxytyrosol, oleuropein, deguelin, PD98059 or wedelolactone, and then stimulated by TNF-α. The levels of M-CSF and IL-6 in the conditioned medium were determined with ELISA. The mRNA expression levels of M-CSF or IL-6 were determined with real-time RT-PCR. The phosphorylation levels of Akt, p44/p42 mitogen-activated protein (MAP) kinase or NF-κB in the cell lysates were determined with Western blot analysis. RESULTS: Hydroxytyrosol and oleuropein attenuated the TNF-α-stimulated M-CSF release. Deguelin, an inhibitor of Akt, significantly suppressed the TNF-α-stimulated M-CSF release, which failed to be affected by the MEK1/2 inhibitor PD98059 or the IκB inhibitor wedelolactone. Hydroxytyrosol and oleuropein suppressed the TNF-α-induced phosphorylation of Akt and p44/p42 MAP kinase. Hydroxytyrosol and oleuropein attenuated the TNF-α-stimulated IL-6 release. Hydroxytyrosol suppressed the TNF-α-induced mRNA expressions of M-CSF and IL-6. Hydroxytyrosol or oleuropein failed to affect the cell viability. CONCLUSION: Our present findings strongly suggest that olive oil polyphenols hydroxytyrosol and oleuropein down-regulates TNF-α signaling at the points upstream of Akt and p44/p42 MAP kinase in osteoblasts, leading to the attenuation of M-CSF and IL-6 synthesis.

RNAscope CSF1 chromogenic in situ hybridization: a potentially useful tool in the differential diagnosis of tenosynovial giant cell tumors.

Colony stimulating factor-1 (CSF1) upregulation and CSF1/colony-stimulating factor 1 receptor (CSF1R) signaling pathway is central to the tumorigenesis of tenosynovial giant cell tumors (TGCT) of both localized (LTGCT) and diffuse (DTGCT) types, and has been demonstrated in a small number of malignant tumors (MTGCT) as well. In situ hybridization for CSF1 mRNA has been shown to be potentially useful in the diagnosis of TGCT, although only a relatively small number of cases have been studied. We studied CSF1 mRNA expression using RNAscope chromogenic in situ hybridization (CISH) in standard tissue sections from 31 TGCT and 26 non-TGCT, and in tumor microarray slides (Pantomics normal MN0341, Pantomics tumor MTU391, Pantomics melanoma MEL961). Among normal tissues, CSF1 mRNA expression was invariably present in synovium (10/10, 100%) and absent in all other normal tissues. All LTGCT and DTGCT were positive (24/24, 100%), exclusively in large, eosinophilic synoviocytes. MTGCT contained large clusters of CSF1-positive malignant synoviocytes (8/8, 100%); malignant spindled cells were also positive. Among non-TGCT, CSF1 CISH was less often positive with high specificity (90%). CSF1-positive cases included leiomyosarcoma, giant cell tumor of bone and of soft parts, pulmonary carcinoma and others. The sensitivity and specificity of RNAscope CSF1 mRNA CISH for the diagnosis of TGCT were 100% and 90%, respectively. We conclude that RNAscope CSF1 CISH may be a valuable adjunct for the diagnosis of TGCT of all types, especially those with atypical or malignant morphologic features. Detection of CSF1 mRNA expression may also have predictive significance in cases where use of the CSF1 inhibitor pexidartinib is considered.

Oct4 promotes M2 macrophage polarization through upregulation of macrophage colony-stimulating factor in lung cancer.

BACKGROUND: Expression of Oct4 maintains cancer stem cell (CSC)-like properties in lung cancer cells and is correlated with poor prognosis of lung adenocarcinoma. M2-type tumor-associated macrophages (TAMs) promote cancer cell migration and metastasis. Tumor microenvironments promote monocyte differentiation into M2 TAMs via a complex cytokine-based connection. We explored the role of Oct4 in cytokine secretion in lung cancer and its impact on M2 TAM polarization. METHODS: Monocytes co-cultured with the conditioned medium from Oct4-overexpressing lung cancer cells were used to investigate M2 TAM differentiation. The inflammatory factors in the conditioned medium of Oct4-overexpressing A549 cells were examined using human inflammation antibody arrays. The correlations of Oct4, macrophage colony-stimulating factor (M-CSF), and M2 TAMs were validated in lung cancer cells, syngeneic mouse lung tumor models, and clinical samples of non-small cell lung cancer (NSCLC). RESULTS: Oct4-overexpressing A549 cells expressed elevated levels of M-CSF, which contributed to increased M2 macrophages and enhanced tumor migration. Overexpression of Oct4 enhanced tumor growth and reduced the survival of lung tumor-bearing mice, which was correlated with increased number of M2 macrophages in lung cancer. Notably, NSCLC patients with high expression levels of Oct4, M-CSF, and M2 TAMs had the poorest recurrence-free survival. A positive correlation between Oct4, M-CSF, and M2 TAMs was observed in the tumor tissue of NSCLC patient. Treatment with all-trans retinoic acid exerted anti-tumor effects and reduced M2 TAMs in tumor-bearing mice. CONCLUSIONS: Our results indicate that Oct4 expressed by lung cancer cells promotes M2 macrophage polarization through upregulation of M-CSF secretion, leading to cancer growth and metastasis. Our findings also implicate that the Oct4/M-CSF axis in M2 macrophage polarization may be potential therapeutic targets for lung cancer.

Malt1 deficient mice develop osteoporosis independent of osteoclast-intrinsic effects of Malt1 deficiency.

This study tested the hypothesis that mucosa associated lymphoid tissue 1 (Malt1) deficiency causes osteoporosis in mice by increasing osteoclastogenesis and osteoclast activity. A patient with combined immunodeficiency (CID) caused by MALT1 deficiency had low bone mineral density resulting in multiple low impact fractures that was corrected by hematopoietic stem cell transplant (HSCT). We have reported that Malt1 deficient Mϕs, another myeloid cell type, are hyper-responsive to inflammatory stimuli. Our objectives were to determine whether Malt1 deficient mice develop an osteoporosis-like phenotype and whether it was caused by Malt1 deficiency in osteoclasts. We found that Malt1 deficient mice had low bone volume by 12 weeks of age, which was primarily associated with reduced trabecular bone. Malt1 protein is expressed and active in osteoclasts and is induced by receptor activator of NF-κB ligand (RANKL) in preosteoclasts. Malt1 deficiency did not impact osteoclast differentiation or activity in vitro. However, Malt1 deficient (Malt1-/- ) mice had more osteoclasts in vivo and had lower levels of serum osteoprotegerin (OPG), an endogenous inhibitor of osteoclastogenesis. Inhibition of Malt1 activity in Mϕs induced MCSF production, required for osteoclastogenesis, and decreased OPG production in response to inflammatory stimuli. In vitro, MCSF increased and OPG inhibited osteoclastogenesis, but effects were not enhanced in Malt1 deficient osteoclasts. These data support the hypothesis that Malt1 deficient mice develop an osteoporotic phenotype with increased osteoclastogenesis in vivo, but suggest that this is caused by inflammation rather than an effect of Malt1 deficiency in osteoclasts.

Sleep modulates haematopoiesis and protects against atherosclerosis.

Sleep is integral to life1. Although insufficient or disrupted sleep increases the risk of multiple pathological conditions, including cardiovascular disease2, we know little about the cellular and molecular mechanisms by which sleep maintains cardiovascular health. Here we report that sleep regulates haematopoiesis and protects against atherosclerosis in mice. We show that mice subjected to sleep fragmentation produce more Ly-6Chigh monocytes, develop larger atherosclerotic lesions and produce less hypocretin-a stimulatory and wake-promoting neuropeptide-in the lateral hypothalamus. Hypocretin controls myelopoiesis by restricting the production of CSF1 by hypocretin-receptor-expressing pre-neutrophils in the bone marrow. Whereas hypocretin-null and haematopoietic hypocretin-receptor-null mice develop monocytosis and accelerated atherosclerosis, sleep-fragmented mice with either haematopoietic CSF1 deficiency or hypocretin supplementation have reduced numbers of circulating monocytes and smaller atherosclerotic lesions. Together, these results identify a neuro-immune axis that links sleep to haematopoiesis and atherosclerosis.

Modulation of Macrophage Polarization by Human Glomerular Mesangial Cells in Response to the Stimuli in Renal Microenvironment.

Mesangial cell (MC) activation and macrophage infiltration are 2 major events closely related with each other in mesangial proliferative glomerulonephritis. In the anti-Thy 1 nephritis model, macrophages mediate the damage and also the expansion of mesangium through secreting various inflammatory factors; however, in glomerular microenvironment how MCs affect macrophage activity in the presence of various stimuli have not yet been understood. In the present study, we found that resting human MCs (HMCs) constitutively expressed chemokine [C-C motif] ligand 2 (CCL-2) and interleukin (IL)-6 and induced M2 polarization of macrophages in the coculture system. HMC proliferation and migration and expression of IL-6, CCL-2, and macrophage colony-stimulating factor in HMCs were enhanced after platelet-derived growth factor (PDGF)-BB stimulation, among which CCL-2 was responsible for inducing the M2 polarization of macrophages. Furthermore, PDGF-BB-stimulated HMCs alleviated the classical activation of macrophages and drove more intensified M2 polarization of macrophages than resting HMCs did. However, lipopolysaccharide and interferon-γ (IFN-γ) stimulated HMCs maintained the M1 phenotype of cocultured macrophages. In conclusion, MCs actively participated in glomerular inflammation through influencing macrophage polarization. The interplay between MCs and infiltrated macrophages is finely modulated by secretory factors such as PDGF-BB and IFN-γ in response to the renal inflammatory microenvironment.

Cysteinyl leukotriene receptor 1 (cysLT1R) regulates osteoclast differentiation and bone resorption.

Excessive bone resorption induced by abnormal osteoclast differentiation has been associated with bone microstructure damage and bone-associated disorders, including osteoporosis. Here, we investigated the physiological roles of the type 1 cysteinyl leukotriene receptor (cysLTR-1) and the pharmacological functions of the specific cysLTR-1 antagonist montelukast on M-CSF- and RANKL-induced osteoclast differentiation. We showed that cysLTR-1 but not cysLTR-2 is expressed in osteoclast precursor cells: mouse bone marrow-derived macrophages (BMMs). We also found that treatment with M-CSF and RANKL significantly increased expression of cysLTR-1. Overexpression of cysLTR-1 promoted osteoclast differentiation of BMMs by increasing NFATc1 and TRAP. In contrast, treatment with montelukast prevented M-CSF- and RANKL-induced osteoclast differentiation of BMMs. Mechanically, our findings demonstrate that montelukast treatment attenuated activation of the ERK1/2, p38, JNK and NF-κB signalling pathways. Additionally, we reported that montelukast treatment ameliorated the generation of ROS and calcium signalling. Importantly, the co-immunoprecipitation assay displayed that montelukast treatment prevented the interaction of RANK and TRAF6. Finally, in vivo experiments indicated that montelukast rescued the reduction of bone volume as well as trabecular number in an ovariectomy mouse model.

Overexpressed miR-145 inhibits osteoclastogenesis in RANKL-induced bone marrow-derived macrophages and ovariectomized mice by regulation of Smad3.

BACKGROUND: MicroRNAs (miRs) play an important role in osteoclastogenesis. However, no study has investigated the underlying molecular mechanisms of miR-145 in this process. The purpose of the present study was to investigate the role of miR-145 and its post-transcriptional mechanism in the progression of osteoclast differentiation. METHODS: Macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) were used to induce osteoclastogenesis originated from bone marrow-derived macrophages (BMMs). Female C57BL/6J mice were divided into sham, OVX, OVX + NC-agomir and OVX + miR-145-agomir groups. Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify osteoclasts in-vitro and in-vivo. The mRNA and protein levels in osteoclast and tibia were assayed by qRT-PCR and western blotting, respectively. RESULTS: miR-145 expression was inhibited in RANKL-induced osteoclastogenesis, whereas overexpression of miR-145 attenuated it. We further found that Smad3 is a direct target gene of miR-145 by binding with its 3'-UTR. Overexpression of miR-145 significantly suppressed Smad3 mRNA and protein expression. In-vivo, miR-145 agomir treatment inhibited osteoclast activity in OVX mice by inhibiting Smad3 expression. CONCLUSION: We provide the evidence that over-expression of miR-145 could inhibit osteoclast differentiation, at least partially, by decreasing Smad3 expression.

Long-term alterations in monocyte function after elective cardiac surgery.

Optimal immunological homoeostasis determines the long-term recovery of patients in the postoperative period. The functional adaptability of monocytes plays a pivotal role in adjusting the host's response to an insult, immunostasis and long-term health, and may help to determine successful recovery. We undertook a longitudinal analysis of the functional adaptability of monocytes in 20 patients undergoing heart surgery with cardiopulmonary bypass, as a model of severe stress. Using each patient's pre-cardiopulmonary bypass data as a baseline, we investigated the characteristics of peripheral blood monocytes' functional plasticity in-vitro before elective bypass, and three months afterwards. Approximately 30% of subjects showed diminished monocyte plasticity, as demonstrated by decreased monocyte differentiation into dendritic cells three months after bypass. Diminished monocyte functional plasticity was related to over-production of macrophage colony-stimulating factor. Adding a neutralising antibody to macrophage colony-stimulating factor corrected the monocytes' differentiation defect. Finally, patients with reduced monocyte plasticity had significantly elevated serum C-reactive protein, with a concomitant increase in cytomegalovirus IgG antibody titres, suggestive of the acquisition of immuno-suppressive traits. Our study shows that severe surgical stress resulted in a lasting immunological defect in individuals who had seemingly recovered.

Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

The formation of the mRNP complex is a critical component of translational regulation and mRNA decay. Both the 5' and 3'UTRs of CSF-1 mRNA are involved in post-transcriptional regulation. In CSF-1 mRNA, a small hairpin loop structure is predicted to form at the extreme 5' end (2-21nt) of the 5'UTR. Nucleolin binds the hairpin loop structure in the 5'UTR of CSF-1 mRNA and enhances translation, while removal of this hairpin loop nucleolin binding element dramatically represses translation. Thus in CSF-1 mRNA, the hairpin loop nucleolin binding element is critical for translational regulation. In addition, nucleolin interacts with the 3'UTR of CSF-1 mRNA and facilitates the miRISC formation which results in poly (A) tail shortening. The overexpression of nucleolin increases the association of CSF-1 mRNA containing short poly (A)n≤26, with polyribosomes. Nucleolin both forms an mRNP complex with the eIF4G and CSF-1 mRNA, and is co-localized with the eIF4G in the cytoplasm further supporting nucleolin's role in translational regulation. The distinct foci formation of nucleolin in the cytoplasm of ovarian and breast cancer cells implicates the translational promoting role of nucleolin in these cancers.

Macrophage Colony Stimulating Factor Derived from CD4+ T Cells Contributes to Control of a Blood-Borne Infection.

Dynamic regulation of leukocyte population size and activation state is crucial for an effective immune response. In malaria, Plasmodium parasites elicit robust host expansion of macrophages and monocytes, but the underlying mechanisms remain unclear. Here we show that myeloid expansion during P. chabaudi infection is dependent upon both CD4+ T cells and the cytokine Macrophage Colony Stimulating Factor (MCSF). Single-cell RNA-Seq analysis on antigen-experienced T cells revealed robust expression of Csf1, the gene encoding MCSF, in a sub-population of CD4+ T cells with distinct transcriptional and surface phenotypes. Selective deletion of Csf1 in CD4+ cells during P. chabaudi infection diminished proliferation and activation of certain myeloid subsets, most notably lymph node-resident CD169+ macrophages, and resulted in increased parasite burden and impaired recovery of infected mice. Depletion of CD169+ macrophages during infection also led to increased parasitemia and significant host mortality, confirming a previously unappreciated role for these cells in control of P. chabaudi. This work establishes the CD4+ T cell as a physiologically relevant source of MCSF in vivo; probes the complexity of the CD4+ T cell response during type 1 infection; and delineates a novel mechanism by which T helper cells regulate myeloid cells to limit growth of a blood-borne intracellular pathogen.

Thermally induced osteocyte damage initiates pro-osteoclastogenic gene expression in vivo.

Bone is often subject to harsh temperatures during orthopaedic procedures resulting in thermally induced bone damage, which may affect the healing response. Postsurgical healing of bone is essential to the success of surgery, therefore, an understanding of the thermally induced responses of bone cells to clinically relevant temperatures in vivo is required. Osteocytes have been shown to be integrally involved in the bone remodelling cascade, via apoptosis, in micro-damage systems. However, it is unknown whether this relationship is similar following thermal damage. Sprague-Dawley rat tibia were exposed to clinically relevant temperatures (47°C or 60°C) to investigate the role of osteocytes in modulating remodelling related factors. Immunohistochemistry was used to quantify osteocyte thermal damage (activated caspase-3). Thermally induced pro-osteoclastogenic genes (Rankl, Opg and M-csf), in addition to genes known to mediate osteoblast and osteoclast differentiation via prostaglandin production (Cox2), vascularization (Vegf) and inflammatory (Il1a) responses, were investigated using gene expression analysis. The results demonstrate that heat-treatment induced significant bone tissue and cellular damage. Pro-osteoclastogenic genes were upregulated depending on the amount of temperature elevation compared with the control. Taken together, the results of this study demonstrate the in vivo effect of thermally induced osteocyte damage on the gene expression profile.

Interleukin-33 stimulates GM-CSF and M-CSF production by human endothelial cells.

Interleukin (IL)-33, a member of the IL-1 family of cytokines, is involved in various inflammatory conditions targeting amongst other cells the endothelium. Besides regulating the maturation and functions of myeloid cells, granulocyte macrophage-colony stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) have been shown to play a role in such pathologies too. It was the aim of our study to investigate a possible influence of IL-33 on GM-CSF and M-CSF production by human endothelial cells. IL-33, but not IL-18 or IL-37, stimulated GM-CSF and M-CSF mRNA expression and protein production by human umbilical vein endothelial cells (HUVECs) and human coronary artery ECs (HCAECs) through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in an IL-1-independent way. This effect was inhibited by the soluble form of ST2 (sST2), which is known to act as a decoy receptor for IL-33. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor fluvastatin could also be shown to moderately reduce the IL-33-mediated effect on M-CSF, but not on GM-CSF expression. In addition, IL-33, IL-1ß, GM-CSF and M-CSF were detected in endothelial cells of human carotid atherosclerotic plaques using immunofluorescence. Upregulation of GM-CSF and M-CSF production by human endothelial cells, an effect that appears to be mediated by NF-κB and to be independent of IL-1, may be an additional mechanism through which IL-33 contributes to inflammatory activation of the vessel wall.

Role of macrophage colony-stimulating factor in the development of neointimal thickening following arterial injury.

Evidence suggests that macrophage colony-stimulating factor (M-CSF) participates critically in atherosclerosis; little is known about the role of M-CSF in the development of neointimal hyperplasia following mechanical vascular injury. We examined the expression of M-CSF and its receptor, c-fms, in rodent and rabbit models of arterial injury. Injured rat carotid arteries expressed 3- to 10-fold higher levels of M-CSF and c-fms mRNA and protein following balloon injury as compared to uninjured arteries. In the rabbit, M-CSF protein expression was greatest in neointimal smooth muscle cells (SMCs) postinjury, with some expression in medial SMCs. M-CSF-positive SMCs exhibited markers of proliferation. At 30days postinjury, neointimal SMCs in the adjacent healed area near the border between injured and uninjured zone lost both proliferative activity and overexpression of M-CSF. The presence of induced M-CSF and c-fms expression correlated with the initiation of SMCs proliferation. M-CSF stimulated incorporation of [(3)H] thymidine in human aortic smooth muscle cells in a concentration-dependent manner. Serum-free conditioned medium from aortic SMCs also promoted DNA synthesis, and this effect was blocked by M-CSF specific antibody. To test further the role of M-CSF in vivo, we induced arterial injury by placing a periadventitial collar around the carotid arteries in compound mutant mice lacking apolipoprotein apoE (apoE(-/-)) and M-CSF. Loss of M-CSF abolished the neointimal hyperplastic response to arterial injury in apoE(-/-) mice. Local delivery of M-CSF to the injured artery restored neointimal proliferation, suggesting a critical role of M-CSF for the development of neointimal thickening following arterial injury.

miR-1207-5p suppresses lung cancer growth and metastasis by targeting CSF1.

We previously reported that miR-1207-5p can inhibit epithelial-mesenchymal transition (EMT) induced by growth factors such as EGF and TGF-ß, but the exact mechanism is unclear. Here we identified that Colony stimulating factor 1 (CSF1) is a target gene of miR-1207-5p. CSF1 controls the production, differentiation and function of macrophage and promotes the release of proinflammatory chemokines. We showed that miR-1207-5p inhibited lung cancer cell A549 proliferation, migration and invasion in vitro, and suppressed the STAT3 and AKT signalings. miR-1207-5p overexpression can increase HUVEC angiogenesis, and can modulate the M2 phenotype of macrophage. miR-1207-5p also significantly inhibited A549 cells metastasis in a nude mouse xenograft model. miR-1207-5p and CSF1 expression levels and their relationship with lung cancer survival and metastasis status were assayed by means of a lung cancer tissue microarray. Macrophage is an essential part of the tumor microenvironment, thus the miR-1207-5p-CSF1 axis maybe a new regulator of lung cancer development through modulating the tumor microenvironment.

CSF1 Overexpression Promotes High-Grade Glioma Formation without Impacting the Polarization Status of Glioma-Associated Microglia and Macrophages.

Current therapies for high-grade gliomas extend survival only modestly. The glioma microenvironment, including glioma-associated microglia/macrophages (GAM), is a potential therapeutic target. The microglia/macrophage cytokine CSF1 and its receptor CSF1R are overexpressed in human high-grade gliomas. To determine whether the other known CSF1R ligand IL34 is expressed in gliomas, we examined expression array data of human high-grade gliomas and performed RT-PCR on glioblastoma sphere-forming cell lines (GSC). Expression microarray analyses indicated that CSF1, but not IL34, is frequently overexpressed in human tumors. We found that while GSCs did express CSF1, most GSC lines did not express detectable levels of IL34 mRNA. We therefore studied the impact of modulating CSF1 levels on gliomagenesis in the context of the GFAP-V12Ha-ras-IRESLacZ (Ras*) model. Csf1 deficiency deterred glioma formation in the Ras* model, whereas CSF1 transgenic overexpression decreased the survival of Ras* mice and promoted the formation of high-grade gliomas. Conversely, CSF1 overexpression increased GAM density, but did not impact GAM polarization state. Regardless of CSF1 expression status, most GAMs were negative for the M2 polarization markers ARG1 and CD206; when present, ARG1(+) and CD206(+) cells were found in regions of peripheral immune cell invasion. Therefore, our findings indicate that CSF1 signaling is oncogenic during gliomagenesis through a mechanism distinct from modulating GAM polarization status. Cancer Res; 76(9); 2552-60. ©2016 AACR.

Modulation of Decidual Macrophage Polarization by Macrophage Colony-Stimulating Factor Derived from First-Trimester Decidual Cells: Implication in Preeclampsia.

During human pregnancy, immune tolerance of the fetal semiallograft occurs in the presence of abundant maternal leukocytes. At the implantation site, macrophages comprise approximately 20% of the leukocyte population and act as primary mediators of tissue remodeling. Decidual macrophages display a balance between anti-inflammatory and proinflammatory phenotypes. However, a shift to an M1 subtype is reported in preeclampsia. Granulocyte-macrophage colony-stimulating-factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are major differentiating factors that mediate M1 and M2 polarization, respectively. Previously, we observed the following: i) the preeclamptic decidua contains an excess of both macrophages and GM-CSF, ii) the preeclampsia-associated proinflammatory cytokines, IL-1ß and tumor necrosis factor-α, markedly enhance GM-CSF and M-CSF expression in cultured leukocyte-free first-trimester decidual cells (FTDCs), iii) FTDC-secreted GM-CSF polarizes macrophages toward an M1 subtype. The microenvironment is a key determinant of macrophage phenotype. Thus, we examined proinflammatory stimulation of FTDC-secreted M-CSF and its role in macrophage development. Immunofluorescence staining demonstrated elevated M-CSF-positive decidual cell numbers in preeclamptic decidua. In FTDCs, IL-1ß and tumor necrosis factor-α signal through the NF-κB pathway to induce M-CSF production, which does the following: i) enhances differentiation of and elevates CD163 expression in macrophages, ii) increases macrophage phagocytic capacity, and iii) inhibits signal-regulatory protein α expression by macrophages. These findings suggest that FTDC-secreted M-CSF modulates the decidual immune balance by inducing M2 macrophage polarization and phagocytic capacity in response to proinflammatory stimuli.

Understanding the Role of Cellular Molecular Clocks in Controlling the Innate Immune Response.

The importance of the 24-h daily cycle, termed circadian, on immune function has been highlighted by a number of recent studies. Immune parameters such as the response to bacterial challenge or immune cell trafficking change with time of day and disruption of circadian rhythms has been linked to inflammatory pathologies. We are beginning to uncover that the key proteins that comprise the molecular clock, most notably BMAL1, CLOCK, and REV-ERBα, also control fundamental aspects of the immune response. Given the ubiquitous nature of the molecular clock in controlling many other types of physiologies such as metabolism and cardiovascular function, a more thorough understanding of the daily rhythm of the immune system may provide important insight into aspects of patient care such as vaccinations and how we manage infectious and inflammatory diseases. In this chapter, we describe a series of experiments to look at circadian expression and function in immune cells. The experiments described herein may provide an initial assessment of the role of the molecular clock on an immune response from any cell type of interest.

Mouse host unlicensed NK cells promote donor allogeneic bone marrow engraftment.

Natural killer (NK) cells exist as subsets based on expression of inhibitory receptors that recognize major histocompatibility complex I (MHCI) molecules. NK cell subsets bearing MHCI binding receptors for self-MHCI have been termed as "licensed" and exhibit a higher ability to respond to stimuli. In the context of bone marrow transplantation (BMT), host licensed-NK (L-NK) cells have also been demonstrated to be responsible for the acute rejection of allogeneic and MHCI-deficient BM cells (BMCs) in mice after lethal irradiation. However, the role of recipient unlicensed-NK (U-NK) cells has not been well established with regard to allogeneic BMC resistance. After NK cell stimulation, the prior depletion of host L-NK cells resulted in a marked increase of donor engraftment compared with the untreated group. Surprisingly, this increased donor engraftment was reduced after total host NK cell depletion, indicating that U-NK cells can actually promote donor allogeneic BMC engraftment. Furthermore, direct coculture of U-NK cells with allogeneic but not syngeneic BMCs resulted in increased colony-forming unit cell growth in vitro, which was at least partially mediated by granulocyte macrophage colony-stimulating factor (GM-CSF) production. These data demonstrate that host NK cell subsets exert markedly different roles in allogeneic BMC engraftment where host L- and U-NK cells reject or promote donor allogeneic BMC engraftment, respectively.

Nonredundant roles of keratinocyte-derived IL-34 and neutrophil-derived CSF1 in Langerhans cell renewal in the steady state and during inflammation.

IL-34 and colony-stimulating factor 1 (CSF1) are two alternative ligands for the CSF1 receptor that play nonredundant roles in the development, survival, and function of tissue macrophages and Langerhans cells (LCs). In this study, we investigated the spatio-temporal production of IL-34 and its impact on skin LCs in the developing embryo and adult mice in the steady state and during inflammation using Il34(LacZ) reporter mice and newly generated inducible Il34-knockout mice. We found that IL-34 is produced in the developing skin epidermis of the embryo, where it promotes the final differentiation of LC precursors. In adult life, LCs required IL-34 to continually self-renew in the steady state. However, during UV-induced skin damage, LC regeneration depended on neutrophils infiltrating the skin, which produced large amounts of CSF1. We conclude that LCs require IL-34 when residing in fully differentiated and anatomically intact skin epidermis, but rely on neutrophil-derived CSF1 during inflammation. Our demonstration that neutrophils are an important source of CSF1 during skin inflammation may exemplify a mechanism through which neutrophils promote their subsequent replacement with mononuclear phagocytes.